Serum PYCARD may become a new diagnostic marker for rheumatoid arthritis patients.

BACKGROUND: The objective of this investigation is to analyze the levels and clinical relevance of serum PYCARD (Pyrin and CARD domain-containing protein, commonly known as ASC-apoptosis-associated speck-like protein containing a caspase activation and recruitment domain), interleukin-38 (IL-38), and interleukin-6 (IL-6) in individuals afflicted with rheumatoid arthritis (RA). METHODS: Our study comprised 88 individuals diagnosed with RA who sought medical attention at the Affiliated Hospital of Chengde Medical University during the period spanning November 2021 to June 2023, constituting the test group. Additionally, a control group of 88 individuals who underwent health assessments at the same hospital during the aforementioned timeframe was included for comparative purposes. The study involved the assessment of IL-38, IL-6, PYCARD, anti-cyclic citrullinated peptide antibody (anti-CCP), and erythrocyte sedimentation rate (ESR) levels in both groups. The research aimed to explore the correlations and diagnostic efficacy of these markers, employing pertinent statistical analyses for comprehensive evaluation. RESULTS: The test group had higher expression levels of PYCARD, IL-6, and IL-38 than the control group (P < 0.05). Based on the correlation analysis, there was a strong relationship between PYCARD and IL-38 (P < 0.01). The receiver operating characteristic (ROC) curve analysis revealed area under the curve (AUC) values of 0.97, 0.96, and 0.96 when using combinations of PYCARD and anti-CCP, IL-38 and anti-CCP, and IL-6 and anti-CCP for predicting RA, respectively. Importantly, all three of these pairs demonstrated superior AUC values compared to PYCARD, IL-38, IL-6, ESR, or anti-CCP used as standalone diagnostic indicators. CONCLUSION: PYCARD, IL-6, and IL-38 exhibit promising potential as novel diagnostic markers and may constitute valuable tools for supporting the diagnosis of RA.

Arbuscular mycorrhizal fungi and phosphorus supply accelerate main medicinal component production of Polygonum cuspidatum.

The medicinal plant Polygonum cuspidatum Sieb. Et Zucc is rich in stilbenes (e.g., polygonin and resveratrol) and anthraquinones (e.g., emodin) for the therapy of human diseases, while how to increase the growth and medicinal composition concentrations of P. cuspidatum has become an urgent issue. The aim of the present study was to evaluate the effects of inoculation with an arbuscular mycorrhizal (AM) fungus, Funneliformis mosseae, on plant growth, phosphorus (P) acquisition, medicinal component concentrations, and expressions of resveratrol synthesis-associated enzyme genes of P. cuspidatum at two P levels (0 M and 0.2 M). P supply (0.2 M) stimulated root AM fungal colonization rate. F. mosseae inoculation significantly improved growth performance (height, diameter, and biomass) and root morphology (diameter, length, and projected area), irrespectively of substrate P levels. P supply and F. mosseae distinctly increased soil acid and neutral phosphatase activities, as well as root P concentrations. P supply increased root physcion and resveratrol concentrations in inoculated and uninoculated plants, along with up-regulated expressions of PcCHS1, PcCRS1, PcRS11, and PcSTS. AM plants represented significantly higher root aloe-emodin, chrysophanol, emodin, physcion, polydatin, and resveratrol concentrations than non-AM plants irrespective of P levels, coupled with up-regulated expressions of PcCHS1, PcCHS2, PcRS11, PcRS, and PcSTS. It is concluded that 0.2 M P supply and F. mosseae inoculation promoted chrysophanol, physcion, polydatin, and resveratrol concentrations of P. cuspidatum, with the increase in resveratrol associated with up-regulated expressions of related genes.

[Identification, biological characteristics, and screening of effective fungicides of Phoma rhei causing leaf spot on Polygonum cuspidatum].

