Superfamily Domain Research Articles

Substrate binding-induced conformational transitions in the omega-3 fatty acid transporter MFSD2A

Major Facilitator Superfamily Domain containing 2 A (MFSD2A) is a transporter that is highly enriched at the blood-brain and blood-retinal barriers, where it mediates Na+-dependent uptake of ω−3 fatty acids in the form of lysolipids into the brain and eyes, respectively. Despite recent structural insights, it remains unclear how this process is initiated, and driven by Na+. Here, we perform Molecular Dynamics simulations which demonstrate that substrates enter outward facing MFSD2A from the outer leaflet of the membrane via lateral openings between transmembrane helices 5/8 and 2/11. The substrate headgroup enters first and engages in Na+ -bridged interactions with a conserved glutamic acid, while the tail is surrounded by hydrophobic residues. This binding mode is consistent with a “trap-and-flip” mechanism and triggers transition to an occluded conformation. Furthermore, using machine learning analysis, we identify key elements that enable these transitions. These results advance our molecular understanding of the MFSD2A transport cycle.

Genome-Wide Identification and Expression Profiling of Pathogenesis-Related Protein 1 (PR-1) Genes in Durum Wheat (Triticum durum Desf.).

Pathogen-related proteins (PRs) are diversified proteins with a low molecular weight implicated in plant response to biotic and abiotic stress as well in regulating different functions in plant maturation. Interestingly, no systematical study has been conducted in durum wheat (Triticum turgidum subsp. durum). In the present study, 12 PR-1 genes encoding a CAP superfamily domain were identified in the genome of Triticum turgidum subsp. durum, which is an important cereal, using in silico approaches. Additionally, phylogenetic analysis showed that the PR-1 genes were classified into three groups based on their isoelectric point and the conserved motif domain. Moreover, our analysis showed that most of the TdPR-1 proteins presented an N-terminal signal peptide. Expression patterns analysis showed that the PR-1 gene family presented temporal and spatial specificity and was induced by different abiotic stresses. This is the first report describing the genome-scale analysis of the durum wheat PR-1 gene family, and these data will help further study the roles of PR-1 genes during stress responses, leading to crop improvement.

Overexpression of the mango MiGF6A and MiGF6B genes promotes early flowering in transgenic Arabidopsis plants

14-3-3 genes are universal regulators that play important roles in regulating flower development and stress responses. In this study, two homologous 14-3-3 genes, MiGF6A and MiGF6B, were obtained from the genome of SiJiMi mango. Sequence analysis showed that both MiGF6A and MiGF6B have a fully conserved 14-3-3 superfamily domain. MiGF6A and MiGF6B were expressed in tissues at all stages of development, especially in the flowers and buds. Both MiGF6A and MiGF6B were expressed in response to low temperature, NaCl and polyethylene glycol (PEG) treatments. Subcellular localization analysis showed that MiGF6A and MiGF6B were located in the nucleus. Overexpression of MiGF6A and MiGF6B in Arabidopsis thaliana resulted in an early-flowering phenotype and significant upregulation of the flowering-related genes FLOWERING LOCUS T (AtFT), AtFD1, and LEAFY (AtLFY). Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses showed that MiGF6A and MiGF6B interact with the MiFD1, MiFD2 and MiSVP3 proteins. These results suggest that MiGF6A and MiGF6B may play positive roles in the mango flowering process.

Mfsd2a utilizes a flippase mechanism to mediate omega-3 fatty acid lysolipid transport

