EGR1 mediates MDR1 transcriptional activity regulating gemcitabine resistance in pancreatic cancer.

BACKGROUND: Gemcitabine is a cornerstone drug for the treatment of all stages of pancreatic cancer and can prolong the survival of patients with pancreatic cancer, but resistance to gemcitabine in pancreatic cancer patients hinders its efficacy. The overexpression of Early growth response 1(EGR1) in pancreatic ductal adenocarcinoma as a mechanism of gemcitabine chemoresistance in pancreatic cancer has not been explored. The major mechanisms of gemcitabine chemoresistance are related to drug uptake, metabolism, and action. One of the common causes of tumor multidrug resistance (MDR) to chemotherapy in cancer cells is that transporter proteins increase intracellular drug efflux and decrease drug concentrations by inducing anti-apoptotic mechanisms. It has been reported that gemcitabine binds to MDR1 with high affinity. The purpose of this research was to investigate the potential mechanisms by which EGR1 associates with MDR1 to regulate gemcitabine resistance in pancreatic cancer cells. METHODS: The following in vitro and in vivo techniques were used in this research to explore the potential mechanisms by which EGR1 binds to MDR1 to regulate gemcitabine resistance in pancreatic cancer cells. Cell culture; in vitro and in vivo study of EGR1 function by loss of function analysis. Binding of EGR1 to the MDR1 promoter was detected using the ChIP assay. qRT-PCR, Western blot assays to detect protein and mRNA expression; use of Annexin V apoptosis detection assay to test apoptosis; CCK8, Edu assay to test cell proliferation viability. The animal model of pancreatic cancer subcutaneous allograft was constructed and the tumours were stained with hematoxylin eosin and Ki-67 expression was detected using immunohistochemistry. FINDINGS: We revealed that EGR1 expression was increased in different pancreatic cancer cell lines compared to normal pancreatic ductal epithelial cells. Moreover, gemcitabine treatment induced upregulation of EGR1 expression in a dose- and time-dependent manner. EGR1 is significantly enriched in the MDR1 promoter sequence.Upon knockdown of EGR1, cell proliferation was impaired in CFPAC-1 and PANC-1 cell lines, apoptosis was enhanced and MDR1 expression was decreased, thereby partially reversing gemcitabine chemoresistance. In animal experiments, knockdown of EGR1 enhanced the inhibitory effect of gemcitabine on tumor growth compared with the sh-NC group. CONCLUSIONS: Our study suggests that EGR1 may be involved in the regulation of MDR1 to enhance gemcitabine resistance in pancreatic cancer cells. EGR1 could be a novel therapeutic target to overcome gemcitabine resistance in pancreatic cancer.

High resistance risk of Phytophthora colocasiae to azoxystrobin in southeastern of China.

Quinone outside inhibitor (QoI) has been applied to manage taro leaf blight caused by Phytophthora colocasiae in southeastern of China for many years. The risk of P. colocasiae to QoI and the potential resistant mechanism remain unknown. In this study, the 74 P. colocasiae strains were sampled from southeastern of China. Sequence analysis of the QoI target Cytb showed one nucleotide variant in the fragment of this gene in this population, producing two haplotypes. The nucleotide variant leads to codon change at 142 (GGT to GCT) producing A142 (alanine) and G142 (glycine) in Hap_1 and Hap_2 strains, respectively. The sensitivity differentiation to azoxystrobin of two haplotypes were observed in vitro. The Hap_1 and Hap_2 strains were confirmed resistant and sensitive by control efficacy of label rate fungicide application, which was 3.0% and 88.8% treated with 500 µg/mL azoxystrobin, respectively. In addition, 10.0 µg/mL azoxystrobin plus 50 µg/mL salicylhydroxamic acid (SHAM) supplemented in PDA medium was identified as a discriminatory dose for differentiation of these two phenotype strains. The azoxystrobin resistant frequency reached 86.5%, indicating prevalence of QoI resistance in the field. Further fitness related features showed that no significant difference in temperature sensitivity, mycelial growth rate, sporangia production, zoospore release and aggressiveness between azoxystrobin-resistant and sensitive strains indicating no potential fitness cost for azoxystrobin resistance. Taken together, azoxystrobin resistance need to be taken into consideration to manage taro leaf blight in southeastern of China.

