Validation and development of eDNA metabarcoding primers for comprehensive assessment of Chinese amphibians.

Environmental DNA (eDNA) metabarcoding has emerged as a powerful, non-invasive tool for biodiversity assessments. However, the accuracy and limitations of these assessment techniques are highly dependent on the choice of primer pairs being used. Although several primer sets have been used in eDNA metabarcoding studies of amphibians, there are few comparisons of their reliability and efficiency. Here, we employed lab- and field-tested sets of publicly available and de novo-designed primers in amplifying 83 species of amphibian from all three orders (Anura, Caudata, and Gymnophiona) and 13 families present in China to evaluate the versatility and specificity of these primers sets in amphibian eDNA metabarcoding studies. Three pairs of primers were highly effective, as they could successfully amplify all the major clades of Chinese amphibians in our study. A few non-amphibian taxa were also amplified by these primers, which implies that further optimization of amphibian-specific primers is still needed. The simultaneous use of three primer sets can completely cover all the species obtained by conventional survey methods and has even effectively distinguished quite a number of species (n = 20) in the Wenshan National Nature Reserve. No single primer set could individually detect all of the species from the studied region, indicating that multiple primers might be necessary for a comprehensive survey of Chinese amphibians. Besides, seasonal variations in amphibian species composition were also revealed by eDNA metabarcoding, which was consistent with traditional survey methods. These results indicate that eDNA metabarcoding has the potential to be a powerful tool for studying spatial and temporal community changes in amphibian species richness.

First Report of Leaf Rot Caused by Apiospora paraphaeosperma on Lily in Wuhan, China.

Lily (Lilium spp.) is a valuable ornamental bulb flower plant in Liliaceae, and its bulbs have high edible and medicinal value. Compared with bulb propagation of other lilies, seed propagation and short growth period are the most significant characteristics of Lilium×formolongi. In 2023, leaf rot disease (LRD) was observed on approximately 70% of the Lilium×formolongi seedlings sown in an experimental greenhouse in Wuhan, Hubei province, China. Irregular brown water-soaked spots were discovered in the early stages of infected seedlings. Then, spots spread throughout the leaves and caused the leaves to brown, soften, and wilted. A pathogen associated with symptoms was isolated by incubating sterilized leaves on potato dextrose agar plates at 25 ℃ for 2-3 days. Then, a pure single colony was isolated through a single hyphal tip isolation method. The fungal colony was white with abundant aerial mycelium and produced a yellow pigment diffusible into the agar. Microscopically, isolated mycelia were reticulate and pale yellow, while conidia were dark brown, smooth, and spherical, 7.31 to 6.98 × 4.03 to 3.87µm (average 5.44×5.41µm; n=30); oval in lateral view, and had a light stripe in the middle. To identify the species of the fungus at the molecular level, ITS and EF-1α genes were amplified and sequenced using primers ITS1/ITS4 (M Gardes et al. 1993) and 758F/986R (Carbone and Kohn 1999). The BLAST results in GenBank showed that the ITS(OR523578) and EF-1α(PP066842) sequences of LRD shared 99.82% and 99.24% identity with the distinct Apiospora paraphaeosperma strains (GenBank accession MT040110, ON806628.1, respectively). Combined with the morphology of the colony and conidium, the fungus was identified as Ap. paraphaeosperma. In the pathogenicity test, six healthy leaves were inoculated with mycelium disc and then kept in an incubator (22 ℃, 90% humidity, 16h light /8h darkness). The inoculated leaves showed necrosis and wilt symptoms similar to those observed in the greenhouse, while the control leaves were asymptomatic. A re-isolation, morphology identification and DNA sequencing of the fungus confirmed its infection with Ap. paraphaeosperma in Lilium spp. At present, rot caused by Ap. paraphaeosperma has only been reported in Thailand and South Korea, both of which are found on bamboo stems (Hyde et al. 2016; Sun Lul Kwon et al. 2022). As far as we know, this is the first report of leaf rot of lily caused by Ap. paraphaeosperma in China. This report can help identify this disease and further develop effective control measures.

