Indirect Labeling Research Articles

Integration of antibody-antigen and receptor-ligand reactions to establish a gold strip biosensor for detection of 33 β-lactam antibiotics

A novel lateral flow immunoassay biosensor, combining the receptor-ligand reaction and antigen-antibody reaction was developed for the detection of β-lactam antibiotics in milk. The receptor protein, the carboxy-terminal domain of β-lactam sensor-transducer mutant from Bacillus. licheniformis, served as the recognition element. Colloidal gold (CG)-labelled receptor antibody-receptor ternary complex was applied as a signal transducer probe. The presence of β-lactams was identified through a color change in the test zone, where the reaction between the complex of CG/anti-receptor antibody/receptor and β-lactam antigen on the test line could be inhibited by β-lactam residues in the sample, thus leading to a reduction in color signal. Based on the indirect labeling of the receptor with an antibody as a linker, the ability to detect 33 β-lactams was below or near the corresponding maximum residue limit. The proposed lateral flow immunoassay biosensor appears to be an excellent field-based screening tool for the qualitative screening of β-lactams in milk.

Abstract PO007: Tumor-immune communication via extracellular vesicles

Abstract Emerging evidence suggests that tumor-derived extracellular vesicles (tEVs) can influence immune cell behavior both locally within the tumor microenvironment and remotely by accessing the lymphatic and systemic circulation. The in vivo effects of such interactions before and after immunotherapy remain largely unappreciated. In this study, we aimed to develop a sensitive but stringent approach to identify host cells that interact with tEVs, with the ultimate goal of studying how tEV influence immune cells in their native microenvironment. Investigation of tEVs’ roles in cancer has been hampered by the need to isolate them before intravenous reinfusion in animals, which can introduce biases such as homogenization of tEV diversity, judgement of amount to reinfuse and assumptions on blood vs lymph biodistribution. To circumvent these issues, we and others pioneered the use of genetically engineered tumor cells to express membrane-bound reporters, which allow to label untouched, endogenously released tEVs and follow their interactions with immune cells. Still, the amount of reporter proteins that tEVs can carry is relatively small and thus the sensitivity of the approach is suboptimal. Here we addressed this issue by engineering tEVs to display a membrane-bound form of Sortase A, a bacterial transpeptidase that catalyzes the transfer of reporter proteins on the much bigger surface of tEV-binding cells. SrtA catalyzes the formation of a peptide bond between a consensus peptide and an N-terminal glycine of a nearby cell surface proteins (e.g. MHC-I, MHC-II, VE-Cadherin, CD19, integrins). We tested 4 different SrtA protein designs and selected the best performing construct for tEV studies. We genetically encoded SrtA in parental tumor cell lines, which then release SrtA as a tEV-bound transmembrane protein. Once a SrtA+ tEV interacts with a target cell in the presence of the consensus peptide (conjugated to a fluorescent reporter), SrtA catalyzes the formation of a covalent bond between the reporter and surface proteins of the tEV-binding cell. As expected, we observed significant increase in labeling when target cells were engineered to express a surface protein carrying 5 N-terminal glycine residues, which confirms that labeling of tEV-binding cells is due to actual transfer of fluorescent reporter – and not due to acyl intermediate formation. As compared to indirect labeling of EV-binding cells (e.g. using the EV marker CD63 fused to GFP), SrtA-based approach shows 1-2 log increase in sensitivity, depending on the EV-producing cell type. Overall, the catalytic nature of our EV reporter system lowers the amount of tEVs a host cell needs to bind before being detectable, thereby increasing sensitivity. We are currently testing our SrtA-based approach in vivo, where endogenous tEVs mainly accumulate in sentinel lymph nodes. We expect to unearth the full set of lymph node immune cells interacting with native tEVs and to identify whether immunecheckpoint blockade therapy affects tEV tropism toward immune cells. Citation Format: Ferdinando Pucci, Nicklas Hamilton, Natalie Claudio, Randall Armstrong. Tumor-immune communication via extracellular vesicles [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO007.

