Gamifying cell culture training: The 'Seru-Otchi' experience for undergraduates.

Working in a stem cell laboratory necessitates a thorough understanding of complex cell culture protocols, the operation of sensitive scientific equipment, adherence to safety standards, and general laboratory etiquette. For novice student researchers, acquiring the necessary specialized knowledge before their initial laboratory experience can be a formidable task. Similarly, for experienced laboratory personnel, efficiently and uniformly training new trainees to a rigorous standard presents a significant challenge. In response to these issues, we have developed an educational and interactive virtual cell culture environment. This interactive virtual lab aims to equip students with foundational knowledge in maintaining cortical brain organoids and to instill an understanding of pertinent safety procedures and laboratory etiquette. The gamification of this training process seeks to provide laboratory supervisors in highly specialized fields with an effective tool to integrate students into their work environments more rapidly and safely.

Who is pregnant? Defining real-world data-based pregnancy episodes in the National COVID Cohort Collaborative (N3C).

Objectives: To define pregnancy episodes and estimate gestational age within electronic health record (EHR) data from the National COVID Cohort Collaborative (N3C). Materials and Methods: We developed a comprehensive approach, named Hierarchy and rule-based pregnancy episode Inference integrated with Pregnancy Progression Signatures (HIPPS), and applied it to EHR data in the N3C (January 1, 2018-April 7, 2022). HIPPS combines: (1) an extension of a previously published pregnancy episode algorithm, (2) a novel algorithm to detect gestational age-specific signatures of a progressing pregnancy for further episode support, and (3) pregnancy start date inference. Clinicians performed validation of HIPPS on a subset of episodes. We then generated pregnancy cohorts based on gestational age precision and pregnancy outcomes for assessment of accuracy and comparison of COVID-19 and other characteristics. Results: We identified 628 165 pregnant persons with 816 471 pregnancy episodes, of which 52.3% were live births, 24.4% were other outcomes (stillbirth, ectopic pregnancy, abortions), and 23.3% had unknown outcomes. Clinician validation agreed 98.8% with HIPPS-identified episodes. We were able to estimate start dates within 1 week of precision for 475 433 (58.2%) episodes. 62 540 (7.7%) episodes had incident COVID-19 during pregnancy. Discussion: HIPPS provides measures of support for pregnancy-related variables such as gestational age and pregnancy outcomes based on N3C data. Gestational age precision allows researchers to find time to events with reasonable confidence. Conclusion: We have developed a novel and robust approach for inferring pregnancy episodes and gestational age that addresses data inconsistency and missingness in EHR data.

Race, poverty, and the lack of follow-up for Arkansas students that fail vision screenings: a cross-sectional study over 7 years.

PURPOSE: To analyze rates of follow-up eye care for students that failed school vision screenings over a 7-year period in 238 Arkansas school districts. METHODS: In this cross-sectional study, vision screening, demographic, socioeconomic, academic, and eye care provider data were collected. The main outcomes were referral rates, rates of follow-up eye care for students with failed vision screenings, and estimated associations between the rate of follow-up and school district and county-level characteristics, such as race, poverty, insurance coverage, academic achievement, and the number of eye care providers. RESULTS: A total of 1,744,805 vision screenings over 7 academic years (2013-2020) were included. The average screening rate was 35.4% across the study years. The screening failure rate ranged from 8.0% to 9.4%. Two-thirds of districts had a follow-up rate between 20% and 50%. 91% had follow-up rates of <60%. School districts with higher concentrations of White students (P < 0.001), higher graduation rates (P = 0.024), higher percentages of students on government-assisted insurance (P = 0.035), and higher standardized scores (P < 0.001) had higher rates of follow-up. There were no statistically significant relationships between the rate of follow-up eye care and the number of school nurses per school district or the number of ophthalmologists or optometrists per county. CONCLUSIONS: Arkansas children in our study cohort that failed vision screenings had inadequate follow-up eye care. Follow-up rates were associated with several key indicators of socioeconomic status.

