Isoform X1 Research Articles

Comparative analysis of the transcriptomes from regenerated plants and root explants of endangered Oplopanax elatus.

Oplopanax elatus is a plant of therapeutic significance in oriental medicine; however, its mass cultivation is limited owing to the difficulties in propagating it from seeds. In this study, we investigated the transcriptome profiles and transcriptional regulatory factors expressed during plantlet regeneration from root tissues of the endangered O. elatus. The RNA-seq results for the control and regenerated plants cultured in liquid medium for 8weeks showed that the clean length of the control group was 11,901,667,912 and that of the 8-week sample was 10,115,155,171, indicating a clean value of 97% for both samples. The number of mapped paired-end reads was 63,922,480 for the control group and 54,146,902 for the 8-week sample. The number of genes for which at least one clean data point was mapped was 43,177 in the control group and 42,970 in the 8-week sample. The results of the differentially expressed gene analysis indicate that the number of upregulated genes in the 8-week sample was 158, and the number of downregulated genes was 424. Gene Ontology (GO) analysis of the upregulated genes revealed that GO terms were classified into 14 categories, and genes expressed in the biological process category occurred most frequently. GO terms of the downregulated genes were evenly distributed into two categories: biological process and molecular function. From the upregulated genes, eight reference genes with significant differences in expression were selected and analyzed using real-time PCR. The Oe38836 gene (late embryogenesis abundant protein M17-like isoform X1) showed the highest expression rate that was more than tenfold that of the control. Oe40610 (auxin-responsive protein SAUR21-like) and Oe07114 (glucose-1-phosphate adenyl transferase-like protein) genes showed expression levels that were increased eightfold relative to the control. The RNA sequencing (RNA-seq) results from the plants regenerated through liquid culture of O. elatus root tissue were confirmed using real-time PCR, indicating their reliability.

Nitrogen deficiency differentially affects lignin biosynthesis genes and flavanols accumulation in tolerant and susceptible tea genotypes (Camellia sinensis (L.) Kuntze)

The mechanisms of the nitrogen deficiency (ND) response are complex and not sufficiently studied in evergreen tree crops. The aim of this study was to investigate the nitrogen deficiency response in two contrasting tea genotypes to reveal molecular crosstalk between tea quality and tolerance to ND. The transcriptional response to two- and four-month nitrogen deficiency was analyzed in tolerant (cv. Karatum) and susceptible (cv. Kolkhida) tea genotypes. Both GO and KEGG analyses indicated that phenylpropanoid pathway was significantly enriched under nitrogen deficiency in both cultivars. Most of the transcription factor DEGs were related to ABA-mediated stress responses; the following transcription factors were upregulated in both genotypes and in both stress periods: TEAK026346 (bZip23), TEAK015869 (RADIALIS-like 3 isoform X1), TEAK022547 (bHLH78) and one was downregulated TEAK030189 (MYB family transcription factor EFM like) indicating their important role for regulation of nitrogen deficiency response. Gene network of phenylpropanoid pathway DEGs indicated the abandoned edges in lignin biosynthesis DEGs. Generally, the results suggest greater stability of the cell wall metabolism and secondary metabolism in tolerant genotype under long term nitrogen deficiency. The revealed lignin biosynthesis genes can be new candidates for molecular breeding to develop tolerant tea genotypes.

Antioxidant Capacity, Enzyme Activities Related to Energy Metabolism, and Transcriptome Analysis of Crassostrea hongkongensis Exposed to Hypoxia.

Crassostrea hongkongensis (C. hongkongensis) is one of the three most commonly cultivated oyster species in China. Seasonal hypoxia is one of the most serious threats to its metabolism, reproductive behavior, and survival. To investigate the effects of hypoxia stress on the antioxidant capacity and energy metabolism of C. hongkongensis, the total antioxidant capacity (T-AOC), glycogen content, and enzyme activities (phosphofructokinase, PFK; pyruvate kinase, PK; phosphoenolpyruvate carboxykinase, PEPCK) of oysters were determined under normoxic (DO 6 ± 0.2 mg/L) and hypoxic (DO 1.5 mg/L) conditions at 0 h, 6 h, 48 h, and 72 h. We also determined the T-AOC, glycogen content, and enzyme activities of oysters under reoxygenation (recovered to normoxia for 24 h). To further examine the potential molecular regulatory mechanism of hypoxic adaptation, a transcriptome analysis was conducted on the gill of C. hongkongensis under normoxia (N, 72 h), hypoxia (H, 72 h), and reoxygenation (R). After being exposed to hypoxia for 6 h, the T-AOC, glycogen content, and enzyme activities of PK, PFK, and PEPCK in C. hongkongensis were significantly decreased. However, after prolonging the duration of hypoxia exposure for 72 h, the T-AOC, glycogen content, and enzyme activities increased compared to that of 48 h. After 24 h reoxygenation, the T-AOC, glycogen content, and enzyme activity of PK and PFK returned to close to initial levels. In addition, a transcriptome analysis discovered 6097 novel genes by mapping the C. hongkongensis genome with the clean reads. In total, 352 differentially expressed genes (DEGs) were identified in the H vs. N comparison group (235 upregulated and 117 downregulated genes). After recovery to normoxia, 292 DEGs (134 upregulated and 158 downregulated genes) were identified in the R vs. N comparison group, and 632 DEGs were identified (253 upregulated and 379 downregulated genes) in the R vs. H comparison group. The DEGs included some hypoxia-tolerant genes, such as phosphoenolpyruvate carboxykinase (PEPCK), mitochondrial (AOX), tyramine beta-hydroxylase (TBH), superoxide dismutase (SOD), glutathione S-transferase (GST), and egl nine homolog 1 isoform X2 (EGLN1). Additionally, DEGs were significantly enriched in the KEGG pathways that are involved in hypoxia tolerance, including the metabolism of xenobiotics by cytochrome P450 pathways and the HIF-1 signaling pathway. Then, we selected the five hypoxic-tolerant candidate DEGs for real-time quantitative polymerase chain reaction (RT-qPCR) validation, and the results were consistent with the transcriptome sequencing data. These discoveries have increased our understanding of hypoxia tolerance, recovery ability after reoxygenation, and molecular mechanisms governing the responses to hypoxia in C. hongkongensis.

