Flow Cytometric Method Research Articles

Determination of antibiotic susceptibility of bacteria by flow cytometric method

In this study, it was aimed to determine the antibiotic susceptibility of bacterial strains by using flow cytometry method by comparing them with current standardized methods.Eleven clinical isolates and 6 standard bacterial strains were included in the study. MIC values were determined by broth microdilution method (BMD), VITEK 2® automated system and flow cytometric method (FCM). FCM was performed with the Accuri C6 flow cytometer.For all strains except P. aeuruginosa ATCC 27853 [BMD-FCM:r = 0.557(p = 0.048); VITEK 2-FCM:r = 0.529(p = 0.063)], E. faecalis ATCC 29212 [BMD-FCM:r = 0.393(p = 0.295); BMD-VITEK 2:r = 0.393(p = 0.295)], and vancomycin-resistant E. faecium clinical isolate [BMD-FCM:r = 0.452(p = 0.063)] r values were in the range of 0.802-0.969 for BMD-FCM (p < 0.001), 0.655-0.941 for BMD-VITEK 2 (p < 0.005) and 0.667-0.953 for FCM-VITEK 2 (p < 0.005).Correlation values of antibiotic susceptibility test results between three methods for Gram-negative bacteria were found as follows; r = 0.927(p < 0.001) for BMD-FCM, r = 0.851(p < 0.001) for BMD-VITEK 2, r = 0.807(p < 0.001) for VITEK 2-FCM. Correlation values were found as follows for Gram positive bacteria; r = 0.848(p < 0.001) for BMD-FCM, r = 0.877(p < 0.001) for BMD-VITEK 2, r = 0.800(p < 0.001) for VITEK 2-FCM. When all bacteria included in the study were evaluated as a total; it was r = 0.911(p < 0.001) for BMD-FCM, r = 0.888(p < 0.001) for BMD-VITEK 2, r = 0.835(p < 0.001) for VITEK 2-FCM. The methicillin resistance of the clinical methicillin resistant S. aureus isolate could not be detected by FCM.It was determined that there was a high level of correlation between methods. FCM shortens the duration of antibiotic susceptibility tests by 12-14h and gives results within the same day. However, it has not been standardized to be widely used in microbiology laboratories and experienced personnel are needed for its implementation.

Effects of radiofrequency radiation on apoptotic and antiapoptotic factors in colorectal cancer cells

ABSTRACT In this study, it is aimed to investigate the effect of radiofrequency radiation (RFR) on apoptotic and antiapoptotic factors under different exposure conditions in human colonic adenocarcinoma cells (Caco-2). We analyzed the effects of 2.5 GHz continuous wave and 3 GPP modulated radiofrequency radiation exposure (15 min on, 15 min off) for 1 h and (1 h on, 1 h off) for 3 hours on Caco-2 cell lines. The cell viability of Caco-2 cells was determined by XTT method. Then, the cells were analyzed by flow cytometry to determine the effects on apoptosis staining with AnnexinV-FITC and PI. Protein expression levels of Bcl-2, Bax, Caspase-3 and Survivin were subsequently analyzed by using flow cytometric methods. Bax, Caspase 8, and Survivin protein levels were also analyzed by western blot. The cell viability rates were not significantly different after 2.5 GHz of RFR exposure for 1 h, but RFR exposure for 3 h at 2.5 GHz frequencies caused a decrease on cell viability of Caco-2 cells. RFR exposure for 1 and 3 hours at 2.5 GHz frequencies resulted in an apoptotic response. Protein analyses of Bcl-2, Bax, Survivin, Caspase-3, and Caspase-8 showed that RFR led to increase the levels of proapoptotic Bax, Caspase-3, and Caspase 8 in Caco-2 cells under different exposure conditions. However, 3-h exposure caused a decrease in antiapoptotic survivin levels. The results of our study indicate that RFR exposure affects the cell death mechanism due to apoptotic pathway.

