Early Detection of Ventilator-Associated Pneumonia from Exhaled Breath in ICU Patients.

OBJECTIVES: Evaluate associations between volatile organic compounds (VOCs) in heat and moisture exchange (HME) filters and the presence of ventilator-associated pneumonia (VAP). SUMMARY BACKGROUND DATA: Clinical diagnostic criteria for VAP have poor inter-observer reliability, and cultures are slow to result. Exhaled breath contains VOCs related to Gram-negative bacterial proliferation, the most identified organisms in VAP. We hypothesized that exhaled VOCs on HME filters can predict nascent VAP in mechanically ventilated ICU patients. METHODS: Gas chromatography-mass spectrometry (GC-MS) was used to analyze 111 heat and moisture exchange (HME) filters from 12 intubated patients who developed VAP. Identities and relative amounts of VOCs were associated with dates of clinical suspicion and culture confirmation of VAP. Matched pairs t-tests were performed to compare VOC abundances in HME filters collected within three days pre- and post-clinical suspicion of VAP (pneumonia days), versus outside of these days (non-pneumonia days). A ROC curve was generated to determine the diagnostic potential of VOCs. RESULTS: Carbon disulfide, associated with the proliferation of certain Gram-negative bacteria, was found in samples collected during pneumonia days for 11 of 12 patients. Carbon disulfide levels were significantly greater (P=0.0163) for filters on pneumonia days. The AUROC for carbon disulfide was 0.649 (95%CI 0.419-0.88). CONCLUSIONS: Carbon disulfide associated with Gram-negative VAP can be identified on HME filters up to three days prior to the initial clinical suspicion, and approximately a week prior to culture confirmation. This suggests VOC sensors may have potential as an adjunctive method for early detection of VAP.

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.

Point-of-Care Molecular Test for the Detection of 14 High-Risk Genotypes of Human Papillomavirus in a Single Tube.

Cervical cancers constitute a large disease burden in developing countries, with the human papillomavirus (HPV) being responsible for most cervical lesions. Many regions in low-resource countries lack adequate access to sensitive point-of-care (POC) screening tools, preventing timely diagnosis and treatment. To reduce screening barriers, we developed a POC HPV molecular test that detects 14 high-risk HPV types in 30 min in a single assay. We introduced innovations to the underlying amplification (recombinase polymerase amplification) and detection methodologies such as improved probe design, reagent lyophilization, and pipette-less processing to increase sensitivity while enabling minimally trained personnel to conduct reproducible testing. Based on 198 clinically derived samples, we demonstrated a sensitivity of 93% and a specificity of 73% compared to an FDA-approved polymerase chain reaction-based clinical method. Our modified pipette-less simplified assay had a sensitivity of 96% and a specificity of 83%. The application of our assay is intended as a near-patient screening tool with further evaluation by a clinician for confirmation.

A Portable, Encapsulated Microbial Whole-Cell Biosensing System for the Detection of Bioavailable Copper (II) in Soil.

A portable, field deployable whole-cell biosensor was developed that can withstand the complex matrices of soil and requires minimal to no sample preparation to monitor bioavailable concentrations of the essential micronutrient copper (II). Conventional measurement of micronutrients is often complex, laboratory-based, and not suitable for monitoring their bioavailable concentration. To address this need, we developed a fluorescence based microbial whole-cell biosensing (MWCB) system encoding for a Cu2+-responsive protein capable of generating a signal upon binding to Cu2+. The sensing-reporting protein was designed by performing circular permutation on the green fluorescent protein (GFP) followed by insertion of a Cu2+ binding motif into the structure of GFP. The design included insertion of several binding motifs and creating plasmids that encoded the corresponding sensing proteins. The signal generated by the sensing-reporting protein is directly proportional to the concentration of Cu2+ in the sample. Evaluation of the resulting biosensing systems carrying these plasmids was performed prior to selection of the optimal fluorescence emitting Cu2+-binding protein. The resulting optimized biosensing system was encapsulated in polyacrylate-alginate beads and embedded in soil for detection of the analyte. Once exposed to the soil, the beads were interrogated to measure the fluorescence signal emitted by the sensing-reporting protein using a portable imaging device. The biosensor was optimized for detection of Cu2+ in terms of selectivity, sensitivity, matrix effects, detection limits, and reproducibility in both liquid and soil matrices. The limit of detection (LoD) of the optimized encapsulated biosensor was calculated as 0.27 mg/L and 1.26 mg/kg of Cu2+ for Cu2+ in solution and soil, respectively. Validation of the portable imaging tools as a potential biosensing device in the field was performed.

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.

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.

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.

Opioid Antagonist Nanodrugs Successfully Attenuate the Severity of Ischemic Stroke.

