Rapid Amplification Of cDNA Ends Research Articles

수온 스트레스에 의한 명태의 Heat Shock Factor 1의 분자생물학적 특성과 Heat Shock Protein 70의 단백질 발현 패턴

This study evaluated the levels of mRNA expression of HSF1 and HSP70 expression patterns in walleye pollock, Gasus chalcogrammus according to water temperature. Walleye pollack HSF1, which has not been reported, was used as a Rapid Amplification of cDNA Ends (RACE)-PCR strategy. The HSF1 amino acid sequence of walleye pollack showed 93% identification with the Atlantic cod. On the other hand, it showed relatively low identification with 50% in zebrafish. However, in the phylogenetic analysis, fish HSF1 formed a single clade group. The relative mRNA expression of HSF1 in the liver, intestine, gill, kidney, and heart of juveniles and immatures was investigated. The tissues in juvenile and immature, HSF1 mRNA expression was the highest at 14℃, and HSF1 mRNA levels increased significantly from 11℃ compared to 5 and 8℃. The mRNA levels of HSF1 were particularly high in the gill both juveniles and immatures. The expression of HSP70 was also evident in 11℃ and 14℃. These results indicate that water temperature of 11°C or higher is exposed to stressed-condition in walleye pollock. Therefore, HSF1 and HSP70 can be considered as important indicator for water temperature stress in walleye pollock.

B-Cell Receptor Features and Database Establishment in Recovered COVID-19 Patients by Combining 5'-RACE with PacBio Sequencing.

Antibodies induced by viral infection can not only prevent subsequent virus infection, but can also mediate pathological injury following infection. Therefore, understanding the B-cell receptor (BCR) repertoire of either specific neutralizing or pathological antibodies from patients convalescing from Coronavirus disease 2019 (COVID-19) infection is of benefit for the preparation of therapeutic or preventive antibodies, and may provide insight into the mechanisms of COVID-19 pathological injury. In this study, we used a molecular approach of combining 5' Rapid Amplification of cDNA Ends (5'-RACE) with PacBio sequencing to analyze the BCR repertoire of all 5 IgH and 2 IgL genes in B-cells harvested from 35 convalescent patients after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We observed numerous BCR clonotypes within most COVID-19 patients, but not in healthy controls, which validates the association of the disease with a prototypical immune response. In addition, many clonotypes were found to be frequently shared between different patients or different classes of antibodies. These convergent clonotypes provide a resource to identify potential therapeutic/prophylactic antibodies, or identify antibodies associated with pathological effects following infection with SARS-CoV-2.

Enhanced Expression of a Novel Lamin A/C Splice Variant in Idiopathic Pulmonary Fibrosis Lung.

In idiopathic pulmonary fibrosis (IPF), the normal delicate lung architecture is replaced with rigid extracellular matrix (ECM) as a result of the accumulation of activated myofibroblasts and excessive deposition of ECM. Lamins have a role in fostering mechanosignaling from the ECM to the nucleus. Although there is a growing number of studies on lamins and associated diseases, there are no prior reports linking aberrations in lamins with pulmonary fibrosis. Here, we discovered, through analysis of RNA sequencing data, a novel isoform of lamin A/C that is more highly expressed in IPF compared with control lung. This novel LMNA (lamin A/C) splice variant includes retained introns 10 and 11 and exons 11 and 12 as documented by rapid amplification of cDNA ends. We found that this novel isoform is induced by stiff ECM. To better clarify the specific effects of this novel isoform of lamin A/C and how it may contribute to the pathogenesis of IPF, we transduced the lamin transcript into primary lung fibroblasts and alveolar epithelial cells and found that it impacts several biological effects, including cell proliferation, senescence, cell contraction, and the transition of fibroblasts to myofibroblasts. We also observed that type II epithelial cells and myofibroblasts in the IPF lung exhibited wrinkled nuclei, and this is notable because this has not been previously described and is consistent with laminopathy-mediated cellular effects.

Cloning and functional analysis of flavanone 3-hydroxylase gene in Rhododendron hybridum Hort.

