Characterization of fire investigators' polyaromatic hydrocarbon exposures using silicone wristbands.

BACKGROUND: Exposures to polyaromatic hydrocarbons (PAHs) contribute to cancer in the fire service. Fire investigators are involved in evaluations of post-fire scenes. In the US, it is estimated that there are up to 9000 fire investigators, compared to approximately 1.1 million total firefighting personnel. This exploratory study contributes initial evidence of PAH exposures sustained by this understudied group using worn silicone passive samplers. OBJECTIVES: Evaluate PAH exposures sustained by fire investigators at post-fire scenes using worn silicone passive samplers. Assess explanatory factors and health risks of PAH exposure at post-fire scenes. METHODS: As part of a cross-sectional study design, silicone wristbands were distributed to 16 North Carolina fire investigators, including eight public, seven private, and one public and private. Wristbands were worn during 46 post-fire scene investigations. Fire investigators completed pre- and post-surveys providing sociodemographic, occupational, and post-fire scene characteristics. Solvent extracts from wristbands were analyzed via gas chromatography-mass spectrometry (GC-MS). Results were used to estimate vapor-phase PAH concentration in the air at post-fire scenes. RESULTS: Fire investigations lasted an average of 148 minutes, standard deviation ± 93 minutes. A significant positive correlation (r=0.455, p<.001) was found between investigation duration and PAH concentrations on wristbands. Significantly greater time-normalized PAH exposures (p=0.039) were observed for investigations of newer post-fire scenes compared to older post-fire scenes. Regulatory airborne PAH exposure limits were exceeded in six investigations, based on exposure to estimated vapor-phase PAH concentrations in the air at post-fire scenes. DISCUSSION: Higher levels of off-gassing and suspended particulates at younger post-fire scenes may explain greater PAH exposure. Weaker correlations are found between wristband PAH concentration and investigation duration at older post-fire scenes, suggesting reduction of off-gassing PAHs over time. Exceedances of regulatory PAH limits indicate a need for protection against vapor-phase contaminants, especially at more recent post-fire scenes.

Directing Cell Delivery to Murine Atherosclerotic Aortic Lesions via Targeting Inflamed Circulatory Interface using Nanocarriers.

Stem cell therapy holds significant potential for many inflammatory diseases and regenerative medicine applications. However, delivery of therapeutic cells to specific disease sites after systemic administration without indiscriminate trafficking to other non-target tissues is a major limitation of current cell therapies. Here, we describe a novel nanocarrier-directed targeted cell delivery system that enables cell surface coating with dendrimer nanocarriers containing adhesion moieties to serve as a global positioning system "GPS" to guide circulating cells to targeted lesions and mediate the anchoring of cells at the inflammation site. By exploiting cell surface ligands/receptors selectively and/or molecular moieties that are highly expressed on activated endothelium in pathologic disease states, nanocarrier-coated cells containing the counterpart binding receptors/ligands can be enabled to specifically traffic to and dock at vasculature within target lesions. We demonstrate the efficacy of the I-domain fragment of LFA-1 ( id LFA-1) complexed to modified nanocarriers to facilitate homing of mesenchymal stem cells (MSCs) to inflamed luminal endothelial cells on which ICAM-1 is highly expressed in a murine model of aortic atherosclerosis. Our method can overcome challenges imposed by the high velocity and dynamic circulatory flow of the aorta to successfully deliver MSCs to atherosclerotic regions and allow for docking of the potentially therapeutic and immunomodulating cells. This targeted cell-delivery platform can be tailored for selective systemic delivery of various types of therapeutic cells to different disease areas.

Accelerating the Screening of Small Peptide Ligands by Combining Peptide-Protein Docking and Machine Learning.

