GPCR targeting of E3 ubiquitin ligase MDM2 by inactive ß-arrestin.

E3 ubiquitin ligase Mdm2 facilitates ß-arrestin ubiquitination, leading to the internalization of G protein-coupled receptors (GPCRs). In this process, ß-arrestins bind to Mdm2 and recruit it to the receptor; however, the molecular architecture of the ß-arrestin-Mdm2 complex has not been elucidated yet. Here, we identified the ß-arrestin-binding region (ABR) on Mdm2 and solved the crystal structure of ß-arrestin1 in complex with Mdm2ABR peptide. The acidic residues of Mdm2ABR bind to the positively charged concave side of the ß-arrestin1 N-domain. The C-tail of ß-arrestin1 is still bound to the N-domain, indicating that Mdm2 binds to the inactive state of ß-arrestin1, whereas the phosphorylated C-terminal tail of GPCRs binds to activate ß-arrestins. The overlapped binding site of Mdm2 and GPCR C-tails on ß-arrestin1 suggests that the binding of GPCR C-tails might trigger the release of Mdm2. Moreover, hydrogen/deuterium exchange experiments further show that Mdm2ABR binding to ß-arrestin1 induces the interdomain interface to be more dynamic and uncouples the IP6-induced oligomer of ß-arrestin1. These results show how the E3 ligase, Mdm2, interacts with ß-arrestins to promote the internalization of GPCRs.

Building Pandemic-Resilient Primary Care Systems: Lessons Learned From COVID-19.

On January 30, 2023, the Biden Administration announced its intention to end the existing COVID-19 public health emergency declaration. The transition to a "postpandemic" landscape presents a unique opportunity to sustain and strengthen pandemic-era changes in care delivery. With this in mind, we present 3 critical lessons learned from a primary care perspective during the COVID-19 pandemic. First, clinical workflows must support both in-person and internet-based care delivery. Second, the integration of asynchronous care delivery is critical. Third, planning for the future means planning for everyone, including those with potentially limited access to health care due to barriers in technology and communication. While these lessons are neither unique to primary care settings nor all-encompassing, they establish a grounded foundation on which to construct higher-quality, more resilient, and more equitable health systems.

Structural and Functional Implication of Natural Variants of Gαs.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are among the most important cellular signaling components, especially G protein-coupled receptors (GPCRs). G proteins comprise three subunits, Gα, Gß, and Gγ. Gα is the key subunit, and its structural state regulates the active status of G proteins. Interaction of guanosine diphosphate (GDP) or guanosine triphosphate (GTP) with Gα switches G protein into basal or active states, respectively. Genetic alteration in Gα could be responsible for the development of various diseases due to its critical role in cell signaling. Specifically, loss-of-function mutations of Gαs are associated with parathyroid hormone-resistant syndrome such as inactivating parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) signaling disorders (iPPSDs), whereas gain-of-function mutations of Gαs are associated with McCune-Albright syndrome and tumor development. In the present study, we analyzed the structural and functional implications of natural variants of the Gαs subtype observed in iPPSDs. Although a few tested natural variants did not alter the structure and function of Gαs, others induced drastic conformational changes in Gαs, resulting in improper folding and aggregation of the proteins. Other natural variants induced only mild conformational changes but altered the GDP/GTP exchange kinetics. Therefore, the results shed light on the relationship between natural variants of Gα and iPPSDs.

A continuous-wave and pulsed X-band electron spin resonance spectrometer operating in ultra-high vacuum for the study of low dimensional spin ensembles.

We report the development of a continuous-wave and pulsed X-band electron spin resonance (ESR) spectrometer for the study of spins on ordered surfaces down to cryogenic temperatures. The spectrometer operates in ultra-high vacuum and utilizes a half-wavelength microstrip line resonator realized using epitaxially grown copper films on single crystal Al2O3 substrates. The one-dimensional microstrip line resonator exhibits a quality factor of more than 200 at room temperature, close to the upper limit determined by radiation losses. The surface characterizations of the copper strip of the resonator by atomic force microscopy, low-energy electron diffraction, and scanning tunneling microscopy show that the surface is atomically clean, flat, and single crystalline. Measuring the ESR spectrum at 15 K from a few nm thick molecular film of YPc2, we find a continuous-wave ESR sensitivity of 2.6 × 1011 spins/G · Hz1/2, indicating that a signal-to-noise ratio of 3.9 G · Hz1/2 is expected from a monolayer of YPc2 molecules. Advanced pulsed ESR experimental capabilities, including dynamical decoupling and electron-nuclear double resonance, are demonstrated using free radicals diluted in a glassy matrix.

