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1.
Otolaryngol Head Neck Surg ; 170(4): 1190-1194, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38230445

RESUMO

Robotic-assisted surgery has gained popularity for otolaryngology procedures. It provides high-definition images and surgical precision to perform diverse procedures. It is an alternative to the operating microscope, endoscope, or exoscope when reaching hidden anatomical structures in the ear. In this proof-of-concept study, we aim to demonstrate the possibility of using a robotic-assisted device to perform ear surgery in conjunction with the microscope or the endoscope. In total, there were 9 ear and lateral skull base procedures performed with the use of robotic-assisted surgery. All surgeons underwent surveys to assess the performance and workload of the device compared to the microscope or endoscope. There were no postoperative complications. Robotic-assisted surgery was optimal for providing high image quality, ergonomics, and maintaining surgical performance. The size of the device and mental demand were higher compared to the microscope or endoscope. Robotic-assisted surgery can be an adjuvant to perform otologic and neurotologic surgery.


Assuntos
Procedimentos Cirúrgicos Otológicos , Procedimentos Cirúrgicos Robóticos , Humanos , Procedimentos Cirúrgicos Robóticos/métodos , Procedimentos Neurocirúrgicos/métodos , Base do Crânio/cirurgia , Endoscópios , Procedimentos Cirúrgicos Otológicos/métodos
2.
PLoS Pathog ; 19(10): e1011682, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37782657

RESUMO

Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34+ hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34+ hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 -RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics.


Assuntos
Citomegalovirus , Transdução de Sinais , Animais , Camundongos , Humanos , Citomegalovirus/fisiologia , Latência Viral , Diferenciação Celular , Células-Tronco Hematopoéticas
3.
Curr Opin Chem Biol ; 77: 102402, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37801755

RESUMO

PARPs encompass a small yet pervasive group of 17 enzymes that catalyze a post-translational modification known as ADP-ribosylation. PARP1, the founding member, has received considerable focus; however, in recent years, the spotlight has shifted to other members within the PARP family. In this opinion piece, we first discuss surprising findings that some FDA-approved PARP1 inhibitors activate innate immune signaling in cancer cells that harbor mutations in the DNA repair pathway. We then discuss hot-off-the-press genetic and pharmacological studies that reveal roles for PARP7, PARP11, and PARP14 in immune signaling in both tumor cells and tumor-associated immune cells. We conclude with thoughts on tuning PARP1-inhibitor-mediated innate immune activation and explore the unrealized potential for small molecule modulators of other PARP family members as next-generation immuno-oncology drugs.


Assuntos
Adenosina Difosfato Ribose , Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Adenosina Difosfato Ribose/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Microambiente Tumoral , Processamento de Proteína Pós-Traducional
4.
Artigo em Inglês | MEDLINE | ID: mdl-37822130

RESUMO

Prior work has demonstrated improved accuracy in otitis media diagnosis based on otoscopy using artificial intelligence (AI)-based approaches compared to clinician evaluation. However, this difference in accuracy has not been shown in a setting resembling the point-of-care. In this study, we compare the diagnostic accuracy of a machine-learning model to that of pediatricians using standard handheld otoscopes. We find that the model is more accurate than clinicians (90.6% vs 59.4%, P = .01). This is a step towards validation of AI-based diagnosis under more real-world conditions. With further validation, for example on different patient populations and in deployment, this technology could be a useful addition to the clinician's toolbox in accurately diagnosing otitis media.

5.
Neuropsychologia ; 191: 108650, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-37517462

RESUMO

Relations among behavioral, psychological, and electrophysiological correlates of Linguistic Empathy were examined in two experiments using lateralized stimuli. Linguistic Empathy is defined as a linguistic manifestation of the point of view the speaker assumes toward the content of the utterance, and of the speaker's attitude toward/identification with the referents therein. Linguistic choices made by the speaker among multiple logically and referentially synonymous lexical and grammatical options reveal the speaker's perspectives. In experiment 1, acceptability ratings were measured for Context-Target sentence pairs that did or did not violate two Empathy Hierarchies (Person Empathy Hierarchy and Topic Empathy Hierarchy); the Empathy Quotient (EQ) test of Psychological Empathy was also administered. Ratings were lower for sentence pairs that violated both hierarchies than for those violating neither and were intermediate for sentences violating only one hierarchy. Linguistic Empathy (LE) was operationalized as the difference in ratings between sentences violating both vs. neither empathy hierarchy; this measure correlated positively with EQ. Experiment 2 replicated those results with new participants and measured reaction time and EEG during ratings. While there were no effects of hemisphere or visual field on the linguistic variables, the amplitude of a positive event-related potential deflection at 380 ms provided a partial electrophysiological correlate for LE. Its difference measure correlated with behavioral LE but not with EQ. Though preliminary, these experiments show that Linguistic Empathy may share information processing computations with Psychological Empathy and have an electrophysiological correlate.


