Some studies have found that PPI had a certain antitumor effect in the proliferation and apoptosis of tumor cells. But the effects of environmental pH on the growth of GC cells and its mechanism are unknown. Therefore, we hoped to find the effects of culture medium pH on the biological behavior of GC cells by in vitro experiments and provide guidance for the use of acid-suppressing drugs in GC patients.  Proton pump inhibitor (PPI) and other acid-suppressing drugs are widely used in the treatment of gastrointestinal ulcer, upper gastrointestinal bleeding, gastritis, and gastric cancer (GC). About 80% of GC patients receive acid suppression treatment. PPI suppresses the production of gastric acid by inhibiting the function of H We aimed to observe the effects of pH changes in GC cell culture medium on the cell biological behavior of cancer cells and to analyze the potential mechanisms. We hoped to find out the effect of acid suppression on the growth of GC cells. α) at pH 6, pH 7, and pH 8.  The GC cell lines (SGC-7901 and MKN45) were used as the research object. We adjusted the pH value in the cell culture medium to observe the changes in cell viability (MTT), apoptosis (flow cytometry), and invasion (Transwell) at pH 6, pH 7, and pH 8. qRT-PCR and western blot (WB) assays were used to determine the expression changes of genes and proteins . Flow cytometry results showed that the apoptosis of SGC-7901 and MKN45 in the pH 8.0 group was more obvious than that in the pH 6.0 or pH 7.0 group (P < 0.001). Flow cytometry results showed that the apoptosis of SGC-7901 and MKN45 in the pH 8.0 group was more obvious than that in the pH 6.0 or pH 7.0 group (P < 0.001). Flow cytometry results showed that the apoptosis of SGC-7901 and MKN45 in the pH 8.0 group was more obvious than that in the pH 6.0 or pH 7.0 group (α) at pH 6, pH 7, and pH 8. P < 0.001). Flow cytometry results showed that the apoptosis of SGC-7901 and MKN45 in the pH 8.0 group was more obvious than that in the pH 6.0 or pH 7.0 group ( The results of MTT showed that the viability of SGC-7901 and MKN45 in the pH 8.0 group was significantly weaker than that in the pH 6.0 or pH 7.0 group (α) but promoted the apoptosis of GC cells and thus inhibited the growth of GC.α) at pH 6, pH 7, and pH 8.  Compared with the microacid environment, the microalkaline environment inhibited the viability, invasion, and expression of genes and proteins (mTOR, AKT, Wnt, Glut, and HIF-1 In Chinpurposes . Some repurposes –5, whilepurposes , 7. Unde2.Human GC cell lines SGC-7901 and MKN45 were provided by the Laboratory of Department of Gastroenterology, the First Affiliated Hospital of Nanjing Medical University . The cells were cultured in RPMI-1640 medium with 10% FBS and penicillin-streptomycin at 37°C in an atmosphere containing 5% COIn each of the following experiments, the pH of the GC cell culture medium was adjusted by sodium bicarbonate, and the pH value of the culture solution was monitored by a pH meter. The experiments were divided into three groups in which the pH was maintained at pH 6.0, pH 7.0, and pH 8.0, respectively, and pH 7.0 was set in the control group.β-Actin was purchased from ImmunoWay (USA). Goat anti-mouse IgG/HRP and goat anti-rabbit IgG/HRP were purchased from Jackson (USA). Antibodies against Glut1 , AKT , mTOR , HIF-1α , and β-actin  were purchased from Cell Signaling Technology , and antibody for wnt3a  was purchased from Abcam .RPMI-1640 was purchased from GIBCO (USA). Penicillin-streptomycin and 0.25% Trypsin-EDTA were purchased from Jiangsu Kaiji Biotechnology Co., Ltd. . Calf serum was purchased from Hangzhou Sijiqing Biological Engineering Materials . Fetal bovine serum was purchased from ExCell Biology (USA). TRIzol was purchased from Invitrogen (USA). WB wash buffer, WB blocking buffer, WB primary antibody diluent, WB second antibody dilution, and WB stripping buffer were purchased from Nanjing Lufei Biotechnology Co., Ltd. . 5 cells per well. The plates were incubated at 37°C in an atmosphere containing 5% CO2 for 24 h. The drug was diluted to the desired concentration with complete medium. Then, 100 μL drug-containing medium was added to each well. A negative control group and a positive control group were set up. The plates were incubated at 37°C in an atmosphere containing 5% CO2 for 72 h. Next, 20 μL MTT solution was added. 4 h later, 150 μL DMSO solution was added and the optical density was 490 nm as shown by a microplate reader.MTT assay was used to evaluate GC cell viability according to the manufacturer's instructions. GC cells were placed in 96-well plates at a density of 3~5 × 10To detect the apoptosis of the GC cells, the cultured SGC-7901, and MKN45 in the logarithmic growth phase were digested with 0.25% trypsin. GC cells were washed with PBS and stained with Annexin V together with propidium iodide for 15 min at room temperature in the dark as recommended by the manufacturer. Then, the apoptosis of GC cells was analyzed by a flow cytometer.5 cells/mL. Then, 100 μL cell suspension was added to the Transwell chamber, whereas the lower chamber was filled with 500 μL of medium supplemented with 20% fetal bovine serum (FBS). The cells were then incubated at 37°C in an atmosphere containing 5% CO2 and 1% O2. The cells in the upper chamber were wiped with a cotton swab and stained with 0.1% crystal violet for 30 min. Finally, cell numbers were counted under a microscope at a magnification of ×200.Transwell invasion assay was used to determine the invasion ability of the cells according to the manufacturer's instructions. The assays on cell invasion ability were performed in 24-well Transwell chambers, which were coated with Matrigel. SGC-7901 and MKN45 were first treated with 0.25% trypsin and then hung in incomplete medium for 24 h. The cell density was adjusted to 5 × 10α primer (142 bp), sense primer: 5-TGCTGATTTGTGAACCCATT-3, antisense primer: 5-TCTGGCTCATATCCCATCAA-3). Relative gene expression levels were detected and calculated using the 2ΔΔCt- comparative method.Total RNA was extracted from GC cells using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Subsequently, they were reversely transcribed to obtain cDNA using the First Strand cDNA Synthesis Kit. qRT-PCR was conducted using the SYBR Green qPCR Master Mix based on the manufacturer's protocol. Primers were designed and synthesized by Nanjing Jinsrui Technology Co., Ltd.  (human-GAPDH primer (132 bp): sense primer: 5-TGGTATCGTGGAAGGACTCA-3, antisense primer: 5-CCAGTAGAGGCAGGGATGAT-3; human-mTOR primer (130 bp), sense primer: 5-ATTCCGACCTTCTGCCTTCA-3, antisense primer: 5-CACAGCCACAGAAAGTAGCC-3; human-AKT primer (136 bp), sense primer: 5-GCGTGACCATGAACGAGTTT-3, antisense primer: 5-TTGGCCACGATGACTTCCTT-3; human-Wnt primer (144 bp), sense primer: 5-TTCTCCCAGTCTCTGTCGTG-3, antisense primer: 5-CATCCAAACTCGTGGCTCTG-3; human-Glut primer (126 bp), sense primer: 5-GGGCATGTGCTTCCAGTATG-3, antisense primer: 5-AAGGTCCGGCCTTTAGTCTC-3; and human-HIF-1α proteins. Protein extraction in each group was performed according to the protein extraction steps. The protein concentration was estimated using the BCA Protein Assay Kit. Subsequently, protein was electrophoresed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to PVDF membranes. Blots were blocked with 5% skim milk for 1 h and incubated overnight with the primary antibody at 4°C. The next day, the membranes were incubated with the corresponding IgG–HRP secondary antibody (1 : 5000) for 1-2 h at room temperature. Finally, the exposure was developed using a developing mixture, and the signals were normalized using β-actin control.WB was used to detect the expression of mTOR, AKT, Wnt, Glut, and HIF-1P < 0.05 was considered statistically significant, and ns meant P > 0.05, ∗ meant P < 0.05, ∗∗ meant P < 0.01, and ∗∗∗ meant P < 0.001.The data were presented as mean + standard deviation (SD). SPSS 22.0 software  was used for statistical analyses. Significant differences between groups were assessed using GraphPad Prism 5 software . The groups were compared using one-way analysis of variance (ANOVA), and the LSD method was used to determine the differences between groups. P > 0.05). Compared with the pH 6.0 or pH 7.0 group, the cell viability rate of the pH 8.0 group decreased (P < 0.01). There were significant differences in cell viability between pH 6.0 vs. pH 7.0, pH 7.0 vs. pH 8.0, and pH 6.0 vs. pH 8.0 (P < 0.01) after 24 h or 48 h of culturing. We also found that the differences in cell viability of GC cells between pH 6.0 vs. pH 7.0, pH 7.0 vs. pH 8.0, and pH 6.0 vs. pH 8.0 were the most obvious after being cultured for 48 h (P < 0.001)  .P < 0.01). Compared with pH 7.0, the apoptosis rate of GC cells increased significantly at pH 8.0 (P < 0.001). Compared with pH 6.0, the apoptosis rate of GC cells increased significantly at pH 8.0 (P < 0.001). The results showed that with the increase of pH, the apoptotic rate of GC cells increased gradually, and the alkaline environment promoted apoptosis and inhibited tumor progression (Flow cytometry was used to detect the apoptosis of GC cells (SGC-7901 and MKN45). The results showed that after treatment with different pH media for 48 h, GC cells showed different degrees of apoptosis, which was mainly early apoptosis (LR). Compared with the pH 6.0 group, the apoptosis rate of GC cells increased at pH 7.0 (gression .P > 0.05); compared with pH 7.0, the number of GC cells passing through Matrigel at pH 8.0 was significantly different (P < 0.001). Compared with pH 6.0, the number of GC cells passing through Matrigel at pH 8.0 was significantly different (P < 0.001). This indicated that with the increase of pH, the invasion ability of GC cells decreased, and the alkaline environment inhibited the invasion of GC cells and tumor progression (The invasion ability of tumor cells was closely related to tumor metastasis. Transwell invasion assay results showed that no significant differences in the number of GC cells passing through Matrigel (mature gel) at pH 7.0 compared with pH 6.0  in GC cells (SGC-7901 and MKN45) was detected at the gene level at pH 6.0, pH 7.0, and pH 8.0, respectively. qRT-PCR results showed no significant differences between the expression of genes  in SGC-7901 at pH 7.0 and that at pH 6.0 (P > 0.05). The expression of mTOR, AKT, HIF-1α, and Wnt in SGC-7901 at pH 8.0 was not different from that at pH 7.0 (P > 0.05), but the expression of Glut was different (P < 0.05). The expression of mTOR, AKT, HIF-1α, Wnt, and Glut in MKN45 was different at pH 7.0 from that at pH 6.0 (P < 0.05). The expression of mTOR, AKT, Wnt, and Glut in MKN45 at pH 8.0 was not different from that at pH 7.0 (P > 0.05); however, the expression of HIF-1α was different (P < 0.05). In GC cells (SGC-7901 and MKN45), the expression of mTOR, AKT, HIF-1α, Wnt, and Glut at pH 8.0 was different from that at pH 6.0 (P < 0.05). This indicated that with the increase of pH, the expression of genes  in GC cells (SGC-7901 and MKN45) decreased, and the alkaline environment inhibited the gene expression in GC cells , but the Wnt protein was significantly different (P < 0.001). The expression of mTOR, HIF-1α, and Glut proteins in SGC-7901 at pH 8.0 was not different from that at pH 7.0 (P > 0.05); however, the AKT and Wnt proteins were different (P < 0.05). The expression of mTOR, AKT, HIF-1α, Wnt, and Glut proteins in SGC-7901 at pH 8.0 was different from that at pH 6.0 (P < 0.05). The expression of HIF-1α and Glut proteins in MKN45 at pH 7.0 was different from that at pH 6.0 (P < 0.05), but the expression of mTOR, AKT, and Wnt proteins was not different (P > 0.05). The expression of HIF-1α and Glut proteins in MKN45 at pH 8.0 was significantly different from that at pH 7.0 (P < 0.01), but the expression of mTOR, AKT, and Wnt proteins was not different (P > 0.05). The expression of mTOR, HIF-1α, Wnt, and Glut proteins in MKN45 at pH 8.0 was significantly different from that at pH 6.0 (P < 0.01), but the expression of AKT was not different (P > 0.05). With an increase of pH, the expression of proteins  in GC cells (SGC-7901 and MKN45) decreased, but the AKT protein was not different in MKN45 at pH 6.0 vs. pH 8.0 (P > 0.05)  .