Visuo-Haptic VR and AR Guidance for Dental Nerve Block Education.

The inferior alveolar nerve block (IANB) is a dental anesthetic injection that is critical to the performance of many dental procedures. Dental students typically learn to administer an IANB through videos and practice on silicone molds and, in many dental schools, on other students. This causes significant stress for both the students and their early patients. To reduce discomfort and improve clinical outcomes, we created an anatomically informed virtual reality headset-based educational system for the IANB. It combines a layered 3D anatomical model, dynamic visual guidance for syringe position and orientation, and active force feedback to emulate syringe interaction with tissue. A companion mobile augmented reality application allows students to step through a visualization of the procedure on a phone or tablet. We conducted a user study to determine the advantages of preclinical training with our IANB simulator. We found that in comparison to dental students who were exposed only to traditional supplementary study materials, dental students who used our IANB simulator were more confident administering their first clinical injections, had less need for syringe readjustments, and had greater success in numbing patients.

Freedom, Rights, and Vaccine Refusal: The History of an Idea.

We analyzed how activists opposed to vaccination have used arguments related to freedom, liberty, and individual rights in US history. We focused on the period from the 1880s through the 1920s, when the first wave of widespread and sustained antivaccination activism in this country occurred. During this era, activists used the language of liberty and freedom most prominently in opposition to compulsory vaccination laws, which the activists alleged violated their constitutionally protected rights. Critics attacked vaccination with liberty-based arguments even when it was not mandatory, and they used the language of freedom expansively to encompass individuals' freedom to choose their health and medical practices, freedom to raise their children as they saw fit, and freedom from the quasicoercive influence of scientific and medical experts and elite institutions. Evidence suggests that in recent years, vaccine refusal has increasingly been framed as a civil right. We argue that this framing has always lain at the heart of resistance to vaccination and that it may prove consequential for the rollout of COVID-19 vaccines. (Am J Public Health. 2022;112(2):234-241. https://doi.org/10.2105/AJPH.2021.306504).

BCL-2 inhibitors sensitize therapy-resistant chronic lymphocytic leukemia cells to VSV oncolysis.

Many primary cancers including chronic lymphocytic leukemia (CLL) are resistant to vesicular stomatitis virus (VSV)-induced oncolysis due to overexpression of the antiapoptotic and antiautophagic members of the B-cell lymphoma-2 (BCL-2) family. In the present study, we investigated the mechanisms of CLL cell death induced as a consequence of VSV infection in the presence of BCL-2 inhibitors, obatoclax, and ABT-737 in primary ex vivo CLL patient samples. Microarray analysis of primary CD19⁺ CD5⁺ CLL cells treated with obatoclax and VSV revealed changes in expression of genes regulating apoptosis, the mechanistic target of rapamycin (mTOR) pathway, and cellular metabolism. A combined therapeutic effect was observed for VSV and BCL-2 inhibitors in cells from untreated patients and from patients unresponsive to standard of care therapy. In addition, combination treatment induced several markers of autophagy--LC3-II accumulation, p62 degradation, and staining of autophagic vacuoles. Inhibition of early stage autophagy using 3-methyladenine (3-MA) led to increased apoptosis in CLL samples. Mechanistically, a combination of BCL-2 inhibitors and VSV disrupted inhibitory interactions of Beclin-1 with BCL-2 and myeloid cell leukemia-1 (MCL-1), thus biasing cells toward autophagy. We propose a mechanism in which changes in cellular metabolism, coupled with pharmacologic disruption of the BCL-2-Beclin-1 interactions, facilitate induction of apoptosis and autophagy to mediate the cytolytic effect of VSV.

Linear ubiquitination of NEMO negatively regulates the interferon antiviral response through disruption of the MAVS-TRAF3 complex.

The RIG-I/Mda5 sensors recognize viral intracellular RNA and trigger host antiviral responses. RIG-I signals through the adaptor protein MAVS, which engages various TRAF family members and results in type I interferon (IFNs) and proinflammatory cytokine production via activation of IRFs and NF-κB, respectively. Both the IRF and NF-κB pathways also require the adaptor protein NEMO. We determined that the RIG-I pathway is differentially regulated by the linear ubiquitin assembly complex (LUBAC), which consists of the E3 ligases HOIL-1L, HOIP, and the accessory protein SHARPIN. LUBAC downregulated virus-mediated IFN induction by targeting NEMO for linear ubiquitination. Linear ubiquitinated NEMO associated with TRAF3 and disrupted the MAVS-TRAF3 complex, which inhibited IFN activation while stimulating NF-κB-dependent signaling. In SHARPIN-deficient MEFs, vesicular stomatitis virus replication was decreased due to increased IFN production. Linear ubiquitination thus switches NEMO from a positive to a negative regulator of RIG-I signaling, resulting in an attenuated IFN response.