Assuntos
Empatia , Linguística , Humanos , Idioma , Potenciais Evocados/fisiologia
6.
Am J Otolaryngol ; 44(6): 103971, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37437333

RESUMO

OBJECTIVE: To assess the impact of an education specialist in a multidisciplinary pediatric hearing loss clinic. STUDY DESIGN: Retrospective review and cross-sectional survey. SETTING: Single tertiary care center. METHODS: Consultations held between an education specialist and families of pediatric deaf or hard of hearing (DHH) children within a two-year period were reviewed. Reasons for referral and services provided to each patient and family who subsequently worked with the educational specialist were assessed. Parents of patients who had previously worked with the education specialist were invited to complete a survey evaluating their experience. RESULTS: 102 patients were referred to the educational specialist in a two-year period. Most common reasons for referral included need for special education plans to accommodate their hearing deficit (32) or family request to support for revisions to such plans (37). 14 patient families completed our survey. 76.9 % of respondents confirmed that the education specialist recommended resources they had not been introduced to before. Given a scale of 1 ("completely dissatisfied") and 10 being "completely satisfied," the average rating of the 14 respondents was 9.0. CONCLUSION: The role of an education specialist in a pediatric hearing loss clinic is to optimize patient and family access to resources that could benefit their DHH child's academic development over time. Future studies should prospectively investigate the impact of education specialist services on the educational progress of DHH patients compared to outcomes without these supports.


Assuntos
Surdez , Perda Auditiva , Criança , Humanos , Estudos Retrospectivos , Estudos Transversais , Perda Auditiva/terapia , Pais
7.
Otol Neurotol ; 44(5): e273-e280, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37167444

RESUMO

OBJECTIVE: To determine the relationship between hearing loss etiology, cochlear implant (CI) programming levels, and speech perception performance in a large clinical cohort of pediatric CI recipients. STUDY DESIGN: Retrospective chart review. SETTING: Tertiary care hospitals. PATIENTS: A total of 136 pediatric CI recipients (218 ears) were included in this study. All patients had diagnoses of either enlarged vestibular aqueduct (EVA) or GJB2 (Connexin-26) mutation confirmed via radiographic data and/or genetic reports. All patients received audiologic care at either Boston Children's Hospital or Massachusetts Eye and Ear in Boston, MA, between the years 1999 and 2020. MAIN OUTCOME MEASURES: Electrode impedances and programming levels for each active electrode and speech perception scores were evaluated as a function of etiology (EVA or GJB2 mutation). RESULTS: Children with EVA had significantly higher impedances and programming levels (thresholds and upper stimulation levels) than the children with GJB2 mutation. Speech perception scores did not differ as a function of etiology in this sample; rather, they were positively correlated with duration of CI experience (time since implantation). CONCLUSIONS: Differences in electrode impedances and CI programming levels suggest that the electrode-neuron interface varies systematically as a function of hearing loss etiology in pediatric CI recipients with EVA and those with GJB2 mutation. Time with the CI was a better predictor of speech perception scores than etiology, suggesting that children can adapt to CI stimulation with experience.


