BD Biosciences (United States)

The Metaproteomics Initiative was officially launched in 2021 to strengthen collaboration, promote knowledge exchange, and support and lead standardization efforts within the growing metaproteomics community. Over the past five years, the Initiative has developed into a structured, global network of researchers. It has launched community-driven benchmark studies, helped shape emerging metadata and reporting standards, developed practical guidance and training materials, organized international symposia, and fostered connections across the microbiome research landscape (https://metaproteomics.org/). We outline the Initiative's organization, activities, achievements, and ongoing efforts, and reflect on how sustained, community-led coordination has shaped the development of metaproteomics as a field. We further position the Grand Metaproteome Challenges as a next step toward coordinated, community-scale biological research, aimed at advancing functional microbiome studies across clinical, industrial, and environmental application domains, and invite engagement from the wider microbiome and omics communities.

Multiparametric flow cytometry (MFC) enables rapid and sensitive quantification of measurable residual disease (MRD) in patients with acute myeloid leukaemia (AML), providing valuable prognostic information and guiding post-remission therapeutic strategies. Given the clinical utility of MFC AML MRD assessment and its inherent technical and analytical complexity, inter-laboratory standardisation is essential to ensure consistency of practice, diagnostic accuracy and reproducibility of results. However, limited options for external quality assessment exist. Representatives from all diagnostic laboratories in Australia and New Zealand currently performing AML MRD testing were invited to attend an in-person workshop to review site-specific practices and develop harmonisation processes. All six participating laboratories demonstrated a high level of concordance with respect to pre-analytical testing; however, greater variation was observed in post-analytical reporting, and a number of consensus recommendations were formulated for harmonisation. Ongoing meetings have also been established to promote continued sharing of expertise between AML MRD laboratories, align analytical strategies and enhance assay validation in accordance with regulatory requirements. These collaborative efforts provide guidance to existing MRD laboratories and those seeking to establish a new MRD service, facilitating sustainable provision of a high-quality regional MFC AML MRD testing network to meet current and anticipated increases in clinical demand.

A spray-dried powderformulation of 5-azacytidine (5-AZA) recentlyshowed significantly improved tumor reduction and systemic exposurefor the potential treatment of metastatic lung cancer. To supportclinical use, the target product profile required double the activeloading. Here, a series of powders were spray dried to increase 5-AZAloading in the formulation, and process parameters were varied toidentify variables controlling powder properties. Formulations weresprayed via in-line mixing, where separate dimethyl sulfoxide (DMSO)and aqueous solutions were mixed immediately upstream of atomization.A core–shell structure was observed matching previous reportsof spray-dried l-leucine with sugar from water and ethanol.To the best of our knowledge, this is the first study investigatingthe process and structure of spray-dried l-leucine and trehaloseusing DMSO and water as processing solvents. Powders with glass transitiontemperatures (Tg) below room temperaturewere successfully manufactured with high yields, attributed to thecrystalline l-leucine shell surrounding the low Tg core. The residual DMSO was controlled by the DMSO:waterratio and the composition of the core. Perturbed-Chain StatisticalAssociating Fluid Theory (PC-SAFT) was employed to understand dropletdrying processes by developing ternary phase diagrams and drying trajectories.This work enables the manufacture of both dry inhalable 5-AZA powderfor the treatment of lung cancer and low Tg materials previously thought to be unmanufacturable via spray drying.It also highlights the importance of aligning experimental data andtheoretical models to improve our understanding of complex processes.

