Real-Time Monitoring of Bacteria Clearance From Blood in a Murine Model.

Bloodstream infections, especially those that are antibiotic resistant, pose a significant challenge to health care leading to increased hospitalization time and patient mortality. There are different facets to this problem that make these diseases difficult to treat, such as the difficulty to detect bacteria in the blood and the poorly understood mechanism of bacterial invasion into and out of the circulatory system. However, little progress has been made in developing techniques to study bacteria dynamics in the bloodstream. Here, we present a new approach using an in vivo flow cytometry platform for real-time, noninvasive, label-free, and quantitative monitoring of the lifespan of green fluorescent protein-expressing Staphylococcus aureus and Pseudomonas aeruginosa in a murine model. We report a relatively fast average rate of clearance for S. aureus (k = 0.37 ± 0.09 min-1 , half-life ~1.9 min) and a slower rate for P. aeruginosa (k = 0.07 ± 0.02 min-1 , half-life ~9.6 min). We also observed what appears to be two stages of clearance for S. aureus, while P. aeruginosa appeared only to have a single stage of clearance. Our results demonstrate that an advanced research tool can be used for studying the dynamics of bacteria cells directly in the bloodstream, providing insight into the progression of infectious diseases in circulation. © 2019 International Society for Advancement of Cytometry.

Detection of Apoptotic Circulating Tumor Cells Using in vivo Fluorescence Flow Cytometry.

Most cancer patients die from metastatic disease as a result of a circulating tumor cell (CTC) spreading from a primary tumor through the blood circulation to distant organs. Many studies have demonstrated the tremendous potential of using CTC counts as prognostic markers of metastatic development and therapeutic efficacy. However, it is only the viable CTCs capable of surviving in the blood circulation that can create distant metastasis. To date, little progress has been made in understanding what proportion of CTCs is viable and what proportion is in an apoptotic state. Here, we introduce a novel approach toward in situ characterization of CTC apoptosis status using a multicolor in vivo flow cytometry platform with fluorescent detection for the real-time identification and enumeration of such cells directly in blood flow. The proof of concept was demonstrated with two-color fluorescence flow cytometry (FFC) using breast cancer cells MDA-MB-231 expressing green fluorescein protein (GFP), staurosporine as an activator of apoptosis, Annexin-V apoptotic kit with orange dye color, and a mouse model. The future application of this new platform for real-time monitoring of antitumor drug efficiency is discussed. © 2018 International Society for Advancement of Cytometry.

In vivo noninvasive analysis of graphene nanomaterial pharmacokinetics using photoacoustic flow cytometry.

Graphene-based nanomaterials (GBNs) are quickly revolutionizing modern electronics, energy generation and storage, clothing and biomedical devices. Due to GBN's variety of physical and chemical parameters that define their toxicity and their aggregation in suspension, interpreting its toxicology without accurate information on graphene's distribution and behavior in live organisms is challenging. In this work, we present a laser-based optical detection methodology for noninvasive detection and pharmacokinetics analysis of GBNs directly in blood flow in mice using in vivo photoacoustic (PA) flow cytometry (PAFC). PAFC provides unique insight on how chemical modifications of GBNs affect their distribution in blood circulation and how quickly they are eliminated from the flow. Overall, PAFC provided unique data crucial for understanding GBN toxicity through real-time detection of GBNs using their intrinsic light absorption contrast. Copyright © 2017 John Wiley & Sons, Ltd.

Photoacoustic in vitro flow cytometry for nanomaterial research.

Conventional flow cytometry is a versatile tool for drug research and cell characterization. However, it is poorly suited for quantification of non-fluorescent proteins and artificial nanomaterials without the use of additional labeling. The rapid growth of biomedical applications for small non-fluorescent nanoparticles (NPs) for drug delivery and contrast and therapy enhancement, as well as research focused on natural cell pigments and chromophores, demands high-throughput quantification methods for the non-fluorescent components. In this work, we present a novel photoacoustic (PA) fluorescence flow cytometry (PAFFC) platform that combines NP quantification though PA detection with conventional in vitro flow cytometry sample characterization using fluorescence labeling. PAFFC simplifies high-throughput analysis of cell-NP interactions, optimization of targeted nanodrugs, and NP toxicity assessment, providing a direct correlation between NP uptake and characterization of toxicity markers for every cell.

Spaser as a biological probe.