Major Facilitator Superfamily Domain containing 2a (Mfsd2a) is a sodium-dependent lysophosphatidylcholine (LPC) transporter expressed at the blood-brain barrier that constitutes the main pathway by which the brain obtains omega-3 fatty acids, such as docosahexanoic acid. Mfsd2a deficiency in humans results in severe microcephaly, underscoring the importance of LPC transport by Mfsd2a for brain development. Biochemical studies and recent cryo-electron microscopy (cryo-EM) structures of Mfsd2a bound to LPC suggest that Mfsd2a transports LPC via an alternating access mechanism between outward-facing and inward-facing conformational states in which the LPC inverts during transport between the outer and inner leaflet of a membrane. However, direct biochemical evidence of flippase activity by Mfsd2a has not been demonstrated and it is not understood how Mfsd2a could invert LPC between the outer and inner leaflet of the membrane in a sodium-dependent manner. Here, we established a unique invitro assay using recombinant Mfsd2a reconstituted in liposomes that exploits the ability of Mfsd2a to transport lysophosphatidylserine (LPS) coupled with a small molecule LPS binding fluorophore that allowed for monitoring of directional flipping of the LPS headgroup from the outer to the inner liposome membrane. Using this assay, we demonstrate that Mfsd2a flips LPS from the outer to the inner leaflet of a membrane bilayer in a sodium-dependent manner. Furthermore, using cryo-EM structures as guides together with mutagenesis and a cell-based transport assay, we identify amino acid residues important for Mfsd2a activity that likely constitute substrate interaction domains. These studies provide direct biochemical evidence that Mfsd2a functions as a lysolipid flippase.

Molecular and functional characterization of two IgM subclasses in large yellow croaker (Larimichthys crocea)

As the predominant immunoglobulin (Ig) isotype, IgM plays a crucial role in the acquired immunity of vertebrates. There is only one Igμ gene in mammals, except cattle, while the number of Igμ gene varies among teleost fish. In the current study, we found two functional Igμ genes (Igμ1 and Igμ2) and a pseudo Cμ gene (ψIgμ) in large yellow croaker (Larimichthys crocea). Both Igμ1 and Igμ2 genes possessed two transcript variants, which encoded the heavy chains of secreted (sIgM1 and sIgM2) and membrane-bound IgM1 and IgM2 (mIgM1 and mIgM2), respectively. Both the heavy chains of sIgM1 and sIgM2 consisted of a variable Ig domain, four constant Ig domains (CH1, CH2, CH3 and CH4) and a secretory tail, while those of mIgM1 and mIgM2 consisted of a variable Ig domain, three constant Ig domains (CH1, CH2 and CH3), a transmembrane domain and a short cytoplasmic tail. Cysteine residues that are necessary for the formation of intrachain and interchain disulfide bonds and tryptophan residues that are important for the folding of the Ig superfamily domain were well conserved in large yellow croaker IgM1 and IgM2. Interestingly, large yellow croaker IgM2 had an extra cysteine (C94) in the CH1 domain compared with IgM1, which may cause the structural difference between IgM1 and IgM2. A liquid chromatography–tandem mass spectrometry analysis revealed that both IgM1 and IgM2 were present at the protein level in large yellow croaker serum. Both the Igμ1 and Igμ2 genes were mainly expressed in systemic immune tissues, such as head kidney and spleen, but the expression level of Igμ2 was much lower than that of Igμ1. After Pseudomonas plecoglossicida infection, the expression levels of Igμ1 and Igμ2 in both the spleen and head kidney were significantly upregulated, with a higher upregulation of Igμ2 than that of Igμ1. These results suggested that Igμ1 and Igμ2 may play a differential role in the immune response of large yellow croaker against bacterial infection.

In Silico Characterization of Lycopene Beta Cyclase (LCYB) and Lycopene Epsilon Cyclase (LCYE) Genes from DH-Pahang (Musa acuminata, A Genome) and DH-PKW (Musa balbisiana, B Genome)

Alpha (α) and beta (β)-carotene are among the nutritious contents of bananas, with the unique feature of a high ratio of α-/β-carotene. Research on the gene and protein of the key enzymes determining the ratio of α-/β-carotene in bananas, namely lycopene beta cyclase (LCYB) and lycopene epsilon cyclase (LCYE), is currently not well defined. Hence, this study aimed to compare the characters of the LCYB and LCYE genes and their putative proteins from Musa acuminata 'DH-Pahang' and Musa balbisiana 'DH-PKW'. The corresponding nucleotide sequences from both species were aligned to detect similarities in the gene structure. Their protein products were characterized at the primary and tertiary levels. The phylogenetic tree was constructed based on nucleotide and protein sequences. The result showed that the gene structure between these two species is similar in LCYB in chromosome 9 but different in LCYB in chromosome 7 and LCYE. The presence of cis-acting regulatory elements in response to light dominated the 2000 nucleotide region of the 5'UTR of LCYB and LCYE genes in both species. Based on protein alignment and domain analysis, the NADB_Rossmann superfamily domain was detected in both LCYB and LCYE. Alignment of the three-dimensional protein structure showed a significant difference between MaLCYB.c07 and MbLCYB.c07 only. The phylogenetic tree based on protein sequences indicated the distant relationship of MaLCYB.c07 and MbLCYB.c07 with other LCYB ingroup OTUs. The results of this study could provide a molecular basis related to the exploration of bananas as a promising functional food to meet the needs of provitamin A.

