Removal of signal peptide variants by cation exchange chromatography: A case study.

Signal peptide (SP) is required for secretion of recombinant proteins and typically cleaved by signal peptidase at its C-region to generate the mature proteins. Miscleavage of the SP is reported occasionally, resulting in a truncated- or elongated-terminal sequence. In the present work, we demonstrated that cation exchange (CEX) chromatography is an effective means for removing SP variants with a case study. With the selected resin/conditions, the chromatographic performance is comparable between runs performed at the low end and high end of load density and elution range. The procedure described in this work can be used as a general approach for resin selection and optimization of chromatographic conditions to remove byproducts that bind more strongly than the product to the selected resin.

[Control status and follow-up of acute attacks in children with bronchial asthma with normal pulmonary ventilation function]. / è‚ºé€šæ°”åŠŸèƒ½æ­£å¸¸çš„å„¿ç«¥æ”¯æ°”ç®¡å“®å–˜æŽ§åˆ¶æƒ å†µåŠæ€¥æ€§å‘ä½œéšè®¿çš„ç ”ç©¶.

OBJECTIVES: To investigate the control status of bronchial asthma (referred to as "asthma") in school-age children with normal pulmonary ventilation function and the occurrence of acute attacks within 1 year of follow-up. METHODS: A retrospective analysis was conducted on clinical data of 327 children aged 6-14 years with bronchial asthma and normal pulmonary ventilation function from April to September 2021. Based on the measured value of one second rate (FEV1/FVC), the children were divided into the ≥80% group (267 cases) and the <80% group (60 cases). The pulmonary ventilation function, asthma control level, and occurrence of acute attacks within 1 year were compared between the two groups. RESULTS: The baseline pulmonary ventilation function in the <80% group was lower than that in the ≥80% group, and the proportion of small airway dysfunction was higher than that in the ≥80% group (P<0.05). After standardized treatment for 1 year, the small airway function indices in the <80% group improved but remained lower than those in the ≥80% group (P<0.05). The rate of incomplete asthma control at baseline was 34.6% (113/327), and the asthma control level in the <80% group was lower than that in the ≥80% group (P<0.05). After standardized treatment for 1 year, the asthma control level in the <80% group remained lower than that in the ≥80% group, and the proportion of acute asthma attacks was higher than that in the ≥80% group (P<0.05). CONCLUSIONS: Approximately one-third of school-age children with asthma still have incomplete asthma control when their pulmonary ventilation function is normal. Among them, children with measured FEV1/FVC<80% have an increased risk of acute asthma attacks and require close follow-up and strengthened asthma management.

Comprehensive Characterization of Androgen-Responsive lncRNAs Mediated Regulatory Network in Hormone-Related Cancers.

The AR signaling pathway plays an important role in initiation and progression of many hormone-related cancers including prostate, bladder, kidney, lung, and breast cancer. However, the potential roles of androgen-responsive long noncoding RNAs (lncRNAs) in hormone-related cancers remained unclear. In the present study, we identified 469 novel androgen-responsive lncRNAs using microarray data. After validating the accuracy of the array data, we constructed a transcriptional network which contained more than 30 transcriptional factors using ChIP-seq data to explore upstream regulators of androgen-responsive lncRNAs. Next, we conducted bioinformatics analysis to identify lncRNA-miRNA-mRNA regulatory network. To explore the potential roles of androgen-responsive lncRNAs in hormone-related cancers, we performed coexpression network and PPI network analyses using TCGA data. GO and KEGG analyses showed these lncRNAs were mainly involved in regulating signal transduction, transcription, development, cell adhesion, immune response, cell differentiation, and MAPK signaling pathway. We also highlight the prognostic value of HPN-AS1, TPTEP1, and LINC00623 in cancer outcomes. Our results suggest that androgen-responsive lncRNAs played important roles in regulating hormone-related cancer progression and could be novel molecular biomarkers.

Crosstalk Between AR and Wnt Signaling Promotes Castration-Resistant Prostate Cancer Growth.

