Combination Of Assays Research Articles

Abstract 1723: Spatial analysis of tumor-infiltrated immune cells with highly specific RNAscope™ RNA-protein Co-detection assays

Abstract Interrogating complex tumor microenvironment requires a multi-omics approach that can provide high level of sensitivity and specificity. Identifying immune cell subsets within the tumor can be vital for predicting response and determining therapeutic efficacy. Detecting target immune cell markers using immunohistochemistry/Immunofluorescence (IHC/IF) and visualizing cytokine expression with in situ hybridization (ISH) can provide comprehensive information about the activation states of immune cells. Here, we demonstrate a newly developed integrated ISH and IHC/IF workflow compatible with manual and automated platforms that can substantially improve RNA-protein co-detection. We demonstrate the use of our RNA-Protein Co-detection assay in combination with the automated RNAscope Multiplex Fluorescent v2 assay, automated RNAscope Chromogenic Duplex assay and manual RNAscope Multiplex Fluorescent v2 assay to detect T cell markers, macrophage markers and checkpoint markers in the tumor microenvironment by using a microarray with different tumor samples. We identified CD4+ helper T cells and CD8+ cytotoxic T lymphocytes. Additionally, we determine the activation states of CD8+ T cells by visualizing IFNG, GZMB and IL-2 expression. We were also able to identify macrophages detected by CD68 protein expression and the M1 and M2 subsets were differentiated by using the M2-specific marker, CD163. We could also delineate tumor-stroma border in the samples by using the Pan-CK probe which distinctly marks the tumor cells and visualize the expression of immunoregulatory receptors PD-L1 and CTLA4 in the tumor cells. This assay is enabled for multiplexing by combining with our fluorescent assay platforms. Similarly, for researchers interested in detecting target expression while retaining morphological context, the codetection assay should be combined with our chromogenic assays. Overall, the new RNAscope-ISH-IHC co-detection workflow and reagents enable optimized simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies with minimal optimization. Citation Format: Anushka Dikshit, Sayantani Basak, Emerald Doolittle, Ilya Kovalenko, Michaeline Bunting. Spatial analysis of tumor-infiltrated immune cells with highly specific RNAscope™ RNA-protein Co-detection assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1723.

Abstract 1977: Extending the capabilities of a high-parameter immunophenotyping assay with cytoplasmic staining applications for mass cytometry

Abstract Mass cytometry, empowered by CyTOF® technology, utilizes monoisotopic metal-tagged antibodies and a high-sensitivity mass cytometer to allow high-dimensional single-cell analysis in complex biological samples. The 30-marker panel Maxpar® Direct™ Immune Profiling Assay™ (Cat. No. 201325) for suspension mass cytometry provides an unprecedented sample-to-answer solution for detecting and analyzing 30 surface markers in a single experiment. With 18 open mass channels for additional biological markers, the Maxpar Direct Assay facilitates panel expansion and enables flexibility for higher multiplexity and applications. Among the potential complementary applications with the Maxpar Direct Assay, intracellular cytokine staining (ICS) is of particular interest as it may be used to assess infiltrating immune cell phenotypes in the tumor microenvironment. However, for the purpose of assessing cell viability in this workflow, the effectiveness of the Cell-ID™ Intercalator-Rh (Cat. No. 201103) that is included in the Maxpar Direct Assay is in question, since cell permeabilization during ICS can potentially damage the DNA-intercalator bondIn this study, we investigated the compatibility of the Cell-ID Intercalator-Rh (103Rh) with intracellular staining. To do this, we stained either human peripheral blood mononuclear cell (PBMC) or whole blood samples (FLDM-400287) with the Maxpar Direct Assay followed by intracellular staining for the detection of expressed cytokines. The intercalator was evaluated for its ability to discriminate live and dead cells when the sample undergoes surface antibody staining. The monoisotopic Cell-ID Cisplatin-194Pt (Cat. No. 201194) was used as the control to provide benchmark measurement of cell viability of the samples. For both sample types, known percentages of heat-killed PBMC were spiked into the samples in order to evaluate the influence of dead cells. We demonstrate that 103Rh provides equivalent functionality as a cell viability indicator during intracellular staining for cytoplasmic proteins compared to Cell-ID Cisplatin-194Pt. This work was designed to support the use of the Maxpar Direct Immune Profiling Assay in combination with additional intracellular markers. Overall, these findings expand the applicability of Cell-ID Intercalator-Rh (103Rh) to processes that involve cytoplasmic staining. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Noah Saederup, Huihui Yao, Michael Cohen, Leslie Fung. Extending the capabilities of a high-parameter immunophenotyping assay with cytoplasmic staining applications for mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1977.