This research introduces a novel pipeline that couples machine learning (ML), and molecular docking for accelerating the process of small peptide ligand screening through the prediction of peptide-protein docking. Eight ML algorithms were analyzed for their potential. Notably, Light Gradient Boosting Machine (LightGBM), despite having comparable F1-score and accuracy to its counterparts, showcased superior computational efficiency. LightGBM was used to classify peptide-protein docking performance of the entire tetrapeptide library of 160,000 peptide ligands against four viral envelope proteins. The library was classified into two groups, 'better performers' and 'worse performers'. By training the LightGBM algorithm on just 1% of the tetrapeptide library, we successfully classified the remaining 99%with an accuracy range of 0.81-0.85 and an F1-score between 0.58-0.67. Three different molecular docking software were used to prove that the process is not software dependent. With an adjustable probability threshold (from 0.5 to 0.95), the process could be accelerated by a factor of at least 10-fold and still get 90-95% concurrence with the method without ML. This study validates the efficiency of machine learning coupled to molecular docking in rapidly identifying top peptides without relying on high-performance computing power, making it an effective tool for screening potential bioactive compounds.

Targeted Bioluminescent Imaging of Pancreatic Ductal Adenocarcinoma Using Nanocarrier-Complexed EGFR-Binding Affibody-Gaussia Luciferase Fusion Protein.

In vivo imaging has enabled impressive advances in biological research, both preclinical and clinical, and researchers have an arsenal of imaging methods available. Bioluminescence imaging is an advantageous method for in vivo studies that allows for the simple acquisition of images with low background signals. Researchers have increasingly been looking for ways to improve bioluminescent imaging for in vivo applications, which we sought to achieve by developing a bioluminescent probe that could specifically target cells of interest. We chose pancreatic ductal adenocarcinoma (PDAC) as the disease model because it is the most common type of pancreatic cancer and has an extremely low survival rate. We targeted the epidermal growth factor receptor (EGFR), which is frequently overexpressed in pancreatic cancer cells, using an EGFR-specific affibody to selectively identify PDAC cells and delivered a Gaussia luciferase (GLuc) bioluminescent protein for imaging by engineering a fusion protein with both the affibody and the bioluminescent protein. This fusion protein was then complexed with a G5-PAMAM dendrimer nanocarrier. The dendrimer was used to improve the protein stability in vivo and increase signal strength. Our targeted bioluminescent complex had an enhanced uptake into PDAC cells in vitro and localized to PDAC tumors in vivo in pancreatic cancer xenograft mice. The bioluminescent complexes could delineate the tumor shape, identify multiple masses, and locate metastases. Through this work, an EGFR-targeted bioluminescent-dendrimer complex enabled the straightforward identification and imaging of pancreatic cancer cells in vivo in preclinical models. This argues for the targeted nanocarrier-mediated delivery of bioluminescent proteins as a way to improve in vivo bioluminescent imaging.

A Bioluminescent Protein-Graphene Oxide Donor-Quencher Pair in DNA Hybridization Assays.

Despite fluorescent quenching with graphene oxide (GO) having shown great success in various applications - bioluminescent quenching has not yet been demonstrated using GO as a quencher. To explore the ability of GO to quench bioluminescence, we used Gaussia luciferase (Gluc) as a donor and GO as a quencher and demonstrated its application in sensing of two target analytes, HIV-1 DNA and IFN-γ. We demonstrated that the incubation of Gluc conjugated HIV-1 and IFN-γ oligonucleotide probes with GO provided for monitoring of probe-target interactions based on bioluminescence measurement in a solution phase sensing system. The limits of detection obtained for IFN-γ and HIV-1 DNA detection were 17 nM and 7.59 nM, respectively. Both sensing systems showed selectivity toward the target analyte. The detection of IFN-γ in saliva matrix was demonstrated. The use of GO as a quencher provides for high sensitivity while maintaining the selectivity of designed probes to their respective targets. The use of GO as a quencher provides for an easy assay design and low cost, environmentally friendly reporter.

Metal organic framework encapsulated tamavidin-Gluc reporter: application in COVID-19 spike antigen bioluminescent immunoassay.

