[The association between aflatoxin exposure and primary hepatocellular carcinoma risks: a case-control study in Chongqing].

Objective: To investigate the association between aflatoxin exposure and primary hepatocellular carcinoma (PHC) development. Methods: From December 2013 to May 2016, we selected 214 patients newly diagnosed with PHC as cases, and 214 patients as controls from three hospitals in Chongqing. Cases were confirmed with PHC diagnosis standard. And cases caused by clear reasons such as drug-induced liver injury, alcoholic liver damage, fatty liver and gallstones etiology, were excluded. Controls were included with no cancer and no digestive system disease, and recruited simultaneously with cases. Cases and controls were frequency-matched (1∶1) by same gender and age (±3 years). Peripheral blood and random urine samples were collected and analyzed for serum HBsAg status by biochemistry analyzer, and serum AFB(1)-ALB adduct and urinary AFB(1)-N(7)-GUA adduct by ELISA. Basic information, living habits and history of disease for patients were obtained by questionnaires. We used wilcoxon rank sum test to compare the median of serum AFB(1)-ALB adduct and urinary AFB(1)-N(7)-GUA adduct in cases and controls. Logistic regression analyses were performed to assess risk factors for PHC, and synergism index (S) of aflatoxin with other factors was estimated by the method of Andersson. Results: There was no significant difference in age between PHC cases (50.74±9.67) years and controls (51.15±9.90) years. Logistic regression showed that the odds ratio of HBV infection for PHC development was 46.3 (95% CI: 23.3-88.0). There was a significant difference in median concentrations of serum AFB(1)-ALB adduct (cases vs controls: 146.23 vs 74.42 ng/g albumin, P<0.001), but no difference in median concentrations of urinary AFB(1)-N(7)-GUA adduct was observed (cases vs controls: 0.17 vs 0.14 ng/mg creatinine, P<0.210). The odd ratios for PHC risk after adjustment were 1.9 (95%CI: 1.1-3.4) for AFB(1)-ALB adduct, and 2.1 (95%CI: 1.0-4.2) for AFB(1)-N(7)-GUA adduct. Moreover, we observed a positive interaction of aflatoxin exposure with HBV, alcohol drinking, and diabetes. The S was 4.7 (95%CI: 2.8-7.9), 3.5 (95%CI: 1.0-12.0), and 12.4 (95%CI: 1.8-84.2), respectively for serum AFB(1)-ALB adduct with each of the three factors mentioned, and was 1.9 (95%CI:1.1-3.1), 2.0 (95%CI: 1.1-3.6), and 2.0 (95%CI: 1.1-3.6), respectively for urinary AFB(1)-N(7)-GUA adduct with each of the three factors mentioned. Conclusion: HBV was still the main risk factor, and AFB(1) exposure was also an independent risk factor for PHC in Chongqing. There was a positive interaction of aflatoxin with HBV, alcohol drinking, and diabetes.

In-depth analysis of internal control genes for quantitative real-time PCR in Brassica oleracea var. botrytis.

Quantitative reverse-transcription PCR (qRT-PCR) is a versatile technique for the analysis of gene expression. The selection of stable reference genes is essential for the application of this technique. Cauliflower (Brassica oleracea L. var. botrytis) is a commonly consumed vegetable that is rich in vitamin, calcium, and iron. Thus far, to our knowledge, there have been no reports on the validation of suitable reference genes for the data normalization of qRT-PCR in cauliflower. In the present study, we analyzed 12 candidate housekeeping genes in cauliflower subjected to different abiotic stresses, hormone treatment conditions, and accessions. geNorm and NormFinder algorithms were used to assess the expression stability of these genes. ACT2 and TIP41 were selected as suitable reference genes across all experimental samples in this study. When different accessions were compared, ACT2 and UNK3 were found to be the most suitable reference genes. In the hormone and abiotic stress treatments, ACT2, TIP41, and UNK2 were the most stably expressed. Our study also provided guidelines for selecting the best reference genes under various experimental conditions.

