The Development of an Isotope Dilution Mass Spectrometry Method for Interleukin-6 Quantification.

Inflammatory responses and tumor developments are closely related, with interleukin-6 (IL-6) playing important roles in both processes. IL-6 has been extensively identified as a potential tumor biomarker. This study developed an isotope dilution mass spectrometry (IDMS) method for quantifying IL-6 based on signature peptides. These peptides were screened by excluding those with missed cleavage or post-translational modification. The method's accuracy was verified using amino acid-based IDMS, in which purified IL-6 protein samples were quantified after hydrolyzing them into amino acids, and no significant difference was observed (p-value < 0.05). The method demonstrated good linearity and sensitivity upon testing. The specificity and matrix effect of the method were verified, and a precision study showed that the coefficient of variation was less than 5% for both the intra-day and inter-day tests. Compared to immunoassays, this method offers distinct advantages, such as the facilitation of multi-target analysis. Furthermore, the peptides used in this study are much more convenient for storage and operation than the antibodies or purified proteins typically used in immunoassays.

Ferroptosis biomarkers predict tumor mutation burden's impact on prognosis in HER2-positive breast cancer.

BACKGROUND: Ferroptosis has recently been associated with multiple degenerative diseases. Ferroptosis induction in cancer cells is a feasible method for treating neoplastic diseases. However, the association of iron proliferation-related genes with prognosis in HER2+ breast cancer (BC) patients is unclear. AIM: To identify and evaluate fresh ferroptosis-related biomarkers for HER2+ BC. METHODS: First, we obtained the mRNA expression profiles and clinical information of HER2+ BC patients from the TCGA and METABRIC public databases. A four-gene prediction model comprising PROM2, SLC7A11, FANCD2, and FH was subsequently developed in the TCGA cohort and confirmed in the METABRIC cohort. Patients were stratified into high-risk and low-risk groups based on their median risk score, an independent predictor of overall survival (OS). Based on these findings, immune infiltration, mutations, and medication sensitivity were analyzed in various risk groupings. Additionally, we assessed patient prognosis by combining the tumor mutation burden (TMB) with risk score. Finally, we evaluated the expression of critical genes by analyzing single-cell RNA sequencing (scRNA-seq) data from malignant vs normal epithelial cells. RESULTS: We found that the higher the risk score was, the worse the prognosis was (P < 0.05). We also found that the immune cell infiltration, mutation, and drug sensitivity were different between the different risk groups. The high-risk subgroup was associated with lower immune scores and high TMB. Moreover, we found that the combination of the TMB and risk score could stratify patients into three groups with distinct prognoses. HRisk-HTMB patients had the worst prognosis, whereas LRisk-LTMB patients had the best prognosis (P < 0.0001). Analysis of the scRNA-seq data showed that PROM2, SLC7A11, and FANCD2 were significantly differentially expressed, whereas FH was not, suggesting that these genes are expressed mainly in cancer epithelial cells (P < 0.01). CONCLUSION: Our model helps guide the prognosis of HER2+ breast cancer patients, and its combination with the TMB can aid in more accurate assessment of patient prognosis and provide new ideas for further diagnosis and treatment.

TMEM241 is a UDP-N-acetylglucosamine transporter required for M6P modification of NPC2 and cholesterol transport.

Accurate intracellular cholesterol traffic plays crucial roles. Niemann Pick type C (NPC) proteins NPC1 and NPC2, are two lysosomal cholesterol transporters that mediate the cholesterol exit from lysosomes. However, other proteins involved in this process remain poorly defined. Here, we find that the previously unannotated protein TMEM241 is required for cholesterol egressing from lysosomes through amphotericin B-based genome-wide CRISPR-Cas9 KO screening. Ablation of TMEM241 caused impaired sorting of NPC2, a protein utilizes the mannose-6-phosphate (M6P) modification for lysosomal targeting, resulting in cholesterol accumulation in the lysosomes. TMEM241 is a member of solute transporters 35 nucleotide sugar transporters family and localizes on the cis-Golgi network. Our data indicate that TMEM241 transports UDP-N-acetylglucosamine (UDP-GlcNAc) into Golgi lumen and UDP-GlcNAc is used for the M6P modification of proteins including NPC2. Furthermore, Tmem241-deficient mice display cholesterol accumulation in pulmonary cells and behave pulmonary injury and hypokinesia. Taken together, we demonstrate that TMEM241 is a Golgi-localized UDP-GlcNAc transporter and loss of TMEM241 causes cholesterol accumulation in lysosomes because of the impaired M6P-dependent lysosomal targeting of NPC2.