Artificial Intelligence-Generated Draft Replies to Patient Inbox Messages.

Importance: The emergence and promise of generative artificial intelligence (AI) represent a turning point for health care. Rigorous evaluation of generative AI deployment in clinical practice is needed to inform strategic decision-making. Objective: To evaluate the implementation of a large language model used to draft responses to patient messages in the electronic inbox. Design, Setting, and Participants: A 5-week, prospective, single-group quality improvement study was conducted from July 10 through August 13, 2023, at a single academic medical center (Stanford Health Care). All attending physicians, advanced practice practitioners, clinic nurses, and clinical pharmacists from the Divisions of Primary Care and Gastroenterology and Hepatology were enrolled in the pilot. Intervention: Draft replies to patient portal messages generated by a Health Insurance Portability and Accountability Act-compliant electronic health record-integrated large language model. Main Outcomes and Measures: The primary outcome was AI-generated draft reply utilization as a percentage of total patient message replies. Secondary outcomes included changes in time measures and clinician experience as assessed by survey. Results: A total of 197 clinicians were enrolled in the pilot; 35 clinicians who were prepilot beta users, out of office, or not tied to a specific ambulatory clinic were excluded, leaving 162 clinicians included in the analysis. The survey analysis cohort consisted of 73 participants (45.1%) who completed both the presurvey and postsurvey. In gastroenterology and hepatology, there were 58 physicians and APPs and 10 nurses. In primary care, there were 83 physicians and APPs, 4 nurses, and 8 clinical pharmacists. The mean AI-generated draft response utilization rate across clinicians was 20%. There was no change in reply action time, write time, or read time between the prepilot and pilot periods. There were statistically significant reductions in the 4-item physician task load score derivative (mean [SD], 61.31 [17.23] presurvey vs 47.26 [17.11] postsurvey; paired difference, -13.87; 95% CI, -17.38 to -9.50; P < .001) and work exhaustion scores (mean [SD], 1.95 [0.79] presurvey vs 1.62 [0.68] postsurvey; paired difference, -0.33; 95% CI, -0.50 to -0.17; P < .001). Conclusions and Relevance: In this quality improvement study of an early implementation of generative AI, there was notable adoption, usability, and improvement in assessments of burden and burnout. There was no improvement in time. Further code-to-bedside testing is needed to guide future development and organizational strategy.

A New Cell Line Derived from the Caudal Fin of the Dwarf Gourami (Trichogaster lalius) and Its Susceptibility to Fish Viruses.

The detection of megalocytiviruses, especially the infectious spleen and kidney necrosis virus (ISKNV), in ornamental fish has increased with the rapid growth of the ornamental fish industry. In this study, dwarf gourami fin (DGF) cells derived from the caudal fin of the dwarf gourami (Trichogaster lalius), which is highly susceptible to red sea bream iridovirus (RSIV) and ISKNV, were established and characterized. The DGF cells were grown at temperatures ranging from 25 °C to 30 °C in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum and were subcultured for more than 100 passages, predominantly with epithelial-like cells. DGF cells had a diploid chromosome number of 2n = 44. Although the initial purpose of this study was to establish a cell line for the causative agents of red sea bream iridoviral disease (RSIV and ISKNV), DGF cells were also susceptible to rhabdoviruses (viral hemorrhagic septicemia virus, hirame rhabdovirus, and spring viraemia of carp virus), exhibiting a significant cytopathic effect characterized by cell rounding and lysis. Additionally, viral replication and virion morphology were confirmed using virus-specific conventional polymerase chain reaction and transmission electron microscopy. Furthermore, both RSIV and ISKNV were replicated at high concentrations in DGF cells compared to other cell lines. Notably, the DGF cells maintained a monolayer during ISKNV infection, indicating the possibility of persistent infection. Thus, DGF can be used for viral diagnosis and may play a critical role in advancing our understanding of ISKNV pathogenesis.

Fucoidan and topography modification improved in situ endothelialization on acellular synthetic vascular grafts.

