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Current antimicrobial susceptibility testing (AST) requires 16–24 hours, delaying initiation of appropriate antibiotics. Hence, there is a need for rapid AST. This study aims to develop and evaluate the feasibility of a rapid flow cytometric AST assay to determine minimum inhibitory concentration (MIC) for carbapenem-resistant Acinetobacter baumannii (CRAB). Antibiotic exposure causes increased intracellular reactive oxygen species (ROS) in bacteria. We hypothesized that ROS can be used as a marker to determine MIC. We assessed three CRAB clinical isolates across fifteen antibiotics at various concentrations in a customized 96-well microtiter plate. The antibiotics assessed include amikacin, beta-lactams (ampicillin/sulbactam, aztreonam, cefepime, ceftolozane/tazobactam, doripenem, imipenem, meropenem, and piperacillin/tazobactam), levofloxacin, polymyxin B, rifampicin, trimethoprim/sulfamethoxazole, and tetracyclines (tigecycline and minocycline). These clinical CRAB isolates were assessed for ROS after antibiotic treatment. Increased ROS levels indicated by increased RedoxSensorTM Green (RSG) fluorescence intensity was assessed using flow cytometry (FCM). MIC was set as the lowest antibiotic concentration that gives a ≥1.5-fold increase in mode RSG fluorescence intensity (MICRSG). Accuracy of MICRSG was determined by comparing against microtiter broth dilution method performed under CLSI guidelines. ROS was deemed accurate in determining the MICs for β-lactams (83.3% accuracy) and trimethoprim/sulfamethoxazole (100% accuracy). In contrast, ROS is less accurate in determining MICs for levofloxacin (33.3% accuracy), rifampicin (0% accuracy), amikacin (33.3% accuracy), and tetracyclines (33.3% accuracy). Collectively, this study described an FCM-AST assay to determine antibiotic susceptibility of CRAB isolates within 5 hours, reducing turnaround time up to 19 hours. [ABSTRACT FROM AUTHOR]

The synthesis and characterisation of five new boron-based coumarin fluorophores are reported, with key structural variations involving the linker at the C3-position (hydrazone or imine) of the 7-(diethylamino)-coumarin (7DEAC) core and the terminal boron moiety (i.e. boronic acid or closo -1,2-carborane). All the coumarin derivatives were found to display significant bathochromic shifts relative to the parent 7DEAC , with conjugate ICCb displaying the greatest overall shift. Confocal microscopy studies with A549 lung cancer cells showed clear differences in the observed intra-cellular distributions of the fluorophores. The polar boronic acid species (HCoBA , HCmBA and HCpBA) were found to localise in the endoplasmic reticulum. In contrast, the lipophilic closo -1,2-carborane derivatives (HCCb and ICCb) were found to localise within lipid droplets (LDs), showcasing the future potential for these probes to be utilised as stains for LD observations by means of confocal microscopy. Novel boron-containing hydrazone and imino-coumarin derivatives were prepared and their intracellular localisations were evaluated by means of confocal microscopy. Carborane conjugates (HCCb and ICCb) were found to be effective lipid droplet stains, and the boronic acid probes (HCoBA , HCmBA and HCpBA) targeted the endoplasmic reticulum (ER). [ABSTRACT FROM AUTHOR]

The synthesis and characterisation of five new boron-based coumarin fluorophores are reported, with key structural variations involving the linker at the C3-position (hydrazone or imine) of the 7-(diethylamino)-coumarin (7DEAC) core and the terminal boron moiety (i.e. boronic acid or closo -1,2-carborane). All the coumarin derivatives were found to display significant bathochromic shifts relative to the parent 7DEAC , with conjugate ICCb displaying the greatest overall shift. Confocal microscopy studies with A549 lung cancer cells showed clear differences in the observed intra-cellular distributions of the fluorophores. The polar boronic acid species (HCoBA , HCmBA and HCpBA) were found to localise in the endoplasmic reticulum. In contrast, the lipophilic closo -1,2-carborane derivatives (HCCb and ICCb) were found to localise within lipid droplets (LDs), showcasing the future potential for these probes to be utilised as stains for LD observations by means of confocal microscopy. Novel boron-containing hydrazone and imino-coumarin derivatives were prepared and their intracellular localisations were evaluated by means of confocal microscopy. Carborane conjugates (HCCb and ICCb) were found to be effective lipid droplet stains, and the boronic acid probes (HCoBA , HCmBA and HCpBA) targeted the endoplasmic reticulum (ER). [ABSTRACT FROM AUTHOR]