PB1841: A NEW APPROACH IN ACUTE MYELOID LEUKEMIA (AML): SAMATYA-PREDICTING SCORE

Background: Since it is not possible to access all methods simultaneously in determination of prognosis, it is important to use different methods together in acute myeloid leukemia (AML). Aims: We aimed to examine the efficiency for prediction of prognosis and response to induction therapy in non-APL AML cases of the “Samatya-predicting score” that we developed using clinical, cytogenetic and flow cytometric features Methods: A total of 213 patients diagnosed between January 2010-December 2020 were examined. Mortality and response to induction therapy (complete, incomplete, failure) were recorded. The subgroups were formed as follows: Responder: Complete response (CR); non-responder: Incomplete or failure. Patients who were not eligible for the study were excluded. Results: Of the 158 patients included in the study, the median value of risk score was determined as 2,5. The area under the curve for the median risk score of 2,5 in ROC analysis was 0,635 (0,541-0,729; 95% confidence interval, p=0,006) for exitus; while it was 0,605 (0,517-0,692; p=0,024) for being responder. The sensitivity for mortality was 88%, the specificity 42%, the positive predictive value (PPV) 90,1%, and the negative predictive value (NPV) was 24,7%. In terms of being non-responder to induction therapy, the sensitivity was 90,1%, the specificity 25,3%, the PPV 89,8%, and the NPV was 32%. OS was shorter in those with high risk scores. Image:Summary/Conclusion: This practical and usable scoring system we developed can be supported also by new parameters.

Measurable Residual Disease in Chronic Lymphocytic Leukemia: Experience in Real-Life Setting with Dry Tube Flow Cytometric Method

Measurable Residual Disease in Chronic Lymphocytic Leukemia: Experience in Real-Life Setting with Dry Tube Flow Cytometric Method

Peiminine regulates the biological characteristics of colorectal cancer cells via P13K/Akt/mTOR and oxidative stress pathways

Purpose: To investigate the influence of peiminine on biological characteristics of colorectal cancer cells, and the underlying mechanism.Methods: Two groups of cultured human colorectal cancer HCT-116 cells were used: peiminine and control groups. Peiminine group cells were exposed to the drug at a final concentration of 100 μmol/L. The effect of peiminine on cell proliferation was determined with CCK-8 method, while its effect on apoptosis was determined with flow cytometric method. Cell migration was determined with scratch test. The effect of peiminine on the expressions of proteins associated with the P13K/Akt/mTOR pathway and Wnt/β-catenin pathway in HCT-116 cells was determined with Western blotting assay.Results: Cell proliferation was markedly reduced in the peiminine group, relative to control (p &lt; 0.05). There was higher percentage cell apoptosis in peiminine-treated cells than in control. Moreover, cell migration potential was significantly lower in the peiminine-treated cells. There were significantly downregulated levels of p-P13K, p-Akt and p-mTOR expressions in peiminine group, relative to the corresponding control expressions (p &lt; 0.05). However, there were significantly higher relative expression of Wnt in peiminine group than in control cells, but β-catenin level was reduced, relative to the corresponding control level (p &lt; 0.05).Conclusion: These data indicate that peiminine suppresses the proliferative, apoptotic and migratory potential of colorectal carcinoma HCT-116 cells via regulation of P13K/Akt/mTOR and oxidative stress pathways.

Extracellular Inflammasome Particles Are Released After Marathon Running and Induce Proinflammatory Effects in Endothelial Cells.

