Analysis of ingredient list and nutrient composition of plant-based burgers available in the global market.

The nutrient composition of plant-based burgers is a key factor when making their purchase/consumption decision to maintain a balanced diet. For this reason, ingredient list and nutritional information of burgers launched in the global market were retrieved from their labels. Products were classified based on the technology development, market position and region of the manufacturer. From the ingredient analysis, we observed a high heterogeneity in the ingredients used, a predominance of soy and wheat as main sources of proteins, and the increasing use of new protein sources (e.g. peas, other types of beans and pseudo-cereals). Oil was the most cited ingredient followed by salt. Nutritional composition varied mainly depending on the region with no clear pattern among countries. To less extent, technology development resulted in traditional products with lower amounts of protein and higher amounts of carbohydrates. Vegan and vegetarian products showed limited differences due to the high intra-heterogenicity.

Development and Validation of Nerve-Targeted Bacteriochlorin Sensors.

We report an innovative approach to producing bacteriochlorins (bacs) via formal cycloaddition by subjecting a porphyrin to a trimolecular reaction. Bacs are near-infrared probes with the intrinsic ability to serve in multimodal imaging. However, despite their ability to fluoresce and chelate metal ions, existing bacs have thus offered limited ability to label biomolecules for target specificity or have lacked chemical purity, limiting their use in bio-imaging. In this work, bacs allowed a precise and controlled appending of clickable linkers, lending the porphyrinoids substantially more chemical stability, clickability, and solubility, rendering them more suitable for preclinical investigation. Our bac probes enable the targeted use of biomolecules in fluorescence imaging and Cerenkov luminescence for guided intraoperative imaging. Bacs' capacity for chelation provides opportunities for use in non-invasive positron emission tomography/computed tomography. Herein, we report the labeling of bacs with Hs1a, a (NaV1.7)-sodium-channel-binding peptide derived from the Chinese tarantula Cyriopagopus schmidti to yield Bac-Hs1a and radiolabeled Hs1a, which shuttles our bac sensor(s) to mouse nerves. In vivo, the bac sensor allowed us to observe high signal-to-background ratios in the nerves of animals injected with fluorescent Bac-Hs1a and radiolabeled Hs1a in all imaging modes. This study demonstrates that Bac-Hs1a and [64Cu]Cu-Bac-Hs1a accumulate in peripheral nerves, providing contrast and utility in the preclinical space. For the chemistry and bio-imaging fields, this study represents an exciting starting point for the modular manipulation of bacs, their development and use as probes for diagnosis, and their deployment as formidable multiplex nerve-imaging agents for use in routine imaging experiments.

Genome mining reveals secondary metabolites of Antarctic bacterium Streptomyces albidoflavus related to antimicrobial and antiproliferative activities.

The urgent need for new antimicrobials arises from antimicrobial resistance. Actinobacteria, especially Streptomyces genus, are responsible for production of numerous clinical antibiotics and anticancer agents. Genome mining reveals the biosynthetic gene clusters (BGCs) related to secondary metabolites and the genetic potential of a strain to produce natural products. However, this potential may not be expressed under laboratory conditions. In the present study, the Antarctic bacterium was taxonomically affiliated as Streptomyces albidoflavus ANT_B131 (CBMAI 1855). The crude extracts showed antimicrobial activity against both fungi, Gram-positive and Gram-negative bacteria and antiproliferative activity against five human tumor cell lines. Whole-genome sequencing reveals a genome size of 6.96 Mb, and the genome mining identified 24 BGCs, representing 13.3% of the genome. The use of three culture media and three extraction methods reveals the expression and recovery of 20.8% of the BGCs. The natural products identified included compounds, such as surugamide A, surugamide D, desferrioxamine B + Al, desferrioxamine E, and ectoine. This study reveals the potential of S. albidoflavus ANT_B131 as a natural product producer. Yet, the diversity of culture media and extraction methods could enhance the BGCs expression and recovery of natural products, and could be a strategy to intensify the BGC expression of natural products.