Severe leaf spot on Polygonum cuspidatum was found in the planting base of P. cuspidatum in Fangxian county, Shiyan of Hubei province. To clarify the types of pathogens and their pathogenesis, the present study isolated and purified the pathogen of leaf spot disease of P. cuspidatum according to Koch's postulates, determined the pathogenicity of the pathogen, and investigated its biological characteristics. Meanwhile, the inhibitory effects of 11 types of fungicides on the bacteria were determined according to the mycelium growth rate, and suitable prevention and control drugs were selected. The results showed that the pathogen isolated from the diseased leaves of P. cuspidatum was Phoma rhei by morphological and molecular identification. The colony morphology and microscopic characteristics were the same as those of Ph. rhei. The homology of rDNA-ITS and TEF gene sequences with Ph. rhei reached 99.96% and 99.43%, respectively. The optimal growth temperature of Ph. rhei was 25 ℃, and the optimal pH was 7-10. Furthermore, Ph. rhei grew faster under dark or light conditions. In fungicides, 0.3% eugenol, 250 g·L~(-1) propiconazole, and 33.5% quinoline copper had significant inhibitory effects on the pathogen with EC_(50) values of 57.54, 59.58, 88.69 µg·mL~(-1), respectively. Eugenol is a botanical fungicide, which can be used as a green and environmentally friendly fungicide in the prevention and control of P. cuspidatum. This study reported for the first time that the pathogen of P. cuspidatum leaf spot was Ph. rhei. investigated the biological characteristics of the pathogen, and screened the indoor chemicals, which provided a theoretical basis for the prevention and control of P. cuspidatum leaf spot in production.

Root Endophytic Fungi Regulate Changes in Sugar and Medicinal Compositions of Polygonum cuspidatum.

Polygonum cuspidatum Sieb. et Zucc is an important industrial crop because it contains a large amount of medicinal secondary metabolites (such as polydatin, resveratrol, chrysophanol, and emodin). However, it is unclear whether root endophytic fungi increase the content of secondary metabolites in the plant. This study aimed to analyze the effects of Funneliformis mosseae (Fm) and Piriformospora indica (Pi) alone or in combination on plant growth, root morphology, thirteen sugars concentrations, and six secondary metabolites (physcion, chrysophanol, emodin, aloe-emodin, polydatin, and resveratrol) concentrations of P. cuspidatum. After 11 weeks of the fungal inoculation, the roots could be colonized by Fm and Pi single or in combination, along with the higher root colonization frequency of Fm > Pi > Fm + Pi in the descending order. In addition, Fm and Pi improved plant growth performance (plant height, stem diameter, leaf number, and shoot and root biomass) and root morphology (average diameter, maximum diameter, total length, area, and volume) to varying degrees, depending on fungal inoculations, in which Pi displayed a relatively better effect on plant growth. Single Fm and Pi inoculation significantly increased three disaccharides (sucrose, maltose, and trehalose) accumulation, while dual inoculum (Fm + Pi) only elevated sucrose concentrations. Most monosaccharides concentrations, such as D-arabinose, D-galactose, D-sorbitol, D-fructose, glucose, and L-rhamnose were not altered or inhibited by the endophytic fungi, except the increase in L-fucose and inositol. All fungal treatments significantly increased root chrysophanol and resveratrol concentrations, while decreased aloe-emodin concentrations. In addition, single Pi and dual Fm + Pi increased emodin concentrations, and single Fm and dual Fm + Pi elevated physcion and polydatin concentrations. It was concluded that Fm and Pi promoted the growth of P. cuspidatum, and the combination of Fm and Pi was more conducive to the production of some secondary metabolites than single inoculation.

Down-regulation apoptosis signal-regulating kinase 1 gene reduced the Litopenaeus vannamei hemocyte apoptosis in WSSV infection.

Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, is crucial in various cellular responses. In the present study, we identified and characterized an ASK1 homolog from Litopenaeus vannamei (LvASK1). The full-length cDNA of LvASK1 was 5400 bp long, with an open reading frame encoding a putative 1420 amino acid protein. LvASK1 was highly expressed in muscle, hemocyte, eyestalk and heart. Real-time RT-PCR analysis showed that the expression of the LvASK1 was upregulated during the white spot syndrome virus (WSSV) challenge. The knocked-down expression of LvASK1 by RNA interference significantly reduced the apoptotic ratio of the hemocytes collected from WSSV-infected L. vannamei. Furthermore, the down-regulation of LvASK1 also decreased the cumulative mortality of WSSV-infected L. vannamei. These results suggested that down-regulation of LvASK1 decreased the apoptotic rate of hemocytes in WSSV-infected shrimp, and that it could contribute to the reduction of cumulative mortality in WSSV-infected L. vannamei.

Transcriptome analysis of the unfolded protein response in hemocytes of Litopenaeus vannamei.

In this study, Litopenaeus vannamei was injected with double-stranded RNA (dsRNA) against L. vannamei immunoglobulin heavy chain binding protein (LvBip) to activating UPR in the hemocytes, shirmps injected dsRNA against enhanced green fluorescence protein (eGFP) as control group. And genes expression in hemocytes of then were analyzed using Illumina Hiseq 2500 (PE100). By comparing the analyzed results, 1418 unigenes were significantly upregulated, and 596 unigenes were significantly down-regulated upon UPR. Analysis of the differentially expressed genes against known databases indicated that the distribution of gene pathways between the upregulated and down-regulated genes were substantially different. A total of 208 genes of UPR system were obtained, and 69 of them were differentially expressed between the two groups. Results also showed that L. vannamei UPR was involved in various metabolic processes, such as glycometabolism, lipid metabolism, amino acid metabolism, and nucleic acid metabolism. In addition, UPR was emgaged in immune-assicoated signaling pathways, such as NF-κB signaling pathway, NOD-like receptor signaling pathway, Hippo signaling pathway, p38 MAPK signaling pathway and Wnt signaling pathway in L. vannamei. These results improved our current understanding of the L. vannamei UPR, and highlighted its importance in cell homeostasis upon environmental stress.

Identification and characterization of a mitochondrial unfolded protein response transcription factor ATFS-1 in Litopenaeus vannamei.

A mitochondrial specific stress response termed mitochondrial unfolded protein response (UPR(mt)) is activated in responding to disturbance of protein homeostasis in mitochondria. The activating transcription factor associated with stress-1 (designated as ATFS-1) is the key regulator of UPR(mt). To investigating the roles of ATFS-1 (LvATFS-1) in Litopenaeus vannamei mitochondrial stress remission and immunity, it's full length cDNA was cloned. The open reading frame of LvATFS-1 was 1, 557 bp in length, deducing to a 268 amino acids protein. LvATFS-1 was highly expressed in muscle, hemocytes and eyestalk. Subcellular location assays showed that N-terminal of LvATFS-1 contained a mitochondrial targeting sequence, which could directed the fused EGFP located to mitochondria. And the C-terminal of LvATFS-1, which had a nuclear localization signal, expressed in nucleus. The in vitro experiments verified that LvATFS-1 could reduced the level of intracellular reactive oxygen species (ROS). And results of real-time RT-PCR indicated that LvATFS-1 might scavenge excess ROS via ROS-eliminating genes regulation. Reporter gene assays showed that LvATFS-1 could upregulated the expression of the antimicrobial peptide genes in Drosophila Schneider 2 cells. Results of real-time RT-PCR showed that Vibrio alginolyticus or white spot syndrome virus (WSSV) infection induced the expression of LvATFS-1. And knocked-down LvATFS-1 by RNAi resulted in a higher cumulative mortality of L. vannamei upon V. alginolyticus or WSSV infection. These results suggested that LvATFS-1 not only rolled in mitochondrial specific stress responding, but also important for L. vannamei immunologic defence.

