Efflux Transporter Genes Research Articles

A Comprehensive Review of Nano-sized Silica’s Effects on Plant Growth, Molecular Responses, and Biochemical Changes

Silicon (Si) is a tetravalent metalloid and second most abundant on earth approximately 28%. Slags were used in agriculture as a silica fertilizer (10–28% Si); which improvise degraded crop soils, as well as crop growth and disease resistance. Si improves abiotic stress in crop lodging, UV tolerance, improves water uptakes and blocking the NaCl toxicity was observed in Gramineae family. It showed tolerance to biotic stress in terms of physical strengthening of plants against microbial infestation and activation of defensive enzymes such as peroxidase, polyphenol oxidase, lipoxygenase, phenylalanine ammonia lyase. There was an enhanced vegetative growth and germination percentage in varied vegetable crops with 100 to 500ppm range of silica nanoparticles. The plants available Si (H4SiO4) form in soil solution ranges about 0.1 to 0.6 mM and it get absorbed from roots via xylem to shoot containing a specialized cell surface protein called as Nod26-like major intrinsic protein (NIP) of aquaporin family. The list of total genes responsible for uptake of silica form are Low silica genes (Lsi1, Lsi2, Lsi3, Lsi6) and Si efflux transporter genes (SIET3, SIET4, SIET5). The nano-sized silica have been proven to be more validated impact on genes responsiveness via morpho-physiological parameter relationship. It is concluding that, use of silica in commercial crops have deep root to influence the genes and a bright future in its crop growth development aspects.

IPSC-derived blood-brain barrier modeling reveals APOE isoform-dependent interactions with amyloid beta

BackgroundThree common isoforms of the apolipoprotein E (APOE) gene - APOE2, APOE3, and APOE4 - hold varying significance in Alzheimer’s Disease (AD) risk. The APOE4 allele is the strongest known genetic risk factor for late-onset Alzheimer’s Disease (AD), and its expression has been shown to correlate with increased central nervous system (CNS) amyloid deposition and accelerated neurodegeneration. Conversely, APOE2 is associated with reduced AD risk and lower CNS amyloid burden. Recent clinical data have suggested that increased blood-brain barrier (BBB) leakage is commonly observed among AD patients and APOE4 carriers. However, it remains unclear how different APOE isoforms may impact AD-related pathologies at the BBB.MethodsTo explore potential impacts of APOE genotypes on BBB properties and BBB interactions with amyloid beta, we differentiated isogenic human induced pluripotent stem cell (iPSC) lines with different APOE genotypes into both brain microvascular endothelial cell-like cells (BMEC-like cells) and brain pericyte-like cells. We then compared the effect of different APOE isoforms on BBB-related and AD-related phenotypes. Statistical significance was determined via ANOVA with Tukey’s post hoc testing as appropriate.ResultsIsogenic BMEC-like cells with different APOE genotypes had similar trans-endothelial electrical resistance, tight junction integrity and efflux transporter gene expression. However, recombinant APOE4 protein significantly impeded the “brain-to-blood” amyloid beta 1–40 (Aβ40) transport capabilities of BMEC-like cells, suggesting a role in diminished amyloid clearance. Conversely, APOE2 increased amyloid beta 1–42 (Aβ42) transport in the model. Furthermore, we demonstrated that APOE-mediated amyloid transport by BMEC-like cells is dependent on LRP1 and p-glycoprotein pathways, mirroring in vivo findings. Pericyte-like cells exhibited similar APOE secretion levels across genotypes, yet APOE4 pericyte-like cells showed heightened extracellular amyloid deposition, while APOE2 pericyte-like cells displayed the least amyloid deposition, an observation in line with vascular pathologies in AD patients.ConclusionsWhile APOE genotype did not directly impact general BMEC or pericyte properties, APOE4 exacerbated amyloid clearance and deposition at the model BBB. Conversely, APOE2 demonstrated a potentially protective role by increasing amyloid transport and decreasing deposition. Our findings highlight that iPSC-derived BBB models can potentially capture amyloid pathologies at the BBB, motivating further development of such in vitro models in AD modeling and drug development.