Abstract 3023: Fatty acid synthase (FASN) inhibitors synergize with carfilzomib (CFZ) in acute myeloid leukemia (AML) and multiple myeloma (MM)

Abstract Background: Fatty acid synthase (FASN) upregulation during conditions of oxidative stress contributes to tumor proliferation and survival, which appears to be a mechanism of proteasome inhibitor resistance. Here we demonstrate that acute myeloid leukemia (AML) cell lines resistant to carfilzomib (CFZ), a second-generation proteasome inhibitor, have higher basal FASN expression and targeting FASN with small molecule inhibitors enhances the cytotoxic effect of CFZ in both AML and multiple myeloma (MM) cell lines. Methods: In a proliferation assay, human AML and MM cell lines were treated with a single dose of CFZ for 7 days. Inhibition of proliferation was defined using an IC50 cutoff for CFZ of 10nM for AML and 5nM for MM as the threshold for sensitivity. Sensitive and resistant cell lines were subjected to apoptosis and cell cycle analyses by flow cytometry after being exposed to CFZ for 72 hours. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA). For CFZ and FASN inhibitor combination assays, AML and MM cell lines with varying sensitivities to CFZ were exposed to CFZ and FASN inhibitors, orlistat or TVB-3166, simultaneously, and the apoptosis rate were analyzed by flow cytometry. For western blots, selected AML and MM cell lines were incubated with compounds for 24 hours, and the lysates were probed for selected targets. Results: Single-agent CFZ induced apoptosis in sensitive AML and MM cell lines, while apoptotic rates remained low in resistant cell lines. Cell cycle analysis showed increased sub-G1 population in sensitive cell lines compared to resistant cell lines. RPPA revealed that FASN, a key enzyme involved in lipogenesis, correlated with CFZ sensitivity, and CFZ resistant lines trended towards higher basal FASN levels. When CFZ was combined with FASN inhibitors, orlistat or TVB-3166, significant synergy was observed in the apoptosis assays in the AML and MM cell lines. Western blot analyses showed FASN inhibitors enhanced the anti-proliferation and pro-apoptotic effects of CFZ. Conclusion: CFZ demonstrated single agent activity in nanomolar ranges in human AML and MM cell lines. When combined with agents targeting lipid-metabolism, CFZ showed synergistic effect in apoptosis, suggesting this combination could potentially be a new therapeutic strategy for AML and MM. Citation Format: Maoyu Peng, Sanaz Noelle Ghafouri, Martina SJ McDermott, Dennis J. Slamon, Sarah M. Larson. Fatty acid synthase (FASN) inhibitors synergize with carfilzomib (CFZ) in acute myeloid leukemia (AML) and multiple myeloma (MM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3023.

Cell tension controlling pathways and nutrient availability regulate plasma membrane ATP synthase trafficking

Introduction: The interplay between mechanical stress and cell metabolism is an emerging topic. However, the mechanotransduction pathways coordinating cell metabolism to the tensional status of the cell are poorly understood. Here, we provide several evidences suggesting that caveolae – mechanosensitive plasma membrane invaginations-, nutrient availability and cell tension controlling conditions are important for the biology of mitochondrial ATP synthase. Material and Methods: For achieving the caveolar interactome, plasma membrane biotinylation and co-localization experiments, we have used human retinal pigment epithelial-1 (RPE-1) cells, murine vascular aortic smooth muscle (MOVAS) cells and mouse embryonic fibroblasts (MEF). We used proximity-dependent biotin identification (BioID) and mass spectrometry to obtain the different interactomes of caveolar components. For plasma membrane (PM) localization of the ATP synthase we used PM biotinylation under different metabolic and mechanical conditions (nutrient starving, cell confluence). To study co-localization, we performed Proximity Ligation Assays (PLAs) in combination with mitochondrial and caveolar markers. Results: First, we have identified the interactome of all core caveolar components. Among the interactors, we identified subunits α and β of the mitochondrial ATP synthase, which interact with most of the caveolar components. Apart from its main localization at the inner mitochondrial membrane, the ATP synthase has also been detected in the PM, facing the extracellular space; this pool is known as ecto-ATP synthase. We show that the interaction with Caveolin-1 (Cav1), one of the main components of caveolae, occurs outside caveolae and mitochondria. In addition, the presence of ecto- ATP synthase at the PM is dependent on Cav1. Furthermore, conditions regulating cell tension, and metabolic challenges that promote autophagy, impinge on ecto-ATP synthase trafficking Conclusions: This study has characterized the interactome of caveolar components and has focused on the subunits α and β of the ATP synthase, which are also present at the PM. These subunits interact with Cav-1, but this interaction seems to occur in intracellular trafficking vesicles. Moreover, this process could be altered by mechanical cues and nutrient deprivation. Thus, our study suggests that trafficking routes regulating PM residents, and regulated by several caveolar components, are linked to nutrient starvation and mitochondrial biology.