Abstract MP60: MSC Interaction With Peripheral Blood Monocytes From Stroke Patients is Dependent on Timing After Stroke

Background: Systemic administration of bone marrow derived stromal cells (MSCs) releases a broad range of trophic factors mediating stroke recovery. How MSCs release these factors may depend on the state of the circulation after systemic administration. We assessed whether the secretomes of MSCs, upon exposure to peripheral blood monocytes (Mo), differs depending on the temporal course when the Mo were isolated after stroke. Methods: Peripheral blood mononuclear cells (PBMCs) from stroke patients was isolated at 24h (N=10) and day 5-7 (N=7) after stroke, and from healthy controls (N=5) using Ficoll gradient. CD14+ Mo were isolated using indirect magnetic beads labelling. Contact and Trans-well co-culture assays were established using 100,000 CD14+ Mo and MSCs each in a 0.8um transwell system. After 24h, media was collected and analyzed for secretome using multiplex cytokine assay. Subtypes of Mo were evaluated using flow cytometry. Results: There was a significant increase in non-classical (CD14+CD16++) Mo and a significant decrease in classical (CD14++CD16-) Mo at Day5-7 after stroke as compared to 24h after stroke (Fig). When Day5-7 CD14+ Mo were co-cultured with MSCs, we found a significant reduction in inflammatory IL-1β and TNF-α, and a significant increase in anti-inflammatory IL-4 as compared to co-cultures of MSCs with healthy Mo. This effect was more pronounced in transwell (vs contact) co-cultures. Co-cultures of MSCs and Mo from controls or from patients 24h after stroke led to reductions in TNF-α and upregulation in IL-4 compared with Mo alone. There were no differences in effect of MSCs co-cultures with healthy vs 24h stroke Mo. Conclusion: Our study indicates that the immunomodulatory effect of MSCs may be dependent on the phenotype of Mo in the peripheral circulation of stroke patients. Hence, depending on the temporal course after stroke, the systemic circulation of stroke patients may differentially regulate MSCs to release soluble factors.

Radiolabeling strategies and pharmacokinetic studies for metal based nanotheranostics.

Radiolabeled metal-based nanoparticles (MNPs) have drawn considerable attention in the fields of nuclear medicine and molecular imaging, drug delivery, and radiation therapy, given the fact that they can be potentially used as diagnostic imaging and/or therapeutic agents, or even as theranostic combinations. Here, we present a systematic review on recent advances in the design and synthesis of MNPs with major focuses on their radiolabeling strategies and the determinants of their in vivo pharmacokinetics, and together how their intended applications would be impacted. For clarification, we categorize all reported radiolabeling strategies for MNPs into indirect and direct approaches. While indirect labeling simply refers to the use of bifunctional chelators or prosthetic groups conjugated to MNPs for post-synthesis labeling with radionuclides, we found that many practical direct labeling methodologies have been developed to incorporate radionuclides into the MNP core without using extra reagents, including chemisorption, radiochemical doping, hadronic bombardment, encapsulation, and isotope or cation exchange. From the perspective of practical use, a few relevant examples are presented and discussed in terms of their pros and cons. We further reviewed the determinants of in vivo pharmacokinetic parameters of MNPs, including factors influencing their in vivo absorption, distribution, metabolism, and elimination, and discussed the challenges and opportunities in the development of radiolabeled MNPs for in vivo biomedical applications. Taken together, we believe the cumulative advancement summarized in this review would provide a general guidance in the field for design and synthesis of radiolabeled MNPs towards practical realization of their much desired theranostic capabilities. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Diagnostic Tools > Diagnostic Nanodevices Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Two new genetically modified mouse alleles labeling distinct phases of retinal ganglion cell development by fluorescent proteins.

