Field evaluation of an automated mosquito surveillance system which classifies Aedes and Culex mosquitoes by genus and sex.

BACKGROUND: Mosquito-borne diseases are a major concern for public and veterinary health authorities, highlighting the importance of effective vector surveillance and control programs. Traditional surveillance methods are labor-intensive and do not provide high temporal resolution, which may hinder a full assessment of the risk of mosquito-borne pathogen transmission. Emerging technologies for automated remote mosquito monitoring have the potential to address these limitations; however, few studies have tested the performance of such systems in the field. METHODS: In the present work, an optical sensor coupled to the entrance of a standard mosquito suction trap was used to record 14,067 mosquito flights of Aedes and Culex genera at four temperature regimes in the laboratory, and the resulting dataset was used to train a machine learning (ML) model. The trap, sensor, and ML model, which form the core of an automated mosquito surveillance system, were tested in the field for two classification purposes: to discriminate Aedes and Culex mosquitoes from other insects that enter the trap and to classify the target mosquitoes by genus and sex. The field performance of the system was assessed using balanced accuracy and regression metrics by comparing the classifications made by the system with those made by the manual inspection of the trap. RESULTS: The field system discriminated the target mosquitoes (Aedes and Culex genera) with a balanced accuracy of 95.5% and classified the genus and sex of those mosquitoes with a balanced accuracy of 88.8%. An analysis of the daily and seasonal temporal dynamics of Aedes and Culex mosquito populations was also performed using the time-stamped classifications from the system. CONCLUSIONS: This study reports results for automated mosquito genus and sex classification using an optical sensor coupled to a mosquito trap in the field with highly balanced accuracy. The compatibility of the sensor with commercial mosquito traps enables the sensor to be integrated into conventional mosquito surveillance methods to provide accurate automatic monitoring with high temporal resolution of Aedes and Culex mosquitoes, two of the most concerning genera in terms of arbovirus transmission.

Decision-support tools to build climate resilience against emerging infectious diseases in Europe and beyond.

Climate change is one of several drivers of recurrent outbreaks and geographical range expansion of infectious diseases in Europe. We propose a framework for the co-production of policy-relevant indicators and decision-support tools that track past, present, and future climate-induced disease risks across hazard, exposure, and vulnerability domains at the animal, human, and environmental interface. This entails the co-development of early warning and response systems and tools to assess the costs and benefits of climate change adaptation and mitigation measures across sectors, to increase health system resilience at regional and local levels and reveal novel policy entry points and opportunities. Our approach involves multi-level engagement, innovative methodologies, and novel data streams. We take advantage of intelligence generated locally and empirically to quantify effects in areas experiencing rapid urban transformation and heterogeneous climate-induced disease threats. Our goal is to reduce the knowledge-to-action gap by developing an integrated One Health-Climate Risk framework.

A horizon scan exercise for aquatic invasive alien species in Iberian inland waters.

As the number of introduced species keeps increasing unabatedly, identifying and prioritising current and potential Invasive Alien Species (IAS) has become essential to manage them. Horizon Scanning (HS), defined as an exploration of potential threats, is considered a fundamental component of IAS management. By combining scientific knowledge on taxa with expert opinion, we identified the most relevant aquatic IAS in the Iberian Peninsula, i.e., those with the greatest geographic extent (or probability of introduction), severe ecological, economic and human health impacts, greatest difficulty and acceptability of management. We highlighted the 126 most relevant IAS already present in Iberian inland waters (i.e., Concern list) and 89 with a high probability of being introduced in the near future (i.e., Alert list), of which 24 and 10 IAS, respectively, were considered as a management priority after receiving the highest scores in the expert assessment (i.e., top-ranked IAS). In both lists, aquatic IAS belonging to the four thematic groups (plants, freshwater invertebrates, estuarine invertebrates, and vertebrates) were identified as having been introduced through various pathways from different regions of the world and classified according to their main functional feeding groups. Also, the latest update of the list of IAS of Union concern pursuant to Regulation (EU) No 1143/2014 includes only 12 top-ranked IAS identified for the Iberian Peninsula, while the national lists incorporate the vast majority of them. This fact underlines the great importance of taxa prioritisation exercises at biogeographical scales as a step prior to risk analyses and their inclusion in national lists. This HS provides a robust assessment and a cost-effective strategy for decision-makers and stakeholders to prioritise the use of limited resources for IAS prevention and management. Although applied at a transnational level in a European biodiversity hotspot, this approach is designed for potential application at any geographical or administrative scale, including the continental one.

