Search Results (16)

Background/Objectives: High-risk neuroblastoma patients are treated with approved anti-ganglioside GD2 antibodies of moderate (dinutuximab beta; DB) and higher binding affinity (naxitamab; NAXI). We evaluated the functional potency of DB compared to NAXI and investigated the target-mediated drug disposition (TMDD). Methods: Tumor spheroids were generated from neuroblastoma cells with varying GD2 expression, stably expressing iRFP680 as a viability marker. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) were assessed in a long-term life-cell viability assay using serial dilutions of the GD2 antibodies. Binding activity was determined by flow cytometry. Processes involved in TMDD were analyzed, including antibody binding to dead tumor cells and to soluble GD2 (sGD2), antibody internalization into tumor and immune cells and the impact of sGD2 on DB and NAXI-mediated ADCC. Results: DB and NAXI mediated a concentration-dependent ADCC response against GD2-positive spheroids and no response against GD2-negative spheroids. DB showed a significantly higher ADCC potency than NAXI in all GD2-positive spheroid models. Binding activity of DB and NAXI was not significantly different. However, the decrease of anti-GD2 antibody binding to viable GD2-positive tumor cells following co-incubation with dead GD2-positive tumor cells or sGD2 was significantly higher for NAXI than DB. Additionally, we found an increased internalization of NAXI compared to DB by tumor cells and particularly CD64+ monocytes. Finally, sGD2 impaired NAXI-mediated ADCC to a significantly greater extent than DB-mediated ADCC. Conclusions: DB has a higher ADCC potency over NAXI at clinically relevant concentrations, attributed to stronger TMDD effects of NAXI compared to DB. Full article

3D object detection plays a pivotal role in achieving accurate environmental perception, particularly in complex traffic scenarios where single-modal detection methods often fail to meet precision requirements. This highlights the necessity of multi-modal fusion approaches to enhance detection performance. However, existing camera-LiDAR intermediate fusion methods suffer from insufficient interaction between local and global features and limited fine-grained feature extraction capabilities, which results in inadequate small object detection and unstable performance in complex scenes. To address these issues, the multi-modal 3D object detection algorithm with pointwise and voxelwise fusion (MPVF) is proposed, which enhances multi-modal feature interaction and optimizes feature extraction strategies to improve detection precision and robustness. First, the pointwise and voxelwise fusion (PVWF) module is proposed to combine local features from the pointwise fusion (PWF) module with global features from the voxelwise fusion (VWF) module, enhancing the interaction between features across modalities, improving small object detection capabilities, and boosting model performance in complex scenes. Second, an expressive feature extraction module, improved ResNet-101 and feature pyramid (IRFP), is developed, comprising the improved ResNet-101 (IR) and feature pyramid (FP) modules. The IR module uses a group convolution strategy to inject high-level semantic features into the PWF and VWF modules, improving extraction efficiency. The FP module, placed at an intermediate stage, captures fine-grained features at various resolutions, enhancing the model’s precision and robustness. Finally, evaluation on the KITTI dataset demonstrates a mean Average Precision (mAP) of 69.24%, a 2.75% improvement over GraphAlign++. Detection accuracy for cars, pedestrians, and cyclists reaches 85.12%, 48.61%, and 70.12%, respectively, with the proposed method excelling in pedestrian and cyclist detection. Full article