+ out of the cell by the proton pump, forming an acid-base gradient, that is, the extracellular pH is acidic and the intracellular pH is alkaline [+-H+ exchanger 1 (NHE-1) increases, and the excessive H+ in the cells is eliminated by Na+-H+ exchange. The expression of anion exchanger-2 (AE-2) reduces, and the Cl−/HCO3− exchange is inhibited, which eliminates excessive HCO3− in the cells and thereby promotes the formation of pH gradient [A weak acid environment, which is common in solid tumor tissues, enhances the viability and invasion of tumor cells. The acidic microenvironment may be related to the “Warburg” effect of tumor cells, that is, under aerobic or anoxic conditions, the cells initiate glycolysis to convert glucose into lactic acid, a process in which energy is produced . Then, talkaline . In addigradient , 12. Howgradient –15.P < 0.001) (P < 0.001)  . Flow cy< 0.001) . Xu et aP < 0.001)  . The resn et al.  found thα at the gene and protein levels in SGC-7901 and MKN45 at different pH by qRT-PCR and western blot, respectively, and tried to analyze the possible mechanism underlying the effect of environmental pH on GC cells. We found that at the gene expression level, the expressions of mTOR, AKT, HIF-1α, Wnt, and Glut at pH 8.0 were lower than that at pH 6.0. At the protein expression level, the AKT protein was not different in MKN45 at pH 6.0 vs. pH 8.0 (P > 0.05), and the expression trends of other proteins were consistent with the trend of gene expression. This might be a result bias caused by a small sample size. At pH 8.0, the protein expression of mTOR, Wnt, Glut, and HIF-1α was significantly inhibited (P < 0.05)  . Therefoα (HIF-1α) and increases its local accumulation. HIF-1α is a key transcriptional regulator that mediates the adaptation of cells to hypoxic microenvironment and plays an important role in tumor cell energy metabolism, growth invasion, and angiogenesis [α induces angiogenesis and glycolysis by activating the expression of vascular endothelial growth factor (VEGF), glucose transporter (Glut), and glycolysis-related enzymes to control the transfer of oxygen and nutrients and improve the growth and proliferation of cancer cells. The HIF-1α-Glut pathway plays an important role in tumor progression [The adaptation of tumor cells to hypoxia and changes in glucose metabolism are the biological characteristics of tumor cells. Through the “Warburg” effect, tumor cells produce large amounts of lactic acid to form a weak acid environment, which is also a hallmark of hypoxic tissues and many solid tumors. Hypoxia in tumor tissues inhibits the degradation of hypoxia-inducible factor-1ogenesis –27. HIF-gression .α is dependent on the phosphatidylinositol 3-kinase (PI3K)/AKT pathway [α may be related to Wnt/β-catenin signaling pathway. In gastrointestinal tumors, the Wnt signaling pathway is regulated by intracellular β-catenin level. A high level of β-catenin activates the Wnt pathway, which affects cell proliferation, differentiation, and microenvironmental adaptation [α competes with Tcf-4 for binding to β-catenin and activates a typical Wnt/β-catenin signaling pathway to adapt to hypoxia [α might also regulate the invasiveness of cancer cells by altering the expression of intermediate filament , extracellular matrix component (fibronectin), and protease (MMP2 and urokinase plasminogen activator receptor). The HIF-1α pathway plays an important role in tumor progression, but the specific mechanism of HIF-1α promoting tumor cell proliferation is not yet fully understood.The stability of HIF-1 pathway , which raptation . HIF-1α  hypoxia . In addi hypoxia  suggesteα, thereby promoting the expression of VEGF and inducing angiogenesis. The mTOR signal of some growth factors regulates mTOR activity through the PI3K/Akt pathway [α. In the next experimental study, we will further study the exact mechanism pathway to confirm the conjecture.mTOR is a central regulatory kinase that induces the expression of related proteins in cell proliferation and metabolism. mTOR also promotes the expression of HIF-1 pathway . Another pathway . Recent  pathway . We specα genes and proteins, promoted tumor cell apoptosis, and inhibited GC progression. Hopefully, these findings can provide guidance for the use of acid-suppressing drugs in GC patients, but the specific mechanism of pH affecting GC cells needs further study. There were still some defects in our study, for example, our data were obtained in vitro SGC-7901 and MKN45 cells, and an in vivo animal model were missing. In the future, we will do some research on nude mice transplanted tumor model and analyze the effects of different feeding solution pH value and PPI on gastric cancer transplanted tumor.This study investigated the effects of different pH on GC cells. The results showed that alkaline microenvironment inhibited the viability and invasion of GC cells and the expression of mTOR, AKT, Wnt, Glut, and HIF-1


Fusarium sheath rot (CFSR), diseased samples were collected from 21 county-level regions in 12 prefecture-level districts of Sichuan Province from 2015 to 2018 in the present study. In the field, two symptom types appeared including small black spots with a linear distribution and wet blotches with a tawny or brown color. One hundred thirty-seven Fusarium isolates were identified based on morphological characteristics and phylogenetic analysis (EF1-α), and Koch’s postulates were also assessed. The results identified the isolates as 8 species in the Fusarium genus, including F. verticillioides, F. proliferatum, F. fujikuroi, F. asiaticum, F. equiseti, F. meridionale, F. graminearum and F. oxysporum, with isolation frequencies of 30.00, 22.67, 15.33, 7.33, 6.00, 5.33, 3.33 and 1.33%, respectively. Fusariumverticillioides and F. proliferatum were the dominant and subdominant species, respectively. Two or more Fusarium species such as F. verticillioides and F. proliferatum were simultaneously identified at a mixed infection rate of 14.67% in the present study. The pathogenicity test results showed that F. proliferatum and F. fujikuroi exhibited the highest virulence, with average disease indices of 30.28 ± 2.87 and 28.06 ± 1.96, followed by F. equiseti and F. verticillioides, with disease indices of 21.48 ± 2.14 and 16.21 ± 1.84, respectively. Fusarium asiaticum, F. graminearum and F. meridonale showed lower virulence, with disease indices of 13.80 ± 2.07, 11.57 ± 2.40 and 13.89 ± 2.49, respectively. Finally, F. orysporum presented the lowest virulence in CFSR, with a disease index of 10.14 ± 1.20. To the best of our knowledge, this is the first report of F. fujikuroi, F. meridionale and F. asiaticum as CFSR pathogens in China.To elucidate the symptoms and pathogens diversity of corn  Zea mays L.) is one of the largest food staples worldwide and is one of the most economically important crops in China1. Maize yields as high as 260.77 billion kilograms have been attained in China, greatly contributing to ensuring food safety and increasing farmer income4. In China, the maize-sowing area reached approximately 44.96 million hm2 in 2015, after which it decreased to 42.39 million hm2 in 20175. Chinese maize production of 259 million tons has been reported, accounting for 39% of Chinese cereal crop production and 22.8% of the global maize output6. However, several diseases caused by fungi, bacteria and viruses are a major factor limiting maize production. Among these diseases, Fusarium spp. can cause ear rot, stalk rot, seedling blight and root rot7.Maize (Fusarium sheath rot (CFSR) is one of the most serious crop diseases in China9. The results of previous studies suggest that Fusarium proliferatum can not only affect maize production but also produces the toxin fumonisins, posing great risks to human and livestock10. F. graminearum, F. verticillioides and F. equiseti have also been identified as causal agents of CFSR11. In fields, these pathogens primarily infect the sheath from the late growth period to the grain-forming stage11. Initial symptoms appear as irregular circular brown necrotic spots, after which the entire sheath gradually appears water-soaked and finally dies13. A severely infected sheath can eventually reduce lodging resistance and yield. According to statistical data, lodging results in major economic losses of approximately 15–25% and can even result in total crop failure14. A positive correlation has been demonstrated between yield loss and disease severity15. Additionally, wounds caused by aphid feeding can exacerbate the sheath rot severity of maize16.Corn 17. However, the disease has not been reported in Sichuan Province, a major maize-producing region in China. Therefore, the goal of our present study was to characterize the disease severity, symptoms and Fusarium spp. pathogens of CFSR, which will provide an important basis for effective integrated control of this disease.Studies have shown that CFSR has occurred in more than 12 provinces in ChinaIn the present study, we investigated the occurrence of CFSR from 2015 to 2018. CFSR is characterized by two primary types of disease symptoms in the field , F. fujikuroi species complex (FFSC), F. incarnatum-equiseti species complex (FIESC), F. oxysporum and F. verticillioides in the databases of NCBI (http://www.ncbi.nlm.nih.gov) and the FUSARIUM-ID (http://isolate.fusariumdb.org/guide.php).One hundred thirty-seven dia Fig. . The feaEF-1α gene sequence was amplified, generating a 700-bp band. For further phylogenetic analysis, a neighbor-joining tree based on the EF-1α gene was constructed, which included 137 Fusarium isolates, 17 reference isolates and 1 outgroup isolate of Bipolaris oryzae   and wet blotches with a tawny color (caused by F. orysporum) were observed after inoculation, while the control plants showed no significant symptoms , with average disease indices of 30.28 ± 2.87 and 28.06 ± 1.96 , including F. verticillioides, F. proliferatum, F. fujikuroi, F. asiaticum, F. equiseti, F. meridionale, F. graminearum and F. oxysporum, with observed isolation frequencies of 30.00, 22.67, 15.33, 7.33, 6.00, 5.33, 3.33 and 1.33%, respectively. Many studies have shown that Fusarium species are consistently isolated and identified in mixed infections with other Fusarium species or fungi on many crops in the field22. In the present study, F. verticillioides and F. proliferatum were consistently observed in mixed infections in CFSR on maize, with a mixed infection rate of 14.67%.Previous studies demonstrated that a complex of five Fusariumproliferatum is a ubiquitous, polyphagous, highly adaptable fungal pathogen of different plant species that attacks plants both in the field and during postharvest storage, causing blights, rots, and wilts on maize, garlic, soybean, tomato and Aloe vera26. Interestingly, several studies have shown that F. proliferatum is also the predominant pathogen of some commercial crops, such as Polygonatum cyrtonema, date palm and Cymbidium29. F. proliferatum was observed as the dominant fungus in infected garlic bulbs, with a high disease incidence of 35.40%, and it was confirmed as the causal agent of dry rot in garlic postharvest30. F. proliferatum was also highly pathogenic, and significant symptoms were also observed 2 weeks after being inoculated on onion31. On soybean, F. proliferatum easily infected seeds, with an observed disease severity index of 43.33–49.16%32. In our present study, F. proliferatum exhibited the highest virulence among the evaluated species, with a disease index of 30.28 in four different maize varieties, which is consistent with results of previous studies16. Additionally, F. proliferatum was also widely distributed, with an isolation frequency of 24.82%. F. equiseti, F. verticillioides and F. graminearum were successively reported as pathogens in CFSR33. Interestingly, F. fujikuroi, F. meridionale and F. asiaticum species were recorded as causing CFSR for the first time in China in the present study  samples, sometimes even 100%38. In addition, the frequency of F. graminearum isolated from ears ranged from 30 to 71% with an average of 57%, and from stalks ranged from 43 to 81%, with the average of 65%39. Although the isolation frequency of F. graminearum from