Electrochemical Synthesis of Selenosulfonates from Diselenides and Sulfonyl Hydrazides.

The electrochemical oxidative radical-radical cross-coupling of sulfonyl hydrazides with diselenides for the synthesis of selenosulfonates was successfully accomplished. The method is applicable to a wide range of aromatic/aliphatic sulfonyl hydrazides and diselenides, providing products in good to excellent yields. Notably, this protocol stands out for its green and sustainable nature, as it does not rely on transition metals and oxidizing agents, and the starting materials are cost-effective and readily available.

Visible-Light Induced Radical Addition-Elimination Reaction for Constructing Allylic Sulfones from Sulfonyl Chlorides and Allyl Bromides.

Allyl sulfones are commonly present in bioactive compounds and organic building blocks. This work introduces a photocatalytic radical addition-elimination reaction involving readily accessible sulfonyl chlorides and allyl bromides. It delivers structurally diverse allylic sulfones in moderate to excellent yields, showcasing a high tolerance to functional groups. Notably, this method operates under mild reaction conditions without the need for oxidants, stoichiometric reducing metals, or additives.

Bioinformatics analysis identifies potential autophagy key genes and immune infiltration in preeclampsia.

BACKGROUND: Preeclampsia (PE) is a disease that seriously threatens maternal and fetal health. Appropriate autophagy can shield the placenta from oxidative stress, but its role in PE is unclear. OBJECTIVE: To identify potential autophagy-related genes in PE. METHODS: Microarray datasets from the Gene Expression Omnibus database, compassing the test dataset GSE10588, along with validation datasets GSE4707 and GSE60438 GPL10558, were utilized. Differentially expressed genes (DEGs) were identified using the limma R package, intersected with autophagy-related genes. Hub genes were obtained using the Cytoscape software and analyzed via gene set enrichment analysis (GSEA). The diagnostic capability of hub genes was evaluated using receiver operating characteristic (ROC) curve analysis. Analysis of immune cell infiltration was conducted using single-sample gene set enrichment analysis (ssGSEA) and CIBERSORT methods. Placental tissues were collected from 10 normal pregnant women and 10 preeclamptic pregnant women, and the expression of hub genes was validated through immunohistochemistry and western blot analysis. RESULTS: Analysis of the microarray data identified 2224 DEGs, among which 26 were autophagy-related DEGs identified through intersection with autophagy genes. Ten hub genes were identified. Immune cell infiltration analysis suggested the potential involvement of T regulatory cells (Tregs), natural killer cells, neutrophils, and T follicular helper cells in the pathogenesis of PE. ROC curve analysis indicated promising diagnostic capabilities for EGFR and TP53. Additionally, levels of EGFR and TP53 were significantly higher in placental tissue from PE pregnancies compared to normal pregnancies. CONCLUSION: EGFR and TP53 may play a role in PE by influencing autophagy.

Visible Light-Induced Hydroacylation of Benzylidenemalononitriles with Aroyl Chlorides Using Silane as a Hydrogen Donor.

A novel photoredox-catalyzed direct hydroacylation of benzylidenemalononitriles is described. In this method, aroyl chlorides are employed as a readily available and affordable source of acyl groups, while commercially available tris(trimethylsilyl)silane acts as both the hydrogen atom donor and electron donor. By eliminating the requirement for complex synthesis of acyl precursors and hydrogen atom-transfer (HAT) reagents, this approach offers a convenient and efficient strategy for the hydroacylation of benzylidenemalononitriles.

Genome-wide DNA methylation pattern in whole blood of patients with Hashimoto thyroiditis.

