Immunosuppressants are clinically approved drugs to treat the potential rejection of transplanted organs and require frequent monitoring due to their narrow therapeutic window. Immunophilins are small proteins that bind immunosuppressants with high affinity, yet there are no examples of fluorogenic immunophilins and their potential application as optical biosensors for immunosuppressive drugs in clinical biosamples. In the present work, we designed novel diazonium BODIPY salts for the site-specific labeling of tyrosine residues in peptides via solid-phase synthesis as well as for late-stage functionalization of whole recombinant proteins. After the optimization of a straightforward one-step labeling procedure for immunophilins PPIA and FKBP12, we demonstrated the application of a fluorogenic analogue of FKBP12 for the selective detection of the immunosuppressant drug tacrolimus, including experiments in urine samples from patients with functioning renal transplants. This chemical methodology opens new avenues to rationally design wash-free immunophilin-based biosensors for rapid therapeutic drug monitoring.
Photodynamic therapy is an anti-cancer treatment that requires illumination of photosensitizers to induce local cell death. Current near-infrared organic photosensitizers are built from large and non-modular structures that cannot be tuned to improve safety and minimize off-target toxicity. This work describes a novel chemical platform to generate enzyme-activatable near-infrared photosensitizers. We optimized the Se-bridged hemicyanine scaffold to include caging groups and biocompatible moieties, and generated cathepsin-triggered photosensitizers for effective ablation of human glioblastoma cells. Furthermore, we demonstrated that enzyme-activatable Se-bridged hemicyanines are effective photosensitizers for the safe ablation of microtumors in vivo, creating new avenues in the chemical design of targeted anti-cancer photodynamic therapy agents.
During the winter of 2023, Chile faced a complex situation related to the respiratory syncytial virus (RSV). After experiencing a decline in RSV circulation during the years of the SARS-CoV-2 pandemic, a late outbreak was observed in the spring of 2022 and an early onset of the outbreak in 2023, with a significant increase in the number of serious cases. The ineffectiveness of strategic planning and risk communication contributed to the complexity of the situation. To avoid the above next winter, measures such as active surveillance, unification of definitions for acute respiratory infections, identification of RSV variants, public education about infections and advance preparation regarding hospital beds and health personnel are suggested. The importance of immunization and intersectoral collaboration to acquire new preventive alternatives is highlighted, as well as the need for early communication about the importance of immunization and identification of high-risk groups, improvement in training of medical personnel and strategic planning of the Ministry of Health. seeking a proactive and collaborative approach to address the complex RSV situation in future winters. The Chilean Immunization Advisory Committee has already carried out an analysis and recommendation on a new prevention alternative. This working group will support any decision of the Ministry of Health in public policies that attempt a change in the paradigm of control of this disease for the health of the children of our country.
Respiratory Syncytial Virus Infections , Respiratory Syncytial Virus, Human , Respiratory Tract Infections , Child , Humans , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Virus Infections/epidemiology , Respiratory Syncytial Virus Infections/prevention & control , Immunization , Vaccination
Grapevine trunk diseases (GTDs) attack the vine's wood, devastating vineyards worldwide. Chile is the world's fourth-largest wine exporter, and Cabernet Sauvignon is one of the most economically important red wine varieties. Botryosphaeria dieback is an important GTD, and Diplodia seriata is one of the main pathogenic species. Biocontrol studies of these pathogens are commonly carried out at different incubation times but at a single temperature. This study aimed to evaluate the biocontrol effect of Chilean PGPB and grapevine endophytic bacteria against D. seriata at different temperatures. We analyzed the biocontrol effect of Pseudomonas sp. GcR15a, Pseudomonas sp. AMCR2b and Rhodococcus sp. PU4, with three D. seriata isolates (PUCV 2120, PUCV 2142 and PUCV 2183) at 8, 22 and 35 °C. Two dual-culture antagonism methods (agar plug diffusion and double plate) were used to evaluate the in vitro effect, and an in vivo test was performed with Cabernet Sauvignon cuttings. In vitro, the greatest inhibitions were obtained using the agar plug diffusion method and at a temperature of 8 °C, where Rhodococcus sp. PU4 obtains a 65% control (average) and Pseudomonas sp. GcR15a a 57% average. At 22 °C, only strains of Pseudomonas sp. show control. At 35 °C, one Pseudomonas strain shows the highest control (38%), on average, similar to tebuconazole (33%), and then Rhodococcus sp. (30%). In vivo, a biocontrol effect is observed against two D. seriata isolates, while the PUCV 2142 proves to be more resistant to control. The biocontrol ability at low temperatures is promising for effective control in the field, where infections occur primarily in winter.
