Preparation of magnetic amphiphilic resin microspheres via the one-step polymerization method and extraction of four glucocorticoids for HPLC-MS analysis.

Amphiphilic materials can be used for sample preparation of chromatography or mass spectrometry. Amphiphilic materials with magnetic properties in combination with magnetic suction devices allow for automated sample preparation. However, conventional synthesis methods are cumbersome and not suitable for the mass production of the material. In this study, a micro-suspension polymerization method was developed to synthesize magnetic amphiphilic resin microspheres (MARMs), providing new ideas for the preparation of amphiphilic microspheres. MARMs with particle sizes ranging from 3 to 6 µm were successfully prepared, with BET surface area up to 653.2 m2/g. A magnetic solid-phase extraction method based on MARM-5 was developed for the extraction of four glucocorticoids including Cortisone, Hydrocortisone, Cortodoxone, and Corticosterone. This method had a very short adsorption time of 0.5 min and a total extraction time of only 13 min. The limit of detection for the four glucocorticoids ranged from 0.22 to 0.82 ng/L. There was a good linear relationship between sample concentration and peak area in the range of 25∼500 ng/L. Relative recovery of 98 %∼108 % and internal standard normalized matrix effect factors of 95∼114 % were obtained, and the relative standard deviation was between 2.3 % and 6.3 %. The MARMs would be used as excellent solid extraction material for glucocorticoids.

A benchmarking framework for the accurate and cost-effective detection of clinically-relevant structural variants for cancer target identification and diagnosis.

BACKGROUND: Accurate clinical structural variant (SV) calling is essential for cancer target identification and diagnosis but has been historically challenging due to the lack of ground truth for clinical specimens. Meanwhile, reduced clinical-testing cost is the key to the widespread clinical utility. METHODS: We analyzed massive data from tumor samples of 476 patients and developed a computational framework for accurate and cost-effective detection of clinically-relevant SVs. In addition, standard materials and classical experiments including immunohistochemistry and/or fluorescence in situ hybridization were used to validate the developed computational framework. RESULTS: We systematically evaluated the common algorithms for SV detection and established an expert-reviewed SV call set of 1,303 tumor-specific SVs with high-evidence levels. Moreover, we developed a random-forest-based decision model to improve the true positive of SVs. To independently validate the tailored 'two-step' strategy, we utilized standard materials and classical experiments. The accuracy of the model was over 90% (92-99.78%) for all types of data. CONCLUSION: Our study provides a valuable resource and an actionable guide to improve cancer-specific SV detection accuracy and clinical applicability.

Multi-omics analyses reveal spatial heterogeneity in primary and metastatic oesophageal squamous cell carcinoma.

BACKGROUND: Biopsies obtained from primary oesophageal squamous cell carcinoma (ESCC) guide diagnosis and treatment. However, spatial intra-tumoral heterogeneity (ITH) influences biopsy-derived information and patient responsiveness to therapy. Here, we aimed to elucidate the spatial ITH of ESCC and matched lymph node metastasis (LNmet ). METHODS: Primary tumour superficial (PTsup ), deep (PTdeep ) and LNmet subregions of patients with locally advanced resectable ESCC were evaluated using whole-exome sequencing (WES), whole-transcriptome sequencing and spatially resolved digital spatial profiling (DSP). To validate the findings, immunohistochemistry was conducted and a single-cell transcriptomic dataset was analysed. RESULTS: WES revealed 15.72%, 5.02% and 32.00% unique mutations in PTsup , PTdeep and LNmet , respectively. Copy number alterations and phylogenetic trees showed spatial ITH among subregions both within and among patients. Driver mutations had a mixed intra-tumoral clonal status among subregions. Transcriptome data showed distinct differentially expressed genes among subregions. LNmet exhibited elevated expression of immunomodulatory genes and enriched immune cells, particularly when compared with PTsup (all P < .05). DSP revealed orthogonal support of bulk transcriptome results, with differences in protein and immune cell abundance between subregions in a spatial context. The integrative analysis of multi-omics data revealed complex heterogeneity in mRNA/protein levels and immune cell abundance within each subregion. CONCLUSIONS: This study comprehensively characterised spatial ITH in ESCC, and the findings highlight the clinical significance of unbiased molecular classification based on multi-omics data and their potential to improve the understanding and management of ESCC. The current practices for tissue sampling are insufficient for guiding precision medicine for ESCC, and routine profiling of PTdeep and/or LNmet should be systematically performed to obtain a more comprehensive understanding of ESCC and better inform treatment decisions.

