Inhibition of Cpeb3 ribozyme elevates CPEB3 protein expression and polyadenylation of its target mRNAs and enhances object location memory.

A self-cleaving ribozyme that maps to an intron of the cytoplasmic polyadenylation element-binding protein 3 (Cpeb3) gene is thought to play a role in human episodic memory, but the underlying mechanisms mediating this effect are not known. We tested the activity of the murine sequence and found that the ribozyme's self-scission half-life matches the time it takes an RNA polymerase to reach the immediate downstream exon, suggesting that the ribozyme-dependent intron cleavage is tuned to co-transcriptional splicing of the Cpeb3 mRNA. Our studies also reveal that the murine ribozyme modulates maturation of its harboring mRNA in both cultured cortical neurons and the hippocampus: inhibition of the ribozyme using an antisense oligonucleotide leads to increased CPEB3 protein expression, which enhances polyadenylation and translation of localized plasticity-related target mRNAs, and subsequently strengthens hippocampal-dependent long-term memory. These findings reveal a previously unknown role for self-cleaving ribozyme activity in regulating experience-induced co-transcriptional and local translational processes required for learning and memory.

Stored within DNA are the instructions cells need to make proteins. In order for proteins to get made, the region of DNA that codes for the desired protein (known as the gene) must first be copied into a molecule called messenger RNA (or mRNA for short). Once transcribed, the mRNA undergoes further modifications, including removing redundant segments known as introns. It then travels to molecular machines that translate its genetic sequence into the building blocks of the protein. Following transcription, some RNAs can fold into catalytic segments known as self-cleaving ribozymes which promote the scission of their own genetic sequence. One such ribozyme resides in the intron of a gene for CPEB3, a protein which adds a poly(A) tail to various mRNAs, including some involved in learning and memory. Although this ribozyme is found in most mammals, its biological role is poorly understood. Previous studies suggested that the ribozyme cleaves itself at the same time as the mRNA for CPEB3 is transcribed. This led Chen et al. to hypothesize that the rate at which these two events occur impacts the amount of CPEB3 produced, resulting in changes in memory and learning. If the ribozyme cleaves quickly, the intron is disrupted and may not be properly removed, leading to less CPEB3 being made. However, if the ribozyme is inhibited, the intron remains intact and is efficiently excised, resulting in higher levels of CPEB3 protein. To test how the ribozyme impacts CPEB3 production, Chen et al. inhibited the enzyme from cutting itself with antisense oligonucleotides (ASOs). The ASOs were applied to in vitro transcription systems, neurons cultured in the laboratory and the brains of living mice in an area called the hippocampus. The in vitro and cell culture experiments led to higher levels of CPEB3 protein and the addition of more poly(A) tails to mRNAs involved in neuron communication. Injection of the ASOs into the brains of mice had the same effect, and also improved their memory and learning. The findings of Chen et al. show a new mechanism for controlling protein production, and suggest that ASOs could be used to increase the levels of CPEB3 and modulate neuronal activity. This is the first time a biological role for a self-cleaving ribozyme in mammals has been identified, and the approach used could be applied to investigate the function of two other self-cleaving ribozymes located in introns in humans.

Clinically significant findings in a decade-long retrospective study of prenatal chromosomal microarray testing.

BACKGROUND: Chromosomal microarray (CMA) is commonly utilized in the obstetrics setting. CMA is recommended when one or more fetal structural abnormalities is identified. CMA is also commonly used to determine genetic etiologies for miscarriages, fetal demise, and confirming positive prenatal cell-free DNA screening results. METHODS: In this study, we retrospectively examined 523 prenatal and 319 products-of-conception (POC) CMA cases tested at Nationwide Children's Hospital from 2011 to 2020. We reviewed the referral indications, the diagnostic yield, and the reported copy number variants (CNV) findings. RESULTS: In our cohort, the diagnostic yield of clinically significant CNV findings for prenatal testing was 7.8% (n = 41/523) compared to POC testing (16.3%, n = 52/319). Abnormal ultrasound findings were the most common indication present in 81% of prenatal samples. Intrauterine fetal demise was the common indication identified in POC samples. The most common pathogenic finding observed in all samples was isolated trisomy 21, detected in seven samples. CONCLUSION: Our CMA study supports the clinical utility of prenatal CMA for clinical management and identifying genetic etiology in POC arrays. In addition, it provides insight to the spectrum of prenatal and POC CMA results as detected in an academic hospital clinical laboratory setting that serves as a reference laboratory.