VSV oncolysis in combination with the BCL-2 inhibitor obatoclax overcomes apoptosis resistance in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), overexpression of antiapoptotic B-cell leukemia/lymphoma 2 (BCL-2) family members contributes to leukemogenesis by interfering with apoptosis; BCL-2 expression also impairs vesicular stomatitis virus (VSV)-mediated oncolysis of primary CLL cells. In the effort to reverse resistance to VSV-mediated oncolysis, we combined VSV with obatoclax (GX15-070)-a small-molecule BCL-2 inhibitor currently in phase 2 clinical trials-and examined the molecular mechanisms governing the in vitro and in vivo antitumor efficiency of combining the two agents. In combination with VSV, obatoclax synergistically induced cell death in primary CLL samples and reduced tumor growth in severe combined immunodeficient (SCID) mice-bearing A20 lymphoma tumors. Mechanistically, the combination stimulated the mitochondrial apoptotic pathway, as reflected by caspase-3 and -9 cleavage, cytochrome c release and BAX translocation. Combination treatment triggered the release of BAX from BCL-2 and myeloid cell leukemia-1 (MCL-1) from BAK, whereas VSV infection induced NOXA expression and increased the formation of a novel BAX-NOXA heterodimer. Finally, NOXA was identified as an important inducer of VSV-obatoclax driven apoptosis via knockdown and overexpression of NOXA. These studies offer insight into the synergy between small-molecule BCL-2 inhibitors such as obatoclax and VSV as a combination strategy to overcome apoptosis resistance in CLL.

Phosphatidylinositol 3-kinase gamma is a critical mediator of myocardial ischemic and adenosine-mediated preconditioning.

Ischemic preconditioning (IPC) is a potent cellular protective mechanism whereby brief periods of sublethal ischemia protect the myocardium from prolonged ischemia-induced injury. We demonstrate the selective role of phosphatidylinositol 3-kinase (PI3K) isoforms in IPC. Hearts from PI3Kgamma knockout mice (PI3Kgamma(-/-)) displayed poorer functional recovery and greater tissue injury following IPC compared to wild-type and PI3Kgamma(+/-) hearts. Examination of the cell-signaling pathways revealed restored phosphorylation levels of Akt and glycogen synthase kinase (GSK)3beta in wild-type hearts, which were abolished in PI3Kgamma(-/-) hearts subjected to IPC. Inhibition of GSK3beta by LiCl reversed the loss in protection in PI3Kgamma(-/-) hearts. In contrast, mice expressing a cardiac-specific kinase-deleted PI3Kalpha (PI3KalphaDN) were resistant to injury induced by 30 minutes of ischemia followed by 40 minutes of reperfusion. Furthermore, the resistance of PI3KalphaDN hearts to ischemia/reperfusion correlated with the persistent expression of p110gamma and was blocked by the PI3K inhibitor wortmannin, suggesting the possible enhanced cell signaling through the PI3Kgamma pathway. These results demonstrate the importance of the PI3Kgamma-Akt-GSK3beta signaling pathway in IPC. Selective activation of myocardial PI3Kgamma may be an attractive target for the treatment of ischemic heart disease.

Phosphorylation of the human retinoid X receptor alpha at serine 260 impairs coactivator(s) recruitment and induces hormone resistance to multiple ligands.