Identification and functional characterization of a solute carrier family 15, member 4 gene in Litopenaeus vannamei.

Innate immunity in shrimp is important in resisting bacterial infection. The NF-κB pathway is pivotal in such an immune response. This study cloned and functionally characterized the solute carrier family (SLC) 15 member A 4 (LvSLC15A4) gene in Litopenaeus vannamei. The open reading frame of LvSLC15A4 is 1, 902 bp long and encodes a putative 633-amino acid protein, which is localized in the plasma membrane and intracellular vesicular compartments. Results of the reporter gene assay showed that LvSLC15A4 upregulated NF-κB target genes, including the immediate-early gene 1 of white spot syndrome virus, as well as several antimicrobial peptide genes, such as pen4, CecA, AttA, and Mtk in S2 cells. Moreover, knocked-down expression of LvSLC15A4 reduced pen4 expression in L. vannamei. LvSLC15A4 down-regulation also increased the cumulative mortality of Vibrio parahemolyticus-infected L. vannamei. Furthermore, LvSLC15A4 expression was induced by unfolded protein response (UPR) in L. vannamei hematocytes. These results suggest that LvSLC15A4 participates in L. vannamei innate immunity via the NF-κB pathway and thus may be related to UPR.

Identification and functional characterization of an endoplasmic reticulum oxidoreductin 1-α gene in Litopenaeus vannamei.

In the current study, full-length sequence of endoplasmic reticulum oxidoreductin 1-α (LvERO1-α) was cloned from Litopenaeus vannamei. Real-time RT-PCR results showed that LvERO1-α was highly expressed in hemocytes, gills, and intestines. White spot syndrome virus (WSSV) challenge was performed, and the expression of LvERO1-α and two other downstream genes of the double-stranded RNA-activated protein kinase-like ER kinase-eIF2α (PERK-α) pathway, namely, homocysteine-induced endoplasmic reticulum protein (LvHERP) and acylamino-acid-releasing enzyme (LvAARE), strongly increased in the hemocytes. Flow cytometry assay results indicated that the apoptosis rate of L. vannamei hemocytes in the LvERO1-α knockdown group was significantly lower than that of the controls. Moreover, shrimps with knockdown expression of LvERO1-α exhibited decreased cumulative mortality upon WSSV infection. Downregulation of L. vannamei immunoglobulin-binding protein (LvBip), which had been proven to induce unfolded protein response (UPR) in L. vannamei, did not only upregulate LvERO1-α, LvHERP, and LvAARE in hemocytes, but also increased their apoptosis rate, as well as the shrimp cumulative mortality. Furthermore, reporter gene assay results showed that the promoter of LvERO1-α was activated by L. vannamei activating transcription factor 4, thereby confirming that LvERO1-α was regulated by the PERK-eIF2α pathway. These results suggested that LvERO1-α plays a critical role in WSSV-induced apoptosis, which likely occurs through the WSSV-activated PERK-eIF2α pathway.

Effects of Common Mycorrhizal Network on Plant Carbohydrates and Soil Properties in Trifoliate Orange-White Clover Association.

Common mycorrhizal network (CMN) allows nutrients and signals to pass between two or more plants. In this study, trifoliate orange (Poncirus trifoliata) and white clover (Trifolium repens) were planted in a two-compartmented rootbox, separated by a 37-µm nylon mesh and then inoculated with an arbuscular mycorrhizal fungus (AMF), Diversispora spurca. Inoculation with D. spurca resulted in formation of a CMN between trifoliate orange and white clover, whilst the best AM colonization occurred in the donor trifoliate orange-receptor white clover association. In the trifoliate orange-white clover association, the mycorrhizal colonization of receptor plant by extraradical hyphae originated from the donor plant significantly increased shoot and root fresh weight and chlorophyll concentration of the receptor plant. Enzymatic activity of soil ß-glucoside hydrolase, protease, acid and neutral phosphatase, water-stable aggregate percentage at 2-4 and 0.5-1 mm size, and mean weight diameter in the rhizosphere of the receptor plant also increased. The hyphae of CMN released more easily-extractable glomalin-related soil protein and total glomalin-related soil protein into the receptor rhizosphere, which represented a significantly positive correlation with aggregate stability. AMF inoculation exhibited diverse changes in leaf and root sucrose concentration in the donor plant, and AM colonization by CMN conferred a significant increase of root glucose in the receptor plant. These results suggested that CMN formed in the trifoliate orange-white clover association, and root AM colonization by CMN promoted plant growth, root glucose accumulation, and rhizospheric soil properties in the receptor plant.