Automated detection of the yellow-legged hornet (Vespa velutina) using an optical sensor with machine learning.

BACKGROUND: The yellow-legged hornet (Vespa velutina) is native to Southeast Asia and is an invasive alien species of concern in many countries. More effective management of populations of V. velutina could be achieved through more widespread and intensive monitoring in the field, however current methods are labor intensive and costly. To address this issue, we have assessed the performance of an optical sensor combined with a machine learning model to classify V. velutina and native wasps/hornets and bees. Our aim is to use the results of the present work as a step towards the development of a monitoring solution for V. velutina in the field. RESULTS: We recorded a total 935 flights from three bee species: Apis mellifera, Bombus terrestris and Osmia bicornis; and four wasp/hornet species: Polistes dominula, Vespula germanica, Vespa crabro and V. velutina. The machine learning model achieved an average accuracy for species classification of 80.1 ± 13.9% and 74.5 ± 7.0% for V. velutina. V. crabro had the highest level of misclassification, confused mainly with V. velutina and P. dominula. These results were obtained using a 14-value peak and valley feature derived from the wingbeat power spectral density. CONCLUSION: This study demonstrates that the wingbeat recordings from a flying insect sensor can be used with machine learning methods to differentiate V. velutina from six other Hymenoptera species in the laboratory and this knowledge could be used to help develop a tool for use in integrated invasive alien species management programs. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Imaging of Clear Cell Renal Carcinoma with Immune Checkpoint Targeting Aptamer-Based Probe.

Immune checkpoint targeting immunotherapy has revolutionized the treatment of certain cancers in the recent years. Determination of the status of immune checkpoint expression in particular cancers may assist decision making. Here, we describe the development of a single-stranded aptamer-based molecular probe specifically recognizing human PD-L1. Target engaging aptamers are selected by iterative enrichment from a random ssDNA pool and the binding is characterized biochemically. Specificity and dose dependence is demonstrated in vitro in the cell culture using human kidney tumor cells (786-0), human melanoma cells (WM115 and WM266.4) and human glioblastoma LN18 cancer cells. The utility of the probe in vivo is demonstrated using two mouse tumor models, where we show that the probe exhibits excellent potential in imaging. We postulate that further development of the probe may allow universal imaging of different types of tumors depending on their PD-L1 status, which may find utility in cancer diagnosis.

A novel optical sensor system for the automatic classification of mosquitoes by genus and sex with high levels of accuracy.

BACKGROUND: Every year, more than 700,000 people die from vector-borne diseases, mainly transmitted by mosquitoes. Vector surveillance plays a major role in the control of these diseases and requires accurate and rapid taxonomical identification. New approaches to mosquito surveillance include the use of acoustic and optical sensors in combination with machine learning techniques to provide an automatic classification of mosquitoes based on their flight characteristics, including wingbeat frequency. The development and application of these methods could enable the remote monitoring of mosquito populations in the field, which could lead to significant improvements in vector surveillance. METHODS: A novel optical sensor prototype coupled to a commercial mosquito trap was tested in laboratory conditions for the automatic classification of mosquitoes by genus and sex. Recordings of > 4300 laboratory-reared mosquitoes of Aedes and Culex genera were made using the sensor. The chosen genera include mosquito species that have a major impact on public health in many parts of the world. Five features were extracted from each recording to form balanced datasets and used for the training and evaluation of five different machine learning algorithms to achieve the best model for mosquito classification. RESULTS: The best accuracy results achieved using machine learning were: 94.2% for genus classification, 99.4% for sex classification of Aedes, and 100% for sex classification of Culex. The best algorithms and features were deep neural network with spectrogram for genus classification and gradient boosting with Mel Frequency Cepstrum Coefficients among others for sex classification of either genus. CONCLUSIONS: To our knowledge, this is the first time that a sensor coupled to a standard mosquito suction trap has provided automatic classification of mosquito genus and sex with high accuracy using a large number of unique samples with class balance. This system represents an improvement of the state of the art in mosquito surveillance and encourages future use of the sensor for remote, real-time characterization of mosquito populations.