Characterization of the tumor microenvironment and identification of spatially predictive biomarkers associated with beneficial neoadjuvant chemoradiotherapy in locally advanced rectal cancer.

Neoadjuvant chemoradiotherapy (nCRT) has become the standard treatment for patients with locally advanced rectal cancer (LARC). However, 20-40% of patients with LARC show little to no response to nCRT. Thus, comprehensively understanding the tumor microenvironment (TME), which might influence therapeutic efficacy, and identifying robust predictive biomarkers is urgently needed. Pre-treatment tumor biopsy specimens from patients with LARC were evaluated in detail through digital spatial profiling (DSP), public RNA sequencing datasets, and multiplex immunofluorescence (mIF). DSP analysis revealed distinct characteristics of the tumor stroma compared to the normal stroma and tumor compartments. We identified high levels of human leukocyte antigen-DR/major histocompatibility complex class II (HLA-DR/MHC-II) in the tumor compartment and B cells in the stroma as potential spatial predictors of nCRT efficacy in the Discovery cohort. Public datasets validated their predictive capacity for clinical outcomes. Using mIF in an independent nCRT cohort and/or the total cohort, we validated that a high density of HLA-DR/MHC-II+ cells in the tumor and CD20 + B cells in the stroma was associated with nCRT efficacy (all p ≤ 0.021). Spatial profiling successfully characterized the LARC TME and identified robust biomarkers with the potential to accurately predict nCRT response. These findings have important implications for individualized therapy.

Does acupuncture improve the metabolic outcomes of obese/overweight children and adolescents?: A systematic review and meta-analysis.

BACKGROUND: Although increasing evidence has revealed the efficacy of acupuncture in obesity/overweight, actual improvement in metabolism in children and adolescents is unclear. Therefore, we conducted a meta-analysis to evaluate this correlation. METHODS: A comprehensive search was conducted using multiple databases, including Medline, Cochrane, Embase, Web of Science, Chinese Biomedical Literature Database, China National Knowledge Infrastructure, Chinese Scientific Journal Database, and Wan-fang Data, to identify relevant randomized controlled trials published before February 1, 2023. General information and data for the descriptive and quantitative analyses were extracted. RESULTS: Fifteen randomized controlled trials of 1288 obese/overweight children and teenagers were included. All the trials were conducted in China and South Korea. Regarding quality assessment, no other significant risk of bias was found. The acupuncture groups were more likely to have improved metabolic indicators of obesity/overweight than the control groups, in terms of body mass index (standardized mean difference [SMD] = -0.45, 95% confidence interval [CI]: -0.69 to -0.21, I2 = 71.4%), body weight (SMD = -0.48, 95% CI: -0.92 to -0.05, I2 = 84.9%), and serum leptin (SMD = -0.34, 95% CI: -0.58 to -0.10, I2 = 91.8%). The subgroup analysis showed that for body mass index, the results were consistent regardless of the intervention duration, body acupuncture or auricular acupuncture combined with other interventions. CONCLUSION: Our results suggest that acupuncture is effective in improving metabolic outcomes of obese/overweight children and adolescents. Owing to the limited number of trials included in this study, the results should be interpreted with caution.

Proteomic Profiling and Tumor Microenvironment Characterization Reveal Molecular and Immunological Hallmarks of Left-Sided and Right-Sided Colon Cancer Tumorigenesis.

