cDNA Sequence Research Articles

Optimizing Tris(2-Carboxyethyl)phosphine and Mercaptohexanol Concentrations for Thiolated Oligonucleotide Immobilization on Platinum Electrodes in Microfluidic Platforms.

In this study, we propose a strategy to explore the impact of the proportion of tris(2-carboxyethyl)phosphine (TCEP) and 6-mercaptohexanol (MCH) on the efficiency of oligonucleotide functionalization on PDMS microfluidic channels equipped with pairs of homemade microfabricated platinum microelectrodes. We identified an optimal concentration of these compounds that enables the effective orientation and distribution of probes, thereby facilitating subsequent target hybridization. The experiment included optimizing sample injection into microfluidic channels. We used TCEP as a reducing agent to help the DNA probes adhere to the channel electrode better. This stopped the formation of disulfide bonds during the probe immobilization step. We found the optimal TCEP/MCH mixture ratio (5 mM TCEP and 50 mM MCH), which led to a more uniform distribution and orientation of the DNA probes on the platinum electrode. These optimized conditions resulted in a more compact DNA monolayer and enhanced detection capabilities. The biosensor's performance was evaluated by the detection of the hybridization of complementary DNA sequences in the presence of equimolar Fe(CN)63-/Fe(CN)64-. The detection of the synthetic GP8 resistance gene is facilitated by a measurable decrease in the electron transfer rate, which is directly proportional to its concentration. Under the optimized conditions, the DNA biosensor showed excellent sensitivity (with a detection limit of 10-17 M) and high specificity when tested against noncomplementary DNA strands.

Long-term therapeutic efficacy and safety profiles of hpCas13d RNA editing in treating early-onset hypertrophic cardiomyopathy

AimsThrough evolving a precise RNA nuclease, hpCas13d, we have successfully inhibited hypertrophic cardiomyopathy in a compound heterozygous model. However, further investigation is needed to assess the long-term therapeutic effects and safety profiles of hpCas13d treatment. Materials and methodsAAV-hpCas13d[RQ] was subcutaneously injected into neonatal Myh6RH/RQ mice. Sequential echocardiography analyses were conducted at 4 months and 12 months to evaluate the sustained therapeutic effects of hpCas13d. Electrocardiography was employed to assess cardiac arrhythmias, and mice were euthanized at 12 months. Quantification of Myh6RQ degradation induced by hpCas13d[RQ] was performed using digital droplet PCR and cDNA sequencing. Histological analysis, RNA sequencing, and proteomic analyses were utilized to examine the inhibitory effects on pathological phenotypes and downstream signaling pathways. Biodistribution, tissue damage, and host immune response to AAV-hpCas13d[RQ] were assessed to evaluate long-term safety profiles. Key findingsThe allele-specific RNA degradation persisted for 12 months in AAV-hpCas13d[RQ]-treated Myh6RH/RQ mice. Partial degradation of pathogenic Myh6RQ transcripts proved adequate for the long-term inhibition of cardiac hypertrophy, arrhythmias, fibrosis, and cellular apoptosis in Myh6RH/RQ mice. RNA sequencing and proteomic analyses revealed that hpCas13d[RQ] treatment impeded hypertrophy and fibrosis, mitochondrial dysfunction, and abnormalities in ion channels downstream of mutant Myh6. Prolonged treatment with AAV-hpCas13d from the neonatal stage did not induce significant tissue damage, liver toxicity, humoral responses, or cellular immune reactions against the AAV9 capsid and bacterial hpCas13d. SignificanceThese results underscore the promising translational potential of AAV-hpCas13d in treating cardiovascular diseases and advancing in vivo gene therapy.

