Generation of human cerebral organoids with a structured outer subventricular zone.

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.

Living and post-mortem CT scans in the gross anatomy lab: A study investigating differences in first-year medical students' exam performance and perceptions.

Basic competency in radiological imaging is essential for physicians to identify and manage diseases. An optimal place in which to include imaging in the medical curriculum is during anatomy as students can correlate the 3D anatomy from their body donors with the 2D cross-sectional anatomy. The goal of this project was to enhance first-year medical students' knowledge of cross-sectional imaging in the gross anatomy lab and to investigate whether there are benefits to learning cross sectional imaging via scans from body donors versus living individuals. Student participant performance was evaluated on laboratory practical examinations, CT image questions and spatial anatomical knowledge in the thorax and abdomen sections of gross anatomy. Students learned the cross-sectional imaging during dissections where they accessed the images relevant to their study on Pacsbin, a web-based Digital Imaging and Communication in Medicine viewer, via iPads. Results showed no statistically significant differences in practical examination scores, spatial anatomical knowledge, or identification of anatomical structures on CT image questions between participants who learned from images on body donors versus living individuals. In a questionnaire given at the end of the course, participants cited that the CT images improved their anatomical and imaging knowledge and that they felt better prepared to use imaging software and interpret diagnostic imaging results upon entering clerkships. While there were no differences in academic performance between the groups, positive outcomes regarding student perceptions of anatomical and imaging knowledge and preparedness for use of imaging software were identified in this study.

An epigenetic barrier sets the timing of human neuronal maturation.

The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.

Combined small-molecule treatment accelerates maturation of human pluripotent stem cell-derived neurons.

The maturation of human pluripotent stem cell (hPSC)-derived neurons mimics the protracted timing of human brain development, extending over months to years for reaching adult-like function. Prolonged in vitro maturation presents a major challenge to stem cell-based applications in modeling and treating neurological disease. Therefore, we designed a high-content imaging assay based on morphological and functional readouts in hPSC-derived cortical neurons which identified multiple compounds that drive neuronal maturation including inhibitors of lysine-specific demethylase 1 and disruptor of telomerase-like 1 and activators of calcium-dependent transcription. A cocktail of four factors, GSK2879552, EPZ-5676, N-methyl-D-aspartate and Bay K 8644, collectively termed GENtoniK, triggered maturation across all parameters tested, including synaptic density, electrophysiology and transcriptomics. Maturation effects were further validated in cortical organoids, spinal motoneurons and non-neural lineages including melanocytes and pancreatic ß-cells. The effects on maturation observed across a broad range of hPSC-derived cell types indicate that some of the mechanisms controlling the timing of human maturation might be shared across lineages.

Randomized controlled trial of urinE chemiStry guided aCute heArt faiLure treATmEnt (ESCALATE): Rationale and design.

Diuresis to achieve decongestion is a central aim of therapy in patients hospitalized for acute decompensated heart failure (ADHF). While multiple clinical trials have investigated initial diuretic strategies for a designated period of time, there is a paucity of evidence to guide diuretic titration strategies continued until decongestion is achieved. The use of urine chemistries (urine sodium and creatinine) in a natriuretic response prediction equation accurately estimates natriuresis in response to diuretic dosing, but a randomized clinical trial is needed to compare a urine chemistry-guided diuresis strategy with a strategy of usual care. The urinE chemiStry guided aCute heArt faiLure treATmEnt (ESCALATE) trial is designed to test the hypothesis that protocolized diuretic therapy guided by spot urine chemistry through completion of intravenous diuresis will be superior to usual care and improve outcomes over the 14 days following randomization. ESCALATE will randomize and obtain complete data on 450 patients with acute heart failure to a diuretic strategy guided by urine chemistry or a usual care strategy. Key inclusion criteria include an objective measure of hypervolemia with at least 10 pounds of estimated excess volume, and key exclusion criteria include significant valvular stenosis, hypotension, and a chronic need for dialysis. Our primary outcome is days of benefit over the 14 days after randomization. Days of benefit combines patient symptoms captured by global clinical status with clinical state quantifying the need for hospitalization and intravenous diuresis. CLINICAL TRIAL REGISTRATION: NCT04481919.