Genome-wide identification, phylogenetic relationships, and expression analysis of the carotenoid cleavage oxygenase gene family in pepper.

Carotenoid cleavage oxygenases (CCOs) are a family of dioxygenases, which specifically catalyze the cleavage of conjugated double bonds in carotenoids and apocarotenoids in plants. In this study, genome-wide analysis of CCO genes in pepper plants was performed using bioinformatic methods. At least 11 members of the CCO gene family were identified in the pepper genome. Phylogenetic analysis showed that pepper and tomato CCO genes could be divided into two groups (CCDs and NCEDs). The CCD group included five sub-groups (CCD1, CCD4, CCD7, CCD8, and CCD-like). These results indicate that there is a close genetic relationship between the two species. Sequence analysis using the online tool, Multiple Expectation Maximization for Motif Elicitation (MEME), showed that the CCO proteins comprise multiple conserved motifs, with 20 to 41 amino acids. In addition, multiple cis-acting elements in the promoter of CCO genes were identified using the online tool PlantCARE, and were found to be involved in light responsiveness, plant hormone regulation, and biotic and abiotic stresses, suggesting potential roles of these proteins under different conditions. RNA-seq analysis revealed that the CCO genes exhibit distinct patterns of expression in the roots, stems, leaves, and fruit. These findings suggest that the CCO genes have important roles in the vegetative and reproductive development of pepper plants.

[Treatment status of tyrosine kinase inhibitor for newly-diagnosed chronic myeloid leukemia: a domestic multi-centre retrospective real-world study].

Objective: To retrospectively analyze the treatment status of tyrosine kinase inhibitors (TKI) in newly diagnosed patients with chronic myeloid leukemia (CML) in China. Methods: Data of chronic phase (CP) and accelerated phase (AP) CML patients diagnosed from January 2006 to December 2022 from 77 centers, ≥18 years old, and receiving initial imatinib, nilotinib, dasatinib or flumatinib-therapy within 6 months after diagnosis in China with complete data were retrospectively interrogated. The choice of initial TKI, current TKI medications, treatment switch and reasons, treatment responses and outcomes as well as the variables associated with them were analyzed. Results: 6 893 patients in CP (n=6 453, 93.6%) or AP (n=440, 6.4%) receiving initial imatinib (n=4 906, 71.2%), nilotinib (n=1 157, 16.8%), dasatinib (n=298, 4.3%) or flumatinib (n=532, 7.2%) -therapy. With the median follow-up of 43 (IQR 22-75) months, 1 581 (22.9%) patients switched TKI due to resistance (n=1 055, 15.3%), intolerance (n=248, 3.6%), pursuit of better efficacy (n=168, 2.4%), economic or other reasons (n=110, 1.6%). The frequency of switching TKI in AP patients was significantly-higher than that in CP patients (44.1% vs 21.5%, P<0.001), and more AP patients switched TKI due to resistance than CP patients (75.3% vs 66.1%, P=0.011). Multi-variable analyses showed that male, lower HGB concentration and ELTS intermediate/high-risk cohort were associated with lower cytogenetic and molecular responses rate and poor outcomes in CP patients; higher WBC count and initial the second-generation TKI treatment, the higher response rates; Ph(+) ACA at diagnosis, poor PFS. However, Sokal intermediate/high-risk cohort was only significantly-associated with lower CCyR and MMR rates and the poor PFS. Lower HGB concentration and larger spleen size were significantly-associated with the lower cytogenetic and molecular response rates in AP patients; initial the second-generation TKI treatment, the higher treatment response rates; lower PLT count, higher blasts and Ph(+) ACA, poorer TFS; Ph(+) ACA, poorer OS. Conclusion: At present, the vast majority of newly-diagnosed CML-CP or AP patients could benefit from TKI treatment in the long term with the good treatment responses and survival outcomes.

[Regulatory effects of the Nocardia rubra cell wall skeleton on the biological function of human neutrophils].