The retinoid X receptor alpha (RXRalpha) is a member of the nuclear receptor superfamily that regulates transcription of target genes through heterodimerization with several partners, including peroxisome proliferator-activated receptor, retinoic acid receptor, thyroid receptor, and vitamin D receptor (VDR). We have shown previously that signaling through VDR.RXRalpha heterodimers was attenuated in ras-transformed keratinocytes due to phosphorylation of serine 260 of the RXRalpha via the activated Ras-Raf-MAPK cascade in these cells. In this study we demonstrate that phosphorylation at serine 260, a site located in the omega loop-AF-2 interacting domain of RXRalpha, inhibits signaling through several heterodimeric partners of the RXRalpha. The inhibition of signaling results in reduced transactivational response to ligand presentation and the reduced physiological response of growth inhibition not only of 1,25-dihydroxyvitamin D3 but also of retinoic acid receptor alpha ligands and LG1069 (an RXRalpha ligand). This partial resistance to ligands could be reversed by inhibition of MAPK activity or by overexpression of a non-phosphorylable RXRalpha mutant at serine 260 (RXRalpha Ser-260-->Ala). Importantly, phosphorylation of RXRalpha at serine 260 impaired the recruitment of DRIP205 and other coactivators to the VDR.RXRalpha complex. Chromatin immunoprecipitation and pulldown assays further demonstrated that coactivator recruitment to the VDR.RXR complex could be restored by treatment with a MAPK inhibitor. Our data suggest that phosphorylation at serine 260 plays a critical role in inducing hormone resistance of RXRalpha-mediated signaling likely through structural changes in the H1-H3 omega loop-AF2 coactivator(s) interacting domain.

Alternative splicing of the APC gene in the neural retina and retinal pigment epithelium.

PURPOSE: Hypertrophy and hyperplasia of the retinal pigment epithelium (RPE) is associated with an inherited predisposition to human familial adenomatous polyposis coli, suggesting that expression of the adenomatous polyposis coli (APC) tumor suppressor may regulate RPE proliferation/differentiation. Distinctive APC isoforms exist in different cell types due to alternative splicing of the APC transcripts. We hypothesize that differences in expression patterns of APC protein isoforms are critical to RPE proliferation/differentiation. METHODS: To investigate these relationships, APC gene expression was characterized in the retinas and RPE from fetal and adult human and mouse, and in the epiretinal membranes (ERM) from 5 patients with proliferative vitreoretinopathy (PVR). Expression patterns of alternative splice-forms of APC transcripts were evaluated by comparative quantitative RT-PCR. Exon 1 of APC encodes a heptad repeat that confers the ability of APC to homodimerize. APC protein isoforms containing or lacking this heptad were characterized by western blot analysis and immunohistochemistry. RESULTS: Comparative quantitative RT-PCR demonstrated a predominant exon 1 containing, conventional APC splice-form in the early developing fetal RPE and retina, and in all the tested ERM samples from patients with PVR. This method also demonstrated an increased level of exon 1 lacking APC splice-form in the mature RPE and retina. Western blot analysis and immunofluorescence microscopy demonstrated the conventional APC only in the RPE, and the APC isoform without the first heptad repeat in both the retina and RPE. Immunofluorescence microscopy also demonstrated only the conventional APC in the ERM samples tested. CONCLUSIONS: These results suggest that alternative splicing of APC leads to differential APC expression with potentially unique functions. APC isoform without the first heptad repeat may play a role in cell cycle cessation in the adult retina and RPE, and the down regulation of this APC isoform may contribute to the potential of RPE to migrate and proliferate.

HGF regulation of RPE proliferation in an IL-1beta/retinal hole-induced rabbit model of PVR.

PURPOSE: To understand molecular events that lead to retinal pigment epithelial (RPE) cell proliferation and migration during the early phases of proliferative vitreoretinopathy (PVR) in a rabbit model. METHODS: Retinal holes were created and interleukin-1beta(IL-1beta) was injected intravitreally. Eyes were examined by indirect ophthalmoscopy and eyecup pieces containing retinal holes were analyzed at different times after the surgery up to 4 weeks. RPE proliferation and migration were examined by immunohistochemistry. Tyrosine phosphorylation of extracellular signal regulated kinase (ERK) and hepatocyte growth factor receptor (HGFR or c-met) was determined by immunoprecipitation and western blot analysis. Tyrosine phosphorylation of c-met and morphological studies was performed on vitreous treated ARPE-19 cells. Expression of c-jun was determined by Northern blot analysis. Matrix metalloproteinase (MMP) content in vitreous was assessed by zymography. RESULTS: Indirect ophthalmoscopy identified formation of epiretinal membrane and immunohistochemistry identified proliferative and migratory RPE and other cells in the posterior segment containing retinal holes at 4 weeks post-surgery. Tyrosine phosphorylation of ERK and c-met occurred in this segment within 30 min of surgery. ARPE-19 cells treated with vitreous from the 24 h post-surgical eyes, but not with control vitreous or IL-1beta, showed morphological changes and tyrosine phosphorylation of c-met. Northern blot analysis in this segment identified upregulation of c-jun within 30 min of surgery and the expression peaked at 72 h. Zymographic analysis of vitreous identified MMP-9 in 12-72 h post-surgery. CONCLUSIONS: These data suggest that the presence of retinal holes and IL-1beta may lead to activation of HGF, mitogen activated protein kinases (MAPK), c-jun and extracellular matrix remodeling, resulting in proliferative and migratory cells in the wounded retina.

