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Spätzle is a crucial ligand for Toll-like receptor (TLR) that triggers the activation of TLR signal pathway in insects. In this study, open reading frames (ORFs) of two spätzles were cloned from Portunus trituberculatus (PtSpz1 and PtSpz2). Both of PtSpzs contained the typical cystine-knot domain of spätzle. Tissue distribution analysis showed that both of PtSpzs were predominantly expressed in the gills. Transcriptional levels of the two PtSpzs in hemocytes and gill rapidly increased at 3 h and 6 h post Vibrio alginolyticus challenge, respectively. The two PtSpzs could bind to several pathogen-associated molecules including lipopolysaccharide (LPS), peptidoglycan (PGN) and envelope proteins of white spot syndrome virus (WSSV). Moreover, the two PtSpzs could directly interact with the extracellular leucine-rich repeats (LRR) domain of TLR. This study revealed that spätzle could interact with pathogen-associated molecules and TLR of host, which may be two important steps for spätzle to deliver signals into host cells.

Lysozyme is relevant to the innate immune system as a vital protein for crustaceans. In the present study, we cloned and characterized a novel c-type lysozyme gene (LvLYZ) from the Pacific white shrimp (Litopenaeus vannamei). The obtained full-length cDNA of LvLYZ was 990 bp and contained an open reading frame of 693 bp. Its deduced amino acid sequence consisted of 230 amino acids (aa) with a 17 aa signal peptide at the N-terminal and 130 aa functional domains. The multiple sequence alignment (MSA) indicated that the typical active sites in LvLYZ were similarly conserved as c-type lysozymes from other species. The transcription of LvLYZ appeared in all detected tissues and had relatively higher expression levels in hemocytes, hepatopancreas, gill and intestine. The mRNA expression profiles of LvLYZ were up-regulated in hemocyte and hepatopancreas post the stimulation of Vibrio parahaemolyticus or white spot syndrome virus (WSSV), respectively. The recombinant protein of LvLYZ (rLvLYZ) exhibited antibacterial activities against various microbes, including Escherichia coli, Vibrio splendidus, Micrococcaus luteus, Vibrio parahaemolyticus and Staphylococcus aureus. These results indicated that LvLYZ could cope with bacteria in L. vannamei and may play a significant role in immune response against invading pathogens.

RNA aptamers are oligonucleotides with high binding affinity and specificity for target molecules and are expected to be a new generation of therapeutic molecules and targeted delivery materials. The tertiary structure of RNA molecules and RNA-protein interaction sites are increasingly important as potential targets for new drugs. The pathological mechanisms of diseases must be understood in detail to guide drug design. In developing RNA aptamers as drugs, information about the interaction mechanisms and structures of RNA aptamer-target protein complexes are useful. We constructed a database, RNA aptamer 3D-structural modeling (RNAapt3D), consisting of RNA aptamer data that are potential drug candidates. The database includes RNA sequences and computationally predicted RNA tertiary structures based on secondary structures and implements methods that can be used to predict unknown structures of RNA aptamer-target molecule complexes. RNAapt3D should enable the design of RNA aptamers for target molecules and improve the efficiency and productivity of candidate drug selection. RNAapt3D can be accessed at https://rnaapt3d.medals.jp.

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disorder caused by various variants in the IDS gene. It is known that genomic recombinants between IDS and its homologous pseudogene IDSP1 account for a small number of patients, for whom genetic diagnosis usually relies on restriction enzyme digestion at specific loci. Nevertheless, such approach cannot reveal the impact of rearrangements on IDS transcription, which is crucial for the interpretation of the pathogenicity of rearrangement variants.RNA sequencing (RNA-seq) was explored to analyze transcriptional alterations in four male MPS II patients who were negative for Sanger sequencing of the IDS gene. Reverse transcription-polymerase chain reaction and TA clone sequencing were used to validate RNA-seq analysis results. The IDS-IDSP1 recombinant was determined by sequencing the indicated loci in genome.Differential expression analysis showed the expression levels of IDS gene in patients were largely reduced compared to the healthy individuals. Differential splicing analysis revealed skipping of exons 8 and 9 of IDS, without any splice-junction defects at the genomic level. In addition, two types of fusion transcripts, IDS_EOLA1 and IDS_EOLA1-DT_EOLA1 were identified by gene fusion analysis. Sequencing of the known rearrangement alleles showed these four patients have the same type of IDS-IDSP1 recombinant.We establish an RNA-seq workflow to analyze transcriptional characteristics of IDS gene from multiple perspectives. Our study validates the diagnostic value of RNA-seq in MPS II, including the discovery of transcriptional alterations and the potential to suggest genome-level rearrangements in IDS.

Tracking SARS-CoV-2 variants of concern (VOC) by genomic sequencing is time-consuming. The rapid screening of VOCs is necessary for clinical laboratories. In this study, we developed a rapid screening method based on multiplex RT-PCR by extended S-gene target failure (eSGTF), a false negative result caused by S-gene mutations.Three S-gene target (SGT) regions (SGT1, codons 65-72; SGT2, codons 152-159; and SGT3, codons 370-377) and an N-gene region (for internal control) were detected in single-tube. Four types of VOC (Alpha, Delta, Omicron BA.1, and Omicron BA.2) are classified by positive/negative patterns of 3 S-gene regions (eSGTF pattern).The eSGTF patterns of VOCs were as follows (SGT1, SGT2, SGT3; P, positive; N, negative): Alpha, NPP; Delta, PNP; Omicron BA.1, NPN pattern; and Omicron BA.2, PPN. As compared with the S-gene sequencing, eSGTF patterns were identical to the specific VOCs (concordance rate = 96.7%, N = 206/213). Seven samples with discordant results had a minor mutation in the probe binding region. The epidemics of VOCs estimated by eSGTF patterns were similar to those in Japan.Multiplex RT-PCR and eSGTF patterns enable high-throughput screening of VOCs. It will be useful for the rapid determination of VOCs in clinical laboratories.

Glutaredoxin (Grx) is a class molecule oxidoreductase, which plays a key role in maintaining redox homeostasis and regulating cell survival pathways. However, the expression pattern and function of Grx remain unknown in the mud crab (Scylla paramamosain). In the present study, a novel full-length of Grx 5 from the mud crab (designated as Sp-Grx 5) was cloned and characterized. The open reading frame of Sp-Grx 5 was 441 bp, which encoded a putative protein of 146 amino acids. The amino acid sequence of Sp-Grx 5 contained a typical C-G-F-S redox active motif and several GSH binding sites. Sp-Grx 5 widely existed in all tested tissues with a high-level expression in hepatopancreas. Subcellular localization showed that Sp-Grx 5 was located in the cytoplasm and nucleus. The expression of Sp-Grx 5 was significantly up-regulated after Vibrio parahaemolyticus infection and cadmium exposure, suggesting that Sp-Grx 5 was involved in innate immunity and detoxification. Furthermore, overexpression of Sp-Grx 5 could improve cells viability after H

Ficolins, belonging to the fibrinogen-related protein superfamily, are important pattern recognition receptors in innate immunity. Here, a ficolin gene Ptficolin was characterized from the swimming crab Portunus trituberculatus. The completed cDNA sequence of Ptficolin encoded a signal peptide, a coiled-coil region and a fibrinogen-like domain but without the typical collagen region of vertebrate ficolins. Ptficolin showed higher expression in stomach and hepatopancreas, and presented a time-dependent response after pathogen challenge and injury stimulation. The recombinant Ptficolin (rPtficolin) could bind to various PAMPs and microorganisms, and agglutinate microorganisms and rabbit erythrocytes in a Ca

Cold shock protein D (CspD) is one of the homologous proteins of cold shock protein A (CspA), inhibiting DNA replication by binding to single-stranded DNA. We found that CspD from Vibrio cholerae (VcCspD) possesses one heme regulatory motif (HRM) sequence and specifically binds heme with a stoichiometry of 1:1. The binding of a synthetic single-stranded DNA oligomer (ssDNA) was followed by fluorescence quenching of Trp. The fluorescence quenching associated with the addition of ssDNA was suppressed in the presence of heme, indicating that heme binding to VcCspD inhibited the formation of the VcCspD-ssDNA complex. Such heme-induced inhibition was not observed for the VcCspD mutant with replacement of Cys22 in the HRM with alanine (C22A). Heme binding at Cys22 is, therefore, essential for the inhibition of ssDNA binding for VcCspD. The growth of Escherichia coli at 37 °C was slowed when VcCspD was overexpressed, indicating that VcCspD hampers the growth of E. coli. When the production of heme in cells was promoted by the addition of a heme precursor, δ-aminolevulinic acid, the growth of E. coli expressing VcCspD was decelerated, but the growth of E. coli expressing the C22A mutant was not decelerated. These observations allow us to conclude that heme specifically binds to the HRM region in VcCspD and inhibits the binding of target ssDNA, which suggests that heme functions as a regulatory molecule for DNA replication.