Left-sided and right-sided colon cancer (LSCC and RSCC) display different biological and clinical characteristics. However, the differences in their tumorigenesis and tumor microenvironment remain unclear. In this study, we profiled the proteomic landscapes of LSCC and RSCC with data-independent acquisition mass spectrometry (DIA-MS) using fresh tumor and adjacent normal tissues from 24 patients. A total of 7403 proteingroups were primarily identified with DIA-MS. After quality control, 7212 proteingroups were used for further analysis. Through comparing the difference in proteomic profiles between LSCC and RSCC samples, 2556 commonly and 1982 region-type-specific regulated proteingroups were characterized. During the development of LSCC and RSCC, metabolic, growth, cell division, cell adhesion, and migration pathways were found to be significantly dysregulated (P < 0.05), which was further confirmed by transcriptome data from TCGA. Compared to RSCC, most parts of the immune-related signatures, immune cell infiltration scores, and overall immune scores of LSCC were higher. The systematic elucidation of proteomic and transcriptomic profiles in this work improves our understanding of tumorigenesis and immune microenvironment characteristics of LSCC and RSCC.

The Mutational, Prognostic, and Therapeutic Landscape of Neuroendocrine Neoplasms.

BACKGROUND: Neuroendocrine neoplasms (NENs) represent clinically and genetically heterogeneous malignancies, thus a comprehensive understanding of underlying molecular characteristics, prognostic signatures, and potential therapeutic targets is urgently needed. METHODS: Next-generation sequencing (NGS) and immunohistochemistry were applied to acquire genomic and immune profiles of NENs from 47 patients. RESULTS: Difference was distinguished based on differentiation grade and primary localization. Poorly differentiated neuroendocrine carcinomas (NECs) and well-differentiated neuroendocrine tumors (NETs) harbored distinct molecular features; we observed that tumor mutational burden (TMB) and tumor neoantigen burden (TNB) were significantly higher in NECs versus NETs. Notably, we identified a 7-gene panel (MLH3, NACA, NOTCH1, NPAP1, RANBP17, TSC2, and ZFHX4) as a novel prognostic signature in NENs; patients who carried mutations in any of the 7 genes exhibited significantly poorer survival. Furthermore, loss of heterozygosity (LOH) and germline homogeneity in human leukocyte antigen (HLA) are common in NENs, accounting for 39% and 36%, respectively. Notably, HLA LOH was an important prognostic biomarker for a subgroup of NEN patients. Finally, we analyzed clinically actionable targets in NENs, revealing that TMB high (TMB-H) or gene mutations in TP53, KRAS, and HRAS were the most frequently observed therapeutic indicators, which granted eligibility to immune checkpoint blockade (ICB) and targeted therapy. CONCLUSION: Our study revealed heterogeneity of NENs, and identified novel prognostic signatures and potential therapeutic targets, which directing improvements of clinical management for NEN patients in the foreseeable future.

Integrated investigation of the prognostic role of HLA LOH in advanced lung cancer patients with immunotherapy.

Although multiple studies have shown that loss of heterozygosity (LOH) at the human leukocyte antigen (HLA) locus is one of the mechanisms of immune escape, the effect of HLA LOH on the immunotherapy response of patients is still unclear. Based on the data of 425 Chinese lung cancer patients, the genomic characteristics with different HLA LOH statuses were analyzed. The driver genes mutation frequency, oncogenic signaling pathways mutation frequency, tumor mutational burden (TMB) and chromosomal instability (CIN) score in the HLA LOH high group was significantly higher than in the HLA LOH negative group. Transcriptome analyses revealed that pre-existing immunologically active tumor microenvironment (TME) was associated with HLA LOH negative patients. Non-small cell lung cancer (NSCLC) patients, especially for lung squamous cell carcinomas (LUSC), with HLA LOH negative have a longer survival period than those with HLA LOH. In addition, the combination of HLA LOH with TMB or programmed cell death-Ligand 1 (PD-L1) expression can further distinguish responders from non-responders. Furthermore, a comprehensive predictive model including HLA LOH status, TMB, PD-L1 expression and CD8+ T cells was constructed and exhibited a higher predictive value, which may improve clinical decision-making.

Systematic assessment and optimizing algorithm of tumor mutational burden calculation and their implications in clinical decision-making.