During development, all retinal cell types arise from retinal progenitor cells (RPCs) in a step-wise fashion. Atoh7 and Pou4f2 mark, and function in, two phases of retinal ganglion cell (RGC) genesis; Atoh7 functions in a subpopulation of RPCs to render them competent for the RGC fate, whereas Pou4f2 participates in RGC fate specification and RGC differentiation. Despite extensive research on their roles, the properties of the two phases represented by these two factors have not been well studied, likely due to the retinal cellular heterogeneity. In this report, we describe two novel knock-in mouse alleles, Atoh7zsGreenCreERT2 and Pou4f2FlagtdTomato , which labeled retinal cells in the two phases of RGC development by fluorescent proteins. Also, the Atoh7zsGreenCreERT2 allele allowed for indirect labeling of RGCs and other cell types upon tamoxifen induction in a dose-dependent manner. Further, these alleles could be used to purify retinal cells in the different phases by fluorescence assisted cell sorting (FACS). Single cell RNA-seq analysis of purified cells from Atoh7zsGreenCreERT2 retinas further validated that this allele labeled both transitional/competent RPCs and their progenies including RGCs. Thus, these two alleles are very useful tools for studying the molecular and genetic mechanisms underlying RGC formation.

Docetaxel-loaded block copolymer micelles labeled with 188Re for combined radiochemotherapy

Block copolymer micelles (BCMs), nano-self-assemblies of amphiphilic copolymers with a hydrophobic core surrounded by a hydrophilic corona, were explored for the simultaneous delivery of a lipophilic chemotherapeutic drug and radiation to tumors. Docetaxel (DTX)-functionalized BCMs were radiolabeled with the β− emitter 188Re by two different approaches (direct and indirect labelling) affording in both cases 188Re-Pz-DTX-BCMs with high purity (>98%). The 188Re-labeled BCMs are stable in PBS pH 7.4 and in cell culture medium, at 37 °C. Cell uptake of 188Re-Pz-DTX-BCMs in MDA-MB-231 metastatic breast cancer cells and MNNG/HOS osteosarcoma cells presented a maximal uptake of ca. 12% at 24 h incubation time. Biodistribution studies in tumor-bearing nude mice (MDA-MB-231 human xenografts) has shown prolonged circulation lifetime in the bloodstream and moderate uptake in the tumor (1.8 ± 0.8% I.A./g at 24 h p.i.). The prolonged blood circulation lifetime together with the in vivo stability, suggests that the micelles are potentially useful as drug delivery system to tumors by the EPR effect. As far as we are aware, we describe herein the first example of BCMs, 188Re-Pz-DTX-BCMs, as versatile nanoplatforms for the simultaneous delivery of beta radiation (188Re) and the chemotherapeutic drug (DTX) potentially useful for cancer therapy. The expected synergistic effect of such combination in pain therapy and/or cancer progression holds promise when compared to the conventional sequential treatment.

In vivo stem cell tracking using scintigraphy in a canine model of DMD

One of the main challenges in cell therapy for muscle diseases is to efficiently target the muscle. To address this issue and achieve better understanding of in vivo cell fate, we evaluated the relevance of a non-invasive cell tracking method in the Golden Retriever Muscular Dystrophy (GRMD) model, a well-recognised model of Duchenne Muscular Dystrophy (DMD). Mesoangioblasts were directly labelled with 111In-oxine, and injected through one of the femoral arteries. The scintigraphy images obtained provided the first quantitative mapping of the immediate biodistribution of mesoangioblasts in a large animal model of DMD. The results revealed that cells were trapped by the first capillary filters: the injected limb and the lung. During the days following injection, radioactivity was redistributed to the liver. In vitro studies, performed with the same cells prepared for injecting the animal, revealed prominent cell death and 111In release. In vivo, cell death resulted in 111In release into the vasculature that was taken up by the liver, resulting in a non-specific and non-cell-bound radioactive signal. Indirect labelling methods would be an attractive alternative to track cells on the mid- and long-term.

Sensitive lateral flow immunoassay of an antibiotic neomycin in foodstuffs.