The water–gas shift (WGS) reaction is one of the most significant reactions in hydrogen technology since it can be used directly to produce hydrogen from the reaction of CO and water; it is also a side reaction taking place in the hydrocarbon reforming processes, determining their selectivity towards H₂ production. The development of highly active WGS catalysts, especially at temperatures below ~450 °C, where the reaction is thermodynamically favored but kinetically limited, remains a challenge. From a fundamental point of view, the reaction mechanism is still unclear. Since specific nanoshapes of CeO₂-based supports have recently been shown to play an important role in the performance of metal nanoparticles dispersed on their surface, in this study, a comparative study of the WGS is conducted on Pt nanoparticles dispersed (with low loading, 0.5 wt.% Pt) on CeO₂ and gadolinium-doped ceria (GDC) supports of different nano-morphologies, i.e., nanorods (NRs) and irregularly faceted particle (IRFP) CeO₂ and GDC, produced by employing hydrothermal and (co-)precipitation synthesis methods, respectively. The results showed that the support’s shape strongly affected its physicochemical properties and in turn the WGS performance of the dispersed Pt nanoparticles. Nanorod-shaped CeO_2,NRs and GDC_NRs supports presented a higher specific surface area, lower primary crystallite size and enhanced reducibility at lower temperatures compared to the corresponding irregular faceted CeO_2,IRFP and GDC_IRFP supports, leading to up to 5-fold higher WGS activity of the Pt particles supported on them. The Pt/GDC_NRs catalyst outperformed all other catalysts and exhibited excellent time-on-stream (TOS) stability. A variety of techniques, namely N₂ physical adsorption–desorption (the BET method), scanning and transmission electron microscopies (SEM and TEM), powder X-ray diffraction (PXRD) and hydrogen temperature programmed reduction (H₂-TPR), were used to identify the texture, structure, morphology and other physical properties of the materials, which together with the in situ diffuse reflectance Fourier transform infrared spectroscopy (DRIFTS) and detailed kinetic studies helped to decipher their catalytic behavior. The enhanced metal–support interactions of Pt nanoparticles with the nanorod-shaped CeO_2,NRs and GDC_NRs supports due to the creation of more active sites at the metal–support interface, leading to significantly improved reducibility of these catalysts, were concluded to be the critical factor for their superior WGS activity. Both the redox and associative reaction mechanisms proposed for WGS in the literature were found to contribute to the reaction pathway. Full article

Leishmania infantum is the vector-borne trypanosomatid parasite causing visceral leishmaniasis in the Mediterranean basin. This neglected tropical disease is treated with a limited number of obsolete drugs that are not exempt from adverse effects and whose overuse has promoted the emergence of resistant pathogens. In the search for novel antitrypanosomatid molecules that help overcome these drawbacks, drug repurposing has emerged as a good strategy. Nitroaromatic compounds have been found in drug discovery campaigns as promising antileishmanial molecules. Fexinidazole (recently introduced for the treatment of stages 1 and 2 of African trypanosomiasis), and pretomanid, which share the nitroimidazole nitroaromatic structure, have provided antileishmanial activity in different studies. In this work, we have tested the in vitro efficacy of these two nitroimidazoles to validate our 384-well high-throughput screening (HTS) platform consisting of L. infantum parasites emitting the near-infrared fluorescent protein (iRFP) as a biomarker of cell viability. These molecules showed good efficacy in both axenic and intramacrophage amastigotes and were poorly cytotoxic in RAW 264.7 and HepG2 cultures. Fexinidazole and pretomanid induced the production of ROS in axenic amastigotes but were not able to inhibit trypanothione reductase (TryR), thus suggesting that these compounds may target thiol metabolism through a different mechanism of action. Full article

Two-photon excitation fluorescence laser-scanning microscopy is the preferred method for studying dynamic processes in living organ models or even in living organisms. Thanks to near-infrared and infrared excitation, it is possible to penetrate deep into the tissue, reaching areas of interest relevant to life sciences and biomedicine. In those imaging experiments, two-photon excitation spectra are needed to select the optimal laser wavelength to excite as many fluorophores as possible simultaneously in the sample under consideration. The more fluorophores that can be excited, and the more cell populations that can be studied, the better access to their arrangement and interaction can be reached in complex systems such as immunological organs. However, for many fluorophores, the two-photon excitation properties are poorly predicted from the single-photon spectra and are not yet available, in the literature or databases. Here, we present the broad excitation range (760 nm to 1300 nm) of photon-flux-normalized two-photon spectra of several fluorescent proteins in their cellular environment. This includes the following fluorescent proteins spanning from the cyan to the infrared part of the spectrum: mCerulean3, mTurquoise2, mT-Sapphire, Clover, mKusabiraOrange2, mOrange2, LSS-mOrange, mRuby2, mBeRFP, mCardinal, iRFP670, NirFP, and iRFP720. Full article

