Conjugation of a Reactive Thiol at the Nucleotide Binding Site for Site-Specific Antibody Functionalization

Described here is a UV photo-cross-linking method that utilizes the NBS (nucleotide binding site) for site-specific covalent functionalization of antibodies with reactive thiol moieties (UV-NBS^Thiol), while preserving antibody activity. By synthesizing an indole-3-butyric acid (IBA) conjugated version of cysteine we site-specifically photo-cross-linked a reactive thiol moiety to antibodies at the NBS. This thiol moiety can then be used as an orthogonally reactive location to conjugate various types of functional ligands that possess a thiol reactive group through disulfide bond formation or reaction with a maleimide functionalized ligand. Our results demonstrate the utility of the UV-NBS^Thiol method by successfully functionalizing a prostate specific antigen antibody (IgG^PSA) with IBA-Thiol and subsequent reaction with maleimide-fluorescein. An optimal UV energy of 0.5–1.5 J/cm² was determined to yield the most efficient photo-cross-linking and resulted in 1–1.5 conjugations per antibody while preserving antibody/antigen binding activity and Fc recognition. Utilizing the IBA-Thiol ligand allows for an efficient means of site-specifically conjugating UV sensitive functionalities to antibody NBS that would otherwise not have been amenable by the previously described UV-NBS photo-cross-linking method. The UV-NBS^Thiol conjugation strategy can be utilized in various diagnostic and therapeutic applications with nearly limitless potential for the preparation of site-specific covalent conjugation of affinity tags, fluorescent molecules, peptides, and chemotherapeutics to antibodies

Tumor-Derived Extracellular Vesicles Breach the Intact Blood???Brain Barrier via Transcytosis

The restrictive nature of the blood???brain barrier (BBB) creates a major challenge for brain drug delivery with current nanomedicines lacking the ability to cross the BBB. Extracellular vesicles (EVs) have been shown to contribute to the progression of a variety of brain diseases including metastatic brain cancer and have been suggested as promising therapeutics and drug delivery vehicles. However, the ability of native tumor-derived EVs to breach the BBB and the mechanism(s) involved in this process remain unknown. Here, we demonstrate that tumor-derived EVs can breach the intact BBB in vivo, and by using state-of-the-art in vitro and in vivo models of the BBB, we have identified transcytosis as the mechanism underlying this process. Moreover, high spatiotemporal resolution microscopy demonstrated that the endothelial recycling endocytic pathway is involved in this transcellular transport. We further identify and characterize the mechanism by which tumor-derived EVs circumvent the low physiologic rate of transcytosis in the BBB by decreasing the brain endothelial expression of rab7 and increasing the efficiency of their transport. These findings identify previously unknown mechanisms by which tumor-derived EVs breach an intact BBB during the course of brain metastasis and can be leveraged to guide and inform the development of drug delivery approaches to deliver therapeutic cargoes across the BBB for treatment of a variety of brain diseases including, but not limited to, brain malignancies

Prevalence of Anosmia in 10.157 Pediatric COVID-19 Cases: Multicenter Study from Turkey.

Introduction: COVID-19-related anosmia is a remarkable and disease-specific finding. With this multicenter cohort study, we aimed to determine the prevalence of anosmia in pediatric cases with COVID-19 from Turkey and make an objective assessment with a smell awareness questionnaire. Material and Methods: This multicenter prospective cohort study was conducted with pediatric infection clinics in 37 centers in 19 different cities of Turkey between October 2020 and March 2021. The symptoms of 10.157 COVID-19 cases 10-18 years old were examined. Age, gender, other accompanying symptoms, and clinical severity of the disease of cases with anosmia and ageusia included in the study were recorded. The cases were interviewed for the smell awareness questionnaire at admission and one month after the illness. Results: Anosmia was present in 12.5% (1.266/10.157) of COVID-19 cases 10-18 years of age. The complete records of 1053 patients followed during the study period were analyzed. The most common symptoms accompanying symptoms with anosmia were ageusia in 885 (84%) cases, fatigue in 534 cases (50.7%), and cough in 466 cases (44.3%). Anosmia was recorded as the only symptom in 84 (8%) of the cases. One month later, it was determined that anosmia persisted in 88 (8.4%) cases. In the smell awareness questionnaire, the score at admission was higher than the score one month later (P < 0.001). Discussion: With this study, we have provided the examination of a large case series across Turkey. Anosmia and ageusia are specific symptoms seen in cases of COVID-19. With the detection of these symptoms, it should be aimed to isolate COVID-19 cases in the early period and reduce the spread of the infection. Such studies are important because the course of COVID-19 in children differs from adults and there is limited data on the prevalence of anosmia