5-Iodotubercidin Inhibits the Growth of Insulinoma Cells by Inducing Apoptosis.

INTRODUCTION: 5-Iodotubercidin, a type of purine derivative, has attracted increasing attention in tumor chemotherapy because of its potential as an antitumor agent in recent years. In this study, we confirmed the effects on apoptosis in insulinoma cell lines induced by 5-iodotubercidin and tried to illuminate the underlying mechanisms. METHODS: We used 5-iodotubercidin in the treatment of insulinoma cells and the cell proliferation was examined using CCK-8 assay, colony-forming assays, and insulinoma animal models. Cell apoptosis was examined using TUNEL assays and Western blotting. Cellular DNA damage was shown by comet assay and immunofluorescence. The expression of apoptosis-regulating proteins and DNA damage biomarker was investigated by Western blotting. Subcutaneous inoculation of the insulinoma cells into nude mice was to measure blood glucose, insulin levels, and tumor growth. ATM siRNA and p53 siRNA were used as loss-of-function targets to evaluate 5-iodotubercidin treatment. RESULTS: 5-Iodotubercidin inhibited the proliferation of insulinoma cells and induced DNA damage and cell apoptosis. Moreover, 5-iodotubercidin induced ATM and p53 activated. In vivo, 5-iodotubercidin inhibited the growth of Ins-1 and Min-6 cells xenografts in nude mice. CONCLUSION: 5-Iodotubercidin induces DNA damage leading to insulinoma cells apoptosis by activating ATM/p53 pathway. Therefore, this is a potential strategy for treating insulinoma.

Investigation on the in vitro metabolism of bicyclol using liver microsomes, hepatocytes and human recombinant cytochrome P450 enzymes.

In vitro metabolism of bicyclol was studied using liver microsomes, hepatocytes and human recombinant cytochrome P450 enzymes. Liquid chromatography-benchtop orbitrap mass spectrometry technique was utilised to identify the metabolites.A total of 19 metabolites, including 5 new metabolites (M2, M3, M4, M5 and M16) were tentatively identified. Among these metabolites, M6&M8 (demethylenation), M9&M10 (demethylation) and M19 (glucuronidation) were the major metabolites.In glutathione (GSH)-supplemented liver microsomes, 5 new GSH conjugates were found and tentatively identified. The formation was assumed to be through demethylenation of methylenedioxyphenyl to form catechol derivatives, which further underwent oxidation to form ortho-quinone intermediates, reacting with GSH to form stable adducts.CYP3A4 and 2C19 were demonstrated to be the major enzymes responsible for the bioactivation of bicyclol.This study provided valuable information on the metabolic fate of bicyclol in liver microsomes and hepatocytes, and the bioactivation pathways were reported for the first time, which would be helpful for us to understand the potential drug-drug interactions and the possible side effect of this drug.

First report of brown rot caused by Monilia mumecola on Chinese plum (Prunus salicina) in Fujian, China.

