Multidrug Resistance Phenomenon Research Articles

Genomic analysis of the nomenclatural type strain of the nematode-associated entomopathogenic bacterium Providencia vermicola

BackgroundEnterobacteria of the genus Providencia are mainly known as opportunistic human pathogens but have been isolated from highly diverse natural environments. The species Providencia vermicola comprises insect pathogenic bacteria carried by entomoparasitic nematodes and is investigated as a possible insect biocontrol agent. The recent publication of several genome sequences from bacteria assigned to this species has given rise to inconsistent preliminary results.ResultsThe genome of the nematode-derived P. vermicola type strain DSM_17385 has been assembled into a 4.2 Mb sequence comprising 5 scaffolds and 13 contigs. A total of 3969 protein-encoding genes were identified. Multilocus sequence typing with different marker sets revealed that none of the previously published presumed P. vermicola genomes represents this taxonomic species. Comparative genomic analysis has confirmed a close phylogenetic relationship of P. vermicola to the P. rettgeri species complex. P. vermicola DSM_17385 carries a type III secretion system (T3SS-1) with probable function in host cell invasion or intracellular survival. Potentially antibiotic resistance-associated genes comprising numerous efflux pumps and point-mutated house-keeping genes, have been identified across the P. vermicola genome. A single small (3.7 kb) plasmid identified, pPVER1, structurally belongs to the qnrD-type family of fluoroquinolone resistance conferring plasmids that is prominent in Providencia and Proteus bacteria, but lacks the qnrD resistance gene.ConclusionsThe sequence reported represents the first well-supported published genome for the taxonomic species P. vermicola to be used as reference in further comparative genomics studies on Providencia bacteria. Due to a striking difference in the type of injectisome encoded by the respective genomes, P. vermicola might operate a fundamentally different mechanism of entomopathogenicity when compared to insect-pathogenic Providencia sneebia or Providencia burhodogranariea. The complete absence of antibiotic resistance gene carrying plasmids or mobile genetic elements as those causing multi drug resistance phenomena in clinical Providencia strains, is consistent with the invertebrate pathogen P. vermicola being in its natural environment efficiently excluded from the propagation routes of multidrug resistance (MDR) carrying genetic elements operating between human pathogens. Susceptibility to MDR plasmid acquisition will likely become a major criterion in the evaluation of P. vermicola for potential applications in biological pest control.

Novel Strategies to Combat the Emerging Drug Resistance in Human Pathogenic Microbes.

The major health-care burden for the developing world are infectious diseases where antimicrobial agents prove to be the magical drugs to combat this. But the phenomenon of antimicrobial resistance (AMR) represents a challenging global issue, which requires to be addressed effectively. The antimicrobial treatment for the emerging multidrug-resistant bacterial (e.g. TB, Cholera) and fungal (e.g. Candidiasis) infections is very limited, and there are multiple causes and reasons responsible for the evolution of such resistance. Considering the critical issues of increasing AMR, there is an urgent requirement of the identification, development, validation, and progression of novel strategies and approaches that can easily be utilized for overcoming this serious issue. Immunotherapy represents a significant way to improve host defenses and combat the issue of antimicrobial drug resistance. Similarly, drug combination therapy represents another promising approach for reducing the evolution of resistance and enhancing the longevity of the antimicrobial agents. Bacteriophage therapy also acts as a novel therapeutic option to control the development of the multidrug resistance (MDR) phenomenon. Besides, CRISPR, an innovative genome editing technology, offers multiple applications to safeguard host defenses to overcome different resistance challenges. The novel approaches/ strategies like combination therapy, bacteriophage therapy, immunotherapy, and CRISPR/Cas discussed here presents an overview of some of the novel strategies/approaches to be adopted against the pathogenic microbes/microbial invasions along with advanced knowledge of different drug resistance mechanisms adopted by the microbial pathogens to gain resistance against different antimicrobial agents. Therefore, understanding the novel control plans/approaches and different drug resistance mechanisms will help achieve the goals of the successful development of potential antimicrobial drugs and their respective targets and eventually help curtail the problem of increasing antimicrobial drug resistance menace in various human pathogenic microbes.