Effect of photobiomodulation associated with strengthening pelvic floor muscles in volunteers with urinary incontinence: a randomized, double-blinded, and placebo-controlled clinical trial.

The dysfunctions of the female pelvic floor have a great influence on the quality of life of women, in all areas, social, psychological, and sexual. Stress urinary incontinence is the clinical condition in which the woman involuntarily loses urine to efforts, such as coughing and sneezing, causing great embarrassment and affecting her quality of life. The physiotherapeutic treatments include muscle strengthening; however, muscle fatigue is present when performing the exercises. Here we investigate the effects of photobiomodulation to prevent muscle fatigue in the pelvic floor in the treatment of stress urinary incontinence, associated with a muscle strengthening exercise protocol. We used an infrared laser (808 nm, 100 mW) and 3 J/point and fluence of 107.1 J/cm2. The application was performed at 3 points on the vaginal introits and at another 3 points inside the vaginal canal cavity for the treatment of stress urinary incontinence associated with strengthening exercises with vaginal cones. Twenty-two volunteers participated in the study, divided into two groups: group 1 (laser therapy + strengthening) and group 2 (placebo laser + strengthening). In the group 1 quality of life score, the assessment (11.63 ± 4.33) was the highest score at 17 and in the reevaluation (7.81 ± 5.14) the lowest was 0 (p < 0.05). The muscular strength increased considerably (p < 0.05) for group 1, where the vast majority of patients gained more than twice the strength in the pelvic apparatus (8.36 ± 6.65 before X 13.81 ± 8.92 after). The volunteers acquired an increase in the contraction of the muscles of the pelvic apparatus (p < 0.05) (3.45 ± 1.07); after laser application, this number increased considerably (4.27 ± 0.61). Endurance had an increase of almost 50% compared to placebo, demonstrating the resistance gain in the perineal muscles (3.90 ± 2.35 X 5 ± 1.90). We concluded that photobiomodulation treatment showed significant efficacy in relation to muscle fatigue in the pelvic apparatus right after a strengthening program in women with stress urinary incontinence.

[18F]PARPi Imaging Is Not Affected by HPV Status In Vitro.

Background: Human papillomavirus- (HPV-) associated oropharyngeal squamous cell carcinomas (OPSCCs) are clinically and pathologically distinct from HPV-negative tumors. Here, we explore whether HPV affects functional biomarkers, including γH2AX, RAD51, and PARP1. Moreover, the role of [18F]PARPi as a broadly applicable imaging tool for head and neck carcinomas is investigated. Methods: HPV-positive and HPV-negative cell lines were used to evaluate the γH2AX, RAD51, and PARP1 expression with immunoblotting and immunofluorescence. Effects of external beam ionizing radiation were investigated in vitro, and survival was investigated via colony-formation assay. [18F]PARPi uptake experiments were performed on HPV-negative and HPV-positive cell lines to quantify PARP1 expression. PARP1 IHC and γH2AX foci were quantified using patient-derived oropharyngeal tumor specimens. Results: Differences in DNA repair were detected, showing higher RAD51 and γH2AX expression in HPV-positive cell lines. Clonogenic assays confirm HPV-positive cell lines to be significantly more radiosensitive. PARP1 expression levels were similar, irrespective of HPV status. Consequently, [18F]PARPi uptake assays demonstrated that this tracer is internalized in cell lines independently from their HPV status. Conclusion: The HPV status, often used clinically to stratify patients, did not affect PARP1 levels, suggesting that PARP imaging can be performed in both HPV-positive and HPV-negative patients. This study confirms that the PET imaging agent [18F]PARPi could serve as a general clinical tool for oropharyngeal cancer patients.

A phase I study of a PARP1-targeted topical fluorophore for the detection of oral cancer.