The United States is in the midst of an opioid epidemic that is linked to a number of serious health issues, including an increase in cerebrovascular events, namely, stroke. Chronic prescription opioid use exacerbates the risk and severity of ischemic stroke, contributing to stroke as the fifth overall cause of death in the United States and costing the US health care system over $30 billion annually. Pathologically, opioids challenge the integrity of the blood-brain barrier (BBB), resulting in a dysregulation of tight junction (TJ) proteins that are crucial in maintaining barrier homeostasis. Despite this, treatment options for ischemic stroke are limited, and there are no pharmacological options to attenuate TJ damage, including in incidents that are linked to opioid use. Herein, we have generated carrier-free, pure "nanodrugs" or nanoparticles of naloxone and naltrexone with enhanced therapeutic properties compared to the original (parent) drugs. The generated nanoformulations of both opioid antagonists exhibited successful attenuation of morphine- or oxycodone-induced alterations of TJ protein expression and reduced oxidative stress to a greater extent than the parent drugs (non-nano). As a proof of concept, we then proceeded to evaluate the therapeutic effectiveness of the generated nanodrugs in an ischemic stroke model of mice exposed to morphine or oxycodone. Our results demonstrate that the opioid antagonist nanoformulations reduced stroke severity in mice. Overall, this research implements advances in nanotechnology-based repurposing of FDA-approved therapeutics, and the obtained results also suggest underlying pharmacological mechanisms of opioid antagonists, further supporting these opioid antagonists and their respective nanoformulations as potential therapeutic agents for ischemic stroke.

On-site detection of food and waterborne bacteria - current technologies, challenges, and future directions.

With the rise in outbreaks of pathogenic bacteria in both food and water resulting in an increased instance of infection, there is a growing public health problem in both developed and developing countries. In this increasing threat the most effective method for control and prevention is rapid and cost-effective detection. Research has shifted in recent years towards the development of rapid and on-site assays for the detection of these kinds of bacteria. However, there are still some limitations in the implementation of these assays in the field. This article discusses the current on-site detection methods. Current scope of advancements and limitations in the development or use of these on-site technologies for food and waterborne bacterial detection is evaluated in this study. With the continued development of these technologies, on-site detection will continue to impact many areas of public health. As these methods continue to improve and diversify further, on-site detection could become more widely implemented in food and water analysis.

Design of a mediator-free, non-enzymatic electrochemical biosensor for glutamate detection.

A mediator-free, non-enzymatic electrochemical biosensor was constructed by covalent immobilization of a genetically engineered periplasmic glutamate binding protein onto gold nanoparticle-modified, screen-printed carbon electrodes (GluBP/AuNP/SPCE) for the purpose of direct measurement of glutamate levels. Glutamate serves as the predominant excitatory neurotransmitter in the central nervous system. As high levels of glutamate are an indicator of many neurologic disorders, there is a need for advancements in glutamate detection technologies. The biosensor was evaluated for glutamate detection by cyclic voltammetry. Binding of glutamate to the immobilized glutamate binding protein results in a conformational change of the latter that alters the microenvironment on the surface of the sensor, which is manifested as a change in signal. Dose-response plots correlating the electrochemical signal to glutamate concentration revealed a detection limit of 0.15 µM with a linear range of 0.1-0.8 µM. Selectivity studies confirmed a strong preferential response of the biosensor for glutamate against common interfering compounds.

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.

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.

Evaluation of silicone-based wristbands as passive sampling systems using PAHs as an exposure proxy for carcinogen monitoring in firefighters: Evidence from the firefighter cancer initiative.

Compared to the general population, firefighters are known to sustain greater levels of exposure to hazardous compounds, despite their personal protective equipment, also known as turnout gear. Among the most significant toxins that firefighters are chronically exposed to are polycyclic aromatic hydrocarbons (PAHs). Additionally, firefighters have also been noted to exhibit an increased incidence of certain types of cancer. Considering a probable link between exposure to PAHs and increased rates of cancer in the fire service, we aim to document ambient chemical concentrations in the firefighter work environment. Our strategy involves the use of silicone-based wristbands that have the capacity to passively sorb PAHs. To determine if wristbands can serve as an effective chemical monitoring device for the fire service, silicone wristbands were pilot-tested as personal sampling devices for work environment risk monitoring in active-duty firefighters. Recovered wristbands underwent multiple extraction steps, followed by GC-MS analysis to demonstrate their efficacy in monitoring PAHs in the firefighter environment. Initial findings from all wristband samples taken from firefighters showed multiple exposures to various PAHs of concern for the health of the firefighters when in a fire environment. In addition to PAH monitoring, we examined known and potential sources of PAH contamination in their work environment. To that end, profiles of elevated PAH concentrations were documented at various fire stations throughout South Florida, for individual firefighters both during station duties and active fire response.

Bioluminescent detection of zearalenone using recombinant peptidomimetic Gaussia luciferase fusion protein.