Selective gene regulation is mediated by recognition of specific DNA sequences by transcription factors (TFs). The extremely challenging task of searching out specific cognate DNA binding sites among several million putative sites within the eukaryotic genome is achieved by complex molecular recognition mechanisms. Elements of this recognition code include the core binding sequence, the flanking sequence context, and the shape and conformational flexibility of the composite binding site. To unravel the extent to which DNA flexibility modulates TF binding, in this study, we employed experimentally guided molecular dynamics simulations of ternary complex of closely related Hox heterodimers Exd-Ubx and Exd-Scr with DNA. Results demonstrate that flexibility signatures embedded in the flanking sequences impact TF binding at the cognate binding site. A DNA sequence has intrinsic shape and flexibility features. While shape features are localized, our analyses reveal that flexibility features of the flanking sequences percolate several basepairs and allosterically modulate TF binding at the core. We also show that lack of flexibility in the motif context can render the cognate site resistant to protein-induced shape changes and subsequently lower TF binding affinity. Overall, this study suggests that flexibility-guided DNA shape, and not merely the static shape, is a key unexplored component of the complex DNA-TF recognition code.

Ficolin, a member of the fibrinogen-related proteins family (FREPs), functions as a pattern recognition receptor (PRR) in vertebrates and in invertebrates as a novel lectin. In this study, we discovered the Ficolin homolog of Chinese mitten crab (Eriocheir sinensis), which we named EsFicolin. The obtained sequence showed that it has a highly conserved C-terminal fibrinogen-related domain (FReD) and a coiled-coil structure for trimer formation. EsFicolin was up-regulated in hemocytes after being stimulated by bacteria. Recombinant EsFicolin protein binds to gram-negative and gram-positive bacteria and agglutinates bacteria through pathogen-associated molecular patterns. In-depth study found that recombinant EsFicolin could effectively remove bacteria and showed direct antibacterial activity. EsFicolin could also promote the phagocytosis of hemocytes to enhance bacterial clearance. These findings suggest that EsFicolin plays an important role in the crab antibacterial immune response.

The sea cucumber Apostichopus japonicus is one of the most dominant and economically important aquaculture species in China. Saponin, which possesses notable biological and pharmacological properties, is a key determinant of the nutritional and health value of A. japonicus. In the present study, we amplified the full-length cDNA of a phosphomevalonate kinase (PMK) gene (named AjPMK) using rapid amplification of cDNA ends (RACE). Subsequently, we engineered a recombinant AjPMK (rAjPMK) protein and assessed its enzymatic activity by enzyme-linked immunosorbent assay (ELISA). Proteins that interact with rAjPMK were screened and identified via pull-down assay combined with liquid chromatography with tandem mass spectrometry (LC-MS/MS). We found that the full-length cDNA of AjPMK contained 1354 bp and an open reading frame (ORF) of 612 bp. The AjPMK protein was predicted not to contain a signal peptide but to contain a phosphonolate kinase domain seen in higher eukaryotes and a P-loop with a relatively conserved nucleoside triphosphate hydrolase domain. The molecular weight of the AjPMK protein was estimated to be 23.81 kDa, and its isoelectric point was predicted to be 8.72. Phylogenetic analysis showed that AjPMK had a closer evolutionary relationship with genes from starfish than with those of other selected species. Besides, we found that rAjPMK synthesized mevalonate-5-diphosphate, interacted either directly or indirectly with crucial pattern recognition receptors (PRRs) and was regulated by immune-related processes, including antioxidative reactions, stress resistance responses and enzyme hydrolysis. Moreover, AjPMK also interacted with farnesyl pyrophosphate synthase, an enzyme reported to be involved in saponin biosynthesis. Together, our findings implied that AjPMK may be directly involved in saponin biosynthesis and the regulation of various innate immune processes.

Members of the E74-like factor (ELF) subfamily are involved in the immune stress process of organisms by regulating immune responses and the development of immune-related cells. PmE74 of Penaeus monodon was characterized and functionally analyzed in this study. The full length of PmE74 was 3106 bp, with a 5'-UTR of 297 bp, and a 3'-UTR of 460 bp. The ORF (Open reading frame) was 2349 bp and encoded 782 amino acids. Domain analysis showed that PmE74 contains a typical Ets domain. Multiple sequence alignment and phylogenetic tree analysis showed that PmE74 clustered with Litopenaeus vannamei E74 and displayed significant similarity (98.98%). PmE74 was expressed in all tissues tested in P. monodon, with the highest levels of expression observed in the testis, intestine, and epidermis. Different pathogen stimulation studies have revealed that PmE74 expression varies in response to different pathogen stimuli. A 96-h acute low salt stress study revealed that PmE74 in the hepatopancreas was upregulated and downregulated in the salinity 17 group and considerably downregulated in the salinity 3 group, whereas PmE74 in gill tissue was considerably downregulated in both groups. Further, by knocking down PmE74 and learning the trends of its linkage genes PmAQP1, PmNKA, PmE75, PmFtz-f1, PmEcR, and PmRXR in response to low salt stress, it was further indicated that PmE74 could have a vital role in the regulation of low salt stress. The SNP test revealed that PmE74-In1-53 was significantly associated with low salt tolerance traits in P. monodon (P 0.05). The findings of this study can aid in the advancement of molecular marker-assisted breeding in P. monodon, as well as provide fundamental data and methodologies for further investigation of its low salt tolerance strains in P. monodon.

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a crucial role in the Toll-like receptor/IL-1R signal pathway, which mediates the downstream signal transduction involved in innate and adaptive immunity. In the present study, an IRAK4 homologue (named as MaIRAK4) from blunt snout bream (Megalobrama amblycephala) was cloned and characterized. The open reading frame (ORF) of MaIRAK4 contains 1422 nucleotides, encoding a putative protein of 473 amino acids. Protein structural analysis revealed that MaIRAK4 has an N-terminal death domain (DD) and a central kinase domain (S_TKc), similar to those of mammals and other fishes. Multiple sequence alignment demonstrated that MaIRAK4 is highly homologous with that of grass carp (97.67%). The qRT-PCR analysis showed that MaIRAK4 expressed widely in all examined tissues, including heart, liver, spleen, kidney, head-kidney, gill, intestine and muscle, with the highest expression in the liver and spleen. After stimulation with LPS, MaIRAK4 expression upregulated significantly and reached a peak at 6 h and 12 h post LPS stimulation in the spleen and head-kidney, respectively. After challenge with Aeromonas hydrophila, MaIRAK4 expression peaked at 48 h and 72 h in spleen/head-kidney and liver, respectively. These results implied that MaIRAK4 is involved in the host defense against bacterial infection. Subcellular localization analysis indicated that MaIRAK4 distributed in the cytoplasm. Co-immunoprecipitation and subcellular co-localization assay revealed that MaIRAK4 can combine with MaMyD88 through DD domain. MaIRAK4 overexpression can induce slightly the NF-κB promoter activity in HEK 293 cells. However, the activity of NF-κB promoter was dramatically enhanced after co-transfection with MaIRAK4 and MaMyD88 plasmids. The results showed that MaIRAK4 was involved in NF-κB signal pathway mediated by maMyD88. The expression level of pro-inflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α) decreased significantly after the siRNA-mediated knockdown of MaIRAK4. Together, these results suggest that MaIRAK4 plays an important function in the innate immunity of M. amblycephala by inducing cytokines expression.