Assuntos
Implante Coclear , Implantes Cocleares , Surdez , Perda Auditiva Neurossensorial , Percepção da Fala , Aqueduto Vestibular , Criança , Humanos , Conexinas/genética , Surdez/genética , Surdez/cirurgia , Perda Auditiva Neurossensorial/cirurgia , Mutação , Estudos Retrospectivos , Aqueduto Vestibular/cirurgia
8.
Int J Pediatr Otorhinolaryngol ; 170: 111598, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37207373

RESUMO

INTRODUCTION: The COVID-19 pandemic has caused unexpected disruptions in patient care, including adherence to the Early Hearing Detection and Intervention (EHDI) 1-3-6 guidelines. These guidelines mandate newborn hearing screening (NHS) by 1 month of age, diagnosis of hearing loss (HL) by 3 months, and referral to Early Intervention by 6 months. The objective of this study was to investigate the impact of COVID-19 on EHDI benchmarks in a major US city to help clinicians address current needs and prepare for future disruptive events. METHODS: Retrospective review was performed for all patients who did not pass NHS at two tertiary care centers between March 2018 and March 2022. Patients were divided into three cohorts based on the periods of time before, during, and after the COVID-19 Massachusetts State of Emergency (SOE). Demographics, medical history, NHS results, Auditory Brainstem Response results, and hearing aid (HA) intervention data were collected. Two-sampled independent t-tests and analysis of variance were used to compute rate and time outcomes. RESULTS: 30,773 newborns underwent NHS and 678 failed NHS. There was no difference in 1-month benchmark NHS rates, increased 3-month benchmark HL diagnosis rate post-SOE COVID (91.7%; p = 0.002), and increased 6-month benchmark HA intervention rate post-SOE COVID compared to pre-COVID (88.9% vs. 44.4%; p = 0.027). Mean time to NHS was lower during SOE COVID compared to pre-COVID (1.9 days vs. 2.0 days; p = 0.038) and mean time to HL diagnosis was higher during SOE COVID (47.5 days; p < 0.001). Lost to follow-up (LTF) rate at HL diagnosis decreased post-SOE (4.8%; p = 0.008). CONCLUSION: No differences in EHDI 1-3-6 benchmark rates between pre-COVID and SOE COVID patients were observed. However, increased 3-month benchmark HL diagnosis and 6-month benchmark HA intervention rates and a decreased LTF rate at 3-month benchmark HL diagnosis were observed post-SOE COVID.


Assuntos
COVID-19 , Surdez , Perda Auditiva , Recém-Nascido , Humanos , Lactente , Pandemias , Triagem Neonatal/métodos , Perda Auditiva/diagnóstico , Perda Auditiva/epidemiologia , Perda Auditiva/terapia , Testes Auditivos/métodos , Teste para COVID-19
9.
Elife ; 122023 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-37092974

RESUMO

Controlled protein synthesis is required to regulate gene expression and is often carried out in a cell type-specific manner. Protein synthesis is commonly measured by labeling the nascent proteome with amino acid analogs or isotope-containing amino acids. These methods have been difficult to implement in vivo as they require lengthy amino acid replacement procedures. O-propargyl-puromycin (OPP) is a puromycin analog that incorporates into nascent polypeptide chains. Through its terminal alkyne, OPP can be conjugated to a fluorophore-azide for directly visualizing nascent protein synthesis, or to a biotin-azide for capture and identification of newly-synthesized proteins. To achieve cell type-specific OPP incorporation, we developed phenylacetyl-OPP (PhAc-OPP), a puromycin analog harboring an enzyme-labile blocking group that can be removed by penicillin G acylase (PGA). Here, we show that cell type-specific PGA expression in Drosophila can be used to achieve OPP labeling of newly-synthesized proteins in targeted cell populations within the brain. Following a brief 2 hr incubation of intact brains with PhAc-OPP, we observe robust imaging and affinity purification of OPP-labeled nascent proteins in PGA-targeted cell populations. We apply this method to show a pronounced age-related decline in neuronal protein synthesis in the fly brain, demonstrating the capability of PhAc-OPP to quantitatively capture in vivo protein synthesis states. This method, which we call POPPi (PGA-dependent OPP incorporation), should be applicable for rapidly visualizing protein synthesis and identifying nascent proteins synthesized under diverse physiological and pathological conditions with cellular specificity in vivo.