MAP kinase and beta-catenin signaling in HGF induced RPE migration.

PURPOSE: Hepatocyte growth factor (HGF) has been implicated in retinal pigment epithelial (RPE) cell proliferation and migration that occurs in proliferative retinal diseases such as proliferative vitreoretinopathy (PVR). The aim of this study is to investigate HGF induced signaling pathways that lead to RPE cell migration. METHODS: Localization of beta-catenin was determined by immunofluorescence. HGF induced migration of ARPE-19 cells was studied using a quantitative migration assay after wounding in the presence of a DNA polymerase inhibitor, and in the presence or absence of a mitogen activated protein kinase (MAP kinase) kinase inhibitor. C-jun expression was determined by semi-quantitative RT-PCR and by Northern blot analysis. P42/p44 MAP kinase activity was determined by western blot and by an immunoprecipitation kinase assay. Tyrosine phosphorylation of the HGF receptor (HGFR or c-met) and beta-catenin was determined by immunoprecipitation and western blot analysis. Transactivation activity of beta-catenin was determined by luciferase reporter gene analysis. RESULTS: Beta-catenin and E-cadherin were co-localized on the basal surface of the RPE in vivo. Diffusion of the cell surface-localized beta-catenin occurs in migratory cells in vitro in the presence of HGF. HGF induced a MAP kinase dependent ARPE-19 cell migration, which is accompanied with a transient increase of c-jun expression and concomitant increases of MAP kinase activity, tyrosine phosphorylation of HGFR and beta-catenin, increased cytosolic levels of beta-catenin, and transactivation activity of beta-catenin. Tyrosine phosphorylation of HGFR and beta-catenin occurs in the primary or passaged RPE cultures or proliferative ARPE-19 cells, but not freshly isolated RPE or differentiated ARPE-19 cells. CONCLUSIONS: This study defines the signal transduction pathways activated by HGF in RPE cells, leading to an increase in the MAP kinase activity and free pool of beta-catenin, and changes in gene expression. These findings are consistent with the hypothesis that both beta-catenin and MAP kinases are components of the HGF induced RPE migration that occurs in proliferative retinal diseases.

Effects of sustained hyperoxia on revascularization in experimental retinopathy of prematurity.

PURPOSE: To investigate the effects of prolonged hyperoxia on vascular recovery and glia survival after experimentally induced retinopathy of prematurity (ROP) in the mouse. METHODS: The effects of hyperoxia on revascularization and vitreous neovascularization were compared between mice raised in 75% oxygen from postnatal day (P)7 to P12, followed by room air recovery and mice raised in 75% oxygen from P7 to P27. The status of astrocytes and Müller cells was evaluated by glial fibrillary acidic protein (GFAP) immunohistochemistry on retinal wholemounts and serial sections. A window of susceptibility to oxygen-induced vaso-obliteration was defined by comparing the extent of retinal vaso-obliteration resulting from 2 days of hyperoxia beginning on P7, P9, P11, P13, or P15. RESULTS: Oxygen-induced vaso-obliteration of retinal capillaries was limited to the period between birth and P15. Paradoxically, revascularization was markedly accelerated and neovascularization markedly reduced in mice maintained in prolonged hyperoxia (P7-P27) compared with mice recovering in room air. The extended use of 75% oxygen during the recovery period was associated with preservation of astrocytes and Müller cells in the avascular retina. CONCLUSIONS: The antiangiogenic effect of hyperoxia on retinal capillaries is strongly dependent on postnatal age. A protocol of continuous 75% supplemental oxygen accelerates recovery of inner retinal vasculature and prevents vitreous neovascularization, by a mechanism that may involve preservation of inner retinal glia.