Ultraviolet light oxidation of fresh hemoglobin eliminates aggregate formation seen in commercially sourced hemoglobin.

Elevated levels of circulating cell-free hemoglobin (CFH) are an integral feature of several clinical conditions including sickle cell anemia, sepsis, hemodialysis and cardiopulmonary bypass. Oxidized (Fe3+, ferric) hemoglobin contributes to the pathophysiology of these disease states and is therefore widely studied in experimental models, many of which use commercially sourced CFH. In this study, we treated human endothelial cells with commercially sourced ferric hemoglobin and observed the appearance of dense cytoplasmic aggregates (CAgg) over time. These CAgg were intensely autofluorescent, altered intracellular structures (such as mitochondria), formed in multiple cell types and with different media composition, and formed regardless of the presence or absence of cells. An in-depth chemical analysis of these CAgg revealed that they contain inorganic components and are not pure hemoglobin. To oxidize freshly isolated hemoglobin without addition of an oxidizing agent, we developed a novel method to convert ferrous CFH to ferric CFH using ultraviolet light without the need for additional redox agents. Unlike commercial ferric hemoglobin, treatment of cells with the fresh ferric hemoglobin did not lead to CAgg formation. These studies suggest that commercially sourced CFH may contain stabilizers and additives which contribute to CAgg formation.

Inorganic Complexes and Metal-Based Nanomaterials for Infectious Disease Diagnostics.

Infectious diseases claim millions of lives each year. Robust and accurate diagnostics are essential tools for identifying those who are at risk and in need of treatment in low-resource settings. Inorganic complexes and metal-based nanomaterials continue to drive the development of diagnostic platforms and strategies that enable infectious disease detection in low-resource settings. In this review, we highlight works from the past 20 years in which inorganic chemistry and nanotechnology were implemented in each of the core components that make up a diagnostic test. First, we present how inorganic biomarkers and their properties are leveraged for infectious disease detection. In the following section, we detail metal-based technologies that have been employed for sample preparation and biomarker isolation from sample matrices. We then describe how inorganic- and nanomaterial-based probes have been utilized in point-of-care diagnostics for signal generation. The following section discusses instrumentation for signal readout in resource-limited settings. Next, we highlight the detection of nucleic acids at the point of care as an emerging application of inorganic chemistry. Lastly, we consider the challenges that remain for translation of the aforementioned diagnostic platforms to low-resource settings.

Magnetically-enabled biomarker extraction and delivery system: towards integrated ASSURED diagnostic tools.

Diagnosis of asymptomatic malaria poses a great challenge to global disease elimination efforts. Healthcare infrastructure in rural settings cannot support existing state-of-the-art tools necessary to diagnose asymptomatic malaria infections. Instead, lateral flow immunoassays (LFAs) are widely used as a diagnostic tool in malaria endemic areas. While LFAs are simple and easy to use, they are unable to detect low levels of parasite infection. We have developed a field deployable Magnetically-enabled Biomarker Extraction And Delivery System (mBEADS) that significantly improves limits of detection for several commercially available LFAs. Integration of mBEADS with leading commercial Plasmodium falciparum malaria LFAs improves detection limits to encompass an estimated 95% of the disease reservoir. This user-centered mBEADS platform makes significant improvements to a previously cumbersome malaria biomarker enrichment strategy by improving reagent stability, decreasing the processing time 10-fold, and reducing the assay cost 10-fold. The resulting mBEADS process adds just three minutes and less than $0.25 to the total cost of a single LFA, thus balancing sensitivity and practicality to align with the World Health Organization's ASSURED criteria for point-of-care (POC) testing.

Cigarette Smoking and Prostate Cancer Mortality in Four US States, 1999-2010.