Objective: To investigate the regulatory effects and mechanism of Nocardia rubra cell wall skeleton (Nr-CWS) on the biological function of human neutrophils. Methods: The experimental research method was used. Fifteen healthy adult volunteers (7 males and 8 females, aged 24 to 45 years) were recruited from Suzhou Physical Examination Center for physical examination from May to October 2022, the peripheral venous blood was collected, and neutrophils were extracted by immunomagnetic bead sorting. The cells were divided into normal control group without any treatment, Nr-CWS alone group treated with Nr-CWS of final mass concentration 60 ng/mL alone, endotoxin/lipopolysaccharide (LPS) alone group stimulated with LPS of final mass concentration 1 µg/mL alone, and LPS+Nr-CWS group stimulated with LPS first and then treated with Nr-CWS as before. After 1 h of culture, the chemotaxis distance, chemotactic cell percentage, chemotactic index, maximum chemotactic speed, and chemotactic function score of neutrophils were detected using the modified agarose chemotactic model; the proportion and fluorescence intensity of phagocytosis cells, the level of reactive oxygen species (ROS), the protein expression levels of granular protein CD35, CD66b, and CD63, and the concentrations of inflammatory cytokines of interleukin 2 (IL-2), IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor alpha (TNF-α), and interferon-γ in cell culture supernatant were detected by flow cytometry. The number of samples in each group in the above experiments was 15. Data were statistically analyzed with analysis of variance for factorial design and independent sample t test. Results: After 1 h of culture, the chemotactic function score of cells in normal control group, Nr-CWS alone group, LPS alone group, and LPS+Nr-CWS group were 15.0, 14.5±0.5, 1.5±0.5, 12.0±1.5, respectively. Compared with those in normal control group, the chemotaxis distance, chemotactic cell percentage, chemotactic index, maximum chemotactic speed, and chemotactic function score of cells were significantly decreased in LPS alone group and LPS+Nr-CWS group (with t values of 18.36, 18.88, 54.28, 18.36, 46.77, 10.58, 14.74, 6.84, 10.58, and 4.24, respectively, P<0.05); compared with those in LPS alone group, the five chemotactic function indexes as above in LPS+Nr-CWS group were significantly increased (with t values of 11.47, 14.65, 11.62, 11.47, and 13.75, respectively, P<0.05). After 1 h of culture, compared with those in normal control group, the proportion and fluorescence intensity of phagocytosis cells were significantly increased in Nr-CWS alone group (with t values of 6.86 and 6.73, respectively, P<0.05), and the above two indexes were significantly decreased in LPS alone group (with t values of 7.35 and 22.72, respectively, P<0.05) and LPS+Nr-CWS group (with t values of 21.37 and 13.10, respectively, P<0.05). After 1 h of culture, compared with that in normal control group, the level of ROS of cells in LPS alone group was significantly increased (t=6.64, P<0.05); compared with that in LPS alone group, the level of ROS of cells in LPS+Nr-CWS group was significantly decreased (t=5.46, P<0.05). After 1 h of culture, compared with those in normal control group, the protein expressions of CD35, CD66b, and CD63 of cells were significantly increased in LPS alone group and LPS+Nr-CWS group (with t values of 16.75, 17.45, 10.82, 5.70, 19.35, and 15.37, respectively, P<0.05); compared with those in LPS alone group, the protein expressions of CD35, CD66b, and CD63 of cells were significantly decreased in LPS+Nr-CWS group (with t values of 4.92, 5.72, and 3.18, respectively, P<0.05). After 1 h of culture, compared with those in normal control group, the concentrations of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α, and interferon-γ in cell culture supernatant were significantly increased in LPS alone group (with t values of 22.10, 9.50, 7.21, 10.22, 24.88, 8.43, and 47.48, respectively, P<0.05), and the concentrations of IL-6, IL-10, IL-17A, TNF-α, and interferon-γ in cell culture supernatant were significantly increased in LPS+Nr-CWS group (with t values of 4.68, 5.12, 8.02, 5.58, and 7.13, respectively, P<0.05); compared with those in LPS alone group, the concentrations of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α, and interferon-γ in cell culture supernatant were significantly decreased in LPS+Nr-CWS group (with t values of 5.39, 2.83, 5.79, 2.90, 5.87, 4.88, and 39.64, respectively, P<0.05). Conclusions: Nr-CWS can enhance the phagocytosis ability of neutrophils in normal condition and improve the chemotactic function, ROS level, degranulation protein level, and inflammatory factor level of human neutrophils in infectious condition. Nr-CWS can enhance the anti-infection ability of human neutrophils by regulating its biological behavior in innate immunity.