CFSR was only 3.65% in our present study, the potential risk of mycotoxins produced by F. graminearum to human health cannot be ignored40.Several studies have shown that Fusarium meridonale is a member of the FGSC that is well-known to cause FHB in wheat and barley worldwide41. In addition, F. meridionale was also recently reported as pathogen in root rot on soybean under monoculture and ear rot on maize43. Moreover, F. asiaticum has been reported as a major and dominant causal agent of FHB on wheat and barley in China46 and was also detected as a pathogen causing Gibberella ear rot of maize and seedborne diseases of soybean48. Although F. meridonale and F. asiaticum were not predominant species in the present study, with low isolation frequencies, they were reported in China for the first time. On the other hand, F. meridonale and F. asiaticum also exhibited typical pathogenicity, with disease indices of 13.89 and 13.80, respectively.Fusarium were recovered from maize fields in Sichuan Province, China. Interestingly, three species, F. fujikuroi, F. meridionale and F. asiaticum, were reported to cause CFSR in China for the first time. All isolates could infect the maize sheath and had disease indices ranging from 10.14 to 30.28. F. proliferatum and F. fujikuroi were the primary pathogens of CFSR, exhibiting high isolation frequencies and disease indices. The results of the present study provides the theoretical basis for integrated control of CFSR in Sichuan Province of China.In summary, in the present study, eight species of 49. Four fields that were at least 1 km apart were contained in each area, and five sites were evaluated in each field, with 100 ear leaf sheaths sampled per site. In addition, 150 diseased maize sheaths with dark brown spots or bronzing and tawny blotch were collected in valve bags and stored in a large ice box before cultural isolation.Twenty-one county-level regions of 12 municipal administrations were surveyed for sampling between 2015 and 2018 in Sichuan, China. The disease percentage and severity of CFSR under natural conditions were investigated as described by Huang et al.51. Maize sheaths were cut into 0.5 cm × 0.5 cm pieces at the junction between disease and healthy tissue prior to isolation. The sheaths were surface-sterilized in 1% sodium hypochlorite for 1 min, rinsed extensively with sterilized water, soaked in 75% ethanol for 30 s, and then rinsed extensively with sterilized water again. Subsequently, all sample pieces were placed on potato dextrose agar  plates supplemented with 0.2 g chloramphenicol for fungal isolation for 3 days at approximately 25 °C under 12-h lighting.The pathogens were isolated from maize sheaths by tissue and single spore isolation−1 carboxymethyl cellulose sodium, 0.5 g·L−1 yeast extract, 2.5 g·L−1 K2HPO4, and 0.25 g·L−1 MgSO4·7H2O)52. Then, the inoculated cultures were incubated for 5 days in a shaking incubator at 27 °C, 120 rpm. The culture characteristics and conidia of Fusarium species were morphologically assessed under a light microscope . One hundred thirty-seven isolates from 6 groups were identified as Fusarium spp.54.Additionally, all strains were transferred to liquid carboxymethyl cellulose medium  with the default setting for DNA assays.All 137 obtained isolates were sub-cultured on PDA for 10 days at 25 °C with 12-h lighting. Approximately 20 mg of mycelia of each isolate was then scraped from the PDA plates with a sterilized ladle, and mycelia were ground to a powder in liquid nitrogen with a mortar. DNA was extracted using the cetyl-trimethylammonium bromide (CTAB) methodrDNA-ITS56 was performed with PCR primers ITS1 and ITS4 using the amplification conditions described by Schoch et al.57. Amplification of the Fusarium translation elongation factor 1α (EF1-α) gene was performed with the primer pair EF1 and EF2 using the amplification conditions described by O'Donnell et al.58. Molecular identification of Fusarium species was confirmed by PCR amplification using the primers ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (GCTGCGTTCTTCATCGATGC) for the partial rDNA-ITS gene and the primers EF1-728F (CATCGAGAAGTTCGAGAAGG) and EF4-986R (TACTTGAAGGAACCCTTACC) for the partial EF1-α gene. PCR amplification was performed in a final volume of 25 μL containing 12.5 μL of 2 × PCR Master Mix , 0.5 μM of each primer and 10 ng of genomic DNA. The thermocycling conditions used for PCR amplification were as follows: 4 min at 94 °C followed by 35 cycles of 45 s at 94 °C, 60 s at 53 °C (for EF1-α) or 58 °C (for rDNA-ITS), and 1 min at 72 °C, with a final extension at 72 °C for 10 min. PCR products were detected by 1.5% agarose gel electrophoresis and then sequenced with an ABI-PRISM3730 automatic sequencer  by Sangon Biotech Co., Ltd. .PCR amplification of rDNA-ITS region was first performed using BLAST at the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov). Then, the EF-1α sequences from Fusarium species were compared to those in the NCBI database using the DNA BLAST program and the FUSARIUM-ID database (http://isolate.fusariumdb.org/guide.php). The sequences were aligned using the software ClustalX 259. Phylogenetic analyses were performed using MEGA5 software package with the default parameters60. The alignments were manually edited to delete trimmed regions and discard incomplete sequences. Phylogenetic trees for each genomic region and their tandem sequences were constructed using the neighbor-joining (NJ) approach61 with 1000 bootstrap repeats and the pairwise deletion option62. Bipolaris oryzae was used as an outgroup (GenBank accession no. KJ 939510).Sequence analysis of the F. oxysporum with only 2 isolates) were assessed for pathogenicity on maize during the flowering period in 2016–2017. Subsequently, they were cultured on PDA medium at 25 °C under 12 h of ambient room lighting per day for 5 days. Thereafter, five mycelial plugs of each strain (5 mm in diameter) were transferred to 100 mL of CMC liquid medium and cultured for 3 days in a shaker incubator . The conidial suspensions were then filtered and adjusted to a final concentration of 1 × 105 spores/mL. Four maize cultivars  were sown in field plots, with each field plot containing 23 × 4 lines and 40 plants per line. Then, a 2-mL aliquot of a conidial suspension for each strain was injected into the first sheath above the ear leaf in 20 plants of each maize cultivar, and 20 control plants treated following the same procedure but were inoculated with sterile water63. Disease symptoms were assessed 25 days after inoculation, and disease severity was scored based on the average severity in 30 plants as described by Huang et al.49. Then, the disease index was calculated based on the average for each species. To confirm Koch’s postulates, isolation from maize sheaths infected with one representative isolate of each Fusarium species was attempted. Subsequently, symptomatic maize sheath tissue was sectioned, and 0.5-cm pieces were placed on PDA for reisolation under the same culture conditions.Twenty-three isolates from eight identified species  (version 22.0 for Windows). Analysis of variance was performed using the general linear model, and means were compared using Duncan’s New Multiple Range test with SPSS, with differences considered significant at P ≤ 0.05.Supplementary Table 1.Supplementary Table 2.


Macadamia integrifolia remains unclear. Macadamia plants were grown hydroponically with seven P levels to characterize biomass allocation, cluster-root development, and exudation of carboxylates and acid phosphatases. Plant biomass increased with increasing P supply, peaking at 5 μM P, was the same at 5–25 μM P, and declined at 50–100 μM P. Leaf P concentration increased with increasing P supply, but shoot biomass was positively correlated with leaf P concentration up to 0.7–0.8 mg P g–1 dry weight (DW), and declined with further increasing leaf P concentration. The number of cluster roots declined with increasing P supply, with a critical value of leaf P concentration at 0.7–0.8 mg P g–1 DW. We found a similar trend for carboxylate release, with a critical value of leaf P concentration at 0.5 mg g–1 DW, but the activity of acid phosphatases showed a gradually-decreasing trend with increasing P supply. Our results suggest that leaf P concentration regulates the development and functioning of cluster roots, with a critical P concentration of 0.5–0.8 mg g–1, above which macadamia growth is inhibited.Phosphorus (P) deficiency induces cluster-root formation and carboxylate exudation in most Proteaceae. However, how external P supply regulates these root traits in  Phosphorus (P) is an essential nutrient for all life on the earth . PhosphoLupinus albus , which belongs to Proteaceae, is native to Australia ; Ca(NO3)2 (2000 μM); K2SO4 (700 μM); Fe-EDTA (20 μM); H3BO3 (10 μM); MnSO4 (0.5 μM); ZnSO4 (0.5 μM); CuSO4 (0.2 μM); (NH4)6Mo7O24 (0.01 μM). The pot size was 7.8 L, and the initial pH of the nutrient solution was measured with a pH-sensitive microelectrode and adjusted to 5.8 using NaOH or HCl. There were four replicates in each treatment.The experiment was conducted in a greenhouse at China Agricultural University, Beijing . Seeds of macadamia were collected in Yunnan Province, Southern China. Seed P concentration was 1.9 ± 0.6 mg P g3 and 2 mL 30% (v/v) H2O2 during the digestion process.Plants were harvested after 6 months of growth. Plants were divided into roots, stems, young leaves, and old leaves for harvesting. The number of cluster roots was measured in every treatment. All plant samples were oven-dried at 70°C for 72 h to determine DW. Dried material was ground to a powder with a stainless-steel grinder to determine P concentration. Plant powders were digested using the microwave-accelerated reaction system . We used 6 mL HNO2 (200), KCl (100), CaCl2 (600), and H3BO3 (5) and it was adjusted to the same pH as that of the nutrient solution with NaOH or HCl (5.8). After the collection of exudates, two drops of microbial inhibitor Micropur  at 0.01 g L–1 and two drops of concentrated H3PO4 were added to inhibit microbial degradation of root exudates : MgClexudates .Root exudates were stored at −20°C until the analysis of carboxylates by HPLC. Before analysis, root exudation samples were passed through sterile Millex GS Millipore 0.22-μm filters. The analysis method was the same as described by Cluster roots at different developmental stages were differentiated as shown in Section “Results” and taken from the plants grown in the 0 μM P treatment.p-nitrophenyl phosphate (NPP) was added and 0.5 mL deionized water. Centrifuge tubes were placed in a 30°C water bath for 15 min, and then 0.5 mL 0.5 M NaOH was added to terminate the reaction and develop the color. The absorbance of the resulting color was determined spectrophotometrically  at 405 nm.Before plants were harvested, another 0.5 g root sample was taken to measure acid phosphatase (APase). Root samples were washed in deionized water four times. The APase activity on the root surface was analyzed according to In this experiment, we changed the nutrient solution every 7 days, and the solution pH was monitored every day using a pH-sensitive microelectrode . We chose cluster-root segments to measure the rhizosphere pH of different development stages using an agar method with pH indicator. 