Enzyme linked immunosorbent assay (ELISA) is one of the most utilized serological methods to diagnose and identify etiologic agents of many infectious diseases and other physiologically important analytes. ELISA can be used either alone or adjunct to other diagnostic methods such as molecular arrays, and other serological techniques. Most ELISA assays utilize reagents that are proteinaceous in nature, which are not very stable and require cold-chain transport systems. Development of a desirable immunoassay requires stability of reagents used and its ability to be stored at room temperature without sacrificing the activity of the reagents or the protein of interest. Metal organic frameworks (MOFs) are a rapidly emerging and evolving class of porous polymeric materials used in a variety of biosensor applications. In this study, we introduce the use of MOFs to stabilize a universal reporter fusion protein, specifically, avidin-like protein (Tam-avidin2) and the small bioluminescent protein Gaussia luciferase (Gluc) forming the fusion reporter, tamavidin2-Gluc (TA2-Gluc). This fusion protein serves as a universal reporter for any assays that utilize biotin-avidin binding strategy. Using SARS-CoV2 S1 spike antigen as the model target antigen, we demonstrated that encapsulation of TA2-Gluc fusion protein using a nano-porous material, zeolitic imidazolate framework-8 (ZIF-8), allows us to store and preserve this reporter protein at room temperature for over 6 months and use it as a reporter for an ELISA assay. Our optimized assay was validated demonstrating a 0.26 µg mL-1 limit of detection, high reproducibility of assay over days, detection of spiked non-virulent SARS-COV2 pseudovirus in real sample matrix, and detection in real COVID-19 infected individuals. This result can lead to the utilization of our TA2-Gluc fusion protein reporter with other assays and potentially in diagnostic technologies in a point-of-care setting.

Bacterial Quorum-Sensing Molecules in Serum: A Potential Tool for Crohn's Disease Management.

Crohn's disease (CD) is an idiopathic inflammatory condition of the gastrointestinal tract with the primary method of diagnosis and follow-up being colonoscopy. A disturbed host-microbiome interaction, including the presence of pathobionts, is implicated in initiation and perpetuation of inflammation. As such, we hypothesized that bacterial quorum-sensing (QS) molecules (QSMs), small molecules bacteria generate to regulate gene expression, would be elevated in patients with CD. We collected serum at the time of colonoscopy from patients with CD and healthy controls, determining through biosensors for QSMs that patients with CD had significantly elevated levels of QSMs in serum. Expansion of these studies may allow for QSM levels in serum to serve as a biomarker for intestinal inflammation in patients with CD.

Rapid Point-of-Care Test Kit for Bacterial Vaginosis: Detection of Vaginolysin and Clue Cells Using Paper Strips and a Smartphone.

There is an unmet need for a point-of-care test that is accurate, affordable, and simple to diagnose bacterial vaginosis, the most common cause of vaginal symptoms among women. Bacterial vaginosis leaves patients with undesirable vaginal discharge, malodor, and discomfort. Currently, the diagnosis of bacterial vaginosis is inaccurate and complex, leading to high rates of misdiagnosis. Inaccurate diagnoses are unsafe as bacterial vaginosis increases the risks of acquiring sexually transmitted infections as well as the likelihood of miscarriages. To date, the most commonly identified bacteria associated with bacterial vaginosis is Gardnerella vaginalis. We developed a method for the expression, purification, and detection of vaginolysin, the most well-characterized virulence factor of G. vaginalis. Elevated levels of G. vaginalis have been shown to lead to a toxic vaginal environment, facilitating bacterial vaginosis. We have developed an enzyme-linked immunosorbent assay for the detection of vaginolysin, which was translated to a lateral flow assay for use in a rapid, straightforward, cost-effective paper-based diagnostic test for vaginolysin that does not require the use of instrumentation. In conjunction, we have employed a commercially available smartphone microscopy kit to visualize clue cells without the need for equipment or electricity. The combination of these methodologies allows for an accurate and easy approach to diagnose bacterial vaginosis with minimal resources for use in any setting.

Transformation of Amphiphilic Antiviral Drugs into New Dimensional Nanovesicles Structures.