Differential Action of Silver Nanoparticles on ABCB1 (MDR1) and ABCC1 (MRP1) Activity in Mammalian Cell Lines.

Silver nanoparticles (AgNPs), due to their unique properties have been receiving immense attention in recent years. In addition to their antibacterial and antifungal activities, AgNPs also cause apoptosis, mitochondria disfunction, nucleic acid damage and show potent anticancer properties in both multidrug resistance (MDR) and sensitive tumors. The MDR phenomenon, caused by the presence of ATP-binding cassette (ABC) proteins, is responsible for the failure of chemotherapy. Thus, investigating the influence of widely used AgNPs on ABC transporters is crucial. In the present study, we have examined the cytotoxicity of silver nanoparticles of a nominal size of 20 nm (Ag20) on the cell lines of different tissue origins. In addition, we have checked the ATP-binding cassette transporters’ activity and expression under AgNP exposure. The results indicate that Ag20 shows a toxic effect on tested cells, as well as modulating the expression and transport activity of ABC proteins.

CRISPR: The Multidrug Resistance Endgame?

The flexibility of microbes to undergo or adapt to the changes in their physiology and genotypical traits has enabled the microbes acquiring resistance to latest or recently discovered drugs which have consequently led to the menace of multidrug resistance (MDR). There is a surge in the discovery of novel antibiotics to counter the rising MDR phenomena, and in such a quest, for investigating an efficient alternative mechanism or compound to combat MDR, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has piqued the interests of the researchers across the globe. CRISPR-Cas9 technology is a genome-editing tool with successful widespread applications in cell lines, plants, animals, and even in human clinical trials, and it is seriously being considered as a potential candidate for countering MDR. This review encompasses the broad scope of CRISPR-Cas9 along with its various variations, underlying principles, mechanisms, as well as applications. Furthermore, the implications of recent advancements in various disciplines are highlighted to enhance the applicability of this technique. Consequently, its research gaps and challenges are also identified so that they can be addressed in the possible future thereby further expanding the lore of CRISPR-Cas9 technique.

Novel Symmetrical Cage Compounds as Inhibitors of the Symmetrical MRP4-Efflux Pump for Anticancer Therapy.

Within the last decades cancer treatment improved by the availability of more specifically acting drugs that address molecular target structures in cancer cells. However, those target-sensitive drugs suffer from ongoing resistances resulting from mutations and moreover they are affected by the cancer phenomenon of multidrug resistance. A multidrug resistant cancer can hardly be treated with the common drugs, so that there have been long efforts to develop drugs to combat that resistance. Transmembrane efflux pumps are the main cause of the multidrug resistance in cancer. Early inhibitors disappointed in cancer treatment without a proof of expression of a respective efflux pump. Recent studies in efflux pump expressing cancer show convincing effects of those inhibitors. Based on the molecular symmetry of the efflux pump multidrug resistant protein (MRP) 4 we synthesized symmetric inhibitors with varied substitution patterns. They were evaluated in a MRP4-overexpressing cancer cell line model to prove structure-dependent effects on the inhibition of the efflux pump activity in an uptake assay of a fluorescent MRP4 substrate. The most active compound was tested to resentisize the MRP4-overexpressing cell line towards a clinically relevant anticancer drug as proof-of-principle to encourage for further preclinical studies.

Multidrug Resistance in Mammals and Fungi-From MDR to PDR: A Rocky Road from Atomic Structures to Transport Mechanisms.

Multidrug resistance (MDR) can be a serious complication for the treatment of cancer as well as for microbial and parasitic infections. Dysregulated overexpression of several members of the ATP-binding cassette transporter families have been intimately linked to MDR phenomena. Three paradigm ABC transporter members, ABCB1 (P-gp), ABCC1 (MRP1) and ABCG2 (BCRP) appear to act as brothers in arms in promoting or causing MDR in a variety of therapeutic cancer settings. However, their molecular mechanisms of action, the basis for their broad and overlapping substrate selectivity, remains ill-posed. The rapidly increasing numbers of high-resolution atomic structures from X-ray crystallography or cryo-EM of mammalian ABC multidrug transporters initiated a new era towards a better understanding of structure–function relationships, and for the dynamics and mechanisms driving their transport cycles. In addition, the atomic structures offered new evolutionary perspectives in cases where transport systems have been structurally conserved from bacteria to humans, including the pleiotropic drug resistance (PDR) family in fungal pathogens for which high resolution structures are as yet unavailable. In this review, we will focus the discussion on comparative mechanisms of mammalian ABCG and fungal PDR transporters, owing to their close evolutionary relationships. In fact, the atomic structures of ABCG2 offer excellent models for a better understanding of fungal PDR transporters. Based on comparative structural models of ABCG transporters and fungal PDRs, we propose closely related or even conserved catalytic cycles, thus offering new therapeutic perspectives for preventing MDR in infectious disease settings.