Promoters play an irreplaceable role in biological processes and genetics, which are responsible for stimulating the transcription and expression of specific genes. Promoter abnormalities have been found in some diseases, and the level of promoter-binding transcription factors can be used as a marker before a disease occurs. Hence, detecting promoters from DNA sequences has important biological significance, particular, distinguishing strong promoters can help to elucidate differences in gene expression and the mechanisms of specific diseases. With the introduction of third-generation sequencing, it is difficult to match the speed of sequencing to the speed of labeling promoters experimentally. Many computing models have been designed to fill this gap and identify unlabeled DNA. However, their feature representation methods are very singular, which cannot reflect the information contained in the original samples. With the aim of avoiding information loss, we propose a computational model based on multiple descriptors and feature selection to jointly express samples. It is worth mentioning that a new feature descriptor called K-mer word vector is defined. The promoter model of multiple feature descriptors dominated by K-mer word vector achieves similar performance to existing methods, the sensitivity of 85.72% can distinguish the promoter more effectively than other methods. Furthermore, the performance of the promoter strength has surpassed published methods, and accuracy of 77.00% greatly improves the ability to distinguish between strong and weak promoters.

Crustins are a kind of antibacterial peptides (AMP) existing in crustaceans, and their antibacterial abilities are considered to be related to the conserved WAP domain. In this study, a novel type I Crustin gene was identified in Exopalaemon carinicauda, named EcCru. The deduced amino acid sequence revealed that the conserved cysteine at position 7 in the WAP domain was replaced by aspartic acid. The gene is 405 bp in length, encoding 134 amino acids, and is mainly distributed in gills and hepatopancreas. After Vibrio parahaemolyticus and Aeromonas hydrophila stimulation, the expression of EcCru was significantly up-regulated within 12 h, and then returned to normal levels. The recombinant protein was obtained using the Pichia pastoris expression system, and the recombinant protein had neither antibacterial activity against gram-positive or gram-negative bacteria. But the antibacterial ability emerged when Asp

DNA is naturally dynamic and can self-assemble into alternative secondary structures including the intercalated motif (i-motif), a four-stranded structure formed in cytosine-rich DNA sequences. Until recently, i-motifs were thought to be unstable in physiological cellular environments. Studies demonstrating their existence in the human genome and role in gene regulation are now shining light on their biological relevance. Herein, we review the effects of epigenetic modifications on i-motif structure and stability, and biological factors that affect i-motif formation within cells. Furthermore, we highlight recent progress in targeting i-motifs with structure-specific ligands for biotechnology and therapeutic purposes.

Thanks to the tremendous advancement of deep sequencing and large-scale profiling, epitranscriptomics has become a rapidly growing field. As one of the most important parts of epitranscriptomics, ribonucleic acid (RNA) methylation has been focused on for years for its fundamental role in regulating the many aspects of RNA function. Thanks to the big data generated in sequencing, machine learning methods have been developed for efficiently identifying methylation sites. In this review, we comprehensively explore machine learning based approaches for predicting 10 types of methylation of RNA, which include m6A, m5C, m7G, 5hmC, m1A, m5U, m6Am, and so on. Firstly, we reviewed three main aspects of machine learning which are data, features and learning algorithms. Then, we summarized all the methods that have been used to predict the 10 types of methylation. Furthermore, the emergent methods which were designed to predict multiple types of methylation were also reviewed. Finally, we discussed the future perspectives for RNA methylation sites prediction.

Dactylogyridae is one of the most studied families of parasitic flatworms with more than 1000 species and 166 genera described to date including ecto- and endoparasites. Dactylogyrid monogeneans were suggested as model organisms for host-parasite macroevolutionary and biogeographical studies due to the scientific and economic importance of some of their host lineages. Consequently, an array of phylogenetic research into different dactylogyrid lineages has been produced over the past years but the last family-wide study was published 16 years ago. Here, we provide a meta-analysis of the phylogenetic relationships of Dactylogyridae including representatives of all genera with available molecular data (n = 67). First, we investigate the systematic informativeness of morphological characters widely used to diagnose dactylogyrid genera through a parsimony analysis of the characters, character mapping, and phylogenetic comparative methods. Second, we provide an overview of the current state of the systematics of the family and its subfamilies, and summarise potentially poly- and paraphyletic genera. Third, we elaborate on the implications of taxonomic, citation, and confirmation bias in past studies. Fourth, we discuss host range, biogeographical, and freshwater-marine patterns. We found two well-supported macroclades which we assigned to the subfamilies Dactylogyrinae and Ancyrocephalinae. These subfamilies further include 16 well-supported clades with only a few synapomorphies that could be deduced from generic diagnoses in the literature. Furthermore, few morphological characters considered systematically informative at the genus level display a strong phylogenetic signal. However, the parsimony analysis suggests that these characters provide little information on the relationships between genera. We conclude that a strong taxonomic bias and low coverage of DNA sequences and regions limit knowledge on morphological and biogeographical evolutionary patterns that can be inferred from these results. We propose addressing potential citation and confirmation biases through a 'level playing field' multiple sequence alignment as provided by this study. This research was funded by Czech Science Foundation (GACˇ R) standard project GA19-13573S. N.K. (BOF21PD01), A.J.C.L. (BOF19OWB02) and M.P.M.V. (BOF20TT06) are financed by the Special Research Fund of Hasselt University, Belgium. Walter A. Boeger (Universidade Federal do Paraná, Brazil) is thanked for fruitful discussions on this matter

Programmed elimination of DNA during development yields somatic cell nuclei with dramatically different DNA sequence and content relative to germline nuclei, profoundly influencing genome architecture and stability. Whole-genome sequencing has significantly expanded the list of taxa known to exhibit this trait and has revealed the identity of excised genes and transposable elements (TEs) in certain taxa. Here, we compare the diverse mechanisms employed by ciliates, nematodes, copepods, and lamprey to downsize their genomes during development and propose tests of hypotheses about the evolution and/or maintenance of this trait. We explore possible functional roles that programmed DNA elimination (PDE) could play in genomic defense (especially against TEs), regulation of development, sex determination, co-option, and modulating nucleotypic effects, which together argue for a place in the mainstream investigation of genome evolution.

Genome sequencing (GS) can aid clinical management of multiple pediatric conditions. Insurers require accurate cost information to inform funding and implementation decisions. The objective was to compare the laboratory workflows and microcosts of trio GS testing in children with developmental delay (DD) and in children with cardiac conditions.Cost items related to each step in trio GS (child and 2 parents) for both populations were identified and measured. Program costs over 5 years were estimated. Probabilistic and deterministic analyses were conducted.The mean cost per trio GS was CAD$6634.11 (95% CI = 6352.29-6913.40) for DD and CAD$8053.10 (95% CI = 7699.30-8558.10) for cardiac conditions. The 5-year program cost was CAD$28.11 million (95% CI = 26.91-29.29) for DD and CAD$5.63 million (95% CI = 5.38-5.98) for cardiac conditions. Supplies constituted the largest cost component for both populations. The higher cost per sample for the population with cardiac conditions was due to the inclusion of pharmacogenomics, higher bioinformatics labor costs, and a more labor intensive case review.This analysis indicated important variation in trio GS workflow and costs between pediatric populations in a single institution. Enhanced understanding of the clinical utility and costs of GS can inform harmonization and implementation decision-making.