Edwardsiella piscicida Ail1: An outer membrane protein required for host infection

Edwardsiella piscicida is a significant pathogenic microorganism, and can lead to systematic infections in multifold hosts, including teleost, amphibians, and mammals. Ail is a surface protein involved in virulence. However, the underlining mechanism is still to be investigated in E. piscicida. This study examined the functions and immunoprotective properties of an Ail molecule from E. piscicida. Ail1 possessed a conserved Ail/Lom superfamily domain and was exposed to the bacterial surface. Mutation of ail1 significantly reduced the survival rate under acid and oxidizing environment, motility, biofilm formation, cellular infection, tissue dissemination, and causing mortality in infected fish. The loss abilities of TX01Δail1 were rescued by overexpression of the ail1 gene. Recombinant Ail1 (rAil1) could interact with peripheral blood leukocytes from Japanese flounder and affect E. piscicida infection in vitro. Vaccine trial showed that rAil1 was used as a potential subunit vaccine and could induce effective immune protection against E. piscicida infection. Fish vaccinated with rAil1 produced the specific antibody against rAil1 in serum, which increased the bactericidal activity of serum complement. In addition, rAil1-vaccinated fish displayed the higher ROS and acid phosphatase activity of macrophages. The above findings indicated that Ail1 played a pivotal role in the virulence of E. piscicida and may be explored as a subunit vaccine in aquaculture.

Functional characterization of chemosensory proteins in response to artificial light treatment in Thalassodes immissaria.

Chemosensory proteins (CSPs) play a vital role in the response to environmental stimuli in insects. However, the involvement of insect CSPs in the stress response to night-time environmental changes has not been examined. In the current study, four TiCSP genes were first cloned from Thalassodes immissaria by transcriptome and RACE-PCR techniques. TiCSPs had typical characteristics of insect CSPs, including a highly conserved four-cysteine motif and olfactory-specific protein D (OS-D) or OS-D superfamily domains. TiCSP1-4 were clustered classified within different clades in a phylogenetic analysis and were differentially expressed at all developmental stages. Under night-time artificial light stress, the expression levels of TiCSP1 in males were significantly decreased at 24 h, and those of TiCSP2 were decreased in both adult sexes at 48 h. In a molecular docking analysis, TiCSPs showed relatively higher binding affinities with sex pheromone components than with host plant volatile molecules. Taking the reduced expression levels of TiCSPs and binding affinities into account, TiCSP1 and TiCSP2 are involved in the stress response processes of T.immissaria under light treatment. Our study supplies basic data for the evaluation of the effects of light interference control technology - an emerging physical control measure on nontarget pests of lychee orchards. © 2023 Society of Chemical Industry.

Porphyromonas gingivalis bacteremia increases the permeability of the blood-brain barrier via the Mfsd2a/Caveolin-1 mediated transcytosis pathway