High Intrinsic Expression of P-glycoprotein and Breast Cancer Resistance Protein in Canine Mammary Carcinomas Regardless of Immunophenotype and Outcome.

Simple SummaryMultidrug resistance of neoplastic cells to chemotherapeutic drugs is a phenomenon mediated by several molecular mechanisms. Among these, P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) counteract the intracellular load of multiple drugs, preventing their efficacy. The basal (intrinsic) cellular expression can be further stimulated by drug exposure. P-gp and BCRP are a subject of intense investigation both in human and veterinary oncology since a better understanding of how their expression is distributed across different tumors allows planning alternative therapeutic strategies. In canine mammary carcinomas, a phenotypic classification similar to the one widely adopted for breast cancer is currently employed. For Basal- and Normal-like phenotypes, chemotherapy is still the main option. In this study, we observed that canine mammary carcinomas bear a high intrinsic expression of both P-gp and BCRP, regardless of their molecular phenotype, and their presence does not influence the outcome.P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are major actors in multidrug resistance (MDR) phenomenon in both human and canine mammary carcinomas (CMCs). The aim of this study was to investigate an association between the intrinsic expression of P-gp and BCRP compared to the immunophenotypes and outcome in CMCs. Fifty CMCs were evaluated at immunohistochemistry (IHC) for P-gp, BCRP, Estrogen receptor alpha (ER), Progesterone receptors (PR), Human Epidermal Growth Factor Receptor type 2 (HER2), basal cytokeratins 5/6 (CK5/6), Epidermal Growth Factor Receptor 1 (EGFR), and Ki67 proliferation index. P-gp and BCRP positive cases were, respectively, 52% and 74.5%, with a significantly higher expression of BCRP than P-gp. Five immunophenotypes were defined in 37 out of 50 CMCs: 9 (24.3%) Luminal A, 5 (13.5%) Luminal B, 9 (24.3%) HER2 overexpressing, 9 (24.3%) Triple-negative basal-like, and 5 (13.5%) Triple-negative non-basal-like. In all CMCs at least one marker was expressed. Follow-up data were available for 25 animals. The average cancer-specific survival was 739 ± 444 days. A number of CMCs bear a high expression of P-gp and BCRP but no significant association was found between their expression and the immunophenotypes, Ki67 index, the histological grade, and tumor-related death.

The Role of miRNA-7 in the Biology of Cancer and Modulation of Drug Resistance.

MicroRNAs (miRNAs, miRs) are small non-coding RNA (ncRNA) molecules capable of regulating post-transcriptional gene expression. Imbalances in the miRNA network have been associated with the development of many pathological conditions and diseases, including cancer. Recently, miRNAs have also been linked to the phenomenon of multidrug resistance (MDR). MiR-7 is one of the extensively studied miRNAs and its role in cancer progression and MDR modulation has been highlighted. MiR-7 is engaged in multiple cellular pathways and acts as a tumor suppressor in the majority of human neoplasia. Its depletion limits the effectiveness of anti-cancer therapies, while its restoration sensitizes cells to the administered drugs. Therefore, miR-7 might be considered as a potential adjuvant agent, which can increase the efficiency of standard chemotherapeutics.

Molecular insights into the Patched1 drug efflux inhibitory activity of panicein A hydroquinone: a computational study.