BACKGROUND: Visual inspection and biopsy is the current standard of care for oral cancer diagnosis, but is subject to misinterpretation and consequently to misdiagnosis. Topically applied PARPi-FL is a molecularly specific, fluorescent contrast-based approach that may fulfill the unmet need for a simple, in vivo, non-invasive, cost-effective, point-of-care method for the early diagnosis of oral cancer. Here, we present results from a phase I safety and feasibility study on fluorescent, topically applied PARPi-FL. Twelve patients with a histologically proven oral squamous cell carcinoma (OSCC) gargled a PARPi-FL solution for 60 s (15 mL, 100 nM, 250 nM, 500 nM, or 1000 nM), followed by gargling a clearing solution for 60 s. Fluorescence measurements of the lesion and surrounding oral mucosa were taken before PARPi-FL application, after PARPi-FL application, and after clearing. Blood pressure, oxygen levels, clinical chemistry, and CBC were obtained before and after tracer administration. RESULTS: PARPi-FL was well-tolerated by all patients without any safety concerns. When analyzing the fluorescence signal, all malignant lesions showed a significant differential in contrast after administration of PARPi-FL, with the highest increase occurring at the highest dose level (1000 nM), where all patients had a tumor-to-margin fluorescence signal ratio of >3. A clearing step was essential to increase signal specificity, as it clears unbound PARPi-FL trapped in normal anatomical structures. PARPi-FL tumor cell specificity was confirmed by ex vivo tabletop confocal microscopy. We have demonstrated that the fluorescence signal arose from the nuclei of tumor cells, endorsing our macroscopic findings. CONCLUSIONS: A PARPi-FL swish & spit solution is a rapid and non-invasive diagnostic tool that preferentially localizes fluorescent contrast to OSCC. This technique holds promise for the early detection of OSCC based on in vivo optical evaluation and targeted biopsy of suspicious lesions in the oral cavity. TRIAL REGISTRATION: Clinicaltrials.gov -NCT03085147, registered on March 21st, 2017.

Bimodal Imaging of Mouse Peripheral Nerves with Chlorin Tracers.

Almost 17 million Americans have a history of cancer, a number expected to reach over 22 million by 2030. Cancer patients often undergo chemotherapy in the form of antineoplastic agents such as cis-platin and paclitaxel. Though effective, these agents can induce debilitating side effects; the most common neurotoxic effect, chemotherapy-induced peripheral neuropathy (CIPN), can endure long after treatment ends. Despite the widespread and chronic nature of the dysfunction, no tools exist to quantitatively measure chemotherapy-induced peripheral neuropathy. Such a tool would not only benefit patients but their stratification could also save significant financial and social costs associated with neuropathic pain. In our first step toward addressing this unmet clinical need, we explored a novel dual approach to localize peripheral nerves: Cerenkov luminescence imaging (CLI) and fluorescence imaging (FI). Our approach revolves around the targeting and imaging of voltage-gated sodium channel subtype NaV1.7, highly expressed in peripheral nerves from both harvested human and mouse tissues. For the first time, we show that Hsp1a, a radiolabeled NaV1.7-selective peptide isolated from Homoeomma spec. Peru, can serve as a targeted vector for delivering a radioactive sensor to the peripheral nervous system. In situ, we observe high signal-to-noise ratios in the sciatic nerves of animals injected with fluorescently labeled Hsp1a and radiolabeled Hsp1a. Moreover, confocal microscopy on fresh nerve tissue shows the same high ratios of fluorescence, corroborating our in vivo results. This study indicates that fluorescently labeled and radiolabeled Hsp1a tracers could be used to identify and demarcate nerves in a clinical setting.

Fluorescence Imaging of Peripheral Nerves by a Nav1.7-Targeted Inhibitor Cystine Knot Peptide.