Aminoglycosides belong to a class of antibiotics now widely used in agriculture and veterinary medicine and expected to contaminate food products. In this study, a sensitive lateral flow immunoassay (LFIA) of an aminoglycoside neomycin (NEO) was developed. Two methods of immunochromatographic detection based on various techniques of gold nanoparticles (AuNPs) introduction as a label were compared. It was demonstrated that the indirect labeling (a conjugation of anti-species antibodies with a marker) allowed for an increase in assay sensitivity by 80 times. The test system was characterized by an instrumental limit of detection of 0.1ng/mL and the cutoff level of 10ng/mL; the assay duration was 15min. Specificity only toward NEO was demonstrated. The developed LFIA has been tested to detect NEO in different foodstuffs. It has been demonstrated that 70-119% of NEO (coefficients of variations < 10%) can be determined in milk, turkey meat, honey, and eggs using simple procedures of preliminary sample preparation. Testing the samples showed the coincidence of the results for the developed lateral flow assay and for commercial ELISA kit.

Cell Surface Labeling by Engineered Extracellular Vesicles.

Extracellular vesicles (EVs) can mediate local and long-range intercellular communication via cell surface signaling. In order to perform in vivo studies of unmanipulated, endogenously released EVs, sensitive but stringent approaches able to detect EV-cell surface interactions are needed. However, isolation and reinfusion of EVs can introduce biases. A rigorous way to study EVs in vivo is by genetically engineering membrane-bound reporters into parental cells. Still, the amount of reporter molecules that EVs can carry is relatively small, and thus, the sensitivity of the approach is suboptimal. This work addresses this issue by engineering EVs to display a membrane-bound form of Sortase A (SrtA), a bacterial transpeptidase that can catalyze the transfer of reporter molecules on the much bigger surface of EV-binding cells. SrtA design and reaction requirements are optimized and validated. Efficient in vitro labeling of EV-binding cells is achieved, even in the presence of only one N-terminal glycine on cell surface proteins. As compared to indirect labeling of EV-binding cells (e.g., using CD63-GFP fusion), the SrtA-based approach shows 1-2 log increase in sensitivity, depending on the EV source. This novel approach will be useful to identify and study the full set of host cells interacting with native EVs in vivo.

Giving Guidance to Graphs: Evaluating Annotations of Data Visualizations for the News

This study quantifies the effectiveness of information recall with direct and indirect labeling of the annotation layer in a news data visualization. Variations of three data visualizations from The New York Times, The Washington Post, and The Wall Street Journal were presented to participants in a crowdsourced experiment to measure their story comprehension. Our results demonstrate that direct labeling offers no advantage over indirect labeling. More significantly, annotations on visualizations do no better to enhance comprehension than visualizations without them, contradicting data visualization orthodoxy.

Multi-target immunofluorescence by separation of antibody cross-labelling via spectral-FLIM-FRET

In biomedical research, indirect immunofluorescence labelling by use of primary and secondary antibodies is central for revealing the spatial distribution of multiple cellular antigens. However, labelling is regularly restricted to few antigens since species variation of primary and corresponding secondary antibodies is limited bearing the risk of unspecific cross-labelling. Here, we introduce a novel microscopic procedure for leveraging undesirable cross-labelling effects among secondary antibodies thereby increasing the number of fluorophore channels. Under cross-labelling conditions, commonly used fluorophores change chemical-physical properties by ‘Förster resonance energy transfer’ leading to defined changes in spectral emission and lifetime decay. By use of spectral fluorescence lifetime imaging and pattern-matching, we demonstrate precise separation of cross-labelled cellular antigens where conventional imaging completely fails. Consequently, this undesired effect serves for an innovative imaging procedure to separate critical antigens where antibody species variation is limited and allows for multi-target labelling by attribution of new fluorophore cross-labelling channels.

Physics-constrained indirect supervised learning

This study proposes a supervised learning method that does not rely on labels. We use variables associated with the label as indirect labels, and construct an indirect physics-constrained loss based on the physical mechanism to train the model. In the training process, the model prediction is mapped to the space of value that conforms to the physical mechanism through the projection matrix, and then the model is trained based on the indirect labels. The final prediction result of the model conforms to the physical mechanism between indirect label and label, and also meets the constraints of the indirect label. The present study also develops projection matrix normalization and prediction covariance analysis to ensure that the model can be fully trained. Finally, the effect of the physics-constrained indirect supervised learning is verified based on a well log generation problem.

