Machine learning-driven multifunctional peptide engineering for sustained ocular drug delivery.

Sustained drug delivery strategies have many potential benefits for treating a range of diseases, particularly chronic diseases that require treatment for years. For many chronic ocular diseases, patient adherence to eye drop dosing regimens and the need for frequent intraocular injections are significant barriers to effective disease management. Here, we utilize peptide engineering to impart melanin binding properties to peptide-drug conjugates to act as a sustained-release depot in the eye. We develop a super learning-based methodology to engineer multifunctional peptides that efficiently enter cells, bind to melanin, and have low cytotoxicity. When the lead multifunctional peptide (HR97) is conjugated to brimonidine, an intraocular pressure lowering drug that is prescribed for three times per day topical dosing, intraocular pressure reduction is observed for up to 18 days after a single intracameral injection in rabbits. Further, the cumulative intraocular pressure lowering effect increases ~17-fold compared to free brimonidine injection. Engineered multifunctional peptide-drug conjugates are a promising approach for providing sustained therapeutic delivery in the eye and beyond.

Data resource: vasopressin-regulated protein phosphorylation sites in the collecting duct.

Vasopressin controls renal water excretion through actions to regulate aquaporin-2 (AQP2) trafficking, transcription, and degradation. These actions are in part dependent on vasopressin-induced phosphorylation changes in collecting duct cells. Although most efforts have focused on the phosphorylation of AQP2 itself, phosphoproteomic studies have identified many vasopressin-regulated phosphorylation sites in proteins other than AQP2. The goal of this bioinformatics-based review is to create a compendium of vasopressin-regulated phosphorylation sites with a focus on those that are seen in both native rat inner medullary collecting ducts and cultured collecting duct cells from the mouse (mpkCCD), arguing that these sites are the best candidates for roles in AQP2 regulation. This analysis identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. We provide resource web pages at https://esbl.nhlbi.nih.gov/Databases/AVP-Phos/ and https://esbl.nhlbi.nih.gov/AVP-Network/, listing the phosphorylation sites and describing annotated functions of each of the vasopressin-targeted phosphoproteins. Among these sites are 23 consensus protein kinase A (PKA) sites that are increased in response to vasopressin, consistent with a central role for PKA in vasopressin signaling. The remaining sites are predicted to be phosphorylated by other kinases, most notably ERK1/2, which accounts for decreased phosphorylation at sites with a X-p(S/T)-P-X motif. Additional protein kinases that undergo vasopressin-induced changes in phosphorylation are Camkk2, Cdk18, Erbb3, Mink1, and Src, which also may be activated directly or indirectly by PKA. The regulated phosphoproteins are mapped to processes that hypothetically can account for vasopressin-mediated control of AQP2 trafficking, cytoskeletal alterations, and Aqp2 gene expression, providing grist for future studies.NEW & NOTEWORTHY Vasopressin regulates renal water excretion through control of the aquaporin-2 water channel in collecting duct cells. Studies of vasopressin-induced protein phosphorylation have focused mainly on the phosphorylation of aquaporin-2. This study describes 44 phosphoproteins other than aquaporin-2 that undergo vasopressin-mediated phosphorylation changes and summarizes potential physiological roles of each.

Bayesian analysis of dynamic phosphoproteomic data identifies protein kinases mediating GPCR responses.

BACKGROUND: A major goal in the discovery of cellular signaling networks is to identify regulated phosphorylation sites ("phosphosites") and map them to the responsible protein kinases. The V2 vasopressin receptor is a G-protein coupled receptor (GPCR) that is responsible for regulation of renal water excretion through control of aquaporin-2-mediated osmotic water transport in kidney collecting duct cells. Genome editing experiments have demonstrated that virtually all vasopressin-triggered phosphorylation changes are dependent on protein kinase A (PKA), but events downstream from PKA are still obscure. METHODS: Here, we used: 1) Tandem mass tag-based quantitative phosphoproteomics to experimentally track phosphorylation changes over time in native collecting ducts isolated from rat kidneys; 2) a clustering algorithm to classify time course data based on abundance changes and the amino acid sequences surrounding the phosphosites; and 3) Bayes' Theorem to integrate the dynamic phosphorylation data with multiple prior "omic" data sets covering expression, subcellular location, known kinase activity, and characteristic surrounding sequences to identify a set of protein kinases that are regulated secondary to PKA activation. RESULTS: Phosphoproteomic studies revealed 185 phosphosites regulated by vasopressin over 15 min. The resulting groups from the cluster algorithm were integrated with Bayes' Theorem to produce corresponding ranked lists of kinases likely responsible for each group. The top kinases establish three PKA-dependent protein kinase modules whose regulation mediate the physiological effects of vasopressin at a cellular level. The three modules are 1) a pathway involving several Rho/Rac/Cdc42-dependent protein kinases that control actin cytoskeleton dynamics; 2) mitogen-activated protein kinase and cyclin-dependent kinase pathways that control cell proliferation; and 3) calcium/calmodulin-dependent signaling. CONCLUSIONS: Our findings identify a novel set of downstream small GTPase effectors and calcium/calmodulin-dependent kinases with potential roles in the regulation of water permeability through actin cytoskeleton rearrangement and aquaporin-2 trafficking. The proposed signaling network provides a stronger hypothesis for the kinases mediating V2 vasopressin receptor responses, encouraging future targeted examination via reductionist approaches. Furthermore, the Bayesian analysis described here provides a template for investigating signaling via other biological systems and GPCRs. Video abstract.