Improved techniques were applied to formulate drugs into dimensional nanostructures, doped "nanovesicles". These nanovesicles are solely composed of self-assembled amphiphilic antiviral agents used for the treatment of viral infections caused by flaviviruses, such as Zika virus. Studies were done to evaluate the effectiveness of the syntheses, formation, and performance under different experimental conditions, and behavior of the drug nanovesicles in vitro and in vivo. These studies demonstrated that assembling the hydrophobic antiviral drug molecules into nanodrugs is a successful technique for the delivery of the therapeutic agents, otherwise difficult to be supplied. Our studies confirmed that this nanodrug preserved and, in many cases, enhanced the embedded cellular activity of the parental free drug molecules, both in vitro and in vivo. This proposed formulation is highly important as it addresses the issue of insolubility and low bioavailabiity of a wide range of highly potent pharmaceutical drugs-not limited to a specific class of antiviral drugs-that are of high demand for the treatment of medical conditions and emerging pathogens.

Impact of Firefighting Aqueous Film-Forming Foams on Human Cell Proliferation and Cellular Mortality.

OBJECTIVES: Evaluate the toxic effects of Aqueous Film-Forming Foams used by firefighters for Class B fire suppression in human-derived kidney cells (HEK-293). METHODS: Three widely used AFFFs were collected from fire departments and were added to HEK-293 cells in various concentrations. Seventy-two hours post-treatment, cellular proliferation and toxicity were examined using commercially available kits. RESULTS: All AFFFs evaluated induced cellular toxicity and significantly decreased cell proliferation, even when cells were treated with concentrations 10-fold lower than the working concentration used for fire suppression. CONCLUSIONS: Despite the reduced usage of PFAS-containing AFFFs in the firefighter work environment, the evaluated AFFFs demonstrated significantly altered cellular proliferation, while also inducing toxicity, indicating the presence of toxic compounds. Both stronger implementation of PFAS-containing AFFFs restrictions and robust evaluation of fluorine-free and next-generation AFFFs are warranted.

Delivery of therapeutic agents and cells to pancreatic islets: Towards a new era in the treatment of diabetes.

Pancreatic islet cells, and in particular insulin-producing beta cells, are centrally involved in the pathogenesis of diabetes mellitus. These cells are of paramount importance for the endocrine control of glycemia and glucose metabolism. In Type 1 Diabetes, islet beta cells are lost due to an autoimmune attack. In Type 2 Diabetes, beta cells become dysfunctional and insufficient to counterbalance insulin resistance in peripheral tissues. Therapeutic agents have been developed to support the function of islet cells, as well as to inhibit deleterious immune responses and inflammation. Most of these agents have undesired effects due to systemic administration and off-target effects. Typically, only a small fraction of therapeutic agent reaches the desired niche in the pancreas. Because islets and their beta cells are scattered throughout the pancreas, access to the niche is limited. Targeted delivery to pancreatic islets could dramatically improve the therapeutic effect, lower the dose requirements, and lower the side effects of agents administered systemically. Targeted delivery is especially relevant for those therapeutics for which the manufacturing is difficult and costly, such as cells, exosomes, and microvesicles. Along with therapeutic agents, imaging reagents intended to quantify the beta cell mass could benefit from targeted delivery. Several methods have been developed to improve the delivery of agents to pancreatic islets. Intra-arterial administration in the pancreatic artery is a promising surgical approach, but it has inherent risks. Targeted delivery strategies have been developed based on ligands for cell surface molecules specific to islet cells or inflamed vascular endothelial cells. Delivery methods range from nanocarriers and vectors to deliver pharmacological agents to viral and non-viral vectors for the delivery of genetic constructs. Several strategies demonstrated enhanced therapeutic effects in diabetes with lower amounts of therapeutic agents and lower off-target side effects. Microvesicles, exosomes, polymer-based vectors, and nanocarriers are gaining popularity for targeted delivery. Notably, liposomes, lipid-assisted nanocarriers, and cationic polymers can be bioengineered to be immune-evasive, and their advantages to transport cargos into target cells make them appealing for pancreatic islet-targeted delivery. Viral vectors have become prominent tools for targeted gene delivery. In this review, we discuss the latest strategies for targeted delivery of therapeutic agents and imaging reagents to pancreatic islet cells.