Objectives: The intracellular NLRP3 inflammasome is an important regulator of sterile inflammation. Recent data suggest that inflammasome particles can be released into circulation. The effects of exercise on circulating extracellular apoptosis-associated speck-like protein (ASC) particles and their effects on endothelial cells are not known. Methods: We established a flow cytometric method to quantitate extracellular ASC specks in human serum. ASC specks were quantitated in 52 marathon runners 24–72 h before, immediately after, and again 24–58 h after the run. For mechanistic characterization, NLRP3 inflammasome particles were isolated from a stable mutant NLRP3 (p.D303N)-YFP HEK cell line and used to treat primary human coronary artery endothelial cells. Results: Athletes showed a significant increase in serum concentration of circulating ASC specks immediately after the marathon (+52% compared with the baseline, p < 0.05) and a decrease during the follow-up after 24–58 h (12% reduction compared with immediately after the run, p < 0.01). Confocal microscopy revealed that human endothelial cells can internalize extracellular NLRP3 inflammasome particles. After internalization, endothelial cells showed an inflammatory response with a higher expression of the cell adhesion molecule ICAM1 (6.9-fold, p < 0.05) and increased adhesion of monocytes (1.5-fold, p < 0.05). Conclusion: These findings identify extracellular inflammasome particles as novel systemic mediators of cell–cell communication that are transiently increased after acute extensive exercise with a high mechanical muscular load.

Bioelectricity Buzz.

Bioelectricity Buzz.

CD157 Can Replace CD24 and CD14 in a Single-Tube Flow-Cytometric Assay to Detect Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones on Both Neutrophils and Monocytes: A Prospective Study From North India.

IntroductionAs per current guidelines, detection of paroxysmal nocturnal hematuria (PNH) clones on leucocytes requires the demonstration of the loss of at least two glycosyl-phosphatidyl-inositol (GPI)-linked molecules on both neutrophils and monocytes by flow cytometry. CD24 and CD14 are GPI-linked molecules expressed on neutrophils and monocytes respectively, whereas another GPI-linked molecule, CD157, is expressed on both neutrophils and monocytes. This prospective study evaluated the ability of CD157 to replace both CD24 and CD14 in a single-tube flow-cytometric assay to detect PNH clones on both neutrophils and monocytes.Materials and methodsPNH clones were newly detected in 52 patients by an existing “standard” single-tube six-color flow-cytometric method, which was routinely performed in our laboratory at the time of undertaking this study. Six antibodies (CD45/CD15/CD64/CD24/CD14/FLAER) were used in this "standard" technique. Subjects were divided into two groups: (i) PNH disease (n=10), and (ii) aplastic anemia/myelodysplastic syndrome (AA/MDS) (n=42). Diagnosis of PNH disease and AA/MDS were made as per standard literature and guidelines. Results were compared with a single-tube five-color “test” assay using the antibodies CD45/CD15/CD64/CD157/FLAER by flow cytometry. Samples from 20 healthy control subjects were used to calculate cut-off values for the “test” assay.ResultsBy the "test" method, cut-off values for detecting PNH clones obtained from receiver operating-characteristic curve analysis were >0.4% for neutrophils (sensitivity=96.15%, specificity=95%), and >0.9% for monocytes (sensitivity=98.08%, specificity=95%). There was significant correlation between PNH clone sizes measured by both the “standard” and “test” assays in neutrophils (PNH disease: r=0.976, p<0.001; AA/MDS: r=0.980, p<0.001) as well as monocytes (PNH disease: r=0.806, p=0.005; AA/MDS: r=0.915, p<0.001). Bland-Altman analysis showed agreement between both assays in all the 52 patients and in individuals with AA/MDS. The cost of the test to the patients was about 15% less in the “test” method than the ”standard” technique, with improved technical efficiency.ConclusionCD157 can replace both CD24 and CD14 in a single-tube flow-cytometric assay to detect PNH clones on both neutrophils and monocytes, with reduced cost to the patients and improved technical efficiency.