Exploring the Impact of Exosomal Cargos on Osteosarcoma Progression: Insights into Therapeutic Potential.

Osteosarcoma (OS) is a primary malignant bone tumor with high metastasis. Poor prognosis highlights a clinical need for novel therapeutic strategies. Exosomes, also known as extracellular vesicles, have been identified as essential players in the modulation of cancer. Recent studies have suggested that OS-derived exosomes can drive pro-tumorigenic or anti-tumorigenic phenotypes by transferring specific cargos, including proteins, nucleic acids, and metabolites, to neighboring cells, significantly impacting the regulation of cellular processes. This review discusses the advancement of exosomes and their cargos in OS. We examine how these exosomes contribute to the modulation of cellular phenotypes associated with tumor progression and metastasis. Furthermore, we explore the potential of exosomes as valuable biomarkers for diagnostics and prognostic purposes and their role in shaping innovative therapeutic strategies in OS treatment development.

Clinical Cytogenetics: Current Practices and Beyond.

BACKGROUND: Throughout history, the field of cytogenetics has witnessed significant changes due to the constant evolution of technologies used to assess chromosome number and structure. Similar to the evolution of single nucleotide variant detection from Sanger sequencing to next-generation sequencing, the identification of chromosome alterations has progressed from banding to fluorescence in situ hybridization (FISH) to chromosomal microarrays. More recently, emerging technologies such as optical genome mapping and genome sequencing have made noteworthy contributions to clinical laboratory testing in the field of cytogenetics. CONTENT: In this review, we journey through some of the most pivotal discoveries that have shaped the development of clinical cytogenetics testing. We also explore the current test offerings, their uses and limitations, and future directions in technology advancements. SUMMARY: Cytogenetics methods, including banding and targeted assessments like FISH, continue to hold crucial roles in cytogenetic testing. These methods offer a rapid turnaround time, especially for conditions with a known etiology involving recognized cytogenetic aberrations. Additionally, laboratories have the flexibility to now employ higher-throughput methodologies to enhance resolution for cases with greater complexity.

Comparing demographics, clinical characteristics, and hospital outcomes by vaccine uptake status: A single-institution cross-sectional study.

Vaccination against Coronavirus disease 2019 (COVID-19) has been the cornerstone of reducing morbidity and mortality of this disease, as it has been shown to decrease the risk of viral transmission, severity of disease, hospitalization, and intubation. However, true understanding of its impact is skewed by heterogeneous vaccine administration due to lack of equitable access, vaccine hesitancy, and varying social determinants of health. Therefore, this study aims to identify groups that are less likely to be vaccinated and understand whether the resultant differences in vaccination rates affect morbidity and mortality in socially marginalized COVID-19 patients. A retrospective cohort analysis was performed on a randomized and stratified population of 939 COVID-19 patients from January 2021 to December 2021. Bivariate analysis and logistic regression were used to assess demographic and clinical characteristic trends in unvaccinated, partially vaccinated, and fully vaccinated groups. No one age (P = .21), gender (P = .9), race (P = .12), ethnicity (P = .09), or health insurance status (P = .13) group was more vaccinated than the other. Similarly, no subgroup was at increased odds of intubation (P = .08) or death. However, patients with all categories of comorbidities including cardiopulmonary disease (P = <.001, effect size .17), renal disease (P = <.001, effect size 0.138), metabolic disease (P = .04), and immunocompromised (P = .01) states were found to have significantly higher vaccination rates. Our study also shows that full vaccination protects against mortality and decreases the odds of intubation by 55% (adjusted odds ratio = 0.453, P value = .015) compared to no vaccination or partial vaccination. Findings from this study show an encouraging trend that sicker patients had higher rates of vaccination against COVID-19. This trend highlights the need for further identification of motivators that may be applied to vaccine-hesitant populations, which can help guide population-level policy, increase vaccination campaign yield, and reach for health equity.