Assuntos
Drosophila , Proteoma , Animais , Proteoma/metabolismo , Drosophila/metabolismo , Azidas/química , Aminoácidos/metabolismo , Puromicina
10.
Otolaryngol Head Neck Surg ; 169(2): 210-220, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-36939587

RESUMO

OBJECTIVE: To systematically review the literature to determine auditory outcomes of cochlear implantation in children ≤12 months old. DATA SOURCE: PubMed, EMBASE, Medline, CINAHL, Cochrane, Scopus, and Web of Science databases were searched from inception to 9/1/2021 using PRISMA guidelines. REVIEW METHODS: Studies analyzing auditory outcomes after cochlear implantation (CI) in children ≤12 months of age were included. Non-English studies and case reports were excluded. Outcome measures included functional and objective auditory results. Two independent reviewers evaluated each abstract and article. Heterogeneity and bias across studies were evaluated. RESULTS: Of 305 articles identified, 17 met inclusion criteria. There were 642 children ages 2 to 12 months at CI. The most common etiologies of hearing loss were congenital CMV, meningitis, idiopathic hearing loss, and GJB2 mutations and other genetic causes. All studies concluded that early CI was safe. Overall, outcomes improved following early CI: IT-MAIS (9 studies), LittlEARS (4 studies), PTA (3 studies), CAP (3 studies), GASP (3 studies), and LNT (3 studies). Nine studies compared outcomes to an older implantation group (>12 months); of these (n = 450 early CI, n = 1189 late CI), 8 studies showed earlier CI achieved comparable or better auditory outcomes than later implantation, whereas 1 study (n = 120) concluded no differences in speech perception improvement. CONCLUSION: Auditory outcomes were overall improved in children ≤12 months old undergoing CI. Studies that compared early to late CI demonstrated similar or better auditory outcomes in early implantation group. Given the comparable safety profile and critical time period of speech and language acquisition, earlier CI should be considered for infants with hearing loss.


Assuntos
Implante Coclear , Implantes Cocleares , Surdez , Percepção da Fala , Lactente , Criança , Humanos , Implante Coclear/métodos , Surdez/cirurgia , Desenvolvimento da Linguagem , Resultado do Tratamento
11.
JAMA Otolaryngol Head Neck Surg ; 149(6): 555-556, 2023 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-36995729

RESUMO

This diagnostic study examines the application of generative artificial intelligence in clinical tool research and development.


Assuntos
Inteligência Artificial , Membrana Timpânica , Humanos , Membrana Timpânica/diagnóstico por imagem
12.
PLOS Digit Health ; 2(2): e0000202, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36827244

RESUMO

Synthetic clinical images could augment real medical image datasets, a novel approach in otolaryngology-head and neck surgery (OHNS). Our objective was to develop a generative adversarial network (GAN) for tympanic membrane images and to validate the quality of synthetic images with human reviewers. Our model was developed using a state-of-the-art GAN architecture, StyleGAN2-ADA. The network was trained on intraoperative high-definition (HD) endoscopic images of tympanic membranes collected from pediatric patients undergoing myringotomy with possible tympanostomy tube placement. A human validation survey was administered to a cohort of OHNS and pediatrics trainees at our institution. The primary measure of model quality was the Frechet Inception Distance (FID), a metric comparing the distribution of generated images with the distribution of real images. The measures used for human reviewer validation were the sensitivity, specificity, and area under the curve (AUC) for humans' ability to discern synthetic from real images. Our dataset comprised 202 images. The best GAN was trained at 512x512 image resolution with a FID of 47.0. The progression of images through training showed stepwise "learning" of the anatomic features of a tympanic membrane. The validation survey was taken by 65 persons who reviewed 925 images. Human reviewers demonstrated a sensitivity of 66%, specificity of 73%, and AUC of 0.69 for the detection of synthetic images. In summary, we successfully developed a GAN to produce synthetic tympanic membrane images and validated this with human reviewers. These images could be used to bolster real datasets with various pathologies and develop more robust deep learning models such as those used for diagnostic predictions from otoscopic images. However, caution should be exercised with the use of synthetic data given issues regarding data diversity and performance validation. Any model trained using synthetic data will require robust external validation to ensure validity and generalizability.