Monitoring Pathogenic Viable E. coli O157:H7 in Food Matrices Based on the Detection of RNA Using Isothermal Amplification and a Paper-Based Platform.

In recent years, the number of product recalls and contamination incidents involving pathogenic bacteria has significantly increased, and the ensuing infections continue to be an ongoing problem for public health and agriculture. Due to the widespread impact of these pathogens, there is a critical need for rapid, on-site assays that can provide rapid results. In this work, we demonstrate the development of a rapid and simple test based on the combination of reverse transcription with recombinase polymerase amplification followed by lateral flow strip detection of viable Escherichia coli O157:H7 cells by detecting the RNA of the pathogen. The optimized method can be performed for approximately 2 h with a detection limit of 10 CFU/mL of E. coli O157:H7 in buffer, spinach, and ground beef samples. Our assay is sensitive, detecting only E. coli O157:H7 and not nonpathogenic E. coli or other similar pathogens. This strategy was able to distinguish viable from nonviable bacteria and more significantly was able to detect viable but nonculturable bacteria, which is a major issue when using culture-based methods for monitoring pathogenic bacteria. An important advantage of this test is that it can provide timely identification and removal of contaminated consumables prior to distribution without an extensive sample preparation.

Peptide-Functionalized Dendrimer Nanocarriers for Targeted Microdystrophin Gene Delivery.

Gene therapy is a good alternative for determined congenital disorders; however, there are numerous limitations for gene delivery in vivo including targeted cellular uptake, intracellular trafficking, and transport through the nuclear membrane. Here, a modified G5 polyamidoamine (G5 PAMAM) dendrimer-DNA complex was developed, which will allow cell-specific targeting to skeletal muscle cells and transport the DNA through the intracellular machinery and the nuclear membrane. The G5 PAMAM nanocarrier was modified with a skeletal muscle-targeting peptide (SMTP), a DLC8-binding peptide (DBP) for intracellular transport, and a nuclear localization signaling peptide (NLS) for nuclear uptake, and polyplexed with plasmid DNA containing the GFP-tagged microdystrophin (µDys) gene. The delivery of µDys has been considered as a therapeutic modality for patients suffering from a debilitating Duchenne muscular dystrophy (DMD) disorder. The nanocarrier-peptide-DNA polyplexes were prepared with different charge ratios and characterized for stability, size, surface charge, and cytotoxicity. Using the optimized nanocarrier polyplexes, the transfection efficiency in vitro was determined by demonstrating the expression of the GFP and the µDys protein using fluorescence and Western blotting studies, respectively. Protein expression in vivo was determined by injecting an optimal nanocarrier polyplex formulation to Duchenne model mice, mdx4Cv. Ultimately, these nanocarrier polyplexes will allow targeted delivery of the microdystrophin gene to skeletal muscle cells and result in improved muscle function in Duchenne muscular dystrophy patients.

Mapping carcinogen exposure across urban fire incident response arenas using passive silicone-based samplers.

Carcinogens are emitted in significant quantities at fire scenes and are a major contributor in the increased cancer risk observed in firefighters when compared to the general population. A knowledge gap exists in the current understanding of the distribution of these toxic compounds within a localized fire incident response arena. Here, we employ stationary silicone-based passive samplers at controlled live fire trainings to evaluate the deposition behavior of polyaromatic hydrocarbons (PAHs) emitted by fires. Our findings indicate significantly greater total PAH exposure in fires fueled by biomass and wood compared to fires burning cleaner fuels, such as propane. A 22% increase in total PAH deposition and a 68% increase in high molecular weight PAH deposition was recorded for biomass fueled fires compared to propane fueled fires. Furthermore, we observe that heavier molecular weight PAHs exhibit a pronounced deposition front within a certain radius of the hot zone, whereas low molecular weight PAHs are more uniformly distributed throughout the area. These findings highlight that the warm zones and cold zones of fire situations yield elevated levels of carcinogen exposure to first responders within them. We anticipate that these findings will help inform decisions made by emergency personnel when evaluating risk for the hot zone, warm zone, and cold zone of urban fires helping ease the carcinogenic risk experienced.