A PLIN1 polymorphism is associated with fat production in male emus

The emu (Dromaius novaehollandiae) is a novel poultry species that produces meat, eggs, and fat. Although emus have recently been domesticated, genetic improvements to establish strains have scarcely progressed. In this study, we investigated the relationship between production traits and perilipin 1-encoding gene (PLIN1) polymorphisms in the emus. We determined the partial complementary DNA (cDNA) sequence of the PLIN1, which is involved in lipid droplet formation. We identified four nucleotide substitution sites (c.270C>T, c.321T>C, c.587A>T, and c.639C>T) in the PLIN1 gene of emus. Of these, c.587A>T is a non-synonymous substitution that converts lysine to methionine at the 196th codon (p.K196M). Although p.K196M was predicted to affect the production traits of emus, a large deflection in genotype frequency was observed in this study; thus, we could not investigate the relationship between genotypes and phenotypes. In males, the fat yields of the CC, CT, and TT genotypes in c.270C>T were 0.25 ± 0.06, 0.22 ± 0.06, and 0.21 ± 0.07 kg, respectively, while the meat yields of the CC, CT, and TT genotypes in c.270C>T were 0.15 ± 0.01, 0.16 ± 0.02, and 0.16 ± 0.03 kg, respectively. These results indicate that male emus with the CC genotype had a significantly higher fat content and lower meat productivity than male emus with the other genotypes (P < 0.05). Therefore, c.270C>T in PLIN1 affects fat and meat production in males. Our findings may contribute to the effective genetic improvement of the emus.

Extracellular vesicles, RNA sequencing, and bioinformatic analyses: Challenges, solutions, and recommendations.

Extracellular vesicles (EVs) are heterogeneous entities secreted by cells into their microenvironment and systemic circulation. Circulating EVs carry functional small RNAs and other molecular footprints from their cell of origin, and thus have evident applications in liquid biopsy, therapeutics, and intercellular communication. Yet, the complete transcriptomic landscape of EVs is poorly characterized due to critical limitations including variable protocols used for EV-RNA extraction, quality control, cDNA library preparation, sequencing technologies, and bioinformatic analyses. Consequently, there is a gap in knowledge and the need for a standardized approach in delineating EV-RNAs. Here, we address these gaps by describing the following points by (1) focusing on the large canopy of the EVs and particles (EVPs), which includes, but not limited to - exosomes and other large and small EVs, lipoproteins, exomeres/supermeres, mitochondrial-derived vesicles, RNA binding proteins, and cell-free DNA/RNA/proteins; (2) examining the potential functional roles and biogenesis of EVPs; (3) discussing various transcriptomic methods and technologies used in uncovering the cargoes of EVPs; (4) presenting a comprehensive list of RNA subtypes reported in EVPs; (5) describing different EV-RNA databases and resources specific to EV-RNA species; (6) reviewing established bioinformatics pipelines and novel strategies for reproducible EV transcriptomics analyses; (7) emphasizing the significant need for a gold standard approach in identifying EV-RNAs across studies; (8) and finally, we highlight current challenges, discuss possible solutions, and present recommendations for robust and reproducible analyses of EVP-associated small RNAs. Overall, we seek to provide clarity on the transcriptomics landscape, sequencing technologies, and bioinformatic analyses of EVP-RNAs. Detailed portrayal of the current state of EVP transcriptomics will lead to a better understanding of how the RNA cargo of EVPs can be used in modern and targeted diagnostics and therapeutics. For the inclusion of different particles discussed in this article, we use the terms large/small EVs, non-vesicular extracellular particles (NVEPs), EPs and EVPs as defined in MISEV guidelines by the International Society of Extracellular Vesicles (ISEV).

Competitive Binding Strategy for Reliable Signal-off Surface Enhanced Raman Scattering Sensing in Protecting Apples from Patulin without External Interference

Patulin is a toxin that can be found in damaged or moldy fruits, especially apples. Very high detection sensitivity can be achieved using the SERS (Surface Enhanced Raman Scattering) technique, which provides information about molecular vibrations. However, unexpected desorption of target toxins from the surface of the SERS substrate and interference from other substances pose a major challenge. Here we investigate a competitive binding strategy for reliable SERS analysis of PAT. Core-Raman molecule-shell (CMS) structures and programmable, specific DNA interactions are utilized to regulate Raman signal intensity without affecting the embedded Raman molecules. Specfically, aptamer-functionalized CMS structures are combined with metal-organic scaffold nanoparticles that possess complementary DNA sequences. In the presence of PAT, binding of the aptamer to the toxin leads to degradation of the hybrid nano assembly, which can be measured by a change in the SERS signal. This SERS strategy enables turn-off sensing in apple juice over a wide range (10pg/mL to 1μg/mL), with a detection limit as low as 0.02ng/mL. Analysis of real samples gives results consistent with high-performance liquid chromatography (HPLC) test. The excellent selectivity and sensitivity of the SERS sensor demonstrate its potential for a wide range of SERS sensor applications.