PURPOSE: SouthSeq is a translational research study that performed genome sequencing (GS) for infants with symptoms suggestive of a genetic disorder. Recruitment targeted racial/ethnic minorities and rural, medically underserved areas in the Southeastern US that are historically under-represented in genomic medicine research. METHODS: GS and analysis were performed for 367 infants to detect disease-causal variation concurrent with standard of care evaluation and testing. RESULTS: Definitive diagnostic (DD) or likely diagnostic (LD) genetic findings were identified in 30% of infants and 14% harbored an uncertain result. Only 43% of DD/LD findings were identified via concurrent clinical genetic testing suggesting that GS testing is better for obtaining early genetic diagnosis. We also identified phenotypes that correlate with the likelihood of receiving a DD/LD finding, such as craniofacial, ophthalmologic, auditory, skin, and hair abnormalities. We did not observe any differences in diagnostic rates between racial/ethnic groups. CONCLUSION: We describe one of the largest-to-date GS cohorts of ill infants, enriched for African American and rural patients. Our results demonstrate the utility of GS as it provides early in life detection of clinically relevant genetic variation not identified via current clinical genetic testing, particularly for infants exhibiting certain phenotypic features.

Interleukin 12 (IL-12) binds its receptor complex of IL-12 receptor beta 1 (IL-12Rβ1) and IL-12Rβ2 to transduce cellular signaling in mammals. In teleosts, the function of Il-12 is drawing increasing attention, but molecular and functional features of Il-12 receptors remain obscure. Especially, the existence of multiple Il-12 isoforms in some fish species elicits the requirement to clarify their receptors. In this study, we isolated three cDNA sequences as Il-12 receptor candidates from grass carp, entitled as grass carp Il-12rβ1 (gcIl-12rβ1), gcIl-12rβ2a and gcIl-12rβ2b. In silico analysis showed that gcIl-12rβ1 and gcIl-12rβ2a shared the conserved gene locus and similar structure characteristics with their orthologues of zebrafish, frog, chicken, mouse and human, respectively. However, the Il-12rβ2b of grass carp and zebrafish was similar to IL-27Ra in non-fish species. Further locally installed BLAST and gene synteny analysis uncovered three gcIl-12 receptors being single copied genes. Tissue distribution assay revealed that gcil12rβ1 and gcil12rβ2a transcripts were predominantly expressed in head kidney, differing from the even distribution of gcil12rβ2b transcripts in all detected tissues. Subsequently, the binding ability and antagonistic effects of recombinant extracellular region of gcIl-12rβ1 with recombinant grass carp Il-12 (rgcIl-12) isoforms were explored, providing functional evidence of the newly cloned gcIl-12rβ1 being genuine orthologues of mammalian IL-12Rβ1. Moreover, our data showed that gcIl-12rβ1 and gcIl-12rβ2a but not gcIl-12rβ1 and gcIl-12rβ2b mediated the effects of rgcIl-12 isoforms on ifn-γ promoter activity, thereby revealing Il-12 receptor signaling in fish. These results identified grass carp Il-12 receptors, thereby advancing our understanding of Il-12 isoform signaling in fish.

Macrophage receptor with collagenous structure (MARCO) is a scavenger receptor that plays a crucial role in the immune response against microbial infections. To clarify the roles of fish MARCO in Singapore grouper iridovirus (SGIV) infection, we identified and characterized Ec-MARCO in the orange-spotted grouper (Epinephelus coioides). The Ec-MARCO encoded a 370-amino acid protein with transmembrane region, coiled coil region and SR domain, which shared high identities with reported MARCO. The abundant transcriptional level of Ec-MARCO was found in spleen, head kidney and blood. And the Ec-MARCO expression was significantly up-regulated in grouper spleen (GS) cells after infection with SGIV in vitro. Subcellular localization analysis revealed that Ec-MARCO was mainly distributed in the cytoplasm and on the cell membrane. Ec-MARCO knockdown in vitro significantly inhibited SGIV infection in GS cells, as evidenced by reduced decreased SGIV major capsid protein (MCP) transcription and MCP protein expression. Further studies showed that Ec-MARCO knockdown positively regulated proinflammatory cytokines and interferon-stimulated genes, and enhanced IFN and ISRE promoter activities. However, overexpression of Ec-MARCO did not affect SGIV entry into host cells. In summary, our results suggested that Ec-MARCO affected SGIV infection by regulating antiviral innate immune response.

Transmembrane protein channels enable fast and highly sensitive electrical detection of single molecules. Nanopore sequencing of DNA was achieved using an engineered Mycobacterium smegmatis porin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level as compared to other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Asymmetric mutations within subunits of the channel protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, random subunit assembly does not allow control of the channel composition of MspA and other oligomeric protein pores. In this study, we showed that it is feasible to convert octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. We constructed single-chain MspA trimers, pentamers, hexamers and heptamers to demonstrate that it is feasible to alter the subunit stoichiometry and the MspA pore diameter. All single-chain MspA proteins formed functional channels in lipid bilayer experiments. Importantly, we demonstrated that single-chain MspA discriminated all four nucleotides identical to MspA produced from monomers. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications.STATEMENT OF SIGNFICANCENanopore sequencing of DNA is a fast and cheap technology that uniquely delivers multi-kilobase reads. It is currently used world-wide in many applications such as genome sequencing, epigenetics, and surveillance of viral and bacterial pathogens and has started to revolutionize human lives in medicine, agriculture and environmental studies. However, the high base-calling error rates prevent nanopore DNA sequencing from reaching its full potential. In this study, we converted octameric MspA into a single-chain pore enabling asymmetric mutations to fine-tune the pore geometry and chemistry and address the shortcomings of nanopores. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications.

Cleidocranial dysplasia (CCD) represents a rare autosomal dominant skeletal dysplasia caused by mutations that induce haploinsufficiency in RUNX2, the important transcription factor of osteoblasts related to bone/cartilage development and maintenance. Clavicular hypoplasia, which involves aberrant tooth/craniofacial bone/skeletal formation, is a feature of classic CCD. RUNX2 mutations can be found in approximately 60-70% of patients with CCD, and around ∼10% of these mutations are microdeletions. The present paper describes the radiological and clinical characteristics of a 5-year-old girl who showed representative CCD features, including extra teeth, aplasia of clavicles, sloping shoulders, marked calvarial hypomineralization, and osteoporosis.We obtained genomic DNA of her family members and performed whole-genome sequencing (WGS) for samples collected from the proband. Quantitative fluorescent PCR (QF-PCR) and specific PCR plus electrophoresis were then performed as validation assays for all participants. In vitro analysis was performed. Luciferase assay for Runx2 transcription activity and evaluation of mRNA levels of Runx2 downstream osteogenic markers were conducted.WGS identified a 11.38-kb microdeletion in RUNX2 comprising 8-9 exons, which was validated by QF-PCR and specific PCR plus electrophoresis. In vitro experiments confirmed the pathogenicity of this variation.The present study identified a 11.38-kb microdeletion in RUNX2 that causes CCD. The deletion in the PST domain of RUNX2 reduces its transcription activity and reduces osteogenic marker levels, eventually decreasing the differentiation of osteoblasts. These findings clarify the role of the CCD-related mechanism in the development of CCD and suggest that it is important to consider copy number variation for the suspected familial patients early.