0.75% w/v agar and 0.006% (w/v) bromocresol purple were mixed, and the pH was adjusted to 6.0. The agar was heated to boiling temperature and then cooled to 40°C.Cluster roots were washed and placed in a clean Petri dish, and then liquid agar was poured into the Petri dish. After 10 min, the color along the roots changed, yellow representing acidification and purple alkalization, respectively.post hoc means comparisons were done by SAS statistical software . Data were analyzed by least squares fitting method and determined as non-linear regression functions in SigmaPlot 10.0 (United States). P < 0.05 was considered significant.One-way ANOVA was performed, and when appropriate, the Plants produced more shoot biomass than root biomass in all treatments. Both shoot and root biomass increased with increasing P supply, with no further change from 5 to 25 μM P, and then growth was significantly inhibited at 50 and 100 μM P . The gre–1 DW) and roots (from 0.36 to 2.9 mg P g–1 DW) increased with increasing P supply , and for malate and citrate, the rates were 2.1 and 3.4 μmol g–1 FW h–1, respectively  and the buffering capacity of the solution (3– as nitrogen source; uptake of NO3– results in the alkalinization of the rhizosphere (We observed an increased pH in nutrient solution compared with the original pH, mainly due to uptake of NOsolution . In thiszosphere . We alsozosphere . Moreove–1 DW.In conclusion, our results suggest that leaf P concentration in macadamia regulates the development of cluster roots and exudation of carboxylates, which is affected by external P supply, and that the plants produce most shoot biomass and show the greatest cluster-root formation and functioning at a critical leaf P concentration of 0.5–0.8 mg gThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.XZ, HL, and JS designed the study. XZ and YL performed the experiments and collected the data. XZ analyzed the data. XZ, YL, KJ, HL, and JS interpreted the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The in vitro hepatic metabolism of curcumin diethyl disuccinate (CDD) in LS9 of rats, dogs, monkeys, and humans was evaluated. The identity of CDD metabolites and the metabolism kinetic parameters, including degradation rate constant, in vitro/in vivo intrinsic clearance, and half-life, were determined. CDD was rapidly metabolized into monoethylsuccinyl curcumin and curcumin in LS9 of all tested species mainly by carboxylesterases (CESs), including CES1 and CES2, and butyrylcholinesterase. The in vitro intrinsic clearance of CDD was in the order of human > dog > monkey > rat, whereas that of monoethylsuccinyl curcumin in the order of dog > monkey > human > rat; this parameter was not correlated with their respective in vivo clearance, which followed the order of dog > monkey > rat > human. Therefore, in vitro drug metabolism data inferred from LS9 of nonhuman origin, especially from monkeys and dogs, cannot be used as preclinical data for human trials, as humans have a smaller liver-to-body weight ratio than monkeys, dogs, and rats. The in vivo drug metabolism is dictated by the anatomical factors of the test subject.Liver S9 (LS9) is a nearly complete collection of all hepatic drug-metabolizing enzymes. It is a low-cost model for predicting drug metabolic activity. This study aimed to identify the suitability of using LS9 of different animal sources in drug metabolism profiling with respect to the possible translation of the  Curcumin  is a natin vitro and in vivo antitumor  at 4°C for 10 min. For metabolite identification, 120 µl of the supernatant was diluted with 40 µL of water before analysis. The metabolites and CDD in the supernatant were identified against CDD, MSCUR, and curcumin standards using liquid chromatography–quantitative time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS) in a Thermo Dionex UltiMate 3000 RSLCnano system equipped with an LPG Micro Pump  and MicrOTOF-Q II . Using this system, the accurate masses for both parent and fragment ions were evaluated to obtain the elemental formula of each compound.Metabolite identification of CDD was performed with HLS9, MLS9, DLS9, and RLS9. A stock solution of CDD was prepared at a concentration of 0.5 mM in 50% acetonitrile in water. Each LS9 in phosphate buffer pH 7.4 prepared at a protein concentration of 1 mg/ml was preincubated at 37°C for 5 min. Next, 49 µl of preincubated LS9 was spiked with 1 µl of CDD stock solution to obtain a test sample of 10 µM CDD, which was subsequently incubated at 37°C for 0.5 min. The metabolism reaction of each sample was terminated by adding 100 µl of ice-cold acetonitrile, and the sample was then vortexed and centrifuged at 14,000 rpm , which was maintained at 35°C. The isocratic mobile phase was composed of solvents (A) acetonitrile and (B) water with 0.2% (v/v) formic acid delivered at a flow rate of 120 μl/min with an injection volume of 15 µl. The positive electrospray ionization (ESI+) source parameters were set as follows: nebulization nitrogen gas pressure, 2.5 bar; capillary voltage, −4.5 kV; dry nitrogen flow rate, 8 L/min; dry nitrogen temperature, 220°C; and end plate offset voltage, −500 V. MS/MS spectra were acquired at a collision energy of 20 eV and over a range of m/z 50–1,000. System control, data acquisition, and data processing were performed using Bruker Compass Data Analysis 4.0 software, and Bruker SmartFormula software was used to determine the molecular formula of the analytes.The LC-QTOF-MS/MS conditions were set as previously reported . Brieflyg) at 4°C for 10 min. For the analysis of the metabolism profile, 120 µl of the supernatant was diluted with 40 µl of water prior to UHPLC  analysis. The sample at the initial time point (0 min) was prepared as the other samples, except that heat-inactivated HLS9, MLS9, DLS9, and RLS9  were used.The metabolism profiles of CDD were investigated against HLS9, MLS9, DLS9, and RLS9. A stock solution of CDD was prepared at 150 µM in 50% acetonitrile in water before use. The metabolism study was initiated by spiking 735 µl of preincubated LS9 with 15 µl of CDD stock solution, and the mixture was subsequently incubated at 37°C. The final concentrations of CDD and LS9 were 3 μM and 1 mg/ml, respectively. A 50-µl aliquot from the mixture was collected at 0.5, 1, 2, 3, 4, 5, 10, 15, 30, 45, 60, 90, and 120 min and immediately mixed with 100 µl of ice-cold acetonitrile containing 0.45 µM of internal standard DMC to terminate the metabolic reaction. The mixture was vortex-mixed and centrifuged at 14,000 rpm  before use. The mobile phase flow rate was kept at 1.2 ml/min in a gradient mode using the following elution program: 0–3.0 min, initial A-B ; 4.5–7.0 min, isocratic elution A-B . CDD, curcumin, and MSCUR in the supernatant were partitioned using a C8 column  with reference to DMC. The detection wavelength was set at 400 nm (λmax(CDD) = 400 nm, λmax(curcumin) = 427 nm, λmax(MSCUR) = 418 nm, and λmax(DMC) = 421 nm). The column was maintained at 35°C throughout the run, and the supernatant injection volume was set at 20 µl.The UHPLC system consisted of an Agilent series 1290 quaternary pump with an online degasser, autosampler, column oven, and diode array detector. The mobile phase was composed of (A) 0.1% (v/v) formic acid in acetonitrile and (B) 0.1% (v/v) formic acid in water. Each solution was filtered through a nylon filter  values greater than 0.99.The concentrations of CDD, curcumin, and MSCUR in the supernatant were determined from the standard plots, which were prepared by spiking individual working solutions of the compounds to heat-inactivated HLS9, MLS9, DLS9, and RLS9 . These wThe percent content of CDD and the metabolites formed upon its incubation with LS9, namely, curcumin and MSCUR, was plotted against incubation time. Analysis of metabolite profiles suggested that the metabolism of CDD adopted a two-step, consecutive, first-order irreversible reaction, a basic model for prodrug metabolism , as show0 is the CDD concentration at time 0, k1 is the degradation rate constant of CDD, and k2 is the degradation rate constant of MSCUR. k1 and k2 were determined by using 3, respectively, with nonlinear regression analysis using the SOLVER function of Microsoft Excel for Office 365 MSO (16.0.12527.20986) (t1/2) was calculated using The following equations were adopted to describe the changes in CDD and MSCUR content over time (t):7.20986) . The halin vitro intrinsic clearance  of CDD and MSCUR in LS9 was calculated using V]incubation is the incubation volume in ml, and [P]incubation is the amount of LS9.The in vivo intrinsic clearance  was modeled from CLin vitroint, values using physiologically based scaling factors, as described by in vivo intrinsic clearance  was the sum of CLin vivo(CDD)int, and CLin vivo(MSCUR).int,The 21 mg/g) . The liv21 mg/g) . The totH = hepatic blood flow of 20, 44, 40, and 70 ml min−1 kg−1 in humans, monkeys, dogs, and rats, respectively (p (fraction of unbound CDD in plasma), estimated at 0.0594, was derived from p into a pseudo-equilibrium constant. CDD is a neutral compound with a log P of 2.55  at 4°C for 10 min. For hepatic esterase identification, 120 µl of the supernatant was diluted with 40 µl of water prior to the UHPLC analysis of CDD and curcumin. The inhibitor-free control (incubation of CDD in the absence of inhibitors), enzyme-free control (incubation of CDD in the absence of LS9), and organic solvent-free control (incubation of CDD in the absence of organic solvent in the inhibitor solution) were similarly evaluated.The hepatic esterases involved in the hydrolytic cleavage of CDD in the respective LS9 samples were identified by the chemical inhibition approach with nine esterase inhibitors, namely, BNPP, digitonin, loperamide, PMSF, BW284c51, iso-OMPA, PCMB, DTNB, and EDTA. Each esterase inhibitor (5 µl) at a final concentration of 100 μM, except for EDTA at a concentration of 1 mM, was incubated individually with 44 µl of each LS9  for 30 min at 37°C prior to the addition of 1 µl CDD (150 µM) to obtain a final concentration of 3 µM. The mixture was then incubated at 0.5 min for MLS9 and DLS9 or at 1 min for HLS9 and RLS9. The metabolism reaction was terminated by adding 100 µl of ice-cold acetonitrile. The mixture was then vortexed and centrifuged at 14,000 rpm  .+] of m/z 369.1327, 369.1331, 369.1336, and 369.1332 with reference to HLS9, MLS9, DLS9, and RLS9  . In the Identification of Curcumin Diethyl Disuccinate Metabolites” ]; t1/2 of MSCUR [t1/2(MSCUR)]; CLint,in vitro of CDD ; CLint,in vitro of MSCUR ; CLint,in vivo of CDD ; CLint,in vivo of MSCUR ; and CLint,in vivo total were determined, and the results are summarized in k2 values were 10- to 20-fold higher than k1 values, indicating that k1 was the rate-determining parameter. Both CDD and MSCUR were rapidly metabolized by all LS9s, with t1/2 of less than 1.04 and 7.19 × 10–2 min, respectively. The short half-lives of CDD and MSCUR observed in this study were consistent with those of other ester-containing compounds that underwent enzymatic hydrolysis, such as vicagrel (t1/2 of approximately 1.6 min) (t1/2 of approximately 4.0 min) (The metabolism kinetic parameters including the 1.6 min)  and GS-64.0 min) .int,in vitro(CDD) and CLint,in vitro(MSCUR)  values obtained from different LS9 were in the following order: human > dog > monkey > rat. The CLint,in vitro(CDD) in HLS9 was higher than that in DLS9, MLS9, and RLS9 by 1.8-, 1.9-, and 2.2-fold, respectively. In the case of MSCUR, its CLint,in vitro(MSCUR) values followed the order of dog > monkey > human > rat. Unlike that of CDD, the CLint,in vitro(MSCUR) values of DLS9 and MLS9 were approximately two- and 1.7-fold higher than that of HLS9. The CLint,in vitro(MSCUR) value of RLS9 was 0.8-fold lower than that of HLS9. Bioinformatic analysis suggested that the amino acid sequences and structural homology of metabolic enzymes are dependent on the animal species (Interspecies differences were observed in CLo(MSCUR) . The CLi species . This ma species .int,in vivo(CDD) and CLint,in vivo(MSCUR) were predicted using a physiologically based scaling factor , was in the following order: DLS9 > MLS9 > RLS9 > HLS9. Apparently, the in vitro hepatic clearances of CDD and MSCUR in DLS9, MLS9, and RLS9 were parallel to the predicted in vivo total clearance profiles. HLS9, on the other hand, was correlated with a relatively small in vivo total clearance, despite its relatively high in vitro CDD clearance, and had higher in vitro MSCUR clearance than RLS9. This could be due to the fact that the ratio of liver mass to body mass of humans is lower than that of rats. Using the well-stirred model, the order of CLH values in HLS9, MLS9, DLS9, and RLS9 did not correspond to the CLint,in vivototal in any specific manner. Larger hepatic blood flow was translated to higher CLH level. Hepatic blood flow might have governed the hepatic metabolism of CDD. The interplay between hepatic blood flow and enzymes requires further study.CLg factor . The preR2 > 0.98)  was used to ensure that the reaction