Revealing Dynamics of Protein Phosphorylation: A Study on the Cashmere Fineness Disparities in Liaoning Cashmere Goats.

Exploring the landscape of protein phosphorylation, this investigation focuses on skin samples from LCG (Liaoning Cashmere Goats), characterized by different levels of cashmere fineness. Employing LC-MS/MS technology, we meticulously scrutinized FT-LCG (fine-type Liaoning Cashmere Goats) and CT-LCG (coarse-type Liaoning Cashmere Goats). Identifying 512 modified proteins, encompassing 1368 phosphorylated peptide segments and 1376 quantifiable phosphorylation sites, our exploration further revealed consistent phosphorylation sites in both groups. Analysis of phosphorylated peptides unveiled kinase substrates, prominently featuring Protein Kinase C, Protein Kinase B and MAPK3-MAPK1-MAPK7-NLK-group. Differential analysis spotlighted 28 disparate proteins, comprising six upregulated and twenty-two downregulated. Cluster analysis showcased the robust clustering efficacy of the two sample groups. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses underscored the significance of the purine metabolism pathway, suggesting its pivotal role in modulating cashmere fineness in LCG. Notably, through differential protein analysis, two crucial proteins were identified: HSL-X (hormone-sensitive lipase isoform X1) and KPRP (keratinocyte proline-rich protein). Further evidence supports LIPE and KPRP as key genes regulating cashmere fineness, paving the way for promising avenues in further research. These findings not only contribute to a nuanced understanding of protein-level dynamics in cashmere but also provide a theoretical foundation for the selective breeding of superior Liaoning Cashmere Goat strands.

Comparative Proteomic Analysis of Floral Buds before and after Opening in Walnut (Juglans regia L.).

The walnut (Juglans regia L.) is a typical and an economically important tree species for nut production with heterodichogamy. The absence of female and male flowering periods seriously affects both the pollination and fruit setting rates of walnuts, thereby affecting the yield and quality. Therefore, studying the characteristics and processes of flower bud differentiation helps in gaining a deeper understanding of the regularity of the mechanism of heterodichogamy in walnuts. In this study, a total of 3540 proteins were detected in walnut and 885 unique differentially expressed proteins (DEPs) were identified using the isobaric tags for the relative and absolute quantitation (iTRAQ)-labeling method. Among all DEPs, 12 common proteins were detected in all four of the obtained contrasts. GO and KEGG analyses of 12 common DEPs showed that their functions are distributed in the cytoplasm metabolic pathways, photosynthesis, glyoxylate and dicarboxylate metabolism, and the biosynthesis of secondary metabolites, which are involved in energy production and conversion, synthesis, and the breakdown of proteomes. In addition, a function analysis was performed, whereby the DEPs were classified as involved in photosynthesis, morphogenesis, metabolism, or the stress response. A total of eight proteins were identified as associated with the morphogenesis of stamen development, such as stamen-specific protein FIL1-like (XP_018830780.1), putative leucine-rich repeat receptor-like serine/threonine-protein kinase At2g24130 (XP_018822513.1), cytochrome P450 704B1-like isoform X2 (XP_018845266.1), ervatamin-B-like (XP_018824181.1), probable glucan endo-1,3-beta-glucosidase A6 (XP_018844051.1), pathogenesis-related protein 5-like (XP_018835774.1), GDSL esterase/lipase At5g22810-like (XP_018833146.1), and fatty acyl-CoA reductase 2 (XP_018848853.1). Our results predict several crucial proteins and deepen the understanding of the biochemical mechanism that regulates the formation of male and female flower buds in walnuts.