Cell Tracking in Cancer Immunotherapy

The impressive development of cancer immunotherapy in the last few years originates from a more precise understanding of control mechanisms in the immune system leading to the discovery of new targets and new therapeutic tools. Since different stages of disease progression elicit different local and systemic inflammatory responses, the ability to longitudinally interrogate the migration and expansion of immune cells throughout the whole body will greatly facilitate disease characterization and guide selection of appropriate treatment regiments. While using radiolabeled white blood cells to detect inflammatory lesions has been a classical nuclear medicine technique for years, new non-invasive methods for monitoring the distribution and migration of biologically active cells in living organisms have emerged. They are designed to improve detection sensitivity and allow for a better preservation of cell activity and integrity. These methods include the monitoring of therapeutic cells but also of all cells related to a specific disease or therapeutic approach. Labeling of therapeutic cells for imaging may be performed in vitro, with some limitations on sensitivity and duration of observation. Alternatively, in vivo cell tracking may be performed by genetically engineering cells or mice so that may be revealed through imaging. In addition, SPECT or PET imaging based on monoclonal antibodies has been used to detect tumors in the human body for years. They may be used to detect and quantify the presence of specific cells within cancer lesions. These methods have been the object of several recent reviews that have concentrated on technical aspects, stressing the differences between direct and indirect labeling. They are briefly described here by distinguishing ex vivo (labeling cells with paramagnetic, radioactive, or fluorescent tracers) and in vivo (in vivo capture of injected radioactive, fluorescent or luminescent tracers, or by using labeled antibodies, ligands, or pre-targeted clickable substrates) imaging methods. This review focuses on cell tracking in specific therapeutic applications, namely cell therapy, and particularly CAR (Chimeric Antigen Receptor) T-cell therapy, which is a fast-growing research field with various therapeutic indications. The potential impact of imaging on the progress of these new therapeutic modalities is discussed.

Gold nanoparticle based double-labeling of melanoma extracellular vesicles to determine the specificity of uptake by cells and preferential accumulation in small metastatic lung tumors

BackgroundExtracellular vesicles (EVs) have shown great potential for targeted therapy, as they have a natural ability to pass through biological barriers and, depending on their origin, can preferentially accumulate at defined sites, including tumors. Analyzing the potential of EVs to target specific cells remains challenging, considering the unspecific binding of lipophilic tracers to other proteins, the limitations of fluorescence for deep tissue imaging and the effect of external labeling strategies on their natural tropism. In this work, we determined the cell-type specific tropism of B16F10-EVs towards cancer cell and metastatic tumors by using fluorescence analysis and quantitative gold labeling measurements. Surface functionalization of plasmonic gold nanoparticles was used to promote indirect labeling of EVs without affecting size distribution, polydispersity, surface charge, protein markers, cell uptake or in vivo biodistribution. Double-labeled EVs with gold and fluorescent dyes were injected into animals developing metastatic lung nodules and analyzed by fluorescence/computer tomography imaging, quantitative neutron activation analysis and gold-enhanced optical microscopy.ResultsWe determined that B16F10 cells preferentially take up their own EVs, when compared with colon adenocarcinoma, macrophage and kidney cell-derived EVs. In addition, we were able to detect the preferential accumulation of B16F10 EVs in small metastatic tumors located in lungs when compared with the rest of the organs, as well as their precise distribution between tumor vessels, alveolus and tumor nodules by histological analysis. Finally, we observed that tumor EVs can be used as effective vectors to increase gold nanoparticle delivery towards metastatic nodules.ConclusionsOur findings provide a valuable tool to study the distribution and interaction of EVs in mice and a novel strategy to improve the targeting of gold nanoparticles to cancer cells and metastatic nodules by using the natural properties of malignant EVs.