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The lipid content of mammalian cells varies greatly between cell type. Current methods for analysing lipid components of cells are technically challenging and destructive. Here, we report a facile, inexpensive method to identify lipid content - intracellular flow cytometric lipid analysis (IFCLA). Distinct lipid classes can be distinguished by Nile Blue fluorescence, Nile Red fluorescence or violet autofluorescence. Nile Blue is fluorescent in the presence of unsaturated fatty acids with a carbon chain length greater than 16. Cis-configured fatty acids induce greater Nile Blue fluorescence than their trans-configured counterparts. In contrast, Nile Red exhibits greatest fluorescence in the presence of cholesterol, cholesteryl esters, some triglycerides and phospholipids. Multiparametric spanning-tree progression analysis for density-normalized events (SPADE) analysis of hepatic cellular lipid distribution, including vitamin A autofluorescence, is presented. This flow cytometric system allows for the rapid, inexpensive and non-destructive identification of lipid content, and highlights the differences in lipid biology between cell types by imaging and flow cytometry., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The most common cell type in the human body, the red blood cell or erythrocyte, has a life span of approximately 3 months. To compensate for this massive cellular requirement and short life span, the major blood producing tissues contain vast numbers of erythroid progenitor cells. Erythroid progenitors differentiate progressively from hematopoietic stem cells to committed erythroid progenitors to reticulocytes lacking a nucleus and finally to functionally mature erythrocytes in the circulation. Different erythroid progenitor activity, representative of distinct stages of erythropoiesis, can be observed using semisolid colony assays. Distinct stages of erythroid maturation can also be monitored by flow cytometry. Here, we discuss the range of different technical approaches that are used to identify and quantify erythroid progenitors, with particular focus on the mouse as a model system.

The bone marrow is the primary site of erythropoiesis in healthy adult mammals. In the bone marrow, erythroid cells mature within specialized microenvironments termed erythroblastic islands (EBIs). EBIs are multi-cellular clusters comprised of a central macrophage surrounded by red blood cell (erythroid) progenitors. It has been proposed that the central macrophage functions as a "nurse-cell" providing iron, cytokines, and growth factors for the developing erythroid cells. The central macrophage also engulfs and destroys extruded erythroid nuclei. EBIs have recently been shown to play clinically important roles during human hematological disease. The molecular mechanisms regulating this hematopoietic niche are largely unknown. In this chapter, we detail protocols to study isolated EBIs using multiple microscopy platforms. Adhesion molecules regulate cell-cell interactions within the EBI and maintain the integrity of the niche. To improve our understanding of the molecular regulation of erythroid cells in EBIs, we have developed protocols for immuno-gold labeling of erythroid surface antigens to combine with scanning electron microscopy. These protocols have allowed imaging of EBIs at the nanometer scale, offering novel insights into the processes regulating red blood cell production.

Heme oxygenase-1 is critical for iron recycling during red blood cell turnover, whereas its impact on steady-state erythropoiesis and red blood cell lifespan is not known. We show here that in 8- to 14-week old mice, heme oxygenase-1 deficiency adversely affects steady-state erythropoiesis in the bone marrow. This is manifested by a decrease in Ter-119(+)-erythroid cells, abnormal adhesion molecule expression on macrophages and erythroid cells, and a greatly diminished ability to form erythroblastic islands. Compared with wild-type animals, red blood cell size and hemoglobin content are decreased, while the number of circulating red blood cells is increased in heme oxygenase-1 deficient mice, overall leading to microcytic anemia. Heme oxygenase-1 deficiency increases oxidative stress in circulating red blood cells and greatly decreases the frequency of macrophages expressing the phosphatidylserine receptor Tim4 in bone marrow, spleen and liver. Heme oxygenase-1 deficiency increases spleen weight and Ter119(+)-erythroid cells in the spleen, although α4β1-integrin expression by these cells and splenic macrophages positive for vascular cell adhesion molecule 1 are both decreased. Red blood cell lifespan is prolonged in heme oxygenase-1 deficient mice compared with wild-type mice. Our findings suggest that while macrophages and relevant receptors required for red blood cell formation and removal are substantially depleted in heme oxygenase-1 deficient mice, the extent of anemia in these mice may be ameliorated by the prolonged lifespan of their oxidatively stressed erythrocytes., (Copyright© Ferrata Storti Foundation.)