Comparative proteomic analysis of resistant and susceptible cotton genotypes in response to leaf hopper infestation

The cotton leaf hopper is a major pest in cotton, causing a hopper burn in leaves. In this study, a comparative proteomic analysis of NDLH2010 (Resistant) and LRA5166 (Susceptible), infected with leaf hopper, was employed using a nano LC-MS/MS approach. A total of 1402 proteins varied significantly between leaf hopper-infected and control plants. The resistant and susceptible genotypes had differentially expressed proteins (DEPs) of 743 and 659, respectively. Functional annotation of DEPs revealed that the DEPs were primarily associated with stress response, hormone synthesis, photosynthesis, cell wall, and secondary metabolites. Notably, DEPs such as polyphenol oxidase, carboxypeptidase, heat shock proteins, protein BTR1-like isoform X2, chaperone protein ClpB1, and β glucosidase factors associated with environmental stress response were also detected. Quantitative real-time PCR (qRT-PCR) analysis confirmed a positive correlation between protein abundances and transcripts for all genes. Collectively, this study provides the molecular mechanisms associated with cotton defense responses against leaf hopper. Significance statementCotton, a natural fiber, assumes a pivotal role as a raw material for textile industries, thereby bearing significant importance in the global economy. The cotton production sector is considerably affected by both biotic and abiotic stresses. The cotton leaf hopper (Amrasca biguttula biguttula (Ishida)) stands as a polyphagous insect, emerging as a dominant sap-feeding pest of the cotton crop. The continuous onslaught of sap-feeding insects on cotton plants has a detrimental impact, with leaf hoppers potentially causing yield reductions of up to 50%. Therefore, comprehending the molecular interplay between cotton and leaf hopper, elucidated at the proteome level, holds promise for more effective pest management strategies. This approach holds the potential to offer insights that contribute to the development of leaf hopper-resistant cotton varieties.

Morphologic, cytometric, quantitative transcriptomic and functional characterisation provide insights into the haemocyte immune responses of Pacific abalone (Haliotis discus hannai).

In recent years, the abalone aquaculture industry has been threatened by the bacterial pathogens. The immune responses mechanisms underlying the phagocytosis of haemocytes remain unclear in Haliotis discus hannai. It is necessary to investigate the immune mechanism in response to these bacterial pathogens challenges. In this study, the phagocytic activities of haemocytes in H. discus hannai were examined by flow cytometry combined with electron microscopy and transcriptomic analyses. The results of Vibrio parahaemolyticus, Vibrio alginolyticus and Staphylococcus aureu challenge using electron microscopy showed a process during phagosome formation in haemocytes. The phagocytic rate (PP) of S. aureus was higher than the other five foreign particles, which was about 63%. The PP of Vibrio harveyi was about 43%, the PP peak of V. alginolyticus in haemocyte was 63.7% at 1.5h. After V. parahaemolyticus and V. alginolyticus challenge, acid phosphatase, alkaline phosphatase, total superoxide dismutase, lysozyme, total antioxidant capacity, catalase, nitric oxide synthase and glutathione peroxidase activities in haemocytes were measured at different times, differentially expressed genes (DEGs) were identified by quantitative transcriptomic analysis. The identified DEGs after V. parahaemolyticus challenge included haemagglutinin/amebocyte aggregation factor-like, supervillin-like isoform X4, calmodulin-like and kyphoscoliosis peptidase-like; the identified DEGs after V. alginolyticus challenge included interleukin-6 receptor subunit beta-like, protein turtle homolog B-like, rho GTPase-activating protein 6-like isoform X2, leukocyte surface antigen CD53-like, calponin-1-like, calmodulin-like, troponin C, troponin I-like isoform X4, troponin T-like isoform X18, tumor necrosis factor ligand superfamily member 10-like, rho-related protein racA-like and haemagglutinin/amebocyte aggregation factor-like. Some immune-related KEGG pathways were significantly up-regulated or down-regulated after challenge, including thyroid hormone synthesis, Th17 cell differentiation signalling pathway, focal adhesion, melanogenesis, leukocyte transendothelial migration, inflammatory mediator regulation of TRP channels, ras signalling pathway, rap1 signalling pathway. This study is the first step towards understanding the H. discus hannai immune system by adapting several tools to gastropods and providing a first detailed morpho-functional study of their haemocytes.

Integration analysis of transcriptome and proteome profiles brings new insights of somatic embryogenesis of two eucalyptus species

BackgroundSomatic embryogenesis (SE) is recognized as a promising technology for plant vegetative propagation. Although previous studies have identified some key regulators involved in the SE process in plant, our knowledge about the molecular changes in the SE process and key regulators associated with high embryogenic potential is still poor, especially in the important fiber and energy source tree – eucalyptus.ResultsIn this study, we analyzed the transcriptome and proteome profiles of E. camaldulensis (with high embryogenic potential) and E. grandis x urophylla (with low embryogenic potential) in SE process: callus induction and development. A total of 12,121 differentially expressed genes (DEGs) and 3,922 differentially expressed proteins (DEPs) were identified in the SE of the two eucalyptus species. Integration analysis identified 1,353 (131 to 546) DEGs/DEPs shared by the two eucalyptus species in the SE process, including 142, 13 and 186 DEGs/DEPs commonly upregulated in the callus induction, maturation and development, respectively. Further, we found that the trihelix transcription factor ASR3 isoform X2 was commonly upregulated in the callus induction of the two eucalyptus species. The SOX30 and WRKY40 TFs were specifically upregulated in the callus induction of E. camaldulensis. Three TFs (bHLH62, bHLH35 isoform X2, RAP2-1) were specifically downregulated in the callus induction of E. grandis x urophylla. WGCNA identified 125 and 26 genes/proteins with high correlation (Pearson correlation > 0.8 or < -0.8) with ASR3 TF in the SE of E. camaldulensis and E. grandis x urophylla, respectively. The potential target gene expression patterns of ASR3 TF were then validated using qRT-PCR in the material.ConclusionsThis is the first time to integrate multiple omics technologies to study the SE of eucalyptus. The findings will enhance our understanding of molecular regulation mechanisms of SE in eucalyptus. The output will also benefit the eucalyptus breeding program.