was independent of the substrate concentration, and the formation of curcumin over the incubation time was dependent on the level of active enzymes. The kinetics of CDD depletion in different LS9s are summarized in  > 0.98) . The app > 0.98) , and the > 0.98) . In addi > 0.98) . To meetEsterase inhibitors, including BNPP, digitonin, loperamide, PMSF, BW284c51, iso-OMPA, PCMB, DTNB, and EDTA are known to inhibit CESs , CES1 S, CES2 C, serine −1 mg−1 protein, respectively, with a relative curcumin formation rate of 100%. The organic solvent did not exhibit any effect on the degradation profiles of CDD into curcumin. A relative curcumin formation rate of 100% was attained in the organic solvent–free control. These results indicated that CDD was degraded via enzymatic hydrolysis in LS9 of all test species.In the absence of inhibitors, the rates of curcumin formation in HLS9, MLS9, DLS9, and RLS9 were 10.77 ± 0.82, 14.60 ± 1.51, 26.79 ± 2.43, and 12.71 ± 0.25 nmol minIn humans, CDD hydrolysis in HLS9 was inhibited by BNPP, digitonin, loperamide, and PCMB, and to a lesser extent by PMSF, iso-OMPA, and DTNB. The esterases of HLS9 responsible for CDD hydrolysis were primarily CESs (including CES1 and CES2) and CMBL, and partially BChE. AChE and PON did not play a critical role in the hydrolysis of CDD by HLS9. CESs and CMBL play an essential role in the hydrolysis of prodrugs in the liver, as CESs are primal enzymes expressed in the hepatic microsome, whereas CMBL is highly expressed in the hepatic cytosol of human and preclinical species . The invCDD hydrolysis in MLS9 was inhibited by BNPP, and to a lesser extent by digitonin, loperamide, PMSF, and iso-OMPA. BW284c51, PCMB, DTNB, and EDTA showed no enzyme inhibitory effects. The esterases of MLS9 responsible for the hydrolysis of CDD were CESs (including CES1 and CES2) and BChE. In the case of DLS9, the relative contribution of esterases in the hydrolysis of CDD was as follows: CES1 and CES2 > BChE > CMBL. With reference to RLS9, the relative contribution of esterases in the hydrolysis of CDD was in the following order: CES1 and CES2, ≈CMBL > BChE > AChE.CDD can be hydrolyzed by CESs (including CES1 and CES2), AChE, BChE, and CMBL. These enzymes are expressed in the liver of human and preclinical species, and they belong to the α/β hydrolase fold family, which may have broad and overlapping substrate specificities . CESs, AApart from the liver, the gut is another site that plays an important role in the hydrolysis of orally administered ester-based drugs . CESs arIn summary, this study provided comparative data on the hepatic metabolism of CDD in LS9 from rats, dogs, monkeys, and humans. The experiments showed that CDD was rapidly metabolized into MSCUR and curcumin in LS9 from all tested species mainly by CESs, including CES1 and CES2, and BChE. Nevertheless, the hepatic clearances were different between species, which was probably caused by the differences in the expression level and amino acid sequences of esterases as a function of the species origin. Additional studies including gut metabolism, phase I and II metabolisms of CDD metabolites, and the extent of protein binding should be further investigated to map the metabolism profiles of CDD.in vitro hepatic metabolism of LS9, a nearly complete collection of all hepatic drug-metabolizing enzymes from different animal species. CDD was rapidly metabolized into MSCUR and curcumin in HLS9, MLS9, DLS9, and RLS9 mainly by CESs (CES1 and CES2) and butyrylcholinesterase. The in vitro intrinsic clearance of CDD was in the following order: human > dog > monkey > rat LS9. The in vitro intrinsic clearance of MSCUR followed the order of dog > monkey > human > rat LS9. The in vitro intrinsic clearance was not correlated with the in vivo clearance of CDD and MSCUR, with the order of dog > monkey > rat > human LS9. In vitro drug metabolism data inferred from LS9 of nonhuman origin, especially of monkey and dog origins, cannot be used as preclinical data for human trials. Humans are characterized by smaller liver-to-body weight ratio than that of monkeys, dogs, and rats. Smaller liver-to-body weight ratio translates to lower in vivo CDD and MSCUR intrinsic clearance. An in vitro LS9 metabolism study does not consider the anatomical factors of the test subject and is not reflective of the in vivo metabolic outcome.In this study, we investigated the No additional dataset is available for this study. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.in vitro metabolism study, analyzed the data, and wrote the manuscript. PRB synthesized the compounds and wrote the manuscript. WS conducted the LC-QTOF-MS/MS analysis. GQ, SY, JW, and TWW reviewed and edited the manuscript. PR designed the research and reviewed and edited the manuscript.PJ designed the research, carried out the This research was supported by a scholarship from the Graduate School of Chulalongkorn University, the 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship, the 90th Anniversary Chulalongkorn University Fund , and the Chulalongkorn Academic Advancement into its Second Century (CUAASC) Project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this era of precision medicine, prognostic heterogeneity is an important feature of patients with non‐small cell lung cancer (NSCLC) with brain metastases (BM). This multi‐institutional study is aimed to verify the applicability of the adjusted Lung‐molGPA model for NSCLC with BM in a Chinese cohort.This retrospective study included 1903 patients at three hospitals in Southwest China. The performance of the Lung‐molGPA model was compared with that of the adjusted DS‐GPA model in terms of estimating the survival of NSCLC with BM.p < 0.001). The adjusted Lung‐molGPA model was more accurate in predicting survival of adenocarcinoma patients than the adjusted DS‐GPA model (C‐index: 0.615 vs 0.571), and it was not suitable for predicting survival of non‐adenocarcinoma patients .The median OS of this patient cohort was 27.0 months, and the adenocarcinoma survived longer than the non‐adenocarcinoma (28.0 months vs 18.7 months, The adjusted Lung‐molGPA model is better than the DS‐GPA model in predicting the prognosis of adenocarcinoma patients. However, it failed to estimate the prognosis for non‐adenocarcinoma patients. In the era of precision medicine, as rising healthcare costs of cancers, it has become essential for the society as a whole to predict the prognosis of NSCLC with BM, whose survival outcomes present heterogeneity even though new treatments strategies such as SRS, molecular target and immune therapy are being developed. Based on the Lung‐molGPA model, we generated an adjusted Lung‐molGPA model. It is aimed to verify the applicability of the adjusted Lung‐molGPA model for NSCLC with BM in a Chinese cohort that a retrospective study included 1903 patients at three hospitals in Southwest China. To our knowledge, this is the first multi‐center study about the applicability of adjusted Lung‐molGPA and DS‐GPA models in a Chinese cohort that the adjusted Lung‐molGPA model is better than the DS‐GPA model in predicting the prognosis of adenocarcinoma. Considering the medical records were obtained from previous clinical diagnosis and treatment, and exemption from informed consent would not adversely affect the patients’ rights and health, the requirement for informed consent waived upon approval of the ethics committee.We created a multi‐institutional retrospective database, including 1903 patients with primary NSCLC and newly diagnosed BM between 1 January 2008 and 31 May 2018 at Sichuan University West China Hospital, Zunyi Medical University Affiliated Hospital, and Guizhou Provincial People's Hospital. Those patients were excluded, including: (1) with multiple malignant tumors, small cell lung cancer (SCLC), neuroendocrine lung cancer, lung sarcoma, and mixed lung cancer; (2) with meningeal metastasis (MM) and BM along with MM were excluded; (3) only received best supportive care. Histological subtype was ascertained according the lung tumor classification criteria of the World Health Organization (WHO). This present study only included in patients with complete clinicopathological data, containing patient age, sex, smoking status, KPS, BMI, T and N stage, ECM, ECM organ numbers, BM numbers, histological subtype, and gene mutation status.Till last follow‐up on 1 August 2019, 452 patients were still surviving, and 143 ones could not be traced; follow‐up loss rate was 7.5%. Among the 1903 cases, the follow‐up information regarding 317 patients was obtained using medical records or telephone calls, and for 1586 patients, information was collected using the resident identity information system of Huichuan District Public Security Sub‐bureau of Zunyi city, Guizhou province.2.2This study evaluated the correlation between prognostic factors and OS using univariate and multivariate analyses, and stratified the cases by referring to the criteria of the adjusted DS‐GPA and Lung‐molGPA models  and the R software (version 3.3.1).OS, as the primary end point, measured from the time of first diagnosis of BM‐by imaging‐till death by any cause or till end of last the follow‐up. Correlation between prognostic factors and survival analyzed by log‐rank (Mantel‐Cox) test for univariate analyses and Cox regression model for the multivariate analyses. Hazard ratios (HRs) and their 95% confidence intervals (CIs) were reported. Stratified analysis was performed using the log‐rank (Mantel‐Cox) test. The model performance was assessed by Harrell's concordance index (C‐index). 33.1There were 1903 cases with age between 20 and 84 years, and a median age of 57 years. In this cohort, 965 patients (50.7%) were above 57 years, 1126 individuals were men (59.2%), 924 individuals (48.6%) had a smoking history, and 228 individuals (12.0%) had not even quit smoking till the time of BM diagnosis. Of the total 1903 patients, 1085 individuals (57.0%) had KPS ≥90, and only 179 cases (9.4%) were less than 70. This study included 127 patients (6.7%) with malnutrition and 538 individuals (28.3%) with overweight. The vast majority cases were adenocarcinoma , and most patients were T and N stage in advanced. There were 1393 patients (73.2%) with ECM, and 986 cases (51.8%) with 1‐2 ECM organ numbers. There were 1182 individuals (62.1%) exhibited 1‐4 brain lesions and 815 patients (42.8%) presented with EGFR mutation or anaplastic lymphoma kinase (ALK) fusion. (See Table p < 0.001). Univariate analysis revealed that female, KPS ≥90, BMI ≥24.0, adenocarcinoma, EGFR or ALK positive, significantly decreased the death risk of patients, but ECM , BM numbers ≥4, N2~N3, and smoking  increased it. Multivariate analysis revealed that smoking, BMI, KPS, ECM organ numbers, BM numbers, histological type, N‐staging, and gene alteration were independent prognostic factors for NSCLC with BM. Interestingly, age and T‐staging were correlated with survival in neither univariate nor multivariate analysis in the current study . Cases with scores of 0‐1.0, 1.5‐2.0, and 2.5‐3.0 had median OS of 21.2, 29.9, and 40.3 months, severally. Adjacent classes stratification analysis had statistical difference (p = 0.000 for 0‐1.0 vs 1.5‐2.0 and p = 0.020 for 1.5‐2.0 vs 2.5‐3.0). Its C‐index was 0.571. Figure p = 0.002). The corresponding median OS values were 13.8, 17.8, and 31.2 months. Adjacent classes stratification analysis showed different statistical P value (p = 0.056 for 0‐1.0 vs 1.5‐2.0 and p = 0.025 for 1.5‐2.0 vs 2.5‐3.0). Its C‐index was 0.599 . Individuals with scores of 0‐1.0, 1.5‐2.0, 2.5‐3.0, and 3.5‐4.0 had median OS values of 18.4, 23.9, 35.5, and 49.0 months, severally. Each adjacent classes stratification analysis had statistical significance . Its C‐index was 0.615. Figure p = 0.001). Individuals with scores of 0‐1.0, 1.5‐2.0, 2.5‐3.0, and 3.5‐4.0 had median OS values of 10.3, 19.5, 27.1, and 37.0 months, severally. However, stratification analysis showed statistical difference in only one class . Its C‐index was 0.621 . The adjusted Lung‐molGPA model was more accurate in predicting survival of the adenocarcinoma than the adjusted DS‐GPA model (C‐index: 0.615 vs 0.571), but it failed to