INTRODUCTION: Prostate cancer (PCa) is the most commonly diagnosed cancer and the third leading cause of cancer-related death in males in the United States. Despite the initial efficacy of androgen deprivation therapy in prostate cancer (PCa) patients, most patients progress to castration-resistant prostate cancer. However, the mechanisms underlying the androgen-independent progression of PCa remain largely unknown. METHODS: In this study, we established a PCa cell line (LNCaP-AI) by maintaining LNCaP cells under androgen-depleted conditions. To explore the cellular and molecular mechanisms of androgen-independent growth of PCa, we analyzed the gene expression patterns in androgen-independent prostate cancer (AIPC) compared with that in androgen-dependent prostate cancer (ADPC). KEGG pathway analysis revealed that Wnt signaling pathways were activated after androgen deprivation therapy (ADT). In vitro experiments showed that the inhibition of Wnt pathway reduced AIPC cell growth by inhibiting cell cycle progression and promoting apoptosis. Furthermore, WNT5A, LEF1 were identified as direct targets of AR by chromatin immunoprecipitation (ChIP) assay and public ChIP-seq datasets analysis. RESULTS: In the present study, we found a regulatory mechanism through which crosstalk between androgen receptor (AR) and Wnt signals promoted androgen-independent conversion of PCa. The Wnt pathway was inhibited by androgen in androgen-dependent prostate cancer cells, but this blocking effect was not elicited in androgen-independent prostate cancer (AIPC) cells. Moreover, Wnt pathway genes WNT5A and LEF1 were directly downregulated by AR. In vitro experiments showed that inhibition of the Wnt pathways repressed AIPC cell growth by inhibiting cell cycle progression and promoting apoptosis. We found that WNT5A and LEF1 were downregulated in low-grade PCa but upregulated in metastatic PCa. CONCLUSION: In summary, we revealed that crosstalk between AR and Wnt signaling pathways promotes androgen-independent growth of PCa, which may provide novel therapeutic opportunities for castration-resistant prostate cancer.

Novel module and hub genes of distinctive breast cancer associated fibroblasts identified by weighted gene co-expression network analysis.

BACKGROUND: As abundant and heterogeneous stromal cells in tumor microenvironment, carcinoma-associated fibroblasts (CAFs) are critically involved in cancer progression. METHODS: To identify co-expression module and hub genes of distinctive breast CAFs, weighted gene co-expression network analysis (WGCNA) was conducted based on the expression array results of CAFs from seven chemo-sensitive breast cancer (BC) patients and seven chemo-resistant ones before neo-adjuvant chemotherapy. RESULTS: A total of 4916 genes were included in WGCNA, and 12 modules were determined. Module-trait assay showed that the blue module (cor = 0.97, P < 0.001) was associated with CAF-related chemo-resistance, which was enriched mainly as "inflammatory response", "interferon-gamma-mediated signaling" and "NIK/NF-kappaB signaling" pathways. Moreover, CXCL8, CXCL10, CXCL11, PLSCR1, RIPK2 and USP18 were found to be potentially associated with chemo-resistance related to CAFs and prognosis of BC. CONCLUSIONS: Our current data offered valuable insights into the molecular mechanisms of distinctive breast CAFs, which was beneficial for revealing how chemo-resistance of BC was initiated.

The enantioselective enrichment, metabolism, and toxicity of fenoxaprop-ethyl and its metabolites in zebrafish.

To investigate the impacts of the widely used chiral herbicide fenoxaprop-ethyl (FE) on aquatic organisms, the enrichment, metabolism, acute toxicity, and oxidative stress of fenoxaprop-ethyl and its main metabolites fenoxaprop (FA), ethyl-2-(4-hydroxyphenoxy)propanoate (EHPP), 2-(4-hydroxyphenoxy)propanoic acid (HPPA), and 6-chloro-2,3-dihydrobenzoxazol-2-one (CDHB) in zebrafish were studied. The enantioselectivity of fenoxaprop-ethyl and its chiral metabolites was also determined. Fenoxaprop-ethyl quickly degraded in zebrafish by aquatic exposure. FA, HPPA, and CDHB were the main metabolites that were enriched in the zebrafish. In the metabolism experiment, the half-lives of the metabolites were 0.92-1.72 days in zebrafish. The R-enantiomers of FA and HPPA were preferentially enriched and metabolized with enantiomeric fractions (EFs) of 0.65-0.85. According to the 96-h acute toxicity, FA, HPPA, EHPP, and CDHB were less toxic to zebrafish than FE, following the order of FE > CDHB > EHPP > FA > HPPA. The S-enantiomers of FE, FA, CDHB, and EHPP were more toxic than the R-enantiomers. FE and its metabolites could significantly increase catalase (CAT) and superoxide dismutase (SOD) activity and the malondialdehyde (MDA) content in gill and liver tissues, indicating their oxidative stress, and these effects were also enantioselective. This work could supply more information for evaluating the risks of fenoxaprop-ethyl on aquatic organisms concerning their metabolites and enantiomers.

Residue behavior of organochlorine pesticides during the production process of yogurt and cheese.