Peroxiredoxin (Prx) is an antioxidant protein family, which widely exists in organisms and plays an important role in innate immunity. In this study, the full-length cDNA of a Prx gene (NdPrx) was obtained from Neocaridina denticulata sinensis, which contains a 735 bp open reading frame (ORF) and encodes a polypeptide of 244 amino acids. It is inferred that the molecular weight of the encoded amino acid is 27261.20 Da and the theoretical isoelectric point is 6.16. Phylogenetic analysis shows that NdPrx and Prx4 have high homology, so it was named NdPrx4. Multiple alignment analysis showed that the amino acid sequence of NdPrx4 had high homology with Prx4 of other species, and the similarity with Homarus americanus was the highest, 92.86%. Quantitative real-time PCR analysis showed that NdPrx4 was expressed in various tissues of N. denticulata sinensis, and the expression in ovary was the highest. It was speculated that NdPrx4 may be related to maternal immune function. Under the stimulation of Cu

Haliotis discus hannai is a commercially important mollusk species, and the abalone aquaculture sector has been jeopardized by deteriorating environmental circumstances such as bacterial infection and thermal stress during the hot summers. However, due to a paucity of genetic information, such as transcriptome resources, our understanding of their stress adaptation is restricted. In this research, using single-molecule long-read (SMRT) sequencing technology, a library composed of ten tissues (i.e., haemocytes, gills, muscle, hepatopancreas, digestive tract, mantle, mucous gland, ovary, testis and head) was constructed and sequenced. In all, 41,855 high-quality unique transcripts, among which 24,778 were successfully annotated. Additionally, 13,463 SSRs, 1,169 transcription factors, and 18,124 lncRNAs were identified in H. discus hannai transcriptome. Furthermore, multiple immune-related transcripts were identified according to KEGG annotation, and a portion of these transcripts were mapped into several classical immune-related pathways, including the PI3K-AKT signaling pathway and Toll-like receptor signaling pathway. Additionally, 24 typical sequences related to the immunity pathway were detected by RT-PCR; the results showed that most of the immune-related genes showed significantly high expression at 72 h after bacterial challenges and thermal stress, especially the expression level of genes in gills was significantly higher than that in haemocytes under V. parahaemolyticus stress at 24 h. At the same time. The analysis of alternative splicing identified several innate immunity-related functions genes, including CD109 and caspase 2. These results suggest that the complex immune system, particularly the powerful innate immunity system, was crucial for H. discus hannai response to numerous environmental challenges.

Serum amyloid protein (SAA) is known as an acute reactive protein of innate immunity in mammals. However, in invertebrates, the role of SAA in innate immunity is still unclear. In this study, a full-length cDNA of the SAA gene (named TcSAA) was cloned from Tridacna crocea, mollusca. The gene includes a 193 bp 5' untranslated region (UTR) and a 129 bp 3' UTR sequence, and the open reading frame (ORF) with 393 bp nucleotides encodes a polypeptide of 130 amino acids. TcSAA contains a typical signal peptide and an SAA functional domain. The mRNA expression of TcSAA was detected in all 12 selected tissues and 7 different developmental stages. Furthermore, the expression of TcSAA was increased quickly in hemocytes after challenge with V. coralliilyticus or LPS. Furthermore, rTcSAA could bind V. coralliilyticus and V. alginolyticus, and the protein could reduce the lethality rate of the clams from 80% to 55% which caused by V. coralliilyticus about 48 h after injection. In summary, these results indicate that TcSAA may act as a marker for monitoring health and protecting T. crocea.

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare monogenetic disease, which is characterized by susceptibility to some weakly virulent mycobacteria. Here, we explored the pathogenic genes and molecular mechanisms of MSMD patients. We recruited three patients diagnosed with MSMD from two families. Two novel mutations (c.1228A > G, p.K410E and c.2071A > G, p.M691V) in STAT1 gene were identified from two families. The translocation of K410E mutant STAT1 protein into nucleus was not affected. The binding ability between gamma-activating sequence (GAS) and K410E mutant STAT1 protein was significantly reduced, which will reduce the interaction between STAT1 protein with the promoters of target genes. The M691V mutant STAT1 protein cannot translocate into the nucleus after IFN-γ stimulation, which will affect the STAT1 protein form gamma-activating factors (GAF) and bind the GAS in the promoter region of downstream target genes. Taken together, our results showed that the mutation of K410E led to impaired binding of STAT1 to target DNA, and the mutation of M691V prevented the transport of STAT1 into the nucleus, which led to MSMD. Together, we identified two novel mutations (c.1228A > G, p.K410E and c.2071A > G, p.M691V) in STAT1 gene in MSMD patients, and deciphered the molecular mechanism of MSMD caused by STAT1 mutations.

The DAN gene family (DAN, Differential screening-selected gene Aberrant in Neuroblastoma) is a group of genes that is expressed during development and plays fundamental roles in limb bud formation and digitation, kidney formation and morphogenesis and left-right axis specification. During adulthood the expression of these genes are associated with diseases, including cancer. Although most of the attention to this group of genes has been dedicated to understanding its role in physiology and development, its evolutionary history remains poorly understood. Thus, the goal of this study is to investigate the evolutionary history of the DAN gene family in vertebrates, with the objective of complementing the already abundant physiological information with an evolutionary context. Our results recovered the monophyly of all DAN gene family members and divide them into five main groups. In addition to the well-known DAN genes, our phylogenetic results revealed the presence of two new DAN gene lineages; one is only retained in cephalochordates, whereas the other one (GREM3) was only identified in cartilaginous fish, holostean fish, and coelacanth. According to the phyletic distribution of the genes, the ancestor of gnathostomes possessed a repertoire of eight DAN genes, and during the radiation of the group GREM1, GREM2, SOST, SOSTDC1, and NBL1 were retained in all major groups, whereas, GREM3, CER1, and DAND5 were differentially lost.

Inhalation of the fungus Alternaria alternata is associated with an increased risk of allergic asthma development and exacerbations. Recent work in acute exposure animal models suggests that A. alternata-induced asthma symptoms, which include inflammation, mucus overproduction and airway hyperresponsiveness, are due to A. alternata proteases that act via protease-activated receptor-2 (PAR2). However, because other active components present in A. alternata may be contributing to asthma pathophysiology through alternative signaling, the specific role PAR2 plays in asthma initiation and maintenance remains undefined. Airway epithelial cells provide the first encounter with A. alternata and are thought to play an important role in initiating the physiologic response. To better understand the role for PAR2 airway epithelial signaling we created a PAR2-deficient human bronchial epithelial cell line (16HBEPAR(−/−)) from a model bronchial parental line (16HBE14o−). Comparison of in vitro physiologic responses in these cell lines demonstrated a complete loss of PAR2 agonist (2at-LIGRL-NH(2)) response and significantly attenuated protease (trypsin and elastase) and A. alternata responses in the 16HBEPAR(−/−) line. Apical application of A. alternata to 16HBE14o− and 16HBEPAR2(−/−) grown at air-liquid interface demonstrated rapid, PAR2-dependent and independent, inflammatory cytokine, chemokine and growth factor basolateral release. In conclusion, the novel human PAR2-deficient cell line allows for direct in vitro examination of the role(s) for PAR2 in allergen challenge with polarized human airway epithelial cells.

Chondrosarcoma (CHS) is the second most common bone malignant tumor and currently has limited treatment options. We have recently demonstrated that thioredoxin interacting protein (TXNIP) plays a crucial role in the oncogenesis of bone sarcoma, yet its implication in CHS is underdetermined. In the present study, we first found that knockdown of TXNIP promotes the proliferation of CHS cell largely through increasing their glycolytic metabolism, which is well-known as Warburg effect for providing energy. Consistent with our previous report that YAP is fundamental for CHS cell growth, herein we revealed that YAP functioned as an upstream molecule of TXNIP, and that YAP negatively regulated TXNIP mRNA and protein expression both in vitro and in vivo. Mechanistically, although knockdown of YAP upregulated both the nuclear and cytoplasmic TXNIP expression, we did not observe any obvious interaction between YAP and TXNIP; instead, miRNA-524-5p was demonstrated to be required for YAP-regulated TXNIP expression and thus controlling CHS cell growth. Together, our study reveals that TXNIP is a tumor suppressor in terms of CHS, and that the YAP/miRNA-524-5p/TXNIP signaling axis may provide a novel clue for CHS targeted therapy.