13.
Neuropsychologia ; 181: 108489, 2023 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-36669696

RESUMO

One critical approach for promoting the efficiency of memory is to adopt selective encoding strategies to prioritize more valuable information. Past neuroimaging studies have shown that value-directed modulation of verbal memory depends heavily on the engagement of left-lateralized semantic processing regions, particularly in the ventrolateral prefrontal cortex (VLPFC). In the present study, we used high-definition transcranial direct current stimulation (HD-tDCS) to seek evidence for a causal role of left VLPFC in supporting the memory advantage for high-value items. Three groups of healthy young adult participants were presented with lists of words to remember, with each word accompanied by an arbitrarily assigned point value. During the first session, all participants received sham stimulation as they encoded five lists of 30 words each. Two of these lists were immediately tested with free recall, with feedback given to allow participants to develop metacognitive insight and strategies to maximize their point total. The second session had the exact same structure as the first, but the groups differed in whether they received continued sham stimulation (N = 22) or anodal stimulation of the left VLPFC (N = 21) or right VLPFC (N = 20). Those lists not tested with immediate recall were tested with recognition judgments after a one-day delay. Since no brain stimulation was applied during this Day 2 test, any performance differences can be attributed to the effects of stimulation on Day 1 encoding processes. Anodal stimulation of left VLPFC significantly boosted participants' memory encoding selectivity. In comparison, no such effect was seen in participants who received right VLPFC or sham stimulation. Estimates of recollection- and familiarity-based responding revealed that left VLPFC stimulation specifically amplified the effects of item value on recollection. These results demonstrate a causal role for left VLPFC in the implementation of selective value-directed encoding strategies, putatively by boosting deep semantic processing of high-value words. Our findings also provide further evidence on the hemispheric lateralization of value-directed verbal memory encoding.


Assuntos
Estimulação Transcraniana por Corrente Contínua , Adulto Jovem , Humanos , Estimulação Transcraniana por Corrente Contínua/métodos , Córtex Pré-Frontal/diagnóstico por imagem , Córtex Pré-Frontal/fisiologia , Rememoração Mental/fisiologia , Reconhecimento Psicológico/fisiologia , Memória de Curto Prazo
14.
Otolaryngol Head Neck Surg ; 168(4): 856-861, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-35439096

RESUMO

OBJECTIVE: To identify inner and middle ear anomalies in children with 22q11.2 deletion syndrome (22q11DS) and determine associations with hearing thresholds. STUDY DESIGN: Retrospective study. SETTING: Two tertiary care academic centers. METHODS: Children presenting with 22q11DS between 2010 and 2020 were included. Temporal bone imaging with computed tomography or magnetic resonance imaging was reviewed by 2 neuroradiologists. RESULTS: Twenty-two patients (12 female, 10 male) were identified. Forty-four ears were evaluated on imaging. There were 15 (34%) ears with abnormal semicircular canals, 14 (32%) with abnormal vestibules, 8 (18%) with abnormal ossicles, 6 (14%) with enlarged vestibular aqueducts, 4 (9.1%) with abnormal facial nerve canals, and 4 (9.1%) with cochlear anomalies. There were 25 ears with imaging and audiometric data. The median pure tone average (PTA) for ears with any structural abnormality was 41.0 dB, as compared with 28.5 dB for ears without any structural abnormality (P = .21). Of 23 ears with normal imaging, 6 (26%) had hearing loss in comparison with 13 (62%) of 21 ears with abnormalities (P = .02). Total number of anomalies per ear was positively correlated with PTA (Pearson correlation coefficient, R = 0.479, P = .01). PTA was significantly higher in patients with facial nerve canal anomalies (P = .002), vestibular aqueduct anomalies (P = .05), and vestibule anomalies (P = .02). CONCLUSIONS: Semicircular canal, ossicular, vestibular aqueduct, and vestibular anomalies were detected in children with 22q11DS, especially in the setting of hearing loss. Careful evaluation of anatomic anomalies is needed prior to surgical intervention in these patients.


Assuntos
Surdez , Síndrome de DiGeorge , Perda Auditiva Neurossensorial , Perda Auditiva , Doenças Vestibulares , Criança , Humanos , Masculino , Feminino , Síndrome de DiGeorge/complicações , Estudos Retrospectivos , Perda Auditiva Neurossensorial/genética , Canais Semicirculares/anormalidades
15.
Otolaryngol Head Neck Surg ; 169(1): 41-46, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35972815