Syphilis-related Ocular Inflammation in HIV-positive and HIV-negative Patients.

PURPOSE: To evaluate the clinical characteristics and visual outcomes of ocular syphilis in human immunodeficiency virus (HIV)-positive and negative patients. METHODS: We performed a retrospective chart review of all patients with newly diagnosed ocular syphilis between January 2015 and August 2021. RESULTS: 68 eyes of 41 patients (22 eyes in 12 HIV-positive patients, 46 eyes in 29 HIV-negative patients) were included. The most common ophthalmic feature was posterior uveitis in HIV-positive and anterior uveitis in HIV-negative patients; 58.3% (7 out of 12 patients) of HIV-positive and 48.2% (14 out of 29 patients) of HIV-negative patients were diagnosed with neurosyphilis. There were no significant differences in laterality, presence of concurrent rash, rapid plasma reagin (RPR) titers, presenting, and final corrected visual acuity between both groups. CONCLUSION: Nearly one-fifth of eyes may suffer from severe visual loss of ≤ 20/200. HIV co-infection may not result in visual outcomes in syphilis.

Cloud-controlled microscopy enables remote project-based biology education in underserved Latinx communities.

Project-based learning (PBL) has long been recognized as an effective way to teach complex biology concepts. However, not all institutions have the resources to facilitate effective project-based coursework for students. We have developed a framework for facilitating PBL using remote-controlled internet-connected microscopes. Through this approach, one lab facility can host an experiment for many students around the world simultaneously. Experiments on this platform can be run on long timescales and with materials that are typically unavailable to high school classrooms. This allows students to perform novel research projects rather than just repeating standard classroom experiments. To investigate the impact of this program, we designed and ran six user studies with students worldwide. All experiments were hosted in Santa Cruz and San Francisco, California, with observations and decisions made remotely by the students using their personal computers and cellphones. In surveys gathered after the experiments, students reported increased excitement for science and a greater desire to pursue a career in STEM. This framework represents a novel, scalable, and effective PBL approach that has the potential to democratize biology and STEM education around the world.

Modular automated microfluidic cell culture platform reduces glycolytic stress in cerebral cortex organoids.

Organ-on-a-chip systems combine microfluidics, cell biology, and tissue engineering to culture 3D organ-specific in vitro models that recapitulate the biology and physiology of their in vivo counterparts. Here, we have developed a multiplex platform that automates the culture of individual organoids in isolated microenvironments at user-defined media flow rates. Programmable workflows allow the use of multiple reagent reservoirs that may be applied to direct differentiation, study temporal variables, and grow cultures long term. Novel techniques in polydimethylsiloxane (PDMS) chip fabrication are described here that enable features on the upper and lower planes of a single PDMS substrate. RNA sequencing (RNA-seq) analysis of automated cerebral cortex organoid cultures shows benefits in reducing glycolytic and endoplasmic reticulum stress compared to conventional in vitro cell cultures.

Cardiovascular, Lymphatic, and Ocular Health in Space.

Life on Earth has evolved continuously under Earth's 1 G force and the protection of the magnetosphere. Thus, astronauts exhibit maladaptive physiological responses during space travel. Exposure to harmful cosmic radiation and weightlessness are unique conditions to the deep-space environment responsible for several spaceflight-associated risks: visual impairment, immune dysfunction, and cancer due to cosmic radiation in astronauts. The evidence thus reviewed indicates that microgravity and cosmic radiation have deleterious effects on the cardiovascular, lymphatic, and vision systems of astronauts on long-duration space missions. The mechanisms responsible for the decline in these systems are potentially due to cytoskeletal filament rearrangement, endothelial dysfunction, and muscular atrophy. These factors may alter fluid hemodynamics within cardiovascular and lymphatic vasculatures such that greater fluid filtration causes facial and intracranial edema. Thus, microgravity induces cephalad fluid shifts contributing to spaceflight-associated neuro-ocular syndrome (SANS). Moreover, visual impairment via retinal ischemia and altered nitric oxide production may alter endothelial function. Based on rodent studies, cosmic radiation may exacerbate the effects of microgravity as observed in impaired endothelium and altered immunity. Relevant findings help understand the extent of these risks associated with spaceflight and suggest relevant countermeasures to protect astronaut health during deep-space missions.