The prevalence, characteristics, and risk factors of external cervical resorption: a retrospective practice-based study.

OBJECTIVES: External cervical root resorption (ECR) is a poorly understood and aggressive form of resorption. The purpose of this study was to examine the prevalence, characteristics, and risk factors associated with the occurrence of ECR in patients seeking endodontic care from private practice settings. MATERIALS AND METHODS: Records of 343 patients with 390 teeth diagnosed with ECR were identified from 3 private endodontic practices from 2008 to 2022. The patients' demographic information, systemic conditions, and dental history were recorded. The characteristics of the cases including Heithersay classification, pulpal and periapical status, and their management were documented. The association between case severity and potential predisposing factors was examined using chi-square analysis. RESULTS: The overall prevalence of ECR among patients seeking endodontic care was low (< 1%). However, there was a greater than twofold increase in the pooled prevalence from 2016 to 2021 (0.99%) compared to the data from 2010 to 2015 (0.46%). The most commonly affected teeth were anterior teeth (48.7%). Class II (30.0%) and class III (45.4%) defects were the most often identified. Patients with a history of trauma or orthodontic treatment were significantly more likely to be diagnosed with severe cervical resorption (class III or IV) (p < 0.05). CONCLUSIONS: There has been an increase in the prevalence of ECR in patients seeking endodontic care. A history of orthodontic treatment and traumatic dental mechanical injuries may predict the severity of resorption. CLINICAL RELEVANCE: The upward trend in the occurrence of ECR warrants close monitoring of the patients at high risk of developing the condition to facilitate early detection and management.

A retrospective study of initial root canal treatment failure in maxillary premolars via using cone-beam computed tomography.

BACKGROUND: This case-series study examined canal morphology and common factors for endodontic failure in maxillary first and second premolars that were referred for retreatment owing to clinical symptoms or radiographic signs. METHODS: Records were retrospectively searched using Current Dental Terminology codes to identify maxillary first and second premolars with endodontic failure. Periapical and cone-beam computed tomographic images were examined to determine Vertucci classifications and suspected factors related to treatment failure. RESULTS: A total of 235 teeth from 213 patients were included for evaluation. The following Vertucci classification of canal configurations were observed for maxillary first and second premolars: type I (1-1) (4.6% and 32.0%, respectively), type II (2-1) (15.9% and 27.9%, respectively), type III (2-2) (76.1% and 36.1%, respectively), type IV (1-2) (0% and 2%, respectively), and type V (3) (3.4% and 2%, respectively). More treatment failures were noticed in maxillary second premolars than first premolars and in females than in males. The 4 most common factors related to failure were inadequate filling, restorative failure, vertical root fracture, and missed canals. Missed canals were more frequently identified in maxillary second premolars (21.8%) than first premolars (11.4%) (P = .044). CONCLUSIONS: Multiple factors are associated with primary root canal treatment failures in maxillary premolars. Variations in canal morphology appear to be underappreciated in maxillary second premolars. PRACTICAL IMPLICATIONS: Maxillary second premolars have more complicated canal configurations than first premolars. Besides adequate filling, clinicians should give extra attention to anatomic variability in second premolars owing to higher failure incidence.

The 'omics of obesity in B-cell acute lymphoblastic leukemia.