Thrombogenesis remains the primary failure of synthetic vascular grafts. Endothelial coverage is crucial to provide an antithrombogenic surface. However, most synthetic materials do not support cell adhesion, and transanastomotic endothelial migration is limited. Here, a surface modification strategy using fucoidan and topography was developed to enable fast in situ endothelialization of polyvinyl alcohol, which is not endothelial cell-adhesive. Among three different immobilization approaches compared, conjugation of aminated-fucoidan promoted endothelial monolayer formation while minimizing thrombogenicity in both in vitro platelet rich plasma testing and ex vivo non-human primate shunt assay. Screening of six topographical patterns showed that 2 µm gratings increased endothelial cell migration without inducing inflammation responses of endothelial cells. Mechanistic studies demonstrated that fucoidan could attract fibronectin, enabling integrin binding and focal adhesion formation and activating focal adhesion kinase (FAK) signaling, and 2 µm gratings further enhanced FAK-mediated cell migration. In a clinically relevant rabbit carotid artery end-to-side anastomosis model, 60% in situ endothelialization was observed throughout the entire lumen of 1.7 mm inner diameter modified grafts, compared to 0% of unmodified graft, and the four-week graft patency also increased. This work presents a promising strategy to stimulate in situ endothelialization on synthetic materials for improving long-term performance.

In vivo evaluation of compliance mismatch on intimal hyperplasia formation in small diameter vascular grafts.

Small diameter synthetic vascular grafts have high failure rate due to the thrombosis and intimal hyperplasia formation. Compliance mismatch between the synthetic graft and native artery has been speculated to be one of the main causes of intimal hyperplasia. However, changing the compliance of synthetic materials without altering material chemistry remains a challenge. Here, we used poly(vinyl alcohol) (PVA) hydrogel as a graft material due to its biocompatibility and tunable mechanical properties to investigate the role of graft compliance in the development of intimal hyperplasia and in vivo patency. Two groups of PVA small diameter grafts with low compliance and high compliance were fabricated by dip casting method and implanted in a rabbit carotid artery end-to-side anastomosis model for 4 weeks. We demonstrated that the grafts with compliance that more closely matched with rabbit carotid artery had lower anastomotic intimal hyperplasia formation and higher graft patency compared to low compliance grafts. Overall, this study suggested that reducing the compliance mismatch between the native artery and vascular grafts is beneficial for reducing intimal hyperplasia formation.

Rabbit Surgery Protocol for End-to-End and End-to-Side Vascular Graft Anastomosis.

Preclinical testing in animal model is a required stage of vascular device development. Among small animal models, rabbits provide vasculature with relative larger caliber for anastomotic implantation of vascular grafts as preclinical testing before conducting large animal studies. Rabbits have similar hemostatic mechanism with human and can accommodate vascular grafts with various diameters at different locations, and thus provide a valid model to assess small-diameter vascular grafts. This chapter will describe the procedures and materials required to conduct survival surgery in rabbit carotid artery models for implantation of small-diameter tubular grafts with an end-to-side and end-to-end anastomotic technique.

Vascular Imaging in Small Animals Using Clinical Ultrasound Scanners.

Conventional ultrasound with frequency (2-15 MHz) has been a global diagnostic and therapeutic tool in clinical medicine, and high-frequency ultrasound (>30 MHz) has been a powerful investigative device for preclinical studies such as cardiovascular research. In this chapter, we describe the use of conventional ultrasound with a 2.5-10 MHz transducer as an investigative device for the measurement/detection of blood flow in rabbit model. The chapter will describe the procedures for the preparation of sonographer, imaging locations, and the details of the rabbits used as well as detailed imaging steps for the preoperative, immediately after operation, and postoperative follow-up ultrasound for vascular surgery, using a vascular graft implantation as an example. We also provide useful notes to avoid pitfalls for successful imaging. The overall goal of this chapter is to deliver the steps in using low-cost, non-invasive, and highly versatile clinical ultrasound imaging in preclinical small animal testing.

Changing compliance of poly(vinyl alcohol) tubular scaffold for vascular graft applications through modifying interlayer adhesion and crosslinking density.

Poly(vinyl alcohol) (PVA) is a water-soluble polymer and forms a hydrogel that has been studied as a potential small-diameter (<6 mm) vascular graft implant. The PVA hydrogel crosslinked using sodium trimetaphosphate (STMP) has been shown to have many beneficial properties such as bioinert, low-thrombogenicity, and easy surface modification. Compared to conventional synthetic vascular graft materials, PVA has also shown to possess better mechanical properties; however, the compliance and other mechanical properties of PVA grafts are yet to be optimized compared to the native blood vessels. Mechanical compliance has been an important parameter to be studied for small-diameter vascular grafts, as compliance has been proposed to play an important role in intimal hyperplasia formation. PVA grafts are made using dip-casting a cylindrical mold into crosslinking solution. The number of dipping can be used to control the wall thickness of the resulting PVA grafts. In this study, we hypothesized that the number of dip layer and wall thickness, the chemical crosslinking, and interlayer adhesive strength could be important parameters in the fabrication process that would affect compliance. This work provides the relationship between the wall thickness, burst pressure, and compliance of PVA. Furthermore, our data showed that interlayer adhesion as well as chemical and physical crosslinking density can increase the compliance of PVA grafts.