Evaluation of the Abbott Alinity i Thyroid-Stimulating Hormone Receptor Antibody (TRAb) Chemiluminescent Microparticle Immunoassay (CMIA).

Background: We evaluated the performance of the Abbott thyroid-stimulating hormone receptor antibody chemiluminescent microparticle immunoassay (CMIA) on the Alinity i. Methods: Verification studies for precision, linearity, analytical measuring range, diagnostic cut offs for Graves' disease were performed. We compared the Abbott CMIA to an established TRAb assay (Roche electrochemiluminescence immunoassay). Method comparison analysis was performed between serum and plasma samples on the Abbott CMIA. Results: Repeatability (CV%) for TRAb were 4.07, 1.56, 0.71 and within-laboratory imprecision (CV%) were 4.07, 1.90, 0.71 at 3.0, 10.0, 30.0 IU/L of TRAb, respectively. Linearity and analytical measuring range were verified from 1.07-47.9 IU/L. The limit of the blank was 0 IU/L, limit of detection was 0.15 IU/L, and limit of quantification was 0.5 IU/L. Passing-Bablok analysis showed agreement between the two assays; Y-intercept = 0.787, slope = 1.04. Passing-Bablok analysis also showed agreement between the plasma and serum samples run on the Abbott CMIA; Y-intercept -0.17, slope = 0.97. Conclusions: The Abbott TRAb CMIA on the Alinity i performs within the manufacturer claims for assay precision, linearity, analytical measuring range, limit of blank, limit of detection, limit of quantitation and diagnostic cut offs for Graves' disease. Thus, the Abbott TRAb CMIA on the Alinity i is fit for clinical use.

Caspase-9 inhibition confers stronger neuronal and vascular protection compared to VEGF neutralization in a mouse model of retinal vein occlusion.

Purpose: Retinal vein occlusion (RVO) is a sight-threatening condition typically treated with intravitreal injection of vascular endothelial growth factor (VEGF) antagonists. Treatment response to anti-VEGF therapies is highly variable, with poor visual outcomes and treatment response in patients with significant retinal nonperfusion following RVO. Recently, caspase-9 has been identified as a potent regulator of edema, gliosis, and neuronal dysfunction during acute retinal hypoxia. The purpose of this study was to compare the therapeutic effect of caspase-9 inhibition against VEGF-neutralization in an established mouse model of RVO. Methods: Adult male C57Bl/6 J mice were randomized to induction of RVO and treatment with either vehicle, intravitreal injection of anti-VEGF antibody, topical administration of a selective caspase-9 inhibitor (Pen1-XBir3), or a combination therapy. Animals were followed on days 1, 2, and 8 after RVO with fundus retinal imaging, and with optical coherence tomography (OCT) to capture retinal swelling, capillary nonperfusion (measured by disorganization of retinal inner layers, DRIL), hyperreflective foci (HRF), and retinal atrophy. Focal electroretinography (ERG) measurements were performed on day 7. Histology was performed on retinal sections from day 8. Results: Both VEGF neutralization and caspase-9 inhibition showed significant retinal protection from RVO compared to vehicle treatment arm. Retinal reperfusion of occluded veins was accelerated in eyes receiving caspase-9 inhibitor, but not significantly different from vehicle in the anti-VEGF group. Retinal edema was suppressed in all treatment groups, with approximately 2-fold greater edema reduction with caspase-9 inhibition compared to VEGF neutralization. HRF were reduced similarly across all treatment groups compared to vehicle. Retinal detachment was reduced only in eyes treated with caspase-9 inhibitor monotherapy. Caspase-9 inhibition reduced retinal atrophy and preserved ERG response; VEGF neutralization did not prevent neurodegeneration following RVO. Conclusion: Caspase-9 inhibition confers stronger neuronal and vascular protection compared to VEGF neutralization in the mouse laser-induced model of RVO.