A real-time cell-binding assay reveals dynamic features of STxB-Gb3 cointernalization and STxB-mediated cargo delivery into cancer cells.

The interaction between the Shiga toxin B-subunit (STxB) and its globotriaosylceramide receptor (Gb3) has a high potential for being exploited for targeted cancer therapy. The primary goal of this study was to evaluate the capacity of STxB to carry small molecules and proteins as cargo into cells. For this purpose, an assay was designed to provide real-time information about the StxB-Gb3 interaction as well as the dynamics and mechanism of the internalization process. The assay revealed the ability to distinguish the process of binding to the cell surface from internalization and presented the importance of receptor and STxB clustering for internalization. The overall setup demonstrated that the binding mechanism is complex, and the concept of affinity is difficult to apply. Hence, time-resolved methods, providing detailed information about the interaction of STxB with cells, are critical for the optimization of intracellular delivery.

Endometrial Cancer Spheres Show Cancer Stem Cells Phenotype and Preference for Oxidative Metabolism.

This study aimed to characterize endometrial cancer regarding cancer stem cells (CSC) markers, regulatory and differentiation pathways, tumorigenicity and glucose metabolism. Endometrial cancer cell line ECC1 was submitted to sphere forming protocols. The first spheres generation (ES1) was cultured in adherent conditions (G1). This procedure was repeated and was obtained generations of spheres (ES1, ES2 and ES3) and spheres-derived cells in adherent conditions (G1, G2 and G3). Populations were characterized regarding CD133, CD24, CD44, aldehyde dehydrogenase (ALDH), hormonal receptors, HER2, P53 and ß-catenin, fluorine-18 fluorodeoxyglucose ([18F]FDG) uptake and metabolism by NMR spectroscopy. An heterotopic model evaluated differential tumor growth. The spheres self-renewal was higher in ES3. The putative CSC markers CD133, CD44 and ALDH expression were higher in spheres. The expression of estrogen receptor (ER)α and P53 decreased in spheres, ERß and progesterone receptor had no significant changes and ß-catenin showed a tendency to increase. There was a higher 18F-FDG uptake in spheres, which also showed a lower lactate production and an oxidative cytosol status. The tumorigenesis in vivo showed an earlier growth of tumours derived from ES3. Endometrial spheres presented self-renewal and differentiation capacity, expressed CSC markers and an undifferentiated phenotype, showing preference for oxidative metabolism.

Ascorbic Acid Chemosensitizes Colorectal Cancer Cells and Synergistically Inhibits Tumor Growth.

Colorectal cancer (CRC) is continuously classified as one of the most incidental and mortal types of cancer worldwide. The positive outcomes of the conventional chemotherapy are frequently associated with high toxicity, which often leads to the suspension of the treatment. Growing evidences consider the use of pharmacological concentrations of ascorbic acid (AA), better known as vitamin C, in the treatment of cancer. The use of AA in a clinical context is essentially related to the adoption of new therapeutic strategies based on combination regimens, where AA plays a chemosensitizing role. The reduced sensitivity of some tumors to chemotherapy and the highly associated adverse effects continue to be some of the major obstacles in the effective treatment of CRC. So, this paper aimed to study the potential of a new therapeutic approach against this neoplasia with diminished side effects for the patient. This approach was based on the study of the combination of high concentrations of AA with reduced concentrations of drugs conventionally used in CRC patients and eligible for first and second line chemotherapeutic regimens, namely 5-fluorouracilo (5-FU), oxaliplatin (Oxa) or irinotecan (Iri). The evaluation of the potential synergy between the compounds was first assessed in vitro in three CRC cell lines with different genetic background and later in vivo using one xenograft animal model of CRC. AA and 5-FU act synergistically in vitro just for longer incubation times, however, in vivo showed no benefit compared to 5-FU alone. In contrast to the lack of synergy seen in in vitro studies with the combination of AA with irinotecan, the animal model revealed the therapeutic potential of this combination. AA also potentiated the effect of Oxa, since a synergistic effect was demonstrated, in almost all conditions and in the three cell lines. Moreover, this combined therapy (CT) caused a stagnation of the tumor growth rate, being the most promising tested combination. Pharmacological concentrations of AA increased the efficacy of Iri and Oxa against CRC, with promising results in cell lines with more aggressive phenotypes, namely, tumors with mutant or null P53 expression and tumors resistant to chemotherapy.