Cytochrome P450 monooxygenase (P450 or CYP) plays an important role in the metabolism of insecticides and plant allelochemicals by insects. CYP321B1, a novel Spodoptera litura P450 gene, was identified and characterized. CYP321B1 contains a 1488 bp open reading frame (ORF) that encodes a 495 amino acid protein. In fourth instar larvae, the highest CYP321B1 expression levels were found in the midgut and fat body. In the tannin feeding test, tannin can significantly induce the expression of CYP321B1 in the midgut and fat body of 4th instar larvae. To verify the function of CYP321B1, RNA interference and metabolome analysis were performed. The results showed that silencing CYP321B1 significantly reduced the rate of weight gain under tannin induction. Metabolome analysis showed silencing affected 47 different metabolites, mainly involved in secondary metabolite biosynthesis and amino acid metabolism, including amino acids, lipid fatty acids, organic acids and their derivatives. Henoxyacetic acid and cysteamine are the most highly regulated metabolites, respectively. These findings demonstrate that CYP321B1 plays an important role in tannin detoxification and metabolism. Functional knowledge about metabolite detoxification genes in this major herbivorous insect pest can provide new insights into this biological process and provide new targets for agricultural pest control.

Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide. Dysregulation of circular RNAs (circRNAs) appears to be a critical factor in CRC progression. However, mechanistic studies delineating the role of circRNAs in CRC remain limited. In this study, qRT-PCR and western blot assays were used to measure the expression of genes and proteins. Migration, invasion, proliferation, and apoptosis were examined by wound-healing, transwell, CCK-8, colony formation, and flow cytometry assays, respectively. Molecular interactions were validated by a dual-luciferase report system. A xenograft animal model was established to examine in vivo tumor growth and lung metastasis. Our data indicated that circN4BP2L2 expression was increased in CRC tissues and cell lines. Notably, inhibition of circN4BP2L2 effectively inhibited proliferation, migration, and invasion of LoVo cells, and inhibited tumor growth and metastasis in vivo, whereas the forced expression of circN4BP2L2 facilitated the proliferation, migration, and invasion of HT-29 cells. Mechanistic studies revealed that circN4BP2L2 acted as a molecular sponge of miR-340-5p to competitively promote CXCR4 expression. Furthermore, inhibition of miR-340-5p reversed the anti-cancer effects of circN4BP2L2 or CXCR4 silencing. Our data indicated an oncogenic role of circN4BP2L2 in CRC via regulation of the miR-340-5p/CXCR4 axis, which may be a promising biomarker and target for CRC treatment. This study determined that circN4BP2L2 acts as an oncogene that promotes tumor growth and metastasis of colorectal cancer (CRC) by regulating the miR-340-5p/CXCR4 axis. The authors reveal a novel mechanism for the ceRNA regulatory network in CRC progression and identify a potential therapeutic target for CRC treatment.

circ_LPAR3 is an oncogene in esophageal squamous cell carcinoma. However, its role in oral squamous cell carcinoma (OSCC) is unknown.To reveal the functions of circ_LPAR3 in OSCC.Online bioinformatic analysis was performed to disclose the differential expression of circ_LPAR3, VEGFC, AKT1 in OSCC and also the target predictions of miR-513b-5p. Transfection was applied in OSCC cells. RT-qPCR was used to detect the RNA expression and western blot to measure the proteins, VEGFC and phosphor-AKT1 (ser473, p-AKT1). CCK8 kit was used for viability detection and Flow cytometry for apoptosis evaluation. RNA pull-down and luciferase reporter methods were used to validate the binding sites to miR-513b-5p on circ_LPAR3, VEGFC and AKT1. OSCC mice models were established to further unveil the functions of circ_LPAR3 in OSCC in vivo. HE staining and immunohistochemistry (CD34, VEGFC and p-AKT1) were further applied to analyze the pathological changes in association with circ_LPAR3 downregulation.circ_LPAR3 was upregulated in OSCC. Its knockdown in cells could decrease cell survival and mobility and in mice model, could inhibit the tumor growth and angiogenesis. Circ_LPAR3 promoted VEGFC and AKT1 activity by sponging miR-513b-5p in OSCC cells.Knockdown of circ_LPAR3 could inhibit the OSCC progression by sponging miR-513b-5p and activating VEGFC and AKT1.

Cryptocaryon irritans, a holotrichous ciliate parasitic protozoan, can trigger marine white spot disease and cause substantial economic losses in mariculture. However, methods of preventing and curing the disease have negatively affect fish, human, other organisms, and the natural environment. The antiparasitic activity of some antimicrobial peptides (AMPs) has garnered extensive attention of scholars. In this study, we identified and characterised a novel antiparasitic peptide, named So-pis, from Sciaenops ocellatus. The sequence analysis, structural features, and tissue distribution suggested that So-pis is genetically related to the piscidins family. However, So-pis showed a relatively low overall conservation compared with other known piscidins. So-pis is abound in glycine residues (22.7 %) and it has a neutral isoelectric point, weak amphipathicity, relatively long α-helix, and high hydrophobicity. These key elements are responsible for its biological activity. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicated that So-pis is a typically gill-expressed peptide. The expression of So-pis in the gill, skin, spleen, and head kidney could be regulated during C. irritans infection, thereby implicating a role of So-pis in immune defence against C. irritans. The synthetic So-pis had limited or no antimicrobial activity against bacterial and yeasts but exhibited potent antiparasitic activity against C. irritans in vitro. The activity of synthetic So-pis against erythrocytes was less potent than its antiparasitic activity against C. irritans. These results indicated that So-pis might be one of the crucial defence cytokines against C. irritans in the red drum. Cumulatively, our data suggested that So-pis might be a potential candidate for developing a novel, effective, and safe therapeutic agent against marine white spot disease.

Circadian locomotor output cycles kaput protein (CLOCK) plays a crucial role in glucose homeostasis and controlling insulin secretion. However, the mechanism of the CLOCK regulating rhythmic insulin secretion has not been fully understood.Rhythmic expression of the CLOCK in rat pancreatic beta cell was detected. INS-1 cells were transfected with siRNAs to knockdown the CLOCK before the cells were incubated with different concentrations of glucose. Insulin secretion was analyzed by ELISA method. Expression of the L-type calcium channel protein (Cav1.2, Cacna1c) was determined both in the CLOCK-knockdown cells and the control cells. Calcium influx was probed by fluorescent. Chromatin immunoprecipitation (ChIP) test and dual-luciferase reporter gene experiments were applied to verify the relationship between the CLOCK and Cav1.2.The CLOCK is abundantly expressed in rat pancreatic beta cells. Transcription level of the CLOCK showed rhythmicity in the beta cells. Compared to the control group, insulin release was significantly impaired with 25 mM glucose incubation in the CLOCK-knockdown group, but not showed with 2.5 mM glucose incubation. The expression of Cav1.2 and the influx of calcium were significantly decreased in the CLOCK-knockdown group with 25 mM glucose incubation. ChIP test indicted that the CLOCK bound to -444∼-454 region of the Cacna1c promoter of the INS-1 cells, but the binding was significantly reduced following the CLOCK-knockdown. Luciferase experiment was in accordance with the finding of ChIP.The CLOCK mediating Cav

LncRNAs are widely involved in various biological processes of plants. Recent evidences indicated that lncRNAs could act as competing endogenous RNAs (ceRNAs) to adsorb complementary miRNAs in a type of target mimicry, thereby indirectly regulating the target genes of miRNAs. In this study, a lncRNA, lncRNA08489 was identified to be the ceRNA of miR482e-3p in tomato plants. The expression patterns of lncRNA08489 and miR482e-3p showed opposite trends after tomato plants infected with Phytophthora infestans. In tomato leaves overexpressing lncRNA08489 (OE08489), the expression level of miR482e-3p decreased and its target gene, NBS-LRR increased. After infection with P. infestans, the resistance of OE08489 plants was stronger than that of the wild type, and the reactive oxygen species (ROS) scavenging ability of OE08489 plants was significantly improved. Taken together, these results indicated that lncRNA08489 acted as a ceRNA to decoy miR482e-3p and regulate the expression of NBS-LRR to enhance tomato resistance through ROS-scavenging system.