Flavanone 3-hydroxylase (F3H) is a key enzyme in the synthesis of phycocyanidins. In this experiment, the petals of red Rhododendron hybridum Hort. at different developmental stages were used as experimental materials. The R. hybridum flavanone 3-hydroxylase (RhF3H) gene was cloned using reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques, and bioinformatics analyses were performed. Petal RhF3H gene expression at different developmental stages were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A pET-28a-RhF3H prokaryotic expression vector was constructed for the preparation and purification of RhF3H protein. A pCAMBIA1302-RhF3H overexpression vector was constructed for genetic transformation in Arabidopsis thaliana by Agrobacterium-mediated method. The results showed that the R. hybridum Hort. RhF3H gene is 1 245 bp long, with an open reading frame of 1 092 bp, encoding 363 amino acids. It contains a Fe2+ binding motif and a 2-ketoglutarate binding motif of the dioxygenase superfamily. Phylogenetic analysis showed that the R. hybridum RhF3H protein is most closely related to the Vaccinium corymbosum F3H protein. qRT-PCR analysis showed that the expression level of the red R. hybridum RhF3H gene tended to increase and then decrease in the petals at different developmental stages, with the highest expression at middle opening stage. The results of the prokaryotic expression showed that the size of the induced protein of the constructed prokaryotic expression vector pET-28a-RhF3H was about 40 kDa, which was similar to the theoretical value. Transgenic RhF3H Arabidopsis thaliana plants were successfully obtained, and PCR identification and β-glucuronidase (GUS) staining demonstrated that the RhF3H gene was integrated into the genome of A. thaliana plants. qRT-PCR, total flavonoid and anthocyanin contentanalysis showed that RhF3H was significantly higher expressed in the transgenic A. thaliana relative to that of the wild type, and its total flavonoid and anthocyanin content were significantly increased. This study provides a theoretical basis for investigating the function of RhF3H gene, as well as for studying the molecular mechanism of flower color in R. simsiib Planch.

Rapid whole genome sequencing methods for RNA viruses.

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.

Identification and Characterization of 5-HT Receptor 1 from Scylla paramamosain: The Essential Roles of 5-HT and Its Receptor Gene during Aggressive Behavior in Crab Species.

Biogenic amines (BAs) play an important role in the aggressive behavior of crustaceans. In mammals and birds, 5-HT and its receptor genes (5-HTRs) are characterized as essential regulators involved in neural signaling pathways during aggressive behavior. However, only one 5-HTR transcript has been reported in crabs. In this study, the full-length cDNA of the 5-HTR1 gene, named Sp5-HTR1, was first isolated from the muscle of the mud crab Scylla paramamosain using the reverse-transcription polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE) methods. The transcript encoded a peptide of 587 amino acid residues with a molecular mass of 63.36 kDa. Western blot results indicate that the 5-HTR1 protein was expressed at the highest level in the thoracic ganglion. Furthermore, the results of quantitative real-time PCR show that the expression levels of Sp5-HTR1 in the ganglion at 0.5, 1, 2, and 4 h after 5-HT injection were significantly upregulated compared with the control group (p < 0.05). Meanwhile, the behavioral changes in 5-HT-injected crabs were analyzed with EthoVision. After 0.5 h of injection, the speed and movement distance of the crab, the duration of aggressive behavior, and the intensity of aggressiveness in the low-5-HT-concentration injection group were significantly higher than those in the saline-injection and control groups (p < 0.05). In this study, we found that the Sp5-HTR1 gene plays a role in the regulation of aggressive behavior by BAs, including 5-HT in the mud crab. The results provide reference data for the analysis of the genetic mechanism of aggressive behaviors in crabs.

Identification of germ cells in large yellow croaker (Larimichthys crocea) and yellow drum (Nibea albiflora) using RT-PCR and in situ hybridization analyses

Ocean-caught large yellow croaker (Larimichthys crocea) represents an important germplasm resource for the breeding of this species; however, these fish tend to show poor survival in captivity and would be unsuitable breeding purposes. As an alternative to the use of wild-caught croakers, germ cell transplantation has been proposed using the L. crocea specimens as donors and yellow drum (Nibea albiflora) as recipients. In this regard, the identification of L. crocea and N. albiflora germ cells is an essential prerequisite for establishing a germ cell transplantation protocol for these fish. In this study, we cloned the 3ʹ untranslated regions (UTR) of the vasa, dnd, and nanos2 genes in N. albiflora using the rapid amplification of cDNA ends (RACE) method and then aligned and analyzed the sequences of the corresponding genes in L. crocea and N. albiflora. On the basis of gene sequence differences, we designed species-specific primers and probes for RT-PCR analysis and in situ hybridization. RT-PCR analysis revealed that these species-specific primers exclusively amplified DNA from gonads of the respective species, thus confirming that we had six specific primer pairs that could be used to distinguish the germ cells in L. crocea and N. albiflora. Using in situ hybridization analysis, we established that whereas Lcvasa and Nadnd probes showed high species specificity, the probes for Navasa and Lcdnd showed a less specificity. In situ hybridization using Lcvasa and Nadnd thus enabled us to visualize the germ cells in these two species. Using these species-specific primers and probes, we can reliably distinguish the germ cells of L. crocea and N. albiflora, thereby establishing an effective approach for the post-transplantation identification of germ cells when using L. crocea and N. albiflora as donors and recipients, respectively.