A Systematic Review and Meta-Analysis of Randomized Controlled Trials on Supine vs. Nonsupine Endotracheal Intubation.

Background: This systematic review and meta-analysis of randomized controlled trials (RCTs) was performed to compare the safety and efficacy of supine vs. nonsupine positions during intubation. Methods: Based on the literature from inception to October 2020, 13 studies with nonemergent intubation in supine and nonsupine positions were chosen using PRISMA and MOOSE protocols. Pooled estimates were calculated using random-effects models with 95% confidence interval (CI). The primary outcome was a successful intubation, attempt, and duration of intubation. The secondary outcome was adverse events (trauma and hypoxia). Bias was evaluated qualitatively, by visual analysis, and quantitatively through the Egger test. Results: The final analysis included 13 clinical trials with 1,916 patients. The pooled success rates in the supine vs. lateral positions were 99.21% and 98.82%. The supine vs. semierect positions were 99.21% and 98.82%. The 1st attempt success rate in the supine vs. lateral position was 85.35% and 88.56% compared to 91.38% and 90.76% for the supine vs. semierect position. The rate of total adverse events in the supine position was 3.73% vs. 6.74% in the lateral position, and the rate of total adverse events in the supine position was 0.44% vs. 0.93% in semierect position. Low to substantial heterogeneity was noted in our analysis. Discussion. There is no significant difference between total successful intubations and success from 1st intubation attempt between supine and nonsupine positions. However, there are slightly higher rates of adverse events in nonsupine position. Addition of more recent studies on supine vs. nonsupine intubations would improve this study. Given these findings, it is important to develop more studies regarding different intubation positions and techniques with the aim of improving efficacy and decreasing adverse outcomes. Other. This review is not registered in a public database. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Inhibition of CPEB3 ribozyme elevates CPEB3 protein expression and polyadenylation of its target mRNAs, and enhances object location memory.

A self-cleaving ribozyme that maps to an intron of the cytoplasmic polyadenylation element binding protein 3 (CPEB3) gene is thought to play a role in human episodic memory, but the underlying mechanisms mediating this effect are not known. We tested the activity of the murine sequence and found that the ribozyme's self-scission half-life matches the time it takes an RNA polymerase to reach the immediate downstream exon, suggesting that the ribozyme-dependent intron cleavage is tuned to co-transcriptional splicing of the CPEB3 mRNA. Our studies also reveal that the murine ribozyme modulates maturation of its harboring mRNA in both cultured cortical neurons and the hippocampus: inhibition of the ribozyme using an antisense oligonucleotide leads to increased CPEB3 protein expression, which enhances polyadenylation and translation of localized plasticity-related target mRNAs, and subsequently strengthens hippocampal-dependent long-term memory. These findings reveal a previously unknown role for self-cleaving ribozyme activity in regulating experience-induced co-transcriptional and local translational processes required for learning and memory.

Enhancing travel time prediction with deep learning on chronological and retrospective time order information of big traffic data.

With growing environmental concerns and the exploitation of ubiquitous big data, smart transportation is transforming logistics business and operations into a more sustainable approach. To answer questions in intelligent transportation planning, such as which data are feasible, which methods are applicable for intelligent prediction of such data, and what are the available operations for prediction, this paper offers a new deep learning approach called bi-directional isometric-gated recurrent unit (BDIGRU). It is merged to the deep learning framework of neural networks for predictive analysis of travel time and business adoption for route planning. The proposed new method directly learns high-level features from big traffic data and reconstructs them by its own attention mechanism drawn by temporal orders to complete the learning process recursively in an end-to-end manner. After deriving the computational algorithm with stochastic gradient descent, we use the proposed method to perform predictive analysis of stochastic travel time under various traffic conditions (especially for congestions) and then determine the optimal vehicle route with the shortest travel time under future uncertainty. Based on empirical results with big traffic data, we show that the proposed BDIGRU method can (1) significantly improve the predictive accuracy of one-step 30 min ahead travel time compared to several conventional (data-driven, model-driven, hybrid, and heuristics) methods measured with several performance criteria, and (2) efficiently determine the optimal vehicle route in relation to the predictive variability under uncertainty.