Bacteremia induced by periodontal infection is an important factor for periodontitis to threaten general health. P. gingivalis DNA/virulence factors have been found in the brain tissues from patients with Alzheimer’s disease (AD). The blood-brain barrier (BBB) is essential for keeping toxic substances from entering brain tissues. However, the effect of P. gingivalis bacteremia on BBB permeability and its underlying mechanism remains unclear. In the present study, rats were injected by tail vein with P. gingivalis three times a week for eight weeks to induce bacteremia. An in vitro BBB model infected with P. gingivalis was also established. We found that the infiltration of Evans blue dye and Albumin protein deposition in the rat brain tissues were increased in the rat brain tissues with P. gingivalis bacteremia and P. gingivalis could pass through the in vitro BBB model. Caveolae were detected after P. gingivalis infection in BMECs both in vivo and in vitro. Caveolin-1 (Cav-1) expression was enhanced after P. gingivalis infection. Downregulation of Cav-1 rescued P. gingivalis-enhanced BMECs permeability. We further found P. gingivalis-gingipain could be colocalized with Cav-1 and the strong hydrogen bonding between Cav-1 and arg-specific-gingipain (RgpA) were detected. Moreover, P. gingivalis significantly inhibited the major facilitator superfamily domain containing 2a (Mfsd2a) expression. Mfsd2a overexpression reversed P. gingivalis-increased BMECs permeability and Cav-1 expression. These results revealed that Mfsd2a/Cav-1 mediated transcytosis is a key pathway governing BBB BMECs permeability induced by P. gingivalis, which may contribute to P. gingivalis/virulence factors entrance and the subsequent neurological impairments.

AaCaMKs Positively Regulate Development, Infection Structure Differentiation and Pathogenicity in Alternaria alternata, Causal Agent of Pear Black Spot.

Calcium/calmodulin-dependent protein kinase (CaMK), a key downstream target protein in the Ca2+ signaling pathway of eukaryotes, plays an important regulatory role in the growth, development and pathogenicity of plant fungi. Three AaCaMKs (AaCaMK1, AaCaMK2 and AaCaMK3) with conserved PKC_like superfamily domains, ATP binding sites and ACT sites have been cloned from Alternaria alternata, However, their regulatory mechanism in A. alternata remains unclear. In this study, the function of the AaCaMKs in the development, infection structure differentiation and pathogenicity of A. alternata was elucidated through targeted gene disruption. The single disruption of AaCaMKs had no impact on the vegetative growth and spore morphology but significantly influenced hyphae growth, sporulation, biomass accumulation and melanin biosynthesis. Further expression analysis revealed that the AaCaMKs were up-regulated during the infection structure differentiation of A. alternata on hydrophobic and pear wax substrates. In vitro and in vivo analysis further revealed that the deletion of a single AaCaMKs gene significantly reduced the A. alternata conidial germination, appressorium formation and infection hyphae formation. In addition, pharmacological analysis confirmed that the CaMK specific inhibitor, KN93, inhibited conidial germination and appressorium formation in A. alternata. Meanwhile, the AaCaMKs genes deficiency significantly reduced the A. alternata pathogenicity. These results demonstrate that AaCaMKs regulate the development, infection structure differentiation and pathogenicity of A. alternata and provide potential targets for new effective fungicides.

Molecular analysis of chicken interferon-alpha inducible protein 6 gene and transcriptional regulation.

Interferon-alpha inducible protein 6 (IFI6) is an interferon-stimulated gene (ISG), belonging to the FAM14 family of proteins and is localized in the mitochondrial membrane, where it plays a role in apoptosis. Transcriptional regulation of this gene is poorly understood in the context of inflammation by intracellular nucleic acid-sensing receptors and pathological conditions caused by viral infection. In this study, chicken IFI6 (chIFI6) was identified and studied for its molecular features and transcriptional regulation in chicken cells and tissues, i.e., lungs, spleens, and tracheas from highly pathogenic avian influenza virus (HPAIV)-infected chickens. The chIFI6-coding sequences contained 1638 nucleotides encoding 107 amino acids in three exons, whereas the duck IFI6-coding sequences contained 495 nucleotides encoding 107 amino acids. IFI6 proteins from chickens, ducks, and quail contain an IF6/IF27-like superfamily domain. Expression of chIFI6 was higher in HPAIV-infected White Leghorn chicken lungs, spleens, and tracheas than in mock-infected controls. TLR3 signals regulate the transcription of chIFI6 in chicken DF-1 cells via the NF-κB and JNK signaling pathways, indicating that multiple signaling pathways differentially contribute to the transcription of chIFI6. Further research is needed to unravel the molecular mechanisms underlying IFI6 transcription, as well as the involvement of chIFI6 in the pathogenesis of HPAIV in chickens.