Enhancing Prognosis, Quality of Life, and Psychological Well-being in Cerebral Stroke Patients through Meticulous Risk Management: A Comprehensive Study.

Background: Cerebral stroke (CS) remains a leading cause of mortality worldwide. In addition to effective treatment, nursing intervention plays a pivotal role in the rehabilitation of CS patients. Objective: This study aims to assess the impact of meticulous care integrated with risk management on rehabilitating cerebral stroke patients. The objective is to provide valuable clinical insights for the management of CS patients. Methods: A comparative observational study was conducted, including a total of 180 CS patients admitted between February 2020 and February 2022 were selected for this study. Among them, 98 patients received meticulous care combined with risk management (research group), while 82 patients underwent routine nursing intervention (control group). We analyzed the changes in the Fugl-Meyer Motor Assessment scale, National Institute of Health Stroke Scale, Activities of Daily Living scale, and Barthel Index before and after the care interventions. Additionally, we documented nursing risk events during treatment, assessed nursing quality scores, and conducted a quality-of-life survey after a one-year follow-up. Results: The research group exhibited significantly higher post-care scores in the Fugl-Meyer Motor Assessment, Activities of Daily Living, and Barthel Index, along with lower National Institute of Health Stroke Scale scores, compared to the control group (P < .05). Furthermore, the research group experienced a lower incidence of nursing risk events and demonstrated higher nursing quality (P < .05). During the prognostic follow-up, the research group displayed a superior quality of life compared to the control group (P < .05). Conclusions: Meticulous care integrated with risk management enhances the recovery of CS patients and is strongly recommended for clinical implementation.

Biosynthetic Pathways and Functions of Indole-3-Acetic Acid in Microorganisms.

Indole-3-acetic acid (IAA) belongs to the family of auxin indole derivatives. IAA regulates almost all aspects of plant growth and development, and is one of the most important plant hormones. In microorganisms too, IAA plays an important role in growth, development, and even plant interaction. Therefore, mechanism studies on the biosynthesis and functions of IAA in microorganisms can promote the production and utilization of IAA in agriculture. This mini-review mainly summarizes the biosynthesis pathways that have been reported in microorganisms, including the indole-3-acetamide pathway, indole-3-pyruvate pathway, tryptamine pathway, indole-3-acetonitrile pathway, tryptophan side chain oxidase pathway, and non-tryptophan dependent pathway. Some pathways interact with each other through common key genes to constitute a network of IAA biosynthesis. In addition, functional studies of IAA in microorganisms, divided into three categories, have also been summarized: the effects on microorganisms, the virulence on plants, and the beneficial impacts on plants.

Domestication, breeding, omics research, and important genes of Zizania latifolia and Zizania palustris.

Wild rice (Zizania spp.), an aquatic grass belonging to the subfamily Gramineae, has a high economic value. Zizania provides food (such as grains and vegetables), a habitat for wild animals, and paper-making pulps, possesses certain medicinal values, and helps control water eutrophication. Zizania is an ideal resource for expanding and enriching a rice breeding gene bank to naturally preserve valuable characteristics lost during domestication. With the Z. latifolia and Z. palustris genomes completely sequenced, fundamental achievements have been made toward understanding the origin and domestication, as well as the genetic basis of important agronomic traits of this genus, substantially accelerating the domestication of this wild plant. The present review summarizes the research results on the edible history, economic value, domestication, breeding, omics research, and important genes of Z. latifolia and Z. palustris over the past decades. These findings broaden the collective understanding of Zizania domestication and breeding, furthering human domestication, improvement, and long-term sustainability of wild plant cultivation.

The Novel Effector Ue943 Is Essential for Host Plant Colonization by Ustilago esculenta.