Development and validation of a sensitive flow cytometric method for determining CECs in RBC products

Background and AimsSubpopulations of immature red blood cells (RBCs) named CD71+ erythroid cells (CECs) with different properties may contribute to RBC transfusion outcomes. However, it is challenging to quantify CECs in leukoreduced RCCs and whole blood due to their rarity and fragility. Current flow cytometry methods are not applicable to leukoreduced RCCs as there is limited peripheral blood mononuclear cells (PBMCs) to use for determination of CECs. We have developed and validated a flow cytometry method for quantifying CECs in WB. MethodsWe determined optimal PE-Cy7-CD235a, BV711-CD71, APC-CD45 concentrations and instrument setting by titration. Linearity and level of detection was determined by spiking labelled PBMCs from cord blood units into unlabeled WB. Low, medium, high levels of CECs were used to determine intra- and inter -run precision. ResultsDetection of CECs was linear (R2 = 0.98) over the range of concentrations assessed with a limit of detection of 0.005%. The overall CVs for the intra- and inter-run precision were 6.9% and 9.7%. ConclusionWe developed a simple, sensitive, and cost-effective flow cytometric method for quantifying the proportion of CECs in non-manipulated WB, which could help understand the impact of RBC products on recipient transfusion outcomes.

A novel digital PCR-based method to quantify (switched) B cells reveals the extent of allelic involvement in different recombination processes in the IGH locus

B cells fulfill an important role in the adaptive immunity. Upon activation and immunoglobulin (IG) class switching, these cells function in the humoral immunity compartment as plasma cells. For clinical applications, it can be important to quantify (switched) B cells accurately in a variety of body fluids and tissues of benign, inflammatory and malignant origin. For decades, flow cytometry and immunohistochemistry (IHC) have been the preferred methods for quantification. Although these methods are widely used, both depend on the accessibility of B cell epitopes and therefore require intact (fixed) cells. Whenever samples are low in quantity and/or quality, accurate quantification can be difficult. By shifting the focus from epitopes to DNA markers, quantification of B cells remains achievable. During differentiation and maturation, B cells are subjected to programmed genetic recombination processes like VDJ rearrangements and class switch recombination (CSR), which result in deletion of specific sequences of the IGH locus. These cell type-specific DNA “scars” (loss of sequences) in IG genes can be exploited as B cell markers in digital PCR (dPCR) based quantification methods. Here, we describe a novel, specific and sensitive digital PCR-based method to quantify mature and switched B cells in DNA specimens of benign and (copy number unstable) malignant origin. We compared this novel way of B cell quantitation with flow cytometric and immunohistochemical methods. Through cross-validation with flow cytometric sorted B cell subpopulations, we gained quantitative insights into allelic involvement in different recombination processes in the IGH locus. Our newly developed method is accurate and independent of the cellular context, offering new possibilities for quantification, even for (limited) small samples like liquid biopsies.

Investigation of the anticancer and apoptotic effect of Micromeria congesta under in vitro conditions and detection of related genes by real-time PCR.

At the present time cancer is one of the biggest health problems and because of the problems encountered in its treatment, alternative treatment methods of herbal origin are researched. In this study, the cytotoxic effects of the essential oil extracted from the Micromeria congesta plant on various cancer cells (A549, ECC-1, HCT-116, HELA, HGC-27, MDA-MB-231, SNU-423, U20S, DLD-1, PC-3) and normal cells (BEAS-2B, CRL-4010) have been examined. Anticancer mechanism of action has been particularly examined on gastric cancer (HGC-27; IC50: 15.84 µg mL-1), on which essential oil showed a high cytotoxic effect. In the study, the cytotoxic effect and the apoptotic effect have been applied by MTT and flow cytometric annexin-V methods, respectively. The apoptotic gene expression (caspase 3, caspase 9, MMP2, MMP9, ACTB) real-time PCR content analysis has been performed with gas chromatography mass spectrometry (GC-MS). M. congesta essentials oil has the highest cytotoxic effect on gastric cancer (HGC-27) cells, decreases MMP2 and MMP9 expressions, and induces apoptosis with increasing the expression of caspase 3 and caspase 8 genes. In addition, it has been determined that piperitenone oxide (40.00 - 45.00%), pulegone (11.00%) and cyclohexanone (18.00 - 19.00%) are the major components of M. congesta essentials oil. In conclusion, it has been determined that the compounds found in high amounts in M. congesta plant induces apoptosis by affecting the expression of compound genes and thus can have the potential to be an alternative drug in the treatment of gastric cancer.