The obesity pandemic currently affects more than 70 million Americans and more than 650 million individuals worldwide. In addition to increasing susceptibility to pathogenic infections (eg, SARS-CoV-2), obesity promotes the development of many cancer subtypes and increases mortality rates in most cases. We and others have demonstrated that, in the context of B-cell acute lymphoblastic leukemia (B-ALL), adipocytes promote multidrug chemoresistance. Furthermore, others have demonstrated that B-ALL cells exposed to the adipocyte secretome alter their metabolic states to circumvent chemotherapy-mediated cytotoxicity. To better understand how adipocytes impact the function of human B-ALL cells, we used a multi-omic RNA-sequencing (single-cell and bulk transcriptomic) and mass spectroscopy (metabolomic and proteomic) approaches to define adipocyte-induced changes in normal and malignant B cells. These analyses revealed that the adipocyte secretome directly modulates programs in human B-ALL cells associated with metabolism, protection from oxidative stress, increased survival, B-cell development, and drivers of chemoresistance. Single-cell RNA sequencing analysis of mice on low- and high-fat diets revealed that obesity suppresses an immunologically active B-cell subpopulation and that the loss of this transcriptomic signature in patients with B-ALL is associated with poor survival outcomes. Analyses of sera and plasma samples from healthy donors and those with B-ALL revealed that obesity is associated with higher circulating levels of immunoglobulin-associated proteins, which support observations in obese mice of altered immunological homeostasis. In all, our multi-omics approach increases our understanding of pathways that may promote chemoresistance in human B-ALL and highlight a novel B-cell-specific signature in patients associated with survival outcomes.

Managing a Deep Subgingival Cusp Fracture: Case Reports.

Cuspal fractures are relatively common. Fortunately for esthetics, when a cuspal fracture occurs in a maxillary premolar, it most commonly involves the palatal cusp. Fractures with a favorable prognosis may be approached with a minimally invasive treatment to successfully retain the natural tooth. This report describes 3 cases of "cuspidization" to treat maxillary premolars with cuspal fractures. After identifying a palatal cusp fracture, the fractured segment was removed, resulting in a tooth that closely resembles a cuspid. Depending on the extent and location of the fracture, root canal treatment was indicated. Subsequently, conservative restorations sealed the access and covered exposed dentin. Full coverage restorations were neither required nor indicated. The resulting treatment provided practical and functional treatment with a good esthetic outcome. When indicated, the described cuspidization technique can conservatively manage patients with subgingival cuspal fractures. The procedure is minimally invasive and cost-effective and can be done conveniently in routine practice.

Generation of human cerebral organoids with a structured outer subventricular zone.

Mammalian outer radial glia (oRG) emerge as cortical progenitor cells that directly support the development of an enlarged outer subventricular zone (oSVZ) and, in turn, the expansion of the neocortex. The in vitro generation of oRG is essential to model and investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 pathway using LIF, which is not produced in guided cortical organoids, we developed a cerebral organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The structured oSVZ is composed of progenitor cells expressing specific oRG markers such as GFAP, LIFR, HOPX , which closely matches human oRG in vivo . In this microenvironment, cortical neurons showed faster maturation with enhanced metabolic and functional activity. Incorporation of hPSC-derived brain vascular LIF- producing pericytes in cerebral organoids mimicked the effects of LIF treatment. These data indicate that the cellular complexity of the cortical microenvironment, including cell-types of the brain vasculature, favors the appearance of oRG and provides a platform to routinely study oRG in hPSC-derived brain organoids.

Dissection of gastric homeostasis in vivo facilitates permanent capture of isthmus-like stem cells in vitro.

The glandular stomach is composed of two regenerative compartments termed corpus and antrum, and our understanding of the transcriptional networks that maintain these tissues is incomplete. Here we show that cell types with equivalent functional roles in the corpus and antrum share similar transcriptional states including the poorly characterized stem cells of the isthmus region. To further study the isthmus, we developed a monolayer two-dimensional (2D) culture system that is continually maintained by Wnt-responsive isthmus-like cells capable of differentiating into several gastric cell types. Importantly, 2D cultures can be converted into conventional three-dimensional organoids, modelling the plasticity of gastric epithelial cells in vivo. Finally, we utilized the 2D culture system to show that Sox2 is both necessary and sufficient to generate enterochromaffin cells. Together, our data provide important insights into gastric homeostasis, establish a tractable culture system to capture isthmus cells and uncover a role for Sox2 in enterochromaffin cells.

Plasma and cerebrospinal fluid pharmacokinetics of vodobatinib, a neuroprotective c-Abl tyrosine kinase inhibitor for the treatment of Parkinson's disease.