The development of a bioluminescent immunosensor is reported for the determination of zearalenone (ZEA) based on a peptide mimetic identified by phage display. The peptide mimetic GW, with a peptide sequence GWWGPYGEIELL, was used to create recombinant fusion proteins with the bioluminescent Gaussia luciferase (GLuc) that were directly used as tracers for toxin detection in a competitive immunoassay without the need for secondary antibodies or further labeling. The bioluminescent sensor, based on protein G-coupled magnetic beads for antibody immobilization, enabled determination of ZEA with a detection limit of 4.2 ng mL-1 (corresponding to 420 µg kg-1 in food samples) and an IC50 value of 11.0 ng mL-1. The sensor performance was evaluated in spiked maize and wheat samples, with recoveries ranging from 87 to 106% (RSD < 20%, n = 3). Finally, the developed method was applied to the analysis of a naturally contaminated reference matrix material and good agreement with the reported concentrations was obtained.Graphical abstract.

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.

Molecular Aptamer Beacons and Their Applications in Sensing, Imaging, and Diagnostics.

The ability to monitor types, concentrations, and activities of different biomolecules is essential to obtain information about the molecular processes within cells. Successful monitoring requires a sensitive and selective tool that can respond to these molecular changes. Molecular aptamer beacon (MAB) is a molecular imaging and detection tool that enables visualization of small or large molecules by combining the selectivity and sensitivity of molecular beacon and aptamer technologies. MAB design leverages structure switching and specific recognition to yield an optical on/off switch in the presence of the target. Various donor-quencher pairs such as fluorescent dyes, quantum dots, carbon-based materials, and metallic nanoparticles have been employed in the design of MABs. In this work, the diverse biomedical applications of MAB technology are focused on. Different conjugation strategies for the energy donor-acceptor pairs are addressed, and the overall sensitivities of each detection system are discussed. The future potential of this technology in the fields of biomedical research and diagnostics is also highlighted.

Accelerated coronary atherosclerosis not explained by traditional risk factors in 13% of young individuals.

IMPORTANCE: Most individuals who die of sudden cardiac death (SCD) display very advanced lesions of atherosclerosis in their coronary arteries. Thus, we sought to identify and characterize a putative subpopulation of young individuals exhibiting accelerated coronary artery atherosclerosis. OBJECTIVE: Our analysis of the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study-which examined 2651 individuals, obtaining quantitative measurements of traditional risk factors for coronary heart disease (CHD)-aimed to identify individuals with advanced coronary artery lesions, and to determine whether risk factors could account for such rapid disease progression, or not. DESIGN: Using the cross-sectional PDAY study data, an exploratory de facto analysis stratified the population by age and observed number of coronary raised lesions and examined these groups via Poisson regression modeling. A separate de novo approach utilized Poisson mixture modeling to generate low- and high-growth groups based on measurements of traditional risk factors, and identified factors contributing to disease progression. PARTICIPANTS: Participants, n = 2651 individuals aged 15-34, who had died of non-cardiac death, were recruited post mortem. Tissues and other samples were harvested for analysis (details in previously published PDAY studies). Main Outcome(s) and Measure(s). Using quantitative measurements of raised coronary lesions and traditional risk factors of CHD, we sought to identify which risk factors account for disease progression. RESULTS: A group of ~13% of the PDAY population exhibits accelerated coronary atherosclerosis despite their young age. Several traditional risk factors were associated with increased odds of inclusion in this subgroup, reflecting current understanding of these markers of disease. However, only age was a significant contributing factor to the observed coronary lesion burden. CONCLUSIONS: While a range of traditional risk factors contribute to an individual's inclusion to the identified subgroup with accelerated atherosclerosis, these factors, with the exceptions of age, are not able to predict an individual's lesion burden. Moreover, unattributed variances in observations indicate the need to study novel risk factors. SHORT SUMMARY: Hypothesis The extent of coronary atherosclerotic disease is limited and homogeneous within youth, and its progression can be accounted for by traditional risk factors in this population. FINDINGS: A subpopulation (~13%) of the Pathobiological Determinants of Atherosclerosis in Youth cohort exhibited accelerated coronary artery atherosclerosis. While several traditional risk factors contribute to an individual's inclusion in this subgroup, these factors, with the exceptions of age, do not predict accurately an individual's lesions burden. Critically, unattributed variances in observations indicate the need for the identification of novel risk factors. MEANING: Screening of the general population at a young age for high-risk group membership could provide opportunity for disease prevention and avoidance of the worse complications such as myocardial infarction and sudden cardiac death later in life.

Enhanced Delivery of Plasmid DNA to Skeletal Muscle Cells using a DLC8-Binding Peptide and ASSLNIA-Modified PAMAM Dendrimer.