Establishment of an in vitro flow cytometric detection method for lipid-accumulated cells

Eukaryotic cells can store and converse excess lipid to the cytosolic lipid droplets. Adipogenesis of preadipocytes has been often used to study the molecular basis and the effect of obesity drugs on fat cell conversion. Many methods were developed for the detection of the cytosolic lipid droplets as Nile red, BODIPY 493/503 (4, 4-difluoro-1, 3, 5, 7, 8-pentamethyl-4-bora-3a, 4adiaza-s-indacene), BODIPY 665/676, 1,6-diphenylhexatriene (DPH), DAPI, Hoechst, Sudan III, and Oil-red O. The differences in the spectral properties of these lipophilic dyes and their advantages of each are discussed. In this study, an in vitro flow cytometric detection method was established for the detection of lipid-accumulated cells. Commonly, the longer the period of adipogenic induction, the greater the quantity of lipid in fat cell can accumulate. Thus, to determine whether increasing the fat stored within a cell would result in the greater granularity. 3T3-L1 cells in culture were hormonally induced for adipogenesis. Then, these cells were dissociated and analyzed in a flow cytometer at 0, 5, and 10 days post-induction. After adipogenic induction, the cells had become increasingly heterogeneous in their cellular granularity. The cells containing greater granular structure were markedly increased, and this increase in granularity positively correlated with the time of the post-adipogenic induction. On the other analysis, the 0 and 10 days post adipogenic induction of 3T3-L1 cells were gated for 4 regions. The R1 region contains cells with a level of granularity similar to that seen in the control cells (non-adipogenic induction), whereas R2 to R4 regions contain cells with increasing granularity. According to all data, we have successfully established an in vitro flow cytometric detection method for the detection of lipid-accumulated cells. We wish this method will be applied on the research of obesity drugs and the design of therapeutic strategies for obesity in the future.

Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm.f.) Merrill.

Tylophora indica (Burm.f.) Merrill. is a pharmacologically important plant, popular for alkaloidal and non-alkaloidal richness. Large scale propagation of T. indica is difficult in the wild as the seeds are small and the frequency of germination is very poor. In the present study, the genome size estimation of in vitro regenerated (indirect, direct and somatic embryo mediated) T. indica was made by flow cytometric method. Clonal fidelity of the regenerants was assessed using a start codon targeted (SCoT) molecular marker. Initially, the explants were inoculated on Murashige and Skoog basal medium supplemented with various concentrations of plant growth regulators like 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin, 6-benzyl amino purine (BAP) and 1-naphthalene acetic acid either singly or in combinations. The highest callus induction frequency (87.75%) was obtained in 6.7 µM 2,4-D added MS medium which metamorphosed into progressive stages (globular, heart, torpedo, and cotyledonary) of embryos. Mature and healthy somatic embryos efficiently germinated into plantlets on 8.8 µM BAP + 1.4 µM GA3 enriched MS medium. Histological and scanning electron microscopic study confirmed the above developing stages. The regenerated shoots were rooted best in 2.45 µM Indole-3-butyric acid supplemented solid MS medium. The plants were hardened and acclimatized with 90% survivability. The flow cytometric 2C DNA content of indirect, direct and somatic embryo derived plants was 1.896 pg, 1.940 pg and 1.926 pg respectively, very similar to the mother plant (1.928 pg). SCoT marker generated a high percentage of monomorphic bands (94%) revealing similarity with the mother plant, thus ensuring genetic fidelity. To the best of our knowledge, this is perhaps the first ever report of 2C DNA content estimation and SCoT marker based genetic homogeneity study in T. indica.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11240-022-02254-z.