Current understanding of intimal hyperplasia and effect of compliance in synthetic small diameter vascular grafts.

Despite much effort, synthetic small diameter vascular grafts still face limited success due to vascular wall thickening known as intimal hyperplasia (IH). Compliance mismatch between graft and native vessels has been proposed to be one of a key mechanical factors of synthetic vascular grafts that could contribute to the formation of IH. While many methods have been developed to determine compliance both in vivo and in vitro, the effects of compliance mismatch still remain uncertain. This review aims to explain the biomechanical factors that are responsible for the formation and development of IH and their relationship with compliance mismatch. Furthermore, this review will address the current methods used to measure compliance both in vitro and in vivo. Lastly, current limitations in understanding the connection between the compliance of vascular grafts and the role it plays in the development and progression of IH will be discussed.

Calcitonin Receptor N-Glycosylation Enhances Peptide Hormone Affinity by Controlling Receptor Dynamics.

The class B G protein-coupled receptor (GPCR) calcitonin receptor (CTR) is a drug target for osteoporosis and diabetes. N-glycosylation of asparagine 130 in its extracellular domain (ECD) enhances calcitonin hormone affinity with the proximal GlcNAc residue mediating this effect through an unknown mechanism. Here, we present two crystal structures of salmon calcitonin-bound, GlcNAc-bearing CTR ECD at 1.78 and 2.85 Å resolutions and analyze the mechanism of the glycan effect. The N130 GlcNAc does not contact the hormone. Surprisingly, the structures are nearly identical to a structure of hormone-bound, N-glycan-free ECD, which suggested that the GlcNAc might affect CTR dynamics not observed in the static crystallographic snapshots. Hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations revealed that glycosylation stabilized a ß-sheet adjacent to the N130 GlcNAc and the N-terminal α-helix near the peptide-binding site while increasing flexibility of the peptide-binding site turret loop. These changes due to N-glycosylation increased the ligand on-rate and decreased its off-rate. The glycan effect extended to RAMP-CTR amylin receptor complexes and was also conserved in the related CGRP receptor. These results reveal that N-glycosylation can modulate GPCR function by altering receptor dynamics.

Sclerostin inhibits Wnt signaling through tandem interaction with two LRP6 ectodomains.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a coreceptor of the ß-catenin-dependent Wnt signaling pathway. The LRP6 ectodomain binds Wnt proteins, as well as Wnt inhibitors such as sclerostin (SOST), which negatively regulates Wnt signaling in osteocytes. Although LRP6 ectodomain 1 (E1) is known to interact with SOST, several unresolved questions remain, such as the reason why SOST binds to LRP6 E1E2 with higher affinity than to the E1 domain alone. Here, we present the crystal structure of the LRP6 E1E2-SOST complex with two interaction sites in tandem. The unexpected additional binding site was identified between the C-terminus of SOST and the LRP6 E2 domain. This interaction was confirmed by in vitro binding and cell-based signaling assays. Its functional significance was further demonstrated in vivo using Xenopus laevis embryos. Our results provide insights into the inhibitory mechanism of SOST on Wnt signaling.

Antilisterial Bacteriocin from Lactobacillus rhamnosus CJNU 0519 Presenting a Narrow Antimicrobial Spectrum.

A lactic acid bacterium presenting antimicrobial activity against a Lactobacillus acidophilus strain used for eradication of acid inhibition was isolated from a natural cheese. The 16S rRNA gene sequence of the isolate best matched with a strain of L. rhamnosus and was designated L. rhamnosus CJNU 0519. The antimicrobial activity of the partially purified bacteriocin of CJNU 0519 was abolished when treated with a protease, indicating the protein nature of the bacteriocin. The partially purified bacteriocin (rhamnocin 519) displayed a narrow antimicrobial activity against L. acidophilus, Listeria monocytogenes, and Staphylococcus aureus among several tested bacterial and yeast strains. Rhamnocin 519 in particular showed strong bactericidal action against L. monocytogenes.