Subviral agents are nucleic acids which lack the features for classification as a virus. Tombusvirus-like associated RNAs (tlaRNAs) are subviral positive-sense, single-stranded RNAs that replicate autonomously, yet depend on a coinfecting virus for encapsidation and transmission. TlaRNAs produce abundant subgenomic RNA (sgRNA) upon infection. Here, we investigate how the well-studied tlaRNA, ST9, produces sgRNA and its function. We found ST9 is a noncoding RNA, due to its lack of protein coding capacity. We used resistance assays with eukaryotic Exoribonuclease-1 (XRN1) to investigate sgRNA production via incomplete degradation of genomic RNA. The ST9 3' untranslated region stalled XRN1 very near the 5' sgRNA end. Thus, the XRN family of enzymes drives sgRNA accumulation in ST9-infected tissue by incomplete degradation of ST9 RNA. This work suggests tlaRNAs are not just parasites of viruses with compatible capsids, but also mutually beneficial partners that influence host cell RNA biology.

Cancer-associated fibroblasts (CAFs) are the most important stromal cells in the tumor microenvironment (TEM) and have been reported to regulate various cancer development. Exosomes are considered important elements involved in intercellular communication and TME regulation, while the potential function of CAFs in lung cancer immunosuppressive microenvironments remains unknown. CAFs-derived exosomes (CAFs-exo) and normal fibroblasts (NFs)-derived exosomes (NFs-exo) were isolated by ultra-centrifugation and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot analysis. A549 cells were co-cultured with peripheral blood mononuclear cells (PBMCs). Flow cytometry assay was performed to detect the killing role of PBMCs on A549 cells. Bioinformatics and luciferase reporter assays were used to analyze the relationship among microRNA (miRNA), long non-coding RNA (lncRNA) and target gene. BALB/c mice were used to construct the lung cancer model by subcutaneous injection. Programmed death ligand 1 (PD-L1) was up-regulated in lung cancer tissues and cells. PD-L1 also up-regulated in CAFs cell medium-mediated A549 cells. CAFs decreased PBMCs induced-cell apoptosis through increasing PD-L1 in A549 cells. Moreover, CAFs transferred exosomes to lung cancer cells to suppress the killing effect of PBMCs through up-regulating PD-L1. Using microarray assays, opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) level was highly expressed in CAFs-exos. After treatment by CAFs-exos, miR-142-5p level was significantly down-regulated in A549 cells. OIP5-AS1 served as a sponge to target miR-142-5p and negatively regulated miR-142-5p expression in lung cancer cells. In addition, PD-L1 was a direct target of miR-142-5p. CAFs derived exosomal OIP5-AS1 reduced PBMCs induced-cell apoptosis and promoted tumor growth through decreasing miR-142-5p and up-regulating PD-L1. CAFs-derived exosomes suppressed the role of PBMCs induced-killing of lung cancer cells and promoted lung cancer progression by OIP5-AS1/ miR-142-5p/ PD-L1 axis, which provided a potential opportunity for diagnosis and treatment of lung cancer.

Osteosarcoma (OS) is the most common primary malignant bone tumor which has unclear pathobiology. Hence, enlightening the exact molecular mechanism underlying osteosarcoma progression is crucial for developing new treatment strategies. One member of the ARID family of DNA binding proteins is ARID3A that is implicated in osteosarcoma pathogenesis. ARID3A could bind E2F1 and regulate the transcription of E2F1 targets. At the same time, BECN1 is a well-characterized autophagy regulator gene that is a direct target of E2F1. The present study aimed to investigate the effect of ARID3A on the expression of BECN1 in osteosarcoma cells. First, we determined gene expression levels of ARID3A, BECN1, and E2F1 in U-2 OS by qPCR and confirmed with online datasets from GEO database. In addition, the prognostic value of these genes was also evaluated from Kaplan-Meier plotter database. Next, ARID3A was overexpressed and silenced in order to investigate the effect of ARID3A on BECN1 expression and proliferation of U-2 OS cells. Our results demonstrated that BECN1 was negatively correlated with E2F1 and positively correlated with ARID3A based on initial expression and prognostic effect in OS. Overexpression of ARID3A upregulated BECN1 while silenced ARID3A downregulated BECN1 expression in U-2 OS cells. Additionally, silencing of ARID3A promoted colony formation and proliferation, whereas overexpression of ARID3A suppressed colony formation and proliferation of U-2 OS cells. Taken together, these results indicate that ARID3A could function as tumor suppressor and affect the expression level of BECN1 in U-2 OS cells.

Calreticulin (Crt) is a highly conserved and multi-functional protein with lectin-like properties and important immunological activities. In this study, a Crt homolog, namely, ToCrt, was cloned and characterized from the obscure puffer Takifugu obscurus with an open reading frame of 1278 bp encoding a putative protein of 425 amino acids. The deduced amino acid sequence of ToCrt consisted of three conserved structural domains: N-domain, P-domain, and C-terminal domain. In the phylogenetic tree, ToCrt formed a separate cluster with three Crts from other pufferfish species (Takifugu rubripes, Takifugu flavidus, and Takifugu bimaculatus). The mRNA transcript of ToCrt was ubiquitously expressed in all the examined tissues in a decreasing order: liver, spleen, kidney, gills, intestine, and heart. After Vibrio harveyi, Edwardsiella tarda, and Aeromonas hydrophila stimulations, the levels of ToCrt mRNA in the kidney and spleen were significantly upregulated compared with that in the control group. The recombinant calreticulin domain of ToCrt (rToCrt) could bind three Gram-negative bacteria (V. harveyi, E. tarda, and A. hydrophila) and polysaccharides from bacterial cell walls such as lipopolysaccharide and peptidoglycan. Meanwhile, rToCrt could agglutinate different kinds of microorganisms and exhibit antimicrobial activity. These results suggested that T. obscurus ToCrt could serve as an antimicrobial effector in the host immune response against invading microorganisms.

Mycoplasma gallisepticum (MG) is one of the most important pathogens that causes chronic respiratory disease (CRD) in chickens. Exosomes secreted from cells have been well demonstrated to deliver miRNAs to recipient cells to modulate cellular functions. The purpose of this study is to explore the underlying functions and mechanisms of exosomal miR-181a-5p in MG-HS infection. In this study, we found that miR-181a-5p expression in vivo and in vitro was significantly up-regulated after MG-HS infection. It was also upregulated in exosomes, which were derived from MG-HS-infected type-II pneumocytes cells (CP-II). In addition, exosomes secreted by MG-HS-infected CP-II were able to transfer miR-181a-5p to recipient chicken embryo fibroblast cells (DF-1), resulting in a significant upregulation of miR-181a-5p expression in recipient DF-1 cells. We further identified that Mg2+/Mn2+-dependent protein phosphatase 1B (PPM1B) was the target gene of miR-181a-5p. Overexpression of miR-181a-5p or knockdown of PPM1B activated the nuclear factor-κB (NF-κB) signaling pathway, whereas inhibition of miR-181a-5p and overexpression of PPM1B led to the opposite results. Besides, up-regulation of miR-181a-5p significantly increased the expression of toll-like receptor 2 (TLR2), myeloid differentiation factor 88 (MyD88), tumor necrosis factors alpha (TNF-α) and interleukin-1β (IL-1β), whereas inhibition of miR-181a-5p showed a contrary result. Up-regulation of miR-181a-5p promoted cell proliferation, cell cycle progression and inhibited apoptosis to resist MG-HS infection. Moreover, overexpression of miR-181a-5p significantly negative regulated the expression of Mycoplasma gallisepticum adhesin protein (pMGA1.2) by directly inhibiting PPM1B. Thus, we concluded that exosomal miR-181a-5p from CP-II cells activated the TLR2-mediated MyD88/NF-κB signaling pathways by directly targeting PPM1B to promote the expression of pro-inflammatory cytokines for defending against MG-HS infection in recipient DF-1 cells.