An activating transcription factor of Litopenaeus vannamei involved in WSSV genes Wsv059 and Wsv166 regulation.

Members of activating transcription factor/cyclic adenosine 3', 5'-monophosphate response element binding protein (ATF/CREB) family are induced by various stress signals and function as effector molecules. Consequently, cellular changes occur in response to discrete sets of instructions. In this work, we found an ATF transcription factor in Litopenaeus vannamei designated as LvATFß. The full-length cDNA of LvATFß was 1388 bp long with an open reading frame of 939 bp that encoded a putative 313 amino acid protein. The protein contained a basic region-leucine zipper (bZip) domain that was a common feature among ATF/CREB transcription factors. LvATFß was highly expressed in intestines, gills, and heart. LvATFß expression was dramatically upregulated by white spot syndrome virus (WSSV) infection. Pull-down assay revealed that LvATFß had strong affinity to promoters of WSSV genes, namely, wsv059 and wsv166. Dual-luciferase reporter assay showed that LvATFß could upregulate the expression of wsv059 and wsv166. Knocked down LvATFß resulted in decreased expression of wsv059 and wsv166 in WSSV-challenged L. vannamei. Knocked down expression of wsv059 and wsv166 by RNA interference inhibited the replication and reduce the mortality of L. vannamei during WSSV challenge inoculation. The copy numbers of WSSV in wsv059 and wsv166 knocked down group were significant lower than in the control. These results suggested that LvATFß may be involved in WSSV replication by regulating the expression of wsv059 and wsv166.

Activating transcription factor 4 and X box binding protein 1 of Litopenaeus vannamei transcriptional regulated white spot syndrome virus genes Wsv023 and Wsv083.

In response to endoplasmic reticulum (ER) stress, the signaling pathway termed unfolded protein response (UPR) is activated. To investigate the role of UPR in Litopenaeus vannamei immunity, the activating transcription factor 4 (designated as LvATF4) which belonged to a branch of the UPR, the [protein kinase RNA (PKR)-like ER kinase, (PERK)]-[eukaryotic initiation factor 2 subunit alpha (eIF2α)] pathway, was identified and characterized. The full-length cDNA of LvATF4 was 1972 bp long, with an open reading frame of 1299 bp long that encoded a 432 amino acid protein. LvATF4 was highly expressed in gills, intestines and stomach. For the white spot syndrome virus (WSSV) challenge, LvATF4 was upregulated in the gills after 3 hpi and increased by 1.9-fold (96 hpi) compared to the mock-treated group. The LvATF4 knock-down by RNA interference resulted in a lower cumulative mortality of L. vannamei under WSSV infection. Reporter gene assays show that LvATF4 could upregulate the expression of the WSSV gene wsv023 based on the activating transcription factor/cyclic adenosine 3', 5'-monophosphate response element (ATF/CRE). Another transcription factor of L. vannamei, X box binding protein 1 (designated as LvXBP1), has a significant function in [inositol-requiring enzyme-1(IRE1) - (XBP1)] pathway. This transcription factor upregulated the expression of the WSSV gene wsv083 based on the UPR element (UPRE). These results suggest that in L. vannamei UPR signaling pathway transcription factors are important for WSSV and might facilitate WSSV infection.