Utilization of Whole Health and Longitudinal Outcomes After Screening Positive for Possible Depression Documented in Veterans Health Administration's Electronic Health Record.

Objectives: Depression is common among Veterans. Veterans Health Administration (VHA) is transforming into a Whole Health system of care that includes holistic treatment planning, well-being programs, and health coaching. This evaluation explores the impact of Whole Health on improving symptoms of depression among Veterans who screen positive for possible depression diagnosis. Materials and Methods: We examined a cohort of Veterans who started using Whole Health after screening positive for possible depression (having a PHQ-2 score ≥3) at 18 VA Whole Health sites. We compared Whole Health users with non-Whole Health users on their follow-up PHQ-2 scores (9-36 months after baseline), using propensity score matching with multivariable regression to adjust for baseline differences. Results: Of the 13,559 Veterans screening positive for possible depression on the PHQ-2 and having a follow-up PHQ-2, 902 (7%) began using Whole Health after their initial positive PHQ-2. Whole Health users at baseline were more likely than non-Whole Health users to have posttraumatic stress disorder or acute stress (43% vs. 29%), anxiety (22% vs. 12%), ongoing opioid use (14% vs. 8%), recent severe pain scores (15% vs. 8%), or obesity (51% vs. 40%). Both groups improved at follow-up, with mean PHQ-2 scores decreasing from 4.49 to 1.77 in the Whole Health group and 4.46 to 1.46 in the conventional care group, with the Whole Health group significantly higher at follow-up. Also, the proportion continuing to screen positive at follow-up trended higher in the Whole Health group (26% and 21%, respectively). Conclusions: After screening positive for depression, Veterans with more mental and physical health conditions were more likely to subsequently use Whole Health services, suggesting that Whole Health is becoming a tool used in VHA to address the needs of complex patients. Nevertheless, the Whole Health group did not improve compared to the Conventional Care group. Results add to the growing body of literature that Whole Health services may play an important role among patients with complex symptom presentations by promoting self-management of symptoms and targeting "what matters most" to Veterans.

Early amygdala and ERC atrophy linked to 3D reconstruction of rostral neurofibrillary tau tangle pathology in Alzheimer's disease.

Previous research has emphasized the unique impact of Alzheimer's Disease (AD) pathology on the medial temporal lobe (MTL), a reflection that tau pathology is particularly striking in the entorhinal and transentorhinal cortex (ERC, TEC) early in the course of disease. However, other brain regions are affected by AD pathology during its early phases. Here, we use longitudinal diffeomorphometry to measure the atrophy rate from MRI of the amygdala compared with that in the ERC and TEC in cognitively unimpaired (CU) controls, CU individuals who progressed to mild cognitive impairment (MCI), and individuals with MCI who progressed to dementia of the AD type (DAT), using a dataset from the Alzheimer's Disease Neuroimaging Initiative (ADNI). Our results show significantly higher atrophy rates of the amygdala in both groups of 'converters' (CU→MCI, MCI→DAT) compared to controls, with rates of volume loss comparable to rates of thickness loss in the ERC and TEC. We localize atrophy within the amygdala within each of these groups using fixed effects modeling. Controlling for the familywise error rate highlights the medial regions of the amygdala as those with significantly higher atrophy in both groups of converters than in controls. Using our recently developed method, referred to as Projective LDDMM, we map measures of neurofibrillary tau tangles (NFTs) from digital pathology to MRI atlases and reconstruct dense 3D spatial distributions of NFT density within regions of the MTL. The distribution of NFTs is consistent with the spatial distribution of MR measured atrophy rates, revealing high densities (and atrophy) in the amygdala (particularly medial), ERC, and rostral third of the MTL. The similarity of the location of NFTs in AD and shape changes in a well-defined clinical population suggests that amygdalar atrophy rate, as measured through MRI may be a viable biomarker for AD.