[To compare the efficacy and incidence of severe hematological adverse events of flumatinib and imatinib in patients newly diagnosed with chronic phase chronic myeloid leukemia].

Objective: To analyze and compare therapy responses, outcomes, and incidence of severe hematologic adverse events of flumatinib and imatinib in patients newly diagnosed with chronic phase chronic myeloid leukemia (CML) . Methods: Data of patients with chronic phase CML diagnosed between January 2006 and November 2022 from 76 centers, aged ≥18 years, and received initial flumatinib or imatinib therapy within 6 months after diagnosis in China were retrospectively interrogated. Propensity score matching (PSM) analysis was performed to reduce the bias of the initial TKI selection, and the therapy responses and outcomes of patients receiving initial flumatinib or imatinib therapy were compared. Results: A total of 4 833 adult patients with CML receiving initial imatinib (n=4 380) or flumatinib (n=453) therapy were included in the study. In the imatinib cohort, the median follow-up time was 54 [interquartile range (IQR), 31-85] months, and the 7-year cumulative incidences of CCyR, MMR, MR(4), and MR(4.5) were 95.2%, 88.4%, 78.3%, and 63.0%, respectively. The 7-year FFS, PFS, and OS rates were 71.8%, 93.0%, and 96.9%, respectively. With the median follow-up of 18 (IQR, 13-25) months in the flumatinib cohort, the 2-year cumulative incidences of CCyR, MMR, MR(4), and MR(4.5) were 95.4%, 86.5%, 58.4%, and 46.6%, respectively. The 2-year FFS, PFS, and OS rates were 80.1%, 95.0%, and 99.5%, respectively. The PSM analysis indicated that patients receiving initial flumatinib therapy had significantly higher cumulative incidences of CCyR, MMR, MR(4), and MR(4.5) and higher probabilities of FFS than those receiving the initial imatinib therapy (all P<0.001), whereas the PFS (P=0.230) and OS (P=0.268) were comparable between the two cohorts. The incidence of severe hematologic adverse events (grade≥Ⅲ) was comparable in the two cohorts. Conclusion: Patients receiving initial flumatinib therapy had higher cumulative incidences of therapy responses and higher probability of FFS than those receiving initial imatinib therapy, whereas the incidence of severe hematologic adverse events was comparable between the two cohorts.

Characterization of MEK1 phosphorylation by the v-Mos protein.

Activation of MAP kinase/Erk Kinase (MEK) via direct phosphorylation by Mos may be crucial for cellular transformation by the activated c-mos or v-mos gene. Recent studies on a number of different protein kinases showed that phosphorylation within a subdomain of the catalytic domain may represent a common mode of activation. In this regard, activation of MEK1 by Raf involves phosphorylation of serine residues 218 and 222. Here we show that recombinant kinase-inactive MEK1 is phosphorylated by v-Mos with equal efficiency at both Ser 218 and Ser 222 in vitro. Tryptic phosphopeptide analysis of glutathione-S-transferase (GST)-MEK1 K97R and its alanine-for-serine mutants indicated that Ser 222 is the preferred phosphorylation site. Wild-type GST-MEK1 was phosphorylated at the same sites but contained a significantly lower amount of doubly phosphorylated species then its K97R kinase-inactive mutant. The ratio of GST-MEK1 species phosphorylated at two serines to those phosphorylated at one serine was similar in auto-phosphorylated and v-Mos-phosphorylated GST-MEK1. Consistent with the in vitro data, phosphopeptide mapping of MEK1 immunoprecipitated from mos transformed cells showed an increased amount of singly phosphorylated phosphopeptide compared to nontransformed cels. MEK1 was found to be more highly activated in NIH3T3 cells transformed by an activated c-mos or v-mos gene than in cells growing normally in medium containing serum. Our data indicate that Mos activated MEK1 in vitro as well as in vivo by phosphorylating Ser 222.