Twenty million Americans suffer from peripheral nerve injury caused by trauma and medical disorders, resulting in a broad spectrum of potentially debilitating side effects. In one out of four cases, patients identify surgery as the root cause of their nerve injury. Particularly during tumor resections or after traumatic injuries, tissue distortion and poor visibility can challenge a surgeon's ability to precisely locate and preserve peripheral nerves. Intuitively, surgical outcomes would improve tremendously if nerves could be highlighted using an exogeneous contrast agent. In clinical practice, however, the current standard of care-visual examination and palpation-remains unchanged. To address this unmet clinical need, we explored the expression of voltage-gated sodium channel Nav1.7 as an intraoperative marker for the peripheral nervous system. We show that expression of Nav1.7 is high in peripheral nerves harvested from both human and mouse tissue. We further show that modification of a Nav1.7-selective peptide, Hsp1a, can serve as a targeted vector for delivering a fluorescent sensor to the peripheral nervous system. Ex vivo, we observe a high signal-to-noise ratio for fluorescently labeled Hsp1a in both histologically prepared and fresh tissue. Using a surgical fluorescent microscope, we show in a simulated clinical scenario that the identification of mouse sciatic nerves is possible, suggesting that fluorescently labeled Hsp1a tracers could be used to discriminate nerves from their surrounding tissues in a routine clinical setting.

Anaerobic degradation of anionic surfactants by indigenous microorganisms from sediments of a tropical polluted river in Brazil.

Linear alkylbenzene sulfonate (LAS) is widely used in the formulation of domestic and industrial cleaning products, the most synthetic surfactants used worldwide. These products can reach water bodies through the discharge of untreated sewage or non-effective treatments. This study evaluates the ability of the microorganisms found in the Tiete river sediment to degrade this synthetic surfactant. The experiment was conducted in a bioreactor, operated in batch sequences under denitrifying conditions, with cycles of 24 hours and stirring at 150rpm, using 430 mL of sediments and 1 070mL of a synthetic substrate consisting of yeast extract, soluble starch, sodium bicarbonate and sucrose. LAS was added at different concentrations of l5mg/L and 30mg/L. The reactor operation was divided into the biomass adaptation to the synthetic substrate without LAS and three experimental conditions: a) addition of l5mg/L of LAS; b) 50% reduction the co-substrate concentration and 15 mg/L of LAS, and c) addition of 30mg/L of LAS and 100% co-substrate concentration. The results showed that the degradation efficiency of LAS was directly related to the addition of co-substrates and the population of denitrifying bacteria. The removal of LAS and nitrate can be achieved simultaneously in wastewater with low organic loads. The reduction in the co-substrates concentration was directly influenced by the number of denitrifying bacteria (2.2x10(13) to 1.0 x 10(8) MPN/gTVS), and consequently, LAS degradation (60.1 to 55.4%). The sediment microorganisms in the Tiete river can be used as an alternative inoculum in the treatment of wastewater with nitrate and LAS contamination.

A Decrease in Branched-Chain Amino Acids after a Competitive Male Professional Volleyball Game-A Metabolomic-Based Approach.

A competitive volleyball game is a highly metabolic and physically demanding event for professional players. This study aimed to investigate whether a single game at the end of a preseason promotes changes in the biochemical markers of physical exercise responses and the metabolomic profile of professional volleyball players. This cross-sectional study included 13 male Brazilian professional volleyball players. Food intake, body composition, heart rate, physical movement variables, and blood biochemical indicators were evaluated. For non-target metabolomic analysis, serum samples were subjected to 500 MHz Nuclear Magnetic Resonance. Data analysis showed no significant difference in the biochemical indicators after the game (p > 0.05). The level of metabolites present in the groups of the main components (ß-hydroxybutyrate, arginine/lysine, isoleucine, leucine, and valine) had decreased after the game. However, formic acid and histidine levels increased. Among the compounds not part of the main components, hypoxanthine and tyrosine increased, whereas low-density lipoprotein and very low-density lipoprotein levels decreased. After the game, the metabolomic profiles of players showed significant negative variations in essential amino acids (leucine, valine, and isoleucine). These decreases might be influenced by athlete diet and reduced glycogen storage due to lower carbohydrate intake, potentially impacting serum-essential amino acid levels via oxidation in skeletal muscle. The study provides insights for developing metabolic compensation strategies in athletes.

NaV1.7 targeted fluorescence imaging agents for nerve identification during intraoperative procedures.