HIV- and sex work-related stigmas and quality of life of female sex workers living with HIV in South Africa: a cross-sectional study.

BACKGROUND: Environmental quality of life (QoL) assesses individually perceived factors such as physical safety and security, accessibility, quality of healthcare, and physical environment. These factors are particularly relevant in the context of sex work and HIV, where stigma has been identified as an important barrier across several prevention and treatment domains. This study aims to examine the association between different types of HIV- and sex work-related stigmas and environmental QoL among female sex workers (FSW) living with HIV in Durban, South Africa. METHODS: We conducted cross-sectional analyses using baseline data from the Siyaphambili randomized controlled trial. FSW who reported sex work as their primary source of income and had been diagnosed with HIV for ≥ 6 months were enrolled from June 2018-March 2020, in eThekwini, South Africa. We evaluated the association between environmental QoL, dichotomizing the environmental domain score collected by the WHO Quality of Life HIV Brief (WHOQOL-HIV BREF) questionnaire at the median, and stigma using modified robust Poisson regression models. Five stigma subscales were assessed: sex work-related (anticipated, enacted, or internalized stigma) and HIV-related (anticipated or enacted stigma). RESULTS: Among 1373 FSW, the median environmental QoL was 10.5 out of 20 [IQR: 9.0-12.5; range 4.0-19.0], while the median overall QoL was 3 out of 5 [IQR: 2-4; range 1-5]. One-third of FSW (n = 456) fell above the median environmental QoL score, while 67% were above the median overall QoL (n = 917). Reporting anticipated sex work stigma was associated with lower environmental QoL (adjusted prevalence ratio [aPR] 0.74 [95% CI 0.61, 0.90]), as was severe internalized sex work stigma (aPR: 0.64, 95% CI 0.48, 0.86). Reporting enacted HIV stigma versus none was similarly associated with lower environmental QoL (aPR: 0.65, 95% CI 0.49, 0.87). Enacted sex work stigma and anticipated HIV stigma were not statistically associated with environmental QoL. CONCLUSIONS: This study highlights the need to consider the impact of multiple stigmas on FSW's non-HIV related clinical outcomes, including safety and physical well-being. Moreover, these results suggest that addressing underlying structural risks may support the impact of more proximal HIV prevention and treatment interventions. Trial registration NCT03500172 (April 17, 2018).

Critical asthma syndrome in trauma patients - A case report and literature review.

Critical asthma syndrome (CAS) is an umbrella term for many acute, life-threatening, and treatment resistant variants of asthma exacerbation, including refractory asthma, near fatal asthma, and status asthmaticus. The asthma mortality rate has steadily increased through the last decade and disproportionately affects women, African-Americans, patients of low socioeconomic status, and adults over the age of 55. Increased awareness of the diagnosis and therapies for CAS can help establish a therapeutic strategy for asthma beyond corticosteroids, bronchodilators, and other conventional treatments. A 37 year-old African American woman presented to our Level 1 Trauma Center after a high-speed motor vehicle crash and was intubated on arrival for airway protection. The patient developed diffuse wheezing and persistent tachycardia, with elevated peak airway pressures and air trapping on mechanical ventilation. Her symptoms were refractory to inhaled steroids, systemic steroids, intravenous magnesium, continuous albuterol administration and ventilator optimization. Heliox, an admixture of 80:20 percent helium to oxygen, was initiated to assist with laminar flow. Throughout the next 24 h, the patient's air trapping improved, subsequently decreasing intrathoracic pressure, improving venous return and resolving her tachycardia. The patient's multiple orthopedic injuries were treated and she was eventually weaned off of Heliox, steroids, and continuous albuterol. She was extubated and endorsed a history of poorly controlled asthma requiring hospitalizations and multiple intubations. Recognition of CAS can be challenging in the trauma patient with distracting injuries. This case illustrates the utility of a stepwise approach to a trauma patient suffering from CAS, and should encourage further research into novel therapies when conventional treatment fails. Given that the populations most affected by CAS are often also subject to a disproportionate burden of trauma, trauma surgeons should maintain both a vigilance for the syndrome as well as a working knowledge of the treatment modalities available.