RESUMO

OBJECTIVE: We compared the diagnostic performance of human clinicians with that of a neural network algorithm developed using a library of tympanic membrane images derived from children taken to the operating room with the intent of performing myringotomy and possible tube placement for recurrent acute otitis media (AOM) or otitis media with effusion (OME). STUDY DESIGN: Retrospective cohort study. SETTING: Tertiary academic medical center from 2018 to 2021. METHODS: A training set of 639 images of tympanic membranes representing normal, OME, and AOM was used to train a neural network as well as a proprietary commercial image classifier from Google. Model diagnostic prediction performance in differentiating normal vs nonpurulent vs purulent effusion was scored based on classification accuracy. A web-based survey was developed to test human clinicians' diagnostic accuracy on a novel image set, and this was compared head to head against our model. RESULTS: Our model achieved a mean prediction accuracy of 80.8% (95% CI, 77.0%-84.6%). The Google model achieved a prediction accuracy of 85.4%. In a validation survey of 39 clinicians analyzing a sample of 22 endoscopic ear images, the average diagnostic accuracy was 65.0%. On the same data set, our model achieved an accuracy of 95.5%. CONCLUSION: Our model outperformed certain groups of human clinicians in assessing images of tympanic membranes for effusions in children. Reduced diagnostic error rates using machine learning models may have implications in reducing rates of misdiagnosis, potentially leading to fewer missed diagnoses, unnecessary antibiotic prescriptions, and surgical procedures.


Assuntos
Aprendizado Profundo , Otite Média com Derrame , Otite Média , Criança , Humanos , Estudos Retrospectivos , Otite Média/diagnóstico , Otite Média/cirurgia , Otite Média com Derrame/diagnóstico , Otite Média com Derrame/cirurgia , Algoritmos
16.
Cell Chem Biol ; 30(1): 43-54.e8, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36529140

RESUMO

The mono-ADP-ribosyltransferase PARP7 has emerged as a key negative regulator of cytosolic NA-sensors of the innate immune system. We apply a rational design strategy for converting a pan-PARP inhibitor into a potent selective PARP7 inhibitor (KMR-206). Consistent with studies using the structurally distinct PARP7 inhibitor RBN-2397, co-treatment of mouse embryonic fibroblasts with KMR-206 and NA-sensor ligands synergistically induced the expression of the type I interferon, IFN-ß. In mouse colon carcinoma (CT-26) cells, KMR-206 alone induced IFN-ß. Both KMR-206 and RBN-2397 increased PARP7 protein levels in CT-26 cells, demonstrating that PARP7's catalytic activity regulates its own protein levels. Curiously, treatment with saturating doses of KMR-206 and RBN-2397 achieved different levels of PARP7 protein, which correlated with the magnitude of type I interferon gene expression. These latter results have important implications for the mechanism of action of PARP7 inhibitors and highlights the usefulness of having structurally distinct chemical probes for the same target.


Assuntos
Antineoplásicos , Interferon Tipo I , Ácidos Nucleicos , Animais , Camundongos , Fibroblastos , Transdução de Sinais
17.
Methods Mol Biol ; 2609: 387-395, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36515849

RESUMO

PARP7 is an enzyme that catalyzes mono-ADP-ribosylation (MARylation), is a critical regulator of type I interferon signaling, and has emerged as an immune-oncology drug candidate. PARP7 is a labile protein that is regulated in a proteasome-dependent manner. Indeed, endogenous PARP7 levels are undetectable by western blot in most cells. Intriguingly, treatment of cells with orthosteric small molecule inhibitors of PARP7 can increase endogenous PARP7 protein to detectable levels. This characteristic of PARP7 inhibitors could potentially be exploited to assess target engagement-and thus cellular efficacy-of PARP7 inhibitors; however, no method exists to quantitatively monitor endogenous PARP7 levels in a high-throughput manner. In this protocol, we describe an assay using a split Nanoluciferase (NanoLuc) system for quantifying endogenous PARP7 protein levels and PARP7 inhibitor target engagement in cells in a 96-well plate format. We show that this assay can be used to quantify PARP7 protein levels under various cellular treatments and can assess cellular PARP7 inhibitor target engagement. We envision this split NanoLuc PARP7 assay can be used not only for evaluating the cellular efficacy of PARP7 inhibitors in a high-throughput manner but also for uncovering the mechanisms regulating PARP7 protein levels in cells.