Tumor mutation burden (TMB) has been validated as a biomarker to predict the response of immune checkpoint inhibitors (ICIs) treatment in various cancers. However, the effects of different sequencing platforms, cancer types, and calculation algorithms on TMB as well as its cut-off value for predicting immunotherapy efficacy in the East Asian population still need to be further investigated. In this study, the data of 4126 samples generated by targeted panel sequencing or whole-exome sequencing (WES) in different platforms and public sequencing data from 3680 samples that contained targeted panel sequencing, WES and whole-genome sequencing (WGS) were obtained. The impact of different sequencing platforms and methods on TMB calculation was assessed. No significant bias was found in TMB calculated by different platforms. However, TMB calculated from WGS was significantly lower than those calculated from targeted panel sequencing and WES. The distribution of TMB at different sequencing depths and tumor purity were analyzed. There was no significant difference in the distribution of TMB when the sequencing depth was greater than 500, the tumor purity estimated by hematoxylin-eosin (HE) staining was between 0.1-1.0 or estimated by next-generation sequencing (NGS) was greater than 0.4. In addition, the somatic-germline-zygosity (SGZ) algorithm was optimized to calculate TMB from tumor-only sequencing samples in the East Asian population. The correlation coefficient of TMB calculated with the optimized SGZ algorithm and paired normal-tumor sequencing is 0.951. Furthermore, the optimal cut-off value of TMB in East Asian lung cancer patients treated with ICIs was determined to be 7 mut/Mb instead of 10 mut/Mb through the ROC curve and Log-rank analysis in the training cohort and validated in the test cohort. Patients with TMB ≥ 7 mut/Mb had better outcomes than patients with TMB<7 mut/Mb. In conclusion, this study systematically analyzed the factors that influenced the TMB calculation and optimized the SGZ algorithm to calculate TMB from tumor-only sequencing samples in the East Asian population. More importantly, the cut-off value of TMB for predicting immunotherapy efficacy was determined to be 7 mut/Mb instead of 10 mut/Mb in East Asian lung cancer patients, which can help in clinical decision-making.

Improving the thermostability of alginate lyase FlAlyA with high expression by computer-aided rational design for industrial preparation of alginate oligosaccharides.

FlAlyA, a PL7 alginate lyase with industrial potential, is widely applied in the preparation the alginate oligosaccharide because of its high activity of degradation the alginate. However, heat inactivation still limits the industrial application of FlAlyA. To further enhance its thermostability, a group of mutants were designed, according to evaluating the B-factor value and free energy change via computer-aided calculation. 25 single-point mutants and one double-points mutant were carried out by site-directed mutagenesis. The optimal two single-point mutants H176D and H71K showed 1.20 and 0.3°C increases in the values of T m, while 7.58 and 1.73 min increases in the values of half-life (t 1/2) at 50°C, respectively, compared with that of the wild-type enzyme. Interestingly, H71K exhibits the comprehensive improvement than WT, including expression level, thermal stability and specific activity. In addition, the mechanism of these two mutants is speculated by multiple sequence alignment, structural basis and molecular dynamics simulation, which is likely to be involved in the formation of new hydrogen bonds and decrease the SASA of the mutants. These results indicate that B-factor is an efficient approach to improves the thermostability of alginate lyase composed of ß-sheet unit. Furthermore, the highest yield of the mutant reached about 650 mg/L, which was nearly 36 times that of previous studies. The high expression, excellent activity and good thermal stability make FlAlyA a potential candidate for the industrial production of alginate oligosaccharides.

Whole-Genome and Transcriptome Sequencing-Based Characterization of Bacillus Cereus NR1 From Subtropical Marine Mangrove and Its Potential Role in Sulfur Metabolism.