Characterization of phosphate transporter genes and the function of SgPT1 involved in phosphate uptake in Stylosanthes guianensis

Phosphorus (P) is one of the principal macronutrients for plant growth and productivity. Although the phosphate (Pi) transporter (PT) of the PHT1 family has been functionally characterized as participating in Pi uptake and transport in plants, information about PT genes in stylo (Stylosanthes guianensis), an important tropical forage legume that exhibits good adaptability to low-P acid soils, is limited. In this study, stylo root growth was found to be stimulated under P deficiency. The responses of PT genes to nutrient deficiencies and their roles in Pi uptake were further investigated in stylo. Four novel PT genes were identified in stylo and designated SgPT2 to SgPT5. Like SgPT1, which had been previously identified, all five SgPT proteins harboured the major facilitator superfamily (MFS) domain. Variations in tissue-specific expression were observed among the SgPT genes, which displayed diverse responses to deficiencies in nitrogen (N), P and potassium (K) in stylo roots. Four of the five SgPTs exhibited high levels of transcriptional responsiveness to P deficiency in roots. Furthermore, SgPT1, a Pi-starvation-induced gene closely related to legume PT homologues that participate in Pi transport, was selected for functional analysis. SgPT1 was localized to the plasma membrane. Analysis of transgenic Arabidopsis showed that overexpression of SgPT1 led to increased Pi accumulation and promoted root growth in Arabidopsis plants. Taken together, the results of this study suggest the involvement of SgPTs in the stylo response to nutrient deprivation. SgPT1 might mediate Pi uptake in stylo, which is beneficial for root growth during P deficiency.

Genome-Wide Identification and Analysis of the GRAS Transcription Factor Gene Family in Theobroma cacao.

GRAS genes exist widely and play vital roles in various physiological processes in plants. In this study, to identify Theobroma cacao (T. cacao) GRAS genes involved in environmental stress and phytohormones, we conducted a genome-wide analysis of the GRAS gene family in T. cacao. A total of 46 GRAS genes of T. cacao were identified. Chromosomal distribution analysis showed that all the TcGRAS genes were evenly distributed on ten chromosomes. Phylogenetic relationships revealed that GRAS proteins could be divided into twelve subfamilies (HAM: 6, LISCL: 10, LAS: 1, SCL4/7: 1, SCR: 4, DLT: 1, SCL3: 3, DELLA: 4, SHR: 5, PAT1: 6, UN1: 1, UN2: 4). Of the T. cacao GRAS genes, all contained the GRAS domain or GRAS superfamily domain. Subcellular localization analysis predicted that TcGRAS proteins were located in the nucleus, chloroplast, and endomembrane system. Gene duplication analysis showed that there were two pairs of tandem repeats and six pairs of fragment duplications, which may account for the rapid expansion in T. cacao. In addition, we also predicted the physicochemical properties and cis-acting elements. The analysis of GO annotation predicted that the TcGRAS genes were involved in many biological processes. This study highlights the evolution, diversity, and characterization of the GRAS genes in T. cacao and provides the first comprehensive analysis of this gene family in the cacao genome.

Chicken GLUT4 undergoes complex alternative splicing events and its expression in striated muscle changes dramatically during development

Glucose transporter protein 4 (GLUT4) plays an important role in regulating insulin-mediated glucose homeostasis in mammals. Until now, studies on GLUT4 have focused on mammals mostly, while chicken GLUT4 has been rarely investigated. In this study, chicken GLUT4 mRNA sequences were obtained by combining conventional amplification, 5'- and 3'- rapid amplification of cDNA ends technique (RACE), then bioinformatics analysis on its genomic structure, splicing pattern, subcellular localization prediction and homologous comparisons were carried out. In addition, the distribution of GLUT4 was detected by RT-qPCR in bird's liver and striated muscles (cardiac muscle, pectoralis and leg muscle) at different ages, including embryonic day 14 (E14), E19, 7-day-old (D7), D21 and D49 (n=3-4). Results showed that chicken GLUT4 gene produced at least 14 transcripts (GenBank accession No: OP491293-OP491306) through alternative splicing and polyadenylation, which predicted encoding 12 types of amino acid (AA) sequences (with length ranged from 65 AA to 519 AA). These proteins contain typical major facilitator superfamily domain of glucose transporters with length variations, sharing a common sequence of 59 AA, and were predicted to have distinct subcellular localization. The dominant transcript (named as T1) consists of 11 exons with an open reading frame being predicted encoding 519 AA. In addition, analyzing on the spatio-temporal expression of chicken GLUT4 showed it dominantly expressed in pectoralis, leg muscles and cardiac muscle, and the mRNA level of chicken GLUT4 dramatically fluctuated with birds' development in cardiac muscle, pectoralis and leg muscles, with the level at D21 significantly higher than that at E14, E19, and D49 (P < 0.05). These data indicated that chicken GLUT4 undergoes complex alternative splicing events, and GLUT4 expression level in striated muscle was subjected to dynamic regulation with birds' development. Results indicate these isoforms may play overlapping and distinct roles in chicken.