Background: Hashimoto thyroiditis (HT), a prevalent autoimmune disorder, is not yet thoroughly understood, especially when it comes to the influence of epigenetics in its pathogenesis. The primary goal of this research was to probe the DNAm profile across the genome in the whole blood derived from patients suffering from HT. Method: Using the Illumina 850K BeadChip, we conducted a genome-wide DNAm assessment on 10 matched pairs of HT sufferers and healthy individuals. Genes with differential methylation (DMGs) were identified and underwent functional annotation via the databases of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. The transcriptional significance of potential epigenetic biomarker genes was corroborated through qRT-PCR. Results: The DNAm profiling across the genome indicated an overall reduction in methylation in HT subjects in comparison with their healthy counterparts. We detected 283 DMPs (adjusted P < 0.05 and |Δß| > 0.1), among which 152 exhibited hypomethylation and 131 demonstrated hypermethylation. Further analysis exposed a noteworthy concentration of hypermethylated DMPs in the 3´UTR, North Shore, and CpG islands, while there was a significant decrease in the Open Sea (all P < 0.001). The 283 DMPs were broadly distributed from chromosome 1 to 22, with chromosome 6 harboring the most DMPs (n = 51) and chromosome 12 carrying the most DMGs (n = 15). The SLFN12 gene, which presented with extreme hypomethylation in its promoter DMPs among HT patients, was identified as the epigenetic marker gene. Consequently, the SLFN12 mRNA expression was markedly upregulated in HT, displaying a negative relationship with its methylation levels. The area under curve (AUC) value for the SLFN12 gene among HT patients was 0.85 (sensitivity: 0.7, specificity: 0.7), a significant difference compared with healthy controls. The methylation levels of all DMPs in SLFN12 gene were negatively correlated with TSH and one CpG site (cg24470734) was positively assocciated with FT4. Conclusion: This investigation presents an initial comprehensive DNAm blueprint for individuals with HT, which permits clear differentiation between HT subjects and normal controls through an epigenetic lens. The SLFN12 gene plays a pivotal role in the onset of HT, suggesting that the methylation status of this gene could serve as a potential epigenetic indicator for HT.

The whole blood DNA methylation of RAB8A and RAP1A in autoimmune thyroiditis: evidence and validation of iodine exposure in a population from different water iodine areas.

Our study aimed to identify and verify G protein-related methylated genes in AIT patients, while also investigate those genes in AIT patients exposed to iodine in different water iodine areas. Different areas were classified by median water iodine (MWI) concentrations: Iodine-Fortified Areas (IFA, MWI<10µg/L), Iodine-Adequate Areas (IAA, 40≤MWI≤100 µg/L), and Iodine-Excessive Areas (IEA, MWI>100 µg/L). We studied 176 AIT cases and 176 controls, with 89, 40, and 47 pairs in IFA, IAA, and IEA, respectively. Using the Illumina Human Methylation 850k BeadChip, we identified candidate methylated genes. MethylTargetTM and QRT-PCR validated DNA methylation and mRNA expression. Results showed hypomethylation and high expression of RAB8A and RAP1A in all 176 AIT cases. RAB8A's CpG sites were mainly hypomethylated in IFA and IEA, while RAP1A's sites were primarily hypomethylated in IEA. This study underscores how water iodine exposure may influence RAB8A and RAP1A methylation in AIT.

Electrosynthesis of 5-Aminocoumaran Derivatives from Olefins and Aminophenols.

The electrochemical synthesis of 5-aminocoumaran derivatives from easily oxidizable aminophenols and readily available olefins is described. The reaction efficiently produces 5-aminocoumarans in high yield under mild and environmentally friendly conditions without the necessity of catalysts, additives, oxidizing agents, or sacrificial reagents. Hydrogen as the sole byproduct of the reaction makes the method clean, highly atom-efficient, and step-economical.

Boosting Mouse Defense against Lethal Toxoplasma gondii Infection with Full-Length and Soluble SAG1 Recombinant Protein.