Brown rot caused by Monilinia spp. (anamorph Monilia) is a common disease in stone fruits worldwide, but the species in different hosts or regions may vary. Monilia mumecola is a recently identified species, only reported in some regions. Although the pathogen has been found on plum in Yunnan, Hubei, and Zhejiang provinces, and Chongqing municipality in China (Yin et al. 2015), it has not been reported in southeast coast of China. In May 2022, brown rot with grey spores on fruit was observed in a plum orchard with 15% disease incidence in Sanming City, Fujian Province, located in southeast coast of China. Four single-spored isolates were obtained from germinating conidia on the water agar for further investigation. The colonies on potato dextrose agar (PDA) were initially white, gradually turned gray to brown, with lobbed margins and rare sporulation. Average mycelial growth rate ranged from 0.74 to 1.08 cm/day at 25 oC. Conidia were lemon-shaped or subglobose, hyaline, with an average size of 17.64 to 19.35×11.14 to 14.44 µm (n=30). Each isolate produced one to three or four germ tubes. Such characteristics are similar to M. mumecola (Yin et al. 2015). To confirm the identity of the isolates, genomic DNA was extracted and species-specific primers of Hu et al. (2011) were used to amplify the 712 bp sequence. In addition, the ITS region, partial genes of glyceraldehyde-3- phosphate dehydrogenase (GAPDH) and beta-tubulin (TUB2) were also amplified using primers sets ITS1/ITS4 (Glass and Donaldson 1995), Mon-G3pdhF/Mon-G3pdhR and Mon-TubF1/ Mon-TubR1, respectively (Hu et al. 2011). Sequences obtained for those three regions were 478, 762 and 1527 bp, respectively. Each region of all four isolates was identical, so one sequence for each region was submitted to GenBank with accession numbers OQ207672, OQ225251 and OQ225252, respectively, which had 100% identity with M. mumecola HQ908786 (ITS), HQ908784 (GAPDH), and HQ908775 (TUB2) using BLAST analysis in NCBI database, respectively. Pathogenicity was conducted with mycelium plugs from the edge of 7-day-old colony on three mature 'Angeleno' plums fruit (Prunus salicina) creating nine inoculations with three wounds per fruit, and each wound was 5 mm in diameter and 2 mm in depth. The same amount fruit and wounds inoculated with PDA plugs without fungi were used as a control. Brown rot symptoms were observed on all inoculated plums 4 days post-inoculation under room temperature with 100% humidity, whereas control plums remained symptomless. Fungal colonies re-isolated from the lesions showed the same morphological features as the original isolate , thus fulfilling Koch's postulates. To our knowledge, this is the first report of M. mumecola on plum in Fujian Province of China. The findings in this studies have important management implications for local plum growers because more than one Monilia species have been reported, where only M. fructicola was present in this region.

Activatable NIR-II Fluorescent Nanoprobe for Rapid Detection and Imaging of Methylglyoxal Facilitated by the Local Nonpolar Microenvironment.

Closely related to multiple chronic inflammation, especially type-2 diabetes (T2D), methylglyoxal (MGO) may be a potential key to visualize disease progression and treatment effects. On the other hand, lack of convenient and fast analytical methods cannot afford accurate MGO quantitative evaluation. In this work, an activatable second near-infrared region (NIR-II) fluorescent probe TDTCD was synthesized and its reaction mechanism with MGO was discussed. The desired NIR-II product preferred response solvents with small polarity. A novel activatable nanoprobe, MG-SLNP, for MGO was then constructed based on rational packaging to provide a local nonpolar microenvironment. The hydrophobic core of nanoparticles not only successfully improved the stability and water solubility but also greatly promoted the response rate while reacting with MGO. The comparison between NIR-II fluorescence and the traditional high-performance liquid chromatography method for T2D blood samples was discussed. A high-resolution viewing window, quick response, and good biocompatibility led to a satisfactory signal-to-noise ratio of MG-SLNP for real-time MGO bio-detection and imaging in vivo.

A high-resolution mass spectrometric method for identification and characterization of the in vitro metabolites of senkyunolide H.

RATIONALE: Ligusticum chuanxiong Hort is a well-known herb medicine that has been widely prescribed to treat cardiovascular diseases in China for hundreds of years. Senkyunolide H (SNH) is one of the major bioactive ingredients extracted from L. chuanxiong, and it displayed neuroprotective effects. To fully understand its mechanism of action, the metabolism needs to be investigated. METHODS: In vitro studies were conducted by incubating SNH with rat and human hepatocytes, and the metabolites were identified and characterized using liquid chromatography in combination with hybrid quadrupole Orbitrap mass spectrometry (LC-Orbitrap-MS). The structures of the metabolites were proposed by accurate mass analysis of respective precursor ions, indicative product ions, and elemental compositions. RESULTS: Under the current conditions, a total of 10 metabolites were identified, and among these metabolites, M3 and M4 were the most abundant metabolites both in rat and human hepatocytes. Our results demonstrated that hydroxylation, hydration, glucuronidation, and GSH conjugation were the primary metabolic pathways of SNH. CONCLUSIONS: The present study provides new information on the metabolism of SNH, which would help prospects of the disposition of SNH.