#2450 Alternative splicing in podocytes under mechanical stress: a new perspective for the pathogenic effect of glomerular hypertension

Abstract Background and Aims High blood pressure is considered one of the most important risk factors leading to chronic kidney disease. The pathogenesis of hypertension-induced damage to podocytes remains still elusive. Since recent reports have shown that alternative splicing (AS) has a significant impact on disease progression, we investigated alternative splicing of mechanically stretched podocytes, a well-established model for glomerular hypertension, in the Sys_CARE (Systems Medicine Investigation of AS in Cardiac and Renal Diseases) project. Our results provide new insights into the molecular mechanisms that might drive the progression of hypertensive nephropathy. Method Differentiated murine podocytes were exposed to mechanical stretch for 3 days under low and high stretch conditions. Subsequently, mRNA and proteins were analyzed by RNA-Seq and LC-MS/MS, respectively. Enrichment analysis identified transcripts which were classified according to the related biological processes, molecular function, and cellular components. Splicing and transcript expression were evaluated with bioinformatical tools such as MAJIQ, Multivariate Analysis of Transcript Splicing (rMATS), leafcutter, Whippet and IsoformSwitchAnalyzeR. Results Transcriptome analyses of mechanically stretched podocytes by RNA sequencing showed that mechanical stress leads to a high number of differentially expressed genes (1323 with log2 fc ± 1) compared to unstretched podocytes. Detailed gene set enrichment analysis for GO-Terms showed an enrichment among up-regulated transcripts related to mRNA processing and RNA splicing. In contrast, most transcripts with decreased expression upon stretch are associated with cytoskeleton function. To identify AS events, different AS tools were used. We found a wide variety of splice events. The most frequent event was exon skipping (between 63-82% of all AS events), followed by intron retention and alternative 5′ or 3′ splice site. 290 alternatively spliced genes were detected after mechanical stretch by three different splice analysis tools (rMATS, leafcutter, Whippet). Out of these candidates, the IsoformSwitchAnalyzeR identified 17 genes exhibiting significant isoform switches. To prioritize the candidates, we performed a screening that included only those genes that were identified by proteomic analysis and that are expressed in podocytes in vivo or showed an altered expression pattern in glomerular disease. This screening led to the two candidates Shroom3 and Myl6. Shroom3, an actin-binding protein, is crucial for podocyte morphology and function. Genetic variants of Shroom3 have been linked with chronic kidney disease by GWAS. Here we found that two of four Shroom3 isoforms showed significant expression changes due to mechanical stretch. Isoform 2 which showed the highest expression in cultured podocytes, significantly decreased, but the shorter isoform X1 was significantly up-regulated after mechanical stretch. Both changes were verified by qRT-PCR as well as by in situ hybridization in mouse tissue. Myosin light chain 6 (Myl6), a component of the myosin motor protein complex, has two isoforms which are detected in cultured podocytes as well as in vivo. We found an expression switch from isoform 1 to 2 due to mechanical stretch. Our analysis showed that this is accompanied by a change of the amino acid sequence AFVRHILS to ELVRMVLN, sequences that are well conserved in mice, rat, and human. The analysis of the protein structure by FoldX revealed that this change in the amino acid sequence could lead to an impairment of the binding of the motor protein complex to actin and might therefore influence the functionality of the actin cytoskeleton, which is highly important for proper podocyte foot process morphology. Conclusion Mechanical stretch of cultured mouse podocytes, which is an excellent model to simulate hypertensive nephropathy, leads to alterations in isoform expression due to alternative splicing. These changes in the expression of isoforms, such as Shroom 3 and Myl6, have the potential to play a key role in the pathophysiology of hypertension-induced nephropathy.