IoT cloud laboratory: Internet of Things architecture for cellular biology.

The Internet of Things (IoT) provides a simple framework to control online devices easily. IoT is now a commonplace tool used by technology companies but is rarely used in biology experiments. IoT can benefit cloud biology research through alarm notifications, automation, and the real-time monitoring of experiments. We developed an IoT architecture to control biological devices and implemented it in lab experiments. Lab devices for electrophysiology, microscopy, and microfluidics were created from the ground up to be part of a unified IoT architecture. The system allows each device to be monitored and controlled from an online web tool. We present our IoT architecture so other labs can replicate it for their own experiments.

Picroscope: low-cost system for simultaneous longitudinal biological imaging.

Simultaneous longitudinal imaging across multiple conditions and replicates has been crucial for scientific studies aiming to understand biological processes and disease. Yet, imaging systems capable of accomplishing these tasks are economically unattainable for most academic and teaching laboratories around the world. Here, we propose the Picroscope, which is the first low-cost system for simultaneous longitudinal biological imaging made primarily using off-the-shelf and 3D-printed materials. The Picroscope is compatible with standard 24-well cell culture plates and captures 3D z-stack image data. The Picroscope can be controlled remotely, allowing for automatic imaging with minimal intervention from the investigator. Here, we use this system in a range of applications. We gathered longitudinal whole organism image data for frogs, zebrafish, and planaria worms. We also gathered image data inside an incubator to observe 2D monolayers and 3D mammalian tissue culture models. Using this tool, we can measure the behavior of entire organisms or individual cells over long-time periods.

Histological Changes Observed in Placentas Exposed to Medication-Assisted Treatment.

INTRODUCTION: To compare the effects of medication-assisted treatment on the placenta in pregnant women with opioid use disorder and uncomplicated pregnancies. METHODS: This is a case-controlled study of pregnant women utilizing medication-assisted treatment, buprenorphine or methadone, which were matched to healthy uncomplicated controls by gestational age. Placental evaluations and neonatal outcomes were evaluated. Data analysis performed standard statistics and relative risk analysis with a p < 0.05 considered significant. RESULTS: There were 143 women who met the inclusion criteria: 103 utilizing MAT, 41 buprenorphine and 62 methadone, and 40 uncomplicated matched healthy controls. The incidence of delayed villous maturation was 36% in the medication-assisted group compared with 10% in controls (RR 3.6: 95% CI 1.37-9.43; p < 0.01). The placental weight was greater (541 ± 117 g versus 491 ± 117 g; p = 0.02), and the fetoplacental weight ratio was lower (5.70 ± 1.1 versus 7.13 ± 1.4; p < 0.01) in the medication-exposed pregnancies compared with controls. The mean birth weight of the MAT newborns was significantly lower than that of the healthy controls (3018 ± 536 g versus 3380 ± 492 g; p < 0.01). When evaluating the subgroups of the MAT newborns, the birth weight of the methadone-exposed newborns (2886 ± 514 g) was significantly lower than that of the buprenorphine-exposed newborns (3218 ± 512 g; p < 0.01). CONCLUSION: Medication-exposed pregnancies have a greater incidence of delayed villous maturation, a larger placental size, and a decreased fetoplacental weight ratio compared to the healthy controls. Larger long-term follow-up studies to evaluate outcomes with the presence of delayed villous maturation are needed.

Ocular complications of perioperative anesthesia: a review.