The presence of organochlorine pesticides (OCPs) in dairy products can lead to human exposure. This study investigated the behavior of OCP residues in milk during yogurt and cheese production. Gas chromatography with electron-capture detection (GC-ECD) was used to detect α-hexachlorocyclohexane (HCH), hexachlorobenzene (HCB), γ-HCH, g-chlordane, and α-chlordane in fresh milk, yogurt, and cheese. The results showed that fermentation reduced the residual concentration of OCPs in yogurt, with processing factors (PFs) ranging from 0.42 to 0.64. The reductions in residue levels during fermentation were due to the activity of the starter. The cheese making process increased the residual concentration of OCPs in cheese compared to raw milk, with PFs ranging from 2.37 to 4.93. Additionally, milk, yogurt, and cheese samples were purchased from local markets and OCP levels were analyzed. The target OCPs ranged from ND to 16.50 µg/kg in these samples.

Chiral quizalofop-ethyl and its metabolite quizalofop-acid in soils: Enantioselective degradation, enzymes interaction and toxicity to Eisenia foetida.

An enantioselective chromatographic method to analyze enantiomers of quizalofop-ethyl and its metabolite quizalofop-acid was established using a high-performance liquid chromatography (HPLC) on (R, R) Whelk-O 1 column. The enantioselective degradation kinetics of quizalofop-ethyl and quizalofop-acid in three soils were investigated. Moreover, the interaction with urease and catalase in the soils and the acute toxicity to Eisenia foetida of quizalofop-ethyl were also determined in order to assess their metabolism mechanism and environmental risk. From the results, quizalofop-ethyl was configurationally stable and was hydrolyzed rapidly to quizalofop-acid, which also degraded enantioselectively but slowly, and the inversion of the S-(-)-quizalofop-acid into the R-(+)-quizalofop-acid was observed in Xinxiang soil. In addition, quizalofop-ethyl and quizalofop-acid enantioselectively affected urease activity but not catalase. The acute toxicity assays to earthworm indicated that the racemic quizalofop-ethyl and quizalofop-acid were more toxic than quizalofop-p-ethyl and quizalofop-p-acid respectively, dramatically, the toxicity of the metabolite was much higher than the parent compound. These results revealed the enantioselective degradation of quizalofop-ethyl and quizalofop-acid, and the differences of toxicity among the enantiomers of the parent compound and the metabolite, which should be considered in future environmental risk evaluation.

Fate and stereoselective behavior of benalaxyl in a water-sediment microcosm.

The environmental behavior and stereoselectivity of the chiral fungicide benalaxyl and its chiral metabolite benalaxyl acid in water, sediment, and water-sediment microcosms were studied. The microcosms were incubated at 25 °C with light or under darkness. The influencing factors such as light and microorganism were investigated. The results showed that benalaxyl had half-lives of >21 days in the microcosm system and that the metabolite benalaxyl acid could exist in the microcosm for >70 days. Benalaxyl was mainly transformed through microbial degradation, and thus sediment microorganisms played a major role in the dissipation of benalaxyl in the aquatic microcosm. The stereoselective behavior of benalaxyl and benalaxyl acid was also investigated. (-)-Benalaxyl was preferentially degraded in the microcosm, resulting in an enrichment of the more toxic enantiomer (+)-benalaxyl, which may cause higher risk to the aquatic system. Moreover, (-)-benalaxyl acid was preferentially formed in the microcosm. The enantioselectivity of the enantiomers in the microcosm should be taken into consideration for an accurate risk assessment.

Iminosugar derivative WGN-26 suppresses acute allograft rejection via inhibiting the IFN-γ/p-STAT1/T-bet signaling pathway.

This study aimed to investigate the effect of the iminosugar derivative WGN-26 on suppressing acute allograft rejection and to explore the underlying mechanisms. The results demonstrated that WGN-26 (12, 6 and 3mg/kg) significantly prolonged the skin allograft survival time in a dose-dependent manner and minimized the pathological changes. The minimum lethal dose was 320 mg/kg. By exploring the potential cellular and molecular mechanisms, we found that WGN-26 dose-dependently inhibited T lymphocyte proliferation, as determined through the single mixed lymphocyte reaction (sMLR) or the ConA-induced T cell proliferation assay in allograft recipients. The FCM results indicated that WGN-26 particularly reduced the percentage of CD3(+)CD4(+) T cells in allograft recipients. After treatment with WGN-26, the secretion of IFN-γ in allograft recipients was lowered, whereas the IL-4 and IL-17 levels remained stable. Furthermore, we found that WGN-26 inhibited the phosphorylation of STAT1 and accelerated the degradation of T-bet protein in allograft recipients. This study provides the first report that the iminosugar derivative WGN-26 dose-dependently prolongs skin allograft survival and that the possible mechanism is mediated by inhibiting CD4(+) T cell proliferation and suppressing the IFN-γ/p-STAT1/T-bet signaling pathway.