Protein expression patterns and metal metabolites in a protogynous hermaphrodite fish, the ricefield eel (Monopterus albus)

BackgroundThe ricefield eel Monopterus albus undergoes a natural sex change from female to male during its life cycle, and previous studies have shown the potential mechanisms of this transition at the transcriptional and protein levels. However, the changes in protein levels have not been fully explored, especially in the intersexual stage.ResultsIn the present study, the protein expression patterns in the gonadal tissues from five different periods, the ovary (OV), early intersexual stage gonad (IE), middle intersexual stage gonad (IM), late intersexual stage gonad (IL), and testis (TE), were determined by untargeted proteomics sequencing. A total of 5125 proteins and 394 differentially expressed proteins (DEPs) were detected in the gonadal tissues. Of the 394 DEPs, there were 136 between the OV and IE groups, 20 between the IM and IE groups, 179 between the IL and IM groups, and 59 between the TE and IL groups. Three candidate proteins, insulin-like growth factor 2 mRNA-binding protein 3 isoform X1 (Igf2bp3), triosephosphate isomerase (Tpi), and Cu-Zn superoxide dismutase isoform X1 [(Cu-Zn) Sod1], were validated by western blotting to verify the reliability of the data. Furthermore, metal metabolite-related proteins were enriched in the IL vs. IM groups and TE vs. IL groups, which had close relationships with sex change, including Cu2+-, Ca2+-, Zn2+- and Fe2+/Fe3+-related proteins. Analysis of the combined transcriptome data revealed consistent protein/mRNA expression trends for two metal metabolite-related proteins/genes [LOC109953912 and calcium Binding Protein 39 Like (cab39l)]. Notably, we detected significantly higher levels of Cu2+ during the sex change process, suggesting that Cu2+ is a male-related metal metabolite that may have an important function in male reproductive development.ConclusionsIn summary, we analyzed the protein profiles of ricefield eel gonadal tissues in five sexual stages (OV, IE, IM, IL, and TE) and verified the plausibility of the data. After preforming the functional enrichment of metal metabolite-related DEPs, we detected the contents of the metal metabolites Zn2+, Cu2+, Ca2+, and Fe2+/Fe3+ at these five stages and screened for (Cu-Zn) Sod1 and Mmp-9 as possible key proteins in the sex reversal process.

Spermatogenesis in cultured blue mussel (Mytilus edulis) from a cold‐ocean environment: Seasonal spatiotemporal expression of three gamete‐associated molecular markers

AbstractTo understand the seasonality of spermatogenesis in cultured males of Mytilus edulis from a cold‐ocean environment, we investigated the cellular transitions occurring within the spermatogenic epithelium of the testicular acini during early and advancing spermatogenesis, with specific reference to the histology of the epithelium, gene specific spermatogenic response, condition, culture environment, and season. A combination of histological evaluation, qPCR analysis, and in situ hybridization was used to examine the cellular transitions taking place in the germinal epithelium from late winter through to a seasonal spawn in summer. We observed clear seasonal transitionary changes in the spermatogenic cell population making up the germinal epithelium (i.e., spermatogonial stem cells, spermatocytes, spermatids, and spermatozoa) extending from February to July. These seasonal transitions in spermatogenic cell type coincided with significant variation in the spatiotemporal expression of two molecular markers for spermatogenesis (i.e., Kelch‐like protein 10 [KLHL10] and Armadillo repeat‐containing protein 4 isoform X2 [ARMC4]) but not for expression of a gamete‐specific Mitochondrial cytochrome oxidase I (MT‐COI). The spatiotemporal expression of these genes is directly linked to the cellular changes taking place in the germinal epithelium during spermatogenesis. These observations not only corresponded to seasonal changes in physiological condition but also environmental temperature and chlorophyll a, thus further supporting the link between male gametogenesis and environment in higher latitude regions.

Differentially expressed proteins and microbial communities of the skin regulate disease resistance to Chinese tongue sole (Cynoglossus semilaevis).

Vibriosis, caused by Vibrio, seriously affects the health of fish, shellfish, and shrimps, causing large economic losses. Teleosts are represent the first bony vertebrates with both innate and adaptive immune responses against pathogens. Aquatic animals encounter hydraulic pressure and more pathogens, compared to terrestrial animals. The skin is the first line of defense in fish, constituting the skin-associated lymphoid tissue (SALT), which belongs to the main mucosa-associated lymphoid tissues (MALT). However, little is known about the function of immunity related proteins in fish. Therefore, this study used iTRAQ (isobaric tags for relative and absolute quantitation) to compare the skin proteome between the resistant and susceptible families of Cynoglossus semilaevis. The protein integrin beta-2, the alpha-enolase isoform X1, subunit B of V-type proton ATPase, eukaryotic translation initiation factor 6, and ubiquitin-like protein ISG15, were highly expressed in the resistant family. The 16S sequencing of the skin tissues of the resistant and susceptible families showed significant differences in the microbial communities of the two families. The protein-microbial interaction identified ten proteins associated with skin microbes, including immunoglobulin heavy chain gene (IGH), B-cell lymphoma/leukemia 10 (BCL10) and pre-B-cell leukemia transcription factor 1 isoform X2 (PBX2). This study highlights the interaction between skin proteins and the microbial compositions of C. semilaevis and provides new insights into understanding aquaculture breeding research.