estimate the prognosis for the non‐adenocarcinoma .In this retrospective study, we created a data set of 1903 NSCLC patients with BM from the southwestern Chinese population; its median OS was 27.0 months, and score of 3.5‐4.0 reached a median OS of 49.0 months in the adjusted Lung‐molGPA model for lung adenocarcinoma. The adenocarcinoma had better prognosis than the non‐adenocarcinoma . Perhaps, in addition to ECM, determining the ECM organ numbers is a more important predictor of survival for patients with NSCLC and BM. Thus, more research is needed to further evaluate the role of BM numbers, especially BMI and ECM organ numbers, in the prognoses of NSCLC with BM to guide prospective clinical trial stratification and prognostic assessments.In univariate analysis, several factors were found to be associated with survival, including sex, smoking status, KPS, BMI, N‐staging, ECM, ECM organ numbers, BM numbers, histology type, and gene mutation status. Moreover, those factors were correlated with patients prognosis such as KPS, BMI, smoking status, N‐staging, ECM organ numbers, BM numbers, histology type, and gene mutation status in multivariate analysis. Interestingly, as a known predictor of survival, even when the cutoff value was adjusted to the median age of 57 years in this cohort, age was found to be correlated with the prognosis neither of the univariate and multivariate analysis, which was not consistent with the results obtained in previous studies.p = 0.000, C‐index = 0.615) and adjacent stratification analyses  were statistical significance. The adjusted DS‐GPA model  can also predict the prognosis of lung adenocarcinoma with BM, but the adjusted Lung‐molGPA model  was a better estimating tool. Consideration that 16.8% of lung non‐adenocarcinoma individuals with BM were EGFR or ALK positive, we used the adjusted Lung‐molGPA model to assess their survival. Although there was a statistical significance among groups as a whole (p = 0.001), two adjacent classes was not found to be statistically significant in the stratified analysis (p = 0.286 for 1.5‐2.0 vs 2.5‐3.0 and p = 0.410 for 2.5‐3.0 vs 3.5‐4.0). This maybe correlated with the uneven stratification of cases and the fact that only nine patients had score of 3.5‐4.0. It manifested once again that gene mutation status was not a suitable prognostic factor for lung non‐adenocarcinoma with BM. Interestingly, even in the adjusted DS‐GPA model, there was statistical difference in the whole comparison (p = 0.002), but one adjacent classes analysis still had close to no statistical significance (p = 0.056 for 0‐1.0 vs 1.5‐2.0). Overall, the adjusted DS‐GPA model was still a more suitable model for predicting the prognosis of lung non‐adenocarcinoma with BM .As a valuable independent predictor for the prognosis of NSCLC with BM, driver gene alteration has been incorporated into the prognosis assessment models that are widely used.In the current study, there are some shortcomings. First, it is a retrospective study, with inherent selection bias and the treatment factor on prognosis was not considered. Second, it excluded those patients who only received best supportive care, because the number of cases was so small. Third, identification of gene mutations principally by detecting tumor lesions in the plasma or elsewhere outside the brain does not reflect the mutation status in the intracranial metastases due to the existence of the blood‐brain barrier (BBB) and tumor heterogeneity.5The current study proved the applicability of the adjusted Lung‐molGPA model in a Chinese cohort of adenocarcinoma NSCLC with BM. Simultaneously, the adjusted DS‐GPA model was still a good user‐friendly tool to predict survival for non‐adenocarcinoma patients. Thus, more studies are needed to estimate the prognosis for patients of NSCLC with BM to improve future clinical trial stratification and help doctors make optimal treatment decisions in clinical practice.All authors have read and approved the final manuscript. No authors reported a conflict of interest related to the study.TYZ: Project conceptualization, cases collection, data curation, methodology, original draft writing, and funding acquisition. YZ: Project conceptualization, cases collection, data analysis, drawing processing, methodology, and draft review and editing. LZ: Project conceptualization, cases collection, data analysis, methodology, original draft writing, and draft review and editing. SSD: Cases collection and drawing processing. MJH: Cases collection and methodology. YCL: Cases collection and drawing processing. YML: Project conceptualization and cases collection. YL: Project design, data curation, formal analysis, funding acquisition, methodology, supervision, and writing‐review and editing. Other authors: cases collection. All authors read and approved the final manuscript.


Burkholderia territorii, a Gram-negative bacterium, encodes for the ι-class carbonic anhydrase  BteCAι, which was recently characterised. It acts as a good catalyst for the hydration of CO2 to bicarbonate and protons, with a kcat value of 3.0 × 105 s−1 and kcat/KM value of 3.9 × 107 M−1 s−1. No inhibition data on this new class of enzymes are available to date. We report here an anion and small molecules inhibition study of BteCAι, which we prove to be a zinc(II)- and not manganese(II)-containing enzyme, as reported for diatom ι-CAs. The best inhibitors were sulphamic acid, stannate, phenylarsonic acid, phenylboronic acid and sulfamide , whereas diethyldithiocarbamate, tellurate, selenate, bicarbonate and cyanate were submillimolar inhibitors . The halides (except iodide), thiocyanate, nitrite, nitrate, carbonate, bisulphite, sulphate, hydrogensulfide, peroxydisulfate, selenocyanate, fluorosulfonate and trithiocarbonate showed KI values in the range of 3.1–9.3 mM. In most cells, tissues and organs, these enzymes participate in crucial physiologic processes connected to pH regulation, metabolism, secretion of electrolytes, transport of gases and anions, and others, which are also therapeutically relevant ,,,,,,,,,The carbonic anhydrase  superfamily is composed nowadays of eight genetically distinct classes, the α-, β-, γ-, δ-, ζ-, η-, θ-, and ι-CAs, which probably may soon increase when additional families will be reported in other organisms. All of them catalyse a simple but physiologically crucial reaction, the carbon dioxide reversible hydration to bicarbonate and protons COThalassiosira pseudonanaa; the corresponding enzyme has been isolated and reported preferring Mn(II) as a metal cofactor in its active site, and not Zn(II) frequently found therein in other organisms. In the same paper, it has been shown that members of the ι-CA family should be present also in bacteria, as deduced by genome analysis, although such enzymes were not characterised at that moment in such organisms. Recently, we confirmed the finding of Gontero’s groupa, and reported the cloning and biochemical characterisation of the first example of a bacterial ι-CA, which was observed in the Gram-negative bacterium Burkholderia territorii and denominated BteCAιb. The enzyme showed a significant CO2 hydrase activity, with kinetic parameters (kcat of 3.0 × 105 s−1 and kcat/KM of 3.9 × 107 M−1 s−1) comparable to those of highly efficient bacterial and even mammalian isoforms, such as human (h) CA Ib. However, no inhibition studies have been performed so far on this enzyme, which has been demonstrated to be dimeric (by using protonography)b. Here, we prove that this is indeed the case by using atomic absorption spectroscopy, and also report the first inhibition study of the enzyme, with small molecules and anions, a well-known class of CAIsVery recently, a gene coding for a member of the ι-CA family has been originally described to occur in the genome of the marine diatom 2.2.1.Anions and small molecules were commercially available reagents of the highest available purity from Sigma-Aldrich . Purity of tested compounds was higher than 99%.2.2.In various enzyme samples, the content of Zn(II) and Mn(II) was measured with a flame PinAAcle 500 Perkin Elmer instrument, located in the Interdepartmental Service Centre for Biotechnology of Agricultural, Chemical and Industrial Interest (CIBIACI), University of Florence.2.3.b.BteCAι was a recombinant enzyme obtained in-house as described earlier2.4.2 hydration activity4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalysed CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5–10% of the reaction were used to determine the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitors (10 mM) and dilutions up to 0.01 µM were prepared in distilled-deionised water. Inhibitor and enzyme solutions of concentrations ranging between 5 and 10 nM were preincubated together for 15 min, at room temperature, prior to assay, in order to allow for the E-I complex formation. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlierAn Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO3.T. pseudonanaa is active with Mn(II) bound as a metal cofactor within its active site and not with Zn(II), such as most other CA isoforms known to date. However, it should be mentioned that γ-CAs are active with Fe(II)b, both in the presence of zinc as well as manganese salts in order to assay which of the two metal ions are incorporated into the holoenzyme. As seen from a, the bacterial ι-CA was proved to be a zinc-containing enzyme.Gontero’s group reported that the ι-CA isolated from the marine diatom We also investigated the inhibition of the bacterial enzyme BteCAι with a wide range of inorganic anions and small molecule compounds known to interact with the CA family of proteins 19. Altho19, but it is rather surprising for iodide, cyanide, and azide, which have quite a high affinity for metal ions in solution and in the active site of many metalloenzymes19. Indeed, some of these anions show a potent inhibitory action for other CAs, such as the isoform hCA I (19). As no X-ray crystallographic data are available so far for ι-CAs, it is impossible to rationalise these interesting and surprising data.some anions, among which iodide, cyanide, azide, perchlorate, perosmate, diphosphate, divanadate, tetraborate, perrhenate, perruthenate, iminodisulfonate, hexafluorophosphate and trifluoromethanesulfonate, did not inhibit BteCAι significantly up to 50 mM concentration of inhibitor in the assay system. This is not unexpected for anions with low affinity for complexing metal ions, such as perchlorate, hexafluorophosphate and trifluoromethanesulfonateThe following inhibitors showed inhibitory action against BteCAι in the millimolar range: fluoride, chloride, bromide, thiocyanate, nitrite, nitrate, carbonate, bisulphite, sulphate, hydrogensulfide, peroxydisulfate, selenocyanate, fluorosulfonate and trithiocarbonate . As above, some of these data stupefied us: sulphate, for example, is a highly inefficient inhibitor of many α-CAs (e.g. hCA I and II), but it inhibits efficiently the bacterial β- and ι-CAs shown in Even more efficient inhibitory action against BteCAι was registered for the following anions: diethyldithiocarbamate, tellurate, selenate, bicarbonate and cyanate, which were submillimolar inhibitors with KI values ranging between 0.71 and 0.94 mM . The bicThe most efficient BteCAι inhibitors detected so far were stannate, sulphamic acid, phenylarsonic acid, phenylboronic acid and sulfamide, with KI values of 6.2–94 µM . Some ofThe data of 4.B. territorii. Unlike the diatom enzyme cloned from T. pseudonana, the bacterial ι-CA has Zn(II) ions at its active site and not Mn(II) counterparts. We also report here the first inhibition study of BteCAι with a range of inorganic anions and small molecules known to act as CA inhibitors. The most efficient BteCAι inhibitors were stannate, sulphamic acid, phenylarsonic acid, phenylboronic acid and sulfamide, with KI values of 6.2–94 µM. Diethyldithiocarbamate, tellurate, selenate, bicarbonate and cyanate were submillimolar inhibitors, with KIs ranging between 0.71 and 0.94 mM. Fluoride, chloride, bromide, thiocyanate, nitrite, nitrate, carbonate, bisulphite, sulphate, hydrogensulfide, peroxydisulfate, selenocyanate, fluorosulfonate and trithiocarbonate showed KI values in the range of 3.1–9.3 mM, whereas no inhibition was registered for iodide, cyanide, azide, perchlorate, perosmate, diphosphate, divanadate, tetraborate, perrhenate, perruthenate, iminodisulfonate, hexafluorophosphate and trifluoromethanesulfonate. These data may be useful for designing more efficient ι-CA inhibitors.We investigated the nature of the metal ion within the active site of the first bacterial ι-CA described so far, namely BteCAι, whose corresponding gene was found in the genome of the Gram-negative bacterium