BACKGROUND: Preclinical evidence suggests that c-Abl is critical in the pathogenesis of Parkinson's Disease (PD). Vodobatinib (K0706) is a potent, specific Abl kinase inhibitor currently being developed for the treatment of PD. In previously reported studies, nilotinib, a multikinase c-Abl inhibitor, did not show clinical activity as evidenced by no improvement of symptoms or the rate of decline after one to six months of treatment at the maximum permissible dose, presumably because of insufficient CNS penetration. Here we report clinical PK and safety data for vodobatinib. OBJECTIVES: To determine safety, plasma PK, and CSF penetration of vodobatinib in healthy volunteers and PD subjects following oral administration, and compare CSF levels to in vitro concentrations required for c-Abl inhibition relative to data reported for nilotinib. METHODS: Inhibition of c-Abl kinase activity and c-Abl binding affinity were first assessed in vitro. Healthy human volunteers and PD patients received various oral doses of vodobatinib once-daily for seven and fourteen days respectively, to assess safety, and plasma and CSF PK. RESULTS: In in vitro assays, vodobatinib was more potent (kinase IC50 = 0.9 nM) than nilotinib (kinase IC50 = 15-45 nM). Administration of vodobatinib 48, 192 and 384 mg to healthy subjects for 7 days yielded mean Cmax, CSF values of 1.8, 11.6, and 12.2 nM respectively, with the two highest doses exceeding the IC50 over the entire dosing interval. Cavg, CSF values were 6-8 times greater than the IC50. Comparable CSF levels were observed in PD patients. All doses were well tolerated in both cohorts. CONCLUSION: Based on achieved CSF concentrations, the potential for c-Abl inhibition in the brain is substantially higher with vodobatinib than with nilotinib. The CSF PK profile of vodobatinib is suitable for determining if c-Abl inhibition will be neuroprotective in PD patients.

Clinical outcomes of non-surgical root canal obturations using NeoMTA: A retrospective series of case reports.

NeoMTA is a commercially available tricalcium silicate-based cement intended for contact with pulp and periradicular tissues. The purpose of this study was to retrospectively evaluate the outcomes of non-surgical root canal treatments with NeoMTA obturations. Patients were treated in a private endodontic practice between 2015 and 2018. All cases, including initial treatments and retreatments, were either fully obturated with NeoMTA, or using gutta-percha with NeoMTA as an endodontic sealer. Outcomes were assessed using follow-up examination data with digital periapical radiographs with a minimum of a 1-year recall. Teeth were classified based on the clinical examination as: healed/healing (success), or non-healed (failure). 265 teeth were included with an average follow-up time of 1.3 years. The overall success rate was 91.7%. Only the presence of a pre-operative periapical radiolucency was found to significantly affect success. Comparison of obturation techniques demonstrated no effect on outcomes. NeoMTA is suitable for endodontic obturation.

Senior Resident Grand Rounds: Results From a Survey of 15 Years of Radiology Residency Graduates.

OBJECTIVE: Radiology residents in their final year of training at our institution are required to deliver a grand rounds presentation prior to graduation in order to facilitate resident involvement in the medical tradition of ground rounds. We evaluated this requirement by conducting a survey of recent residency alumni on their experience delivering grand rounds and its perceived value in radiology training. MATERIALS AND METHODS: We developed an anonymous, 10-question survey regarding the alumni experience with the grand rounds presentation requirement, which has been required since 2007. Question formats included yes-or-no and 5 point Likert-type formats, as well as a space to provide comments. RESULTS: Eighty-three alumni were contacted and 39 responded (46.9% response rate). Current practice setting was academic in 41.0%, 46.2% have reused material from their presentation, and 46.2% have given a grand rounds presentation since completing residency. When asked if delivering the presentation was a valuable experience, 2.6% disagreed, 23.1% were neutral, 35.9% agreed, and 38.5% strongly agreed. When asked if they would recommend future senior residents to give a grand rounds presentation, 2.6% strongly disagreed, 30.8% were neutral, 30.8% agreed, and 35.9% strongly agreed. CONCLUSIONS: The majority of survey respondents agree or strongly agree their grand rounds presentation experience was valuable and would recommend future residents to continue this process. Based on survey comments, we conclude this experience is valuable for multiple reasons, including the opportunity to demonstrate mastery of a radiology topic, the ability to repurpose presentation material in the future, and the experience of delivering a formal presentation.