Infantile-onset Pompe disease complicated by sickle cell anemia: Case report and management considerations.

Infantile-onset Pompe disease (IOPD) is a rare, severe disorder of lysosomal storage of glycogen that leads to progressive cardiac and skeletal myopathy. IOPD is a fatal disease in childhood unless treated with enzyme replacement therapy (ERT) from an early age. Sickle cell anemia (SCA) is a relatively common hemoglobinopathy caused by a specific variant in the hemoglobin beta-chain. Here we report a case of a male newborn of African ancestry diagnosed and treated for IOPD and SCA. Molecular testing confirmed two GAA variants, NM_000152.5: c.842G>C, p.(Arg281Pro) and NM_000152.5: c.2560C>T, p.(Arg854*) in trans, and homozygosity for the HBB variant causative of SCA, consistent with his diagnosis. An acute neonatal presentation of hypotonia and cardiomyopathy required ERT with alglucosidase alfa infusions preceded by immune tolerance induction (ITI), as well as chronic red blood cell transfusions and penicillin V potassium prophylaxis for treatment of IOPD and SCA. Clinical course was further complicated by multiple respiratory infections. We review the current guidelines and interventions taken to optimize his care and the pitfalls of those guidelines when treating patients with concomitant conditions. To the best of our knowledge, no other case reports of the concomitance of these two disorders was found. This report emphasizes the importance of newborn screening, early intervention, and treatment considerations for this complex patient presentation of IOPD and SCA.

UHRF1 overexpression promotes osteosarcoma metastasis through altered exosome production and AMPK/SEMA3E suppression.

Loss-of-function mutations at the retinoblastoma (RB1) gene are associated with increased mortality, metastasis, and poor therapeutic outcome in several cancers, including osteosarcoma. However, the mechanism(s) through which RB1 loss worsens clinical outcome remains understudied. Ubiquitin-like with PHD and Ring Finger domains 1 (UHRF1) has been identified as a critical downstream effector of the RB/E2F signaling pathway that is overexpressed in various cancers. Here, we determined the role and regulatory mechanisms of UHRF1 in rendering osteosarcoma cells more aggressive. Higher UHRF1 expression correlated with malignancy in osteosarcoma cell lines, clinical samples, and genetically engineered mouse models. Gain- and loss-of-function assays revealed that UHRF1 has cell-intrinsic and extrinsic functions promoting cell proliferation, migration, invasion, angiogenesis, and metastasis. UHRF1 overexpression induced angiogenesis by suppressing AMPK activation and Semaphorin 3E (SEMA3E) expression. Further, UHRF1-mediated migration and metastasis resulted, at least in part, through altered expression of extracellular vesicles and their cargo, including urokinase-type plasminogen activator (uPA). Novel osteosarcoma genetically engineered mouse models confirmed that knocking out Uhrf1 considerably decreased metastasis and reversed the poorer survival associated with Rb1 loss. This presents a new mechanistic insight into RB1 loss-associated poor prognosis and novel oncogenic roles of UHRF1 in the regulation of angiogenesis and exosome secretion, both critical for osteosarcoma metastasis. This provides substantial support for targeting UHRF1 or its downstream effectors as novel therapeutic options to improve current treatment for osteosarcoma.