Highly sensitive lateral flow test with indirect labelling for zearalenone in baby food

ABSTRACT Maximum permissible levels of mycotoxins in baby food may be 1% of those in ordinary food. Therefore, highly sensitive methods of mycotoxin control are in demand. To detect such low amounts, expensive instrumental methods are commonly used. Advantages of immunochromatographic analyses are their low cost and simple sample preparation; however, their sensitivity needs to be increased to contend with instrumental methods. A scheme for competitive immunochromatography with indirect labelling was implemented and developed for the detection of mycotoxin zearalenone (ZEA). Two separate reagents were used for the assay, namely free specific antibodies and antispecies antibodies conjugated with gold nanoparticles. This made it possible to simultaneously increase the sensitivity of the assay and the reliability of measurements. The instrumental detection limit of ZEA in baby food was 5 pg/mL (100 pg/g). Thus, the sensitivity attained is comparable with liquid chromatography characteristics. The duration of the analysis was 17 min.

Preparation and SPECT/CT Imaging of 177Lu-Labeled Peptide Nucleic Acid (PNA) Targeting CITED1: Therapeutic Evaluation in Tumor-Bearing Nude Mice.

PurposeThe expression of Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 1 (CITED1) is upregulated in papillary thyroid carcinoma (PTC) and mediates cell proliferation and migration. To facilitate early diagnosis and monitoring of recurrent or metastatic PTC, we designed 177Lu-labeled antisense peptide nucleic acid (PNA) targeting CITED1 mRNA to evaluate the therapeutic potential, while analyzing its distribution in nude mice and the characteristics withsingle-photon emission-computed tomography/computed tomography (SPECT/CT) imaging.Materials and Methods177Lu-DOTA-anti-CITED1-PNA (177Lu-asPNA) was obtained by indirect labeling. High-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) were used to determine the labeling rate and radiochemical purity. The stability of 177Lu-asPNA was evaluated by TLC, and the radioactivity count was measured by a γ counter to calculate its uptake capacity in K1 cells. To analyze the distribution of 177Lu-asPNA in body tissues and organs of nude mice, static single-photon emission-computed tomography (SPECT) imaging and SPECT/CT image fusion were performed. Then, the therapeutic effects of probes were explored by tumor growth curves and survival analysis.ResultsOur probe showed a radiochemical purity of 96.5±0.15% at 1 hr and specific activity of 8.7±0.53 MBq/μg. The uptake rate in the 177Lu-asPNA group was much higher than that in the 177Lu-DOTA-nonsense-PNA (177Lu-nonsense-PNA) and 177Lu-DOTA groups (P<0.05). The biological distribution showed that the tumor/muscle ratio was at its highest at 24 h (4.98±0.34) post-inoculation, with SPECT/CT imaging showing clear tumor development. By measuring tumor volume of tumor-bearing nude mice, the 177Lu-asPNA group showed a significant difference in tumor size 9 days after injection (P < 0.05). Kaplan-Meier survival curves showed that the overall survival rate in the 177Lu-asPNA group was significantly different from those in the DOTA-anti-CITED1-PNA (asPNA) and saline groups (P = 0.002, log-rank test).Conclusion177Lu-asPNA was developed successfully, showing a high labeling rate and good stability. SPECT/CT imaging demonstrated tumor growth in nude mice, which was effectively inhibited by our probe, thus prolonging survival.

Immunochromatographic tests for the detection of microcystin-LR toxin in water and fish samples

Immunochromatographic analysis of microcystin-LR with indirect antibody labeling by gold nanoparticles has been proposed and successfully applied for fish and water samples.