Revealing the binding properties between resorcinol and DNA.

Resorcinol (1,3-dihydroxybenzene) is a common coupling agent in permanent hair dyes, and has arrested people's attention for its potential hazard to human health. However, the action mechanism of resorcinol and human DNA has not been elucidated. In this research, the binding properties between resorcinol and calf thymus DNA (ct-DNA) were studied for the first time through various spectral and molecular docking techniques. Spectral studies showed that the initial fluorescence quenching of resorcinol against DNA was a static one. The result of ΔH < 0 and ΔS > 0 was produced from thermodynamic experimental data, therefore it could be concluded that electrostatic force was the major driving force, while binding constant Kb was 1.56 × 104 M-1 at 298 K. The electrostatic binding network between resorcinol and ct-DNA was established explicitly through competitive substitution analysis and other spectral approaches. The results of FT-IR absorption spectra indicated that resorcinol had bound to the DNA phosphate skeleton. Molecular docking clearly revealed that binding occurred between hydroxyl groups of resorcinol and phosphorus oxygen bonds (P-O) of the DNA skeleton. These findings may deepen our understanding of the action mechanism between resorcinol and ct-DNA and provide some useful data on the effect of resorcinol on human diseases.

Prevalence of H6Y mutation in ß-tubulin causing thiophanate-methyl resistant in Monilinia fructicola from Fujian, China.

Brown rot disease broke out in stone fruit orchards of Fujian, China in 2019, despite pre-harvest application of methyl benzimidazole carbamate (MBC). To determine the reason, a total of 44 Monilinia fructicola strains were collected from nectarine, plum and peach fruits in this study, among which 79.5% strains were resistant to thiophanate-methyl, indicated by discriminatory dose of 5 µg/mL. The resistance of these strains was confirmed by treating detached peach fruit with label rates of formulated thiophanate-methyl which only completely inhibit infection of the sensitive strains, but not the resistant strains. Further analysis of the mechanism of MBC resistance revealed that all resistant strains carry a H6Y mutation in ß-tubulin protein Tub2, which was only reported previously in the M. fructicola strains from California, USA, and do not display obvious fitness penalties, as no significant defects in mycelial growth rate, sporulation, conidia germination, aggressiveness on detached peach fruit and temperature sensitivity was detected. In addition, we found that diethofencarb, the agent for managing MBC-resistance strains, was unable to inhibit growth of the H6Y strains. Taken together, our study, for the first time, identified a mutation form of H6Y in the ß-tubulin protein of M. fructicola in China, rendering the strains wide resistance to thiophanate-methyl. This mechanism of M. fructicola gaining resistance to MBC fungicides needs to be fully considered, when designing management strategies to control brown rot disease in stone fruit orchards.

First Report of Black Dot Caused by Colletotrichum coccodes on Potato in the Tibet Autonomous Region of China.