Microbial side-chain cleavage of phytosterols by mycobacteria in vegetable oil/aqueous two-phase system.

Microbial side-chain cleavage of natural sterols to 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) by Mycobacteria has received much attention in pharmaceutical industry, while low yield of the reaction owing to the strong hydrophobicity of sterols is a tough problem to be solved urgently. Eight kinds of vegetable oils, i.e., sunflower, peanut, corn, olive, linseed, walnut, grape seed, and rice oil, were used to construct oil/aqueous biphasic systems in the biotransformation of phytosterols by Mycobacterium sp. MB 3683 cells. The results indicated that vegetable oils are suitable for phytosterol biotransformation. Specially, the yield of AD carried out in sunflower biphasic system (phase ratio of 1:9, oil to aqueous) was greatly increased to 84.8 % with 10 g/L feeding concentration after 120-h transformation at 30 °C and 200 rpm. Distribution coefficients of AD in different oil/aqueous systems were also determined. Because vegetable oils are of low cost and because of their eco-friendly characters, there is a great potential for the application of oil/aqueous two-phase systems in bacteria whole cell biocatalysis.

Magneto-induced stress enhancing effect in a colloidal suspension of paramagnetic and superparamagnetic particles dispersed in a ferrofluid medium.

The magneto-induced stress and relative microstructure in a colloidal suspension of paramagnetic and superparamagnetic particles dispersed in a ferrofluid medium is studied using particle-level dynamics simulation. It shows that the stress perpendicular to the direction of an external uniaxial magnetic field can be strongly enhanced by increasing the ratio of paramagnetic particles to approaching that of superparamagnetic particles. The magnetic field-induced net-like or embedded chain-like microstructures formed by paramagnetic and superparamagnetic particles contribute to this stress enhancing effect.

[Assessment on anti-nociception and anti-inflammation pharmacodynamics of Asarum heterotropoides var. mandshuricum and Asarum sieboldii].

OBJECTIVE: To assess the anti-nociception and anti-inflammation pharmacodynamics of Asarum heterotropoides var. mandshuricum and A. sieboldii. METHOD: Both the writhing test and hot plate test were conducted to assess the anti-nociceptive effect of Asarum and Xylene-induced mouse ear edema was conducted to assess the anti-inflammatory effect of Asarum. RESULT: Twelve samples of A. heterotropoides var. mandshuricum and A. sieboldii from different producing areas showed anti-nociceptive and anti-inflammatory effects. Specifically, 27% to 61% of the seven samples of A. heterotropoides var. mandshuricum showed anti-nociceptive effect and while 34% to 48% of A. sieboldi showed anti-nociceptive effect. The inflammatory inhibition rate of A. heterotropoides var. mandshuricum produced in six producing areas (38%-57%) is higher than that of A. heterotropoides var. mandshuricum produced in five producing areas (34%-48%). The same kind of Asarum produced in different areas showed significant differences. A. heterotropoides var. mandshuricum produced in Jilin province (38%-57%) showed better anti-nociceptive effect than sample produced in Heilongjiang province (34%) in writhing test. A. heterotropoides var. mandshuricum produced in Heilongjiang (43%) province showed a better anti-nociceptive effect than samples produced in Liaoning province (29%-36%) in hot plate test. A. sieboldii produced in Shaanxi province (47%-49%) showed a better anti-nociceptive effect than samples produced in Hubei province (40%) in writhing test. A. sieboldii produced in Shaanxi province (45%-59%) showed better anti-nociceptive effect than samples produced in Chongqing (40%) in hot plate test. A. heterotropoides var. mandshuricum produced in Jilin province (51%-63%) showed better anti-inflammatory effect than samples produced in Heilongjiang province (50%). In totality, the results from analysis of geoherbalism showed that famous-region A. heterotropoides var. mandshuricum and A. sieboldii had a better anti-nociception effect than Asarum produced in other producing areas, famous-region A. heterotropoides var. mandshuricum had a better effect than those produced in other producing areas in anti-inflammation. But famous-region A. sieboldii showed no obvious difference from those produced in other producing areas in anti-inflammation. CONCLUSION: All samples of Asarum showed anti-nociceptive and anti-inflammatory effects, but with significant differences among Asarum produced in different areas, indicating the eoherbalism to some extent.