Prevalence of qacEΔ1, qacE, oqxA, oqxB, acrA, cepA and zitB genes among multidrug-resistant Klebsiella pneumoniae isolated in a cardiac hospital

Background. Infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae are the leading cause of mortality worldwide. The widespread use of disinfectants and antiseptics has caused the emergence of K. pneumoniae with reduced sensitivity to them, which, in combination with MDR, can pose a significant epidemiological threat. The aim of the study was to assess the prevalence of efflux pump and transporter genes associated with biocide resistance and their association with antibiotic resistance among K. pneumoniae isolated in a cardiac surgical hospital. Materials and methods. K. pneumoniae isolates (n = 50) from the patients and medical equipment were tested by polymerase chain reaction for the presence of genes of 4 types of efflux pumps (qacEΔ1, qacE, oqxA, oqxB, acrA) and 2 transporters involved in the outflow of cations (cepA) and zinc ions (zitB). Spearman's rank correlation test was used to assess the strength of the association between the efflux pumps, beta-lactamase genes and mobile genetic elements. Results. The occurrence of K. pneumoniae containing qacEΔ1, qacE, oqxA, oqxB, acrA, cepA and zitB was high: 54, 62, 100, 84, 100, 72 и 96% respectively. K. pneumoniae with a combination of all the studied pumps was most often detected (32%), and these isolates were MDR in 100% of cases. The qacE, qacEΔ1 genes were closely associated with resistance to cephalosporins, carbapenems, fluoroquinolones, carbapenemase genes, and integrons. The results of the study showed that the genes of various efflux pumps associated with biocide resistance and their combinations were widely represented among the clinical isolates of MDR K. pneumoniae. Conclusion. The high prevalence of efflux pump genes associated with resistance to quaternary ammonium compounds, chlorhexidine and zinc salts and their significant association with antibiotic resistance in nosocomial K. pneumoniae underlines the importance of further studying the mechanisms of cross-resistance to biocides to improve methods of combating MDR nosocomial pathogens.

Tissue-specific deposition, speciation and transport of antimony in rice.

Rice (Oryza sativa) as a staple food is a potential intake source of antimony (Sb), a toxic metalloid. However, how rice accumulates this element is still poorly understood. Here, we investigated tissue-specific deposition, speciation, and transport of Sb in rice. We found that Sb(III) is the preferential form of Sb uptake in rice, but most Sb accumulates in the roots, resulting in a very low root-to-shoot translocation (less than 2%). Analysis of Sb deposition with laser ablation-inductively coupled plasma-mass spectrometry showed that most Sb deposits at the root exodermis. Furthermore, we found that Sb is mainly present as Sb(III) in the root cell sap after uptake. Further characterization showed that Sb(III) uptake is mediated by Low silicon rice 1 (Lsi1), a Si permeable transporter. Lsi1 showed transport activity for Sb(III) rather than Sb(V) in yeast (Saccharomyces cerevisiae). Knockout of Lsi1 resulted in a significant decrease in Sb accumulation in both roots and shoots. Sb concentration in the root cell sap of two independent lsi1 mutants decreased to less than 3% of that in wild-type rice, indicating that Lsi1 is a major transporter for Sb(III) uptake. Knockout of Lsi1 also enhanced rice tolerance to Sb toxicity. However, knockout of Si efflux transporter genes, including Lsi2 and Lsi3, did not affect Sb accumulation. Taken together, our results showed that Sb(III) is taken up by Lsi1 localized at the root exodermis and is deposited at this cell layer due to lack of Sb efflux transporters in rice.

Utilization of carbon catabolite repression for efficiently biotransformation of anthraquinone O-glucuronides by Streptomyces coeruleorubidus DM.

Carbon catabolite repression (CCR) is a highly conserved mechanism that regulates carbon source utilization in Streptomyces. CCR has a negative impact on secondary metabolite fermentation, both in industrial and research settings. In this study, CCR was observed in the daunorubicin (DNR)-producing strain Streptomyces coeruleorubidus DM, which was cultivated in high concentration of carbohydrates. Unexpectedly, DM exhibited a high ability for anthraquinone glucuronidation biotransformation under CCR conditions with a maximum bioconversion rate of 95% achieved at pH 6, 30°C for 24 h. The co-utilization of glucose and sucrose resulted in the highest biotransformation rate compared to other carbon source combinations. Three novel anthraquinone glucuronides were obtained, with purpurin-O-glucuronide showing significantly improved water solubility, antioxidant activity, and antibacterial bioactivity. Comparative transcript analysis revealed that glucose and sucrose utilization were significantly upregulated as DM cultivated under CCR condition, which strongly enhance the biosynthetic pathway of the precursors Uridine diphosphate glucuronic acid (UDPGA). Meanwhile, the carbon metabolic flux has significantly enhanced the fatty acid biosynthesis, the exhaust of acetyl coenzyme A may lead to the complete repression of the biosynthesis of DNR, Additionally, the efflux transporter genes were simultaneously downregulated, which may contribute to the anthraquinones intracellular glucuronidation. Overall, our findings demonstrate that utilizing CCR can be a valuable strategy for enhancing the biotransformation efficiency of anthraquinone O-glucuronides by DM. This approach has the potential to improve the bioavailability and therapeutic potential of these compounds, opening up new possibilities for their pharmaceutical applications.