Potato black dot causing by Colletotrichum coccodes is a common disease in potato throughout the world, infecting underground stems, tubers, roots and foliage. Potato is becoming the third main food crop produced on ~16,000 ha annually in the Tibet Autonomous Region of China, situated on the world's highest plateau. However, the disease causing by C. coccodes has not been reported in this region. During the disease survey in the Bailang County of Tibet in August, 2020, some potato plants cv. "JiZhang 12" with chlorosis and wilting of foliage were observed. The incidence of affected plants was 20%. Necrotic lesions were also observed on the basal stems of the affected plants. Three affected plants were collected for pathogen isolation and three isolates were obtained for further investigation. The colonies on potato dextrose agar (PDA) were initially white, turning gradually black with age and producing abundant black sclerotia. Conidia were cylindrical, hyaline, aseptate, guttulate, with average size of 13.80 to 18.55×4.94 to 5.35 µm for the three isolates in which 30 conidia each were measured. Such characteristics are similar to C. coccodes (Lees and Hilton, 2003). Mycelial growth rate was 0.69 to 0.74 cm/day at 25 oC over the three isolates. The three isolates were confirmed to be C. coccodes by species-specific PCR using primer set of Cc1NF1/Cc2NR1 producing 350 bp amplicons in the internal transcribed spacer (ITS) region according to Cullen et al. (2002). The Cc1NF1/Cc2NR1 sequences were identical for three isolates, therefore one sequence from isolate BL_JZ_J1 was submitted to the GenBank with accession number OM368349. Additional genes were also sequencing including the actin (ACT), chitin synthase l (CHS1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and another larger ITS region were also amplified from genomic DNA using primers sets ACT512F/ACT783R, CHS-79F/CHS-354R (Carbone and Kohn 1999), GDF1/GDR1 (Templeton et al. 1992) and ITS1/ITS4 (Glass and Donaldson 1995), respectively. Sequences obtained for those four regions were 216 bp, 218bp, 235bp and 576 bp, respectively. Each region in the three isolates were also identical, therefore one sequence for each region was submitted to the GenBank with accession number of OM417059, OM417060, OM417061, and OM349570, respectively, which had 100% similarity with C. coccodes of MN336525 (ACT), KU821274 (CHS1), KU821397 (GAPDH) and KU821175 (ITS), respectively. Phylogenetic analyses based on the concatenated sequences of those four loci showed that the BL_JZ_J1 was close to C. coccodes, a reference isolate of CPOS1 with the accession numbers of GQ856787 (ACT), GQ856723 (CHS1), GQ856744 (GAPDH) and GQ485588 (ITS) (Yang et al. 2009). Pathogenicity was confirmed by inoculating a conidia suspension (100 µl of 105 conidia/ml) on three stems of 6-week-old potato plant cv. 'Favorita' with an artificial wound generated by sterile toothpick for each isolate. An equal volume of sterile water was injected on the wound of three stems as a noninoculated control. Brownish necrotic lesions were observed on all inoculated stems 3 days post-inoculation under natural conditions, whereas control stems remained symptomless. Reisolation of the fungus from all inoculated symptomatic plants confirmed Koch's Postulates. To our knowledge, this is the first report of C. coccodes in potato in Tibet Autonomous Region of China. The finding of black dot in this region has important management implications for the growers since the pathogen can survive for long periods in the field both on potato debris and in soil.

Bioremediation of quinclorac injury on tobacco by a rhizosphere bacterium.

The presence of herbicides residues in soil represents a serious problem for agriculture. Quinclorac is a common herbicide applied in rice field, but its residue can cause abnormal growth in successive crop of tobacco in Southern China. Remediation by microorganisms is considered to be an environmentally friendly method to remove such pollutants injury. The aims of this study were to obtain quinclorac remediation isolates and to investigate the possible mechanism(s) of remediation. Six bacterial isolates were obtained from rhizosphere of rice-tobacco rotation fields, and were found to be capable of degrading quinclorac on a mineral salt medium (MSM), with degradation efficiency ranging from 2.1 to 23.7%. Among these isolates, J5 had the highest degradation efficiency, and was identified as Klebsiella variicola based on phylogenetic analyses and a metabolic profile generating by Biolog GEN III system. Bioremediation of quinclorac injury was confirmed using pot assays with tobacco, in which J5 reversed the detrimental effect of quinclorac on leaf area, leaf number, and plant height. The J5 isolate also seemed to promote plant growth, in terms of tobacco seedling growth and seed germination, which were 2.2 times and 1.6 times higher compared to untreated control, respectively. The mechanisms of plant growth promoting (PGP) traits were found to involve nitrogen-fixing, indole-3-acetic acid (IAA) production, and phosphate solubilization ability. In addition, proteomic analysis and relative quantitative PCR revealed an elevated level of 4-hydroxyphenylacetate 3-monooxygenase (HPMO) in quinclorac-treated J5, suggesting that this enzyme may play an important role in quinclorac remediation. This study showed that the J5 isolate could be exploited to not only assist in soil remediation due to quinclorac residue issues but also promote tobacco growth.