A new expression vector for high level protein production, one step purification and direct isotopic labeling of calmodulin-binding peptide fusion proteins.

Calmodulin-binding peptide (CBP), a peptide of 26 amino acids derived from muscle myosin light chain kinase (MLCK), binds to calmodulin with nanomolar affinity. Proteins fused in frame with CBP can be purified from crude E. coli lysates in a single step using calmodulin affinity chromatography (Stofko-Hahn et al., 1992). Because the binding between CBP and calmodulin is calcium-dependent, the fusion protein can be eluted from the resin with virtually any buffer containing EGTA (2 mM) and used directly for many applications. To take full advantage of this affinity purification system, we constructed the versatile CBP fusion protein expression vector pCAL-n. The CBP coding sequence was positioned for fusion at the N-terminus, an advantage that ensures consistent high level synthesis of fusion proteins due to the efficient translation of the CBP in E. coli. The production of fusion proteins from pCAL-n is controlled by the tightly regulated T7(lac)O promoter. A versatile multiple cloning site (MCS) was included to facilitate the cloning of genes of interest. The protein coding sequence for the enzyme c-Jun N-terminal kinase (JNK) was inserted into the MCS of pCAL-n, and the resulting fusion protein CBP-JNK synthesized in E. coli cells at 15-20 mg/1 culture. CBP-JNK was purified to near homogeneity in one step with calmodulin affinity resin. Purified CBP-JNK is fully active, and the CBP peptide tag can be removed by cleavage with thrombin. We also show that CBP can be efficiently phosphorylated by cAMP-dependent protein kinase. Hence, the purified fusion proteins can be labeled directly with [gamma-32P]ATP and used to probe protein-protein or protein-nucleic acid interactions.

Stable luciferase reporter cell lines for signal transduction pathway readout using GAL4 fusion transactivators.

While GAL4 fusion activators have been widely used for dissecting signal transduction pathways in transient assays, there has been surprisingly little reported on utilizing cell lines with stably integrated fusion activators. To avoid problems with the efficiency and reproducibility inherent to transient transfection, we describe here the generation and characterization of HeLa reporter cell lines, which contain a stably integrated luciferase gene responsive to stably integrated and constitutively expressed GAL4-CREB or GAL4-Elk1 fusion activators. These cell lines exhibited extremely low basal luciferase expression but robust response to various extracellular stimuli or the expression of signaling molecules that resulted in elevated MAP kinase or PKA activities. This integrated two-component reporter system allows one to focus specifically on particular signaling pathway endpoints and the altered transactivation activity of either Elk1 or CREB. With the procedures described here, many novel cell-based assays can be developed by generating new reporter cell lines with medically important but difficult-to-transfect cell types, and by using different reporter genes or different fusion transactivator genes.

Site directed mutagenesis to probe for active site components of liver mitochondrial aldehyde dehydrogenase.

Mutational analysis allowed us to rule out an essential role for the histidine residues and for serine 74 in mammalian aldehyde dehydrogenase. The later though, was found to be important in coenzyme interaction. The function of the serine could not be replaced by threonine or by cysteine. The absolute requirement for cysteine 302 and for glutamate 268 was verified using mutational analysis. The fact that these two residues are completed conserved among all aldehyde dehydrogenases is consistent with their being essential in the catalytic process.

Studies on application of a lead acetate solution as filter to the photochemical reaction of ergosterol to improve the yield of vitamin D.

Since a lead acetate solution can remove most of the ultraviolet (UV) light in the range below 275 nm which usually gives undesirable by-products in the photochemical conversion of ergosterol to vitamin D, it is useful as a filter solution for the reaction to obtain higher yield of vitamin D. When a 5% lead acetate solution was used as the filter, the yield of vitamin D was 20-25% higher than that without using filter solution.