Butyrate, a dietary fiber derivative that improves irinotecan effect in colon cancer cells.

A diet rich in fiber is associated with a low risk of developing colorectal cancer. Dietary fiber fermentation by intestinal microflora results in the production of butyrate, which has been reported as a chemopreventive agent and a histone deacetylase inhibitor (HDACi). Irinotecan is used as second-line treatment and induces adverse effects with serious life-threatening toxicities in at least 36% of patients. Our study intends to find a synergy that could improve the efficacy and decrease the toxicity of chemotherapy. Results demonstrate that milimolar concentrations of butyrate has an anti-proliferative effect in all three colon cancer cell lines under study, leading to a decrease on cell viability, expression of P21, P53 and ß-catenin, being able to modulate P-glycoprotein activity and to induce apoptosis by modulation of BAX/BCL-2 ratio. Combined therapy has a cytotoxic potential, resulting in a synergistic effect, and allows a reduction in irinotecan concentration needed to reduce IC50. This potential was verified in terms of cell viability and death, cell cycle and expression of P21 and P53. Butyrate and irinotecan act synergistically in the three cancer cell lines, despite the different genetic background and location, and inhibited tumor growth in a xenograft model. Butyrate is able to influence the mechanism of LS1034 cell line chemoresistance. Butyrate in combination with chemotherapeutic agents has an important role for the treatment of colorectal cancer. Such understanding can guide decisions about which patients with colorectal cancer may benefit from therapy with butyrate demonstrating the important role of diet in colorectal cancer treatment.

Detecting ligand interactions in real time on living bacterial cells.

Time-resolved analysis assays of receptor-ligand interactions are fundamental in basic research and drug discovery. Adequate methods are well developed for the analysis of recombinant proteins such as antibody-antigen interactions. However, assays for time-resolved ligand-binding processes on living cells are still rare, in particular within microbiology. In this report, the real-time cell-binding assay (RT-CBA) technology LigandTracer®, originally designed for mammalian cell culture, was extended to cover Gram-positive and Gram-negative bacteria. This required the development of new immobilization methods for bacteria, since LigandTracer depends on cells being firmly attached to a Petri dish. The evaluated Escherichia coli CJ236 and BL21 as well as Staphylococcus carnosus TM300 strains were immobilized to plastic Petri dishes using antibody capture, allowing us to depict kinetic binding traces of fluorescently labeled antibodies directed against surface-displayed bacterial proteins for as long as 10-15 h. Interaction parameters, such as the affinity and kinetic constants, could be estimated with high precision (coefficient of variation 9-44%) and the bacteria stayed viable for at least 16 h. The other tested attachment protocols were inferior to the antibody capture approach. Our attachment protocol is generic and could potentially also be applied to other assays and purposes.

Impact of assay temperature on antibody binding characteristics in living cells: A case study.

Kinetic and thermodynamic studies of ligand-receptor interactions are essential for increasing the understanding of receptor activation mechanisms and drug behavior. The characterization of molecular interactions on living cells in real-time goes beyond most current binding assays, and provides valuable information about the dynamics and underlying mechanism of the molecules in a living system. The effect of temperature on interactions in cell-based assays is, however, rarely discussed. In the present study, the effect of temperature on binding of monoclonal antibodies, cetuximab and pertuzumab to specific receptors on living cancer cells was evaluated, and the affinity and kinetics of the interactions were estimated at selected key temperatures. Changes in the behavior of the interactions, particularly in the on- and off-rates were observed, leading to greatly extended time to reach the equilibrium at 21°C compared with at 37°C. However, the observed changes in kinetic characteristics were less than a factor of 10. It was concluded that it is possible to conduct real-time measurements with living cells at different temperatures, and demonstrated that influences of the ambient temperature on the interaction behavior are likely to be less than one order of magnitude.

