Early Detection of Ventilator-associated Pneumonia From Exhaled Breath in Intensive Care Unit Patients.

OBJECTIVE: Evaluate associations between volatile organic compounds (VOCs) in heat and moisture exchange (HME) filters and the presence of ventilator-associated pneumonia (VAP). BACKGROUND: Clinical diagnostic criteria for VAP have poor interobserver 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 intensive care unit patients. METHODS: Gas chromatography-mass spectrometry was used to analyze 111 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 3 days pre and postclinical suspicion of VAP (pneumonia days), versus outside of these days (non-pneumonia days). A receiver operating characteristic 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 Area Under the Curve of the Reciever Operating Characteristic curve (AUC ROC) 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 3 days before the initial clinical suspicion, and approximately a week before 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.

Activation of Stimulator of Interferon Genes (STING): Promising Strategy to Overcome Immune Resistance in Prostate Cancer.

Prostate cancer (PCa) is the most frequent and second-lethal cancer among men. Despite considerable efforts to explore treatments like autologous cellular immunotherapy and immune checkpoint inhibitors, their success remains limited. The intricate tumor microenvironment (TME) and its interaction with the immune system pose significant challenges in PCa treatment. Consequently, researchers have directed their focus on augmenting the immune system's anti-tumor response by targeting the STimulator of the Interferon Genes (STING) pathway. The STING pathway is activated when foreign DNA is detected in the cytoplasm of innate immune cells, resulting in the activation of endoplasmic reticulum (ER) STING. This, in turn, triggers an augmentation of signaling, leading to the production of type I interferon (IFN) and other pro-inflammatory cytokines. Numerous studies have demonstrated that activation of the STING pathway induces immune system rejection and targeted elimination of PCa cells. Researchers have been exploring various methods to activate the STING pathway, including the use of bacterial vectors to deliver STING agonists and the combination of radiation therapy with STING agonists. Achieving effective radiation therapy with minimal side effects and optimal anti-tumor immune responses necessitates precise adjustments to radiation dosing and fractionation schedules. This comprehensive review discusses promising findings from studies focusing on activating the STING pathway to combat PCa. The STING pathway exhibits the potential to serve as an effective treatment modality for PCa, offering new hope for improving the lives of those affected by this devastating disease.

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.

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.

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.

The Anti-Inflammatory Effects of Cannabidiol (CBD) on Acne.

Acne is the most common skin condition in the United States and affects approximately 85% of people ages 12-24. As a multifactorial disease, the pathogenesis of acne involves overproduction of sebum, irregular shedding of the cutaneous cells, accretion of Cutibacterium acnes at the pilosebaceous unit, and inflammation. To date, conventional therapies for acne include topical retinoids, over-the-counter bactericidal agents, and systematic treatments, such as oral antibiotics and isotretinoin. However, the potential for significant side effects and risk of antibiotic resistance remain limitations in these therapies, in turn reducing patient compliance and adherence to acne treatment regimens. Therefore, the use of natural plant-derived treatments or phytotherapeutics as an alternative or adjuvant to conventional treatments is attractive to patients due to their safety and minimal risk for side effects. Cannabidiol (CBD) is a non-psychoactive phytocannabinoid of the Cannabis sativa (hemp) plant. The therapeutic use of CBD has been implicated in many diseases with an inflammatory aspect, including cancers, neurodegeneration, immunological disorders, and dermatological diseases. However, the use of CBD for acne treatment remains a novel window of opportunity. Herein, we summarize the available and relevant data, highlighting the potential use of CBD in acne for its anti-inflammatory properties. To that extent, CBD and other cannabis constituents such as cannabis seeds were found to reduce inflammation and expression of inflammatory cytokines including TNF-α and IL-1ß when evaluated in acne-like conditions. Treatment with these cannabis extracts was also found to be safe and well tolerated, further strengthening the prospect of CBD as an anti-inflammatory therapeutic for acne.

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.

Current salivary biomarkers for detection of human papilloma virus-induced oropharyngeal squamous cell carcinoma.

Human papilloma virus (HPV) infection is a key risk factor and etiology for oropharyngeal squamous cell carcinoma (OPSCC). HPV-induced OPSCC is rapidly increasing in incidence, with men experiencing increased mortality. When identified at an early stage, HPV-induced OPSCC can be successfully treated. Diagnosis of HPV-related OPSCC relies on an expert physical examination and invasive biopsy. Since saliva bathes the oropharyngeal mucosa and can be collected noninvasively, saliva obtained via salivary risings is an attractive body fluid for early detection of HPV-induced OPSCC. A plethora of DNA, RNA, and protein salivary biomarkers have been explored. This review discusses these markers and their robustness for detecting oncogenic HPV in OPSCC saliva samples. Methods detecting HPV DNA were more reliable than those detecting RNA, albeit both require time-consuming analyses. Salivary HPV proteomics are a new, promising focus of HPV detection research, and while more practical, lag behind nucleic acid detection methods in their development.