Lactobacillus plantarum MNC 21. Saccharified sorghum malt extracts were inoculated with 4 log cfu/mL MNC 21 and incubated at 30°C for 24 h. MRS broth was the reference medium. Microbial counts, pH, titratable acidity (TA), free amino nitrogen (FAN), and total sugars were measured. Maximum microbial counts in the extracts and MRS broth were 9 and 10 log cfu/mL, respectively. Maximum growth rate in the extracts was 0.7–0.9 log cfu/mL/h and 0.8 log cfu/mL/h in MRS broth. The final pH of the extracts was 3.5–3.6, with an overall increase in TA of 1.2% in Epuripur and 0.2% in other varieties. Final pH and TA of MRS broth were 4.1 and 1.3%, respectively. Total sugars dropped by 95.2% and FAN by 2.1% in MRS broth. In contrast, total sugars and FAN dropped by 5.6–9.1% and 24.9–32.7% respectively, in the extracts. Sorghum malt extracts can be adopted as alternative low-cost growth media for lactic acid bacteria cultures.Cultivation of lactic acid bacteria cultures is vital for research and commercial production of fermented foods. However, the conventional growth media used are generally costly. Malt extracts from four sorghum varieties  were evaluated as alternative low-cost growth media for  Lactobacillus (L.) rhamnosus GG, L. plantarum 299v, and L. casei Shirota have probiotic properties such as alleviation of rotavirus diarrhea, prevention of traveller's diarrhea and Clostridium difficile colitis, prevention of atopic dermatitis, treatment of irritable bowel syndrome, increase in iron absorption, intestinal flora reposition, and improved digestion [Lactic acid bacteria (LAB) are commonly applied as starter cultures for numerous fermented foods, cereals, tubers, fruits, vegetables, milk, fish, and meat. They impart characteristic flavors and tastes while contributing to food safety and preservation . Some LAigestion .Lactobacillus Selection Broth [The growth of LAB is often restricted to rich-nutrient habitats, so they are cultivated in conventional media such as lactobacilli MRS broth, M17 broth, Micro Inoculum Broth, Rogosa SL Broth, Tomato Juice Broth, APT Broth, Elliker Broth, and on Broth . These mon Broth –18. Sorgon Broth , 20. In on Broth . So, sorL. plantarum are generally regarded as safe owing to their documented history of safe use [L. plantarum MNC 21, an isolate of Obushera, a fermented sorghum-millet beverage from Uganda, is a high acid producer and acid-stable microbe. It is thus a suitable starter culture for Obushera [L. plantarum MNC 21.safe use , 22. TheObushera . It is fFour improved sorghum varieties  were used in this study. SESO 1 and Epuripur are white-grained, whereas SESO 3 is brown-grained. Eyera is a popular local brown-grained variety. All four varieties were obtained from National Semi-Arid Resources Research Institute in Serere district, Uganda.μm screen. The flour was mixed with water to form a mixture of 11% total solids. To convert starch to maltose, the mixture was heated to 75°C, followed by addition of α-amylase  at a rate of 1000 units per mL. The slurry was held at 75°C for 1 h with continuous stirring. To convert maltose to glucose, the slurry temperature was lowered to 55°C, and amyloglucosidase  was added at a rate of 1000 units per mL. The slurry was held at 55°C for 1 h with continuous stirring. The malt extract was cooled to about 25°C, decanted, and filtered using grade filter papers (Whatman No. 1). It was then sterilized at 121°C for 15 min and cooled to 25°C.Sorghum grain was malted following procedures described by Taylor . The malL. plantarum MNC 21 isolated from Obushera by Mukisa [L. plantarum MNC 21 was subcultured thrice after which the cells were recovered by centrifugation at 7,500 x g for 10 min. The cell pellets were suspended in 10 mL of sterile Ringer's solution. Culture purity was checked using a microscope .y Mukisa  was usedL. plantarum MNC 21 and incubated at 30°C for 24 h. Growth characteristics of MNC 21 were evaluated by determining microbial counts, pH, titratable acidity (TA), total sugars, and free amino nitrogen (FAN) at 2 h intervals for 24 h. MRS broth was also inoculated with MNC 21 and used as the control medium. The maximum growth rate (μmax) was calculated as described by [A hundred milliliters of sterile sorghum malt extract were inoculated with about 4 log cfu/mL of ribed by .L. plantarum MNC 21 in MRS agar and incubating at 30°C for 48 h. The pH was determined using a pH meter . Titratable acidity was determined by titrating 10 mL of the extract against 0.1 N NaOH using phenolphthalein indicator [Microbial counts were determined by pour plating selected serial dilutions of the malt extract or MRS broth containing ndicator . FAN wasndicator . Total sndicator .α = 0.05. Mean comparisons were made using the least significant difference (LSD) test. Analyses were done using Statistix (student version 9.0) software.Results were presented as means ± standard deviations (mean ± SD) of three independent experiments. Data were subjected to one-way analysis of variance (ANOVA) to test for significant differences at L. plantarum MNC 21 in MRS broth and malt extracts from different sorghum varieties. The LAB grew in all varieties, reaching a maximum average microbial count of 8.9 ± 0.0 log cfu/mL, one log cycle lower than what was observed in MRS broth. A steep increase in microbial counts in all media occurred in the first 4–18 h of fermentation and then plateaued. Maximum microbial counts were obtained earliest (16 h) in Epuripur and latest in MRS broth (22 h). Maximum growth rate (μmax) was in the order Epuripur > SESO 1 = MRS broth > SESO 3 = Eyera ( = Eyera . The netp > 0.05). X0 = Initial microbial count, Xmax = maximum microbial count, and μmax = growth rate constant.Values are means ± standard deviations. Values in the same column with similar superscripts are not significantly different  bioreactor using a corn steep liquor medium is 10 log cfu/mL . This islus spp. . The brolus spp. , 31. Thelus spp.  and Mukilus spp. . These an et al. , who usemulation , 33. TheP < 0.05) maximum cell counts obtained in the malt extracts could be attributed to the low quality rather than quantity of FAN in the malt extracts since the residual FAN in the broth and malt extracts was still high at the end of the fermentation  than in MRS broth (1.5 log cfu/mg FAN). MRS broth has yeast extract, beef extract, and bacteriological peptone which could be more superior nitrogen sources than the sorghum protein. Therefore, the lower  growth rate but also attained its maximum cell count 6–8 h earlier than the rest of the varieties , rith phase  coincideth phase . Althougth phase , this brth phase . This ism is 3.4 , 42. Indes death . Among tThe changes in total sugars  and FAN in the media are shown in Figures P > 0.05).Values are means ± standard deviations. Values in the same column with similar superscript letters are not significantly different  cell counts by the end of the fermentation  than that attained in MRS broth (Although the final microbial count in the extracts was about 9 log cfu/mL, one log lower (RS broth , there wRS broth . Unlike L. plantarum MNC 21 and possibly other microbial cultures. The carbon and nitrogen sources in the sorghum malt extracts are present in amounts sufficient to support growth of L. plantarum MNC 21. Performance of the sorghum malt extracts could be improved by addition of buffers to minimize the toxicity resulting from lactic acid accumulation.This study showed that sorghum malt extracts can be used as alternative growth medium for propagation of


After the FDNPP accident, radiocaesium was first detected in muscle samples from wild Japanese monkeys exposed to radioactive materials, and haematologic effects, changes in head size, and delayed body weight gain were also reported, but little is known about the distribution of 137Cs in various organ and tissue samples of 10 wild Japanese monkeys inhabiting the forested areas of Fukushima City that were captured between July and August 2012. Among muscle, brain, heart, kidney, liver, lung, and spleen, muscle exhibited the highest and the brain the lowest 137Cs concentration. The concentration (mean ± SD) of 137Cs in muscle, brain, heart, kidney, liver, lung, and spleen was 77 ± 66, 26 ± 22, 41 ± 35, 49 ± 41, 41 ± 38, 53 ± 41, and 53 ± 51 Bq/kg, respectively. These results can help us understand the biological effects of long-term internal radiation exposure in non-human primates.We detected the  Following the massive earthquake that struck eastern Japan on March 11, 2011, a large amount of radioactive material was released into the environment from the damaged reactor of the Fukushima Daiichi Nuclear Power Plant (FDNPP) . To date2 in snowy areas [137Cs in various organs and tissues of wild Japanese monkeys inhabiting the forested areas surrounding Fukushima City that were captured between July and August 2012.The Japanese monkey, one of the world’s northernmost wild primates, is endemic to Japan and has a life span of more than 20 years . Japaneswy areas –21. The wy areas . We begawy areas  and repowy areas  as well wy areas  after thMacaca fuscata) captured between July and August 2012 from the forested areas around Fukushima City. These monkeys were used in a previously reported study to determine the concentrations of 134Cs and 137Cs in the muscles and characterize the changes in concentration over time as well as their relationship with soil contamination levels [Samples of muscle, brain, heart, kidney, liver, lung, and spleen were collected from 10 Japanese monkeys  in Food Allergy Research Laboratories FARL . Data were corrected to the background radiation dose of the measurement environment on an as-needed basis. us study . The rad137Cs concentration between groups were analysed by one-way ANOVA using SPSS, ver. 19 .Differences in mean 137Cs in skeletal muscle and various organ samples obtained from 10 Japanese monkeys captured between July and August 2012. The monkeys inhabited the forested areas surrounding Fukushima City, which is located 70 km from the FDNPP. The average 137Cs concentration in the skeletal muscles and internal organs ranged from 26 to 77 Bq/kg. Among the skeletal muscle, brain, heart, kidney, liver, lung, and spleen, muscle exhibited the highest and the brain the lowest 137Cs concentration. The mean (± SD) 137Cs concentration in the muscle, brain, heart, kidney, liver, lung, and spleen was 77 ± 66, 26 ± 22, 41 ± 35, 49 ± 41, 41 ± 38, 53 ± 41, and 53 ± 51 Bq/kg, respectively. There were no significant differences in mean 137Cs concentration between groups, as determined by one-way ANOVA. The ratio of the concentration of 137Cs in each organ to that in muscle (137Cs in each organ/137Cs in muscle) was 0.34 ± 0.06, 0.53 ± 0.13, 0.64 ± 0.14, 0.52 ± 0.14, 0.70 ± 0.26, and 0.66 ± 0.15 for the brain, heart, kidney, liver, lung, and spleen, respectively.Table 137Cs concentration of the internal organs seemed to be slightly different between species. In particular, the lung and spleen showed high 137Cs concentrations in Japanese monkeys.In this study, we found the highest concentration of radiocaesium in the skeletal muscle and lowest concentration in the brain of Japanese wild monkeys inhabiting forested areas surrounding Fukushima City. These findings agree with those of previous studies in cattle , 9 and w137Cs concentration in organs of two individuals, labelled FF-890 and FF-904, was higher than that in other individuals, which was related to the soil concentration of radiocaesium.The muscle radiocaesium concentration in the monkeys correlated significantly with the level of soil contamination at each capture location . The 137137Cs in the organs and tissues of Japanese monkeys captured in the areas surrounding Fukushima City will enhance our understanding of the biological effects of long-term internal radiation exposure. Continued monitoring of Japanese wild monkeys exposed to radioactive materials following the FDNPP accident would help in understanding the biological effects of long-term internal radiation exposure to the natural world.These data regarding the distribution of 137Cs concentration among the organs of the body and its biological effects under long-term internal exposure.Continued monitoring of Japanese wild monkeys exposed to radioactive materials following the FDNPP accident would help in understanding the variation of the