Targeted Proteins Reveal Cathepsin D as a Novel Biomarker in Differentiating Hepatocellular Carcinoma from Cirrhosis and Other Liver Cancers

Objective:Hepatocellular carcinoma (HCC) represents a global health concern, particularly in Southeast Asia where hepatitis B virus (HBV) infection is common. In this study, we applied tissue-based proteomics to identify novel serological proteins for HCC and validated their performance in serum specimens. Methods:In a discovery set, liver tissue specimens of HBV-related HCC, intrahepatic cholangiocarcinoma (iCCA) and colorectal cancer with liver metastasis (CRLM) were analyzed using mass spectrometry (LTQ-Orbitrap-XL). A subset of proteins that showed highly expressed in HCC were then confirmed by Western blotting. Additionally, clinical significance of selected candidate proteins was tested in serum samples of 80 patients with HBV-related HCC, 50 patients with HBV-related liver cirrhosis and 30 healthy controls. Results:Based on LTQ-Orbitrap-XL mass spectrometer, various differentially expressed proteins (DEPs) between tumor and adjacent non-tumor tissues were identified. These included 77 DEPs for HCC, 77 DEPs for iCCA and 55 DEPs for CRLM. Among selected candidate proteins, annexin A2 and cathepsin D were confirmed to be overexpressed in HCC tissue by Western blot analysis. In a validate cohort, serum cathepsin D level, but not annexin A2, was significantly higher in HCC compared with the non-HCC groups. Serum cathepsin D level was also positively correlated with tumor size and tumor stage. Additionally, the combined assay of serum cathepsin D and alpha-fetoprotein had a high sensitivity in detecting early HCC (83%) and intermediate/advanced HCC (96%). Moreover, patients with low serum cathepsin D (<305 ng/mL) displayed significantly better overall survival than those whose serum levels were high (≥305 ng/mL). Conclusions:Proteomics and subsequent validation revealed cathepsin D as a novel biomarker for HCC. Apart from its diagnostic role, serum cathepsin D might also serve as a prognostic biomarker of HCC. Additional large-scale studies are needed to verify our findings.

A cell-free RNA-based next-generation sequencing (NGS) assay for the detection of actionable gene fusions in patients with non–small cell lung cancer (NSCLC).

3040 Background: Gene fusions and alternate RNA splice forms represent clinically actionable driver and resistance-conferring alterations in NSCLC and other cancers. Where tissue samples are inadequate for molecular testing, liquid biopsies could address the gap in detection of clinically relevant gene fusions. Targeted cell-free (cf) DNA-based NGS methods are typically used for non-invasive blood-based testing of gene fusions, but have limited sensitivity if fusion breakpoints involve long intronic regions or repetitive sequences (e.g. NTRKs and NRG1). RNA-based approaches are not affected by introns, can identify expressed fusion genes and can discriminate splicing. We developed an NGS assay optimized for cfRNA analyte for the detection of actionable gene fusions and exon deletion/skipping events in liquid biopsies. Methods: A highly multiplexed molecular-barcoded primer panel was designed for cfRNA-based detection of actionable fusion genes in NSCLC ( ALK, BRAF, FGFR2, FGFR3, MET (including exon 14 skipping), NRG1, NTRK1/2/3, RET and ROS1) covering &gt; 90% of reported driver and partner gene exons in COSMIC database. The panel also targets housekeeping genes as endogenous sample controls. We developed a custom bioinformatics pipeline to call fusions and exon skipping based on split and spanning reads. Results: In initial analytical validation using fragmented RNA from pre-characterized reference material (representing 32 unique fusions at known copy numbers), the assay could detect as low as 10 fusion copies with a sensitivity of 97.6% and a specificity of 100%. For clinical testing, cfRNA co-eluted with cfDNA in nucleic acid extracts from plasma was used. In plasma samples (n = 103) from advanced NSCLC, 76% (15/21) of fusions (5 ALK, 3 RET, 2 ROS1, 5 MET ex14 skipping, 1 FGFR3-TACC3) detected in a clinically validated cfDNA assay (LiquidHALLMARK) were also detected in corresponding cfRNA. In a subset of samples that were driver-negative untreated or tyrosine kinase-inhibitor treated, the cfRNA assay yielded 9 gene fusions or breakpoints (1 CD74-NRG1, 4 BRAF, 2 MET, 2 FGFR3-TACC3 fusions) that could not be detected by the cfDNA assay. This represents an increase of 8.7% (9/103) of actionable alterations (driver or resistance) identified using cfRNA. Together cfRNA and cfDNA resulted in 30 fusion events to be detected compared to 21 by the cfDNA assay alone, representing a 42.8% (9/21) increase in fusion-specific detection of the combined assay. Conclusions: This novel cfRNA assay can detect actionable gene fusions and exon skipping events in liquid biopsies with high sensitivity. Combining cfRNA with more routine cfDNA testing can increase the total actionable diagnostic information from non-invasive testing in NSCLC patients where tissue samples are lacking, especially for gene fusions not amenable to detection in cfDNA.

