Laser-Induced Graphene Electrochemical Immunosensors for Rapid and Label-Free Monitoring of Salmonella enterica in Chicken Broth

Food-borne illnesses are a growing concern for the food industry and consumers, with millions of cases reported every year. Consequently, there is a critical need to develop rapid, sensitive, and inexpensive techniques for pathogen detection in order to mitigate this problem. However, current pathogen detection strategies mainly include time-consuming laboratory methods and highly trained personnel. Electrochemical in-field biosensors offer a rapid, low-cost alternative to laboratory techniques, but the electrodes used in these biosensors require expensive nanomaterials to increase their sensitivity, such as noble metals (e.g., platinum, gold) or carbon nanomaterials (e.g., carbon nanotubes, or graphene). Herein, we report the fabrication of a highly sensitive and label-free laser-induced graphene (LIG) electrode that is subsequently functionalized with antibodies to electrochemically quantify the food-borne pathogen Salmonella enterica serovar Typhimurium. The LIG electrodes were produced by laser induction on the polyimide film in ambient conditions and, hence, circumvent the need for high-temperature, vacuum environment, and metal seed catalysts commonly associated with graphene-based electrodes fabricated via chemical vapor deposition processes. After functionalization with Salmonella antibodies, the LIG biosensors were able to detect live Salmonella in chicken broth across a wide linear range (25 to 105 CFU mL–1) and with a low detection limit (13 ± 7 CFU mL–1; n = 3, mean ± standard deviation). These results were acquired with an average response time of 22 min without the need for sample preconcentration or redox labeling techniques. Moreover, these LIG immunosensors displayed high selectivity as demonstrated by nonsignificant response to other bacteria strains. These results demonstrate how LIG-based electrodes can be used for electrochemical immunosensing in general and, more specifically, could be used as a viable option for rapid and low-cost pathogen detection in food processing facilities before contaminated foods reach the consumer

Auditory Cortical Detection and Discrimination Correlates with Communicative Significance

Plasticity studies suggest that behavioral relevance can change the cortical processing of trained or conditioned sensory stimuli. However, whether this occurs in the context of natural communication, where stimulus significance is acquired through social interaction, has not been well investigated, perhaps because neural responses to species-specific vocalizations can be difficult to interpret within a systematic framework. The ultrasonic communication system between isolated mouse pups and adult females that either do or do not recognize the calls' significance provides an opportunity to explore this issue. We applied an information-based analysis to multi- and single unit data collected from anesthetized mothers and pup-naïve females to quantify how the communicative significance of pup calls affects their encoding in the auditory cortex. The timing and magnitude of information that cortical responses convey (at a 2-ms resolution) for pup call detection and discrimination was significantly improved in mothers compared to naïve females, most likely because of changes in call frequency encoding. This was not the case for a non-natural sound ensemble outside the mouse vocalization repertoire. The results demonstrate that a sensory cortical change in the timing code for communication sounds is correlated with the vocalizations' behavioral relevance, potentially enhancing functional processing by improving its signal to noise ratio

Hedgehog signaling via a calcitonin receptor-like receptor can induce arterial differentiation independently of VEGF signaling in zebrafish

Multiple signaling pathways control the specification of endothelial cells (ECs) to become arteries or veins during vertebrate embryogenesis. Current models propose that a cascade of Hedgehog (Hh), vascular endothelial growth factor (VEGF), and Notch signaling acts instructively on ECs to control the choice between arterial or venous fate. Differences in the phenotypes induced by Hh, VEGF, or Notch inhibition suggest that not all of the effects of Hh on arteriovenous specification are mediated by VEGF. We establish that full derepression of the Hh pathway in ptc1;ptc2 mutants converts the posterior cardinal vein into a second arterial vessel that manifests intact arterial gene expression, intersegmental vessel sprouting, and HSC gene expression. Importantly, although VEGF was thought to be absolutely essential for arterial fates, we find that normal and ectopic arterial differentiation can occur without VEGF signaling in ptc1;ptc2 mutants. Furthermore, Hh is able to bypass VEGF to induce arterial differentiation in ECs via the calcitonin receptor-like receptor, thus revealing a surprising complexity in the interplay between Hh and VEGF signaling during arteriovenous specification. Finally, our experiments establish a dual function of Hh during induction of runx1+ HSCs