Ocular complications associated with anesthesia in ocular and non-ocular surgeries are rare adverse events which may present with clinical presentations vacillating between easily treatable corneal abrasions to more serious complication such as irreversible bilateral vision loss. In this review, we outline the different techniques of anesthetic delivery in ocular surgeries and highlight the incidence and etiologies of associated injuries. The changes in vision in non-ocular surgeries are mistaken for residual sedation or anesthetics, therefore require high clinical suspicion on part of the treating ophthalmologists, to ensure early diagnosis, adequate and swift management especially in surgeries such as cardiac, spine, head and neck, and some orthopedic procedures, that have a comparatively higher incidence of ocular complications. In this article, we review the literature for reports on the clinical incidence of different ocular complications associated with anesthesia in non-ocular surgeries and outline the current understanding of pathophysiological processes associated with these adverse events.

Perceptions of a Virtual Interview Exercise for Ophthalmology Residency Applicants.

Importance In-person interviews have traditionally been considered a crucial component of the residency application process. Virtual interviews (VIs) became the standard format for the 2020 to 2021 application cycle due to the novel coronavirus disease 2019 (COVID-19) pandemic. VIs offer a new perspective and challenge to this process which warrants unique considerations and further understanding of effects on applicants. Objective This study aimed to assess the perceived efficacy of a VI preparedness exercise for ophthalmology residency applicants in the 2021 residency application cycle. Design, Setting, and Participants A cross-sectional survey was distributed online. All participants in a mock VI exercise conducted via video-telecommunication technology were invited to complete the survey. Data collection occurred from October 12, 2020, to November 2, 2020. A follow-up survey after a match results released was distributed to all participants and data collection occurred from February 18, 2021, to February 25, 2021. Main Outcome and Measures Applicant demographics, comfort, and attitudes toward VIs and VI practice were the primary measurements of this study. Results Responses to the initial survey were received from all 35 participants (100%) in the VI mock interviews. There was a statistically significant difference between the pre- and postinterview responses for "How prepared do you feel for virtual interviews with residency programs?" ( p = 0.0003) and "How likely are you to practice virtual interviews with someone you know?" ( p = 0.0023). Participants reported feeling more prepared for VIs with residency programs after the mock interview ( p = 0.002). A greater proportion of participants responded with "Very Likely" after the mock interview in comparison to before the interview to the questions "How likely are you to practice interviews with someone you know?" ( p = 0.039) and "How likely are you to practice virtual interviews in the same room/area as you will during the official interview season?" ( p = 0.021). Of the 35 original participants, 20 completed the follow-up survey. There were an equal number of participants who responded either "Helped Somewhat" ( n = 9) or "Helped Greatly" ( n = 9) to "How much did the VI mock exercise help you for the actual interview season?" in the follow-up survey. The majority of follow-up survey respondents (17/20) reported that they had additional practice in the virtual environment for interviews after the VI mock exercise. There was no significant difference in perceived helpfulness of the VI mock exercise during the actual interview season between matched and unmatched participants. Conclusion and Relevance As residency applicants prepare for future VIs, practice and adequate preparation will be essential. In this study, implementation of a VI preparedness exercise had a positive impact on applicants' perception of their preparedness and intention to practice the format in the future.

An old disease on the rise: new approaches to syphilis in pregnancy.

PURPOSE OF REVIEW: Maternal and congenital syphilis infection is on the rise in the United States and worldwide. Without adequate testing or provider recognition of infection, treatment can be neglected resulting in significant perinatal morbidity and mortality. This review article discusses the epidemiology of T. pallidum, describes novel diagnostic tests, and considers the need to expand therapeutic options. RECENT FINDINGS: A new chemiluminescence immunoassay for use in the reverse-sequence algorithm is more sensitive and specific in pregnant women than previously noted and is helpful for identifying pregnant women at highest risk for neonatal congenital syphilis. Point-of-care testing may be used to detect early syphilitic disease and provide same-day testing and treatment. Randomized control trials of oral cefixime for treatment of syphilis are paving the way for potential use in pregnant women. Penicillin skin testing, challenge, and desensitization in pregnancy can be done safely. SUMMARY: Congenital syphilis is a preventable disease and treatable infection in the modern world, but we are still met with challenges in its eradication. We should proceed with advancing efficient laboratory testing, expanding medical therapy, and implementing public health measures to curb the rise of the disease.