Translation of a Protease Turnover Assay for Clinical Discrimination of Mucinous Pancreatic Cysts.

The classification of pancreatic cyst fluids can provide a basis for the early detection of pancreatic cancer while eliminating unnecessary procedures. A candidate biomarker, gastricsin (pepsin C), was found to be present in potentially malignant mucinous pancreatic cyst fluids. A gastricsin activity assay using a magnetic bead-based platform has been developed using immobilized peptide substrates selective for gastricsin bearing a dimeric rhodamine dye. The unique dye structure allows quantitation of enzyme-cleaved product by both fluorescence and surface enhanced Raman spectroscopy (SERS). The performance of this assay was compared with ELISA assays of pepsinogen C and the standard of care, carcinoembryonic antigen (CEA), in the same clinical sample cohort. A retrospective cohort of mucinous (n = 40) and non-mucinous (n = 29) classes of pancreatic cyst fluid samples were analyzed using the new protease activity assay. For both assay detection modes, successful differentiation of mucinous and non-mucinous cyst fluid was achieved using 1 µL clinical samples. The activity-based assays in combination with CEA exhibit optimal sensitivity and specificity of 87% and 93%, respectively. The use of this gastricsin activity assay requires a minimal volume of clinical specimen, offers a rapid assay time, and shows improvements in the differentiation of mucinous and non-mucinous cysts using an accurate standardized readout of product formation, all without interfering with the clinical standard of care.

Tissue Expression Analysis, Cloning, and Characterization of the 5'-Regulatory Region of the Bovine LATS1 Gene.

As a member of the large tumor suppressor (LATS) gene family, LATS1 plays an important role in regulating muscle growth and development. In this study, we determined the distinct exhibit patterns of tissue expression of bovine LATS1. Further, we determined the functional proximal minimal promoter of bovine LATS1 and identified the key transcription factors in the core promoter region to elucidate its molecular regulation mechanism. The results showed that bovine LATS1 was highly expressed in the longissimus thoracis and upregulation in infancy muscle. An electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay in combination with site-directed mutation and small interfering RNA (siRNA) interference demonstrated that myogenic differentiation 1 (Myod1) and myocyte enhancer factor 2A (MEF2A) binding in the core promoter region (−298/−123 bp) play important roles in the transcriptional regulation of the bovine LATS1 promoter. Taken together, these interactions provide insight into the regulatory mechanisms of LATS1 transcription in mediating skeletal muscle growth in cattle.