Peptide-Modified Biopolymers for Biomedical Applications.

Polymeric biomaterials have been used in a variety of applications, like cargo delivery and tissue scaffolding, because they are easily synthesized and can be adapted to many systems. However, there is still a need to further enhance and improve their functions to progress their use in the biomedical field. A promising solution is to modify the polymer surfaces with peptides that can increase biocompatibility, cellular interactions, and receptor targeting. In recent years, peptide modifications have been used to overcome many challenges to polymer biomaterial development. This review discusses recent progress in developing peptide-modified polymers for therapeutic applications including cell-specific targeting and tissue engineering. Furthermore, we will explore some of the most frequently studied base components of these biomaterials.

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.

Cannabidiol as a Novel Therapeutic for Immune Modulation.

The immune-suppressive effects of cannabidiol (CBD) are attributed to the modulation of essential immunological signaling pathways and receptors. Mechanistic understanding of the pharmacological effects of CBD emphasizes the therapeutic potential of CBD as a novel immune modulator. Studies have observed that the antagonists of CB1 and CB2 receptors and transient receptor potential vanilloid 1 reverse the immunomodulatory effects of CBD. CBD also inhibits critical activators of the Janus kinase/signal transducer and activator of transcription signaling pathway, as well as the nucleotide-binding oligomerization domain-like receptor signaling pathway, in turn decreasing pro-inflammatory cytokine production. Furthermore, CBD protects against cellular damage incurred during immune responses by modulating adenosine signaling. Ultimately, the data overwhelmingly support the immunosuppressive effects of CBD and this timely review draws attention to the prospective development of CBD as an effective immune modulatory therapeutic.

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.

A Preliminary Study on the Influence of Cannabis and Opioid Use on Weight Loss and Mental Health Biomarkers Post-weight Loss Surgery.

PURPOSE: A subpopulation of weight loss surgery (WLS) patients endorse cannabis and/or opioid use; however, impact on post-WLS anxiety and depression is unclear. This study examined the influence of the independent and combined use of cannabis and opioids on (1) depression and anxiety, (2) duodenum serotonin and cortisol, and (3) total percent weight loss. MATERIALS AND METHODS: A cross-sectional analysis was conducted among patients (N = 18) who had biomarkers of serotonin and cortisol collected from the duodenum during WLS. Cannabis and opioid use was determined by self-reported lifetime, past-year, and past 30-day use. The Beck Anxiety Inventory and Depression Inventory-II assessed depression and anxiety symptoms. Total percent weight loss was calculated from pre-WLS and post-WLS weight (kg). Chi-squared analyses and t tests were conducted. RESULTS: Over a quarter (27.8%) were cannabis-only users and 16.7% used a combination of cannabis and opioids. None reported using only opioids in this sample. Combination users presented with greater depressive symptoms (22.7%, p = 0.01) and greater total percent weight loss (34.1%, p = 0.04) than cannabis users (7.8, 23.2%, respectively). Cannabis users had greater serotonin (p = 0.02) and cortisol (p = 0.01) levels than combination users and never users. CONCLUSIONS: Cannabis users had greater cortisol levels than never users and combination users. Combination users had greater weight loss and depression symptoms than cannabis users. Future studies should consider a larger sample size, utilization of a cohort design to address causality, and examination of the type, dose, and route of cannabis and opioid administration to further understand the impact of the combined use of cannabis and opioids post-WLS.

Computationally Designed Peptides for Zika Virus Detection: An Incremental Construction Approach.

Herein, and in contrast to current production of anti-Zika virus antibodies, we propose a semi-combinatorial virtual strategy to select short peptides as biomimetic antibodies/binding agents for the detection of intact Zika virus (ZIKV) particles. The virtual approach was based on generating different docking cycles of tetra, penta, hexa, and heptapeptide libraries by maximizing the discrimination between the amino acid motif in the ZIKV and dengue virus (DENV) envelope protein glycosylation site. Eight peptides, two for each length (tetra, penta, hexa, and heptapeptide) were then synthesized and tested vs. intact ZIKV particles by using a direct enzyme linked immunosorbent assay (ELISA). As a reference, we employed a well-established anti-ZIKV antibody, the antibody 4G2. Three peptide-based assays had good detection limits with dynamic range starting from 105 copies/mL of intact ZIKV particles; this was one order magnitude lower than the other peptides or antibodies. These three peptides showed slight cross-reactivity against the three serotypes of DENV (DENV-1, -2, and -3) at a concentration of 106 copies/mL of intact virus particles, but the discrimination between the DENV and ZIKV was lost when the coating concentration was increased to 107 copies/mL of the virus. The sensitivity of the peptides was tested in the presence of two biological matrices, serum and urine diluted 1:10 and 1:1, respectively. The detection limits decreased about one order of magnitude for ZIKV detection in serum or urine, albeit still having for two of the three peptides tested a distinct analytical signal starting from 106 copies/mL, the concentration of ZIKV in acute infection.

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.

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.