Glycosylation of rhodopsin is necessary for its stability and incorporation into photoreceptor outer segment discs.

Rhodopsin, a G-protein coupled receptor, most abundant protein in retinal rod photoreceptors, is glycosylated at asparagines-2 and 15 on its N-terminus. To understand the role of rhodopsin's glycosylation in vivo, we generated and characterized a transgenic mouse model that expresses a non-glycosylated form of rhodopsin. We show that lack of glycosylation triggers a dominant form of progressive retinal degeneration. Electron microscopic examination of retinas at postnatal day 17 revealed the presence of vacuolar structures that distorted rod photoreceptor outer segments and became more prominent with age. Expression of non-glycosylated rhodopsin alone showed that it is unstable and is regulated via ubiquitin-mediated proteasomal degradation at the base of outer segments. We observed similar vacuolization in outer segments of transgenic mice expressing human rhodopsin with a T17M mutation (hT17M), suggesting that the mechanism responsible for the degenerative process in mice expressing the non-glycosylated rhodopsin and the RHO(hT17M) mice is likely the cause of phenotype observed in retinitis pigmentosa patients carrying T17M mutation.

Virtual simulated patient encounters: Bridging the gap in maternal mental health training for prelicensure nursing students.

Postpartum mood disorders (PPMDs) can result in significant negative impacts for the mother, baby, and family. Nurses can be trained to effectively screen for and educate patients about PPMDs. However, available data suggest that clinical opportunities for practicing these important conversations in real-time can be limited. Focused practice discussing PPMDs with patients may improve a nurse's confidence to conduct screenings and help reduce stigma regarding PPMDs. When in-person clinical learning opportunities are limited, simulated patient encounters can serve as useful and accessible alternatives. Though traditional simulations are often conducted in-person with an actor, they can also be offered to students via a virtual platform. This article describes the creation of two virtual simulated patient encounters (vSPEs) designed to improve prelicensure nursing students' confidence in screening and caring for patients at risk for PPMDs. These vSPEs were implemented during the COVID-19 pandemic to fulfill required labor and birth/postpartum clinical rotation hours. A thorough description of the vSPE design and implementation is provided to encourage nursing and other health professions educators to explore a virtual approach to training students to screen and care for postpartum patients with suspected PPMDs.

COVID-19 vaccine hesitancy and intent in California registered nurses.

Despite a national vaccination effort prioritizing frontline healthcare workers, COVID-19 vaccination rates among nurses have been lower than necessary to protect workforce and patient health. Historically, nurses have been more vaccine hesitant than other healthcare workers. To assess the vaccine attitudes and COVID-19 vaccine intent of California's registered nurses, we conducted a statewide cross-sectional survey among 603 licensed RNs working in direct patient care. Of 167 respondents (27.7%), 111 met inclusion criteria. Their mean score of 3.01 on a 6-point rating scale on the Vaccine Attitudes Examination scale measuring general vaccine hesitancy was comparable to previous findings among U.S. West Coast adults. Greater vaccine hesitancy was significantly associated with lower COVID-19 vaccine intent, after controlling for relevant confounders. Since nurses make up the largest portion of the healthcare workforce, it is crucial to specifically address this group's vaccine hesitancy.

p53 selectively regulates developmental apoptosis of rod photoreceptors.

Retinal cells become post-mitotic early during post-natal development. It is likely that p53, a well-known cell cycle regulator, is involved in regulating the genesis, differentiation and death of retinal cells. Furthermore, retinal cells are under constant oxidative stress that can result in DNA damage, due to the extremely high level of metabolic activity associated with phototransduction. If not repaired, this damage may result in p53-dependent cell death and ensuing vision loss. In this study, the role of p53 during retinal development and in the post-mitotic retina is investigated. A previously described super p53 transgenic mouse that expresses an extra copy of the mouse p53 gene driven by its endogenous promoter is utilized. Another transgenic mouse (HIP) that expresses the p53 gene in rod and cone photoreceptors driven by the human interphotoreceptor retinoid binding protein promoter was generated. The electroretinogram (ERG) of the super p53 mouse exhibited reduced rod-driven scotopic a and b wave and cone-driven photopic b wave responses. This deficit resulted from a reduced number of rod photoreceptors and inner nuclear layer cells. However, the reduced photopic signal arose only from lost inner retinal neurons, as cone numbers did not change. Furthermore, cell loss was non-progressive and resulted from increased apoptosis during retinal developmental as determined by TUNEL staining. In contrast, the continuous and specific expression of p53 in rod and cone photoreceptors in the mature retinas of HIP mice led to the selective loss of both rods and cones. These findings strongly support a role for p53 in regulating developmental apoptosis in the retina and suggest a potential role, either direct or indirect, for p53 in the degenerative photoreceptor loss associated with human blinding disorders.

Expression and role of p53 in the retina.