Functional analysis of the Candida albicans ECE1 Promoter.

The formation of hyphae is a key virulence attribute of Candida albicans as they are required for adhesion to and invasion of host cells, and ultimately deep-tissue dissemination. Hyphae also secrete the peptide toxin candidalysin, which is crucial for destruction of host cell membranes. The peptide is derived from a precursor protein encoded by the gene ECE1 which is strongly induced during hyphal growth. Previous studies revealed a very complex regulation of this gene involving several transcription factors. However, the promoter of the gene is still not characterized. Here, we present a functional analysis of the intergenic region upstream of the ECE1 gene. Rapid amplification of cDNA ends (RACE)-PCR was performed to identify the 5' untranslated region, which has a size of 49 bp regardless of the hyphae-inducing condition. By using green fluorescent protein (GFP) reporter constructs we further defined a minimal promoter length of 1,500 bp which was verified by RT-qPCR. Finally, we identified the TATA element required for the expression of the gene. It is located 106 to 109 bp upstream of the ECE1 start codon. Our results illustrate that despite a very short 5' UTR, a relatively long promoter is required to secure ECE1 transcription, indicating a complex regulatory machinery tightly controlling the expression of the gene. IMPORTANCE In recent years it was shown that secretion of the toxic peptide candidalysin from hyphae of the major human fungal pathogen Candida albicans contributes heavily to its virulence. The peptide is derived from a precursor protein which is encoded by the ECE1 gene whose transcription is known to be closely associated with formation of hyphae. Here, we used a GFP reporter system to determine the length of the ECE1 promoter and were able to show that it has a minimal size of 1,500 bp. Surprisingly, the gene has a very short 5' UTR of only 49 bp. In accordance with this, the TATA element required for transcription is located 106 to 109 bp upstream of the start codon. This indicates that ECE1 expression is controlled by a very long promoter allowing a complex network of transcription factors to contribute to the gene's regulation.

Role of the Hypothetical Protein BB0563 during Borrelia burgdorferi Infection in Animals.

The alternative sigma factor RpoS in Borrelia burgdorferi, the etiological agent of Lyme disease, has long been postulated to regulate virulence-associated genes other than ospC and dbpA. Here, we demonstrate that bb0563, a gene encoding a hypothetical protein, is regulated by RpoS and contributes to the optimal infectivity of B. burgdorferi. When B. burgdorferi was exposed to environmental stimuli, bb0563 showed similar expression patterns as rpoS, ospC, and dbpA. Expression of bb0563 was significantly downregulated when rpoS was inactivated and was restored in the complemented strain. By using rapid amplification of cDNA ends (RACE) and luciferase reporter assays, a functional promoter was identified in the regulatory region upstream of bb0563. Gene expression from this promoter was drastically decreased in the rpoS mutant. We next investigated the role of bb0563 during animal infection. By using quantitative reverse transcription-PCR (RT-PCR), we found that bb0563 was highly expressed in mouse tissues during infection. We further created a bb0563-deficient mutant in a bioluminescent B. burgdorferi strain and examined infection dynamics using in vivo imaging. Relative to the parental and complemented strains, the mutant showed a delayed infection pattern and bacterial load was reduced. Another bb0563 deletion mutant was also created in the strain 297 background, and quantitative PCR (qPCR) analysis revealed a significantly lower spirochetal burden in tissue samples collected from animals infected with the mutant. In addition, localization studies indicate that BB0563 is not exposed on the cell surface but is associated with outer membrane. Taken together, these results suggest that bb0563 is required for optimal infectivity of B. burgdorferi during experimental infection.