APOE e4 dependent deficits in brain DHA phospholipids and mfsd2a in Alzheimer’s disease patients with severe cerebral amyloid angiopathy

AbstractBackgroundThe apolipoprotein E e4 allele (APOE4) is a major risk factor, contributing to vascular pathologies in AD which include CAA, BBB dysfunction and reduced cerebral vascular integrity. The diminished capacity of APOE4 to transport docosahexaenoic acid (DHA), an essential fatty acid required for the structural and functional maintenance and vascular integrity of the brain, could also contribute to AD pathogenesis. However, it remains undetermined if changes in the brain DHA content of phospholipids (PL) and DHA transporters exist in relation to APOE4, AD diagnosis and CAA pathology.MethodWe performed liquid chromatography/mass spectrometry‐based PL analysis of the cerebrovascular and parenchymal fractions from autopsied human brain of pathologically confirmed AD cases and controls. We performed an antibody‐based examination of the major facilitator superfamily domain containing 2A (mfsd2a) protein in the cerebrovasculature from these subjects and brain homogenates from transgenic mice with human APOE (APOE‐TR) and APOE‐TR crossed with mice with five AD mutations (EFAD). We performed proteomic analyses of the cerebrovascular and parenchymal fractions of the brains from 50‐week‐old APOE‐TR.ResultIn the cerebrovasculature and parenchyma, DHA containing PL species were lower in ε4 carriers with AD compared to control ε4 carriers. We observed an ε4‐dependent decrease in mfsd2a level in the cerebrovasculature of ε4 carriers compared to non‐carriers. Stratification of DHA containing PL by CAA showed that these PL levels were reduced in ε4 positive AD patients with severe CAA. In addition, brain levels of DHA containing PC species and mfsd2a were lower in APOE4‐TR and E4FAD mice compared to other genotypes in each mouse model. Furthermore, protein levels of glucose transporter 1 (Glut1) and a metabolite transporter, monocarboxylate transporter 1 (MCT1), were lower in the cerebrovasculature of APOE4‐TR relative to APOE3‐TR and APOE2‐TR mice.ConclusionThese findings demonstrate that brain DHA deficiencies in ε4 carriers may be due to reduced mfsd2a expression and partly associated with severe CAA. Thus, targeting this transport mechanism may improve the bioavailability of DHA into the brains of ε4 carriers who are at risk of developing AD.

Ovarian expression and localization of a low-density lipoprotein receptor related protein 13 (Lrp13) in orange-spotted grouper (Epinephelus coioides)