Transcriptome analysis with different leaf blades identifies the phloem-specific phosphate transporter OsPHO1;3 required for phosphate homeostasis in rice.

Phosphate (Pi) is essential for plant growth and development. One strategy to improve Pi use efficiency is to enhance Pi remobilization among leaves. Using transcriptome analysis with first (top) and fourth (down) leaf blades from rice (Oryza sativa) in Pi-sufficient and deficient conditions, we identified 1384 genes differentially expressed among these leaf blades. These genes were involved in physiological processes, metabolism, transport, and photosynthesis. Moreover, we identified the Pi efflux transporter gene, OsPHO1;3, responding to Pi-supplied conditions among these leaf blades. OsPHO1;3 is highly expressed in companion cells of phloem, but not xylem, in leaf blades and induced by Pi starvation. Mutation of OsPHO1;3 led to Pi accumulation in second to fourth leaves under Pi-sufficient conditions, but enhanced Pi levels in first leaves under Pi-deficient conditions. These Pi accumulations in leaves of Ospho1;3 mutants resulted from induction of OsPHT1;2 and OsPHT1;8 in root and reduction of Pi remobilization in leaf blades, revealed by the decreased Pi in phloem of leaves. Importantly, lack of OsPHO1;3 caused growth defects under a range of Pi-supplied conditions. These results demonstrate that Pi remobilization is essential for Pi homeostasis and plant growth irrespective of Pi-supplied conditions, and OsPHO1;3 plays an essential role in Pi remobilization for normal plant growth.

PfPIN5 promotes style elongation by regulating cell length in Primula forbesii Franch.

Style dimorphism is one of the polymorphic characteristics of flowers in heterostylous plants, which have two types of flowers: the pin morph, with long styles and shorter anthers, and the thrum morph, with short styles and longer anthers. The formation of dimorphic styles has received attention in the plant world. Previous studies showed that CYP734A50 in Primula determined style length and limited style elongation and that the brassinosteroid metabolic pathway was involved in regulation of style length. However, it is unknown whether there are other factors affecting the style length of Primula. Differentially expressed genes highly expressed in pin morph styles were screened based on Primula forbesii transcriptome data. Virus-induced gene silencing was used to silence these genes, and the style length and anatomical changes were observed 20 days after injection. PfPIN5 was highly expressed in pin morph styles. When PfPIN5 was silenced, the style length was shortened in pin and long-homostyle plants by shortening the length of style cells. Moreover, silencing CYP734A50 in thrum morph plants increased the expression level of PfPIN5 significantly, and the style length increased. The results indicated that PfPIN5, an auxin efflux transporter gene, contributed to regulation of style elongation in P. forbesii. The results implied that the auxin pathway might also be involved in the formation of styles of P. forbesii, providing a new pathway for elucidating the molecular mechanism of style elongation in P. forbesii.

Impact and Diversity of ESBL-Producing Klebsiella pneumoniae Recovered from Raw Chicken Meat Samples in Türkiye.