Skeletal muscle is ideally suited and highly desirable as a target for therapeutic gene delivery because of its abundance, high vascularization, and high levels of protein expression. However, efficient gene delivery to skeletal muscle remains a current challenge. Besides the major obstacle of cell-specific targeting, efficient intracellular trafficking, or the cytosolic transport of DNA to the nucleus, must be demonstrated. To overcome the challenge of cell-specific targeting, herein we develop a generation 5-polyamidoamine dendrimer (G5-PAMAM) functionalized with a skeletal muscle-targeted peptide, ASSLNIA (G5-SMTP). Specifically, to demonstrate the feasibility of our approach, we prepared a complex of our G5-SMTP dendrimer with a plasmid encoding firefly luciferase and investigated its delivery to skeletal muscle cells. Luciferase assays indicated a threefold increase in transfection efficiency of C2C12 murine skeletal muscle cells using G5-SMTP when compared with nontargeting nanocarriers using unmodified G5. To further improve the transfection yield, we employed a cationic dynein light chain 8 protein (DLC8)-binding peptide (DBP) containing an internal sequence known to bind to the DLC8 of the dynein motor protein complex. Complexation of DBP with our targeting nanocarrier, that is, G5-SMTP, and our luciferase plasmid cargo resulted in a functional nanocarrier that showed an additional sixfold increase in transfection efficiency compared with G5-SMTP transfection alone. To our knowledge, this is the first successful use of two different functional nanocarrier components that enable targeted skeletal muscle cell recognition and increased efficiency of intracellular trafficking to synergistically enhance gene delivery to skeletal muscle cells. This strategy of targeting and trafficking can also be universally applied to any cell/tissue type for which a recognition domain exists.

Multiplexing cytokine analysis: towards reducing sample volume needs in clinical diagnostics.

The trend for improved more precise diagnostics and management of disease heavily relies on the measurement of panels of biomarkers in physiological samples of patients. Ideally, the ultimate goal would be to detect as many clinically relevant biomarkers as possible in a single drop of blood, achieving quick, sensitive, reproducible, and affordable detection in small volume physiological samples. Bioluminescent (BL) proteins provide many of the desired characteristics required for such labels, including detection at extremely low concentrations, no interference from physiological fluids leading to excellent detection limits, and compatibility with many miniaturized systems. However, to date the use of BL proteins has been restricted by their limited multiplexing capabilities. BL proteins typically exhibit a single emission profile and decay kinetics making the simultaneous detection of multiple analytes difficult. Recent progresses in this area include the use of two different engineered luminescent proteins to achieve resolved signals via one-dimensional time resolution. This approach, however, to date only lead to a dual analyte detection. Herein, we have demonstrated that using a two-dimensional approach that combines both temporal and spatial resolution, we can expand the multiplexing capabilities of bioluminescent proteins. To that end, the photoprotein aequorin (AEQ) has been employed for the simultaneous detection of three separate analytes in a single well, differentiated through the use of three discrete time/wavelength windows. Through a combination of site-specific mutations and synthetic coelenterazines "semi-synthetic" AEQ variants have been developed with altered emission profiles and decay kinetics. In this study, two AEQ mutant proteins were genetically conjugated to three pro-inflammatory cytokines (tumor necrosis factor alpha, interleukins 6 and 8) resulting in AEQ-labeled cytokines. These fusion proteins were combined with synthetic coelenterazines resulting in proteins with differing emission maxima and half-lives to allow for the simultaneous detection of all three cytokines in a single sample. The validity of the assay was demonstrated in serum by employing human physiological samples and comparing our results with commercially available individual tests for each of the three cytokines.

Design of Gaussia luciferase-based bioluminescent stem-loop probe for sensitive detection of HIV-1 nucleic acids.

Here we describe the design of a bioluminescent stem-loop probe for the sensitive detection of HIV-1 spliced RNA. In this study, we employed Gaussia luciferase (GLuc), a bioluminescent protein that has several advantages over other bioluminescent proteins, including smaller size, higher bioluminescent intensity, and chemical and thermal stability. GLuc was chemically conjugated to the DABCYL-modified stem-loop probe (SLP) and was purified with a 2-step process to remove unconjugated GLuc and SLP. The binding of the target RNA to the loop region of the SLP results in the open conformation separating the stem part of SLP. GLuc conjugated to the stem acts as a reporter that produces light by a chemical reaction upon adding its substrate, coelenterazine in the presence of the target, while DABCYL serves as a quencher of bioluminescence in the closed conformation of SLP in the absence of the target. The optimized GLuc based-SLP assay resulted in a signal-to-background ratio of 47, which is the highest reported with bioluminescent SLPs and is significantly higher compared to traditional fluorescence-based SLPs that yield low signal to background ratio. Moreover, the assay showed an excellent selectivity against a single and double mismatched nucleic acid target, low detection limit, and ability to detect spiked HIV-1 RNA in human serum matrix.