Toxoplasmosis is a major worldwide protozoan zoonosis. The surface antigen 1 (SAG1) of Toxoplasma gondii (T. gondii) has always been recognized as an ideal vaccine candidate antigen. However, the intact and soluble SAG1 protein is usually difficult to acquire in vitro, which is unfavorable for employing the recombinant protein as a vaccine candidate antigen. In the present study, we obtained the full-length SAG1 recombinant protein in soluble form by Escherichia coli Transetta (DE3) cells under optimized expression conditions. The immunogenicity and protective ability of this recombinant protein against T. gondii acute infection were evaluated in a mouse model. Monitoring changes in serum antibody levels and types, the presence of cytokines, and the rate of lymphocyte proliferation in vaccinated mice were used to assess humoral and cellular immune responses. Additional assessments were performed to determine the protective potency of the recombinant protein in combating T. gondii RH tachyzoites. It was found that the titers of both IgG2a and IgG2b were considerably greater in the immunized mice compared to the titers of IgG1 and IgG3. The levels of Th1-type cytokines (IFN-γ, IL-12p70, IL-2, and TNF-α) and Th2-type cytokines (IL-10) significantly increased when splenocytes from immunological group mice were treated with T. gondii lysate antigen. Compared to the control group, a recombinant protein substantially increased the longevity of infected mice, with an average death time prolonged by 14.50 ± 0.34 days (p < 0.0001). These findings suggest that the full-length and soluble SAG1 recombinant protein produced potent immune responses in mice and could be a preferred subunit vaccine candidate for T. gondii, offering a feasible option for vaccination against acute toxoplasmosis.

Photoredox-Catalyzed Radical-Radical Cross-Coupling of α-Ketoesters with Ethers: Access to Sterically Hindered α-Hydroxy Esters.

We describe a photoredox catalysis method for synthesizing sterically hindered α-hydroxy esters from α-ketoesters and ethers through a radical-radical cross-coupling reaction. This approach utilizes commercially available Ir[dF(CF3)ppy]2(dtbbpy)PF6 as a photocatalyst and inexpensive and readily available nBu4NBr as a hydrogen atom transfer catalyst. Unactivated tetrahydrofuran and other ethers effectively react with various α-ketoesters to yield the desired products. The efficiency of this reaction is highlighted by its broad substrate scope, good functional group tolerance, and mild reaction conditions.

Electrochemical C(sp2)-H/N-H "formal" cross-dehydrogenative coupling of olefins with benzotriazoles for synthesis of N-vinyl benzotriazoles.

The paper details an electrochemical method that couples olefins with benzotriazoles to form C(sp2)-N bonds, enabling the synthesis of N-vinyl benzotriazoles in moderate to good yields. nBu4NI functions as both an electrolyte and an iodine mediator, and the method does not require oxidants or metals. It is a highly atom-economical and clean reaction, with hydrogen as the sole byproduct.

Identification and characterization of CYC2-like genes related to floral symmetric development in Tagetes erecta (Asteraceae).

Marigold (Tagetes erecta) is an annual herbaceous flower belonging to Asteraceae, whose capitulum is composed of bilateral symmetry ray florets on the outer periphery and radial symmetry disk florets on the inside. The flower symmetry evolution from radial symmetry to bilateral symmetry has changed the morphology, inflorescence architecture and function of florets among several lineages in Asteraceae. Several studies have identified that CYC2 genes in TCP transcription factor family are the key genes regulating the flower morphogenesis, such as corolla symmetry and stamen development. Here, seven TeCYC2 genes were cloned and phylogenetically grouped into the CYC2 branch of TCP transcription family. TeCYC2c and TeCYC2d were found to be expressed specifically in ray florets, TeCYC2b was strongly expressed in both ray and disk florets, TeCYC2g was significantly higher expressed in ray florets than in disk florets, while TeCYC2a, TeCYC2e1 and TeCYC2e2 were significantly expressed in disk florets, according to an examination of the expression profile. Among the ectopic expression lines of seven TeCYC2 genes in Arabidopsis thaliana, the flower symmetry of all transgenic lines was changed from radial symmetry to bilateral symmetry, and only the reproductive growth of TeCYC2c lines was affected. In TeCYC2c transgenic Arabidopsis, the pollen sac was difficult to crack, and the filaments were shorter than the pistils, resulting in a significant decrease in the seed setting rate. All TeCYC2 proteins were localized in the nucleus. Eight pairs of interactions between TeCYC2 proteins were validated by Y2H and BiFC assays, indicating the possibility of TeCYC2 proteins forming homodimers or heterodimers to improve functional specificity. Our findings verified the main regulatory role of TeCYC2c on the development of corollas and stamen in marigold, and analyzed the interaction network of the formation mechanism of floral symmetry in two florets, which provided more insights into the expansion of CYC2 genes in the evolution of Asteraceae inflorescence and contributed to elucidate the complex regulatory network, as well as the molecular breeding concerning flower form diversity in marigold.