Analysis of the population structure of a gorgonian forest (Placogorgia sp.) using a photogrammetric 3D modeling approach at Le Danois Bank, Cantabrian Sea

The presence of gorgonian forests and deep-sea sponge aggregations in the Le Danois Bank promoted its declaration as the “El Cachucho” Marine Protected Area (MPA) by the Spanish Ministry of Environment, and its inclusion in the Natura 2000 network. Both habitats are considered vulnerable, so follow-up surveys are being performed to monitor their conservation in compliance with the EU Habitats Directive. The use of a non-invasive methodology, which does not cause damage or alterations on benthic communities, is particularly necessary in vulnerable ecosystem studies and MPA monitoring. This study analyzed the assemblage structure of a Placogorgia sp. population using a 3D photogrammetry-based method. The study was carried out through the analysis of the video transects obtained at the Le Danois Bank, using the Politolana underwater towed vehicle during the July 2017 ECOMARG survey. Recent developments in specific software of photogrammetric image analysis allowed extracting valuable information from these video transects. Using the Pix4D Mapper Pro software, 3D point clouds were obtained, and the size and morphometry of yellow fan-shaped gorgonian population structure could be evaluated. Due to gorgonian's high structural complexity, the use of length (i.e. height) as the morphometric descriptor of the real size of the colonies is not appropriate. Instead of length, the fan surface area covered by each gorgonian colony was selected as a suitable parameter of size. The direct measurement of this parameter was possible through a complete 3D reconstruction of the gorgonian forest. A total of 426 colonies of Placogorgia sp. were digitalized to obtain surface measurements and fan spread orientation calculations in 3D models. The results show that gorgonian populations were mostly composed of a high proportion of small colonies (0–0.10 m2). The population structure distribution shows a high proportion (~27%) of recruits (0.5 m2). In 78% of the gorgonian colonies, facing angles were grouped inside the first quadrant (0°-90°), in accordance with the main current direction in this zone. Colony distribution and fan orientation inside the gorgonian forest can be used as data sources to improve monitoring and management programs of these unique habitats in MPAs

New planar light source for the induction and monitoring of photodynamic processes in vitro

We recently developed a new light source that allows for the continuous monitoring of light-induced changes using common spectrophotometric devices adapted for microplate analyses. This source was designed primarily to induce photodynamic processes in cell models. Modern light components, such as LED chips, were used to improve the irradiance homogeneity. In addition, this source forms a small hermetic chamber and thus allows for the regulation of the surrounding atmosphere, which plays a significant role in these light-dependent reactions. The efficacy of the new light source was proven via kinetic measurements of reactive oxygen species generated during the photodynamic reaction of chloroaluminium phthalocyanine disulfonate (ClAlPcS2) in three cell lines: human melanoma cells (G361), human breast adenocarcinoma cells (MCF7), and human fibroblasts (BJ)

Octahedral Molybdenum Cluster-Based Nanomaterials for Potential Photodynamic Therapy

