Method for detecting acetylated PD-L1 in cell lysates v1

Here we present a method that allows detection of acetylated PD-L1 and is applicable to a wide range of cell lines. The method captures >90% of acetylated PD-L1 species, is semi-quantitative and simple to perform in any lab equipped with tissue culture and western blot equipment. The method involves processing cells in a lysis buffer that has been optimized for efficient immunoprecipitation (IP) of acetylated species, an IP enrichment step utilizing an acetyl-lysine affinity matrix and western blot detection of both total and acetylated PD-L1 on the same blot. This technique compliments the alternative IP approach utilizing a PD-L1 antibody as the IP reagent and an anti-acetyl lysine antibody as the detection reagent. However, because the protocol described here enables the detection of both total and acetylated PD-L1 on the same blot, this method has the advantage of allowing quantitation of the percent of PD-L1 that is acetylated, an important parameter for mechanistic interpretation. The method described here utilizes beads that are covalently linked to the affinity antibody, resulting in extremely clean IP results. Western blots can be re-probed with a pan anti-acetyl lysine antibody to visualize the total protein acetylation profile in any given lysate, a property that is useful when examining PD-L1 acetylation in the presence of HDAC inhibitors or other treatments affecting global acetylation.

Development of a qPCR for the detection and quantification of Salmonella spp. in sheep feces and tissues

Salmonella spp. are common causes of disease in intensive livestock production systems, and contamination of foodstuffs is of significant concern for public health. Therefore, the identification and quantification of Salmonella spp. is important for monitoring the level of fecal shedding or tissue colonization in infected animals and animal products. We developed and evaluated a quantitative PCR (qPCR) method on spiked sheep tissue and fecal samples for the detection and quantification of Salmonella spp. Without the use of a pre-enrichment step, the qPCR limit of detection (LOD) results for sheep fecal (4 × 104–6 × 103 cfu/g) and tissue (4 × 105–4 × 103 cfu/g) samples were not adequate for detection purposes. With the inclusion of a 6-h pre-enrichment step in buffered peptone water (BPW), the LOD was 9 cfu/g (2.57 × 101 copies/g) in sheep feces, and 5.4 cfu/g (3.22 copies/g) sheep tissue. Comparison of the 6-h BPW qPCR method with a 24-h mannitol–selenite–cystine broth enrichment culture method using spiked samples revealed a sensitivity of 91% and 92%, respectively, and a specificity of 100% for both methods. The correlation was significant between the quantity (copies/mL) of Salmonella spp. in BPW at 6 h and at 0 h, allowing semiquantitative analysis. Our results demonstrate that, following inclusion of a 6-h pre-enrichment step in BPW, qPCR is semiquantitative with improved LODs of Salmonella spp. in sheep fecal and tissue samples.

Cartography of Free-Living Amoebae in Soil in Guadeloupe (French West Indies) Using DNA Metabarcoding

Free-living amoebae (FLA) are ubiquitous protists. Pathogenic FLA such as N. fowleri can be found in hot springs in Guadeloupe, soil being the origin of this contamination. Herein, we analyzed the diversity and distribution of FLA in soil using a targeted metataxonomic analysis. Soil samples (n = 107) were collected from 40 sites. DNA was extracted directly from soil samples or from FLA cultivated at different temperatures (30, 37 and 44 °C). Metabarcoding studies were then conducted through FLA 18SrDNA amplicons sequencing; amplicon sequence variants (ASV) were extracted from each sample and taxonomy assigned against SILVA database using QIIME2 and SHAMAN pipelines. Vermamoeba were detected in DNA extracted directly from the soil, but to detect other FLA an amoebal enrichment step was necessary. V. vermiformis was by far the most represented species of FLA, being detected throughout the islands. Although Naegleria were mainly found in Basse-Terre region, N. fowleri was also detected in Grand Terre and Les Saintes Islands. Acanthamoeba were mainly found in areas where temperature is approx. 30 °C. Vannella and Vahlkampfia were randomly found in Guadeloupe islands. FLA detected in Guadeloupe include both pathogenic genera and genera that can putatively harbor microbial pathogens, therefore posing a potential threat to human health.

