The naturally occurring flavonoid nobiletin reverses methotrexate resistance via inhibition of P-glycoprotein synthesis

Methotrexate (MTX) is the first-line treatment for rheumatoid arthritis (RA). However, after long-term treatment, some patients develop resistance. P-glycoprotein (P-gp), as an indispensable drug transporter, is essential for mediating this MTX resistance. In addition, nobiletin (NOB), a naturally occurring polymethoxylated flavonoid, has also been shown to reverse P-gp–mediated MTX resistance in RA groups; however, the precise role of NOB in this process is still unclear. Here, we administered MTX and NOB alone or in combination to collagen II-induced arthritic (CIA) mice and evaluated disease severity using the arthritis index, synovial histopathological changes, immunohistochemistry, and P-gp expression. In addition, we used conventional RNA-seq to identify targets and possible pathways through which NOB reverses MTX-induced drug resistance. We found that NOB in combination with MTX could enhance its performance in synovial tissue and decrease P-gp expression in CIA mice compared to MTX treatment alone. In vitro, in MTX-resistant fibroblast-like synoviocytes from CIA cells (CIA-FLS/MTX), we show that NOB treatment downregulated the PI3K/AKT/HIF-1α pathway, thereby reducing the synthesis of the P-gp protein. In addition, NOB significantly inhibited glycolysis and metabolic activity of CIA-FLS/MTX cells, which could reduce the production of ATP and block P-gp, ultimately decreasing the efflux of MTX and maintaining its anti-RA effects. In conclusion, this study shows that NOB overcomes MTX resistance in CIA-FLS/MTX cells through the PI3K/AKT/HIF-1α pathway, simultaneously influencing metabolic processes and inhibiting P-gp–induced drug efflux.     

How do plants defend themselves against pathogens-Biochemical mechanisms and genetic interventions

In agro-ecosystem, plant pathogens hamper food quality, crop yield, and global food security. Manipulation of naturally occurring defense mechanisms in host plants is an effective and sustainable approach for plant disease management. Various natural compounds, ranging from cell wall components to metabolic enzymes have been reported to protect plants from infection by pathogens and hence provide specific resistance to hosts against pathogens, termed as induced resistance. It involves various biochemical components, that play an important role in molecular and cellular signaling events occurring either before (elicitation) or after pathogen infection. The induction of reactive oxygen species, activation of defensive machinery of plants comprising of enzymatic and non-enzymatic antioxidative components, secondary metabolites, pathogenesis-related protein expression (e.g. chitinases and glucanases), phytoalexin production, modification in cell wall composition, melatonin production, carotenoids accumulation, and altered activity of polyamines are major induced changes in host plants during pathogen infection. Hence, the altered concentration of biochemical components in host plants restricts disease development. Such biochemical or metabolic markers can be harnessed for the development of “pathogen-proof” plants. Effective utilization of the key metabolites-based metabolic markers can pave the path for candidate gene identification. This present review discusses the valuable information for understanding the biochemical response mechanism of plants to cope with pathogens and genomics-metabolomics-based sustainable development of pathogen proof cultivars along with knowledge gaps and future perspectives to enhance sustainable agricultural production.     

Antibiotic resistance in faecal bacteria (Escherichia coli,Enterococcus spp.) in feral pigeons

Aims: To determine the presence of antibiotic‐resistant faecal Escherichia coli and Enterococcus spp. in feral pigeons (Columba livia forma domestica) in the Czech Republic. Methods and Results: Cloacal swabs of feral pigeons collected in the city of Brno in 2006 were cultivated for antibiotic‐resistant E. coli. Resistance genes, class 1 and 2 integrons, and gene cassettes were detected in resistant isolates by polymerase chain reaction (PCR). The samples were also cultivated for enterococci. Species status of enterococci isolates was determined using repetitive extragenic palindromic‐PCR. Resistance genes were detected in resistant enterococci by PCR. E. coli isolates were found in 203 of 247 pigeon samples. Antibiotic resistance was recorded in three (1·5%, n_E. coli = 203) isolates. Using agar containing ciprofloxacin, 12 (5%, n_samples = 247) E. coli strains resistant to ciprofloxacin were isolated. No ESBL‐producing E. coli isolates were detected. A total of 143 enterococci were isolated: Ent. faecalis (36 isolates), Ent. faecium (27), Ent. durans (19), Ent. hirae (17), Ent. mundtii (17), Ent. gallinarum (12), Ent. casseliflavus (12) and Ent. columbae (3). Resistance to one to four antibiotics was detected in 45 (31%) isolates. Resistances were determined by tetK, tetL, tetM, tetO, aac(6′)aph(2′′), ant(4′)‐Ia, aph(3′)‐IIIa, ermB, pbp5, vanA and vanC1 genes. Conclusions: Antibiotic‐resistant E. coli and Enterococcus spp. occurred in feral pigeons in various prevalences. Significance and Impact of the Study: Feral pigeon should be considered a risk species for spreading in the environment antimicrobial resistant E. coli and enterococci.     