Surgeries and trauma result in traumatic and iatrogenic nerve damage that can result in a debilitating condition that approximately affects 189 million individuals worldwide. The risk of nerve injury during oncologic surgery is increased due to tumors displacing normal nerve location, blood turbidity, and past surgical procedures, which complicate even an experienced surgeon's ability to precisely locate vital nerves. Unfortunately, there is a glaring absence of contrast agents to assist surgeons in safeguarding vital nerves. To address this unmet clinical need, we leveraged the abundant expression of the voltage-gated sodium channel 1.7 (NaV1.7) as an intraoperative marker to access peripheral nerves in vivo, and visualized nerves for surgical guidance using a fluorescently-tagged version of a potent NaV1.7-targeted peptide, Tsp1a, derived from a Peruvian tarantula. We characterized the expression of NaV1.7 in sensory and motor peripheral nerves across mouse, primate, and human specimens and demonstrated universal expression. We synthesized and characterized a total of 10 fluorescently labeled Tsp1a-peptide conjugates to delineate nerves. We tested the ability of these peptide-conjugates to specifically accumulate in mouse nerves with a high signal-to-noise ratio in vivo. Using the best-performing candidate, Tsp1a-IR800, we performed thyroidectomies in non-human primates and demonstrated successful demarcation of the recurrent laryngeal and vagus nerves, which are commonly subjected to irreversible damage. The ability of Tsp1a to enhance nerve contrast during surgery provides opportunities to minimize nerve damage and revolutionize standards of care across various surgical specialties.

Noninvasive Diagnostic Method to Objectively Measure Olfaction and Diagnose Smell Disorders by a Molecularly Targeted Fluorescence Imaging Agent.

Despite the recent advances in understanding the mechanisms of olfaction, no tools are currently available to noninvasively identify loss of smell. Because of the substantial increase in patients presenting with coronavirus disease 2019-related loss of smell, the pandemic has highlighted the urgent need to develop quantitative methods. Methods: Our group investigated the use of a novel fluorescent probe named Tsp1a-IR800P as a tool to diagnose loss of smell. Tsp1a-IR800P targets sodium channel 1.7, which plays a critical role in olfaction by aiding the signal propagation to the olfactory bulb. Results: Intuitively, we have identified that conditions leading to loss of smell, including chronic inflammation and coronavirus disease 2019, correlate with the downregulation of sodium channel 1.7 expression in the olfactory epithelium, both at the transcript and at the protein levels. We demonstrated that lower Tsp1a-IR800P fluorescence emissions significantly correlate with loss of smell in live animals-thus representing a potential tool for its semiquantitative assessment. Currently available methods rely on delayed subjective behavioral studies. Conclusion: This method could aid in significantly improving preclinical and clinical studies by providing a way to objectively diagnose loss of smell and therefore aid the development of therapeutic interventions.

A Prospective Double-Blinded Comparison of Reflectance Confocal Microscopy with Conventional Histopathology for In Vivo Assessment in Oral Cancer.

PURPOSE: We investigated reflectance confocal microscopy (RCM) as a possible noninvasive approach for the diagnosis of cancer and real-time assessment of surgical margins. EXPERIMENTAL DESIGN: In a phase I study on 20 patients, we established the RCM imaging morphologic features that distinguish oral squamous cell carcinoma (OSCC) from normal tissue with a newly developed intraoral RCM probe. Our subsequent phase II prospective double-blinded study in 60 patients tested the diagnostic accuracy of RCM against histopathology. Five RCM videos from the tumor and five from normal surrounding mucosa were collected on each patient, followed by a 3-mm punch biopsy of the imaged area. An experienced RCM reader, who was blinded to biopsy location and histologic diagnosis, examined the videos from both regions and classified each as "tumor" or "not tumor" based on RCM features established in phase I. Hematoxylin and eosin slides from the biopsies were read by a pathologist who was blinded to RCM results. Using histology as the gold standard, we calculated the sensitivity and specificity of RCM. RESULTS: We report a high agreement between the blinded readers (95% for normal tissue and 81.7% for tumors), high specificity (98.3%) and negative predictive values (96.6%) for normal tissue identification, and high sensitivity (90%) and positive predictive values (88.2%) for tumor detection. CONCLUSIONS: RCM imaging is a promising technology for noninvasive in vivo diagnosis of OSCC and for real-time intraoperative evaluation of mucosal surgical margins. Its inherent constraint, however, stems from the diminished capability to evaluate structures located at more substantial depths within the tissue.