Emerging resistance to current antibiotics is a global threat to human health. Therefore, comprehending the mechanism behind antibiotic resistance holds paramount importance. In the pursuit of finding new antibacterial agents, our group has developed a small molecule, PPEF (2'-(4-ethoxyphenyl)-5-(4-propylpiperazin-1-yl)-1H,1'H-2,5'-bibenzo(d)imidazole), having bisbenzimidazole as a pharmacophore, targeting bacterial type IA topoisomerase, a novel drug target in bacteria. We examined the emergence of mutations leading to PPEF resistance in laboratory-evolved Staphylococcus aureus strains. The growth curve revealed that S. aureus 25923 PPEF-resistant (SA-PR) and methicillin-resistant S. aureus 43300 PPEF-resistant (MRSA-PR) attained stationary phase earlier than their respective reference strains. RNA sequencing analysis revealed that atpD (ATP synthase gene) was downregulated by 2 log2-fold in both SA-PR and MRSA-PR strains, whereas there was 10 to 13 log2-fold downregulation of mecR1 (methicillin resistance-inducing gene), ble (bleomycin resistance-inducing gene), blaZ (beta-lactamase), pbp (penicillin-binding protein gene), ermA (rRNA adenine methyltransferase gene), and kdpB (potassium-transporting ATPase) in the MRSA-PR strain. Quantitative reverse-transcriptase PCR data confirmed these results. Additionally, MRSA-PR showed a 5 log2-fold upregulation of comG and a 9 log2-fold downregulation of topB, indicating increased genomic variability and stress adaptation contributing to resistance. Genomic sequencing revealed deletions of resistance genes, including aac(6')-aph(2''), aadD, mecA, and blaZ in MRSA-PR, resulting in a gain in resistance and a diminishing returns epistasis pattern in PPEF-evolved S. aureus strains. This led to the development of an evolved MRSA-PR strain susceptible to oxacillin, ciprofloxacin, gentamicin, and imipenem. Our findings indicate that adaptation to PPEF has increased antibiotic susceptibility, thereby changing the clinical outcomes of infections.IMPORTANCEThis study investigates how Staphylococcus aureus bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) strain, develop resistance to a new candidate antibacterial compound, PPEF (2'-(4-ethoxyphenyl)-5-(4-propylpiperazin-1-yl)-1H,1'H-2,5'-bibenzo(d)imidazole). The research found that resistant strains grew slower and showed significant changes in the activity of genes related to antibiotic resistance. Some resistance genes were deleted in the resistant MRSA strain, making it more sensitive to other antibiotics like oxacillin and ciprofloxacin. These findings highlight how resistance to PPEF leads to increased sensitivity to conventional antibiotics. This suggests that developing combination therapies of PPEF with other antibiotics could optimize treatment regimens and slow resistance evolution. This study also indicates that the antibiotic regimens could be designed to force resistant bacteria into evolutionary trade-offs, where they lose resistance to widely used antibiotics while gaining resistance to a new compound like PPEF.

Cell populations in flow cytometry are typically identified via visual manual gating, a time-consuming and error-prone approach to select subpopulations based on expression of cellular markers. Batch processing can be used to automate the analysis of bimodally distributed data but underperforms with highly-variable or continuously-expressed markers. We developed a visual pattern recognition automated gating tool, BD ElastiGate Software (hereafter ElastiGate), to recapitulate the visual process of manual gating by automatically adjusting gates to capture local variability. ElastiGate converts histograms and two-dimensional plots into images, then uses elastic B-spline image registration to transform pre-gated training plot images and their gates to corresponding ungated target plot images, thereby adjusting for local variations. ElastiGate was validated with biologically relevant datasets in CAR-T cell manufacturing, tumor-infiltrating immunophenotyping, cytotoxicity assays (> 500 data files), and a high-dimensional dataset. Accuracy was evaluated against corresponding manually gated analysis using F1 score statistics. ElastiGate performed similarly to manual gating, with average F1 scores of > 0.9 across all gates. ElastiGate, accessible as a FlowJo Software plugin or in BD FACSuite Software, uses minimal training samples to automate gating while substantially reducing analysis time and outperforming existing 2D plot autogating solutions in F1 scores and ease of implementation.