Cloning and characterization of nitrate reductase gene in kelp Saccharina japonica (Laminariales, Phaeophyta)

BackgroundBrown macroalgae dominate temperate coastal ecosystems, and their productivity is typically limited by nitrate availability. As an economically important kelp, Saccharina japonica is the most productive farmed seaweed and needs to be supplemented with sufficient nitrate throughout the cultivation process. However, molecular characterization of genes involved in nitrogen assimilation has not been conducted in brown macroalgae.ResultsHere, we described the identification of the nitrate reductase (NR) gene from S. japonica (SjNR). Using two different cloning methods for SjNR, i.e. rapid amplification of cDNA ends (RACE) and cDNA cloning alone, a single fragment was obtained respectively. According to results of sequence analysis between these two fragments, the tentative coding sequence in two clones, SjNR-L and SjNR-S, were suggested to represent two transcripts of the single copy SjNR, and the ATG of SjNR-S was located inside the third exon of SjNR-L. In the 5′ upstream sequence of each transcript, promoter core elements, response elements, especially multiple N response elements which occurred in microalgal NR, were all predicted. Further sequence analysis revealed that both transcripts encoded all five domains conserved in eukaryotic plant NRs. RT-qPCR results showed that the transcription level of SjNR in juvenile sporophytes could be significantly induced by nitrate and inhibited by ammonium, which was in line with plant NRs. The recombinant SjNR-L and SjNR-S were all proved to have NR activity, suggesting that the single-copy gene SjNR might be regulated on transcription level based on alternative promoters and multiple transcriptional start sites. Moreover, both NADH and NADPH were found to be able to act as electron donors for SjNR alone, which is the first confirmation that brown algal NR has a NAD(P)H-bispecific form.ConclusionThese results will provide a scientific basis for understanding the N demand of kelp in various stages of cultivation and evaluating the environmental remediation potential of kelp in eutrophic sea areas.

AjTGFβ alleviates V. splendidus-induced inflammation through SMADs pathway in Apostichopus japonicus

The inhibition of inflammatory response is an essential process to control the development of inflammation and is an important step to protect the organism from excessive inflammatory damage. As a pleiotropic cytokine, transforming growth factor beta (TGF-β) plays a regulatory role in inhibiting inflammation in vertebrates. To investigate the role of TGF-β in the regulation of inflammation in invertebrates, we cloned and characterized the TGF-β gene from Apostichopus japonicus via rapid amplification of cDNA ends, and the sample was designated as AjTGF-β. For Vibrio splendidus-challenged sea cucumbers, the expression of AjTGF-β mRNAs in coelomocytes decreased at 96 h (0.27-fold), which was contrary to the trend of inflammation. AjTGF-β was expressed in all tissues with the highest expression in the body wall. When AjTGF-β was knocked down by using small interfering RNA (siRNA-KD) to 0.45-fold, AjSMAD 2/3 and AjSMAD6 were downregulated to 0.32- and 0.05-fold compared with the control group, respectively. Furthermore, when the damaged sea cucumber was challenged by V. splendidus co-incubated with rAjTGF-β, the damage area had no extensive inflammation, and damaged repair appeared at 72 h compared with the Vs + BSA group, in which the expression of AjSMAD 2/3 was upregulated by 1.35-fold. Under this condition, AjSMAD 2/3 silencing alleviated rAjTGF-β-induced damage recovery. Moreover, rAjTGF-β slightly induced the collagen I expression from 6.13 ng/mL to 7.84 ng/mL, and collagen III was upregulated from 6.23 ng/mL to 6.89 ng/mL compared with the Vs + BSA group. This finding indicates that AjTGF-β negatively regulated the inflammatory progress and accelerated the repair of damage by AjSMADs to regulate the collagens expression.

Characterization and Expression Analysis of Four Cadmium-Tolerance-Associated Genes of Avicennia marina (Forsk.).

Mangroves colonize the intertidal area of estuaries (e.g., Pichavaram, Payardia, and Mai Po) with remarkable cadmium (Cd) pollution. A study on the mechanism of mangrove plant response to Cd pollution can help to understand the adaptive characteristics of plants under Cd stress. This study explored the roles of peroxidase (PRX), pectate lyase (PL), and phytosulfokine (PSK) genes in cadmium tolerance of mangrove Avicennia marina. Full-length sequences of four genes (i.e., AmPRX1, AmPRX2, AmPL, and AmPSK) associated with metal tolerance were identified with suppression subtractive hybridization and rapid amplification of cDNA ends. These genes showed the characteristic features of the respective protein family, indicating functions similar to other plant proteins. Real-time quantitative PCR analysis demonstrated that cadmium exposure resulted in differences in expression patterns among the tissues. Our findings emphasize the complex regulatory mechanism of these four genes in response to trace metal pollution and reveal their functions in metabolic signaling during the stress response.