Dimethyl phthalate induces blood immunotoxicity through oxidative damage and caspase-dependent apoptosis

Dimethyl phthalate (DMP), a low-molecular-weight phthalate ester, exists in ectoparasiticides, plastics, and insect repellants, and has been linked to neurotoxic, reproductive, and endocrine disruptive responses. However, its blood immunotoxic effects and mechanism are still poorly understood. In this study, rats were exposed to gradient concentrations of DMP through intragastric administration to assess the blood immunotoxic effects in the combined assay of biomarker, cytometry, and transcriptomics. DMP treatment altered the redox status of rats, thus causing oxidative damage. Significantly decreased blood cell counts and disordered antibody and cytokine secretion were observed in treated rats, suggesting the suppressed immune defense and destructed inflammatory regulation. Flow cytometry showed that in lymphocytes, especially CD3+CD4+ T cells, the occurrence of apoptosis/necrosis was positively related to DMP exposure level. Transcriptomics revealed an oxidative stress-related mechanism. The overexpression of the Bcl-2 family genes and the activation of the Fas/FasL pathway triggered downstream caspase cascade and caused reactive oxygen species signaling-mediated apoptosis/necrosis. To the best of our knowledge, it was the first report that the exposure to low-molecular-weight phthalate esters potentially triggered blood immunotoxicity. The result and underlying mechanisms can provide an essential basis for understanding phthalate ester toxicity and usage regulation.

Multiphoton Microscopy Reveals DAPK1-Dependent Extracellular Matrix Remodeling in a Chorioallantoic Membrane (CAM) Model.

Simple SummaryThe formation of metastasis is not only intricately orchestrated by cancer cells but is also affected by the surrounding extracellular matrix (ECM). The barrier function of the ECM represents an obstacle that cancer cells have to overcome to disseminate from the primary tumor to form metastasis in distant organs. Here, we demonstrate an approach to studying the remodeling of a collagen-rich ECM by colorectal tumor cells using multiphoton microscopy (MPM). This approach allows the analysis of the invasion front of tumors grown on the CAM in 3D. MPM is superior to conventional histology, which is limited to 2D analysis and needs extensive tissue preparation.Cancer cells facilitate tumor growth by creating favorable tumor micro-environments (TME), altering homeostasis and immune response in the extracellular matrix (ECM) of surrounding tissue. A potential factor that contributes to TME generation and ECM remodeling is the cytoskeleton-associated human death-associated protein kinase 1 (DAPK1). Increased tumor cell motility and de-adhesion (thus, promoting metastasis), as well as upregulated plasminogen-signaling, are shown when functionally analyzing the DAPK1 ko-related proteome. However, the systematic investigation of how tumor cells actively modulate the ECM at the tissue level is experimentally challenging since animal models do not allow direct experimental access while artificial in vitro scaffolds cannot simulate the entire complexity of tissue systems. Here, we used the chorioallantoic membrane (CAM) assay as a natural, collagen-rich tissue model in combination with all-optical experimental access by multiphoton microscopy (MPM) to study the ECM remodeling potential of colorectal tumor cells with and without DAPK1 in situ and even in vivo. This approach demonstrates the suitability of the CAM assay in combination with multiphoton microscopy for studying collagen remodeling during tumor growth. Our results indicate the high ECM remodeling potential of DAPK1 ko tumor cells at the tissue level and support our findings from proteomics.

CRISPR-Cas12a combination to alleviate the false-positive in loop-mediated isothermal amplification-based diagnosis of Neisseria meningitidis

BackgroundLoop isothermal amplification (LAMP) has recently been proposed as a point-of-care diagnostic tool to detect acute infectious pathogens; however, this technique embeds risk of generating false-positive results. Whereas, with abilities to accurately recognize specific sequence, the CRISPR/Cas12a can forms complexes with cognate RNA sensors and cleave pathogen’s DNA targets complimerntary to its cognate RNA, afterward acquiring the collateral activity to unbiasedly cut nearby off-target fragments. Therefore, if relevant fluorescent-quencher-nucleic probes are present in the reaction, the non-specific cleavage of probes releases fluorescences and establish diagnostic read-outs.MethodsThe MetA gene of N. meningitidis was selected as target to optimize the LAMP reaction, whereas pseudo-dilution series of N. meningitidis gemonics DNA was used to establish the detection limit of LAMP/Cas12a combination assay. The diagnostic performance of established LAMP/Cas12a combination assay was validated in comparation with standard real-time PCR on 51 CSF samples (14 N. meningitidis confirmed patients and 37 control subjects).ResultsIn relevant biochemical conditions, CRISPR-Cas12a and LAMP can work synchronously to accurately identify genetics materials of Nesseria menitigistis at the level 40 copies/reaction less than 2 h.ConclusionsIn properly optimized conditions, the CRISPR-Cas12a system helps to alleviate false positive result hence enhancing the specificity of the LAMP assays.

May Chelated Iron Be Pro‐Inflammatory?