Photo/radiosensitizers, such as octahedral molybdenum clusters (Mo6), have been intensively studied for photodynamic applications to treat various diseases. However, their delivery to the desired target can be hampered by its limited solubility, low stability in physiological conditions, and inappropriate biodistribution, thus limiting the therapeutic effect and increasing the side effects of the therapy. To overcome such obstacles and to prepare photofunctional nanomaterials, we employed biocompatible and water-soluble copolymers based on N-(2-hydroxypropyl)methacrylamide (pHPMA) as carriers of Mo6 clusters. Several strategies based on electrostatic, hydrophobic, or covalent interactions were employed for the formation of polymer-cluster constructs. Importantly, the luminescent properties of the Mo6 clusters were preserved upon association with the polymers: all polymer-cluster constructs exhibited an effective quenching of their excited states, suggesting a production of singlet oxygen (O2(1Δg)) species which is a major factor for a successful photodynamic treatment. Even though the colloidal stability of all polymer-cluster constructs was satisfactory in deionized water, the complexes prepared by electrostatic and hydrophobic interactions underwent severe aggregation in phosphate buffer saline (PBS) accompanied by the disruption of the cohesive forces between the cluster and polymer molecules. On the contrary, the conjugates prepared by covalent interactions notably displayed colloidal stability in PBS in addition to high luminescence quantum yields, suggesting that pHPMA is a suitable nanocarrier for molybdenum cluster-based photosensitizers intended for photodynamic applications

Multimodal imaging of breast cancer metastasis targeting and antimetastatic nanotherapy

INTRODUCTION: As opposed to the routine use of nanomedicines for drug targeting to solid tumors, the highest medical need refers to targeting and treating metastasis. Little is known regarding the accumulation of polymers, liposomes and micelles in metastases, and no systematic analyses have been performed comparing drug targeting to different types (and sizes) of metastases. We here employ three different nanocarriers, an optically imageable metastatic mouse model and several different imaging techniques, to assess drug targeting and drug treatment of metastasis. With fluorophore-labeled nanomedicines, we show that metastases can be more efficiently targeted than primary tumors. In addition, we provide convincing proof-of-principle that docetaxel-loaded micelles are able to inhibit metastasis, also providing initial insights into the vascularity of different types of metastases within different organs. METHODS: Female Nu/Nu mice were orthotopically implanted with 2x105 4T1-iRFP cells (exc 680 nm). Mice were non-invasively monitored for local tumor growth and metastatic colonization using hybrid 3D computed tomography - fluorescence molecular tomography (CT-FMT). Upon metastastic detection, fluorophore-labeled (488/750) polymers, liposomes and micelles were i.v. injected. At 72h, primary tumors and metastases were harvested for ex vivo fluorescence reflectance imaging (2D FRI), assessing the colocalization between metastases (680nm) and nanocarriers (750nm). In an initial therapy study, 4T1-iRFP bearing mice were i.v. treated with vehicle, free docetaxel (Taxotere 30 mg/kg), and core-crosslinked polymeric micelles containing docetaxel (CriPec 30 mg/kg). In addition, a single CriPec 90 mg/kg was administered. Animals were CT-FMT scanned for metastasis colonization, organs were harvested for histology and 1 mouse/group was perfused with the vascular casting agent Microfil (for high-resolution ex vivo CT imaging of blood vessels in tumors and metastases). RESULTS AND DISCUSSION: Metastatic colonization was sensitively detected using CT-FMT (Fig.1A-C). Fluorophore-labeled polymers and liposomes efficiently colocalized with metastases (overlap between 680 and 750 signals, Fig.1B-C), with no accumulation in healthy areas in lungs, lymph nodes and ovary (Fig.1C). The therapeutic efficacy of docetaxel-loaded core-crosslinked polymeric micelles was found to be higher than that of the free drug, with smaller primary tumors and less lung metastases, as visualized and quantified using optical imaging, CT imaging and histology (Fig.1D-E). A single 90 mg/kg micelle dose was found to be more efficient than three 30 mg/kg doses of free drug. High-resolution ex vivo μCT provided initial insights on the microvascular network in different types of metastases, showing that those are more extensively and more homogenously vascularized than primary tumors (Fig.1F). CONCLUSION: In summary, we here systematically show that different types of metastases - in particular lung, lymph node, bone and ovary - can be efficiently targeted and treated using nanomedicines. Initial evidence correlating vascular characteristics with metastatic drug targeting is also provided. (Figure Presented)