INTRODUCTION: In the United States, prostate cancer mortality rates have declined in recent decades. Cigarette smoking, a risk factor for prostate cancer death, has also declined. It is unknown whether declines in smoking prevalence produced detectable declines in prostate cancer mortality. We examined state prostate cancer mortality rates in relation to changes in cigarette smoking. METHODS: We studied men aged 35 years or older from California, Kentucky, Maryland, and Utah. Data on state smoking prevalence were obtained from the Behavioral Risk Factor Surveillance System. Mortality rates for prostate cancer and external causes (control condition) were obtained from the Centers for Disease Control and Prevention's Wide-Ranging Online Data for Epidemiologic Research. The average annual percentage change from 1999 through 2010 was estimated using joinpoint analysis. RESULTS: From 1999 through 2010, smoking in California declined by 3.5% per year (-4.4% to -2.5%), and prostate cancer mortality rates declined by 2.5% per year (-2.9% to -2.2%). In Kentucky, smoking declined by 3.0% per year (-4.0% to -1.9%) and prostate cancer mortality rates declined by 3.5% per year (-4.3% to -2.7%). In Maryland, smoking declined by 3.0% per year (-7.0% to 1.2%), and prostate cancer mortality rates declined by 3.5% per year (-4.1% to -3.0%).In Utah, smoking declined by 3.5% per year (-5.6% to -1.3%) and prostate cancer mortality rates declined by 2.1% per year (-3.8% to -0.4%). No corresponding patterns were observed for external causes of death. CONCLUSION: Declines in prostate cancer mortality rates appear to parallel declines in smoking prevalence at the population level. This study suggests that declines in prostate cancer mortality rates may be a beneficial effect of reduced smoking in the population.

Low-Resource Nucleic Acid Extraction Method Enabled by High-Gradient Magnetic Separation.

Nucleic acid-based diagnostic tests often require isolation and concentration of nucleic acids from biological samples. Commercial purification kits are difficult to use in low-resource settings because of their cost and insufficient laboratory infrastructure. Several recent approaches based on the use of magnetic beads offer a potential solution but remain limited to small volume samples. We have developed a simple and low-cost nucleic acid extraction method suitable for isolation and concentration of nucleic acids from small or large sample volumes. The method uses magnetic beads, a transfer pipette, steel wool, and an external magnet to implement high-gradient magnetic separation (HGMS) to retain nucleic acid-magnetic bead complexes within the device's steel wool matrix for subsequent processing steps. We demonstrate the method's utility by extracting tuberculosis DNA from both sputum and urine, two typical large volume sample matrices (5-200 mL), using guanidine-based extraction chemistry. Our HGMS-enabled extraction method is statistically indistinguishable from commercial extraction kits when detecting a spiked 123-base DNA sequence. For our HGMS-enabled extraction method, we obtained extraction efficiencies for sputum and urine of approximately 10 and 90%, whereas commercial kits obtained 10-17 and 70-96%, respectively. We also used this method previously in a blinded sample preparation comparison study published by Beall et al., 2019. Our manual extraction method is insensitive to high flow rates and sample viscosity, with capture of ∼100% for flow rates up to 45 mL/min and viscosities up to 55 cP, possibly making it suitable for a wide variety of sample volumes and types and point-of-care users. This HGMS-enabled extraction method provides a robust instrument-free method for magnetic bead-based nucleic acid extraction, potentially suitable for field implementation of nucleic acid testing.

Rapid concentration and elution of malarial antigen histidine-rich protein II using solid phase Zn(II) resin in a simple flow-through pipette tip format.

Rapid diagnostic tests (RDTs) designed to function at the point of care are becoming more prevalent in malaria diagnostics because of their low cost and simplicity. While many of these tests function effectively with high parasite density samples, their poor sensitivity can often lead to misdiagnosis when parasitemia falls below 100 parasites/µl. In this study, a flow-through pipette-based column was explored as a cost-effective means to capture and elute more Plasmodium falciparum histidine-rich protein II (HRPII) antigen, concentrating the biomarker available in large-volume lysed whole blood samples into volumes compatible with Plasmodium falciparum-specific RDTs. A systematic investigation of immobilized metal affinity chromatography divalent metal species and solid phase supports established the optimal design parameters necessary to create a flow-through column incorporated into a standard pipette tip. The bidirectional flow inherent to this format maximizes mixing efficiency so that in less than 5 min of sample processing, the test band signal intensity was increased up to a factor of twelve from HRPII concentrations as low as 25 pM. In addition, the limit of detection per sample was decreased by a factor of five when compared to the RDT manufacturer's suggested protocol. Both the development process and commercial viability of this application are explored, serving as a potential model for future applications.