The use of non-functional clonotypes as a natural calibrator for quantitative bias correction in adaptive immune receptor repertoire profiling.

High-throughput sequencing of adaptive immune receptor repertoires is a valuable tool for receiving insights in adaptive immunity studies. Several powerful TCR/BCR repertoire reconstruction and analysis methods have been developed in the past decade. However, detecting and correcting the discrepancy between real and experimentally observed lymphocyte clone frequencies are still challenging. Here, we discovered a hallmark anomaly in the ratio between read count and clone count-based frequencies of non-functional clonotypes in multiplex PCR-based immune repertoires. Calculating this anomaly, we formulated a quantitative measure of V- and J-genes frequency bias driven by multiplex PCR during library preparation called Over Amplification Rate (OAR). Based on the OAR concept, we developed an original software for multiplex PCR-specific bias evaluation and correction named iROAR: immune Repertoire Over Amplification Removal (https://github.com/smiranast/iROAR). The iROAR algorithm was successfully tested on previously published TCR repertoires obtained using both 5' RACE (Rapid Amplification of cDNA Ends)-based and multiplex PCR-based approaches and compared with a biological spike-in-based method for PCR bias evaluation. The developed approach can increase the accuracy and consistency of repertoires reconstructed by different methods making them more applicable for comparative analysis.

Small RNA sequencing of Scutellaria baicalensis reveals brassica yellows virus infection and the complementary development of a sensitive and reliable RT-qPCR assay

To identify the potential causal agent that has caused Scutellaria baicalensis to exhibit yellowing and other morbid symptoms, we sampled from this medicinal plant in Gansu Province, Northwest China, namely, that plant's main growing area. All 18 contings obtained from small RNA sequencing (sRNA-Seq) were homologous to brassica yellows virus (BrYV), with 93%–100% nucleotide similarity. A full-length genome (5693 bp) was obtained using segmentation amplification and Rapid Amplification of cDNA Ends (RACE), which was subsequently identified as BrYV-HQ. Genomic similarity between BrYV-HQ and other isolates ranged from 80.64% to 90.58%. Observed variation was mainly in the coding region of the ORF 3–5 fusion proteins. Evolutionary tree analysis indicated that the kinship of BrYV-HQ may be linked to BrYV-NtabQJ (MK057527.1), known to infect tobacco plants produced in China. Recombination analysis indicated that BrYV-HQ may have derived through recombination between BrYV-NtabQJ and a BrYV isolate (KY310572.1) that infects Brassica napus in China. Based on the coat protein (CP) gene design of the RT-qPCR primer set obtained, the linearity of the standard curve equation y = −2.987*Log(x) + 41.75, which we established for quantitative detection, was good (ranging from 4.43 × 1010 copies/μl∼4.43 × 103 copies/μl) for the quantitative detection of BrYV-HQ. This study marks the first time that BrYV has been confirmed to infect S. baicalensis and upon which a quantitative detection method was established, providing a molecular basis for the research and management of S. baicalensis viral disease in China.

Development of a Universal Cloning System for Reverse Genetics of Human Enteroviruses.

Enteroviruses (EVs) have been associated with several human diseases. Due to their continuous emergence and divergence, EV species have generated more than 100 types and recombinant strains, increasing the public health threat caused by them. Hence, an efficient and universal cloning system for reverse genetics (RG) of highly divergent viruses is needed to understand the molecular mechanisms of viral pathology and evolution. In this study, we generated a versatile human EV whole-genome cDNA template by enhancing the template-switching method and designing universal primers capable of simultaneous cloning and rapid amplification of cDNA ends (RACE)-PCR of EVs. Moreover, by devising strategies to overcome limitations of previous cloning methods, we simplified significant cloning steps to be completed within a day. Of note, we successfully verified our efficient universal cloning system enabling RG of a broad range of human EVs, including EV-A (EV-A71), EV-B (CV-B5, ECHO6, and ECHO30), EV-C (CV-A24), and EV-D (EV-D68), with viral titers and phenotypes comparable to those of their wild types. This rapid and straightforward universal EV cloning strategy will help us elucidate molecular characteristics, pathogenesis, and applications of a broad range of EV serotypes for further development of genetic vaccines and delivery tools using various replication systems. IMPORTANCE Due to the broad spread, incidence, and genetic divergence of enteroviruses (EVs), it has been challenging to deal with this virus that causes severe human diseases, including aseptic meningitis, myocarditis, encephalitis, and poliomyelitis. Therefore, an efficient and universal cloning system for the reverse genetics of highly divergent EVs contributes to an understanding of the viral pathology and molecular mechanisms of evolution. We have simplified the important cloning steps, hereby enhancing the template-switching method and designing universal primers, which enable the important cloning steps to be completed in a day. We have also successfully demonstrated recovery of a broad range of human EVs, including EV-A to -D types, using this advanced universal cloning system. This rapid and robust universal EV cloning strategy will aid in elucidating the molecular characteristics, pathogenesis, and applications of a wide range of EVs for further development of genetic vaccines and antiviral screening using various replication systems.