Acquisition and annotation in high resolution in vivo digital biopsy by confocal microscopy for diagnosis in oral precancer and cancer.

Introduction: Scanned fibre endomicroscopes are full point-scanning confocal microscopes with submicron lateral resolution with an optical slice thickness thin enough to isolate individual cell layers, allow active positioning of the optical slice in the z-axis and collection of megapixel images. Here we present descriptive findings and a brief atlas of an acquisition and annotation protocol high resolution in vivo capture of oral mucosal pathology including oral squamous cell carcinoma and dysplasia using a fluorescence scanned fibre endomicroscope with 3 topical fluorescent imaging agents: fluorescein, acriflavine and PARPi-FL. Methods: Digital biopsy was successfully performed via an acquisition protocol in seventy-one patients presenting for investigation of oral mucosal abnormalities using a miniaturized, handheld scanned fibre endoscope. Multiple imaging agents were utilized and multiple time points sampled. Fifty-nine patients had a matched histopathology correlating in location with imaging. The images were annotated back to macrographic location using a purpose-built software, MouthMap™. Results: Acquisition and annotation of cellular level resolved images was demonstrated with all 3 topical agents. Descriptive observations between clinically or histologically normal oral mucosa showed regular intranuclear distance, a regular nuclear profile and fluorescent homogeneity. This was dependent on the intraoral location and type of epithelium being observed. Key features of malignancy were a loss of intranuclear distance, disordered nuclear clustering and irregular nuclear fluorescence intensity and size. Perinuclear fluorescent granules were seen in the absence of irregular nuclear features in lichenoid inflammation. Discussion: High resolution oral biopsy allows for painless and rapid capture of multiple mucosal sites, resulting in more data points to increase diagnostic precision. High resolution digital micrographs can be easily compared serially across multiple time points utilizing an annotation software. In the present study we have demonstrated realization of a high-resolution digital biopsy protocol of the oral mucosa for utility in the diagnosis of oral cancer and precancer..

Polyethylene Glycol 3350 (PEG 3350) as a Practical Vehicle for Rapid Reconstitution of PARPi-FL Formulations for Clinical Use.

PARPi-FL is a molecularly specific fluorescent agent that targets poly ADP-ribose polymerase 1, a DNA repair enzyme overexpressed in the nuclei of tumor cells. This imaging agent is being investigated in a clinical trial (NCT03085147) for the detection of oral cancer. The PARPi-FL mouthwash formulation currently being used in the phase I/II clinical trial comprises 1,000 nM of PARPi-FL dissolved first in 4.5 ml of polyethylene glycol (PEG) 300 and then in 9.5 ml of water. This formulation requires a 2-step process that can be cumbersome for routine clinical use. To minimize errors and simplify the formulation process, we have developed a new one-step formulation, which requires only the direct addition of water into a vial containing a mixture of the PARPi-FL and PEG 3350, which is also a powder. In a series of analytical and preclinical studies, we demonstrate that the new formulation of PARPi-FL is stable over 365 days, sustains its characteristics, and performs similar to the previous formulation. Moving forward, the new formulation of the PARPi-FL will be used for patients accrued in the phase II clinical trial.

Effectiveness of current protein recommendations in adolescent athletes on a low-carbon diet.