A Rapid Assessing Method of Drug Susceptibility Using Flow Cytometry for Mycobacterium tuberculosis Isolates Resistant to Isoniazid, Rifampin, and Ethambutol.

BackgroundThe current conventional drug susceptibility test (DST) for Mycobacterium tuberculosis (Mtb) takes several weeks of incubation to obtain results. As a rapid method, molecular DST requires only a few days to get the results but does not fully cover the phenotypic resistance. A new rapid method based on the ability of viable Mtb bacilli to hydrolyze fluorescein diacetate to free fluorescein with detection of fluorescent mycobacteria by flow cytometric analysis, was recently developed.MethodsTo evaluate this cytometric method, we tested 39 clinical isolates which were susceptible or resistant to isoniazid (INH) or rifampin (RIF), or ethambutol (EMB) by phenotypic or molecular DST methods and compared the results.ResultsThe susceptibility was determined by measuring the viability rate of Mtb and all the isolates which were tested with INH, RIF, and EMB showed susceptibility results concordant with those by the phenotypic solid and liquid media methods. The isolates having no mutations in the molecular DST but resistance in the conventional phenotypic DST were also resistant in this cytometric method. These results suggest that the flow cytometric DST method is faster than conventional agar phenotypic DST and may complement the results of molecular DST.ConclusionIn conclusion, the cytometric method could provide quick and more accurate information that would help clinicians to choose more effective drugs.

Importance of the Diastolic Flow Reversal Parameters on Quantitation of Aortic Regurgitation

Importance of the Diastolic Flow Reversal Parameters on Quantitation of Aortic Regurgitation

Green Synthesized Fe3O4 Nanoparticles as Silymarin Drug Carrier and their Anticancer Activity against Liver-HepG2 and Lung-A549 Cancer Cells

Iron oxide nanoparticles (IONPs) were synthesized by co-precipitation method with Commiphora berryi latex as stabilizing agent. The IONPS were coated with PEG2K and then loaded with silymarin drug. The synthesized IONPs, PEG2K and silymarin loaded nanoparticles were analyzed by FTIR, XRD, SEM and TEM techniques. The FTIR results indicated the coating of plant materials and the drug on the nanoparticles. The XRD results showed that the synthesized particles have a crystalline structure and controlled size of about 14.5 nm. Surface morphology of the iron oxide nanoparticles and drug loaded IONPs from SEM and TEM results show the cubic shaped particles with less agglomeration. The silymarin loaded IONPs were tested against liver and lung cancer cells by MTT assay and fluorescence microscopic analysis using various concentrations such as 10 μL, 25 μL and 50 μL. Results confirmed that the cell apoptosis was done by nucleus fragmentations. Further, the quantitative cell apoptosis was done by flow cytometric method. The results confirmed that the silymarin loaded IONPs disclosed excellent anticancer activity against lung cancer cells with 30.47% of post apoptotic cells over liver cancer cells with 30.27% post apoptosis.

Influence of vascular endothelial growth factor on neutrophil activation in asthmatics.

IntroductionAsthma is a complex syndrome associated with heterogeneity in the type of inflammation that modulates airway hyperresponsiveness and remodelling. Airway inflammation with neutrophils, cytokines like vascular endothelial growth factor (VEGF) and various gene polymorphisms might be relevant for asthma pathogenesis.AimTo investigate the expression of CD69 and CD11b markers on peripheral neutrophils after VEGF in vitro stimulation in asthmatics. Furthermore, the possible influence of a genetic factor (del/ins genotype at -2549 -2567 position in the VEGF-promoter gene) was taken into account.Material and methods122 subjects (82 asthmatics and 40 controls) participated in our study. CD69 and CD11b presence on peripheral blood neutrophils was detected using the flow cytometric method without exogenous stimulation (negative control), after N-formyl-methionine-leucyl-phenylalanine stimulation (fMLP – positive control) and after VEGF stimulation. Genotyping for the VEGF-promoter region was performed by polymerase chain reaction (PCR).ResultsPeripheral neutrophil stimulation with VEGF enhances the activity of these cells and induces CD69 and CD11b expression in a dose-dependent manner compared with unstimulated neutrophils (p > 0.05). CD69 and CD11b markers were slightly presented (p = 0.05 and p = 0.07, respectively) on neutrophils stimulated with fMLP in asthmatics with the ins genotype variant in the VEGF-promoter region.ConclusionsOur results demonstrate that VEGF might insignificantly activate neutrophils in asthmatics. In addition, the modulated expression of CD69 and CD11b on peripheral neutrophils is not related to potential contribution of the VEGF gene polymorphism.