Lactobacilli are a promising natural tool against vaginal dysbiosis and infections. However, new local delivery systems and additional knowledge about their distribution and mechanism of action would contribute to the development of effective medicine. This will be facilitated by the introduction of the techniques for effective, inexpensive, and real-time tracking of these probiotics following their release. Here, we engineered three model vaginal lactobacilli (Lactobacillus crispatus ATCC 33820, Lactobacillus gasseri ATCC 33323, and Lactobacillus jensenii ATCC 25258) and a control Lactobacillus plantarum ATCC 8014 to express fluorescent proteins with different spectral properties, including infrared fluorescent protein (IRFP), green fluorescent protein (GFP), red fluorescent protein (mCherry), and blue fluorescent protein (mTagBFP2). The expression of these fluorescent proteins differed between the Lactobacillus species and enabled quantification and discrimination between lactobacilli, with the longer wavelength fluorescent proteins showing superior resolving power. Each Lactobacillus strain was labeled with an individual fluorescent protein and incorporated into poly (ethylene oxide) nanofibers using electrospinning, as confirmed by fluorescence and scanning electron microscopy. The lactobacilli retained their fluorescence in nanofibers, as well as after nanofiber dissolution. To summarize, vaginal lactobacilli were incorporated into electrospun nanofibers to provide a potential solid vaginal delivery system, and the fluorescent proteins were introduced to distinguish between them and allow their tracking in the future probiotic-delivery studies. Full article

A genuine dilaton $\sigma$ allows scales to exist even in the limit of exact conformal invariance. In gauge theories, these may occur at an infrared fixed point (IRFP) ${\alpha }_{\mathrm{IR}}$ through dimensional transmutation. These large scales at ${\alpha }_{\mathrm{IR}}$ can be separated from small scales produced by ${\theta }_{\mu }^{\mu }$ , the trace of the energy-momentum tensor. For quantum chromodynamics (QCD), the conformal limit can be combined with chiral $SU\left(3\right)\times SU\left(3\right)$ symmetry to produce chiral-scale perturbation theory $\chi$ PT ${}_{\sigma }$ , with $f_0\left(500\right)$ as the dilaton. The technicolor (TC) analogue of this is crawling TC: at low energies, the gauge coupling $\alpha$ goes directly to (but does not walk past) ${\alpha }_{\mathrm{IR}}$ , and the massless dilaton at ${\alpha }_{\mathrm{IR}}$ corresponds to a light Higgs boson at $\alpha \lesssim {\alpha }_{\mathrm{IR}}$ . It is suggested that the $W^{\pm }$ and $Z^0$ bosons set the scale of the Higgs boson mass. Unlike crawling TC, in walking TC, ${\theta }_{\mu }^{\mu }$ produces all scales, large and small, so it is hard to argue that its “dilatonic” candidate for the Higgs boson is not heavy. Full article

The menu offered in restaurants must meet different aspects of quality. Cultural elements are related to their acceptance and can contribute to the preservation of habits, sustainable agricultural systems, and the maintenance of biodiversity and sustainability, among other factors. In this context, this research proposes an instrument for classifying menus regarding the presence/absence of regional foods called the identifier of regional foods presence (IRFP) as a new perspective to evaluate sustainable menus. For this, lists of regional preparations and ingredients were prepared for each Brazilian region. Sequentially, we submitted the dishes/ingredients to a developed decision tree for the classification of foods into regional or national foods. The score, based on the presence/absence of regional foods, considered the components of a menu, with zero attributed to a lack of regional ingredients/dishes. For national dishes/ingredients, researchers attributed a minimum score equal to ten. One regional food gave a score of50 to the menu, and with more than one regional food, a daily menu scored 100. The final menu evaluation was based on the mean scores of the menus in each restaurant. Scores between 0–49.9 were considered inadequate; 50–74.9, adequate; and excellent between 75–100. The IRFP was applied to 111 menus with data collected from all the offered dishes. In total, the study evaluated data from 774 recipes from the menus of 37 restaurants located in the five Brazilian regions with a similar operating system. ANOVA was used to verify if there was a statistical difference between the mean score of each Brazilian region (p < 0.05). The average score obtained by the IRFP in menus from Brazilian community restaurants was 80.3 ± 30.9 (excellent), showing a significant difference between the Northeast and Southeast Regions, with a more significant presence of regional foods in the Northeast (87.7 ± 28.7). The use of the IRFP in menus was shown to be easy in its application, contributing to a stimulation of the use of regional items and, consequently, to the direct and indirect benefits generated for the food system and the local population. Full article