Galectin-8-like isoform X1 mediates antibacterial, antiviral, and antioxidant responses in red-lip mullet (Planiliza haematocheilus) through positive modulation of pro-inflammatory cytokine, chemokine, and enzymatic antioxidant activity

Galectin 8 belongs to the tandem repeat subclass of the galectin superfamily. It possesses two homologous carbohydrate recognition domains linked by a short peptide and preferentially binds to β-galactoside-containing glycol-conjugates in a calcium-independent manner. This study identified Galectin-8-like isoform X1 (PhGal8X1) from red-lip mullet (Planiliza haematocheilus) and investigated its role in regulating fish immunity. The open reading frame of PhGal8X1 was 918bp, encoding a soluble protein of 305 amino acids. The protein had a theoretical isoelectric (pI) point of 7.7 and an estimated molecular weight of 34.078 kDa. PhGal8X1 was expressed in various tissues of the fish, with prominent levels in the brain, stomach, and intestine. PhGal8X1 expression was significantly (p < 0.05) induced in the blood and spleen upon challenge with different immune stimuli, including polyinosinic:polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. The recombinant PhGal8X1 protein demonstrated agglutination activity towards various bacterial pathogens at a minimum effective concentration of 50 μg/mL or 100 μg/mL. Subcellular localization observations revealed that PhGal8X1 was primarily localized in the cytoplasm. PhGal8X1 overexpression in fathead minnow cells significantly (p < 0.05) inhibited viral hemorrhagic septicemia virus (VHSV) replication. The expression levels of four proinflammatory cytokines and two chemokines were significantly (p < 0.05) upregulated in PhGal8X1 overexpressing cells in response to VHSV infection. Furthermore, overexpression of PhGal8X1 exhibited protective effects against oxidative stress induced by H2O2 through the upregulation of antioxidant enzymes. Taken together, these findings provide compelling evidence that PhGal8X1 plays a crucial role in enhancing innate immunity and promoting cell survival through effective regulation of antibacterial, antiviral, and antioxidant defense mechanisms in red-lip mullet.

Transcriptome analysis of mantle and pearl sac of freshwater pearl mussel Lamellidens marginalis to identify biomineralisation-associated genes

ABSTRACT The freshwater pearl mussel, Lamellidens marginalis, is widely distributed in India and is cultured for freshwater pearl production. Understanding the biomineralisation process sheds light on the genes responsible for nacre secretion. However, information on the transcriptome of L. marginalis mantle tissue and pearl sac is lacking. This study generated transcriptome resources for L. marginalis, identifying genes involved in biomineralisation. Illumina paired-end sequencing yielded 11.13 million raw reads, assembled into 133,246 contigs and 26,373 unigenes. Of these, 21,033 unigenes exhibited homology with previously reported molluscan proteins. Genes related to biomineralisation, such as pif, perlucin, calreticulin, calmodulin, chitin synthase, chitin dehydrogenase, carbonic anhydrase, tyrosinase, shell matrix protein, dermatopontin isoform X1, chitinase 3, chitinase domain-containing protein 1, and putative chitinase-1were identified. This baseline information will help to compare the expression levels between the phenotypes of high-quality and low-quality pearls. Further investigations, incorporating large sample sizes and phenotype information, could assist in identifying type I genetic markers linked to pearl quality.

Integrated transcriptome and metabolome analysis reveals anthocyanin biosynthesis mechanisms in pepper (Capsicum annuum L.) leaves under continuous blue light irradiation

BackgroundDifferent metabolic compounds give pepper leaves and fruits their diverse colors. Anthocyanin accumulation is the main cause of the purple color of pepper leaves. The light environment is a critical factor affecting anthocyanin biosynthesis. It is essential that we understand how to use light to regulate anthocyanin biosynthesis in plants.ResultPepper leaves were significantly blue–purple only in continuous blue light or white light (with a blue light component) irradiation treatments, and the anthocyanin content of pepper leaves increased significantly after continuous blue light irradiation. This green-to-purple phenotype change in pepper leaves was due to the expression of different genes. We found that the anthocyanin synthesis precursor-related genes PAL and 4CL, as well as the structural genes F3H, DFR, ANS, BZ1, and F3’5’H in the anthocyanin synthesis pathway, had high expression under continuous blue light irradiation. Similarly, the expression of transcription factors MYB1R1-like, MYB48, MYB4-like isoform X1, bHLH143-like, and bHLH92-like isoform X3, and circadian rhythm-related genes LHY and COP1, were significantly increased after continuous blue light irradiation. A correlation network analysis revealed that these transcription factors and circadian rhythm-related genes were positively correlated with structural genes in the anthocyanin synthesis pathway. Metabolomic analysis showed that delphinidin-3-O-glucoside and delphinidin-3-O-rutinoside were significantly higher under continuous blue light irradiation relative to other light treatments. We selected 12 genes involved in anthocyanin synthesis in pepper leaves for qRT-PCR analysis, and the accuracy of the RNA-seq results was confirmed.ConclusionsIn this study, we found that blue light and 24-hour irradiation together induced the expression of key genes and the accumulation of metabolites in the anthocyanin synthesis pathway, thus promoting anthocyanin biosynthesis in pepper leaves. These results provide a basis for future study of the mechanisms of light quality and photoperiod in anthocyanin synthesis and metabolism, and our study may serve as a valuable reference for screening light ratios that regulate anthocyanin biosynthesis in plants.

Transcriptomic investigation unveils the role of energy metabolism under low phosphorus and salt combined stress in soybean (Glycine max).