The smut fungus Ustilago esculenta obligately parasitizes Zizania latifolia and induces smut galls at the stem tips of host plants. Previous research identified a putative secreted protein, Ue943, which is required for the biotrophic phase of U. esculenta but not for the saprophytic phase. Here, we studied the role of Ue943 during the infection process. Conserved homologs of Ue943 were found in smut fungi. Ue943 can be secreted by U. esculenta and localized to the biotrophic interface between fungi and plants. It is required at the early stage of colonization. The Ue943 deletion mutant caused reactive oxygen species (ROS) production and callose deposition in the host plant at 1 and 5 days post inoculation, which led to failed colonization. The virulence deficiency was restored by overexpressing gene Ue943 or Ue943:GFP. Transcriptome analysis further showed a series of changes in plant hormones following ROS production when the host plant was exposed to ΔUe943. We hypothesize that Ue943 might be responsible for ROS suppression or avoidance of recognition by the plant immune system. The mechanism underlying Ue943 requires further study to provide more insights into the virulence of smut fungi.

Baicalein Resensitizes Multidrug-Resistant Gram-Negative Pathogens to Doxycycline.

As multidrug-resistant pathogens emerge and spread rapidly, novel antibiotics urgently need to be discovered. With a dwindling antibiotic pipeline, antibiotic adjuvants might be used to revitalize existing antibiotics. In recent decades, traditional Chinese medicine has occupied an essential position in adjuvants of antibiotics. This study found that baicalein potentiates doxycycline against multidrug-resistant Gram-negative pathogens. Mechanism studies have shown that baicalein causes membrane disruption by attaching to phospholipids on the Gram-negative bacterial cytoplasmic membrane and lipopolysaccharides on the outer membrane. This process facilitates the entry of doxycycline into bacteria. Through collaborative strategies, baicalein can also increase the production of reactive oxygen species and inhibit the activities of multidrug efflux pumps and biofilm formation to potentiate antibiotic efficacy. Additionally, baicalein attenuates the lipopolysaccharide-induced inflammatory response in vitro. Finally, baicalein can significantly improve doxycycline efficacy in mouse lung infection models. The present study showed that baicalein might be considered a lead compound, and it should be further optimized and developed as an adjuvant that helps combat antibiotic resistance. IMPORTANCE Doxycycline is an important broad-spectrum tetracycline antibiotic used for treating multiple human infections, but its resistance rates are recently rising globally. Thus, new agents capable of boosting the effectiveness of doxycycline need to be discovered. In this study, it was found that baicalein potentiates doxycycline against multidrug-resistant Gram-negative pathogens in vitro and in vivo. Due to its low cytotoxicity and resistance, the combination of baicalein and doxycycline provides a valuable clinical reference for selecting more effective therapeutic strategies for treating infections caused by multidrug-resistant Gram-negative clinical isolates.

[Strategies for exogenous RNA delivery in RNAi-mediated pest management].

Plant diseases and insect pests threaten the safety of crop production greatly. Traditional methods for pest management are challenged by the problems such as environmental pollution, off-target effects, and resistance of pathogens and insects. New biotechnology-based strategies for pest control are expected to be developed. RNA interference (RNAi) is an endogenous process of gene regulation, which has been widely used to study the gene functions in various organisms. In recent years, RNAi-based pest management has received increasing attention. The effective delivery of the exogenous interference RNA into the targets is a key step in RNAi-mediated plant diseases and pest control. Considerable advances were made on the mechanism of RNAi, and various RNA delivery systems were developed for efficient pest control. Here we review the latest advances on mechanisms and influencing factors of RNA delivery, summarize the strategies of exogenous RNA delivery in RNAi-mediated pest control, and highlight the advantages of nanoparticle complexes in dsRNA delivery.

Amino Phenyl Copper Phosphate-Bridged Reactive Phosphaphenanthrene to Intensify Fire Safety of Epoxy Resins.