Melanoma is the most aggressive form of skin cancer. Hypoxia is a feature of the tumor microenvironment that reduces efficacy of immuno- and chemotherapies, resulting in poor clinical outcomes. Lactococcus lactis is a facultative anaerobic gram-positive lactic acid bacterium (LAB) that is Generally Recognized as Safe (GRAS). Recently, the use of LAB as a delivery vehicle has emerged as an alternative strategy to deliver therapeutic molecules; therefore, we investigated whether L. lactis can target and localize within melanoma hypoxic niches. To simulate hypoxic conditions in vitro, melanoma cells A2058, A375 and MeWo were cultured in a chamber with a gas mixture of 5% CO₂, 94% N₂ and 1% O₂. Among the cell lines tested, MeWo cells displayed greater survival rates when compared to A2058 and A375 cells. Co-cultures of L. lactis expressing GFP or mCherry and MeWo cells revealed that L. lactis efficiently express the transgenes under hypoxic conditions. Moreover, multispectral optoacoustic tomography (MSOT), and near infrared (NIR) imaging of tumor-bearing BALB/c mice revealed that the intravenous injection of either L. lactis expressing β-galactosidase (β-gal) or infrared fluorescent protein (IRFP713) results in the establishment of the recombinant bacteria within tumor hypoxic niches. Overall, our data suggest that L. lactis represents an alternative strategy to target and deliver therapeutic molecules into the tumor hypoxic microenvironment. Full article

Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IXα (BV) cofactor as a chromophore are increasingly used in cell biology and biomedicine, since their absorption and fluorescence spectra lie within the so-called optical “transparency window” of biological tissues. However, the quantum yield of BV fluorescence in these biomarkers does not exceed 0.145. The task of generating biomarkers with a higher fluorescence quantum yield remains relevant. To address the problem, we proposed the use of phycocyanobilin (PCB) as a chromophore of biomarkers derived from bacterial phytochromes. In this work, we characterized the complexes of iRFP713 evolved from RpBphP2 and its mutant variants with different location of cysteine residues capable of covalent tetrapyrrole attachment with the PCB cofactor. All analyzed proteins assembled with PCB were shown to have a higher fluorescence quantum yield than the proteins assembled with BV. The iRFP713/V256C and iRFP713/C15S/V256C assembled with PCB have a particularly high quantum yield of 0.5 and 0.45, which exceeds the quantum yield of all currently available near-infrared biomarkers. Moreover, PCB has 4 times greater affinity for iRFP713/V256C and iRFP713/C15S/V256C proteins compared to BV. These data establish iRFP713/V256C and iRFP713/C15S/V256C assembled with the PCB chromophore as promising biomarkers for application in vivo. The analysis of the spectral properties of the tested biomarkers allowed for suggesting that the high-fluorescence quantum yield of the PCB chromophore can be attributed to the lower mobility of the D-ring of PCB compared to BV. Full article

Fluorescence imaging is a well-known method for monitoring fluorescence emitted from the subject of interest and provides important insights about cell dynamics and molecules in mammalian cells. Currently, many solutions exist for measuring fluorescence, but the application methods are complex and the costs are high. This paper describes the design and development of a low-cost, smart and portable fluorimeter for the detection of colorectal cancer cell expressing IRFP702. A flashlight is used as a light source, which emits light in the visible range and acts as an excitation source, while a photodiode is used as a detector. It also uses a longpass filter to only allow the wavelength of interest to pass from the cultured cell. It eliminates the need of both the dichroic mirror and excitation filter, which makes the developed device low cost, compact and portable as well as lightweight. The custom-built sample chamber is black in color to minimize interference and is printed with a 3D printer to accommodate the detector circuitry. An established colorectal cancer cell line (human colorectal carcinoma (HCT116)) was cultured in the laboratory environment. A near-infrared fluorescent protein IRFP702 was expressed in the colorectal cancer cells that were used to test the proof-of-concept. The fluorescent cancer cells were first tested with a commercial imaging system (Odyssey^® CLx) and then with the developed prototype to validate the result in a preclinical setting. The developed fluorimeter is versatile as it can also be used to detect multiple types of cancer cells by simply replacing the filters based on the fluorophore. Full article

Traditional in vivo investigation of fungal infection and new antifungal therapies in mouse models is usually carried out using post mortem methodologies. However, biomedical imaging techniques focusing on non-invasive techniques using bioluminescent and fluorescent proteins have become valuable tools. These new techniques address ethical concerns as they allow reduction in the number of animals required to evaluate new antifungal therapies. They also allow better understanding of the growth and spread of the pathogen during infection. In this review, we concentrate on imaging technologies using different fungal reporter proteins. We discuss the advantages and limitations of these different reporters and compare the efficacy of bioluminescent and fluorescent proteins for fungal research. Full article