A study of programmed death-1/programmed death ligand and iodine-induced autoimmune thyroiditis in NOD.H-2h4 mice.

Excess iodine will trigger the occurrence of autoimmune thyroiditis (AIT), and programmed death-1 (PD-1)/programmed death ligand (PD-L) will also contribute to the development of AIT. The purpose of this study was to explore the role that negative regulatory signals mediated by PD-1/PD-L play in the development of spontaneous autoimmune thyroiditis (SAT) in NOD.H-2h4 mice when they are exposed to iodine. Programmed death ligand 1 (PD-L1) antibody was administered intraperitoneally to NOD.H-2h4 mice. The relevant indicators were determined by flow cytometry, real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, immunohistochemistry, pathological hematoxylin and eosin staining, and arsenic-cerium catalytic spectrophotometry. Results showed that the level of urinary iodine, the level of thyroid lymphocyte infiltration, the level of thyroglobulin antibodies (TgAb) and interferon (IFN-γ)/tumor necrosis factor (TNF-α)/interleukin (IL-2)/IL-17, and the relative expression of PD-1/PD-L1/programmed death-2 (PD-L2) increased with the intervention of excess iodine. After the intervention of the PD-L1 antibody, the expression of PD-1/PD-L1/PD-L2 in different degrees was inhibited, but the level of thyroid lymphocyte infiltration and serum TgAb/IFN-γ/TNF-α/ IL-2/IL-17 did not decrease. Collectively, although PD-1/PD-L participates in the occurrence of SAT and induces inflammation, administration of the PD-L1 antibody does not effectively improve the pathological process of SAT. More research is needed to determine whether PD-1/PD-L intervention can treat autoimmune thyroid disease.

Photoredox-Catalyzed Radical-Radical Cross-Coupling of Sulfonyl Chlorides with Trifluoroborate Salts.

Sulfones are widely found in natural products and drug molecules. Here, we disclose a strategy for direct synthesis of sulfone compounds with diverse structures by visible-light-catalyzed radical-radical cross-coupling of sulfonyl chlorides and trifluoroborate salts. Allyl, benzyl, vinyl, and aryl trifluoroborates can be successfully cross-coupled with (hetero)aryl and alkyl sulfonyl chlorides, respectively. This strategy features redox neutrality, good substrate generality, simple operation, and benign reaction conditions.

Effects of different iodine levels on the DNA methylation of intrinsic apoptosis-associated genes and analysis of gene-environment interactions in patients with autoimmune thyroiditis.

Iodine is an essential nutrient that may change the occurrence of autoimmune thyroiditis (AIT). Apoptosis and DNA methylation participate in the pathogenesis and destructive mechanism of AIT. We detected the methylation and the expression of mRNA of intrinsic apoptosis-associated genes (YWHAG, ING4, BRSK2 and GJA1) to identify the potential interactions between the levels of methylation in these genes and different levels of iodine. 176 adult patients with AIT in Shandong Province, China, were included. The MethylTargetTM assay was used to verify the levels of methylation. We used PCR to detect the mRNA levels of the candidate genes. Interactions between methylation levels of the candidate genes and iodine levels were evaluated with multiplicative and addictive interaction models and GMDR. In the AIT group, YWHAG_1 and six CpG sites and BRSK2_1 and eight CpG sites were hypermethylated, whereas ING4_1 and one CpG site were hypomethylated. A negative correlation was found between methylation levels of YWHAG and mRNA expression. The combination of iodine fortification, YWHAG_1 hypermethylation and BRSK2_1 hypermethylation was significantly associated with elevated AIT risk. A four-locus model (YWHAG_1 × ING4_1 × BRSK2_1 × iodine level) was found to be the best model of the gene-environment interactions. We identified abnormal changes in the methylation status of YWHAG, ING4 and BRSK2 in patients with AIT in different iodine levels. Iodine fortification not only affected the methylation levels of YWHAG and BRSK2 but also interacted with the methylation levels of these genes and may ultimately increase the risk of AIT.