To improve the compatibility between flame retardant and epoxy resin (EP) matrix, amino phenyl copper phosphate-9, 10-dihydro-9-oxygen-10-phospha-phenanthrene-10-oxide (CuPPA-DOPO) is synthesized through surface grafting, which is blended with EP matrix to prepare EP/CuPPA-DOPO composites. The amorphous structure of CuPPA-DOPO is characterized by X-ray diffraction and Fourier-transform infrared spectroscopy. Scanning electron microscope (SEM) images indicate that the agglomeration of hybrids is improved, resisting the intense intermolecular attractions on account of the acting force between CuPPA and DOPO. The results of thermal analysis show that CuPPA-DOPO can promote the premature decomposition of EP and increase the residual amount of EP composites. It is worth mentioning that EP/6 wt% CuPPA-DOPO composites reach UL-94 V-1 level and limiting oxygen index (LOI) of 32.6%. Meanwhile, their peak heat release rate (PHRR), peak smoke production release (PSPR) and CO2 production (CO2P) are decreased by 52.5%, 26.1% and 41.4%, respectively, compared with those of EP. The inhibition effect of CuPPA-DOPO on the combustion of EP may be due to the release of phosphorus and ammonia free radicals, as well as the catalytic charring ability of metal oxides and phosphorus phases.

An Endoglucanase Secreted by Ustilago esculenta Promotes Fungal Proliferation.

Ustilago esculenta is a fungus of two morphological forms, among the filamentous dikaryon that can induce the plant stem to expand to form fleshy stem. In order to establish biotrophy with Zizania latifolia which belongs to the tribe Oryzeae (Poaceae), U. esculenta firstly needs to secrete a bunch of effectors, among them being cell wall degrading enzymes (CWDEs). We have isolated a gene, UeEgl1, which was differentially expressed in MT-type and T-type U. esculenta at an early stage of infection, and specifically induced in the filamentous growth of the T-type. Bioinformatics analysis and enzyme activity assay indicated that UeEgl1 functions outside the cell as a ß-1,4-endoglucanase with a conserved domain of the glycosyl hydrolase family 45 (GH45) which targets the main component of the plant cell wall ß-1,4 linked glycosidic bonds. The phenotype analysis of UeEgl1 deletion mutants and UeEgl1 over-expression transformants showed that UeEgl1 had no significant effect on the budding, cell fusion, and filamentous growth of U. esculenta in vitro. Further study found that over-expression of UeEgl1 promoted the proliferation of mycelia inside Z. latifolia, and raised plant defense responses. The above results show that the UeEgl1 gene may play an important role in the early stage of infection through the decomposition of the plant cell wall.

Transcriptome Comparison between Two Strains of Ustilago esculenta during the Mating.

Ustilago esculenta is a smut fungus that obligately infects Zizania latifolia and stimulates tissue swelling to form galls. Unlike T-type, MT-type U. esculenta can only proliferate within plant tissues and infect the offspring of their host. Production of telispores, haploid life, and plant cuticle penetration are not essential for it, which may lead to the degeneration in these processes. Transcriptome changes during the mating of T- and MT-type U. esculenta were studied. The functions of several secreted proteins were further confirmed by knock-out mutants. Our results showed that MT-type U. esculenta can receive environmental signals in mating and circumstance sensing as T-type does. However, MT-type U. esculenta takes a longer time for conjunction tube formation and cytoplasmic fusion. A large number of genes encoding secreted proteins are enriched in the purple co-expression module. They are significantly up-regulated in the late stage of mating in T-type U. esculenta, indicating their relationship with infecting. The knock-out of g6161 (xylanase) resulted in an attenuated symptom. The knock-out of g943 or g4344 (function unidentified) completely blocked the infection at an early stage. This study provides a comprehensive comparison between T- and MT-type during mating and identifies two candidate effectors for further study.

Idiopathic Granulomatous Mastitis with Skin Rupture: A Retrospective Cohort Study of 200 Patients Who Underwent Surgical and Nonsurgical Treatment.