Human Hedgehog receptor Patched1 (PTCH1) is able to efflux chemotherapeutics of different chemical structure out of cancer cells thus contributing to multidrug resistance phenomena in tumor treatment. A screening of natural compounds purified from marine sponges led to the identification of the first PTCH1 efflux inhibitor, panicein A hydroquinone (PAH), demonstrated to increase doxorubicin toxicity in vitro and vemurafenib toxicity in vitro and in vivo. In this work we combined different computational techniques to gain molecular insights of the inhibitory activity of PAH and some of its active and inactive analogues. We first performed a thorough characterization and druggability analysis of the main putative substrate binding pockets known from available cryo-electron microscopy structures. Further, dynamical descriptors of the active and inactive PAH analogues were extracted from microsecond-long all-atom molecular dynamics simulations in water solution. Finally, a blind ensemble docking methodology coupled with the conformational analysis of compounds enabled rationalization of the interaction between PTCH1 and PAH and derivatives in terms of their intrinsic physico-chemical properties. Our results suggest that the Neck pocket is the preferential binding site for PAH analogues on PTCH1, and that compounds assuming an open cylindric-like shape in solution are most likely to be good binders for PTCH1.

Mutagenesis Analysis of ABCB4 Gene Promoter of Danio rerio

Zebrafish abcb4 gene (ortholog to human ABCB1 gene) serves primarily in multidrug resistance (MDR) mechanism by effluxing chemotherapeutic agents, chemicals, xenobiotics, and numerous anti-cancer drugs out of the cells. This study aims to identify the specific transcription factor binding sites (TFBS) within the promoter region of zebrafish abcb4 gene and determine the functional roles of these factors in abcb4 gene expression regulation via mutagenesis analysis. First, primers were designed to target and amplify the promoter region of zebrafish abcb4 gene through gradient PCR. The zebrafish abcb4 gene promoter was then cloned into pGL3.0 vector and sent for sequencing. The sequencing results revealed high similarity to zebrafish DNA sequence from clone DKEY-24I24 in linkage group 16, indicating a successful cloning of targeted gene. Thereafter, consensus sequence of zebrafish abcb4 gene promoter was generated with the length of 1,392 bp which was close to its expected size during primer design (1,500 bp). Using MATCH tool, 155 TFBSs were found within zebrafish abcb4 gene promoter region. Activator protein 1 (AP-1) TFBS at 1,255 bp was chosen to be mutated through site-directed mutagenesis. Mutagenic primers (forward primer: 5’ GGG CAA GGC AGT ATA AAC GTG 3’ and reverse primer: 5’ TTA TGT TTC TAG GGA TTA CGT CAC 3’) were designed to substitute AGT with GGG to remove the AP-1 TFBS. By mutating the zebrafish abcb4 gene promoter, the MDR phenomenon driven by zebrafish abcb4 gene can be elucidated and this might provide clues to the development of tumor and malignancy in human. The results from this study may enrich the knowledge in chemotherapy and cancer treatments.

INFLUENCE OF POLYSACCHARIDE COMPLEXES OF PLANTS OF CENTRAL RUSSIA ON THE P-GLYCOPROTEIN ACTIVITY IN VITRO

The purpose of this work was to study the effect of polysaccharide complexes isolated from tansy flowers (Tanacetum vulgare L., fam. Asteraceae) and melilotus herb (Melilotus officinalis L., fam. Fabaceae) on P-glycoprotein (Pgp, ABCB1 protein) activity in vitro.
 On Caco-2 cell line the effect of polysaccharide complex isolated from tansy flowers and melilotus herb on Pgp activity was studied. In vitro Pgp activity was assessed by the transport of fexofenadine in the transwell system. High performance liquid chromatography with UV detection at wavelength 220 nm was used to determine fexofenadine concentration in the transport medium.
 It was revealed that when polysaccharide isolated from tansy flowers was added to the transport medium in concentrations 10 and 100 μM the ratio of the apparent permeability coefficients of fexofenadine b-a/a-b decreased by 1.81 and 2.65 times, respectively, compared with the series of isolated transport of fexofenadine, which indicated decreased Pgp functional activity under the polysaccharide action. The polysaccharide complex of the melilotus herb did not change the b-a/a-b ratio in any of the applied concentrations, thus it did not affect the activity of this transporter. It is advisable to continue the study of tansy flower polysaccharide complex as an inhibitor of Pgp transporter protein in order to assess the possibility of its clinical use for the treatment of pharmacoresistant forms of cancer by overcoming the phenomenon of multidrug resistance of cells.