A Novel Ocular Tonometry Task Trainer.

The measurement of intraocular pressure via ocular tonometry is a skill necessary for the evaluation of emergency department patients with ocular complaints. Accurate results inform the use of time-sensitive medications or invasive procedures. We sought to develop and evaluate an affordable, realistic, and reproducible task trainer to allow Emergency Medicine residents and medical students to practice tonometry. We placed an angiocatheter into the vitreous chamber of a swine eye through the optic nerve stump and sealed it with a purse string suture and cyanoacrylate glue. This allowed us to connect intravenous extension tubing and use a saline-filled syringe to repeatedly adjust intraocular pressure in real time. Optionally, this model can be mounted in a polystyrene foam mannequin head to enhance realism and facilitate practice. The task trainer was implemented in medical student and Emergency Medicine resident education at Vanderbilt University Medical Center. Thirty-six learners participated in the study, all of whom completed pre-course and post-course surveys. Among all learners, the mean comfort with performing tonometry improved significantly (3.26 to 7.64 {Z = -4.95, p < 0.005}). The mean confidence in the accuracy of measurements also increased (3.11 to 7.56 {Z = -4.8, p < 0.005}). On a 10-point scale, learners felt this task trainer was highly helpful in increasing their comfort with and the ability to perform tonometry (mean 9.19 {SD 1.19}). We have developed a low-cost and easily constructed ocular tonometry task trainer that resulted in significant improvement in learner comfort and confidence.

A Porcine-Based Task Trainer for the Instruction of Seidel's Test and Ocular Foreign Body Removal.

Ophthalmologic emergencies account for tens of thousands of patients presenting to the emergency department (ED) each year. Studies continue to show that ophthalmology education in medical school is limited, and this leads to many resident physicians who lack confidence in their ophthalmological examination and procedural skills if opportunities to practice these skills are not deliberately sought out during their training. Medical simulation continues to be an excellent way for physicians to learn and master these anxiety-inducing procedures. We present a newly modified, innovative, and realistic porcine-based emergency ophthalmologic procedural task trainer with live television screen projection to further improve Emergency Medicine (EM) resident education on the performance of Seidel's test and ocular foreign body removal.

High-fidelity simulation training improves resident knowledge of contrast reaction management, especially for junior residents.

Rationale and objectives: Contrast reactions are rare but serious events, frequently managed independently by Radiology Residents, who are likely underprepared to lead the acute event response. At our tertiary care center, Radiology Residents are the first responders to contrast reaction scenarios, and previously had didactic only training. We sought to create a High-Fidelity Simulation Training, and to assess whether this improved resident knowledge in managing contrast reactions. Materials and methods: In September of 2020, we administered a didactic only contrast reaction training to 20 residents, with an anonymous 20 question multiple choice pre- and post-test. In January of 2022, we administered a 4-hour, 4-station contrast reaction High-Fidelity Simulation Training to 22 residents, with the same 20 question multiple choice pre- and post-test. Results: The average number of residents answering each question correctly did not significantly improve following the didactic only training (p = 0.116). Following high-fidelity simulation training, however, there was a significant improvement by a mean of 2.45 (p = 0.028), as well as a mean improvement in individual scores of 10.45% (p = 0.0001). Comparing junior and senior residents, there was a significant difference in pre-test scores, with senior residents scoring on average 9.67% better (p = 0.0364); however on post-test scores, there was no significant difference. Conclusion: High-fidelity simulation training improves resident knowledge of contrast reaction management, and allows inexperienced junior residents to attain senior resident level proficiency in these high-stress scenarios.