BACKGROUND: Idiopathic granulomatous mastitis (IGM) can clinically and radiographically mimic an abscess or breast cancer. Although IGM is benign, it can cause the breast skin to appear "riddled with holes" and can even result in the loss of the breast. The optimal treatment has not been established. METHODS: We retrospectively studied the medical records of 200 patients with IGM who were treated for skin rupture from June 2015 to June 2017 in our institute. The patients' treatment modalities (including surgery after steroid therapy and steroid therapy alone), outcomes, and scores of satisfaction questionnaires were analyzed. The time to healing and recurrence rate were compared with a focus on the treatment modalities to identify the most effective treatments for IGM. RESULTS: The median follow-up time was 15.64 months (range, 12-36 months). In total, 156 patients were treated with surgery after steroid therapy and 44 were treated with steroid therapy alone. The median times to healing in the surgical and nonsurgical groups were 25 and 258 days, respectively (p = 0.003). Four of 156 (2.56%) patients developed post-excision wound complications. Eight of 156 patients (5.1%) in the surgical group and 10 of 44 (22.7%) patients in the nonsurgical group developed recurrence (p < 0.01). The scores of the satisfaction questionnaire were 36 ± 4.28 in the surgical group and 24 ± 8.62 in the nonsurgical group (p = 0.017). CONCLUSION: IGM is a benign disease but can have serious consequences. Surgery after steroid therapy is an effective and more satisfactory treatment than steroid therapy alone.

Nevadensin relieves food allergic responses and passive cutaneous anaphylaxis in mice through inhibiting the expression of c-Kit receptors.

Nevadensin (NEV), a natural flavonoid compound derived from Lysionotus pauciflorus Maxim, has numerous biological activities. However, few researchers have examined its potential impact on alleviating allergies. In the present study, NEV was found to upregulate rectal temperature, suppress the development of diarrhea, and decrease the levels of serum specific immunoglobulin E, histamine and mouse MC protease-1 in ovalbumin-allergic mice. Moreover, NEV also alleviated passive cutaneous anaphylaxis reactions and inhibited the release of ß-hexosaminidase and histamine in bone marrow-derived mast cells. Furthermore, we provide the first demonstration that NEV decreases the expression of c-Kit and suppresses the proliferation of bone marrow-derived mast cells and accelerates their apoptosis. These findings indicated that L. pauciflorus-derived NEV might have the potential to alleviate food hypersensitivity.

Viridicatol Isolated from Deep-Sea Penicillium Griseofulvum Alleviates Anaphylaxis and Repairs the Intestinal Barrier in Mice by Suppressing Mast Cell Activation.

Viridicatol is a quinoline alkaloid isolated from the deep-sea-derived fungus Penicillium griseofulvum. The structure of viridicatol was unambiguously established by X-ray diffraction analysis. In this study, a mouse model of ovalbumin-induced food allergy and the rat basophil leukemia (RBL)-2H3 cell model were established to explore the anti-allergic properties of viridicatol. On the basis of the mouse model, we found viridicatol to alleviate the allergy symptoms; decrease the levels of specific immunoglobulin E, mast cell protease-1, histamine, and tumor necrosis factor-α; and promote the production of interleukin-10 in the serum. The treatment of viridicatol also downregulated the population of B cells and mast cells (MCs), as well as upregulated the population of regulatory T cells in the spleen. Moreover, viridicatol alleviated intestinal villi injury and inhibited the degranulation of intestinal MCs to promote intestinal barrier repair in mice. Furthermore, the accumulation of Ca2+ in RBL-2H3 cells was significantly suppressed by viridicatol, which could block the activation of MCs. Taken together, these data indicated that deep-sea viridicatol may represent a novel therapeutic for allergic diseases.

BRCA2 c.8827C>T pathogenic mutation in a consanguineous Chinese family with hereditary breast cancer.

BACKGROUND: Mutations in the BRCA2 DNA repair associated gene (BRCA2) are associated with the development of breast cancer, with different ethnic mutations at different sites. Based on different types of BRCA2 variants, the underlying mechanism remains still elusive. METHODS: Next-generation sequencing (NGS) was performed to detect germ line mutations in BRCA2. The expressions of BRCA2 mRNA and BRCA2 protein were detected by Real-time PCR and Western blot, respectively. RESULTS: In a consanguineous Chinese family with hereditary breast cancer, one woman had unilateral breast cancer, two women had bilateral asynchronous breast cancer, and one man had prostate cancer. We identified a mutation site (NM_000059.4: c.8827C>T, NP_ 000050.3: p.(Gln2943*)) in BRCA2 gene, which was a nonsense mutation that predicted disrupting peptide chain synthesis and limiting BRCA2 protein production, validated by the decreased expressions of both BRCA2 mRNA and BRCA2 protein. CONCLUSION: In this study, we identified a BRCA2 c.8827C>T nonsense mutation with a truncated BRCA2 protein in a consanguineous Chinese Han family, suggesting individuals with this mutation should be regularly screened for malignancies such as breast, prostate, and ovarian cancer. Our study verified the function of this BRCA2 mutation site and provided a new target for the precise treatment of such patients.