The interrelationship between human, animal and environmental sectors leads to the spread of antibiotic resistance due to selective pressures, evolutionary traits and genomic evolution. In particular, the frequent use of antibiotics in livestock inevitably influences the emergence of specific resistance determinants in human strains, associated with reduced treatment options in clinical therapy. In this study, ESBL-producing Klebsiella pneumoniae strains isolated from chicken meat samples were evaluated for public health implications in Türkiye. Whole-genome sequencing was used for genetic dissection and phylogenetic comparison of their genomes. The isolates were assigned to four MLST types (ST147, ST37, ST2747 and ST219); two of them were found to represent the ST147 clone associated with severe human infections worldwide. In addition to cephalosporins, high resistance levels to quinolones/fluoroquinolones were identified phenotypically, caused by acquired resistance genes and chromosomal point variations. One isolate was also found to carry the qacE∆1 efflux transporter gene, which confers tolerance to quaternary ammonium compounds. The detection of virulence genes (i.e., that coding for enterobactin) associated with the pathogenicity of K. pneumoniae suggests a public health impact. Thus, comprehensive information on the occurrence and impact of K. pneumoniae from livestock is needed to derive appropriate management strategies for consumer protection. In this study, it was shown that poultry meat serves as a reservoir of clinically emerging multidrug-resistant high-risk clones.

Genomic insights into the adaptation of Acinetobacter johnsonii RB2-047 to the heavy metal-contaminated subsurface mine environment

The subsurface mine environments characterized by high levels of toxic metals and low nutrient availability represent an extreme threat to bacterial persistence. In recent study, the genomic analysis of the Acinetobacter johnsonii strain RB2-047 isolated from the Rozália Gold Mine in Slovakia was performed. As expected, the studied isolate showed a high level of heavy metal tolerance (minimum inhibitory concentrations were 500 mg/L for copper and nickel, 1,500 mg/L for lead, and 250 mg/L for zinc). The RB2-047 strain also showed noticeable resistance to several antibiotics (ampicillin, kanamycin, chloramphenicol, tetracycline and ciprofloxacin). The genomic composition analysis demonstrated a low number of antibiotic and metal resistance coding genes, but a high occurrence of efflux transporter genes located on the bacterial chromosome. The experimental inhibition of efflux pumps resulted in decreased tolerance to Zn and Ni (but not to Cu and Pb) and to all antibiotics tested. In addition, the H33342 dye-accumulation assay confirmed the high efflux activity in the RB2-047 isolate. These findings showed the important role of efflux pumps in the adaptation of Acinetobacter johsonii strain RB2-047 to metal polluted mine environment as well as in development of multi-antibiotic resistance.

A Novel NCI-H69AR Drug-Resistant Small-Cell Lung Cancer Mini-Tumor Model for Anti-Cancer Treatment Screening.

Small-cell lung cancer is a fast-growing carcinoma with a poor prognosis and a high level of relapse due to multi-drug resistance (MDR). Genetic mutations that lead to the overexpression of efflux transporter proteins can contribute to MDR. In vitro cancer models play a tremendous role in chemotherapy development and the screening of possible anti-cancer molecules. Low-cost and simple in vitro models are normally used. Traditional two-dimensional (2D) models have numerous shortcomings when considering the physiological resemblance of an in vivo setting. Three-dimensional (3D) models aim to bridge the gap between conventional 2D models and the in vivo setting. Some of the advantages of functional 3D spheroids include better representation of the in vivo physiology and tumor characteristics when compared to traditional 2D cultures. During this study, an NCI-H69AR drug-resistant mini-tumor model (MRP1 hyperexpressive) was developed by making use of a rotating clinostat bioreactor system (ClinoStar®; CelVivo ApS, Odense, Denmark). Spheroid growth and viability were assessed over a 25-day period to determine the ideal experimental period with mature and metabolically stable constructs. The applicability of this model for anti-cancer research was evaluated through treatment with irinotecan, paclitaxel and cisplatin for 96 h, followed by a 96 h recovery period. Parameters measured included planar surface area measurements, estimated glucose consumption, soluble protein content, intracellular adenosine triphosphate levels, extracellular adenylate kinase levels, histology and efflux transporter gene expression. The established functional spheroid model proved viable and stable during the treatment period, with retained relative hyperexpression of the MRP1 efflux transporter gene but increased expression of the P-gp transporter gene compared to the cells cultured in 2D. As expected, treatment with the abovementioned anti-cancer drugs at clinical doses (100 mg/m2 irinotecan, 80 mg/m2 paclitaxel and 75 mg/m2 cisplatin) had minimal impact on the drug-resistant mini-tumors, and the functional spheroid models were able to recover following the removal of treatment.

NIGT1 represses plant growth and mitigates phosphate starvation signaling to balance the growth response tradeoff in rice.