Adolescent athletes require adequate energy and nutrient supply to support growth, development, and the demands associated with exercise and training. However, they are susceptible to nutritional inadequacies affecting their health and physical performance. Food choices with nutrient adequacy and environmental protection is crucial for a sustainable diet. Therefore, we aimed to assess the adequacy of low-carbon diets to meet the protein requirements of adolescent athletes. Therefore, a cross-sectional observational study was conducted with 91 adolescent athletes from sports clubs in Rio de Janeiro who underwent anthropometric and food consumption assessments. To estimate the environmental impact of anthropogenic activities, the sustainability indicators carbon footprint (CF) and water footprint (WF) were used. The CF of the athlete's diet was compared with the benchmark of 1,571 g CO2eq/cap/d estimated by the World Wildlife Fund (WWF). Protein recommendations according to the American Dietetic Association (ADA) for athletes and protein food groups according to the low-carbon EAT-Lancet reference diet were used as references. The results were stratified by sport modality, age, sex, and income range. The Mann-Whitney test was performed, followed by the Kruskal-Wallis test with Dunn's post-hoc test to assess the differences between groups using the statistical program GraphPad PRISM® version 8.0. CF and WF were directly associated with total energy intake, total protein intake, animal-origin protein intake, and the food groups of meat and eggs. Significant differences were observed in the environmental impact of diet based on sports groups and gender. The athletes' profile with higher environmental impact was male, middle-income class, and of any age group. The quartiles of CF of the overall diets were above the 1,571 g CO2eq/cap/d benchmark. Additionally, ADA's recommended range of daily protein consumption was met by most athletes, even in the lowest quartile of CF. Thus, a diet with a lower environmental impact can meet protein recommendations in adolescent athletes. The results found are of interest to the sports and food industries. It could help in designing a balanced diet for athletes as well as ensure less negative environmental impacts of food production and consumption.

Non-invasive diagnostic method to objectively measure olfaction and diagnose smell disorders by molecularly targeted fluorescent imaging agent.

The sense of smell (olfaction) is one of the most important senses for animals including humans. Despite significant advances in the understanding mechanism of olfaction, currently, there are no objective non-invasive methods that can identify loss of smell. Covid-19-related loss of smell has highlighted the need to develop methods that can identify loss of olfaction. Voltage-gated sodium channel 1.7 (NaV1.7) plays a critical role in olfaction by aiding the signal propagation to the olfactory bulb. We have identified several conditions such as chronic inflammation and viral infections such as Covid-19 that lead to loss of smell correlate with downregulation of NaV1.7 expression at transcript and protein levels in the olfactory epithelium. Leveraging this knowledge, we have developed a novel fluorescent probe Tsp1a-IR800 that targets NaV1.7. Using fluorescence imaging we can objectively measure the loss of sense of smell in live animals non-invasively. We also demonstrate that our non-invasive method is semiquantitative because the loss of fluorescence intensity correlates with the level of smell loss. Our results indicate, that our probe Tsp1a-IR800, can objectively diagnose anosmia in animal and human subjects using infrared fluorescence. We believe this method to non-invasively diagnose loss of smell objectively is a significant advancement in relation to current methods that rely on highly subjective behavioral studies and can aid in studying olfaction loss and the development of therapeutic interventions.

Combined PARP1-Targeted Nuclear Contrast and Reflectance Contrast Enhance Confocal Microscopic Detection of Basal Cell Carcinoma.

Reflectance confocal microscopy (RCM) with endogenous backscattered contrast can noninvasively image basal cell carcinomas (BCCs) in skin. However, BCCs present with high nuclear density, and the relatively weak backscattering from nuclei imposes a fundamental limit on contrast, detectability, and diagnostic accuracy. We investigated PARPi-FL, an exogenous nuclear poly(adenosine diphosphate ribose) polymerase (PARP1)-targeted fluorescent contrast agent, and fluorescence confocal microscopy toward improving BCC diagnosis. Methods: We tested PARP1 expression in 95 BCC tissues using immunohistochemistry, followed by PARPi-FL staining in 32 fresh surgical BCC specimens. The diagnostic accuracy of PARPi-FL contrast was evaluated in 83 surgical specimens. The optimal parameters for permeability of PARPi-FL through intact skin was tested ex vivo on 5 human skin specimens and in vivo in 3 adult Yorkshire pigs. Results: We found significantly higher PARP1 expression and PARPi-FL binding in BCCs than in normal skin structures. Blinded reading of RCM-and-fluorescence confocal microscopy images by 2 experts demonstrated a higher diagnostic accuracy for BCCs with combined fluorescence and reflectance contrast than for RCM alone. Optimal parameters (time and concentration) for PARPi-FL transepidermal permeation through intact skin were successfully determined. Conclusion: Combined fluorescence and reflectance contrast may improve noninvasive BCC diagnosis with confocal microscopy.