Ovarian cancers (OCs) are the most lethal gynaecological malignancy, with high levels of relapse and acquired chemo-resistance. Whilst the tumour–immune nexus controls both cancer progression and regression, the lack of an appropriate system to accurately model tumour stage and immune status has hampered the validation of clinically relevant immunotherapies and therapeutic vaccines to date. To address this need, we stably integrated the near-infrared phytochrome iRFP720 at the ROSA26 genomic locus of ID8 mouse OC cells. Intrabursal ovarian implantation into C57BL/6 mice, followed by regular, non-invasive fluorescence imaging, permitted the direct visualization of tumour mass and distribution over the course of progression. Four distinct phases of tumour growth and dissemination were detectable over time that closely mimicked clinical OC progression. Progression-related changes in immune cells also paralleled typical immune profiles observed in human OCs. Specifically, we observed changes in both the CD8+ T cell effector (Teff):regulatory (Treg) ratio, as well as the dendritic cell (DC)-to-myeloid derived suppressor cell (MDSC) ratio over time across multiple immune cell compartments and in peritoneal ascites. Importantly, iRFP720 expression had no detectible influence over immune profiles. This new model permits non-invasive, longitudinal tumour monitoring whilst preserving host–tumour immune interactions, and allows for the pre-clinical assessment of immune profiles throughout disease progression as well as the direct visualization of therapeutic responses. This simple fluorescence-based approach provides a useful new tool for the validation of novel immuno-therapeutics against OC. Full article

Near-infrared fluorescent proteins (NIR FPs) based on the complexes of bacterial phytochromes with their natural biliverdin chromophore are widely used as genetically encoded optical probes for visualization of cellular processes and deep-tissue imaging of cells and organs in living animals. In this work, we show that the steady-state and kinetic dependencies of the various spectral characteristics of iRFP713, developed from the bacterial phytochrome RpBphP2 and recorded at protein unfolding induced by guanidine hydrochloride (GdnHCl), guanidine thiocyanate (GTC), and urea, differ substantially. A study of the unfolding of three single-tryptophan mutant forms of iRFP713 expectedly revealed that protein unfolding begins with the dissociation of the native dimer, while the monomers remain compact. A further increase in the denaturant concentration leads to the formation of an intermediate state of iRFP713 having hydrophobic areas exposed on the protein surface (I). The total surface charge of iRFP713 (pI 5.86) changes from negative to positive with an increase in the concentration of GdnHCl and GTC because the negative charge of glutamic and aspartic acids is neutralized by forming salt bridges between the carboxyl groups and GdnH⁺ ions and because the guanidinium cations bind to amide groups of glutamines and asparagines. The coincidence of both the concentration of the denaturants at which the intermediate state of iRFP713 accumulates and the concentration of GdnH⁺ ions at which the neutralization of the surface charge of the protein in this state is ensured results in strong protein aggregation. This is evidently realized by iRFP713 unfolding by GTC. At the unfolding of the protein by GdnHCl, an intermediate state is populated at higher denaturant concentrations and a strong aggregation is not observed. As expected, protein aggregates are not formed in the presence of the urea. The aggregation of the protein upon neutralization of the charge on the macromolecule surface is the main indicator of the intermediate state of protein. The unfolded state of iRFP713, whose formation is accompanied by a significant decrease in the parameter A, was found to have a different residual structure in the denaturants used. Full article

Near-infrared (NIR) fluorescent proteins (FPs) designed from PAS (Per-ARNT-Sim repeats) and GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA transcriptional activator) domains of bacterial phytochromes covalently bind biliverdin (BV) chromophore via one or two Cys residues. We studied BV interaction with a series of NIR FP variants derived from the recently reported BphP1-FP protein. The latter was engineered from a bacterial phytochrome RpBphP1, and has two reactive Cys residues (Cys15 in the PAS domain and Cys256 in the GAF domain), whereas its mutants contain single Cys residues either in the PAS domain or in the GAF domain, or no Cys residues. We characterized BphP1-FP and its mutants biochemically and spectroscopically in the absence and in the presence of denaturant. We found that all BphP1-FP variants are monomers. We revealed that spectral properties of the BphP1-FP variants containing either Cys15 or Cys256, or both, are determined by the covalently bound BV chromophore only. Consequently, this suggests an involvement of the inter-monomeric allosteric effects in the BV interaction with monomers in dimeric NIR FPs, such as iRFPs. Likely, insertion of the Cys15 residue, in addition to the Cys256 residue, in dimeric NIR FPs influences BV binding by promoting the BV chromophore covalent cross-linking to both PAS and GAF domains. Full article