Radioactive polymeric nanoparticles for biomedical application

Nowadays, emerging radiolabeled nanosystems are revolutionizing medicine in terms of diagnostics, treatment, and theranostics. These radionuclides include polymeric nanoparticles (NPs), liposomal carriers, dendrimers, magnetic iron oxide NPs, silica NPs, carbon nanotubes, and inorganic metal-based nanoformulations. Between these nano-platforms, polymeric NPs have gained attention in the biomedical field due to their excellent properties, such as their surface to mass ratio, quantum properties, biodegradability, low toxicity, and ability to absorb and carry other molecules. In addition, NPs are capable of carrying high payloads of radionuclides which can be used for diagnostic, treatment, and theranostics depending on the radioactive material linked. The radiolabeling process of nanoparticles can be performed by direct or indirect labeling process. In both cases, the most appropriate must be selected in order to keep the targeting properties as preserved as possible. In addition, radionuclide therapy has the advantage of delivering a highly concentrated absorbed dose to the targeted tissue while sparing the surrounding healthy tissues. Said another way, radioactive polymeric NPs represent a promising prospect in the treatment and diagnostics of cardiovascular diseases such as cardiac ischemia, infectious diseases such as tuberculosis, and other type of cancer cells or tumors.

Nitrogen and phosphorus uptake from isotope‐labeled fertilizers by sorghum and soil microorganisms

AbstractOn nutrient‐depleted Lixisols from Burkina Faso, nutrient acquisition by crops and soil microbes mainly relies on the limited amounts of mineral and organic fertilizers applied by small‐scale farmers. The objective of this study was to determine simultaneously the uptake of nitrogen (N) and phosphorus (P) contained in organic and mineral fertilizers by sorghum [Sorghum bicolor (L.) Moench ] and soil microbial biomass. Double 15N and 33P direct and indirect labeling techniques were applied in a pot experiment to determine the contributions of different fertilizers to sorghum N and P uptake during 52 d of growth. In parallel, soil respiration, available, and microbial N and P were tracked in an incubation experiment. Sorghum derived 83–90% of P from fertilizers. Nitrogen from cattle manure was poorly available, contributing only 20% of the N taken up by sorghum. Water‐soluble mineral fertilizers increased soil N and P availability, resulting in the highest total N and P uptake by sorghum from fertilizers and soil among all treatments. The application of cowpea [Vigna unguiculata (L.) Walp.] residues induced microbial N and P immobilization, reducing sorghum N and P uptake to the level of the non‐fertilized treatment. The use of double 15N and 33P labeling elucidated the impact of fertilizers on soil nutrient pools. The low plant N/P ratio suggested N limitation for sorghum in the manure treatment. Cowpea residues were inefficient for sorghum nutrition, but they increased soil microbial nutrient pools. This study gives insights on the potential effects of legume residues used as green manure to build soil fertility.

Can composition and preheating improve infiltrant characteristics and penetrability in demineralized enamel?

The composition of infiltrants can influence their physical properties, viscosity and depth of penetration (DP). Strategies are used to increase the DP, such as the addition of diluents or the use of heat. This study aimed to evaluate the effect of preheating and composition on physical properties and DP of infiltrants in demineralized enamel. The groups were assigned, and the following experimental formulations were made: 25%BisEMA +75%TEGDMA; 25%BisEMA +65%TEGDMA +10%ethanol; 25%BisEMA +65%TEGDMA +10%HEMA; 100%TEGDMA; 90%TEGDMA +10%ethanol; 90%TEGDMA +10%HEMA. The samples were photoactivated at two temperatures (25°C and 55°C). Degree of conversion (DC) was performed using an infrared spectrophotometer. Elastic modulus (E), flexural strength (FS) and contact angle (CA) tests were also performed. The DP of an infiltrant in demineralized enamel was determined by confocal laser scanning microscopy (CLSM) using an indirect labeling technique. The data were analyzed by two-way ANOVA and Tukey's test. DC increased after preheating in all the groups; however, 90%TEGDMA+10%ethanol showed the lowest DC for both temperatures, and the lowest E. Preheating did not influence E or FS. The CA increased at 55°C for most groups, but decreased for groups containing HEMA. Temperature did not seem to influence DP, and Icon showed the lowest DP values. The 100%TEGDMA composition showed more homogeneous penetration, whereas Icon showed heterogeneous and superficial penetration. The preheating technique does not improve all properties in all the material compositions. The composition of a material can influence and improve its properties.