Function of PxGrp78 in the Reproduction of Plutella xylostella.

Glucose-regulated protein 78 (Grp78), a crucial molecular chaperone in the endoplasmic reticulum, has been extensively investigated in vertebrates. However, its functional exploration in insects remains limited. This study cloned the full-length cDNA sequence of Grp78 in Plutella xylostella (L.), which is 2583 bp long. The open reading frame (ORF) is 2004 bp in length and encodes a total of 667 amino acids, including three conserved characteristic sequences of the HSP70 family. PxGrp78 is expressed in various developmental stages of P. xylostella, with the highest expression observed in third instar larvae and higher expression in female adults compared to male adults. The interference with PxGrp78 in female adults was found to significantly reduce the quantity of egg laying and the hatching rate, as well as shorten the oviposition period and down-regulate the expression of the PxVg gene. These results suggested an important role for PxGrp78 in the reproduction of P. xylostella.

Identification and functional analysis of the transcriptional factor GeERF4B-1 in Gelsemium elegans

Gelsemium elegans, a vine plant of Loganiaceae, has both medicinal and forage values. However, it is susceptible to low temperatures during growth. Exploring low temperature response genes is of great significance for cold resistance breeding of G. elegans. Ethylene response factors (ERFs) are the transcription factors of the AP2/ERF superfamily and play a crucial role in plant stress response. In this study, based on the unigene GeERF involved in the response to low temperature stress in the transcriptome of G. elegans, a full-length cDNA sequence of the transcription factor GeERF4B-1 was cloned from the leaves of G. elegans by reverse transcription-polymerase chain reaction (RT-PCR). Bioinformatics analysis showed that GeERF4B-1 had an open reading frame of 759 bp, encoding a protein composed of 252 amino acid residues and with a relative molecular weight of 27 kDa. The deduced protein was predicted to be an unstable, alkaline, and hydrophilic protein. The phylogenetic tree showed that GeERF4B-1 was in the same clade as the B-4 subfamily of the ERF family. The results of the subcellular localization experiment revealed that GeERF4B-1 was located in the nucleus. Real time quantitative PCR (RT-qPCR) analysis indicated that GeERF4B-1 was expressed in the root, stem, and leaf of G. elegans, with the highest expression level in the root. Compared with the control, the treatments with a low temperature (4 ℃), methyl jasmonate (MeJA), and abscisic acid (ABA) up-regulated the expression level of GeERF4B-1, which reached the peak at 24-48 h. This result revealed that GeERF4B-1 actively responded to low temperature, MeJA, and ABA stresses. However, both sodium chloride (NaCl) and drought treatments down-regulated the expression of GeERF4B-1. In addition, a prokaryotic expression vector of GeERF4B-1 was constructed, and a fusion protein of approximately 52 kDa was yielded after induced expression. The results of the plate stress assay showed that compared with the control, the prokaryotic strain expressing GeERF4B-1 demonstrated enhanced tolerance to low temperatures and sensitivity to salt and mannitol stresses. This study provides theoretical references and potential genetic resources for breeding G. elegans varieties with stress resistance.

Calnexin interacts with B-cell receptor-associated protein 31 (Bap31) to mediate coelomocyte phagocytosis and Vibrio splendidus clearance in Apostichopus japonicus

Calnexin serves as a lectin chaperone located on the endoplasmic reticulum membrane and functions in glycoprotein folding and synthesis quality control, as well as in Ca2+ storage. Calnexin is extensively documented to participate in host immunity in the endoplasmic reticulum. However, the functions and fundamental mechanisms of calnexin in the invertebrate innate defence remain largely unknown. In this research, the complete cDNA sequence for calnexin from Apostichopus japonicus (Ajcalnexin) was cloned, revealing a 1779 bp open reading frame that codes for 592 amino acids, 113 bp 5′-Untranslated Region (UTR), and 3251 bp 3′-UTR. Upon Vibrio splendidus infection, both AjCalnexin mRNA and protein levels were significantly increased in coelomocytes. Knocking down Ajcalnexin with specific siRNAs significantly decreased coelomocyte phagocytosis, reducing the intracellular load of V. splendidus. By contrast, overexpression of AjCalnexin using recombinant AjCalnexin protein (rAjCalnexin) had the opposite effect. Moreover, B-cell receptor-associated protein 31 of A. japonicus (AjBap31) was identified as an interacting partner of AjCalnexin, which positively regulates AjBap31 expression. Silencing Ajbap31 also decreased coelomocyte phagocytosis and inhibited the intracellular load of V. splendidus. Furthermore, phagocytosis levels and intracellular loads of V. splendidus in the coelomocytes of sea cucumbers treated with rAjCalnexin and siAjBap31 were significantly lower than those in rAjCalnexin- and siNC-treated sea cucumbers. Collectively, we provide the first functional evidence that the AjCalnexin-AjBap31 axis plays a crucial role in host immune defence by mediating coelomocyte phagocytosis in A. japonicus during V. splendidus infection. These findings enhance understanding of the regulatory mechanism of phagocytosis in echinoderms and offer theoretical insights for preventing and controlling skin ulcer syndrome in sea cucumbers.