Assuntos
ADP-Ribosilação , Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Bioensaio
18.
Chem Sci ; 13(46): 13898-13906, 2022 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-36544740

RESUMO

PARP16-the sole ER-resident PARP family member-is gaining attention as a potential therapeutic target for cancer treatment. Nevertheless, the precise function of the catalytic activity of PARP16 is poorly understood. This is primarily due to the lack of inhibitors that are selective for PARP16 over other PARP family members. Herein, we describe a structure-guided strategy for generating a selective PARP16 inhibitor by incorporating two selectivity determinants into a phthalazinone pan-PARP inhibitor scaffold: (i) an acrylamide-based inhibitor (DB008) designed to covalently react with a non-conserved cysteine (Cys169, human numbering) in the NAD+ binding pocket of PARP16 and (ii) a dual-purpose ethynyl group designed to bind in a unique hydrophobic cavity adjacent to the NAD+ binding pocket as well as serve as a click handle. DB008 exhibits good selectivity for PARP16 versus other PARP family members. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) confirmed that covalent labeling of PARP16 by DB008 in cells is dependent on Cys169. DB008 exhibits excellent proteome-wide selectivity at concentrations required to achieve saturable labeling of endogenous PARP16. In-cell competition labeling experiments using DB008 provided a facile strategy for evaluating putative PARP16 inhibitors. Lastly, we found that PARP16 is sequestered into a detergent-insoluble fraction under prolonged amino acid starvation, and surprisingly, treatment with PARP16 inhibitors prevented this effect. These results suggest that the catalytic activity of PARP16 regulates its solubility in response to nutrient stress.

19.
Laryngoscope Investig Otolaryngol ; 7(6): 2103-2111, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36544952

RESUMO

Objectives: Microtia and aural atresia are congenital ear anomalies with a wide-ranging spectrum of phenotypes and varied functional and psychosocial consequences for patients. This study seeks to analyze the management of microtia-atresia patients at our center over a 20-year period and to propose recommendations for advancing microtia-atresia care at a national level. Methods: We performed a retrospective analysis of data from patients presenting to the Massachusetts Eye and Ear (Boston, MA) for initial otolaryngology consultation for congenital microtia and/or aural atresia between 1999 and 2018. Results: Over the 20-year study period, 229 patients presented to our microtia-atresia center at a median age of 7 years. The severity of microtia was most commonly classified as grade III (n = 87, 38%), 61% (n = 140) of patients had complete atresia, the median Jahrsdoerfer grading scale score was 6 (range 0-10), and 81 patients (35%) underwent surgery for microtia repair. For hearing rehabilitation, 30 patients (64%) underwent bone conduction device implantation and 17 patients (36%) underwent atresiaplasty. The implementation of an interdisciplinary, longitudinal care model resulted in an increase in patient (r = 0.819, p < .001) and surgical volume (microtia surgeries, r = 0.521, p = .019; otologic surgeries, r = 0.767, p < .001) at our center over time. Conclusion: An interdisciplinary team approach to microtia-atresia patient care may result in increased patient volume, which could improve aesthetic and hearing outcomes over time by concentrating care and surgical expertise. Future work should aim to establish standardized clinical consensus recommendations to guide the creation of high-quality microtia-atresia care programs. Level of Evidence: 4.

20.
Int J Neonatal Screen ; 8(4)2022 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-36547382

RESUMO

This study sought to assess the current state of screening for congenital cytomegalovirus infection in newborns among birth hospitals and newborn nurseries in the state of Massachusetts. A survey assessing hospital protocols for cytomegalovirus testing in newborns was distributed to all birth hospitals and newborn nurseries in Massachusetts from November 2020 to February 2021. 73.3% of hospitals responded to at least one survey question. Of these, fewer than half (48.5%) had any established approach for neonatal cytomegalovirus screening. Salivary polymerase chain reaction was the most common testing modality. Most hospitals did not perform confirmatory testing for positive test results. Most respondents (87.9%) did not know or did not answer how results of cCMV screening were reported to families and who was responsible for coordinating care for cCMV-infected infants. We conclude that congenital cytomegalovirus screening protocols are absent or incomplete in most Massachusetts birth hospitals and newborn nurseries. A cohesive strategy involving standardized education and screening guidelines is needed to reduce the incidence and burden of congenital cytomegalovirus disease on children and their families.

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