Antibody dynamics post-Comirnaty and CoronaVac vaccination in Malaysia.

Several vaccines have been fast-tracked through clinical trials to mitigate the progression of the SARS­CoV­2 pandemic. We analyzed sequential blood samples from 314 recipients of Comirnaty and CoronaVac in East Malaysia for the spike-binding IgG (IgG-S), nucleocapsid-binding IgG (IgG-N), spike-binding IgM (IgM-S) and serum vitamin D (VitD). A subset of samples was analyzed for the neutralizing antibodies (Ig-RBD). Results showed that IgG-S due to Comirnaty was significantly higher than CoronaVac. IgM-S was detected in 80.0% Comirnaty and 69.5% CoronaVac recipients, while IgG-N was detected in 58.1% CoronaVac but not in Comirnaty recipients. All IgG-S-positive vaccines possessed detectable Ig-RBD after the second dose but with a weak to moderate correlation. The serum VitD levels did not influence the antibody magnitude in both vaccines. In essence, SARS-CoV-2 vaccination is an IgG-S-dominant event, Comirnaty was more effective than CoronaVac in mounting IgG-S and Ig-RBD responses, independent of the patient's VitD level.

Quantification of Immunostained Caspase-9 in Retinal Tissue.

The family of caspases is known to mediate many cellular pathways beyond cell death, including cell differentiation, axonal pathfinding, and proliferation. Since the identification of the family of cell death proteases, there has been a search for tools to identify and expand the function of specific family members in development, health, and disease states. However, many of the currently commercially available caspase tools that are widely used are not specific for the targeted caspase. In this report, we delineate the approach we have used to identify, validate, and target caspase-9 in the nervous system using a novel inhibitor and genetic approaches with immunohistochemical read-outs. Specifically, we used the retinal neuronal tissue as a model to identify and validate the presence and function of caspases. This approach enables the interrogation of cell-type specific apoptotic and non-apoptotic caspase-9 functions and can be applied to other complex tissues and caspases of interest. Understanding the functions of caspases can help to expand current knowledge in cell biology, and can also be advantageous to identify potential therapeutic targets due to their involvement in disease.

Noninvasive Ophthalmic Imaging Measures Retinal Degeneration and Vision Deficits in Ndufs4-/- Mouse Model of Mitochondrial Complex I Deficiency.

Purpose: To characterize postnatal ocular pathology in a Ndufs4-/- mouse model of complex I deficiency using noninvasive retinal imaging and visual testing. Methods: Ndufs4-/- mice and wild-type (WT) littermates were analyzed at 3, 5, and 7 weeks postnatal. Retinal morphology was visualized by optical coherence tomography (OCT). OCT images were analyzed for changes in retinal thickness and reflectivity profiles. Visual function was assessed by electroretinogram (ERG) and optomotor reflex (OMR). Results: Ndufs4-/- animals have normal OCT morphology at weaning and develop inner plexiform layer atrophy over weeks 5 to 7. Outer retinal layers show hyporeflectivity of the external limiting membrane (ELM) and photoreceptor ellipsoid zone (EZ). Retinal function is impaired at 3 weeks, with profound deficits in b-wave, a-wave, and oscillatory potential amplitudes. The b-wave and oscillatory potential implicit times are delayed, but the a-wave implicit time is unaffected. Ndufs4-/- animals have normal OMR at 3 weeks and present with increasing acuity and contrast OMR deficits at 5 and 7 weeks. Physiological thinning of inner retinal layers, attenuation of ELM reflectivity, and attenuation of ERG b- and a-wave amplitudes occur in WT C57BL/6 littermates between weeks 3 and 7. Conclusions: Noninvasive ocular imaging captures early-onset retinal degeneration in Ndufs4-/- mice and is a tractable approach for investigating retinal pathology subsequent to complex I deficiency. Translational Relevance: Ophthalmic imaging captures clinically relevant measures of retinal disease in a fast-progressing mouse model of complex I deficiency consistent with human Leigh syndrome.