Caregiver Preferences for Three-Dimensional Printed or Augmented Reality Craniosynostosis Skull Models: A Cross-Sectional Survey.

BACKGROUND: Recent advances in three-dimensional (3D) printing and augmented reality (AR) have expanded anatomical modeling possibilities for caregiver craniosynostosis education. The purpose of this study is to characterize caregiver preferences regarding these visual models and determine the impact of these models on caregiver understanding of craniosynostosis. METHODS: The authors constructed 3D-printed and AR craniosynostosis models, which were randomly presented in a cross-sectional survey. Caregivers rated each model's utility in learning about craniosynostosis, learning about skull anatomy, viewing an abnormal head shape, easing anxiety, and increasing trust in the surgeon in comparison to a two-dimensional (2D) diagram. Furthermore, caregivers were asked to identify the fused suture on each model and indicate their preference for generic versus patient-specific models. RESULTS: A total of 412 craniosynostosis caregivers completed the survey (mean age 33 years, 56% Caucasian, 51% male). Caregivers preferred interactive, patient-specific 3D-printed or AR models over 2D diagrams (mean score difference 3D-printed to 2D: 0.16, P < 0.05; mean score difference AR to 2D: 0.17, P < 0.01) for learning about craniosynostosis, with no significant difference in preference between 3D-printed and AR models. Caregiver detection accuracy of the fused suture on the sagittal model was 19% higher with the 3D-printed model than with the AR model (P < 0.05) and 17% higher with the 3D-printed model than with the 2D diagram (P < 0.05). CONCLUSIONS: Our findings indicate that craniosynostosis caregivers prefer 3D-printed or AR models over 2D diagrams in learning about craniosynostosis. Future craniosynostosis skull models with increased user interactivity and patient-specific components can better suit caregiver preferences.

"ADPKD-omics": determinants of cyclic AMP levels in renal epithelial cells.

The regulation of cyclic adenosine monophosphate (cAMP) levels in kidney epithelial cells is important in at least 2 groups of disorders, namely water balance disorders and autosomal dominant polycystic kidney disease. Focusing on the latter, we review genes that code for proteins that are determinants of cAMP levels in cells. We identify which of these determinants are expressed in the 14 kidney tubule segments using recently published RNA-sequencing and protein mass spectrometry data ("autosomal dominant polycystic kidney disease-omics"). This includes G protein-coupled receptors, adenylyl cyclases, cyclic nucleotide phosphodiesterases, cAMP transporters, cAMP-binding proteins, regulator of G protein-signaling proteins, G protein-coupled receptor kinases, arrestins, calcium transporters, and calcium-binding proteins. In addition, compartmentalized cAMP signaling in the primary cilium is discussed, and a specialized database of the proteome of the primary cilium of cultured "IMCD3" cells is provided as an online resource (https://esbl.nhlbi.nih.gov/Databases/CiliumProteome/). Overall, this article provides a general resource in the form of a curated list of proteins likely to play roles in determination of cAMP levels in kidney epithelial cells and, therefore, likely to be determinants of progression of autosomal dominant polycystic kidney disease.

Strategy to enhance dendritic cell-mediated DNA vaccination in the lung.

We here introduce a new paradigm to promote pulmonary DNA vaccination. Specifically, we demonstrate that nanoparticles designed to rapidly penetrate airway mucus (mucus-penetrating particle or MPP) enhance the delivery of inhaled model DNA vaccine (i.e. ovalbumin-expressing plasmids) to pulmonary dendritic cells (DC), leading to robust and durable local and trans-mucosal immunity. In contrast, mucus-impermeable particles were poorly taken up by pulmonary DC following inhalation, despite their superior ability to mediate DC uptake in vitro compared to MPP. In addition to the enhanced immunity achieved in mucosal surfaces, inhaled MPP unexpectedly provided significantly greater systemic immune responses compared to gold-standard approaches applied in the clinic for systemic vaccination, including intradermal injection and intramuscular electroporation. We also showed here that inhaled MPP significantly enhanced the survival of an orthotopic mouse model of aggressive lung cancer compared to the gold-standard approaches. Importantly, we discovered that MPP-mediated pulmonary DNA vaccination induced memory T-cell immunity, particularly the ready-to-act effector memory-biased phenotype, both locally and systemically. The findings here underscore the importance of breaching the airway mucus barrier to facilitate DNA vaccine uptake by pulmonary DC and thus to initiate full-blown immune responses.