Selective Binding of a Lower Lysine Methylation State: An N,N-Dimethyllysine Selective Host Molecule and Its Use in Methyl Proteomics

<p>Post-translational modifications (PTMs) are critical controllers of protein functions. One set of important PTMs are <i>N</i>-methylated side chains of lysine and arginine, which exist in several functionally distinct forms. Multiple groups have demonstrated the selective binding of the most hydrophobic family member, trimethyllysine (Kme3), using various macrocyclic hosts, but the selective binding of lower methylation states remains challenging. Herein we report that a new calixarene modification – the installation of a sulfonate ester at the lower rim of <i>p</i>-sulfonatocalix[4]arene —efficiently generates a <i>N,N</i>-dimethyllysine (Kme2)-selective host. We characterize its binding behaviors in solution, and demonstrate its effectiveness in a pan-methyllysine enrichment step that enables the observation of hundreds of otherwise unobservable methylation marks in global proteomics experiments.</p><p>The submission includes a manuscript preprint, supporting information, and a tabulation of proteomics data.</p>

Selective Binding of a Lower Lysine Methylation State: An N,N-Dimethyllysine Selective Host Molecule and Its Use in Methyl Proteomics

<p>Post-translational modifications (PTMs) are critical controllers of protein functions. One set of important PTMs are <i>N</i>-methylated side chains of lysine and arginine, which exist in several functionally distinct forms. Multiple groups have demonstrated the selective binding of the most hydrophobic family member, trimethyllysine (Kme3), using various macrocyclic hosts, but the selective binding of lower methylation states remains challenging. Herein we report that a new calixarene modification – the installation of a sulfonate ester at the lower rim of <i>p</i>-sulfonatocalix[4]arene —efficiently generates a <i>N,N</i>-dimethyllysine (Kme2)-selective host. We characterize its binding behaviors in solution, and demonstrate its effectiveness in a pan-methyllysine enrichment step that enables the observation of hundreds of otherwise unobservable methylation marks in global proteomics experiments.</p><p>The submission includes a manuscript preprint, supporting information, and a tabulation of proteomics data.</p>

Detection and Quantification of FLT3 Internal Tandem Duplication Mutations Do Not Vary Significantly Between Whole Blood and Blast-Enriched Samples

Objectives Many commonly used FLT3 mutational assay protocols require a tedious blast enrichment step. We investigated whether elimination of this step would still give equivalent results and compared the accuracy of variant allele fraction (VAF) between polymerase chain reaction/capillary electrophoresis (PCR/CE) vs next-generation sequencing (NGS) methods. Methods Total leukocyte vs blast-enriched whole-blood aliquots were tested for FLT3 internal tandem duplication (ITD) and tyrosine kinase domain mutations by PCR/CE. VAF of the ITD mutations was also compared with NGS VAF. Results Blast-enriched vs total leukocyte specimens showed 100% concordance in the 25 positive specimens. VAF was consistently lower by NGS, with poorer fidelity to PCR/CE VAF as the ITD size increased. Conclusions Our study supports elimination of the blast enrichment step without compromising results or sensitivity. In addition, since NGS shows a loose correlation with PCR/CE quantitative results, NGS VAF should not be reported for FLT3 ITDs.

Developing a multiplex real-time PCR with a new pre-enrichment to simultaneously detect four foodborne bacteria in milk

Aim: The aim of this study is to formulate a new single nonselective pre-enrichment medium (ELSS) that can support the concurrent growth of four major foodborne pathogens containing E. coli O157: H7, L. monocytogenes, S. aureus and S. enterica serovar Entertidis to develop a multiplex TaqMan Real-time PCR (mRT-PCR). Methods: The mRT-PCR with a new pre-enrichment was carried out for simultaneous detection and quantification of these foodborne bacteria. Results: By using mRT-PCR after 16 h pre-enrichment in ELSS, the detection limit of each pathogen was 1 CFU/25 ml contaminated milk, as well as inclusivity and exclusivity reached 100%. Conclusion: The mRT-PCR assay with pre-enrichment step is a fast and reliable technique for detecting single or multiple pathogens in food products.