The Isolation and Characterization of Perlucin in Pacific Abalone, Haliotis discus hannai: A Shell Morphogenic Protein with Potential Responses to Thermal Stress and Starvation.

Perlucin is a shell matrix protein that plays a significant role in regulating shell biomineralization. This study aimed to isolate and characterize the perlucin gene and analyze its expression to explore its role in shell formation, regeneration, and responses to thermal stress and starvation in Pacific abalone. The isolated full-length cDNA sequence of Hdh-Perlucin is 1002 bp long, encoding a 163-amino-acid polypeptide with a signal peptide. The mature peptide of Hdh-Perlucin contains a C-type lectin domain with signature motif and six conserved cysteine residues. Gene Ontology analysis suggests that Hdh-Perlucin exhibits carbohydrate-binding activity. Significantly higher expression of Hdh-Perlucin was observed during the juvenile, veliger, and trochophore stages, compared with cell division stage during early development. Upregulated expression was recorded from slow to rapid growth phases and during shell biomineralization, while downregulated expression was noted during starvation. Under thermal stress, expression peaked at 30 °C and 25 °C for 6 and 12 h, respectively, while consistently higher levels were observed at 15 °C throughout the experiment. This study provides the first comprehensive structural and expression analysis of Hdh-Perlucin, highlighting its roles in metamorphosis, shell formation and regeneration, and responses to heat stress and starvation in abalone.

Stabilization of a single-stranded DNA of adeno-associated virus by inverted terminal repeats

Parvoviruses have evolved to possess a linear single-stranded DNA (ssDNA) genome ranging from 4 to 6.3 kb. Adeno-associated virus (AAV), a member of the Parvoviridae family, contains approximately 5 kb of linear ssDNA within its capsid. This ssDNA features two 145-base inverted terminal repeats (ITRs) positioned at each end. ITRs have a T-shaped hairpin secondary structure, which plays a crucial role in viral replication. To investigate the impact of ITRs on ssDNA stability, we conducted a DNA denaturation-reannealing assay in 10 mM magnesium acetate, 50 mM potassium acetate, and 20 mM Tris-acetate buffer at pH7.9. Conventional double-stranded DNA (dsDNA) fragments retain a reannealing capability of over 50% for sizes under 8.8 kb, gradually losing this capability as sizes increase; however, dsDNA fragments in rAAV ranging from 0.7 to 6.3 kb did not exhibit a reannealing profile. This suggests that the presence of ITRs at both ends hinders annealing between the complementary strands. These results indicate that ITR structures preferentially induce an ssDNA conformation less than 6.3 kb in size, and that the stability of AAV ssDNA contributes to the viral life cycle, including processes such as infection, replication, and packaging. Considering the size of parvovirus genomes, it appears that their genomes require reversible flexibility in complementary DNA strands; simultaneously, this adaptability needs to be regulated by specific palindromic ITRs at both ends.

Natural Diversity of Heat-Induced Transcription of Retrotransposons in Arabidopsis thaliana.