Inorganic phosphate (Pi) availability is an important factor that affects the growth and yield of crops, thus an appropriate and effective response to Pi-fluctuation is critical. However, how crops orchestrate Pi-signaling and growth under Pi-starvation conditions to optimize the growth defense tradeoff remains unclear. Here we show that a Pi-starvation induced transcription factor NIGT1 (NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1) controls plant growth and prevents a hyperresponse to Pi-starvation by directly repressing the expression of growth-related and Pi-signaling genes to achieve a balance between growth and response under a varying Pi environment. NIGT1 directly binds to the promoters of Pi-starvation signaling marker genes, like IPS1, miR827 and SPX2, under Pi-deficient conditions to mitigate the Pi-starvation responsive (PSR). It also directly represses the expression of vacuolar Pi efflux transporter genes VPE1/2 to regulate plant Pi-homeostasis. We further demonstrate that NIGT1 constrains shoot growth by repressing the expression of growth-related regulatory genes, including brassinolide signal transduction master regulator BZR1, cell division regulator CYCB1;1, and DNA replication regulator PSF3. Our findings reveal the function of NIGT1 in orchestrating plant growth and Pi-starvation signaling, and also provide evidence that NIGT1 acts as a safeguard to avoid hyperresponse during Pi-starvation stress in rice. This article is protected by copyright. All rights reserved.

Variable resistance to zinc intoxication among Streptococcus agalactiae reveals a novel IS1381 insertion element within the zinc efflux transporter gene czcD.

Streptococcus agalactiae, also known as group B Streptococcus, is an important human and animal pathogen. Zinc (Zn) is an essential trace element for normal bacterial physiology but intoxicates bacteria at high concentrations. Molecular systems for Zn detoxification exist in S. agalactiae, however the degree to which Zn detoxification may vary among different S. agalactiae isolates is not clear. We measured resistance to Zn intoxication in a diverse collection of clinical isolates of S. agalactiae by comparing the growth of the bacteria in defined conditions of Zn stress. We found significant differences in the ability of different S. agalactiae isolates to resist Zn intoxication; some strains such as S. agalactiae 18RS21 were able to survive and grow at 3.8-fold higher levels of Zn stress compared to other reference strains such as BM110 (6.4mM vs 1.68mM Zn as inhibitory, respectively). We performed in silico analysis of the available genomes of the S. agalactiae isolates used in this study to examine the sequence of czcD, which encodes an efflux protein for Zn that supports resistance in S. agalactiae. Interestingly, this revealed the presence of a mobile insertion sequence (IS) element, termed IS1381, in the 5' region of czcD in S. agalactiae strain 834, which was hyper-resistant to Zn intoxication. Interrogating a wider collection of S. agalactiae genomes revealed identical placement of IS1381 in czcD in other isolates from the clonal-complex-19 (CC19) 19 lineage. Collectively, these results show a resistance spectrum among S. agalactiae isolates enables survival in varying degrees of Zn stress, and this phenotypic variability has implications for understanding bacterial survival in metal stress.

Differential Expression of ABC Transporter Genes in Brain Vessels vs. Peripheral Tissues and Vessels from Human, Mouse and Rat.

ATP-binding cassette (ABC) transporters comprise a superfamily of genes encoding membrane proteins with nucleotide-binding domains (NBD). These transporters, including drug efflux across the blood-brain barrier (BBB), carry a variety of substrates through plasma membranes against substrate gradients, fueled by hydrolyzing ATP. The expression patterns/enrichment of ABC transporter genes in brain microvessels compared to peripheral vessels and tissues are largely uncharacterized. In this study, the expression patterns of ABC transporter genes in brain microvessels, peripheral tissues (lung, liver and spleen) and lung vessels were investigated using RNA-seq and WesTM analyses in three species: human, mouse and rat. The study demonstrated that ABC drug efflux transporter genes (including ABCB1, ABCG2, ABCC4 and ABCC5) were highly expressed in isolated brain microvessels in all three species studied; the expression of ABCB1, ABCG2, ABCC1, ABCC4 and ABCC5 was generally higher in rodent brain microvessels compared to those of humans. In contrast, ABCC2 and ABCC3 expression was low in brain microvessels, but high in rodent liver and lung vessels. Overall, most ABC transporters (with the exception of drug efflux transporters) were enriched in peripheral tissues compared to brain microvessels in humans, while in rodent species, additional ABC transporters were found to be enriched in brain microvessels. This study furthers the understanding of species similarities and differences in the expression patterns of ABC transporter genes; this is important for translational studies in drug development. In particular, CNS drug delivery and toxicity may vary among species depending on their unique profiles of ABC transporter expression in brain microvessels and BBB.