Various diseases, including cancer, are caused by genetic mutations. A 5'-tailed duplex (TD) DNA, consisting of a long single-stranded (ss) editor DNA and a short (∼35-base) ss assistant oligodeoxyribonucleotide, can introduce a base-substitution in living cells and thus correct mutated genes. Previously, several hundred-base DNAs were employed as the editor DNAs. In this study, 5'-TDs were prepared from various editor DNAs with different lengths and examined for their gene correction abilities, using plasmid DNA bearing a mutated copepod green fluorescent protein (copGFP) gene, in human cells. High-throughput analysis was performed by the reactivated fluorescence of the wild-type protein encoded by the corrected gene as the indicator. The analysis revealed that 5'-TDs with ∼100-base ss editor DNAs enabled gene editing at least as efficiently as those with longer editor DNAs. Moreover, the antisense strand was more effective as the editor than the sense strand, in contrast to the 5'-TDs with longer editor strands. These results indicated that the 5'-TD fragments with shorter editor strands than those used in previous studies are useful nucleic acids for gene correction.

The SOCS/CIS is a family of intracellular proteins distributed widely among living organisms. The members of this family have extensively been studied in mammals and have been shown to regulate various physiological processes. In contrast, the functional roles of SOCS/CIS family proteins are unknown in most invertebrates, including insects. Here, we retrieved a full-length open reading frame (ORF) of SOCS-6 from Chines oak silkworm, Antheraea pernyi (Designated as ApSOCS-6), using the RNA-seq database. The predicted ApSOCS-6 amino acid sequence comprised an N-terminal SH2 domain and a C-terminal SOCS-box domain. It shared the highly conserved structures of the SOCS proteins with other lepidopteran species. ApSOCS-6 mRNA transcript was detected in all the tested tissues of the A. pernyi larvae; however, the highest mRNA levels were found in the larval hemocytes, fat bodies, and integuments. The mRNA transcript levels of ApSOCS-6 were increased in the A. pernyi larval hemocytes and fat bodies after a challenge by the Gram-positive bacteria, M. luteus, Gram-negative bacteria, Escherichia coli, Virus, ApNPV, and Fungus, B. bassiana. After the knockdown of ApSOCS-6, we found a significant increase in bacterial clearance and a decrease in the relative replication of bacteria. To evaluate the possible cause of enhanced antibacterial activity, we measured antimicrobial peptides expression in the fat body of A. pernyi larvae. The production of AMPs was strongly increased in the B. cereus infected larval fat bodies following silencing of ApSOCS-6. Our data indicate that ApSOCS-6 negatively regulates the expression of AMPs in immune tissues in response to the B. cereus challenge.

Transglutaminases (TGases) are widely known to play critical roles in innate immunity, in particular, TGase2, which involves in autophagy process to help degrade protein aggregates under stressful conditions in mammals. Nevertheless, the function of the TGase2 counterpart whether involves in invertebrate autophagy is largely unknown. In this study, a novel TGase2-like homologous gene from the sea cucumber Apostichopus japonicus (named as AjTGase2-like) was cloned using RACE technology and its biological functions were also investigated. The AjTGase2-like gene encoded a peptide of 750 amino acids with the representative domains of Transglut_N domain, TGc domain, and two Transglut_C domains, which exhibited highly conservative with vertebrate TGase2. Multiple sequence alignments and phylogenetic analysis both supported that AjTGase2-like belonged to a new member of TGase2 subfamily. AjTGase2-like was pervasively expressed in all examined tissues, with the largest transcription in muscle, followed by respiratory trees, and intestine. After immersion infection with Vibrio splendidus, the mRNA and protein levels of AjTGase2-like were both significantly induced and reached the highest levels at 24 h, indicating AjTGase2-like plays a key role in immune response. Further functional analysis showed that the ubiquitinated protein level was significantly increased by 1.65-fold (p 0.01) after silencing of AjTGase2-like, and the protein levels of AjLC3-II/I and AjBeclin1 were both obviously decreased by 0.49-fold (p 0.01) and 0.64-fold (p 0.01) at the same time, while the authophagy receptor of Ajp62 was signally up-regulated by 1.40-fold (p 0.01) under same condition. Moreover, the immunofluorescence signals of AjLC3 and Ajp62 were consistent with their protein levels, suggesting knockdown of AjTGase2-like causes a blockage in autophagy. More importantly, the AjLC3 positive signal was not increased after adding with chloroquine in the case of AjTGase2-like interference, indicating AjTGase2-like might play pivotal role in the early step of autophagosome formation. Besides, our results showed that the fluorescence signal of AjTGase2-like was largely co-localized with Ajp62 around the cytoplasm in vivo, and rAjp62 could directly combine with rAjTGase2-like in vitro, indicating AjTGase2-like interacts with Ajp62 during autophagy. Overall, our findings supported that AjTGase2-like served as a positive regulator in sea cucumber authophay.

Cyclohexanecarboxylate (CHCA) is formed by oxidative microbial degradation of n-alkylcycloparaffins and anaerobic degradation of benzoate, and also known to be a synthetic intermediate or the starter unit of biosynthesis of cellular constituents and secondary metabolites. Although two degradation pathways have been proposed, genetic information has been limited to the β-oxidation-like pathway. In this study, we identified a gene cluster, designated chcC1XTC2B1B2RAaAbAc, that is responsible for the CHCA aromatization pathway in Sinomonas (formerly Corynebacterium) cyclohexanicum strain ATCC 51369. Reverse transcription-PCR analysis indicated that the chc gene cluster is inducible by CHCA and that it consists of two transcriptional units, chcC1XTC2B1B2R and chcAaAbAc. Overexpression of the various genes in Escherichia coli, and purification of the recombinant proteins led to the functional characterization of ChcAaAbAc as subunits of a cytochrome P450 system responsible for CHCA hydroxylation; ChcB1 and ChcB2 as trans-4-hydroxyCHCA and cis-4-hydroxyCHCA dehydrogenases, respectively; ChcC1 was identified as a 4-oxoCHCA desaturase containing a covalently bound FAD; and ChcC2 was identified as a 4-oxocyclohexenecarboxylate desaturase. The binding constant of ChcAa for CHCA was found to be 0.37 mM. Kinetic parameters established for ChcB1 indicated that it has a high catalytic efficiency towards 4-oxoCHCA compared to trans- or cis-4-hydroxyCHCA. The Km and Kcat values of ChcC1 for 4-oxoCHCA were 0.39 mM and 44 s−1, respectively. Taken together with previous work on the identification of a pobA gene encoding a 4-hydroxybenzoate hydroxylase, we have now localized the remaining set of genes for the final degradation of protocatechuate before entry into the tricarboxylic acid cycle.

Exonic circular RNAs (circRNAs) are RNA molecules that are covalently closed by back-splicing via canonical splicing machinery. Despite overlapping sequences, exon circularization generates RNA secondary structures through intramolecular base-pairing that are different from the linear transcript. Here we review factors that may affect circRNA structure and how structure affects circRNA function and regulation. We highlight considerations for RNA sequencing and expression measurement to ensure highly structured circRNAs are accurately represented by the data and discuss issues that need to be addressed in generating circRNAs to recapitulate their endogenous structures. We conclude our review by discussing experimental strategies on revealing the varied roles of RNA structure in circRNA biogenesis, function and decay.

Stable Intronic Sequence RNA (sisRNA) is a relatively new class of non-coding RNA. Found in many organisms, these sisRNA produced from their host genes are generally involved in regulatory roles, controlling gene expression at multiple levels through active involvement in regulatory feedback loops. Large scale identification of sisRNA via genome-wide RNA sequencing has been difficult, largely in part due to its low abundance. Done on its own, RNA sequencing often yields a large mass of information that is ironically uninformative; the potential sisRNA reads being masked by other highly abundant RNA species like ribosomal RNA and messenger RNA. In this review, we present a practical workflow for the enrichment of circular sisRNA through the use of transcriptionally quiescent systems, rRNA-depletion, and RNase R treatment prior to deep sequencing. This workflow allows circular sisRNA to be reliably detected. We also present various methods to experimentally validate the circularity and stability of the circular sisRNA identified, as well as a few methods for further functional characterisation.