Clinical characterization of NTCP deficiency in paediatric patients : A case-control study based on SLC10A1 genotyping analysis.

Na+ -taurocholate cotransporting polypeptide deficiency (NTCPD) is a newly described disorder arising from biallelic mutations of the SLC10A1 gene. As a result of a lack of compelling evidence from case-control studies, its genotypic and phenotypic features remain open for in-depth investigation. This study aimed to explore the genotypic and clinical phenotypic characteristics of paediatric patients with NTCPD. The SLC10A1 genotypes of all NTCPD patients were confirmed by screening for the prevalent variant c.800C>T and Sanger sequencing when necessary. The clinical presentations and laboratory changes were collected, reviewed and analysed, and then qualitatively and quantitatively compared with the relevant controls. A total of 113 paediatric NTCPD patients were diagnosed while c.374dupG and c.682_683delCT were detected as two novel pathogenic mutations. Hypercholanemia was observed in 99.12% of the patients. Indirect hyperbilirubinemia in affected neonates exhibited higher positive rates in comparison to controls. Moreover, transient cholestatic jaundice, elevated liver enzymes and 25-hydroxyvitamin D (Vit D) deficiency during early infancy were more commonly observed in patients than in controls. All NTCPD patients exhibited favourable clinical outcomes as a result of symptomatic and supportive treatment. The findings enriched the SLC10A1 mutation spectrum and provided comprehensive insights into the phenotypic characteristics of NTCPD. NTCPD should be considered and SLC10A1 gene should be analysed in patients with above age-dependent clinical features. Furthermore, over investigation and intervention should be avoided in the management of NTCPD patients.

NEAT1 Knockdown Inhibits Keloid Fibroblast Progression by miR-196b-5p/FGF2 Axis.

BACKGROUND: Keloid is a benign fibroproliferative tumor of the skin caused by abnormal wound healing process after skin injury. Long noncoding RNAs have been reported to be involved in the development of keloid. However, the role and mechanism of nuclear enriched abundant transcript 1 (NEAT1) in keloid are still unknown. METHODS: Quantitative real-time polymerase chain reaction was performed to detect the expression of NEAT1, miR-196b-5p, and fibroblast growth factor 2 (FGF2). Western blot was conducted to measure the levels of collagen I, α-smooth muscle actin, fibronectin, and FGF2. Cell Counting Kit-8 assay and transwell assay were used to evaluate cell viability and migration, respectively. Dual-luciferase reporter assay was conducted to verify the targeting relationship between miR-196b-5p and NEAT1 or FGF2. RESULTS: NEAT1 was increased and miR-196b-5p was decreased in keloid tissues and fibroblasts. NEAT1 knockdown or miR-196b-5p overexpression suppressed cell viability, migration, and extracellular matrix (ECM) component production in keloid fibroblasts. MiR-196 b-5p was a target of NEAT1, and NEAT1 overexpression reversed the effect of miR-196b-5p on keloid fibroblast progression. Moreover, we found that miR-196b-5p directly targeted FGF2. FGF2 knockdown suppressed keloid fibroblast viability, migration, and ECM protein production. FGF2 overexpression abolished the effect of miR-196b-5p overexpression on keloid fibroblast development. CONCLUSIONS: NEAT1 silencing suppressed cell viability, migration, and ECM expression in keloid fibroblasts by regulating miR-196b-5p/FGF2 axis, indicating a promising strategy for keloid treatment.

Survival Cost and Diverse Molecular Mechanisms of Magnaporthe oryzae Isolate Resistance to Epoxiconazole.