Association Between ABCG2, ABCB1, ABCC2 Efflux Transporter Single-Nucleotide Variants and Irinotecan Adverse Effects in Patients With Colorectal Cancer: A Real-Life Study.

Among patients treated with irinotecan, homozygous carriers of the UGT1A1*28 allele are at increased risk for neutropenia, but UGT1A1 genotype alone does not account for irinotecan-induced toxicity. Our aim was to study the association between single-nucleotide variants in genes encoding for efflux transporters of irinotecan (ABCG2, ABCB1, and ABCC2) and toxicity in real life. The source population was a cohort of patients with colorectal cancer (CRC) in Northern Israel, who had undergone genome-wide association study. From the source population we chose the patients with CRC prescribed irinotecan, and a comparative cohort of patients with CRC treated with other anticancer systemic therapies. Using Clalit Health Services electronic medical records (including laboratory results) we ascertained hematological and gastrointestinal adverse effects and mortality, within 90 days of the first dose, as a composite outcome. There were 601 patients with CRC who received irinotecan, and 756 patients with CRC treated with other anticancer regimens. The minor allele in rs2231142 (ABCG2) was associated with lower incidence of the composite outcome (odds ratio (OR)=0.54 (0.33, 0.91); P= 0.02) in irinotecan-treated patients with CRC, but not in patients with CRC treated with other regimens. ABCB1 rs1045642 and ABCC2 rs3740066 were not associated with the composite outcome. In a sensitivity analysis, adjusted for UGT1A1 status and for possible demographic and clinical confounders, adjusted OR was 0.56 (0.33, 0.94) for the association between rs2231142 (ABCG2) and the composite outcome. In conclusion, we describe a novel association between the minor allele of rs2231142 in the efflux transporter gene ABCG2 and protection against severe side effects in CRC patients treating with irinotecan.

<i>CDR1, CDR2, MDR1</i> and <i>ERG11</i> expression in azole resistant <i>Сandida albicans</i> isolated from HIV-infected patients in city of Moscow

Candida fungi are common opportunistic microorganisms capable of causing infections of various localization, as well as life-threatening conditions in immunocompromised patients, such as HIV-infected individuals, oncology patients, subjects undergoing HSCT, which number has been steadily increasing in recent years. In addition, resistance to anti-fungal drugs has been spreading as well. Naturally sensitive to azoles, C. albicans possess a variety of mechanisms of acquired resistance, including efflux transporters and target protein-encoding gene amplification. This study was conducted to assess a prevalence of such mechanisms in the isolates sample obtained from HIV-infected patients in the Moscow region of the Russian Federation, characterize a relationship between these mechanisms and patterns of developing drug resistance. 18 strains of C. albicans resistant to fluconazole and voriconazole were isolated from HIV-infected patients with recurrent oropharyngeal candidiasis in the Moscow region. The expression levels of the ERG11, MDR1, CDR1, CDR2 genes involved in the formation of acquired azole resistance were measured using quantitative PCR, the 2CT method with ACT and PMA genes as control genes and reference values of sensitive isolates. Expression levels exceeding the average values of sensitive isolates by more than 3 standard deviations were considered significantly elevated. In most of the isolates, elevated levels of CDR1 and CDR2 gene expression were found: 89% and 78%, respectively. The expression level of the MDR1 gene was increased only in 28% of cases. ERG11 expression levels were significantly elevated in 78% of the isolates. Expression levels of all resistance genes studied were significantly increased in 4 strains. In this sample of C. albicans isolates, acquired resistance is mainly associated with efflux vectors encoded by the CDR1 and CDR2 genes. Also, in most isolates, an increased expression level for the azole target protein gene ERG11 was detected. The expression level of the efflux transporter gene MDR1 was increased in the smallest number of samples. It is also impossible to exclude a potential role of other mechanisms in developing acquired resistance, such as mutations in the ERG11 gene. It can be assumed that the identified mechanisms of resistance result from long-term, widespread, and sometimes uncontrolled use of azoles, including those in treatment and prevention of candidiasis in HIV-infected patients.