We have shown that two iron chelates, used in iron nutrition, promote induction of the oncogenic growth factor Amphiregulin in human gut epithelial cells. Since then, we have investigated several iron compounds on the safe lists of EFSA and USFDA using a human intestinal cell assay in combination with proteomic profiling. Here we will report proteomic cell data for other iron chelates, salts and nanoparticulate iron which suggest that iron chelates may increase the cellular sensitivity to pro‐inflammatory mediators and growth promotors by increasing their receptor levels. We conclude that iron chelates may be pro‐inflammatory and pro‐oncogenic to intestinal cells.

RNA-protein Co-detection using spatial analysis to profile tumor-infiltrated immune cells

Abstract Interrogating complex tumor microenvironment requires a multi-omics approach. Detecting target immune cell markers using immunohistochemistry/Immunofluorescence (IHC/IF) and visualizing cytokine expression with in situ hybridization (ISH) can provide comprehensive information about the activation states of immune cells. Here, we demonstrate a newly developed integrated ISH and IHC/IF workflow compatible with manual and automated platforms with improved RNA-protein co-detection. We demonstrate the use of our RNA-Protein Co-detection assay in combination with the automated and manual RNAscope Multiplex Fluorescent assay as well as the automated RNAscope Chromogenic Duplex assay to detect T cell markers, macrophage markers and checkpoint markers in the tumor microenvironment by using a microarray with different tumor samples. We identified CD4+ helper T cells, CD8+ cytotoxic T cells and determined their activation states by visualizing IFNG, GZMB and IL-2 expression. We were also able to identify macrophages detected by CD68 protein expression and differentiate the M1 and M2 subtypes by using M2-specific marker, CD163. We could also delineate tumor-stroma border in the samples by using the Pan-CK probe which distinctly marks the tumor cells and visualize expression of immunoregulatory receptors PD-L1 and CTLA4 in the tumor. This assay is enables for multiplexing by combining with our fluorescent assays or enables target detection while retaining morphological context when combined with our chromogenic assays. Overall, the new RNAscope-ISH-IHC/IF co-detection workflow and reagents enable simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies with minimal optimization.

How does S‐palmitoylation affect the Organic Anion Transporting Polypeptide 1B1 (OATP1B1)?

BackgroundOrganic Anion Transporting Polypeptide 1B1 (OATP1B1) is an important transporter localized to the basolateral membrane of human hepatocytes that mediates the uptake of numerous therapeutics like statins, antibiotics, and anti‐cancer agents. Previous studies from our lab suggest that OATP1B1 is localized in membrane microdomains, also known as lipid rafts, which contain various components like saturated lipids, cholesterol, and lipidated proteins. Lipidated proteins are frequently the result of S‐palmitoylation, a reversible post translational modification that adds palmitic acid (C16:0) to cysteine residues. Currently, not much is known about the potential impact of S‐palmitoylation on OATP1B1 function and localization. Aim: We hypothesize that OATP1B1 is S‐palmitoylated and that this affects its function and localization.MethodsWe measured uptake of the model substrate estradiol‐17β‐glucuronide in cells expressing OATP1B1. S‐palmitoylation was detected using an acyl‐biotin exchange (ABE) assay in combination with western blotting. To prevent S‐palmitoylation, cells were preincubated with 2‐bromopalmitate (2BP). Expression of OATP1B1 at the plasma membrane was assessed using surface biotinylation.ResultsThe ABE assay demonstrated that OATP1B1 is S‐palmitoylated and that pretreatment of the cells for 18 hours with 2BP reduces S‐palmitoylation. Inhibition of S‐palmitoylation with 2BP for 18 hours also resulted in decreased OATP1B1‐mediated transport of estradiol‐17β‐glucuronide as well as in a reduction of surface expression of OATP1B1.Summary and ConclusionsOur results demonstrate that OATP1B1 is S‐palmitoylated. Inhibition of S‐palmitoylation reduces the function and the surface expression of OATP1B1. Thus, these findings suggest that S‐palmitoylation is a post‐translational modification involved in the regulation of this important hepatic drug uptake transporter.