PARP1: A Potential Molecular Marker to Identify Cancer During Colposcopy Procedures.

Despite efforts in prevention, cervical cancer still presents with a high worldwide incidence and remains a great problem in public health, especially in low-income countries. Screening programs, such as colposcopy with Papanicolaou testing, have greatly improved mortality rates. However, the agents currently used to delineate those lesions (topical application of acetic acid or Lugol iodine) lack specificity and sometimes can lead to unnecessary biopsies or even cervical excisions. A tool to enable in vivo histology to quickly and quantitatively distinguish between tumor, dysplastic tissue, and healthy tissue would be of great clinical interest. Methods: Here, we describe the use of PARPi-FL, a fluorescent inhibitor of poly[adenosine diphosphate-ribose]polymerase 1 (PARP1), which is a nuclear enzyme that is overexpressed in cancer when compared with the normal surrounding tissues. We exploit its use as an optical imaging agent to specifically target PARP1 expression, which was demonstrated to be higher in cervical cancer than the normal surrounding tissue. Results: After topical application of PARPi-FL on freshly excised cone biopsy samples, the nuclei of tumor cells emitted a specific fluorescent signal that could be visualized using a handheld fluorescence confocal microscope. Conclusion: This approach has the potential to improve in vivo identification of tumor cells during colposcopy examination, allowing a rapid, noninvasive, and accurate histopathologic assessment.

Imaging Early-Stage Metastases Using an 18F-Labeled VEGFR-1-Specific Single Chain VEGF Mutant.

PURPOSE: Metastatic breast cancer is the second leading cause of cancer-related death in women. The 5-year survival rate for metastatic breast cancer has remained near 26.9 % for over a decade. The recruitment of hematopoietic stem cells with high expression of the vascular endothelial growth factor receptor 1 (VEGFR-1) has been implicated in early stages of metastasis formation. We propose the use of an 18F-labeled single-chain version of VEGF121, re-engineered to be selective for VEGFR-1 (scVR1), as a positron emission tomography (PET) imaging agent to non-invasively image early-stage metastases. PROCEDURES: scVR1 was 18F-labeled via a biorthogonal click reaction between site-specifically trans-cyclooctene functionalized scVR1 and an Al18F labeled tetrazine-NODA (1,4,7-triazacyclononane-1,4-diiacetic acid). The [18F]AlF-NODA-scVR1 was purified using a PD10 column and subsequently analyzed on HPLC to determine radiochemical purity. Animal experiments were performed in 6-8-week-old female BALB/c mice bearing orthotopic primary 4T1 breast tumors or 4T1 metastatic lesions. The [18F]AlF-NODA-scVR1 tracer was administered via tail vein injection; PET imaging and ex vivo analysis was performed 2 h post-injection. RESULTS: The [18F]AlF-NODA-scVR1 was prepared with a 98.2 ± 1.5 % radiochemical purity and an apparent molar activity of 7.5 ± 1.2 GBq/µmol. The specific binding of scVR1 to VEGFR-1 was confirmed via bead-based assay. The ex vivo biodistribution showed tumor uptake of 3.5 ± 0.5 % ID/g and was readily observable in PET images. Metastasis formation was detected with [18F]AlF-NODA-scVR1 tracer showing colocalization with bioluminescent imaging as well as ex vivo autoradiography and immunofluorescent staining of VEGFR-1. CONCLUSIONS: The diagnostic capabilities of the [18F]AlF-NODA-scVR1 PET tracer was confirmed in both orthotopic and metastatic murine cancer models. These results support the potential use of [18F]AlF-NODA-scVR1 as a PET tracer that could image metastases, providing clinicians with an additional tool to assess a patient's need for adjuvant therapies.