Functional Properties of the MAP Kinase UeKpp2 in Ustilago esculenta.

Ustilago esculenta undergoes an endophytic life cycle in Zizania latifolia. It induces the stem of its host to swell, forming the edible galls called jiaobai in China, which are the second most commonly cultivated aquatic vegetable in China. Z. latifolia raised for jiaobai can only reproduce asexually because the U. esculenta infection completely inhibits flowering. The infection and proliferation in the host plants during the formation of edible gall differ from those of conventional pathogens. Previous studies have shown a close relationship between mitogen-activated protein kinase (MAPK) and fungal pathogenesis. In this study, we explored the functional properties of the MAPK UeKpp2. Cross-species complementation assays were carried out, which indicated a functional complementation between the UeKpp2 of U. esculenta and the Kpp2 of Ustilago maydis. Next, UeKpp2 mutants of the UeT14 and the UeT55 sporidia background were generated; these showed an aberrant morphology of budding cells, and attenuated mating and filamentous growth in vitro, in the context of normal pathogenicity. Interestingly, we identified another protein kinase, UeUkc1, which acted downstream of UeKpp2 and may participate in the regulation of cell shape. We also found a defect of filamentous growth in UeKpp2 mutants that was not related to a defect of the induction of mating-type genes but was directly related to a defect in UeRbf1 induction. Overall, our results indicate an important role for UeKpp2 in U. esculenta that is slightly different from those reported for other smut fungi.

Sulfated oligosaccharide of Gracilaria lemaneiformis protect against food allergic response in mice by up-regulating immunosuppression.

Sulfated oligosaccharide of Gracilaria lemaneiformis (GLSO) was prepared from sulfated polysaccharides which possessed antiallergic activity by degradation with high temperature and pressure combined with vitamin C treatment. The present study demonstrated that GLSO could attenuate food anaphylaxis, and inhibit the production of immunoglobulin E, histamine, and related cytokines in both prevention and therapy ovalbumin-induced mice model. Additionally, the gut microbiota analysis revealed that GLSO markedly rescued OVA-induced changes in the Firmicutes to Bacteroidetes ratio. Following flow cytometry, GLSO was found to suppress the subpopulation of T helper 2 and B cells, and significantly up-regulate regulatory T cells (Tregs) differentiation. Furthermore, GLSO-mediated immunosuppression could be verified by co-culturing Tregs sorted from GLSO-treated mice and CD4+ T cells or mast cells. In a word, GLSO attenuated food anaphylaxis through the regulation of gut microbiota and induction of immunosuppression. GLSO had the potential to be used as a nutrient component against food allergy.

LncRNA WT1-AS Inhibits Triple-Negative Breast Cancer Cell Migration and Invasion by Downregulating Transforming Growth Factor ß1.

Background: Long noncoding RNA WT1-AS has been demonstrated as a potential tumor suppressor in gastric cancer. However, the functions of WT1-AS in other types of cancer remain unclear. Our study was therefore performed to explore the role of WT1-AS in triple-negative breast cancer (TNBC). Materials and Methods: Tissue specimens were obtained from 62 TNBC patients included in this study. A TNBC cell line BT-549 was used as the cell model of TNBC. Gene expression was detected by qPCR and Western blot. Overexpression experiments were used to analyze gene interactions. Transwell assays were used to explore the effects of transfections on cell invasion and migration. Results: We found that WT1-AS was downregulated in TNBC tissues than in nontumor tissues and decreased with increase in clinical stages. Transforming growth factor ß1 (TGF-ß1) was upregulated in TNBC tissues and inversely correlated with WT1-AS. TGF-ß1 overexpression did not significantly affect WT1-AS in BT-549 cells, but WT1-AS negatively regulated the expression of TGF-ß1. WT1-AS overexpression caused inhibited migration and invasion of TNBC cells. TGF-ß1 overexpression showed opposite functions and reduced the effects of WT1-AS overexpression. Conclusion: WT1-AS may downregulate TGF-ß to inhibit the migration and invasion of TNBC cells.