Since it was identified in 1979, p53 has been widely studied for its role in tumor suppression. It is mutated in approximately half of all human cancers, leading to aberrant cell growth. In addition to its role as a tumor suppressor, p53 is activated in response to various cell stress signals, including DNA damage and hypoxia. This activation leads to alterations in target gene expression, giving p53 a regulatory role in diverse cellular functions such as apoptosis, senescence, and cell cycle arrest. Throughout life, the eye is exposed to a multitude of stressors including disease, light-induced damage, and oxidative stress, all of which can lead to debilitating loss of vision. This article examines the role of p53 during ocular development. Finally, the role of p53 is examined in ocular response to intense light exposure, ionizing radiation, oxidative stress, degenerative disorders, and retinoblastoma.

Pattern of expression of p53, its family members, and regulators during early ocular development and in the post-mitotic retina.

PURPOSE: Because of its role in cell cycle regulation and apoptosis, p53 may be involved in maintaining the post-mitotic state of the adult eye. To shed light on the role of p53 in retinal development and maintenance, this study investigated the pattern of expression of p53, its family members, and its regulators during the development of the mouse eye. METHODS: Relative quantitative real-time PCR (qRT-PCR) was used to determine the steady-state levels of target transcripts in RNA extracted from wild-type mouse whole eyes or retinas between embryonic day (E) 15 and post-natal day (P) 30. Immunoblotting was used to compare the steady-state levels of the protein to that of the transcript. RESULTS: Transcript and protein levels for p53 in the eye were highest at E17 and E18, respectively. However, both p53 transcript and protein levels dropped precipitously thereafter, and no protein was detected on immunoblots after P3. Expression patterns of p63, p73, Mdm2, Mdm4, and Yy1 did not follow that of p53. Immunohistochemistry analysis of the developing eye showed that both p53 and Mdm2 are abundantly expressed at E18 in all layers of the retinal neuroblast. CONCLUSIONS: Downregulation of p53 in the post-mitotic retina suggests that, although p53 may be involved in ocular and retinal development, it may play a minimal role in healthy adult retinal function.

Treatment of autoimmune inflammation by a TLR7 ligand regulating the innate immune system.

The Toll-like receptors (TLR) have been advocated as attractive therapeutic targets because TLR signaling plays dual roles in initiating adaptive immune responses and perpetuating inflammation. Paradoxically, repeated stimulation of bone marrow mononuclear cells with a synthetic TLR7 ligand 9-benzyl-8-hydroxy-2-(2-methoxyethoxy) adenine (called 1V136) leads to subsequent TLR hyporesponsiveness. Further studies on the mechanism of action of this pharmacologic agent demonstrated that the TLR7 ligand treatment depressed dendritic cell activation, but did not directly affect T cell function. To verify this mechanism, we utilized experimental allergic encephalitis (EAE) as an in vivo T cell dependent autoimmune model. Drug treated SJL/J mice immunized with proteolipid protein (PLP)(139-151) peptide had attenuated disease severity, reduced accumulation of mononuclear cells in the central nervous system (CNS), and limited demyelination, without any apparent systemic toxicity. Splenic T cells from treated mice produced less cytokines upon antigenic rechallenge. In the spinal cords of 1V136-treated EAE mice, the expression of chemoattractants was also reduced, suggesting innate immune cell hyposensitization in the CNS. Indeed, systemic 1V136 did penetrate the CNS. These experiments indicated that repeated doses of a TLR7 ligand may desensitize dendritic cells in lymphoid organs, leading to diminished T cell responses. This treatment strategy might be a new modality to treat T cell mediated autoimmune diseases.

Selecting optimum eukaryotic integral membrane proteins for structure determination by rapid expression and solubilization screening.

A medium-throughput approach is used to rapidly identify membrane proteins from a eukaryotic organism that are most amenable to expression in amounts and quality adequate to support structure determination. The goal was to expand knowledge of new membrane protein structures based on proteome-wide coverage. In the first phase, membrane proteins from the budding yeast Saccharomyces cerevisiae were selected for homologous expression in S. cerevisiae, a system that can be adapted to expression of membrane proteins from other eukaryotes. We performed medium-scale expression and solubilization tests on 351 rationally selected membrane proteins from S. cerevisiae. These targets are inclusive of all annotated and unannotated membrane protein families within the organism's membrane proteome. Two hundred seventy-two targets were expressed, and of these, 234 solubilized in the detergent n-dodecyl-beta-D-maltopyranoside. Furthermore, we report the identity of a subset of targets that were purified to homogeneity to facilitate structure determinations. The extensibility of this approach is demonstrated with the expression of 10 human integral membrane proteins from the solute carrier superfamily. This discovery-oriented pipeline provides an efficient way to select proteins from particular membrane protein classes, families, or organisms that may be more suited to structure analysis than others.