Virtual Screening of FDA-Approved Drugs against LasR of Pseudomonas aeruginosa for Antibiofilm Potential

Pseudomonas aeruginosa is a Gram-negative pathogenic bacterium that is present commonly in soil and water and is responsible for causing septic shock, pneumonia, urinary tract and gastrointestinal infections, etc. The multi-drug resistance (MDR) phenomenon has increased dramatically in past years and is now considered a major threat globally, so there is an urgent need to develop new strategies to overcome drug resistance by P. aeruginosa. In P. aeruginosa, a major factor of drug resistance is associated to the formation of biofilms by the LasR enzyme, which regulates quorum sensing and has been reported as a new therapeutic target for designing novel antibacterial molecules. In this study, virtual screening and molecular docking were performed against the ligand binding domain (LBD) of LasR by employing a pharmacophore hypothesis for the screening of 2373 FDA-approved compounds to filter top-scoring hit compounds. Six inhibitors out of 2373 compounds were found to have binding affinities close to that of known LasR inhibitors. The binding modes of these compounds to the binding site in LasR-LBD were analyzed to identify the key interactions that contribute to the inhibition of LasR activity. Then, 50 ns simulations of top hit compounds were performed to elucidate the stability of their binding conformations with the LasR-LBD. This study, thus concluded that sulfamerazine showed the highest binding affinity for the LasR-LBD binding pocket exhibiting strong inhibitory binding interactions during molecular dynamics (MD) simulation.

Abstract 3006: Anticancer and multidrug resistance-reversing activities of novel antimicrobial peptides

Abstract Cancer is a growing concern in public health problems worldwide. Although the development of advanced chemotherapies has already partly reduced the cancer death rate and improved patients' prognosis, the phenomenon of multidrug resistance (MDR), which impairs the efficacy of a serial structure-unrelated anticancer agent, casts a large shadow over the bright future of chemotherapies. MDR has various mechanisms to induce acquired drug resistance of cancer cells after a relatively long period of chemotherapy. One of the most common causes of MDR is the expression of ATP-binding cassette (ABC) transporters, which act as efflux pumps on cancer cell membranes and transport anticancer drugs out of cancer cells thereby reducing the intracellular drug concentration. The growing resistance of cancer cells to current anticancer drugs leads to the urgent need to develop anticancer drugs with a new mechanism of action. Over the past decade, antimicrobial peptides have entered the view of scientists as a new generation of anticancer drugs. In this study, the anticancer effects and the reversal activities against ABC transporter mediated MDR of four novel antimicrobial peptides were investigated. The results showed that, at non-toxic concentrations in vitro, peptides #7, #14, #22, and #24 significantly sensitized ABCB1- and ABCC1-overexpressing cell lines, including both drug-selected MDR cancer cell lines and ABCB1 or ABCC1 gene transfected HEK293 cell lines, to the substrate anticancer drugs paclitaxel and vincristine. Among the antimicrobial peptide compounds, #14 and #24 with the most potent reversal ability were selected for further investigation. These peptides increased the intracellular accumulation of [3H]-paclitaxel in ABCB1 overexpressing cells and that of [3H]-vincristine in ABCC1 overexpressing cells by suppressing the efflux function of ABCB1 and ABCC1 transporters, without causing alteration in the expression levels and localization patterns of ABCB1 and ABCC1 in overexpressing cells. Furthermore, the results of the ATPase assay showed that both two peptides stimulated ABCB1 ATPase activity. Taken together, these results demonstrated that peptide #14 and #24 reverse ABCB1- and ABCC1-mediated MDR through blocking the function of ABCB1 and ABCC1 without affecting the transporters' expression and cellular localization, which suggests that antimicrobial peptides may be used as a novel prospective cancer therapeutic strategy in the combination with traditional anticancer agents. Citation Format: Qiu-Xu Teng, Zi-Ning Lei, Xiaofang Luo, Jing-Quan Wang, Zuodong Qin, John N. Wurpel, Zhe-Sheng Chen. Anticancer and multidrug resistance-reversing activities of novel antimicrobial peptides [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3006.