Comparison of buprenorphine and methadone in the management of maternal opioid use disorder in full term pregnancies.

Objectives To compare pregnancy outcomes with medication assisted treatment using. methadone or buprenorphine in term mothers with opioid use disorder. Methods A cohort of women receiving medication assisted treatment with either methadone or buprenorphine were identified from delivery records over a 10-year period. Women were excluded with delivery <37 weeks, multiple gestations, or a known anomalous fetus. Maternal demographics, medications, mode of delivery, birthweight, newborn length of stay, and neonatal abstinence syndrome were extracted. The study was IRB approved and a p-value of <0.05 was significant. Results There were 260 women, 140 (53.8%) with methadone use and 120 (46.2%) with buprenorphine use. Groups were similar for maternal age, race, parity, homeless rate, tobacco use, mode of delivery and incidence of neonatal abstinence syndrome. The methadone group had a lower mean newborn birthweight (2874±459 g) and a greater incidence of low birth weight (11.4%) than the buprenorphine group (3282±452 g; p<0.001 and 2.5%; p=0.006). The incidence of neonatal abstinence syndrome was similar between groups (97% methadone vs. 92.5% buprenorphine; p=0.08). The methadone group had a longer newborn length of stay (11.4+7.4 days) and more newborn treatment with morphine (44.6%) than the buprenorphine group (8.2+4.4 days; p<0.001 and 24.2%; p<0.001). Maternal methadone use was an independent predictor for a newborn length of hospital stay >7 days (OR 3.61; 95% confidence interval 1.32-9.86; p=0.01). Conclusions Medication assisted treatment favors buprenorphine use when compared to. methadone with an increased birthweight, reduced need for newborn treatment, and a shorter newborn length of stay in term infants.

Hemoglobin S/OArab: Retinal Manifestations of a Rare Hemoglobinopathy.

Hemoglobin S/OArab (Hgb S/OArab) disease is a rare hemoglobinopathy which presents similarly to sickle cell retinopathy, with only three prior reports that describe associated retinal findings. In this report, we present ophthalmic examination findings in 2 patients with Hgb S/OArab. One patient exhibited peripheral ischemia and sunburst lesions without neovascular disease, and the other patient developed proliferative retinopathy of both eyes and multiple posterior-pole branch retinal artery occlusions in one eye. To our knowledge, this is the first case of retinal arterial occlusive disease in Hgb S/OArab, and the first report of fundus autofluorescence and OCT angiography in Hgb/OArab retinopathy.

Podocyte-Specific Loss of Krüppel-Like Factor 6 Increases Mitochondrial Injury in Diabetic Kidney Disease.

Mitochondrial injury is uniformly observed in several murine models as well as in individuals with diabetic kidney disease (DKD). Although emerging evidence has highlighted the role of key transcriptional regulators in mitochondrial biogenesis, little is known about the regulation of mitochondrial cytochrome c oxidase assembly in the podocyte under diabetic conditions. We recently reported a critical role of the zinc finger Krüppel-like factor 6 (KLF6) in maintaining mitochondrial function and preventing apoptosis in a proteinuric murine model. In this study, we report that podocyte-specific knockdown of Klf6 increased the susceptibility to streptozotocin-induced DKD in the resistant C57BL/6 mouse strain. We observed that the loss of KLF6 in podocytes reduced the expression of synthesis of cytochrome c oxidase 2 with resultant increased mitochondrial injury, leading to activation of the intrinsic apoptotic pathway under diabetic conditions. Conversely, mitochondrial injury and apoptosis were significantly attenuated with overexpression of KLF6 in cultured human podocytes under hyperglycemic conditions. Finally, we observed a significant reduction in glomerular and podocyte-specific expression of KLF6 in human kidney biopsies with progression of DKD. Collectively, these data suggest that podocyte-specific KLF6 is critical to preventing mitochondrial injury and apoptosis under diabetic conditions.