Molecular characterization of a novel gammacarmovirus infecting cucurbits in India

In this study, we describe the identification of a new gammacarmovirus infecting Cucurbita pepo plants showing a range of mosaic, stunting, yellowing, and wilting symptoms. The virus had a narrow host range and mostly produced chlorotic and necrotic local lesions in the majority of the tested plants. However, Nicotiana benthamiana showed systemic symptoms under laboratory conditions. Using a combination of Sanger sequencing and rapid amplification of cDNA ends (RACE), the complete genome sequence of the virus was determined to be 4274 nucleotides (nt) in length. Its genome organization is similar to that of members of the genus Gammacarmovirus in the family Tombusviridae, consisting of five overlapping open reading frames (ORFs) encoding p28, replicase, p7A, p7B, and coat protein (CP), respectively. The genome is flanked by short 5' and 3' non-coding regions (NCR) at either end. In pairwise comparisons of replicase and CP sequences, the virus showed the highest amino acid sequence identity of 71.55% and 54.86%, respectively, to melon necrotic spot virus (MNSV), the type member of the genus Gammacarmovirus. Since the sequence identity values are below the species demarcation threshold suggested by the International Committee on Taxonomy of Viruses (ICTV), the virus from Cucurbita pepo plants, for which the name "cucurbit carmovirus" (CuCV) is proposed, represents a new species. In phylogenetic analysis based on the replicase and CP amino acid sequences, CuCV clustered with MNSV but formed a distinct branch, further confirming that the virus is a distinct member of the genus Gammacarmovirus.

Molecular characterization and immune functions of lipasin in Nile tilapia (Oreochromis niloticus): Involvement in the regulation of tumor necrosis factor-α secretion

Lipasin, the product of the angiopoietin-like 8 (angptl8) gene, is known as a critical regulator of plasma lipid metabolism. However, its immune function in vertebrates is currently poorly understood. By 5'/3′-rapid amplification of cDNA ends (RACE), we established the structural identity of Nile tilapia (Oreochromis niloticus) angptl8. The transcripts of tilapia angptl8 were widely expressed in various tissues, with the highest levels in the liver. Following lipopolysaccharide in vivo challenges, time-dependent angptl8 gene expression was observed in the head kidney and liver. On the basis of the sequence obtained, we produced recombinant lipasin that inhibited lipoprotein lipase activity. Treatment of head kidney leukocytes with lipasin stimulated tumor necrosis factor-α (TNF-α) secretion and gene expression. In addition, lipasin-induced TNF-α secretion could be prevented by inhibiting the nuclear factor-kappa B (NF-κB) signaling pathway. Furthermore, lipasin enhanced the phosphorylation and degradation of IκBα and promoted translocation of the p65 subunit of NF-κB to the nucleus. Collectively, the current findings suggested that lipasin was involved in the immune response of Nile tilapia and stimulated TNF-α secretion by activating the NF-κB pathway in tilapia head kidney leukocytes.

Transcription factor Ap2b regulates the mouse autosomal recessive polycystic kidney disease genes, Pkhd1 and Cys1.

