Mass cytometry is a revolutionary technology that allows for the simultaneous quantification of >40 different biomarkers with cellular resolution. The biomarkers are detected using metal-labeled antibodies as well as small-molecule probes of cell size, viability, and biochemical status. Barcoding is an important component of sample preparation because it reduces processing time, eliminates sample-to-sample variation, discriminates cell doublets, reduces the amount of antibody needed, and conserves sample. We developed a thiol-reactive tellurium-based barcode, TeMal. TeMal is nontoxic at working concentrations, compatible with metal-labeled antibodies, and can readily be applied to live or fixed cells, making it advantageous and complementary compared to existing barcoding reagents. We have demonstrated the utility of TeMal by barcoding microscale samples in situ to facilitate analysis of cells from an automated cell culture system using mass cytometry.

Mass cytometry (MC) measures metal isotope signals from single cells and bead samples. Since large numbers of isotopes can be employed as labels, mass cytometry is a powerful analytical technique for multiparameter cytometric assays. The calibration protocol in MC is a critical algorithm, which employs metal-encoded microbeads as an internal standard to correct the data for instrumental signal drift. The current generation of commercially available beads carries four lanthanide elements (cerium, europium, holmium, and lutetium). However, this is not sufficient to calibrate the full span of detection channels, ranging from yttrium (89 amu) to bismuth (209 amu), which are now available. To address this issue we prepared polystyrene microbeads encoded with seven elements (yttrium, indium, and bismuth in addition to the four lanthanides) by multistage dispersion polymerization for MC calibration and normalization. The bead synthesis conditions were optimized to obtain microbeads that were uniform in size and generated strong MC signal intensities at similar levels for the eight encoded isotopes. Metal ion leaching from the beads under storage and application conditions was also examined. We demonstrated that the precision of normalized MC signals in the MC detection channels was improved by employing seven-element-encoded microbeads as a standard.

In the field of cell-based therapeutics, there is a great need for high-quality, robust, and validated measurements for cell characterization. Flow cytometry has emerged as a critically important platform due to its high-throughput capability and its ability to simultaneously measure multiple parameters in the same sample. However, to assure the confidence in measurement, well characterized biological reference materials are needed for standardizing clinical assays and harmonizing flow cytometric results between laboratories. To date, the lack of adequate reference materials, and the complexity of the cytometer instrumentation have resulted in few standards. This study was designed to evaluate CD19 expression in three potential biological cell reference materials and provide a preliminary assessment of their suitability to support future development of CD19 reference standards. Three commercially available human peripheral blood mononuclear cells (PBMCs) obtained from three different manufacturers were tested. Variables that could potentially contribute to the differences in the CD19 expression, such as PBMCs manufacturing process, number of healthy donors used in manufacturing each PBMC lot, antibody reagent, operators, and experimental days were included in our evaluation. CD19 antibodies bound per cell (ABC) values were measured using two flow cytometry-based quantification schemes with two independent calibration methods, a single point calibration using a CD4 reference cell and QuantiBrite PE bead calibration. Three lots of PBMC from three different manufacturers were obtained. Each lot of PBMC was tested on three different experimental days by three operators using three different lots of unimolar anti-CD19PE conjugates. CD19 ABC values were obtained in parallel on a selected lot of the PBMC samples using mass spectrometry (CyTOF) with two independent calibration methods, EQ4 and bead-based calibration were evaluated with CyTOF-technology. Including all studied variabilities such as PBMC lot, antibody reagent lot, and operator, the averaged mean values of CD19 ABC for the three PBMC manufacturers (A,B, and C) obtained by flow cytometry were found to be: 7953 with a %CV of 9.0 for PBMC-A, 10535 with a %CV of 7.8 for PBMC-B, and 12384 with a %CV of 16 for PBMC-C. These CD19 ABC values agree closely with the findings using CyTOF. The averaged mean values of CD19 ABC for the tested PBMCs is 9295 using flow cytometry-based method and 9699 using CyTOF. The relative contributions from various sources of uncertainty in CD19 ABC values were quantified for the flow cytometry-based measurement scheme. This uncertainty analysis suggests that the number of antigens or ligand binding sites per cell in each PBMC preparation is the largest source of variability. On the other hand, the calibration method does not add significant uncertainty to the expression estimates. Our preliminary assessment showed the suitability of the tested materials to serve as PBMC-based CD19+ reference control materials for use in quantifying relevant B cell markers in B cell lymphoproliferative disorders and immunotherapy. However, users should consider the variabilities resulting from different lots of PBMC and antibody reagent when utilizing cell-based reference materials for quantification purposes and perform bridging studies to ensure harmonization between the results before switching to a new lot.