Understanding cell biology greatly benefits from the development of advanced diagnostic probes. Here we introduce a 22-nm spaser (plasmonic nanolaser) with the ability to serve as a super-bright, water-soluble, biocompatible probe capable of generating stimulated emission directly inside living cells and animal tissues. We have demonstrated a lasing regime associated with the formation of a dynamic vapour nanobubble around the spaser that leads to giant spasing with emission intensity and spectral width >100 times brighter and 30-fold narrower, respectively, than for quantum dots. The absorption losses in the spaser enhance its multifunctionality, allowing for nanobubble-amplified photothermal and photoacoustic imaging and therapy. Furthermore, the silica spaser surface has been covalently functionalized with folic acid for molecular targeting of cancer cells. All these properties make a nanobubble spaser a promising multimodal, super-contrast, ultrafast cellular probe with a single-pulse nanosecond excitation for a variety of in vitro and in vivo biomedical applications.

In Vivo Flow Cytometry of Circulating Tumor-Associated Exosomes.

Circulating tumor cells (CTCs) demonstrated the potential as prognostic markers of metastatic development. However, the incurable metastasis can already be developed at the time of initial diagnosis with the existing CTC assays. Alternatively, tumor-associated particles (CTPs) including exosomes can be a more valuable prognostic marker because they can be released from the primary tumor long before CTCs and in larger amount. However, little progress has been made in high sensitivity detection of CTPs, especially in vivo. We show here that in vivo integrated photoacoustic (PA) and fluorescence flow cytometry (PAFFC) platform can provide the detection of melanoma and breast-cancer-associated single CTPs with endogenously expressed melanin and genetically engineered proteins or exogenous dyes as PA and fluorescent contrast agents. The two-beam, time-of-light PAFFC can measure the sizes of CTCs and CTPs and identify bulk and rolling CTCs and CTC clusters, with no influence on blood flow instability. This technique revealed a higher concentration of CTPs than CTCs at an early cancer stage. Because a single tumor cell can release many CTPs and in vivo PAFFC can examine the whole blood volume, PAFFC diagnostic platform has the potential to dramatically improve (up to 105-fold) the sensitivity of cancer diagnosis.

Barriers to cervical cancer screening and follow-up care among Black Women in Massachusetts.

OBJECTIVE: To explore factors that might lead to delays in appropriate cervical cancer screening and diagnosis among Black women in Massachusetts. DESIGN: Qualitative using focus groups. SETTING: Hospitals, health centers, and community-based organizations in Boston, Massachusetts. PARTICIPANTS: Sixty-four participants including Black, non-Hispanic women from the general population and cervical cancer survivors, community leaders in women's health, and health care providers. METHODS: Six focus groups. Data were analyzed using methods based on grounded theory. RESULTS: Findings from interviews revealed that inadequate information and education of providers and patients create barriers to appropriate screening and treatment practices for Black women. Fear, cultural beliefs, and compounding factors related to poverty, gender roles, and health system barriers create delays to screening and follow-up care. Also, unconscious bias, therapeutic delays, and miscommunication are important factors affecting continuity of care. CONCLUSION: These results suggest a need for comprehensive, culturally specific cervical cancer prevention education initiatives and interventions for Black women and strategies to improve patient-provider relationships.

Differences in phosphorus and nitrogen delivery to the Gulf of Mexico from the Mississippi River Basin.

Seasonal hypoxia in the northern Gulf of Mexico has been linked to increased nitrogen fluxes from the Mississippi and Atchafalaya River Basins, though recent evidence shows that phosphorus also influences productivity in the Gulf. We developed a spatially explicit and structurally detailed SPARROW water-quality model that reveals important differences in the sources and transport processes that control nitrogen (N) and phosphorus (P) delivery to the Gulf. Our model simulations indicate that agricultural sources in the watersheds contribute more than 70% of the delivered N and P. However, corn and soybean cultivation is the largest contributor of N (52%), followed by atmospheric deposition sources (16%); whereas P originates primarily from animal manure on pasture and rangelands (37%), followed by corn and soybeans (25%), other crops (18%), and urban sources (12%). The fraction of in-stream P and N load delivered to the Gulf increases with stream size, but reservoir trapping of P causes large local- and regional-scale differences in delivery. Our results indicate the diversity of management approaches required to achieve efficient control of nutrient loads to the Gulf. These include recognition of important differences in the agricultural sources of N and P, the role of atmospheric N, attention to P sources downstream from reservoirs, and better control of both N and P in close proximity to large rivers.