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases on rice worldwide. Epoxiconazole is a 14α-demethylation inhibitor (DMI) with excellent control on rice blast; to date, no resistant isolates have been observed in the field. Four mutants resistant to epoxiconazole were generated from three parental isolates via fungicide adaptation. Resistance was stable after 10 weekly consecutive transfers on fungicide-free medium. Three parameters, including growth rate, sporulation in vitro, and aggressiveness, were significantly lower for mutants compared with their parental isolates, with the exception of the low-resistance isolate. Sporulation and aggressiveness were negatively correlated with effective concentration values for 50% inhibition of mycelial growth for parental isolates and mutants (P < 0.05). Cross-resistance was found between epoxiconazole and prochloraz (ρ = 0.863, P = 0.000) or difenoconazole (ρ = 0.861, P = 0.000). The resistance factor for mutants was positively correlated with the relative expression of MoCYP51A in epoxiconazole treatment (r = 0.977, P = 0.02). In addition, two putative amino acid substitutions in MoCYP51A were found in two resistant mutants: Y126F in the high-resistance mutant and I125L in the low-resistance mutant. Mutation Y126F reduced the affinity of MoCYP51A with epoxiconazole, whereas I125L was not in the binding pocket of epoxiconazole. No amino acid change or overexpression in MoCYP51B was found in any of the mutants studied. To our knowledge, this is the first study to report DMI resistance observed in M. oryzae. The survival cost of M. oryzae resistance to epoxiconazole might be the reason why DMI resistance has not yet emerged in field populations worldwide.

Reduced sensitivity of azoxystrobin and thiophanate-methyl resistance in Lasiodiplodia theobromae from papaya.

Stem-end rot caused by Lasiodiplodia theobromae is one of the most devastating diseases of papaya in northeastern Brazil. It is most effectively controlled by applications of fungicides, including site-specific fungicides at risk for resistance development. This study investigated the molecular mechanisms of reduced sensitivity to the QoI fungicide azoxystrobin and resistance to the MBC fungicide thiophanate-methyl in L. theobromae from Brazilian orchards. The EC50 values for azoxystrobin in sixty-four isolates ranged from 0.36 µg/ml to 364.24 µg/ml and the frequency distribution of EC50 values formed a multimodal curve, indicating reduced sensitivity to azoxystrobin. In detached fruit assays reduced sensitive isolates were not controlled as effectively as sensitive isolates at lowest label rate. Partial fragments were obtained from target genes ß-tubulin (751 bp) and Cytb (687 bp) of isolates resistant to thiophanate-methyl and reduced sensitivity to azoxystrobin. Sequence analysis of the ß-tubulin fragment revealed a mutation corresponding to E198K in all thiophanate-methyl-resistant isolates, while reduced sensitivity to axoxystrobin was not attributable to Cytb gene alterations. The target gene-based mechanism conferring resistance to thiophanate-methyl will likely be stable even if selection pressure subsides. However, the mechanism conferring reduced sensitivity to azoxystrobin is not based on target gene modifications and thus may not be as stable as other genotypes with mutations in Cytb gene.

Low evolutionary risk of iprovalicarb resistance in Phytophthora infestans.

Iprovalicarb is a carboxylic acid amide (CAA) fungicide, highly effective in the control of potato late blight, causing by Phytophthora infestans. Due to cross-resistance with other CAA fungicides and moderate resistance risk of P. capsici to iprovalicarb, the evolutionary risk of P. infestans resistance to this fungicide and the contribution of inherited genes and environmental effect was evaluated using a common garden experiment. The results showed that the ratio of heritability and plasticity of iprovalicarb in the seven populations equaled 1.0, indicating both inherited genes and environmental factors were essential to iprovalicarb sensitivity in P. infestans. The pairwise population differentiation determined by SSR loci (FST) between populations ranged from 0.007 to 0.133 and the overall FST was significantly higher than population differentiation in RGR (QST), suggesting constraining selection acting on iprovalicarb sensitivity. We also found a new indicator of growth rate inhibition (GRI) for fungicide sensitivity, which was negatively correlated to growth rate in the absence of iprovalicarb, indicating a trade-off between iprovalicarb resistance and pathogen's growth. The constraining selection plus a trade-off between GRI and growth rate revealed low risk of P. infestans evolving resistance to iprovalicarb.