Intraspecific variation in external and internal pigmentation is common among fishes and explained by a variety of biological and ecological factors. Blue-colored flesh in fishes is relatively rare but has been documented in some species of the sculpin, greenling, and perch families. Diet, starvation, photoprotection, and camouflage have all been suggested as proximate mechanisms driving blue flesh, but causal factors are poorly understood. We evaluated the relative importance of biological and spatial factors that could explain variation in blue coloration in 2021 lingcod (Ophiodon elongatus) captured across their range in the northeastern Pacific, from southeast Alaska to southern California. The probability of having blue flesh was highest for fish that were female, caught in shallower water, and smaller in body size. The incidence of blueness varied by region (4–25% of all fish) but was also confounded by differences in sex ratios of fish caught among regions. We analyzed the multivariate fatty acid composition of a subset of 175 fish from across the sampling range to test for differences in trophic biomarkers in blue lingcod. Lingcod fatty acid composition differed between regions and flesh colors but not between sexes. Blue-fleshed fish had lower concentrations of total fatty acids, 18:1ω-9, 16:1ω-7, 18:1ω-7, and ω-6 fatty acids, suggesting differences in energetics and energy storage in blue fish. While our data indicate potential links between diet and blue flesh in lingcod, important questions remain about the physiological mechanisms governing blueness and its biological consequences.

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In recent years, there have been increasing numbers of bacterial strains emerging that are resistant to the currently available antibiotics. In the search for new antibiotics, attention has been focused on natural antimicrobial peptides that act by selectively disrupting the membranes of bacterial cells, a mechanism that is thought to be nonconducive to the development of resistance. It is desirable to mimic the structures and activities of these peptides while introducing properties such as resistance to proteolytic degradation, which make molecules more ideal for development as drugs. Described here is the design and synthesis of beta-strand mimetic oligomers based on alternating alpha-amino acids and azacyclohexenone units that segregate cationic lysine and hydrophobic valine side chains on opposite faces of the beta-strand. (1)H NMR dilution studies demonstrated that despite the incorporation of alternating d- and l-amino acids in order to obtain facial amphiphilicity, these oligomers are capable of dimerizing to beta-sheet mimics in a manner similar to the oligomers containing all l-amino acids. The ability of the molecules to disrupt phospholipid vesicles mimicking the membranes of both bacterial and mammalian cells was investigated using a fluorescent dye leakage assay. Several of the oligomers were found to exhibit activity and selectivity for the bacterial over mammalian membranes. Overall, these studies demonstrate the promise of this class of molecules for the development of new potential antibiotics and provide information on the structural features that are important for activity.

Fishery-independent hook-and-line surveys are currently being used to assess marine reserve performance in California and Oregon using a regionally standardized approach. Catch compositions generated from these hookand-line surveys (pole-and-line gear) at Oregon's southernmost marine reserve were compared with local commercial landing data. Several species present in the commercial catch were undersampled in the marine reserve hook-and-line dataset, including China Rockfish Sebastes nebulosus, Vermilion Rockfish S. miniatus, Quillback Rockfish S. maliger, Copper Rockfish S. caurinus, and Cabezon Scorpaenichthys marmoratus. We conducted a gear selectivity study to explore whether modified commercial long-lining gear could supplement current hook-and-line efforts. Both gear types were fished simultaneously from a single vessel inside and outside of the reserve. Species composition, catch rate, size distribution, and fish condition between the two gear types were compared. Catch composition differed significantly between longline and hook-and-line gear. Catch rates of nearshore rocky reef fish species were higher for longline than hook-and-line gear for all but two species. Importantly, higher catch rates were significant for three of the species of interest (Cabezon, Vermilion Rockfish, and Copper Rockfish). For four different species, larger individuals were caught on the longline compared with the hook-and-line gear. Incidence of predation and mortality were higher with long-lining but limited to three species groups: Black Rockfish S. melanops, Blue Rockfish S. mystinus and Deacon Rockfish S. diaconus complex, and Canary Rockfish S. pinniger. Symptoms of barotrauma were higher with hook-and-line gear. We demonstrated that longline gear can be used to catch and release species targeted by the local fishery and used simultaneously with hook-and-line gear from a single vessel to broaden both the species and the size ranges sampled. These results underscore the need to consider regionally standardized long-term monitoring approaches in conjunction with locally tailored efforts to generate data for detecting marine reserve effects at both local and regional scales.Fishery-independent data-collected independently from fisheries landings and logbooks-are recognized as important for improving fishery stock assessments (Harms et al. 2010), monitoring for temporal changes in trophic structure (Shackell et al. 2010), and evaluating the performance of spatial fishing closures (Yoklavich et al. 2007). Different sampling methods have been used to generate fishery-independent data, including nonextractive visual surveys (Watson et al. 2005) and extractive surveys using a variety of fishing gear. Extractive surveys are particularly useful in the nearshore waters of the

Mass cytometry is a novel cell-by-cell analysis technique, which uses elemental tags instead of fluorophores. Sample cells undergo rapid ionization in inductively coupled plasma and the ionized elemental tags are then analyzed by means of time-of-flight mass spectrometry. Benefits of the mass cytometry approach are in no need for compensation, the high number of detection channels (up to 100) and low background noise. In this work, we applied a biotinylated aptamer against human PTK7 receptor for characterization of positive (human acute lymphoblastic leukemia) and negative (human Burkitt's lymphoma) cells by a mass cytometry instrument. Our proof of principal experiments showed that biotinylated aptamers in conjunction with metal-labeled neutravidin can be successfully utilized for mass cytometry experiments at par with commercially available antibodies. Graphical abstract Biotinylated aptamers in conjunction with metal-labeled neutravidin bind to cell biomarkers, and then injected into the inductively coupled plasma (ICP) source, where cells are vaporized, atomized, and ionized in the plasma for subsequent mass spectrometry (MS) analysis of lanthanide metals.