Combined dual effect of modulation of human neutrophils' oxidative burst and inhibition of colon cancer cells proliferation by hydroxycinnamic acid derivatives.

Colon cancer is one of the most incident cancers in the Western World. While both genetic and epigenetic factors may contribute to the development of colon cancer, it is known that chronic inflammation associated to excessive production of reactive oxygen and nitrogen species by phagocytes may ultimately initiate the multistep process of colon cancer development. Phenolic compounds, which reveal antioxidant and antiproliferative activities in colon cancer cells, can be a good approach to surpass this problem. In this work, hydroxycinnamic amides and the respective acid precursors were tested in vitro for their capacity to modulate human neutrophils' oxidative burst and simultaneously to inhibit growth of colon cancer cells. A phenolic amide derivative, caffeic acid hexylamide (CAHA) (4) was found to be the most active compound in both assays, inhibiting human neutrophils' oxidative burst, restraining the inflammatory process, inhibiting growth of colon cancer cells and triggering mitochondrial dysfunction that leads cancer cells to apoptosis. Altogether, these achievements can contribute to the understanding of the relationship between antioxidant and anticancer activities and based on the structure-activity relationships (SAR) established can be the starting point to find more effective phenolic compounds as anticancer agents.

Ascorbic acid and colon cancer: an oxidative stimulus to cell death depending on cell profile.

Colorectal cancer is a major health problem worldwide with urgent need for new and effective anti-cancer approaches that allow treating, increasing survival and improving life quality of patients. At pharmacological concentrations, ascorbic acid (AA) exerts a selective cytotoxic effect, whose mechanism of cytotoxicity remains unsolved. It has been suggested that it depends on the production of extracellular hydrogen peroxide, using ascorbate radical as an intermediate. The aim of this study was to evaluate the effects induced by AA in three colon cancer cell lines, as well as, possible cell death mechanisms involved. Our results showed that pharmacological concentrations of AA induce anti-proliferative, cytotoxic and genotoxic effects on three colon cancer cell lines under study. We also found that AA can induce cell death by an increment of oxidative stress, but also mediating a ROS-independent mechanism, as observed in LS1034 cells. This work explores AA anti-tumoral effects and highlights its applicability in the treatment of CC, underlying the importance of proceeding to clinical trials.

The role of immune system exhaustion on cancer cell escape and anti-tumor immune induction after irradiation.

Immune surveillance seems to represent an effective tumor suppressor mechanism. However, some cancer cells survive and become variants, being poorly immunogenic and able to enter a steady-state phase. These cells become functionally dormant or remain hidden clinically throughout. Neoplastic cells seem to be able to instruct immune cells to undergo changes promoting malignancy. Radiotherapy may act as a trigger of the immune response. After radiotherapy a sequence of reactions occurs, starting in the damage of oncogenic cells by multiple mechanisms, leading to the immune system positive feedback against the tumor. The link between radiotherapy and the immune system is evident. T cells, macrophages, Natural Killer cells and other immune cells seem to have a key role in controlling the tumor. T cells may be dysfunctional and remain in a state of T cell exhaustion, nonetheless, they often retain a high potential for successful defense against cancer, being able to be mobilized to become highly functional. The lack of clinical trials on a large scale makes data a little robust, in spite of promising information, there are still many variables in the studies relating to radiation and immune system. The clarification of the mechanisms underlying immune response to radiation exposure may contribute to treatment improvement, gain of life quality and span of patients.

Cholangiocarcinoma: from molecular biology to treatment.

Cholangiocarcinoma is a rare tumor originating in the bile ducts, which, according to their anatomical location, is classified as intrahepatic, extrahepatic and hilar. Nevertheless, incidence rates have increased markedly in recent decades. With respect to tumor biology, several genetic alterations correlated with resistance to chemotherapy and radiotherapy have been identified. Here, we highlight changes in KRAS and TP53 genes that are normally associated with a more aggressive phenotype. Also IL-6 and some proteins of the BCL-2 family appear to be involved in the resistance that the cholangiocarcinoma presents toward conventional therapies. With regard to diagnosis, tumor markers most commonly used are CEA and CA 19-9, and although its use isolated appears controversial, their combined value has been increasingly advocated. In imaging terms, various methods are needed, such as abdominal ultrasound, computed tomography and cholangiopancreatography. Regarding therapy, surgical modalities are the only ones that offer chance of cure; however, due to late diagnosis, most patients cannot take advantage of them. Thus, the majority of patients are directed to other therapeutic modalities like chemotherapy, which, in this context, assumes a purely palliative role. Thus, it becomes urgent to investigate new therapeutic options for this highly aggressive type of tumor.