Transposable elements (TEs) are major components of plant genomes, profoundly impacting the fitness of their hosts. However, technical bottlenecks have long hindered our mechanistic understanding of TEs. Using RNA-Seq and long-read sequencing with Oxford Nanopore Technologies' (ONT) direct cDNA sequencing, we analyzed the heat-induced transcription of TEs in three natural accessions of Arabidopsis thaliana (Cvi-0, Col-0, and Ler-1). In addition to the well-studied ONSEN retrotransposon family, we confirmed Copia-35 as a second heat-responsive retrotransposon family with particularly high activity in the relict accession Cvi-0. Our analysis revealed distinct expression patterns of individual TE copies and suggest different mechanisms regulating the GAG protein production in the ONSEN versus Copia-35 families. In addition, analogously to ONSEN, Copia-35 activation led to the upregulation of flanking genes such as APUM9 and potentially to the quantitative modulation of flowering time. ONT data allowed us to test the extent to which read-through formation is important in the regulation of adjacent genes. Unexpectedly, our results indicate that for both families, the upregulation of flanking genes is not predominantly directly initiated by transcription from their 3' long terminal repeats. These findings highlight the intraspecific expressional diversity linked to retrotransposon activation under stress.

Bacterial expression, purification, and characterization of human cytochrome P450 3A4 without N-terminal modifications

In this communication we reported a bacterial system that over-expressed full-length wild-type (WT) human CYP3A4 in Escherichia coli (E. coli) at a level of 495 nmol/L culture. This level of expression was achieved by cloning the cDNA sequence of CYP3A4 WT to a pLW01-P450 vector and co-expressing it with chaperones GroEL/ES in bacterial C41(DE3) cells. Aided with a C-terminal His5-tag, the expressed CYP3A4 WT was purified to homogeneity with a specific content of 14.3 ± 2.0 nmole P450/mg protein using a single Ni-Penta agarose column. Like the N-terminal modified form (CYP3A4-NF14), CYP3A4 WT binds substrate testosterone with a typical sigmoidal feature at slightly higher affinity. Functional characterization revealed that CYP3A4 WT exhibited lower testosterone 6β-hydroxylase activities than CYP3A4-NF14 in reconstituted phospholipid systems. In addition, it was found that the 6β-hydroxylase activity of CYP3A4 WT was less dependent on excess cytochrome P450 oxidoreductase (POR), compared with CYP3A4-NF14. These results suggest that the N-terminal membrane anchor of CYP3A4 WT enhances its interactions with POR and marginally increases testosterone binding.

High-Throughput Discovery of Substrate Peptide Sequences for E3 Ubiquitin Ligases Using a cDNA Display Method.

Cells utilize ubiquitin as a posttranslational protein modifier to convey various signals such as proteasomal degradation. The disfunction of ubiquitylation or following proteasomal degradation can give rise to the accumulation and aggregation of improperly ubquitylated proteins, which is known to be a general causation of many neurodegenerative diseases. Thus, the characterization of substrate peptide sequences of E3 ligases is crucial in biological and pharmaceutical sciences. In this study, we developed a novel high-throughput screening system for substrate peptide sequences of E3 ligases using a cDNA display method, which enables covalent conjugation between peptide sequences and their corresponding cDNA sequences. First, we focused on the MDM2 E3 ligase and its known peptide substrate as a model to establish the screening method, and confirmed that cDNA display method was compatible with in vitro ubiquitylation. Then, we demonstrated identification of MDM2 substrate sequences from random libraries to identify a novel motif (VKFTGGQLA). Bioinformatics analysis of the hit sequences was performed to gain insight about endogenous substrate proteins.

First report of lily mottle virus naturally infecting lily (Lilium asiatica) in Texas, USA.