Alteration of Lipid Profile Between Subclinical Hypothyroidism and Well-Matched Controls: A Meta-Analysis.

In previous studies, subclinical hypothyroidism (SCH) has been associated with altered lipid profiles. However, since the discrepancy between these study results may reside in the great heterogeneity of the populations studied, this relationship is controversial. This study aimed to explore the changes in total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) between subclinical hypothyroidism (SCH) and well-matched euthyroid (EU) groups. Multiple databases were searched for publications before December 1, 2021, including cross-sectional studies on the association between SCH and lipid profile matched by age, gender, and BMI. Twenty-five articles with 3347 participants were included for meta-analysis. The results showed that the TC, TG, and LDL-c levels of the SCH groups were higher than the EU groups (TC, SMD=0.49, 95% CI 0.27, 0.71, p<0.001) (TG, SMD=0.43, 95% CI 0.21, 0.64, p<0.05 ) (LDL-c, SMD=0.75, 95% CI 0.46, 1.03, p<0.001 ). The HDL-c levels of the SCH group were lower than the control group (SMD=-0.53, 95% CI -0.81, -0.25, p<0.05). SCH has a larger impact on LDL-c than the other three indicators. After subgroup analyses, there was a larger impact on lipid alteration in the subgroup of TSH>10 µIU/ml, especially on LDL-c. This study found that SCH was associated with altered lipid profiles. Appropriate clinical treatment may be needed to prevent dyslipidemia and related diseases.

Electrochemical Synthesis of ß-Iodoesters by 1,2-Iodoesterization of Unactivated Alkenes with Carboxylic Acids and Tetrabutylammonium Iodide.

We report a novel and highly selective electrochemical method for the synthesis of ß-iodoesters via difunctionalization of alkenes. The reaction is carried out in an undivided cell under constant current conditions without any additives, catalysts, oxidants, and sacrificial reagents. Inexpensive and readily available tetrabutylammonium iodide not only acts as an electrolyte but also serves as an iodine source. The reaction shows high selectivity and good functional group tolerance, providing products in yields of up to 98%. This method is applicable not only to the iodofunctionalization of alkenes but also to the chloro- and bromofunctionalization of alkenes. The successful modification of drugs and natural products demonstrates the potential utility of this approach.

Electrochemical Cross-Dehydrogenative Coupling of Isochroman and Unactivated Ketones.

An unprecedented electrochemical cross-dehydrogenative coupling reaction between isochroman and unactivated ketones to directly synthesize α-substituted isochromans has been developed. This strategy provides a facile and efficient procedure to the direct activation of C(sp3)-H bond adjacent to the O atom of isochroman. The method features high atom economy, chemical oxidant-free, and mild conditions, in which methanesulfonic acid (MsOH) acts as both electrolyte and catalyst, making the process more convenient and environmentally friendly. Gram-scale experiment and synthesis of antitumor active compounds demonstrate the great potential of this protocol for practical applications.

Visible Light-Induced Cross-Dehydrocoupling of 3-Cyanocoumarins with Unactivated Aliphatic Aldehydes Enables Access to 4-Acylated Coumarins.

We report visible light-induced cross-dehydrocoupling of 3-cyanocoumarins with unactivated aliphatic aldehydes for the direct synthesis of 4-acylated coumarins. Inexpensive and readily available (n-Bu)4NBr was used as a precursor of the hydrogen atom transfer (HAT) catalyst and [Ir(ppy)2(dtbbpy)PF6] as a photocatalyst. A variety of aldehydes including linear, branched, cyclic, and α,ß-unsaturated aliphatic aldehydes were suitable for this reaction yielding the desired acylated coumarin derivatives in 41-98% yields. This method provides a mild, atom-economical, and environmentally friendly synthetic route for the direct acylation of 3-cyanocoumarins. This is the first example so far of C-4 aliphatic acylation of coumarins.