Pre-treatment Eckardt score is a simple factor for predicting one-year peroral endoscopic myotomy failure in patients with achalasia.

BACKGROUND: Peroral endoscopic myotomy (POEM) is a novel treatment for achalasia with excellent outcomes. But the predictor for treatment failure is not well defined. This study was aimed to prospectively investigate the factors for predicting failed POEM. METHODS: From June 2011 to May 2015, a total of 115 achalasia patients treated by POEM were included for the retrospective cohort study from Nanfang Hospital and the First People's Hospital of Yunnan Province. Patients were followed up with Eckardt score, high-resolution manometry and endoscope. POEM failure was defined as primary failure (Eckardt score failed to decrease to 3 or below) and recurrences (decrease of Eckardt score to 3 or below, then rise to more than 3) during one-year follow-up. Univariate and multivariate Cox regression analyses were performed to assess the predictive factor. For the associated factor, receiver operating characteristic curve (ROC) was utilized to determine the cutoff value of the predicting factor. RESULTS: The failure rate of POEM after 1 year was 7.0% (8/115), including 5 primary failure cases and 3 recurrences. Multivariate analysis showed higher pre-treatment Eckardt score was the single independent factor associated with POEM failure [9.5 (6-12) vs. 7 (2-12), odds ratio (OR) 2.24, 95 confidence interval (95% CI) 1.39-3.93, p = 0.001]. The cutoff value (Eckardt score ≥9) had 87.5 sensitivity (95% CI 47.3-99.7%) and 73.8% specificity (95% CI 64.4-81.9%) for predicting failed POEM. CONCLUSIONS: Pre-treatment Eckardt score could be a predictive factor for failed POEM. Eckardt score ≥9 was associated with high sensitivity and specificity for predicting POEM failure.

Development of a carboxymethyl chitosan functionalized nanoemulsion formulation for increasing aqueous solubility, stability and skin permeability of astaxanthin using low-energy method.

In this research, firstly astaxanthin (ASX)-loaded nanoemulsions (NEs) were produced using a convenient low-energy emulsion phase inversion method. The optimised ASX-NEs were prepared in the presence of Cremophor® EL and Labrafil® M 1944 CS, with a surfactant-to-oil ratio of 4:6. The ASX-NE droplets were spherical with a mean droplet diameter below 100 nm and a small negative surface charge. The system was stable without alteration of mean droplet diameter for three months. Then, the ASX-NE was functionalised with carboxymethyl chitosan (CMCS) through direct CMCS (0.02%) incorporation during the preparation process. The ASX chemical stability and skin permeability increased in the following order: ASX solution control < ASX-NE < CMCS-ASX-NE. Cell viability assays on L929 cells revealed low cytotoxicity of blank NE, ASX-NE and CMCS-ASX-NE in the range from 5 to 500 µg mL-1. In conclusion, the CMCS-ASX-NE might be a promising delivery vehicle in dermal and transdermal products.

[Phenotypic and genetic analysis of a family affected with microvillus inclusion disease].

OBJECTIVE: To explore the clinical features and mutations of MYO5B gene in a family affected with microvillus inclusion disease. METHODS: Clinical data of an infant affected with microvillus inclusion disease was collected. Genomic DNA was extracted from peripheral blood samples from the patient and her parents. PCR amplification and Sanger sequencing were performed to analyze all the exons and their flanking sequences of the MYO5B gene. RESULTS: The patient presented with complicated manifestations including respiratory distress syndrome, dehydration, acidosis, bowel dilatation, liver and kidney dysfunction, and severe and intractable diarrhea. A compound mutation of the MYO5B gene, i.e., IVS37-1G>C/c.2729_2731delC (p.R911Afs916X), was discovered in the patient. The former was a splice-site mutation inherited from the mother, while the latter was a frameshift mutation inherited from the father. Both were not reported previously. CONCLUSION: Based on the clinical and molecular evidence, the patient was diagnosed with microvillus inclusion disease. Above finding has expanded the mutation spectrum of the MYO5B gene, which can provide valuable information for genetic counseling for the family.