Soybean (Glycine max) is economically significant, but the mechanisms underlying its adaptation to simultaneous low phosphorus and salt stresses are unclear. We employed the Shennong 94-1-8 soybean germplasm to conduct a comprehensive analysis, integrating both physiochemical and transcriptomic approaches, to unravel the response mechanisms of soybean when subjected to simultaneous low phosphorus and salt stresses. Remarkably, the combined stress exhibited the most pronounced impact on the soybean root system, which led to a substantial reduction in total soluble sugar (TSS) and total soluble protein (TSP) within the plants under this treatment. A total of 20,953 differentially expressed genes were identified through pairwise comparisons. Heatmap analysis of genes related to energy metabolism pathways demonstrated a significant down-regulation in expression under salt and low phosphorus + salt treatments, while low phosphorus treatment did not exhibit similar expression trends. Furthermore, the weighted gene co-expression network analysis (WGCNA) indicated that the blue module had a strong positive correlation with TSS and TSP. Notably, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase 1, FCS-Like Zinc finger 8, auxin response factor 18 isoform X2, and NADP-dependent malic enzyme emerged as hub genes associated with energy metabolism. In summary, our findings indicate that soybean roots are more adversely affected by salt and combined stress than by low phosphorus alone due to reduced activity in energy metabolism-related pathways and hub genes. These results offer novel insights into the adaptive mechanisms of soybeans when facing the combined stress of low phosphorus and salinity.

Sperm proteome and sperm parameters of captive Amazon squirrel monkeys (Saimiri collinsi Osgood, 1916) during the non-fatted and fatted condition

The aims of this work were (1) to characterize the proteome profile of Saimiri collinsi (Amazon squirrel monkey) sperm, and (2) to evaluate if there are meaningful differences in sperm quality and sperm protein landscape during the non-fatted and fatted conditions of the animals. Semen of adult males (n=4) was collected by electroejaculation and diluted in ACP-118® extender. Sperm concentration, motility, vigor, plasma membrane integrity and functionality, and sperm morphology were evaluated. Sperm proteome was characterized by mass spectrometry. The non-fatted and fatted conditions were defined according to the fattening index. There were increases in seminal volume, pH, and sperm motility of S. collinisi during the fatted in comparison with the non-fatted condition (p < 0.05). Animals in the fatted condition had greater body mass (909 ± 23.98 g) in comparison with the non-fatted condition (818.8 ± 5.97 g, p = 0.0001). There were 1161 proteins identified in the sperm of the monkeys. Thirty-seven proteins were upregulated (p < 0.05) in non-fatted condition (such as coiled-coil domain-containing protein 105, protein kinase C alpha type isoform X1, pyruvate dehydrogenase protein X component – mitochondrial and heat shock 70 kDa protein 1-like) and they were involved in cellular and metabolic processes, catalytic activity and binding. Twenty-eight sperm proteins proteins were upregulated (p < 0.05) in fatted condition (such as Cytochrome b-c1 complex subunit 2, mitochondrial; calcium-binding mitochondrial carrier protein SCaMC-1-like; heat shock 70 kDa protein 13, and izumo sperm-egg fusion protein 1), participating in cellular processes, metabolic processes, and localization, binding, catalytic activity, ATP-dependent activity, and transport activity. Functional annotations of sperm proteins upregulated in non-fatted condition were described as hydrogen ion transport, chaperone, cilium, flagellum, and cell projection. In in fatted conditions, such annotations were described as fertilization, spermatogenesis and transport, chaperone, and motor protein. The present study brings an unprecedent contribution to our understanding about the reproductive biology of adult Saimiri collinsi. It also provides support for conservation and development of assisted reproductive technologies for Neotropical primates.

Differentially expressed genes in orbital adipose/connective tissue of thyroid-associated orbitopathy.

Thyroid-associated orbitopathy (TAO) is a disease associated with autoimmune thyroid disorders and it can lead to proptosis, diplopia, and vision-threatening compressive optic neuropathy. To comprehensively understand the molecular mechanisms underlying orbital adipogenesis in TAO, we characterize the intrinsic molecular properties of orbital adipose/connective tissue from patients with TAO and control individuals. RNA sequencing analysis (RNA-seq) was performed to measure the gene expression of orbital adipose/connective tissues of TAO patients. Differentially expressed genes (DEGs) were detected and analyzed through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Gene Set Enrichment Analysis (GSEA). The protein-protein interaction (PPI) network was constructed using the STRING database, and hub genes were identified by the Cytoscape plug-in, cytoHubba. We validated several top DEGs through quantitative real-time polymerase chain reaction (qRT-PCR). We identified 183 DEGs in adipose tissue between TAO patients (n=3) and control patients (n=3) through RNA sequencing, including 114 upregulated genes and 69 downregulated genes. The PPI network of these DEGs had 202 nodes and 743 edges. PCR-based validation results of orbital adipose tissue showed multiple top-ranked genes in TAO patients (n=4) are immune and inflammatory response genes compared with the control individual (n=4). They include ceruloplasmin isoform x3 (CP), alkaline tissue-nonspecific isozyme isoform x1 (ALPL), and angiotensinogen (AGT), which were overrepresented by 2.27- to 6.40-fold. Meanwhile, protein mab-21-like 1 (MAB21L1), phosphoinositide 3-kinase gamma-subunit (PIK3C2G), and clavesin-2 (CLVS2) decreased by 2.6% to 32.8%. R-spondin 1 (RSPO1), which is related to oogonia differentiation and developmental angiogenesis, was significantly downregulated in the orbital muscle tissues of patients with TAO compared with the control groups (P=0.024). Our results suggest that there are genetic differences in orbital adipose-connective tissues derived from TAO patients. The upregulation of the inflammatory response in orbital fat of TAO may be consistent with the clinical phenotype like eyelid edema, exophthalmos, and excess tearing. Downregulation of MAB21L1, PIK3C2G, and CLVS2 in TAO tissue demonstrates dysregulation of differentiation, oxidative stress, and developmental pathways.