Overcoming Multidrug Resistance: Flavonoid and Terpenoid Nitrogen-Containing Derivatives as ABC Transporter Modulators.

Multidrug resistance (MDR) in cancer is one of the main limitations for chemotherapy success. Numerous mechanisms are behind the MDR phenomenon wherein the overexpression of the ATP-binding cassette (ABC) transporter proteins P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance protein 1 (MRP1) is highlighted as a prime factor. Natural product-derived compounds are being addressed as promising ABC transporter modulators to tackle MDR. Flavonoids and terpenoids have been extensively explored in this field as mono or dual modulators of these efflux pumps. Nitrogen-bearing moieties on these scaffolds were proved to influence the modulation of ABC transporters efflux function. This review highlights the potential of semisynthetic nitrogen-containing flavonoid and terpenoid derivatives as candidates for the design of effective MDR reversers. A brief introduction concerning the major role of efflux pumps in multidrug resistance, the potential of natural product-derived compounds in MDR reversal, namely natural flavonoid and terpenoids, and the effect of the introduction of nitrogen-containing groups are provided. The main modifications that have been performed during last few years to generate flavonoid and terpenoid derivatives, bearing nitrogen moieties, such as aliphatic, aromatic and heterocycle amine, amide, and related functional groups, as well as their P-gp, MRP1 and BCRP inhibitory activities are reviewed and discussed.

Homology Modeling of the Human P-glycoprotein (ABCB1) and Insights into Ligand Binding through Molecular Docking Studies.

The ABCB1 transporter also known as P-glycoprotein (P-gp) is a transmembrane protein belonging to the ATP binding cassette super-family of transporters; it is a xenobiotic efflux pump that limits intracellular drug accumulation by pumping the compounds out of cells. P-gp contributes to a decrease of toxicity and possesses broad substrate specificity. It is involved in the failure of numerous anticancer and antiviral chemotherapies due to the multidrug resistance (MDR) phenomenon, where it removes the chemotherapeutics out of the targeted cells. Understanding the details of the ligand–P-gp interaction is therefore crucial for the development of drugs that might overcome the MRD phenomenon and for obtaining a more effective prediction of the toxicity of certain compounds. In this work, an in silico modeling was performed using homology modeling and molecular docking methods with the aim of better understanding the ligand–P-gp interactions. Based on different mouse P-gp structural templates from the PDB repository, a 3D model of the human P-gp (hP-gp) was constructed by means of protein homology modeling. The homology model was then used to perform molecular docking calculations on a set of thirteen compounds, including some well-known compounds that interact with P-gp as substrates, inhibitors, or both. The sum of ranking differences (SRD) was employed for the comparison of the different scoring functions used in the docking calculations. A consensus-ranking scheme was employed for the selection of the top-ranked pose for each docked ligand. The docking results showed that a high number of π interactions, mainly π–sigma, π–alkyl, and π–π type of interactions, together with the simultaneous presence of hydrogen bond interactions contribute to the stability of the ligand–protein complex in the binding site. It was also observed that some interacting residues in hP-gp are the same when compared to those observed in a co-crystallized ligand (PBDE-100) with mouse P-gp (PDB ID: 4XWK). Our in silico approach is consistent with available experimental results regarding P-gp efflux transport assay; therefore it could be useful in the prediction of the role of new compounds in systemic toxicity.

MDR1 Gene Polymorphisms and Its Association With Expression as a Clinical Relevance in Terms of Response to Chemotherapy and Prognosis in Ovarian Cancer.