Extending the capabilities of a high-parameter immunophenotyping assay with cytoplasmic staining applications for mass cytometry

Abstract Maxpar® Direct™ Immune Profiling Assay™ (Cat. No. 201325) is a 30-marker panel for suspension mass cytometry. This panel provides an unprecedented sample-to-answer solution for detecting and analyzing 30 surface markers in a single experiment. The 18 open mass channels in the Maxpar Direct Assay facilitate panel expansion and enable flexibility for higher multiplexity and applications. Among the potential complementary applications, intracellular cytokine staining (ICS) is of particular interest as it may be used to assess infiltrating immune cell phenotypes in the tumor microenvironment. However, for the purpose of assessing cell viability in this workflow, the effectiveness of the Cell-ID™ Intercalator-Rh (103Rh, Cat. No. 201103) included in the Maxpar Direct Assay is in question, as cell permeabilization during ICS can potentially damage the DNA-intercalator bond. In this study, we investigated the compatibility of 103Rh with intracellular staining. We stained either human peripheral blood mononuclear cell or whole blood samples with the Maxpar Direct Assay followed by intracellular staining for the detection of expressed cytokines. We demonstrate that 103Rh provides equivalent functionality as a cell viability indicator during intracellular staining for cytoplasmic proteins compared to the benchmark Cell-ID Cisplatin-194Pt (Cat. No. 201194). This work was designed to support use of the Maxpar Direct Immune Profiling Assay in combination with additional intracellular markers. Overall, these findings expand the applicability of Cell-ID Intercalator-Rh (103Rh) to processes that involve cytoplasmic staining. For Research Use Only. Not for use in diagnostic procedures.

Antioxidant and Antibacterial potential of Lycium shawii Roem. and Schult (Awsaj) and Tetraena qatarensis (Hadidi) Beier and Thulin leaves: A Herbal approach to Combat Drug Resistance and Oxidative Stress

The present investigation deals with the antioxidant and antibacterial activity of chloroform and methanolic extracts of Lycium shawii and Tetraena qatarensis leaves. Antioxidant activity was evaluated using DPPH and FRAP assay. In the DPPH assay, the lowest IC50 values 45.63 and 46.17µg/ml were observed with methanolic and chloroform extract Lycium shawii leaves, respectively. In FRAP assay most of the extracts showed optical density &gt;0.5 at 40µg/ml. In the antibacterial activity test, all tested extracts were found active towards all bacterial strains with MIC ranged between 0.03125 to 0.12mg/mL. Subsequently, in the combined effect assay, the highest fold increase (92.31%) was observed against Salmonella typhi with methanol extract of Lycium shawii leaves. The examined plants could be used as a treatment for microbial infection and oxidative stress after detailed follow-up investigations.

Antifungal, antibiofilm and anti-resistance activities of Brazilian monofloral honeys against Candida spp.

Candida is a fungus commonly involved in diseases of the skin and mucous membranes and has a high resistance to antifungal drugs. Moreover, there are few therapeutic options to treat these infections. Honey is a popular product that is easily accessible and has a diverse chemical composition, such as phenolic compounds that can have an antimicrobial effect in various metabolic pathways. Therefore, in this study, we aimed to characterize the antifungal activity of tree popular Brazilians honey against Candida species. Aroeira, Eucalyptus and Assa-Peixe honey was characterized by total phenolics and its antifungal activity on Candida species was tested by the microdilution method, antibiofilm activity, yeast to hypha (Y–H) transition, combination assays, multistage resistance study, and antibi-scanning electron microscopy of the fungi. Aroeira honey showh potent antifungal activity in vitro against planktonic cells of C. albicans (MIC 20–30% w/v) and non-albicans (MIC 30–40% w/v) and also breaks down mature biofilms formed by these yeasts at 40% w/v. Urethral catheters sensitized with 30% Aroeira honey reduced adhesion and biofilm formation by C. albicans. Aroeira honey did not induce resistance in C. albicans during the 21-day exposure. Its ability to alter fungal membrane structure contributes to the synergistic effects of Aroeira honey with commercial antifungal agents (miconazole, ketoconazole, itraconazole). Aroeira honey inhibits yeast-to-alpha transition in C. albicans, and its prior exposure for 1 h reverses the azole resistance profile of a highly resistant strain of C. albicans. Taken together, these results indicate that Aroeira honey is a promising therapeutic agent against superficial candidiasis.