Lily (Lilium asiatica) is an important plant grown for its range of flower colors and heavy scent. In March 2024, potyvirus-like symptoms consisting of light-yellow mottling and mosaic were noticed on 12/20 lily plants in a private property in Weslaco, Hidalgo County, Texas. Two symptomatic plants (WTX1 and WTX2) were sampled randomly for virus diagnosis. The leaf extracts of both samples were negative for the potyvirus group using Agdia's Poty ImmunoStrip® (Agdia, Inc., Elkhart, IN, USA). However, rub-inoculation of the extracts onto healthy Nicotiana benthamiana and Vigna unguiculata plants (n=4, each) induced mild mottle symptoms on the systemic leaves of both herbaceous test plants 20 to 28 days post inoculation, indicating the presence of a mechanically transmissible agent in the samples. No virus-like symptoms were observed on the mock-inoculated plants (n=1, each) of both species. To test for suspected potyvirus infection, 2-µg of total nucleic acid extracts (Dellaporta et al. 1983) from WTX1 and WTX2 were used for complimentary DNA (cDNA) synthesis with Oligo(dT) primer and the PrimeScript 1st strand cDNA synthesis kit (Takara Bio, USA). One microliter aliquot of each cDNA served as template in 12.5-µl conventional PCR reaction volumes with 5 U Taq polymerase (Roche Diagnostics, Indianapolis, IN), and two pairs of degenerate primers targeting the partial cylindrical inclusion (CI) gene and the helper component protease (HC-Pro) of potyviruses (Ha et al. 2008). The expected ~700-bp DNA product of each gene target was amplified from both samples. The amplicons were excised, gel eluted (Zymoclean™ Gel DNA Recovery kit) and cloned individually into the pJET1.2/Blunt vector (Life Technologies). Recombinant plasmids from two transformed Escherichia coli cells per cloned DNA insert were Sanger sequenced. BLASTn analysis of each gene-specific nucleotide (nt) sequence fragment revealed their identities (83 to 92 % nt) as lily mottle virus (LMoV; genus Potyvirus) at 93 to 98% query coverage. In pairwise comparison, the obtained sequences of LMoV isolates from TX specific to the CI (GenBank accession nos. PQ037260-61) and the HC-Pro (PQ037262-63) shared ~99% nt and 100% amino acid (aa) identities with each other and ~91-92% nt and 98-100% aa identities with the closest isolate, Bate5 (JN127341) from Australia. Based on these results, a pair of LMoV-specific primers (LM_v109-F: 5'-GGCCAGTAATGTGCACAAGC-3' and LM_c527-R: 5'-TCGCTGTAGCTAGCGACGTAC-3') was newly designed to target the partial CI gene, internal to the 700-bp fragment obtained above with the generic primers. The primer pair was used to screen each of the mechanically inoculated test plants by RT-PCR as previously described above. The expected 438-bp fragment of the LMoV CI gene was amplified from all the inoculated plants of both N. benthamiana and V. unguiculata; the results were confirmed by Sanger sequencing as described above. The mock-inoculated plant of each experimental host plant tested negative for LMoV. LMoV is a quarantine pest and was previously reported in the U.S. (Brierley and Smith, 1944) and other lily-producing parts of the world, such as the Netherlands, China, Korea, and Brazil. To our knowledge, this is the first confirmed report of the virus in Texas, thus expanding its geographical range. Testing of lily foundation stocks before propagation is essential to prevent further spread of LMoV via planting material.

CDC42 enhances phagocytic activity and exhibits antimicrobial functions through the recruitment of IQGAP1 in Apostichopus japonicus

Cell Division Cycle 42 protein (CDC42) is a pivotal member of the Rho family of small GTPases, integral to cell signaling and the regulation of cell polarity. CDC42 is extensively documented to participate in host immunity, particularly in processes such as phagocytosis and cell migration. Nevertheless, the precise mechanisms by which CDC42 affects host-bacteria interactions remain unclear in Echinoderms. In this study, we have isolated and characterized the CDC42 gene from Apostichopus japonicus, referred to as AjCDC42. The complete cDNA sequence of AjCDC42 extends 1282 base pairs, including a typical classical RHO domain, spanning amino acids from 6 to 179. Spatial expression analysis demonstrated that AjCDC42 is ubiquitously expressed across all examined tissues. Following infection with Vibrio splendidus and stimulation with LPS, the protein and mRNA levels of AjCDC42 were significantly upregulated in coelomocytes. Moreover, the knockdown of AjCDC42 significantly decreased V. splendidus-induced phagocytic activity in coelomocytes, leading to a reduced intracellular load of V. splendidus, as evidenced by flow cytometry and bacterial plate counting assays. Mechanistically, AjCDC42 was identified as an interacting partner of IQ Motif Containing GTPase Activating Protein 1 (AjIQGAP1) through the use of co-immunoprecipitation, immunofluorescence, and GST pull-down assays combined with mass spectrometry techniques. Additional functional analyses revealed that the knockdown of AjIQGAP1 similarly decreased the phagocytic activity and the intracellular load of V. splendidus in coelomocytes. Furthermore, we detected that decreased levels of AjCDC42 and AjIQGAP1 impaired cytoskeletal rearrangement and delayed lysosomal clearance of V. splendidus. In summary, our findings underscore the pivotal role of the AjCDC42-AjIQGAP1 axis in regulating coelomocytes phagocytosis in A. japonicus during V. splendidus infection.