Role of granulocyte colony-stimulating factor in paclitaxel-induced intestinal barrier breakdown and bacterial translocation in rats.

BACKGROUND: Chemotherapy causes breakdown of the intestinal barrier, which may lead to bacterial translocation. Paclitaxel, an anti-tubulin agent, has many side effects; however, its effect on the intestinal barrier is unknown. Previous studies show that granulocyte colony-stimulating factor (G-CSF) plays an important role in modulating intestinal barrier function, but these studies are not conclusive. Here, we investigated the effects of paclitaxel on the intestinal barrier, and whether G-CSF could prevent paclitaxel-induced bacterial translocation. METHODS: Twenty-four male Sprague-Dawley rats were divided into three groups: control group, paclitaxel group and paclitaxel + G-CSF group. Intestinal permeability was measured by the urinary excretion rates of lactulose and mannitol administered by gavage. The mesenteric lymph nodes, spleen and liver were aseptically harvested for bacterial culture.Endotoxin levels and white blood cell (WBC) counts were measured and bacterial quantification performed using relative real-time PCR. Jejunum samples were also obtained for histological observation. Intestinal apoptosis was evaluated using a fragmented DNA assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate(dUTP)-biotin nick end-labeling staining. One-way analysis of variance and Fisher's exact test were used to compare differences between groups. RESULTS: Paclitaxel induced apoptosis in 12.5% of jejunum villus cells, which was reduced to 3.8% by G-CSF treatment.Apoptosis in the control group was 0.6%. Paclitaxel treatment also resulted in villus atrophy, increased intestinal permeability and a reduction in the WBC count. G-CSF treatment resulted in increased villus height and returned WBC counts to normal levels. No bacterial translocation was detected in the control group, whereas 6/8, 8/8, and 8/8 rats in the paclitaxel group were culture-positive in the liver, spleen and mesenteric lymph nodes, respectively. Bacterial translocation was partially inhibited by G-CSF. CONCLUSIONS: Paclitaxel disrupts the intestinal barrier, resulting in bacterial translocation. G-CSF treatment protects the intestinal barrier, prevents bacterial translocation, and attenuates paclitaxel-induced intestinal side-effects.

The chiral separation of triazole pesticides enantiomers by amylose-tris (3,5-dimethylphenylcarbamate) chiral stationary phase.

The amylose-tris(3,5-dimethylphenylcarbamate) chiral stationary phase was synthesized and used to separate the enantiomers of triazole pesticides by high-performance liquid chromatography. The mobile phase was n-hexane-isopropanol applying a flow rate of 1.0 mL/min. Six triazole pesticides were enantioselectively separated. Myclobutanil, paclobutrazol, tebuconazole, and uniconazole obtained complete separation with the resolution factors of 5.73, 2.99, 1.72, and 2.07, respectively, and imazalil and diniconazole obtained partial separation with the resolution factors of 0.79 and 0.77 under the optimized conditions. The effect of the content of isopropanol as well as column temperature on the separation was investigated. A circular dichroism detector was used to identify the enantiomers and determine the elution orders. The results showed the low temperature was good for the chiral separation except for diniconazole. The thermodynamic parameters calculated based on linear Van't Hoff plots showed the chiral separations were controlled by enthalpy.

The chiral resolution of pesticides on amylose-tris(3,5-dimethylphenylcarbamate) CSP by HPLC and the enantiomeric identification by circular dichroism.

Amylose-tris(3,5-dimethylphenylcarbamate) (ADMPC) was synthesized and coated on gamma-aminopropylsilica to prepare a chiral stationary phase (CSP). The chiral resolutions of seven pesticide enantiomers including fenoxaprop-ethyl, quizalofop-ethyl, lactofen, metalaxyl, benalaxyl, hexythiazox and fluroxypyr-meptyl on the CSP by high-performance liquid chromatography were performed. Mobile phase was n-hexane and isopropanol with a flow rate of 1.0 ml/min. The influences of isopropanol content in the mobile phase and temperature on the resolutions were investigated. Under the optimized conditions the enantiomers could obtain complete resolutions except that metalaxyl got partial resolution. Decreasing the content of isopropanol increased the retention and the resolutions. Temperature was an important chromatographic parameter for optimization, and the results showed that low temperature was not always good to the resolutions. The enantiomers were identified by a circular dichroism (CD) detector which could provide the CD signals [(+) or (-)] and the CD spectra in the range of 220-420 nm by online scanning.