Characterising the morphological characters and carbohydrate metabolism of oat culms and their association with lodging resistance

• Lodging resistance can be improved by enhancing the mechanical strength of culms, and culm carbohydrates could improve this mechanical strength. Culm carbohydrates can regulate development of the culm and affect its toughness.
• The present study determines the relationship between lodging and carbohydrate content in oat culms. Field experiments were conducted in alpine regions in 2017 and 2018 using three oat varieties with different lodging resistance. Lodging‐related morphological characteristics were directly determined and culm carbohydrate content and enzyme activity related to cellulose synthesis and sucrose metabolism were evaluated with ultraviolet spectrophotometry.
• Results showed that the lower the gravity height or the lower ratio of gravity height to plant height, the stronger the lodging resistance of the varieties. Higher culm nonstructural (NSC) and structural (SC) carbohydrate content contributed to the ability of culms to resist lodging, especially the content of cellulose and sucrose. PCA showed that sucrose metabolism and SC content were closely related to lodging resistance. Correlation analysis showed that the lodging index (LI) was significantly negatively correlated with NSC. Sucrose content was highly and significantly positively correlated with NSC. Additionally, the activities of sucrose phosphate synthase (SPS) and sucrose synthase (SS) were highly and significantly positively correlated with sucrose and cellulose content.
• The relationship between field characters and oat lodging, as well as the regulatory mechanism of carbohydrate content on lodging resistance of the culm are discussed.

     

Multiple drug resistance and strength of attachment to surfaces in Pseudomonas aeruginosa isolates

Aims: To investigate the presence of a relationship between the strength of attachment of Pseudomonas aeruginosa to stainless steel surfaces and their observed multiple drug resistance. Methods and Results: Multiple drug resistance of clinical and environmental isolates of Ps. aeruginosa was evaluated using disc diffusion method. The blot succession technique was used to quantify the strength of attachment of Ps. aeruginosa isolates. Different multiple drug–resistant Ps. aeruginosa isolates exhibited variable attachment strength. Although the highest multiple drug–resistant clinical isolate was shown to have the least attachment strength among clinical isolates, a weak correlation was found between attachment strength and multiple resistance among our investigated Ps. aeruginosa isolates. Conclusions: There is a weak correlation between multiple drug resistance and strength of attachment to stainless steel surfaces. Significance and Impact of the Study: Even low‐resistant Ps. aeruginosa could have the potential of attaching firmly to surfaces and forming biofilm.     

Heat stress adaptation of Escherichia coli under dynamic conditions: effect of inoculum size*

Aims: When subjected to dynamic temperatures surpassing the expected maximum growth temperature, Escherichia coli K12 MG1655 shows disturbed growth curves. These irregular population dynamics were explained by considering two subpopulations, i.e. a thermoresistant and a thermosensitive one ( Van Derlinden et al. 2010a ). In this paper, the influence of the initial cell concentration on the subpopulations’ dynamics is evaluated. Methods and Results: Experiments were performed in a bioreactor with the temperature increasing from 42 to 65·2°C (1 and 4°C h^?1) with varying initial cell concentrations [6, 12 and 18 ln(CFU ml^?1)]. When started from the highest cell concentration, the population was characterized by a higher overall maximum growth temperature and a higher inactivation temperature. For all experimental set‐ups, resistant cells were still growing at the final temperature of 65·2°C. Conclusions: The initial cell concentration had no effect on temperature resistance. The increase in temperature resistance of the sensitive subpopulation was because of the change of the physiological state to the stationary phase. Significance and Impact of the Study: A higher initial cell concentration leads to higher heat stress adaptation when cultures reach a maximum cell concentration. The observed growth at a temperature of 65·2°C is very important for food safety and the temperature treatment of micro‐organisms.     

Regulation of antioxidant metabolism by translation initiation factor 2alpha

Oxidative stress and highly specific decreases in glutathione (GSH) are associated with nerve cell death in Parkinson's disease. Using an experimental nerve cell model for oxidative stress and an expression cloning strategy, a gene involved in oxidative stress-induced programmed cell death was identified which both mediates the cell death program and regulates GSH levels. Two stress-resistant clones were isolated which contain antisense gene fragments of the translation initiation factor (eIF)2alpha and express a low amount of eIF2alpha. Sensitivity is restored when the clones are transfected with full-length eIF2alpha; transfection of wild-type cells with the truncated eIF2alpha gene confers resistance. The phosphorylation of eIF2alpha also results in resistance to oxidative stress. In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca(2+). In contrast, the resistant clones maintain high GSH levels and show no elevation in peroxides or Ca(2+) when stressed, and the GSH synthetic enzyme gamma-glutamyl cysteine synthetase (gammaGCS) is elevated. The change in gammaGCS is regulated by a translational mechanism. Therefore, eIF2alpha is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.     

Temporal patterns of genetic variation for resistance and infectivity in a Daphnia-microparasite system

Theoretical studies have indicated that the population genetics of host-parasite interactions may be highly dynamic. with parasites perpetually adapting to common host genotypes and hosts evolving resistance to common parasite genotypes. The present study examined temporal variation in resistance of hosts and infectivity of parasites within three populations of Daphnia magna infected with the sterilizing bacterium Pasteuria ramosa. Parasite isolates and host clones were collected in each of two years (1997, 1998) from one population; in two other populations, hosts were collected from both years, but parasites from only the first year. We then performed infection experiments (separately for each population) that exposed hosts to parasites from the same year or made combinations involving hosts and parasites from different years. In two populations, patterns were consistent with the evolution of host resistance: either infectivity or the speed with which parasites sterilized hosts declined from 1997 to 1998. In another population, infectivity, virulence, and parasite spore production did not vary among host-year or parasite-year. For this population, we also detected strong within-population genetic variation for resistance. Thus, in this case, genetic variability for fitness-related traits apparently did not translate into evolutionary change. We discuss a number of reasons why genetic change may not occur as expected in parasite-host systems, including negative correlations between resistance and other traits, gene flow, or that the dynamic process itself may obscure the detection of gene frequency changes.