Microbial metabolite restricts 5-fluorouracil-resistant colonic tumor progression by sensitizing drug transporters via regulation of FOXO3-FOXM1 axis.

The survival rate of colorectal cancer patients is adversely affected by the selection of tumors resistant to conventional anti-cancer drugs such as 5-fluorouracil (5FU). Although there is mounting evidence that commensal gut microbiota is essential for effective colon cancer treatment, the detailed molecular mechanisms and the role of gut microbial metabolites remain elusive. The goal of this study is to decipher the impact and mechanisms of gut microbial metabolite, urolithin A (UroA) and its structural analogue, UAS03 on reversal of 5FU-resistant (5FUR) colon cancers.Methods: We have utilized the SW480 and HCT-116 parental (5FU-sensitive) and 5FUR colon cancer cells to examine the chemosensitization effects of UroA or UAS03 by using both in vitro and in vivo models. The effects of mono (UroA/UAS03/5FU) and combinatorial therapy (UroA/UAS03 + 5FU) on cell proliferation, apoptosis, cell migration and invasion, regulation of epithelial mesenchymal transition (EMT) mediators, expression and activities of drug transporters, and their regulatory transcription factors were examined using molecular, cellular, immunological and flowcytometric methods. Further, the anti-tumor effects of mono/combination therapy (UroA or UAS03 or 5FU or UroA/UAS03 + 5FU) were examined using pre-clinical models of 5FUR-tumor xenografts in NRGS mice and azoxymethane (AOM)-dextran sodium sulfate (DSS)-induced colon tumors.Results: Our data showed that UroA or UAS03 in combination with 5FU significantly inhibited cell viability, proliferation, invasiveness as well as induced apoptosis of the 5FUR colon cancer cells compared to mono treatments. Mechanistically, UroA or UAS03 chemosensitized the 5FUR cancer cells by downregulating the expression and activities of drug transporters (MDR1, BCRP, MRP2 and MRP7) leading to a decrease in the efflux of 5FU. Further, our data suggested the UroA or UAS03 chemosensitized 5FUR cancer cells to 5FU treatment through regulating FOXO3-FOXM1 axis. Oral treatment with UroA or UAS03 in combination with low dose i.p. 5FU significantly reduced the growth of 5FUR-tumor xenografts in NRGS mice. Further, combination therapy significantly abrogated colonic tumors in AOM-DSS-induced colon tumors in mice.Conclusions: In summary, gut microbial metabolite UroA and its structural analogue UAS03 chemosensitized the 5FUR colon cancers for effective 5FU chemotherapy. This study provided the novel characteristics of gut microbial metabolites to have significant translational implications in drug-resistant cancer therapeutics.