In spite of the significant advancements in the treatment modalities, 30% of advanced stage ovarian cancer (OC) patients do not respond to the standard chemotherapeutic regimen and most of the responders finally relapse over time due to the escalation of multidrug resistance (MDR) Phenomenon. Our present study evaluated chemotherapeutic sensitivity response among 47 ovarian tumor patients of which we found 37 (78.8%) sensitive and remaining 10 (21.2%) resistant. Among the resistant, seven tumor samples were found to be platinum resistant or refractory to platinum (CB/TX), one to carboplatin, and two to 5FU. Notably, all these resistant cases were observed in the disease recurrence group of patients identified at stage III or IV. The stage III resistant cases revealed heterozygous mutation (C/T) in exon 12 (C1236T) and 26 (C3435T) and increased level of mRNA, whereas homozygous mutation (T/T) was found at stage IV tumor patients. The genotypic difference was found to be significant (p = 0.03) for exon 12, and p = 0.003 for exon 26 mutant genotypes. No significant association between genotypes of different exons with tumor stages and tumor grade was observed (p > 0.05). However, a significant association was observed between the genotype of exon-12 and histopathology of tumor tissue (p = 0.028). Statistically, the chemotherapy response was found to be significantly associated with the tumor stage (p = 0.019). We also observed a significant difference in PFS (P = 0.019) and OS (P = 0.047) between tumor grades 1 and 3. Notably, the highest mRNA expression was observed in resistant tumor sample T-32, where interestingly we found homozygosity TT in all of the exons 12, 21, and 26. Thus, we suggest that exons 12 (C1236T) and exon 26 (C3435T) polymorphism may play a role in inducing drug resistance by altering the expression level of the MDR1 gene. To summarize, we suggest that the expression of MDR1 in OC is influenced by tumor stage and genotype variants as well as by chemotherapeutic drugs. Thus our findings suggest that inter individual variability in platinum based therapy may be anticipated by MDR1 genotypes. Further studies on a large number of samples shall eventually lead to provide beneficial information for the individualized chemotherapy.

Overcoming Doxorubicin Resistance with Lipid-Polymer Hybrid Nanoparticles Photoreleasing Nitric Oxide.

We report on tailored lipid-polymer hybrid nanoparticles (NPs) delivering nitric oxide (NO) under the control of visible light as a tool for overcoming doxorubicin (DOX) resistance. The NPs consist of a polymeric core and a coating. They are appropriately designed to entrap DOX in the poly(lactide-co-glycolide) core and a NO photodonor (NOPD) in the phospholipid shell to avoid their mutual interaction both in the ground and excited states. The characteristic red fluorescence of DOX, useful for its tracking in cells, is well preserved upon incorporation within the NPs, even in the copresence of NOPD. The NP scaffold enhances the NO photoreleasing efficiency of the entrapped NOPD when compared with that of the free compound, and the copresence of DOX does not significantly affect such enhanced photochemical performance. Besides, the delivery of DOX and NOPD from NPs is also not mutually influenced. Experiments carried out in M14 DOX-resistant melanoma cells demonstrate that NO release from the multicargo NPs can be finely regulated by excitation with visible light, at a concentration level below the cytotoxic doses but sufficient enough to inhibit the efflux transporters mostly responsible for DOX cellular extrusion. This results in increased cellular retention of DOX with consequent enhancement of its antitumor activity. This approach, in principle, is not dependent on the type of chemotherapeutic used and may pave the way for new treatment modalities based on the photoregulated release of NO to overcome the multidrug resistance phenomenon and improve cancer chemotherapies.

Effect of chlorogenic and gallic acids combined with azoles on antifungal susceptibility and virulence of multidrug-resistant Candida spp. and Malassezia furfur isolates.

Chlorogenic acid (CHA) and gallic acid (GA) are safe natural phenolic compounds that are used as enhancers of some drugs in influencing antioxidant, anticancer, and antibacterial activities. Among fungi, Candida spp. and Malassezia spp. are characterized by an increasing prevalence of multidrug resistance phenomena and by a high morbidity and mortality of their infections. No data are available about the efficacy of CHA and GA combined with azoles on the antifungal susceptibility and on the virulence of both fungi. Therefore, their antifungal and antivirulence effects have been tested in combination with fluconazole (FLZ) or ketoconazole (KTZ) on 23 Candida spp. and 8M. furfur isolates. Broth microdilution chequerboard, time-kill studies, and extracellular enzymes (phospholipase and hemolytic) activities were evaluated, displaying a synergistic antifungal action between CHA or GA and FLZ or KTZ on C. albicans, C. bovina, and C. parapsilosis, and antagonistic antifungal effects on M. furfur and Pichia kudriavzevii (Candida krusei) isolates. The time-kill studies confirmed the chequerboard findings, showing fungicidal inhibitory effect only when the GA was combined with azoles on Candida strains. However, the combination of phenolics with azoles had no effect on the virulence of the tested isolates. Our study indicates that the combination between natural products and conventional drugs could be an efficient strategy for combating azole resistance and for controlling fungistatic effects of azole drugs.