Untying the regulatory roles of miRNAs in CuO-NPs stress response mechanism in maize: A genome-wide sRNA transcriptome analysis

MicroRNAs (miRNAs), a group of tiny non-coding RNAs play pivotal role in plant responses to environmental stress. The present small RNA transcriptome study aims to untie the role of miRNAs in CuO-NPs stress adaptation in maize seedlings. Restricted seedling growth, enhanced ROS generation and higher membrane damage were recorded under CuO-NPs [<50 nm, 8 mM] treatment. Deep sequencing reveals 7 up- and 36 down-regulated known miRNAs from CuO-NPs challenged leaves. Gene ontology study demonstrates involvement of CuO-NPs responsive miRNAs in a variety of biological processes including plant growth (miR159a, miR159b), redox homeostasis (miR156e, miR395a), detoxification of heavy metals (miR156e, miR827), signal transduction (miR156e, miR156d), and cell signalling (miR167b-3p, miR393a). Enhanced transcriptional abundance of ABC transporter G family member 41 isoform X2 and HM-associated isoprenylated plant protein 45 isoform X1 might be involved in sequestration and detoxification of excess Cu, essential for metal homeostasis in maize. The miR528-5p mediated up-regulation of superoxide dismutase does not give much protection against CuO-NPs induced oxidative stress damages as evident after histochemical staining with NBT. Moreover, CuO-NPs stress mediated down regulation of miR396 could be an underlying cause of the restricted seedling growth. Taken together, our findings provide insights into the miRNA-guided stress regulatory networks involved in plant's adaptive responses to CuO-NPs stress.

PSXIV-17 Examining Mammary Gland Transcriptomic Profiles Using Rna-Seq in Slick and Wild-Type Haired Holstein Cattle Under Tropical Conditions

Abstract In lactating dairy cows, heat stress reduces feed intake, alters the metabolism of the animal, and compromises milk production. Holstein cattle carrying the prolactin receptor gene mutation (SLICK) have been shown to have superior performance under tropical conditions by exhibiting increased milk yield compared with wild-type haired Holstein cattle (WT). Recent research indicated that SLICK cattle have greater mammary gland blood flow and lower vaginal temperatures, while maintaining prolonged voluntary exposure to direct solar radiation than their WT counterparts. Nevertheless, differences in molecular mechanisms between genotypes at the mammary gland level have been unexplored. The objective of the current study was to investigate possible differences in the mammary gland transcriptome from SLICK (n = 7) and WT (n = 6) Puerto Rican Holstein cattle. At 160 ± 3 days in milk (DIM), ultrasound guided mammary biopsies were collected from the left rear quarter of each animal and samples were snap frozen. Unstranded RNA-seq libraries were created from the frozen mammary tissue and sequenced using Illumina Novaseq 2x150bp. Quantification of expression was done by salmon [v1.9.0] using the Bos taurus (ARS UCD1.3) transcriptome database. Testing for significant differential expression (DE) was done using a pipeline consisting of the R packages tximport [v1.26.1] and edgeR [v3.40.2]. The fry method from edgeR was used to do gene set enrichment analysis (GSEA) for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms. A single gene (GeneID:513329, major allergen Equ c 1 isoform X2) showed significant (B.H. adjusted P-value ≤ 0.05) differential expression. Aligning the protein sequence for this gene using the BLASTp against the non-redundant (NR) database revealed high similarity to two pheromones, salivary lipocalin 2 and salivary lipocalin-like. Both the KEGG pathway and GO term GSEA showed no statistically significant enrichment at B.H. adjusted P-value ≤ 0.05; however, using a less stringent criteria with a normal P-value ≤ 0.05, multiple relevant pathways were identified. For example, the KEGG pathway analysis revealed that the arachidonic acid metabolism pathway (bta00590) was enriched, and the tight junction function pathway (bta4530) was depleted in the SLICK Holstein samples. Among the GO analysis, genes were found to be downregulated in response to cold (GO:0009409), hair follicle development (GO:0001942), and vasoconstriction (GO:0042310), whereas positive regulation of oxytocin production (GO:0140668), and defense response to Gram-positive bacterium (GO:0050830) were upregulated. In conclusion, differences in gene expression were limited, but after enrichment interesting pathways were revealed and deserve further exploration to aid in elucidating different molecular mechanisms between SLICK and WT cattle.