Volumetric modulated arc therapy (VMAT) adopted in post-mastectomy radiation therapy (PMRT) has the capacity to achieve highly conformal dose distributions. The research aims to evaluate the impact of positioning errors in the dosimetry of VMAT left-sided PMRT.∆) of the mean dose (Dm) and DVH endpoints Vx and Dy  were used to compare the dosimetry of the reference vs perturbed plans.A total of 18 perturbations where introduced in 11 VMAT treatment plans that shifted the isocenter from its reference position of 3, 5, 10 mm in six directions. The thoracic wall and supraclavicular clinical target volumes (CTVs), the heart and the left lung dose volume histograms (DVHs) of 198 perturbed plans were calculated. The absolute differences , only isocenter shifts in the right, posterior and inferior directions worsen the plan dosimetry, nevertheless not negligible lung Inaccuracy in isocenter positioning for VMAT left-sided PMRT irradiation may impact the dosimetry of the CTVs and OARs to a different extent, depending on the directions and magnitude of the perturbation. The acquired information could be useful for planning strategies to guarantee the accuracy of the treatment delivered. Post-mastectomy radiation therapy (PMRT) is technically difficult, given the complexity of the target volume and its proximity to critical structures, including the heart, lung, brachial plexus, and contralateral breast –3. More To buffer these daily setup errors and ensure adequate dosing to target tissue, the planning treatment volume incorporates a margin to the clinical target volume taking into consideration the systematic and random errors of daily setup . This stρ = 1 g·cm − 3) with dimensions of 28 × 28 × 1 cm3, placed over the skin in correspondence with the treatment field was used during the planning computed tomography (CT) and for all treatments; as many institutions reported [This retrospective study included the analysis of 198 perturbed treatment plans and corresponding dose volume histograms (DVHs) of 11 cases of left-sided breast cancer patients who underwent PMRT. The patients were immobilized in the supine position using a WingSTEP breast board with head holder and a KneeSTEP knee support . A sheet of water-equivalent bolus .The DVHs of the target’s, left lung and heart were obtained for each perturbation introduced and case studied. The absolute difference of the mean dose (Dm) and of the dose volume histogram endpoints Vx and Dy , were used to compare the dosimetry of reference versus the perturbed plans; particularly Table ∆ D95, ∆ D98, and ∆ Dm of 14.4% (range 3.4–27.1), 19.1% (range 4.5–35.7), and 3.7% (range 1.5–6.2), respectively; while for CTVsv ∆ D95, ∆ D98, and ∆ Dm mean decreases of 11.2% (range 5.0–22.1), 16.9% (range 9.1–30.2), and 2.1% (range 1.5–2.7), respectively, were registered. The maximum dose for both targets was only slightly affected and only for the anterior and right directions with a maximum increase of 1.7 Gy. For 5 mm isocenter shifts, ∆ D95, ∆ D98, and ∆ Dm decreased to 3.8% (range 1.8–7.6), 6.1% (range 3.0–11.6), and 0.6% (range 0.0–1.2), respectively, for CTVth and to 2.6% (range 1.4–4.8), 4.4% (range 1.4–10.7), and 1.2% (range 0.8–1.4), respectively, for CTVsv. For 3 mm isocenter shifts, lower mean differences were found: ∆ D95, ∆ D98, and ∆ Dm decreased to 1.2% (range 0.2–2.8), 2.0% (range 0.6–5.6), and 0.4% (range 0.0–0.8), respectively, for CTVth, and to 0.8% (range 0.2–1.4), 1.4% (range 1.0–2.8), and 0.8% (range 0.6–1.0), respectively, for CTVsv.Significant absolute percentage differences were registered for isocenter shifts of 10 mm with a mean decrease for CTVth ∆ V20, ∆ V40, and ∆ Dm of + 8.4%, 7.8% and 3.6 Gy, + 3.9%, 3.8%, and 1.7 Gy, and + 2.3%, + 2.1%, and + 1.1 Gy, were registered for 10, 5, 3 mm isocenter shifts. For the heart, the higher difference was registered for isocenter shifts in the posterior direction with mean ∆ V25 and ∆ Dm of + 7.0%, and 3.0 Gy, of + 3.0%, and + 1.4 Gy, and of + 1.6% and + 0.8 Gy, respectively, for 10, 5, 3 mm isocenter shifts.For the OARs, only isocenter shifts in the right, posterior, and inferior directions worsen the plan dosimetry; particularly, for the left lung means In this study, the impact of positioning errors on the plan dosimetry of a VMAT PMRT was investigated for three different magnitudes and six different directions of isocenter shifts. The results obtained make it possible to share some considerations. As expected, the impact on the plan dosimetry resulting from a misalignment of the isocenter increases with the magnitude of the isocenter shift, generally for both targets and OARs; nevertheless, shifts of 3 mm that slightly affect the target coverage could be relevant for the increase of dose of the OARs as shows Fig. The directions that most affect the coverage of both targets  do not affect the organs at risk as they move them away from the treatment field, other directions  that only slightly affect the variation in target coverage lead to nonnegligible overdoses of the organs at risk as they significantly increase the portion of the lung and heart irradiated. Moreover, isocenter misalignments of 3 mm, with a negligible impact on the target’s dose coverage, maintain positive lung absolute dose differences The higher the dose of the incidental radiation to the heart, the higher the probability of an event of cardiovascular complication or generally cardiotoxic effects –25; thisThe results of this study were obtained using the patient data derived from the clinical practice of our department. The research does not have the value of a multi-institutional quality- assurance program, therefore the results cannot be generalized. Nevertheless, the findings obtained show that OARs dose deviations from the initial treatment conditions can arise for isocenter misplacement that slightly affect the target coverage.Knowledge of dose coverage variation in chest wall tissue, adjacent lung, and heart are necessary to properly manage the accuracy of the treatment delivered along the treatment course. Isocenter positioning variations can be seen where the online match had failed on an individual day, although the neighbouring fractions were well matched. The use of custom margins in specific “critical” directions, for OARs, combined with the margins already in use by PTV, would help to limit the impact of possible positioning errors that may occur when setup support technologies  are not available or cannot be scheduled on a daily basis. Investigations performed with own equipment can highlight the impact on the plan dosimetry of isocenter positioning variations in specific directions; asymmetrical action levels could be considered for the daily verification.Inaccuracy in isocenter positioning for VMAT left-sided PMRT irradiation may impact the dosimetry of the CTVs and OARs to a different extent, depending on the directions and magnitude of the perturbation. The acquired information could be useful for planning strategies to guarantee the accuracy of the treatment delivered.


Linum bienne, the sister species of the crop L. usitatissimum, to provide molecular genetic tools for the investigation of L. bienne genetic diversity and structure.Nuclear microsatellite markers were developed for L. bienne by means of genome skimming, and 44 loci successfully amplified. Of these, 16 loci evenly spread across the L. usitatissimum reference nuclear genome were used for genotyping six L. bienne populations. Excluding one monomorphic locus, the number of alleles per locus ranged from two to 12. Four out of six populations harbored private alleles. The levels of expected and observed heterozygosity were 0.076 to 0.667 and 0.000 to 1.000, respectively. All 16 loci successfully cross‐amplified in L. usitatissimum.Fifty microsatellite loci were identified in L. bienne, and 28 additional loci that successfully amplified are available for further testing.The 16 microsatellite loci developed here can be used for population genetic studies in  Linum L. (Linaceae) includes 180–200 species, with most species diversity concentrated in the Mediterranean Basin. It has become an important plant group to investigate the evolution of breeding systems and genome duplication events  markers, but from a limited geographical range. Here, we screen 50 microsatellite markers that will serve to investigate genetic diversity and structure of L. bienne.L. bienne individuals from different locations (Appendix L. usitatissimum nuclear genome reference (GenBank IDs CP027619.1–CP027633.1) in BWA version 0.7.17 . The PCR program consisted of an initial denaturation of 2 min at 94°C; 35 cycles of 1 min at 94°C, 1 min at 56°C , and 2 min at 72°C; and a final extension step of 10 min at 72°C. For 12 out of 50 primer pairs, these conditions did not lead to amplification or produced multiple bands. When multiple bands were obtained, we tested the primers again by increasing Ta by 1°C. In situations where no initial amplification occurred, we decreased Ta by 1°C. In total, 44 loci amplified successfully at the end of this process  were estimated with the R package hierfstat version 0.04‐22 , departure from Hardy–Weinberg equilibrium (HWE), linkage disequilibrium, and number of private alleles were calculated using the R package poppr version 2.8.3 . Genetic diversity analyses are presented in Table  Goudet, . UnbiaseL. bienne (1.23% of missing data on average), but cross‐amplification was successful only in L. usitatissimum. In L. bienne, locus ssr14.3 was monomorphic and therefore excluded from the analyses. Locus ssr2.2 included two different microsatellite regions that were then treated as independent loci (ssr2a.2 and ssr2b.2). The number of alleles per locus varied between two and 12 over all six L. bienne populations. All populations harbored one to three private alleles for one or more loci, except for populations VIL and IOW2. Depending on the population, 12 to 16 loci significantly deviated from HWE (P < 0.05). When loci were in HWE, it was mostly due to fixed alleles  (Appendix All 16 loci selected for genotyping amplified in Appendix .Ho and frequent deviation from HWE (Table Linum, which was also observed by Cloutier et al. (The high WE Table  might arr et al. . If duplMicrosatellite loci are ideal for providing fine‐scale geographic and temporal information about population genetic processes such as relatedness. The set of loci developed here are distributed across the genome and will therefore be useful to distinguish between genome‐wide processes caused by demography and locus‐specific processes such as adaptation. However, putative paralogy needs investigation. The sequencing of different alleles and additional analysis of the genomic data set could serve to discriminate between paralog copies.R.P.B., A.C.B., B.L., and R.G.A. collected the plant material; R.G., B.L., and J.V. conducted the lab work; B.L. and J.V. implemented the genome skimming pipeline; B.L. analyzed the data and wrote the manuscript; R.P.B. and F.X.P. provided the funding and coordinated the work; all authors contributed to reviewing the manuscript.APPENDIX S1. Script for de novo genome assembly using six Linum bienne individuals of different geographical origin, subsequent microsatellite loci mining and primer design, and in silico genotyping.Click here for additional data file.APPENDIX S2. Index of association for 16 polymorphic loci included in this study.Click here for additional data file.