The essential functions that cytokine/immune cell interactions play in tissue homeostasis and during disease have prompted the molecular design of targeted fluorophores to monitor their activity in real time. Whereas activatable probes for imaging immune-related enzymes are common, many immunological functions are mediated by binding events between cytokines and their cognate receptors that are hard to monitor by live-cell imaging. A prime example is interleukin-33 (IL-33), a key cytokine in innate and adaptive immunity, whose interaction with the ST2 cell-surface receptor results in downstream signaling and activation of NF-κB and AP-1 pathways. In the present work, we have designed a chemical platform to site-specifically introduce OFF-to-ON BODIPY fluorophores into full cytokine proteins and generate the first nativelike fluorescent analogues of IL-33. Among different incorporation strategies, chemical aminoacylation followed by bioorthogonal derivatization led to the best labeling results. Importantly, the BODIPY-labeled IL-33 derivatives-unlike IL-33-GFP constructs-exhibited ST2-specific binding and downstream bioactivity profiles comparable to those of the wild-type interleukin. Real-time fluorescence microscopy assays under no wash conditions confirmed the internalization of IL-33 through ST2 receptors and its intracellular trafficking through the endosomal pathway. We envision that the modularity and versatility of our BODIPY labeling platform will facilitate the synthesis of minimally tagged fluorogenic cytokines as the next generation of imaging reagents for real-time visualization of signaling events in live immune cells.
Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of the protein subcellular location and function. Some proteins, however, show altered localization or function when labeled using direct fusions to fluorescent proteins, making them difficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to â¼200 nm, which is 2 orders of magnitude larger than the size of most proteins. To circumvent these challenges, we previously developed LIVE-PAINT, a live-cell super-resolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-of-interest. Here, we successfully use LIVE-PAINT to image yeast membrane proteins that do not tolerate the direct fusion of a fluorescent protein by using peptide tags as short as 5-residues. We also demonstrate that it is possible to resolve multiple proteins at the nanoscale concurrently using orthogonal peptide interaction pairs.
Peptides , Proteins , Diagnostic Imaging , Saccharomyces cerevisiae , Fluorescent Dyes/chemistry
During the COVID-19 pandemic, the importance of vaccinating children against SARS-CoV-2 was rapidly established. This study describes the safety of CoronaVac® in children and adolescents between 3- and 17-years-old in a multicenter study in Chile with two vaccine doses in a 4-week interval. For all participants, immediate adverse events (AEs), serious AEs (SAEs), and AEs of special interest (AESIs) were registered throughout the study. In the safety subgroup, AEs were recorded 28 days after each dose. COVID-19 surveillance was performed throughout the study. A total of 1139 individuals received the first and 1102 the second dose of CoronaVac®; 835 were in the safety subgroup. The first dose showed the highest number of AEs: up to 22.2% of participants reported any local and 17.1% systemic AE. AEs were more frequent in adolescents after the first dose, were transient, and mainly mild. Pain at the inoculation site was the most frequent AE for all ages. Fever was the most frequent systemic AE for 3-5 years old and headache in 6-17 years old. No SAEs or AESIs related to vaccination occurred. Most of the COVID-19 cases were mild and managed as outpatients. CoronaVac® was safe and well tolerated in children and adolescents, with different safety patterns according to age.
T cells are an essential part of the immune system with crucial roles in adaptive response and the maintenance of tissue homeostasis. Depending on their microenvironment, T cells can be differentiated into multiple states with distinct functions. This myriad of cellular activities have prompted the development of numerous smart probes, ranging from small molecule fluorophores to nanoconstructs with variable molecular architectures and fluorescence emission mechanisms. In this Tutorial Review, we summarize recent efforts in the design, synthesis and application of smart probes for imaging T cells in tumors and inflammation sites by targeting metabolic and enzymatic biomarkers as well as specific surface receptors. Finally, we briefly review current strategies for how smart probes are employed to monitor the response of T cells to anti-cancer immunotherapies. We hope that this Review may help chemists, biologists and immunologists to design the next generation of molecular imaging probes for T cells and anti-cancer immunotherapies.