Integrating central nervous system metagenomics and host response for diagnosis of tuberculosis meningitis and its mimics

The epidemiology of infectious causes of meningitis in sub-Saharan Africa is not well understood, and a common cause of meningitis in this region, Mycobacterium tuberculosis (TB), is notoriously hard to diagnose. Here we show that integrating cerebrospinal fluid (CSF) metagenomic next-generation sequencing (mNGS) with a host gene expression-based machine learning classifier (MLC) enhances diagnostic accuracy for TB meningitis (TBM) and its mimics. 368 HIV-infected Ugandan adults with subacute meningitis were prospectively enrolled. Total RNA and DNA CSF mNGS libraries were sequenced to identify meningitis pathogens. In parallel, a CSF host transcriptomic MLC to distinguish between TBM and other infections was trained and then evaluated in a blinded fashion on an independent dataset. mNGS identifies an array of infectious TBM mimics (and co-infections), including emerging, treatable, and vaccine-preventable pathogens including Wesselsbron virus, Toxoplasma gondii, Streptococcus pneumoniae, Nocardia brasiliensis, measles virus and cytomegalovirus. By leveraging the specificity of mNGS and the sensitivity of an MLC created from CSF host transcriptomes, the combined assay has high sensitivity (88.9%) and specificity (86.7%) for the detection of TBM and its many mimics. Furthermore, we achieve comparable combined assay performance at sequencing depths more amenable to performing diagnostic mNGS in low resource settings.

VaxArray immunoassay for the multiplexed quantification of poliovirus D-antigen

Next generation poliovirus vaccines are critical to reaching global poliovirus eradication goals. Recent efforts have focused on creating inactivated vaccines using attenuated Sabin strains that maintain patient safety benefits and immunogenicity of conventional inactivated vaccines while increasing manufacturing safety and lowering production costs, and on developing novel oral vaccines using modified Sabin strains that provide critical mucosal immunity but are further attenuated to minimize risk of reversion to neurovirulence. In addition, there is a push to improve the analytical tools for poliovirus vaccine characterization. Conventional and Sabin inactivated poliovirus vaccines typically rely on standard plate-based ELISA as in vitro D-antigen potency assays in combination with WHO international standards as calibrants. While widely utilized, the current D-antigen ELISA assays have a long time to result (up to 72 h), can suffer from lab-to-lab inconsistency due to non-standardized protocols and reagents, and are inherently singleplex. For D-antigen quantitation, we have developed the VaxArray Polio Assay Kit, a multiplexed, microarray-based immunoassay that uses poliovirus-specific human monoclonal antibodies currently under consideration as standardized reagents for characterizing inactivated Sabin and Salk vaccines. The VaxArray assay can simultaneously quantify all 3 poliovirus serotypes with a time to result of less than 3 h. Here we demonstrate that the assay has limits of quantification suitable for both bioprocess samples and final vaccines, excellent reproducibility and precision, and improved accuracy over an analogous plate-based ELISA. The assay is suitable for adjuvanted combination vaccines, as common vaccine additives and crude matrices do not interfere with quantification, and is intended as a high throughput, standardized quantitation tool to aid inactivated poliovirus vaccine manufacturers in streamlining vaccine development and manufacturing, aiding the global polio eradication effort.

Sonidegib potentiates the cancer cells’ sensitivity to cytostatic agents by functional inhibition of ABCB1 and ABCG2 in vitro and ex vivo

Sonidegib (LDE-225) is a Hedgehog pathway inhibitor used for the therapy of basal cell carcinoma. In addition, the drug is a subject of clinical trials for the treatment of other solid tumors including non-small cell lung cancer (NSCLC). In this study, we explored the potential of sonidegib to act as a perpetrator of drug-drug interactions (DDIs) and modulator of transporter- and enzyme-mediated multidrug resistance (MDR). First, we found that transport functions of ABCB1 and ABCG2 were effectively inhibited by sonidegib in accumulation studies. In contrast, the drug did not cause fluctuations in mRNA levels of tested efflux transporters. In drug combination assays, sonidegib synergistically enhanced the cytotoxicity of daunorubicin and mitoxantrone in ABCB1- and ABCG2-overexpressing cells, respectively. Notably, similar phenomena were also observed in explant tumor cultures derived from NSCLC-suffering patients. In addition, the anticancer effects of sonidegib were not hampered by the expression of the ABC transporters associated with MDR. Last, sonidegib had no significant influence on the activity of CYP3A4 isoform in vitro. In summary, our work suggests that sonidegib can be considered a potential perpetrator of clinical DDIs on ABCB1 and ABCG2. After in vivo evaluation, its chemosensitizing properties might be projected into efficient and safe treatment regimen for the clinical management of NSCLC patients with high ABCB1/ABCG2 expression.