A new class of sensing elements for sensors: Clamp peptides for Zika virus.

The design of a new class of selective and high affinity antibody mimetics termed clamp peptide (CP) that incorporate three short peptides structurally and mechanically mimicking a clamp is proposed as sensing elements for a reliable detection sensor platform. The CPs consist of two short peptides functioning as arms that recognize two different epitopes in the target protein and are connected by a third short peptide that acts as a hinge between the peptide arms. For the construction of CPs, we employed a rational design combined with computational methods. To illustrate our approach, we designed a CP that binds selectively to the envelope protein of the Zika virus (ZIKV). The virtual docking cycles were run maximizing the discrimination between ZIKV and Dengue virus (DENV) envelope proteins. DENV was chosen among the flavivirus family because it has high structural similarity with ZIKV. When employed in a colorimetric binding assay or in label-free electrochemical impedance sensor format, the CP was selective for ZIKV vs DENV particles showing detection limit under 104 copies/mL, comparable to anti-ZIKV antibodies. Apparent dissociation binding constants (Kd) confirmed a better performance of CPs than mono-arm peptides (Kd of best CP = 162 nM ± 23 nM; Kd of best mono-arm peptide = 11.15 ± 2.76 µM). The performance of the assays based on CPs was also verified in serum and urine (diluted 1:10 and 1:1 respectively). The detection limits of CPs decreased about one order of magnitude for ZIKV detection in serum or urine, with a distinct analytical signal starting from 105 copies/mL of ZIKV.

Understanding the Role and Clinical Applications of Exosomes in Gynecologic Malignancies: A Review of the Current Literature.

BACKGROUND: Gynecologic malignancies are those which arise in the female reproductive organs of the ovaries, cervix, and uterus. They carry a great deal of morbidity and mortality for patients, largely due to challenges in diagnosis and treatment of these cancers. Although advances in technology and understanding of these diseases have greatly improved diagnosis, treatment, and ultimately survival for patients with gynecologic malignancies over the last few decades, there is still room for improvements in diagnosis and treatment, for which exosomes may be the key. This paper reviews the current knowledge regarding gynecologic tumor derived-exosomal genetic material and proteins, their role in cancer progression, and their potential for advancing the clinical care of patients with gynecologic cancers through novel diagnostics and therapeutics. LITERATURE REVIEW: Ovarian tumor derived exosome specific proteins are reviewed in detail, discussing their role in ovarian cancer metastasis. The key microRNAs in cervical cancer and their implications in future clinical use are discussed. Additionally, uterine cancer-associated fibroblast (CAF)-derived exosomes which may promote endometrial cancer cell migration and invasion through a specific miR-148b are reviewed. The various laboratory techniques and commercial kits for the isolation of exosomes to allow for their clinical utilization are described as well. CONCLUSION: Exosomes may be the key to solving many unanswered questions, and closing the gaps so as to improve the outcomes of patients with gynecologic cancers around the world. The potential utilization of the current knowledge of exosomes, as they relate to gynecologic cancers, to advance the field and bridge the gaps in diagnostics and therapeutics highlight the promising future of exosomes in gynecologic malignancies.

Identification of a Signaling Mechanism by Which the Microbiome Regulates Th17 Cell-Mediated Depressive-Like Behaviors in Mice.