Abstract P061: De novo design of importin-a-specific NLS sequences for nuclear-targeted therapeutics

Abstract Background: Research focused on the application of nuclear localization sequence (NLS)-based therapeutics has been a topic of intense interest to medicine since the core principles governing nuclear transport were awarded the Nobel Prize in 1999. Despite these efforts, efficient nuclear localization has been difficult. A major obstacle is that NLSs have cationic-net charges (abundance of lysines/arginines) required for appropriate interactions with the nuclear transporter importin-a (Impa). Once in the blood stream, cationic charges can cause strong non-specific cellular uptake and are rapidly cleared from the plasma compartment, which prevents sufficient tumor cell uptake and, hence, nuclear localization. Here, describe a de novo computational approach for generating 43 novel NLSs (based on 3 different NLS classes) that contained amino acid substitutions to achieve net-neutral charge states. We also introduce a novel nuclear isolation-quantitative flow cytometric method for determining nuclear localization efficiency. Material and methods An algorithm was created based on complete interaction binding affinity strengths for 20 x 20 pairs of octapeptides consisting of the 20 common amino acids. 9 PDB files were selected as templates and were comprised of viral, RNA processing, and transcription initiation protein NLSs bound to Impa. In silico alanine scanning on all NLS templates generated rankings on amino acid sensitivities for each position in the sequences. Non-sensitive residues were then subjected to residue scanning for generating net-neutral charged NLSs. Computational docking studies generated predictive binding scores relative to wild type NLSs. Favorable NLS candidates were genetically fused to GFP. In contrast to utilizing fluorescence microscopy, which cannot determine nuclear localization efficiency, we created a method to isolate nuclei from transiently transfected CHOK1 cells and quantify nuclear localization by flow cytometry. Results 2-8 net-neutral NLSs with good binding for Imga could be generated for each PDB file. The net-neutral mutants often contained an abundance of glutamic and/or aspartic acid substitutions, and were able to bind to Impa residues to compensate for the replaced amino acids. Transfected GFP-NLS constructs displayed variable fluorescence expression kinetics. Therefore, we created an in-house plasma membrane lysis protocol to isolate intact nuclei. Quantitative evaluations are currently underway by evaluating nuclei fluorescence by flow cytometry at various time points. Thus far, the tested GFP-NLSs are able to localize to the nucleus. Conclusions An important objective of computational protein design is the generation of high affinity peptides as a precursor to the development of therapeutics, and as a tool to aid researchers in understanding governing interaction principles of specific complexes. We have achieved both the development of potential NLS peptides for overcoming the cationic-sequestration barrier, and for understanding novel NLS principles to further advance the NLS-therapeutics field. Citation Format: Olga Bednova, Alexis Rioux-Chevalier, Dipika Patel, Mathieu Boudreau, Jeffrey Victor Leyton. De novo design of importin-a-specific NLS sequences for nuclear-targeted therapeutics [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P061.

A flow cytometric method for enumeration and speciation of coccidia affecting broiler chickens

Production losses, mortality, and control measures associated with coccidiosis, caused by Eimera species, cost the broiler industry over $14 billion a year. Current means to distinguish Eimeria species such as oocyst morphology, pre-patent period and site of infection are subjective, labor intensive or unsuitable for high-throughput applications. Although Polymerase Chain Reaction (PCR) techniques have been validated, the target gene cannot differentiate relative abundance of each species in mixed infections. In this study, we developed a non-antibody-based flow cytometry high throughput method to simultaneously enumerate and speciate four Eimeria species, E. acervulina, E. mitis, E. maxima, and E. tenella, using commercial coccidia vaccine as well as field fecal samples. Our findings showed that the four Eimeria oocyst populations could be distinctly speciated based on their size and granularity (shape) via scatter plotting. These distinct populations were sorted and confirmed by quantitative real-time PCR assay. Finally, the flow cytometry findings were applied to enumerate and speciate oocysts from fecal samples collected from commercial broiler flocks vaccinated for coccidiosis at day of hatch and the results were validated against the conventional manual method of floatation and microscopic examination. Collectively, the findings of this study suggested that non-antibody based Flow Cytometry technique can be successful in the simultaneous enumeration and speciation of coccidia. Further development and validation is needed to make this diagnostic tool useful for field applications at a much larger scale as well as to speciate other Eimeria species.