Design of a Duplex-to-Complex Structure-Switching Approach for the Homogeneous Determination of Marine Biotoxins in Water.

In this report, we describe a fluorescent assay for the detection of six marine toxins in water. The mechanism of detection is based on a duplex-to-complex structure-switching approach. The six aptamers specific to the targeted cyanotoxins were conjugated to a fluorescent dye, carboxyfluorescein (FAM). In parallel, complementary DNA (cDNA) sequences specific to each aptamer were conjugated to a fluorescence quencher BHQ1. In the absence of the target, an aptamer-cDNA duplex structure is formed, and the fluorescence is quenched. By adding the toxin, the aptamer tends to bind to its target and releases the cDNA. The fluorescence intensity is consequently restored after the formation of the complex aptamer-toxin, where the fluorescence recovery is directly correlated with the analyte concentration. Based on this principle, a highly sensitive detection of the six marine toxins was achieved, with the limits of detection of 0.15, 0.06, 0.075, 0.027, 0.041, and 0.026 nM for microcystin-LR, anatoxin-α, saxitoxin, cylindrospermopsin, okadaic acid, and brevetoxin, respectively. Moreover, each aptameric assay showed a very good selectivity towards the other five marine toxins. Finally, the developed technique was applied for the detection of the six toxins in spiked water samples with excellent recoveries.

The Inhibitory Effect and Mechanism of the Histidine-Rich Peptide rAj-HRP from Apostichopus japonicus on Human Colon Cancer HCT116 Cells.

Colon cancer is a common and lethal malignancy, ranking second in global cancer-related mortality, highlighting the urgent need for novel targeted therapies. The sea cucumber (Apostichopus japonicus) is a marine organism known for its medicinal properties. After conducting a bioinformatics analysis of the cDNA library of Apostichopus japonicus, we found and cloned a cDNA sequence encoding histidine-rich peptides, and the recombinant peptide was named rAj-HRP. Human histidine-rich peptides are known for their anti-cancer properties, raising questions as to whether rAj-HRP might exhibit similar effects. To investigate whether rAj-HRP can inhibit colon cancer, we used human colon cancer HCT116 cells as a model and studied the tumor suppressive activity in vitro and in vivo. The results showed that rAj-HRP inhibited HCT116 cell proliferation, migration, and adhesion to extracellular matrix (ECM) proteins in vitro. It also disrupted the cytoskeleton and induced apoptosis in these cells. In vivo, rAj-HRP significantly inhibited the growth of HCT116 tumors in BALB/c mice, reducing tumor volume and weight without affecting the body weight of the tumor-bearing mice. Western blot analysis showed that rAj-HRP inhibited HCT116 cell proliferation and induced apoptosis by upregulating BAX and promoting PARP zymogen degradation. Additionally, rAj-HRP inhibited HCT116 cell adhesion and migration by reducing MMP2 levels. Further research showed that rAj-HRP downregulated EGFR expression in HCT116 cells and inhibited key downstream molecules, including AKT, P-AKT, PLCγ, P38 MAPK, and c-Jun. In conclusion, rAj-HRP exhibits significant inhibitory effects on HCT116 cells in both in vitro and in vivo, primarily through the EGFR and apoptosis pathways. These findings suggest that rAj-HRP has the potential as a novel targeted therapy for colon cancer.

Functional Analysis of CPSF30 in Nilaparvata lugens Using RNA Interference Reveals Its Essential Role in Development and Survival.