Sulfur, organosulfur compounds, and sulfides are essential parts of life. Microbial sulfate assimilation is among the most active and ancient metabolic activities in the sulfur cycle that operates in various ecosystems. We analyzed the molecular basis of bacterial characterization. NR1 was isolated and purified from mangrove sediments. Whole-genome sequencing indicated that the NR1 isolate was closely related to Bacillus cereus. The genome contained 5,305 functional genes with a total length of 5,420,664 bp, a GC content of 35.62%, 42 rRNA, and 107 tRNA. DBT-grown cultures exhibited DBT utilization, fleeting emergence of DBT sulfone (DBTO2), and formation of 2-hydroxybiphenyl (2-HBP). Molecular analysis of the PCR products' dsz operon revealed the presence of dszA, dszB, and dszC genes, which encoded for NR1's 90% DBT desulfurization activity. Furthermore, 17 sulfur metabolism-related genes, including genes involved in assimilation sulfate reduction, APS and PAPS, and the cys, ssu, and TST gene families, were identified. In sulfate media, alkenesulfonate was converted to sulfite and inhibited ssu enzymes. Downregulated cysK variants were associated with nrnA expression and the regulation of L-cysteine synthesis. These findings established a scientific foundation for further research and application of bacteria to mangrove rehabilitation and ecological treatment by evaluating the bacterial characterization and sulfur degradation metabolic pathway. We used whole-genome and transcriptome sequencing to examine their genetic characteristics.

An enhanced genetic mutation-based model for predicting the efficacy of immune checkpoint inhibitors in patients with melanoma.

Background: Programmed death ligand 1 (PD-L1) and tumor mutation burden (TMB) have been developed as biomarkers for the treatment of immune checkpoint inhibitors (ICIs). However, some patients who are TMB-high or PD-L1-high remained resistant to ICIs therapy. Therefore, a more clinically applicable and effective model for predicting the efficacy of ICIs is urgently needed. Methods: In this study, genomic data for 466 patients with melanoma treated with ICIs from seven independent cohorts were collected and used as training and validation cohorts (training cohort n = 300, validation cohort1 n = 61, validation cohort2 n = 105). Ten machine learning classifiers, including Random Forest classifier, Stochastic Gradient Descent (SGD) classifier and Linear Support Vector Classifier (SVC), were subsequently evaluated. Results: The Linear SVC with a 186-gene mutation-based set was screened to construct the durable clinical benefit (DCB) model. Patients predicted to have DCB (pDCB) were associated with a better response to the treatment of ICIs in the validation cohort1 (AUC=0.838) and cohort2 (AUC=0.993). Compared with TMB and other reported genetic mutation-based signatures, the DCB model showed greater predictive power. Furthermore, we explored the genomic features in determining the benefits of ICIs treatment and found that patients with pDCB were associated with higher tumor immunogenicity. Conclusion: The DCB model constructed in this study can effectively predict the efficacy of ICIs treatment in patients with melanoma, which will be helpful for clinical decision-making.

Epitranscriptomic editing of the RNA N6-methyladenosine modification by dCasRx conjugated methyltransferase and demethylase.

N6-methyladenosine (m6A) is a common modification on endogenous RNA transcripts in mammalian cells. Technologies to precisely modify the RNA m6A levels at specific transcriptomic loci empower interrogation of biological functions of epitranscriptomic modifications. Here, we developed a bidirectional dCasRx epitranscriptome editing platform composed of a nuclear-localized dCasRx conjugated with either a methyltransferase, METTL3, or a demethylase, ALKBH5, to manipulate methylation events at targeted m6A sites. Leveraging this platform, we specifically and efficiently edited m6A modifications at targeted sites, reflected in gene expression and cell proliferation. We employed the dCasRx epitranscriptomic editor system to elucidate the molecular function of m6A-binding proteins YTHDF paralogs (YTHDF1, YTHDF2 and YTHDF3), revealing that YTHDFs promote m6A-mediated mRNA degradation. Collectively, our dCasRx epitranscriptome perturbation platform permits site-specific m6A editing for delineating of functional roles of individual m6A modifications in the mammalian epitranscriptome.

SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2.

Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.

CRISPR Screening of CAR T Cells and Cancer Stem Cells Reveals Critical Dependencies for Cell-Based Therapies.