Piezoelectric biosensors assisted with electroacoustic impedance spectroscopy: a tool for accurate quantitative molecular recognition analysis.

In this work, electroacoustic impedance analysis based on a modified Butterworth-Van Dyke (BVD) model is used to complement resonance frequency measurements of piezoelectric crystal sensors for the identification and removal of interfering signals. This approach enables the accurate use of the Sauerbrey correlation to establish a direct relationship between mass deposited at the sensor surface and measured frequency variations. Kinetic models can thus be evaluated and binding constants estimated directly from the measured data. We further demonstrate the usefulness of this approach by applying it to the study of the formation of 11-hydroxy-1-undecanothiol self-assembled monolayers (SAM) as well as to the binding of streptavidin to immobilized biotin. Kinetic and equilibrium parameters were estimated from transient analysis, adsorption isotherms, Scatchard and Hill plots obtained from the frequency data for both the alkanethiol and streptavidin films. This strategy based on electroacoustic impedance assisted quartz-crystal microbalance (QCM) biosensors is expected to be a major contribution for the use of these piezoelectric devices as a reliable and cheap detection system that can easily be integrated into analytical techniques.

Piezoelectric biosensors for biorecognition analysis: application to the kinetic study of HIV-1 Vif protein binding to recombinant antibodies.

In this work three piezoelectric sensors modified with anti-HIV-1 Vif (virion infectivity factor) single fragment antibodies (4BL scFV), single domains (VH) and camelized single domains (VHD) were constructed and used to detect HIV1 Vif in liquid samples. Dithio-bis-succinimidyl-undecanoate (DSU) and 11-hydroxy-1-undecanethiol (HUT) mixed self assembled monolayers (SAM) were generated at the sensors surface onto which the antibodies were immobilized. All sensors detected specifically the target HIV1-Vif antigen in solution and no unspecific binding was monitored. Impedance analysis was performed to quantify electroacoustic and viscoelastic interferences during antibody immobilization and antigen recognition. The elimination of such interferences enabled the quantitative use of the piezoelectric immunosensors to estimate the antibody surface density as well as antigen binding and equilibrium constants. In spite of the possible limitation regarding mass transport and other related molecular phenomena, which were not considered in the binding model used, this work demonstrates the usefulness of piezoelectric biosensors in biorecognition analysis and evidences the advantages on using simultaneous impedance analysis to bring analytical significance to measured data, and thus to improve piezoelectric sensors sensitivity and applicability.

Recombinant single-chain variable fragment and single domain antibody piezoimmunosensors for detection of HIV1 virion infectivity factor.

In this paper recombinant single-chain fragments (scFv-4BL), and single domain antibodies (4BL-V(H)) and (4BL-V(H)D) generated against HIV1 virion infectivity factor (Vif) are used to develop piezoimmunosensors for HIV1 recognition. Mixed self assembled monolayers were generated at the surface of gold coated crystal sensors to which scFv-4BL, 4BL-V(H), or 4BL-V(H)D were immobilized. Impedance analysis was used to discriminate interfering signals from frequency variation data and to increase the sensor sensitivity. The elimination of interfering signals enabled the quantification of the amount of immobilized protein and gave some indication on the viscoelasticity of immobilized biofilms. All the modified sensors were able to specifically recognize HIV1 Vif in liquid samples. The results indicate that lower sensitivities are obtained with 4BL-V(H) single domain antibodies, possibly due to its higher hydrophobic character. The sensitivity obtained when using scFv-4BL was reestablished when using the more hydrophilic 4BL-V(H)D single domain. 4BL-V(H)D piezoimunosensors were effective in recognizing HIV1 Vif from protein mixtures and from cell extracts of human embryonic kidney cells expressing HIV1 Vif. The results presented in this paper demonstrate the potential applicability of the developed piezoimmunosensors to monitor HIV1 infection evolution.