Transcription factor Ap2b (TFAP2B), an AP-2 family transcription factor, binds to the palindromic consensus DNA sequence, 5'-GCCN3-5GGC-3'. Mice lacking functional Tfap2b gene die in the perinatal or neonatal period with cystic dilatation of the kidney distal tubules and collecting ducts, a phenotype resembling autosomal recessive polycystic kidney disease (ARPKD). Human ARPKD is caused by mutations in PKHD1, DZIP1L, and CYS1, which are conserved in mammals. In this study, we examined the potential role of TFAP2B as a common regulator of Pkhd1 and Cys1. We determined the transcription start site (TSS) of Cys1 using 5' Rapid Amplification of cDNA Ends (5'RACE); the TSS of Pkhd1 has been previously established. Bioinformatic approaches identified cis-regulatory elements, including two TFAP2B consensus binding sites, in the upstream regulatory regions of both Pkhd1 and Cys1. Based on reporter gene assays performed in mouse renal collecting duct cells (mIMCD-3), TFAP2B activated the Pkhd1 and Cys1 promoters and electromobility shift assay (EMSA) confirmed TFAP2B binding to the in silico identified sites. These results suggest that Tfap2b participates in a renal epithelial cell gene regulatory network that includes Pkhd1 and Cys1. Disruption of this network impairs renal tubular differentiation, causing ductal dilatation that is the hallmark of recessive PKD.

Alternative splicing is not a key source of chemerin isoforms diversity

BackgroundChemerin is a chemoattractant protein with adipokine and antimicrobial properties encoded by the retinoic acid receptor responder 2 (RARRES2) gene. Chemerin bioactivity largely depends on carboxyl-terminal proteolytic processing that generates chemerin isoforms with different chemotactic, regulatory, and antimicrobial potentials. While these mechanisms are relatively well known, the role of alternative splicing in generating isoform diversity remains obscure.Methods and resultsUsing rapid amplification of cDNA ends (RACE) PCR, we determined RARRES2 transcript variants present in mouse and human tissues and identified novel transcript variant 4 of mouse Rarres2 encoding mChem153K. Moreover, analyses of real-time quantitative PCR (RT-qPCR) and publicly-available next-generation RNA sequencing (RNA-seq) datasets showed that different alternatively spliced variants of mouse Rarres2 are present in mouse tissues and their expression patterns were unaffected by inflammatory and infectious stimuli except brown adipose tissue. However, only one transcript variant of human RARRES2 was present in liver and adipose tissue.ConclusionOur findings indicate a limited role for alternative splicing in generating chemerin isoform diversity under all tested conditions.

A novel picorna-like virus identified in the cotton boll weevil Anthonomus grandis (Coleoptera: Curculionidae).

The cotton boll weevil (CBW; Anthonomus grandis; Coleoptera: Curculionidae) is considered the major insect pest of cotton, causing considerable losses in yield and fiber quality. An increase in the boll weevil population due to increasingly inefficient chemical control measures is of great concernamong cotton producers. The absence of conventional or transgenic cultivars with minimal resistance to CBW has stimulated the search for new molecular and biological tools for efficient control of this insect pest. In this study, we used a metagenomic approach based on RNA deep sequencing to investigate the presence of viruses and coding viral RNA in apparently healthy native adult CBW insects collected from cotton crops in Mato Grosso state, Brazil. Using an Illumina HiSeq 2000 paired-end platform, 138,798 virus-related reads were obtained, and a consensus sequence of a putative new virus, 10,632 nucleotides in length, was assembled. The sequences of the 5' and 3' untranslated regions (UTRs) were determined by rapid amplification of cDNA ends (RACE), followed by Nanopore sequencing. The complete genome sequence included a 5'-UTR (1,158 nucleotides), a 3'-UTR (561 nucleotides), and a single ORF of 8,913 nucleotides encoding a large polyprotein. Sequence analysis of the putative polyprotein showed several regions with high sequence similarity to structural and non-structural proteins of viruses of the family Iflaviridae. Pairwise alignments of polyprotein amino acid sequences showed the highest sequence identity (32.13%) to a partial polyprotein sequence of a putative iflavirus (QKN89051.1) found in samples from wild zoo birds in China. Phylogenetic analysis based on full polyprotein sequences of different iflaviruses indicated that this new picorna-like virus is most closely related to iflaviruses found in lepidopteran insects, and it was therefore tentatively named "Anthonomus grandis iflavirus 1" (AgIV-1). This is, to our knowledge, the first complete viral genome sequence found in CBW, and it could provide a basis for further studies about the infectivity and transmission of this virus and its possible association with symptoms or acute disease. AgIV-1 could potentially be used to develop biological or molecular tools, such as a viral vector to carry interfering RNA molecules for CBW control.