OBJECTIVE: Microbiota dysbiosis has been linked to major depressive disorder, but the mechanisms whereby the microbiota modulates mood remain poorly understood. The authors tested whether specific changes in the microbiome modulate depressive-like behaviors. METHODS: Stools from learned helpless, non-learned helpless, and non-shocked mice were analyzed by V4 16S RNA sequencing to identify gut bacteria associated with learned helplessness and to quantify the level of the quorum-sensing molecule autoinducer-2 (AI-2). T cells were analyzed by flow cytometry, and serum amyloid proteins (SAA) were analyzed by quantitative real-time polymerase chain reaction. Fecal transfer approach and administration of oleic acid and AI-2 were used to determine the effects of the microbiome and quorum-sensing molecules on depressive-like behaviors. RESULTS: Mice deficient in segmented filamentous bacteria (SFB) were resilient to the induction of depressive-like behavior, and were resensitized when SFB was reintroduced in the gut. SFB produces the quorum-sensing AI-2 and promotes the production of SAA1 and SAA2 by the host, which increases T helper 17 (Th17) cell production. Th17 cells were required to promote depressive-like behaviors by AI-2, as AI-2 administration did not promote susceptibility to depressive-like behaviors or SAA1 and SAA2 production in Th17-deficient mice after stress. Oleic acid, an AI-2 inhibitor, exhibited antidepressant properties, reducing depressive-like behavior, intestinal SAA1 and SAA2 production, and hippocampal Th17 cell accumulation. Stool samples from 10 people with current depressive symptoms and 10 matched healthy control subjects were analyzed as well. Patients with current major depressive disorder exhibited increased fecal interleukin 17A, SAA, and SFB levels. CONCLUSIONS: The study results reveal a novel mechanism by which bacteria alter mood.

An Intact Cell Bioluminescence-Based Assay for the Simple and Rapid Diagnosis of Urinary Tract Infection.

Urinary tract infection (UTI) is one of the most common infections, accounting for a substantial portion of outpatient hospital and clinic visits. Standard diagnosis of UTI by culture and sensitivity can take at least 48 h, and improper diagnosis can lead to an increase in antibiotic resistance following therapy. To address these shortcomings, rapid bioluminescence assays were developed and evaluated for the detection of UTI using intact, viable cells of Photobacterium mandapamensis USTCMS 1132 or previously lyophilized cells of Photobacterium leiognathi ATCC 33981™. Two platform technologies-tube bioluminescence extinction technology urine (TuBETUr) and cellphone-based UTI bioluminescence extinction technology (CUBET)-were developed and standardized using artificial urine to detect four commonly isolated UTI pathogens-namely, Escherichia coli, Proteus mirabilis, Staphylococcus aureus, and Candida albicans. Besides detection, these assays could also provide information regarding pathogen concentration/level, helping guide treatment decisions. These technologies were able to detect microbes associated with UTI at less than 105 CFU/mL, which is usually the lower cut-off limit for a positive UTI diagnosis. Among the 29 positive UTI samples yielding 105-106 CFU/mL pathogen concentrations, a total of 29 urine specimens were correctly detected by TuBETUr as UTI-positive based on an 1119 s detection window. Similarly, the rapid CUBET method was able to discriminate UTIs from normal samples with high confidence (p ≤ 0.0001), using single-pot conditions and cell phone-based monitoring. These technologies could potentially address the need for point-of-care UTI detection while reducing the possibility of antibiotic resistance associated with misdiagnosed cases of urinary tract infections, especially in low-resource environments.

Bioluminescent Protein-Inhibitor Pair in the Design of a Molecular Aptamer Beacon Biosensing System.

Although bioluminescent molecular beacons designed around resonance quenchers have shown higher signal-to-noise ratios and increased sensitivity compared with fluorescent beacon systems, bioluminescence quenching is still comparatively inefficient. A more elegant solution to inefficient quenching can be realized by designing a competitive inhibitor that is structurally very similar to the native substrate, resulting in essentially complete substrate exclusion. In this work, we designed a conjugated anti-interferon-γ (IFN-γ) molecular aptamer beacon (MAB) attached to a bioluminescent protein, Gaussia luciferase (GLuc), and an inhibitor molecule with a similar structure to the native substrate coelenterazine. To prove that a MAB can be more sensitive and have a better signal-to-noise ratio, a bioluminescence-based assay was developed against IFN-γ and provided an optimized, physiologically relevant detection limit of 1.0 nM. We believe that this inhibitor approach may provide a simple alternative strategy to standard resonance quenching in the development of high-performance molecular beacon-based biosensing systems.