Low-density lipoprotein receptor (LDLR) superfamily is an ancient gene family composed of different genes encoding membrane receptors involved in ligand binding and endocytosis. These receptors typically consist of class A ligand-binding repeats (LBRs), epidermal growth factor precursor-like (EGF) domain, class b YWXD motifs, transmembrane (TM) domain, and cytoplasmic (CP) domain. Low-density lipoprotein receptor related protein 13 (Lrp13), a vitellogenin receptor with a unique domain structure, belonging to the LDLR superfamily, would play an important role in vitellogenesis in teleosts. In this study, the full-length cDNA of Ec-lrp13 was isolated from the ovary of orange-spotted grouper (Epinephelus coioides), an economically important aquaculture species in China. The open reading frame (ORF) of Ec-lrp13 is 3831 bp in length, encoding 1276 amino acids. The Ec-Lrp13 includes several typical domains of the LDLR superfamily and contains the specific structure of the Lrp13 protein. Ec-lrp13 is predominantly expressed in ovary. During ovarian development, Ec-lrp13 was highly expressed in stage II and III ovaries but its mRNA level was significantly decreased in stage IV ovary. In situ hybridization (ISH) analysis revealed that Ec-lrp13 mRNA signals were obviously observed in the ooplasm of PG and EPV oocytes, but decreased in the LPV oocytes, and weakly found in the VG oocytes. Immunohistochemistry (IHC) detection showed that Ec-Lrp13 protein signals were only observed in the LPV and VG oocytes. Our data provides preliminary evidence that Lrp13 may be involved in the Vtg uptake during ovarian development of orange-spotted grouper.

Genetic Diversity and Characterization of Circular Replication (Rep)-Encoding Single-Stranded (CRESS) DNA Viruses.

The CRESS-DNA viruses are the ubiquitous virus detected in almost all eukaryotic life trees and play an essential role in the maintaining ecosystem of the globe. Still, their genetic diversity is not fully understood. Here, we bring to light the genetic diversity of replication (Rep) and capsid (Cap) proteins of CRESS-DNA viruses. We divided the Rep protein of the CRESS-DNA virus into 10 clusters using CLANS and phylogenetic analyses. Also, most of the Rep protein in Rep cluster 1 (R1) and R2 (Circoviridae, Smacoviridae, Nanoviridae, and CRESSV1-5) contain the Viral_Rep superfamily and P-loop_NTPase superfamily domains, while the Rep protein of viruses in other clusters has no such characterized functional domain. The Circoviridae, Nanoviridae, and CRESSV1-3 viruses contain two domains, such as Viral_Rep and P-loop_NTPase; the CRESSV4 and CRESSV5 viruses have only the Viral_Rep domain; most of the sequences in the pCRESS-related group have only P-loop_NTPase; and Smacoviridae do not have these two domains. Further, we divided the Cap protein of the CRESS-DNA virus into 20 clusters using CLANS and phylogenetic analyses. The Rep and Cap proteins of Circoviridae and Smacoviridae are grouped into a specific cluster. Cap protein of CRESS-DNA viruses grouped with one cluster and Rep protein with another cluster. Further, our study reveals that selection pressure plays a significant role in the evolution of CRESS-DNA viruses' Rep and Cap genes rather than mutational pressure. We hope this study will help determine the genetic diversity of CRESS-DNA viruses as more sequences are discovered in the future. IMPORTANCE The genetic diversity of CRESS-DNA viruses is not fully understood. CRESS-DNA viruses are classified as CRESSV1 to CRESSV6 using only Rep protein. This study revealed that the Rep protein of the CRESS-DNA viruses is classified as CRESSV1 to CRESSV6 groups and the new Smacoviridae-related, CRESSV2-related, pCRESS-related, Circoviridae-related, and 1 to 4 outgroups, according to the Viral_Rep and P-loop_NTPase domain organization, CLANS, and phylogenetic analysis. Furthermore, for the first time in this study, the Cap protein of CRESS-DNA viruses was classified into 20 distinct clusters by CLANS and phylogenetic analysis. Through this classification, the genetic diversity of CRESS-DNA viruses clarifies the possibility of recombinations in Cap and Rep proteins. Finally, it has been shown that selection pressure plays a significant role in the evolution and genetic diversity of Cap and Rep proteins. This study explains the genetic diversity of CRESS-DNA viruses and hopes that it will help classify future detected viruses.

Effect of Maternal Body Mass Index on the Transcriptomic Network of Human First-Trimester Chorionic Villi.