Molecular Probes , T-Lymphocytes , T-Lymphocytes/metabolism , Fluorescent Dyes , Immunotherapy , Optical Imaging
Late-stage diversification of structurally complex amino acids and peptides provides tremendous potential for drug discovery and molecular imaging. Specifically, labeling peptides with fluorescent tags is one of the most important methods for visualizing their mode of operation. Despite major recent advances in the field, direct molecular peptide labeling by C-H activation is largely limited to dyes with relatively short emission wavelengths, leading to high background signals and poor signal-to-noise ratios. In sharp contrast, here we report on the fluorescent labeling of peptides catalyzed by non-toxic manganese(i) via C(sp2)-H alkenylation in chemo- and site-selective manners, providing modular access to novel near-infrared (NIR) nitrobenzodiazole-based peptide fluorogenic probes.
The G protein-coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid-resistant BODIPY-based amino acid (Trp-BODIPY PLUS), and its implementation for solid-phase synthesis of fluorescent bioactive peptides. Trp-BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn-on fluorescence emission upon target binding for wash-free imaging. Finally, we employed Trp-BODIPY PLUS to prepare some of the first fluorogenic kisspeptin-based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging.
Islets of Langerhans , Kisspeptins , Mice , Animals , Humans , Kisspeptins/chemistry , Kisspeptins/metabolism , Peptides/chemistry , Islets of Langerhans/diagnostic imaging , Islets of Langerhans/metabolism , Receptors, G-Protein-Coupled/metabolism , Optical Imaging , Amino Acids/metabolism
The multiple applications of super-resolution microscopy have prompted the need for minimally invasive labeling strategies for peptide-guided fluorescence imaging. Many fluorescent reporters display limitations (e.g., large and charged scaffolds, non-specific binding) as building blocks for the construction of fluorogenic peptides. Herein we have built a library of benzodiazole amino acids and systematically examined them as reporters for background-free fluorescence microscopy. We have identified amine-derivatized benzoselenadiazoles as scalable and photostable amino acids for the straightforward solid-phase synthesis of fluorescent peptides. Benzodiazole amino acids retain the binding capabilities of bioactive peptides and display excellent signal-to-background ratios. Furthermore, we have demonstrated their application in peptide-PAINT imaging of postsynaptic density protein-95 nanoclusters in the synaptosomes from mouse brain tissues.
Amino Acids , Peptides , Animals , Mice , Amines , Fluorescent Dyes/chemistry , Optical Imaging/methods , Solid-Phase Synthesis Techniques
Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.
Fluorescent Dyes , T-Lymphocytes, Cytotoxic , Animals , Humans , Mice , Granzymes , Killer Cells, Natural , Mice, Knockout
The multiple applications of super-resolution microscopy have prompted the need for minimally invasive labeling strategies for peptide-guided fluorescence imaging. Many fluorescent reporters display limitations (e.g., large and charged scaffolds, non-specific binding) as building blocks for the construction of fluorogenic peptides. Herein we have built a library of benzodiazole amino acids and systematically examined them as reporters for background-free fluorescence microscopy. We have identified amine-derivatized benzoselenadiazoles as scalable and photostable amino acids for the straightforward solid-phase synthesis of fluorescent peptides. Benzodiazole amino acids retain the binding capabilities of bioactive peptides and display excellent signal-to-background ratios. Furthermore, we have demonstrated their application in peptide-PAINT imaging of postsynaptic density protein-95 nanoclusters in the synaptosomes from mouse brain tissues.
Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.
The G protein-coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid-resistant BODIPY-based amino acid (Trp-BODIPY PLUS), and its implementation for solid-phase synthesis of fluorescent bioactive peptides. Trp-BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn-on fluorescence emission upon target binding for wash-free imaging. Finally, we employed Trp-BODIPY PLUS to prepare some of the first fluorogenic kisspeptin-based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging.