Glioblastoma (GBM) contains self-renewing GBM stem cells (GSC) potentially amenable to immunologic targeting, but chimeric antigen receptor (CAR) T-cell therapy has demonstrated limited clinical responses in GBM. Here, we interrogated molecular determinants of CAR-mediated GBM killing through whole-genome CRISPR screens in both CAR T cells and patient-derived GSCs. Screening of CAR T cells identified dependencies for effector functions, including TLE4 and IKZF2. Targeted knockout of these genes enhanced CAR antitumor efficacy. Bulk and single-cell RNA sequencing of edited CAR T cells revealed transcriptional profiles of superior effector function and inhibited exhaustion responses. Reciprocal screening of GSCs identified genes essential for susceptibility to CAR-mediated killing, including RELA and NPLOC4, the knockout of which altered tumor-immune signaling and increased responsiveness of CAR therapy. Overall, CRISPR screening of CAR T cells and GSCs discovered avenues for enhancing CAR therapeutic efficacy against GBM, with the potential to be extended to other solid tumors. SIGNIFICANCE: Reciprocal CRISPR screening identified genes in both CAR T cells and tumor cells regulating the potency of CAR T-cell cytotoxicity, informing molecular targeting strategies to potentiate CAR T-cell antitumor efficacy and elucidate genetic modifications of tumor cells in combination with CAR T cells to advance immuno-oncotherapy.This article is highlighted in the In This Issue feature, p. 995.

Cryptotanshinone from Salvia miltiorrhiza Bunge (Danshen) inhibited inflammatory responses via TLR4/MyD88 signaling pathway.

BACKGROUND: Cryptotanshinone (CPT), as a major component of Salvia miltiorrhiza Bunge (Danshen), displays many pharmacological activities including anti-inflammatory effects. However, the exact cellular and molecular mechanisms of the anti-inflammatory activities of CPT remain to be elucidated. The present study was aimed to clarify its mechanisms on lipopolysaccharide (LPS)-induced inflammatory responses in mouse macrophages, RAW264.7 cells. METHODS: In the current study, the anti-inflammatory properties of CPT were evaluated using LPS-stimulated RAW264.7 cell model. MTT assay was used to determine the viability of RAW264.7 cells. The anti-inflammatory effects of CPT were measured based on the detection of nitric oxide (NO) production (Griess and flow cytometry assay), and tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release (ELISA). Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzyme expressions were also determined by western blotting. Besides, by using flow cytometry, we also evaluated the effect of CPT on LPS-induced calcium influx. Finally, the underlying anti-inflammatory mechanisms of CPT were investigated using western blotting to assess the protein levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phosphatidylinositol 3-kinase (PI3K)/AKT, nuclear factor erythroid 2 related factor 2 (Nrf2), mitogen-activated protein kinase (MAPK), and nuclear factor-kappa B (NF-κB) pathways. RESULTS: Our data showed that CPT inhibited LPS-induced pro-inflammatory cytokine release like IL-6, and TNF-α, as well as NO production. It displayed a significant inhibitory effect on the protein expressions such as iNOS, COX-2, NF-κB pathway like inhibitor of kappa B kinase (IKK)α/ß, inhibitor of kappa B (IκB)-α and NF-κB/p65, PI3K/AKT pathway like PI3K and AKT, and MAPK pathway like c-Jun N-terminal kinase (JNK)1/2, extracellular signal-regulated kinase (ERK)1/2, and p38, in LPS-stimulated RAW264.7 macrophages. Moreover, the immunofluorescence results indicated that CPT suppressed NF-κB/p65 translocation from the cytoplasm into the nucleus. Further investigations showed that CPT treatment increased NAD(P)H quinone oxidoreductase-1 (NQO1) and heme oxygenase-1 (HO-1) expressions together with its upstream mediator, Nrf2. In addition, CPT inhibited LPS-induced toll-like receptor 4 (TLR4) and MyD88 expressions in RAW264.7 macrophages. CONCLUSIONS: Collectively, we suggested that CPT exerted significant anti-inflammatory effects via modulating TLR4-MyD88/PI3K/Nrf2 and TLR4-MyD88/NF-κB/MAPK pathways.

[Effects of exogenous IBA and fungal elicitor on growth of in vitro roots culture of Dysosma versipellis and production of podophyllotoxin].