EPEN-09. Multi-omics characterization of the blood-brain barrier in molecular groups of ependymoma

Abstract BACKGROUND: A major challenge in the treatment of brain tumors is the limited penetration of many drugs through the blood-brain barrier (BBB). BBB characteristics include endothelial cells connected by tight junction proteins (e.g. Claudin5) and expression of efflux transporters (e.g.P-gp) with the physiological role to limit the accumulation of potentially toxic substances in brain tissue. While BBB permeability shows anatomical region-specific characteristics and changes with age, brain tumors can also impact its integrity. To achieve therapeutically relevant drug concentrations in the tumor, characterization of the pathophysiological BBB is fundamental when developing (pre)clinical trials. Our study seeks to increase our understanding of the BBB composition in various molecular groups of ependymoma. RESULTS: Transcriptional BBB characteristics were assessed for primary ependymoma (n=440), cell lines (n=3), mouse models (n=22), and healthy brain controls (n=200). T-distributed stochastic neighbor embedding (tSNE)-based clustering analyses based on most relevant tight junction and efflux transporter gene sets revealed distinct molecular ependymoma group-specific expression patterns. While patient-derived xenografts models (PDX, n=20) showed high similarity with patient tumor samples, in utero electroporation-based (IUE, n=2) mouse models did not fully recapitulate these BBB characteristics. Supratentorial ependymoma with ZFTA fusions revealed a higher transcriptional expression level of important tight junctions (e.g.Claudin5) compared to other groups and normal brain which was confirmed at protein level in respective PDX models by using both western blot and ultra-high content imaging. CONCLUSION: Analyses of important BBB markers revealed significant differences between molecular groups of ependymoma, which may partly explain drug resistance of ependymoma with ZFTA fusion caused by a low BBB permeability. BBB characteristics of corresponding models suggest that IUE and PDX models representing ZFTA fusion-driven tumors cannot equally be used within preclinical drug trials. Findings will be further validated in preclinical studies while molecular BBB characterization will be expanded to other brain tumors.

Zap1 is required for Candida glabrata response to fluconazole

The increasing prevalence of fluconazole-resistant clinical isolates of Candida spp. strongly hinders the widespread use of the drug. To tackle this problem, great efforts have been made to fully understand the fungal response to fluconazole. In this work, we show that the role of Zap1 in Candida glabrata goes beyond regulating yeast adaptation to zinc deficiency. In line with our previous observation that deletion of ZAP1 makes yeast cells more sensitive to fluconazole, we found that the mutant CgΔzap1 accumulates higher levels of the drug, which correlates well with its lower levels of ergosterol. Surprisingly, Zap1 is a negative regulator of the drug efflux transporter gene CDR1 and of its regulator, PDR1. The apparent paradox of drug accumulation in cells where genes encoding transporters relevant for drug extrusion are being overexpressed led us to postulate that their activity could be impaired. In agreement, Zap1-depleted cells present, in addition to decreased ergosterol levels, an altered composition of membrane phospholipids, which together should impact membrane function and impair the detoxification of fluconazole. Overall, our study brings to light Zap1 as an important hub in Candida glabrata response to fluconazole.

Nitric oxide alleviates salt-induced stress damage by regulating the ascorbate–glutathione cycle and Na+/K+ homeostasis in Nitraria tangutorum Bobr

Nitric oxide (NO) is an important signaling molecule involved in mediation of salt stress induced physiological responses in plants. In this study, we investigated the effect of NO on Nitraria tangutorum seedlings exposed to salt stress. Exogenous application of NO donor, sodium nitroprusside (SNP) increased fresh weight, shoot and root elongation and decreased electrolyte leakage and malondialdehyde (MDA) content in N. tangutorum seedlings under salt stress. Simultaneously, leaf senescence and root damage induced by salt stress were alleviated. SNP effectively increased NO content both in leaves and roots of plants under salt stress. Meanwhile, SNP activated the ascorbate-glutathione (AsA-GSH) cycle by increasing antioxidants contents, antioxidant enzymes activities, and related genes expression, thereby scavenging reactive oxygen species (ROS) and alleviating oxidative damage caused by salt stress. SNP alleviated salt stress induced ion toxicity by promoting Na+ efflux and ion transporter gene expression and reducing Na+ content and the Na+/K+ ratio. In addition, application of NO specific scavenger cPTIO and mammalian NO synthase inhibitor L-NAME sifnificantly aggravated stress damage in plant under salt stress. These results show the beneficial impacts of NO as a stress-signaling molecule that positively regulates defense response in N. tangutorum to salt stress.