The occurrence of the multidrug resistance (MDR) and the prevalence of virulence genes and QACs resistance genes in E. coli isolated from environmental and avian sources

Colibacillosis is a major disease affecting poultry leads to high morbidity and mortality which causing tremendous economic losses worldwide. These economic disparities are amplified among low and middle-income where sanitation and hygiene are challenged by the increasing demand for quality sources of animal protein. With a view to investigating the prevalence of virulence genes and QACs resistance genes as well as monitoring the antibiogram of E. coli strains, a total of 368 specimens were collected from diseased broiler chickens (n = 226) and environmental sources (n = 142) at large-scale poultry farms in Ismailia Governorate, Egypt. The bacteriological examination proved that E. coli prevalence was 26.76% and 50.44% in the farm environment and diseased broilers, respectively. In tandem, the isolated E. coli strains were serogrouped, determining the most common serotypes were O78, O1:H7, O91:H21 and O126. Isolates were tested for antimicrobial susceptibility against 12 antibiotics, screened for 4 virulence genes (iss, papC, eaeA, and cfaI), and screened for 3 QACs resistance genes (qacEΔ1, qacA/B, and qacC/D). All the tested strains were positive for iss and papC genes, only 20.3% of the tested strains were positive for eaeA gene, moreover, the examined strains were negative to CFAI gene. Furthermore, all the tested strains were positive for qacEΔ1, qacA/B, and qacC/D genes. In conclusion; virulence genes (iss, papC) as well as QACs resistance genes are common in avian Pathogenic E. coli and environmental strains and are mainly associated with multi-drug resistance phenomena.

Predicting the artificial immunity induced by RUTI\xae vaccine against tuberculosis using universal immune system simulator (UISS)

BackgroundTuberculosis (TB) represents a worldwide cause of mortality (it infects one third of the world’s population) affecting mostly developing countries, including India, and recently also developed ones due to the increased mobility of the world population and the evolution of different new bacterial strains capable to provoke multi-drug resistance phenomena. Currently, antitubercular drugs are unable to eradicate subpopulations of Mycobacterium tuberculosis (MTB) bacilli and therapeutic vaccinations have been postulated to overcome some of the critical issues related to the increase of drug-resistant forms and the difficult clinical and public health management of tuberculosis patients. The Horizon 2020 EC funded project “In Silico Trial for Tuberculosis Vaccine Development” (STriTuVaD) to support the identification of new therapeutic interventions against tuberculosis through novel in silico modelling of human immune responses to disease and vaccines, thereby drastically reduce the cost of clinical trials in this critical sector of public healthcare.ResultsWe present the application of the Universal Immune System Simulator (UISS) computational modeling infrastructure as a disease model for TB. The model is capable to simulate the main features and dynamics of the immune system activities i.e., the artificial immunity induced by RUTI® vaccine, a polyantigenic liposomal therapeutic vaccine made of fragments of Mycobacterium tuberculosis cells (FCMtb). Based on the available data coming from phase II Clinical Trial in subjects with latent tuberculosis infection treated with RUTI® and isoniazid, we generated simulation scenarios through validated data in order to tune UISS accordingly to STriTuVaD objectives. The first case simulates the establishment of MTB latent chronic infection with some typical granuloma formation; the second scenario deals with a reactivation phase during latent chronic infection; the third represents the latent chronic disease infection scenario during RUTI® vaccine administration.ConclusionsThe application of this computational modeling strategy helpfully contributes to simulate those mechanisms involved in the early stages and in the progression of tuberculosis infection and to predict how specific therapeutical strategies will act in this scenario. In view of these results, UISS owns the capacity to open the door for a prompt integration of in silico methods within the pipeline of clinical trials, supporting and guiding the testing of treatments in patients affected by tuberculosis.