Abstract Disclosure: P.N. Surampudi: Research Investigator; Self; Spruce Biosciences. A.H. Hamrahian: Consulting Fee; Self; Spruce Biosciences. Research Investigator; Self; Spruce Biosciences. R. Charlton: Employee; Self; Spruce Biosciences. P. Ramtin: Employee; Self; Spruce Biosciences. M.E. Geffner: Consulting Fee; Self; Spruce Biosciences, Ferring Pharmaceuticals, Adrenas, Neurocrine Biosciences, Novo Nordisk, Eton, Pfizer, Inc., Nutritional Growth Solutions, Nutritional Growth Solutions. Research Investigator; Self; Novo Nordisk. Background: Relative to the general population, patients with classic congenital adrenal hyperplasia (CAH) have increased risks for morbidity and mortality which require specialized management across life stages[1]. Endocrine care, particularly as part of multi-disciplinary teams, can help mitigate risks of developing adrenal crises as well as the impact of glucocorticoid under- or overexposure, including adrenal insufficiency; cardiometabolic risk associated with insulin resistance and excess weight gain; fertility issues associated with irregular menses and gonadal adrenal rest tumors; diminished quality of life; increased mental health issues, including anxiety, depression, and sleep disturbance; and increased fracture risk associated with decreased bone mineral density. Study Objectives and Design: We hypothesized that the utilization of endocrine and multi-disciplinary care by patients with CAH in the United States (U.S.) could be adversely affected by non-therapeutic factors related to geographic location and access to health care. In this cross-sectional study, we utilized the Definitive Healthcare database (Nov. 2020-Nov. 2022) to gain insight into these factors in adults with CAH. Study Findings: Analysis of healthcare provider utilization revealed that only ∼30% of U.S. adults with CAH received care from endocrinologists. The rest utilized primary-care providers (PCPs: IM, FP, NP, PA, and Ob-Gyn) to obtain care. A review of insurance coverage of U.S. adults with CAH suggests that ∼70% had access to commercial insurance and over 50% resided in states with the largest populations (California, Texas, New York, Florida, Ohio, Michigan, and Illinois), both factors which should ordinarily increase accessibility of subspecialty endocrine and multi-disciplinary care. Conclusions: In our study, only 30% of U.S. CAH adults received care from an endocrinologist. Limited utilization of specialty care does not appear to be due to either diverse geographical distribution or access to healthcare. The gap in expert care may adversely affect the management of CAH and contribute to associated comorbidities. Increased partnership between PCPs and endocrinologists, and increased awareness and education regarding specialty care among patients with CAH are needed to improve health outcomes and thereby reduce the risk of morbidity and mortality in adults with CAH.

Abstract In this study, we introduce an innovative microwell-based approach, offering the capability to capture up to 500,000 single cells and show stability of a partially used 8-lane microwell cartridge up to 6 months after first use. Stability of microwell cartridges used for single-cell capture and the integrity of the individual lanes are crucial factors in genomics research to ensure the accuracy and reliability of generated data. Stability is paramount to ensure consistent results over multiple experiments. High-quality materials and manufacturing processes are essential to prevent wear and tear, ensuring that the microwell cartridges function optimally for an extended period. Integrity between the individual lanes must also be intact to prevent cross-contamination between samples. To demonstrate partial use stability of the 8-lane microwell cartridge, multiple assay combinations were performed on different lanes of the cartridge, collected at different time points. Whole transcriptome amplification (WTA) together with AbSeq antibody-oligonucleotide conjugates for cellular indexing of transcriptomes and epitopes (CITE-Seq) was performed on peripheral blood mononuclear cells (PBMCs), loaded and captured in Lanes 1 and 2, on Day 1; sample multiplexing was used with targeted mRNA assay on Jurkat and Ramos cells, loaded and captured in Lanes 3 and 4, after four months; and WTA together with the AbSeq Assay was performed on PBMCs, loaded and captured in Lanes 5 and 6, after six months. The different time points collected were compared to a single-lane microwell cartridge. Our data showed that assay performance was not compromised with the storage of a partially used cartridge, and there was no contamination detected between the lanes. High-throughput cell load and capture using WTA and the AbSeq Assay also showed that each lane of the 8-lane microwell cartridge was able to process up to 65,000 cells with no limit of detection on assay sensitivity. By maintaining cartridge stability and stringent measures to prevent contamination, researchers can ensure the reliability and accuracy of their data. This is vital for various applications such as developing targeted immunotherapies and precision medicine approaches, ultimately enhancing our capacity to combat a wide range of diseases. For Research Use Only. Not for use in diagnostic or therapeutic procedures.BD and BD Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved. Citation Format: Hye-Won Song, Ricelle A. Acob, Zorine Hlathu, Xueying Zhao, Jamie Moskwa, Larry Wang, Aruna Ayer. Stability and performance of a partially used 8-lane microwell cartridge on single-cell capture and immune response profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 333.

We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment.We outline the biological insight that can be gained from the simultaneous measurement of such a large number of proteins and propose that this approach provides a unique opportunity for the comprehensive exploration of the immune status in tissue biopsies and other human samples with a limited number of cells. Of note, we tested the panel to be compatible with cell sorting for further downstream applications. Furthermore, to facilitate the wide-spread implementation of such a panel across different cohorts and samples, we established a trimmed-down 45-color version which can be used with different spectral cytometry platforms.Finally, to generate this panel, we utilized not only existing panel design guidelines, but also developed new metrics to systematically identify the optimal combination of 50 fluorochromes and evaluate fluorochrome-specific resolution in the context of a 50-color unmixing matrix.