Using the White as basic medium, the effects of the exogenous IBA and endophytic fungal elicitor on the growth of in vitro roots cultures of Dysosma versipellis and production of podophyllotoxin were investigated in this study. The results showed that the IBA and the endophytic fungus Zasmidium syzygii elicitor could increase the content of podophyllotoxin of in vitro roots of D. versipellis after 3 weeks. The White medium added with 3 mg·L~(-1) IBA induced the highest increase of podophyllotoxin(1 830.86 µg·g~(-1)), which was 2.07 folds greater than the control, and followed by 1.5 mg·L~(-1) IBA, fungal elicitor, 1 mg·L~(-1) IBA, 0.5 mg·L~(-1) IBA and 4.5 mg·L~(-1) IBA, which was 1.82, 1.71, 1.63, 1.43 and 1.1 folds greater than the control, respectively. The results also showed that the growth of roots was certain positively correlated with the change of IBA concentration. Therefore, 3 mg·L~(-1) IBA was the most suitable for the production of podophyllotoxin in the in vitro roots of D. versipellis, and the stimulating effect of Z. syzygii fungal elicitor was between 1.5 mg·L~(-1) and 1 mg·L~(-1) IBA, which was a potential natural elicitor to induce the accumulation of podophyllotoxin in future production.

[Inhibitory effect of endophytic fungi from Dysosma versipellis on HIV-1 IN-LEDGF/p75 interaction].

To determine the inhibitory effect of endophytic fungi from Dysosma versipellis on HIV-1 IN-LEDGF/p75 interaction,the protein-protein interaction between human immunodeficiency virus type 1( HIV-1) integrase and lens epithelial growth factor p75 protein( LEDGF/p75) was used as a target. The homogeneous time-resolved fluorescence( HTRF) technique was used in the inhibitory activity assay. The results showed that eight endophytic fungi with anti-IN-LEDGF/p75 interaction activity were screened out from fifty-three strains with different morphological characteristic. Among them,106 strain showed strong inhibitory activity against HIV-1 IN-LEDGF/p75 interaction with IC50 value of 5. 23 mg·L-1,and was identified as a potential novel species of Magnaporthaceae family by the analyses of ITS-rDNA,LSU and RPB2 sequences data. This study demonstrated that potential natural active ingredients against the HIV-1 IN-LEDGF/p75 interaction exist in the endophytic fungi of D. versipellis. These results may provide available candidate strain resources for the research and development of new anti-acquired immunodeficiency syndrome drugs.

Author Correction: Diversity and bioactive potential of culturable fungal endophytes of Dysosma versipellis; a rare medicinal plant endemic to China.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Simultaneous purification of dihydrotanshinone, tanshinone I, cryptotanshinone, and tanshinone IIA from Salvia miltiorrhiza and their anti-inflammatory activities investigation.

Dihydrotanshinone, tanshinone I, cryptotanshinone, and tanshinone IIA are major lipid-soluble constituents isolated from Salvia miltiorrhiza Bunge (Danshen). In the present study, a systematic method was developed to simultaneously isolate and purify those compounds using macroporous adsorption resins and semi-preparative HPLC with a dynamic axial compress (DAC) system. The Danshen extract (95% alcohol) was divided into three fractions using different concentrations of alcohol (0%, 45%, and 90%) on D101 column. The content of total tanshinones of 90% alcohol eluent (TTS) was over 97%. Furthermore, the anti-inflammatory effects of those samples were investigated on LPS-stimulated RAW264.7 cells and three animal models. The results showed that the anti-inflammatory effect of TTS in vitro was superior to the one of any other sample including 0% and 45% eluent, and total tanshinones capsules. In addition, TTS exhibited a stronger anti-inflammatory effect than that of dihydrotanshinone, tanshinone IIA, cryptotanshinone, and tanshinone I, respectively. For animal models, TTS could significantly suppress xylene-induced ear oedema and rescue LPS-induced septic death and acute kidney injury in mice. In summary, the separation process developed in the study was high-efficiency, economic, and low-contamination, which was fit to industrial producing. TTS is a potential agent for the treatment of inflammatory diseases.