The relationship between fertility and maternal body weight is shaped like an inverted "U," meaning that fertility is negatively affected in overweight or underweight women. Timely and appropriate maternal-fetal interaction is a crucial part of successful pregnancy. However, it is not clear how body weight affects maternal-fetal interaction. Placental villi are the bridge for maternal-fetal interaction. Therefore, we collected villi from pregnant women with different body mass indexes (BMI), who voluntarily underwent induced abortion, to construct a molecular network via RNA-seq. Surprisingly, based on global and significant gene network analysis, we found that dysregulation of inflammatory reaction, cell adhesion, and immune response were the most significantly enriched pathways. We also conducted dynamic gene expression analysis with BMI as a variable, and identified several distinct clusters. Among them, cluster 9 showed an inverted "U" shape and genes in it were mainly enriched in chemical synaptic transmission and cell-cell adhesion via plasma-membrane adhesion molecules. Additionally, genes in the "U" shaped cluster (cluster 5) were enriched in regulation of adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains and negative regulation of immune response. We thus conclude that maternal body weight can affect maternal-fetal interaction through alterations or aberrant activation of inflammatory reaction and immune response. Regulating inflammatory reaction may be a potential therapeutic strategy to improve fertility of overweight and underweight people.

PDZK1-interacting protein 1(PDZK1IP1) promotes subcutaneous preadipocyte proliferation in goats

PDZK1-interacting protein 1(PDZK1IP1), also known as MAP17, is encoded by the PDZK1IP1 gene and is a membrane-associated protein. PDZK1IP1 have been proven to be a potent regulator of cancer cell proliferation. However, the role of PDZK1IP1 in regulating goat subcutaneous preadipocyte proliferation is unknown. Here, we cloned the full-length coding sequence of PDZK1IP1 gene, investigated the potential functional of PDZK1IP1 in goat subcutaneous preadipocyte proliferation by gaining or losing function in vitro. Our results indicated that goat PDZK1IP1 gene consists of 345 bp, encoding a protein of 114 amino acids containing a typical PDZK1IP1 (MAP17) super family domain. Overexpression of PDZK1IP1 significantly increased the number of EdU-positive cells and cell viability, and also upregulated mRNA expression of cell proliferation-associated genes including CCND1 and CDK2 in vitro cultured cells. Conversely, knockdown of PDZK1IP1 mediated by siRNA technique significantly inhibited subcutaneous preadipocyte proliferation and downregulated mRNA expression of cell proliferation-associated genes including CCNE1, CCND1 and CDK2. Collectively, these results suggested that PDZK1IP1 can promote proliferation of goat subcutaneous preadipocyte.

Insight into the molecular structure and function of peptidoglycan recognition protein SC2 (PGRP-SC2) from Amphiprion clarkii: Investigating the role in innate immunity

Peptidoglycan recognition proteins (PGRPs) belong to the pattern recognition receptor (PRR) family and are conserved from insects to mammals. PGRPs show specific binding abilities to peptidoglycans (PGNs) in various microbes. In this study, molecular and functional analyses of PGRP-SC2 from Amphiprion clarkii (AcPGRP-SC2) were conducted. The 492 bp ORF of AcPGRP-SC2 encoded a protein of 164 amino acids with a molecular weight of 17.58 kDa and pI of 8.9. The PGRP superfamily domain was identified from the protein sequence of AcPGRP-SC2 and sequence similarities were observed with homologous proteins. Quantitative polymerase chain reaction (qPCR) analysis revealed that AcPGRP-SC2 transcripts were ubiquitously expressed in all tested tissues, with high levels in the skin, and transcript expression was significantly modulated by immune stimulation with lipopolysaccharide (LPS), Polyinosinic:polycytidylic acid (poly I:C), and Vibrio harveyi post-immune challenge. Recombinant AcPGRP-SC2 with the maltose-binding protein fusion (rAcPGRP-SC2) was used to evaluate LPS-, PGN-, and bacterial-binding activities and to conduct bacterial agglutination assays, and the results demonstrated that AcPGRP-SC2 exhibited bacterial recognition, binding, and colonization abilities to a range of Gram-positive and Gram-negative bacterial strains. Moreover, rAcPGRP-SC2-pre-treated Fat Head Minnow (FHM) cells exhibited significant upregulation in NF-ĸB1, NF-ĸB2, and stat3 expression upon treatment with killed bacteria. Taken together, our findings suggest that AcPGRP-SC2 plays an important role in the immune response against microbial pathogens in A. clarkii.