Azole and echinocandin resistance mechanisms and genotyping of Candida tropicalis in Japan: cross-boundary dissemination and animal–human transmission of C. tropicalis infection

ObjectivesTo assess the prevalence and genetic basis of antifungal resistance mechanisms as well as the genotyping of Candida tropicalis from clinical and non-clinical sources in Japan. MethodsEighty C. tropicalis isolates, including 32 clinical isolates recovered from 29 patients and 48 non-clinical isolates recovered from 24 different sources (animals and the environment) were evaluated. All isolates were tested phenotypically for resistance to a wide range of antifungals and genotypically for resistance mechanisms to azole and echinocandin. Furthermore, all the isolates were genotyped by multilocus sequence typing (MLST). ResultsPhenotypically, 30.2% (16/53) of the isolates were azole-resistant, with high levels of azole resistance among clinical isolates (51.7%; 15/29) and low levels (4.2%; 1/24) among non-clinical isolates. None of the isolates were reported as echinocandin resistant, with 60.4% (32/53) of the isolates intermediate to caspofungin. Azole resistance was basically attributed to high expression levels of drug efflux transporter genes (CDR2 and CDR3), transcription factors (TAC1 and UPC2) and ergosterol biosynthesis pathway HMG gene. No FKS1 hot spot 1 (HS1) or HS2 missense mutations were detected in any of the isolates. MLST analysis revealed 36 different sequence types (STs), with the first identification of 23 new STs. Phylogenetic analysis confirmed the close relationship between the clinical and non-clinical isolates, with identifications of ST232 and ST933 among patients and marine mammals. ConclusionOur results confirmed the emergence of azole resistance in C. tropicalis in Japan. Furthermore, phylogenetic analysis confirmed the transboundary dissemination and cross-transmission of C. tropicalis between humans and animals.

Messenger RNA Expression of Albumin, Transferrin, Transthyretin, Asialoglycoprotein Receptor, Cytochrome P450 Isoform, Uptake Transporter, and Efflux Transporter Genes as a Function of Culture Duration in Prolonged Cultured Cryopreserved Human Hepatocytes as Collagen-Matrigel Sandwich Cultures: Evidence for Redifferentiation upon Prolonged Culturing.

Hepatic gene expression as a function of culture duration was evaluated in prolonged cultured human hepatocytes. Human hepatocytes from seven donors were maintained as near-confluent collagen-Matrigelsandwich cultures, with messenger RNA expression for genes responsible for key hepatic functions quantified by real-time polymerase chain reaction at culture durations of 0 (day of plating), 2, 7, 9, 16, 23, 26, 29, 36, and 43 days. Key hepatocyte genes were evaluated, including the differentiation markers albumin, transferrin, and transthyretin; the hepatocyte-specific asialoglycoprotein receptor 1 cytochrome P450 isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A7; uptake transporter isoforms SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, and SLCO2B1; efflux transporter isoforms ATP binding cassette (ABC)B1, ABCB11, ABCC2, ABCC3, ABCC4, and ABCG2; and the nonspecific housekeeping gene hypoxanthine ribosyl transferase 1 (HPRT1). The well established dedifferentiation phenomenon was observed on day 2, with substantial (>80%) decreases in gene expression in day 2 cultures observed for all genes evaluated except HPRT1 and efflux transporters ABCB1, ABCC2, ABCC3 (<50% decrease in expression), ABCC4 (>400% increase in expression), and ABCG2 (no decrease in expression). All genes with a >80% decrease in expression were found to have increased levels of expression on day 7, with peak expression observed on either day 7 or day 9, followed by a gradual decrease in expression up to the longest duration evaluated of 43 days. Our results provide evidence that cultured human hepatocytes undergo redifferentiation upon prolonged culturing. SIGNIFICANCE STATEMENT: This study reports that although human hepatocytes underwent dedifferentiation upon 2 days of culture, prolonged culturing resulted in redifferentiation based on gene expression of differentiation markers, uptake and efflux transporters, and cytochrome P450 isoforms. The observed redifferentiation suggests that prolonged (>7 days) culturing of human hepatocyte cultures may represent an experimental approach to overcome the initial dedifferentiation process, resulting in "stabilized" hepatocytes that can be applied toward the evaluation of drug properties requiring an extended period of treatment and evaluation.