The brown planthopper (Nilaparvata lugens) is a major pest threatening global rice production, significantly reducing yields annually. As N. lugens increasingly develops resistance to conventional control methods, such as chemical pesticides, there is an urgent need for innovative and sustainable pest management strategies. Cleavage and Polyadenylation Specificity Factor 30 (CPSF30) is a key protein involved in mRNA 3' end processing, yet its function in N. lugens remains poorly understood. This study aims to elucidate the role of CPSF30 in the growth and development of N. lugens and evaluate its potential as a target for RNA interference (RNAi)-based pest control strategies. We cloned and characterized the cDNA sequence of NlCPSF30, which encodes a protein of 341 amino acids containing five CCCH zinc-finger domains and two CCHC zinc-knuckle domains. Sequence alignment revealed that NlCPSF30 is highly conserved among insect species, particularly in the zinc-finger domains essential for RNA binding and processing. Phylogenetic analysis showed that NlCPSF30 is closely related to CPSF30 proteins from other hemipteran species. Expression analysis indicated that NlCPSF30 is most highly expressed in the fat body and during the adult stage, with significantly higher expression in females than in males. RNAi-mediated silencing of NlCPSF30 in third-instar nymphs resulted in severe phenotypic abnormalities, including disrupted molting and increased mortality following injection of double-stranded RNA (dsRNA) targeting NlCPSF30. Moreover, it influenced the expression of genes associated with hormone regulation, namely NlHry, NlE93, and NlKr-h1. These results suggest that NlCPSF30 is integral to critical physiological processes, with its disruption leading to increased mortality. Our findings identify NlCPSF30 as an essential gene for N. lugens' survival and a promising target for RNAi-based pest management strategies. This study provides a valuable molecular target and theoretical insights for developing RNAi-based control methods against N. lugens.

CD spectra reveal the state of G-quadruplexes and i-motifs in repeated and other DNA sequences

The B DNA of the genome contains numerous sequences that can form various noncanonical structures including G-quadruplex (G4), formed by two or more stacks of four guanine residues in a plane, and intercalating-motif (i-motif, iM) formed by alternately arranged C-C+ pairs. One of the easy yet sensitive methods to study G4s and iMs is circular dichroism (CD) spectroscopy which generates characteristic G4 and iM peaks. We have analyzed and compared the effects of various environmental factors including pH, buffer composition, temperature, flanking sequences, complimentary DNA strands, and single-stranded DNA binding protein (SSB) on the CD patterns of G4s and iMs generated by two groups of DNA molecules, one containing tandem repeats of GGGGCC and CCCCGG from the C9ORF72 gene associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), and the second containing polyG/polyC clusters from oncogene promoter-proximal regions without such tandem repeats. Changes in pH caused drastic changes in CCCCGG-iM and GGGGCC-G4 and the changes were dependent on repeat numbers and G:C base-pairing. In contrast, with the DNA sequences from the promoter-proximal regions of oncogenes, iMs disassembled upon pH changes with the peak slowly shifting to lower wavelength but the G4s did not show significant change. Complementary DNA strands and flanking DNA sequences also regulate G4 and iM formation. The single-stranded DNA binding protein, SSB, disassembled both G4s and iMs formed by almost all sequences suggesting an in vivo role for SSBs in the disassembly of G4s and iMs during DNA replication and other DNA transactions.

Artemin molecular chaperone from Artemia urmiana improves tolerance of Arabidopsis thaliana to abiotic stress.

Artemia is a genus of aquatic microcrustaceans that belong to the class Branchiopoda. Encysted Artemia urmiana embryos are resistant to harsh environmental stressors, including repeated desiccation, prolonged anoxia, extreme temperatures, and high levels of UV radiation. The protein artemin has a chaperone activity and is believed to play a crucial role in protecting the organism against such stresses. To elucidate the potential functional roles of artemin in plants, the cDNA sequence of artemin was cloned into the pZPY122 binary plant expression vector. Agrobacterium -mediated transformation and the floral-dip technique were used to introduce this construct into Arabidopsis thaliana . Three independent transgenic lines (art1 , art2 , art3 ) were generated and subjected to heat stress at 45°C. Results showed a significant interaction between heat stress and genotype for germination rate, germination speed, vigor index, and seedling and root length. The transgenic lines with the artemin transgene (ART ) exhibited remarkable heat stress tolerance compared with wild-type plants. They also had markedly lower levels of electrolyte leakage, hydrogen peroxide content, higher activities of catalase, superoxide dismutase and peroxidase, greater total protien content, and increased accumulation of proline. Under heat stress conditions, the expression of two key abiotic stress-responsive genes, DREB2A and HSFA3 , was significantly upregulated in the ART lines compared to the wild-type . These findings suggest that the ART gene from A. urmiana may act as molecular chaperone when expressed in Arabidopsis , thereby enhancing the plant's tolerance to heat stress.