Multiple vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been evaluated in clinical trials. However, trials addressing the immune response in the pediatric population are scarce. The inactivated vaccine CoronaVac has been shown to be safe and immunogenic in a phase 1/2 clinical trial in a pediatric cohort in China. Here, we report interim safety and immunogenicity results of a phase 3 clinical trial for CoronaVac in healthy children and adolescents in Chile. Participants 3 to 17 years old received two doses of CoronaVac in a 4-week interval until 31 December 2021. Local and systemic adverse reactions were registered for volunteers who received one or two doses of CoronaVac. Whole-blood samples were collected from a subgroup of 148 participants for humoral and cellular immunity analyses. The main adverse reaction reported after the first and second doses was pain at the injection site. Four weeks after the second dose, an increase in neutralizing antibody titer was observed in subjects relative to their baseline visit. Similar results were found for activation of specific CD4+ T cells. Neutralizing antibodies were identified against the Delta and Omicron variants. However, these titers were lower than those for the D614G strain. Importantly, comparable CD4+ T cell responses were detected against these variants of concern. Therefore, CoronaVac is safe and immunogenic in subjects 3 to 17 years old, inducing neutralizing antibody secretion and activating CD4+ T cells against SARS-CoV-2 and its variants. (This study has been registered at ClinicalTrials.gov under no. NCT04992260.) IMPORTANCE This work evaluated the immune response induced by two doses of CoronaVac separated by 4 weeks in healthy children and adolescents in Chile. To date, few studies have described the effects of CoronaVac in the pediatric population. Therefore, it is essential to generate knowledge regarding the protection of vaccines in this population. Along these lines, we reported the anti-S humoral response and cellular immune response to several SARS-CoV-2 proteins that have been published and recently studied. Here, we show that a vaccination schedule consisting of two doses separated by 4 weeks induces the secretion of neutralizing antibodies against SARS-CoV-2. Furthermore, CoronaVac induces the activation of CD4+ T cells upon stimulation with peptides from the proteome of SARS-CoV-2. These results indicate that, even though the neutralizing antibody response induced by vaccination decreases against the Delta and Omicron variants, the cellular response against these variants is comparable to the response against the ancestral strain D614G, even being significantly higher against Omicron.
COVID-19 , SARS-CoV-2 , Adolescent , Humans , Child , Child, Preschool , Antibodies, Neutralizing , Vaccines, Inactivated , Antibodies, Viral
The lifetime of proteins in synapses is important for their signaling, maintenance, and remodeling, and for memory duration. We quantified the lifetime of endogenous PSD95, an abundant postsynaptic protein in excitatory synapses, at single-synapse resolution across the mouse brain and lifespan, generating the Protein Lifetime Synaptome Atlas. Excitatory synapses have a wide range of PSD95 lifetimes extending from hours to several months, with distinct spatial distributions in dendrites, neurons, and brain regions. Synapses with short protein lifetimes are enriched in young animals and in brain regions controlling innate behaviors, whereas synapses with long protein lifetimes accumulate during development, are enriched in the cortex and CA1 where memories are stored, and are preferentially preserved in old age. Synapse protein lifetime increases throughout the brain in a mouse model of autism and schizophrenia. Protein lifetime adds a further layer to synapse diversity and enriches prevailing concepts in brain development, aging, and disease.
Longevity , Synapses , Mice , Animals , Synapses/physiology , Neurons/physiology , Brain/physiology , Disks Large Homolog 4 Protein/metabolism
Increased levels of tumor-associated macrophages (TAMs) are indicators of poor prognosis in most cancers. Although antibodies and small molecules blocking the recruitment of macrophages to tumors are under evaluation as anticancer therapies, these strategies are not specific for macrophage subpopulations. Herein we report the first enzyme-activatable chemokine conjugates for effective targeting of defined macrophage subsets in live tumors. Our constructs exploit the high expression of chemokine receptors (e.g., CCR2) and the activity of cysteine cathepsins in TAMs to target these cells selectively over other macrophages and immune cells (e.g., neutrophils, T cells, B cells). Furthermore, we demonstrate that cathepsin-activatable chemokines are compatible with both fluorescent and therapeutic cargos, opening new avenues in the design of targeted theranostic probes for immune cells in the tumor microenvironment.
Cysteine , Tumor-Associated Macrophages , Cathepsins , Chemokines , Receptors, Chemokine , Tumor Microenvironment
Immunotherapy promotes the attack of cancer cells by the immune system; however, it is difficult to detect early responses before changes in tumor size occur. Here, we report the rational design of a fluorogenic peptide able to detect picomolar concentrations of active granzyme B as a biomarker of immune-mediated anticancer action. Through a series of chemical iterations and molecular dynamics simulations, we synthesize a library of FRET peptides and identify probe H5 with an optimal fit into granzyme B. We demonstrate that probe H5 enables the real-time detection of T cell-mediated anticancer activity in mouse tumors and in tumors from lung cancer patients. Furthermore, we show image-based phenotypic screens, which reveal that the AKT kinase inhibitor AZD5363 shows immune-mediated anticancer activity. The reactivity of probe H5 may enable the monitoring of early responses to anticancer treatments using tissue biopsies.
Immunotherapy , Lung Neoplasms , Animals , Biopsy , Granzymes , Humans , Lung Neoplasms/drug therapy , Mice , Peptides , Research