Krüppel-Like Factor 15 Mediates Glucocorticoid-Induced Restoration of Podocyte Differentiation Markers.

Podocyte injury is the inciting event in primary glomerulopathies, such as minimal change disease and primary FSGS, and glucocorticoids remain the initial and often, the primary treatment of choice for these glomerulopathies. Because inflammation is not readily apparent in these diseases, understanding the direct effects of glucocorticoids on the podocyte, independent of the immunomodulatory effects, may lead to the identification of targets downstream of glucocorticoids that minimize toxicity without compromising efficacy. Several studies showed that treatment with glucocorticoids restores podocyte differentiation markers and normal ultrastructure and improves cell survival in murine podocytes. We previously determined that Krüppel-like factor 15 (KLF15), a kidney-enriched zinc finger transcription factor, is required for restoring podocyte differentiation markers in mice and human podocytes under cell stress. Here, we show that in vitro treatment with dexamethasone induced a rapid increase of KLF15 expression in human and murine podocytes and enhanced the affinity of glucocorticoid receptor binding to the promoter region of KLF15 In three independent proteinuric murine models, podocyte-specific loss of Klf15 abrogated dexamethasone-induced podocyte recovery. Furthermore, knockdown of KLF15 reduced cell survival and destabilized the actin cytoskeleton in differentiated human podocytes. Conversely, overexpression of KLF15 stabilized the actin cytoskeleton under cell stress in human podocytes. Finally, the level of KLF15 expression in the podocytes and glomeruli from human biopsy specimens correlated with glucocorticoid responsiveness in 35 patients with minimal change disease or primary FSGS. Thus, these studies identify the critical role of KLF15 in mediating the salutary effects of glucocorticoids in the podocyte.

DNA methylation of the serotonin transporter gene (SLC6A4) is associated with brain function involved in processing emotional stimuli.

BACKGROUND: The aim of the present study was to investigate the association of fMRI blood oxygen-level dependent (BOLD) reactivity with the level of epigenetic methylation of SLC6A4 in blood DNA from a sample of healthy participants and patients with major depressive disorder (MDD). METHODS: We investigated patients with MDD and healthy controls using fMRI and an emotional attention-shifting task. We assessed site-specific DNA methylation of a previously characterized SLC6A4 region in peripheral blood DNA using pyrosequencing. RESULTS: Our study involved 25 patients with MDD and 35 healthy controls. Activation in the anterior insula elicited by negative emotional content was significantly positively associated with the degree of SLC6A4 methylation. Significantly negative associations were observed between activation in the posterior insula and the degree of SLC6A4 methylation when judging the geometry of pictures after seeing negative in contrast to positive emotional stimuli. Healthy controls with a high degree of SLC6A4 methylation depicted significantly more activity elicited by positive stimuli in limbic regions and more activity elicited by negative stimuli in limbic as well as cognitive control regions than those with a low degree of SLC6A4 methylation. LIMITATIONS: It is impossible to measure methylation directly in the brain and thus we assessed peripheral methylation of SLC6A4. Since the association was cross-sectional, no conclusion about cause and effect can be drawn. CONCLUSION: Our study provides further support to the hypothesis that particular DNA methylation states that are associated with brain function during emotion processing are detectable in the periphery.

DNA methylation of the serotonin transporter gene in peripheral cells and stress-related changes in hippocampal volume: a study in depressed patients and healthy controls.

Serotonin plays an important role in the etiology of depression. Serotonin is also crucial for brain development. For instance, animal studies have demonstrated that early disruptions in the serotonin system affect brain development and emotion regulation in later life. A plausible explanation is that environmental stressors reprogram the serotonin system through epigenetic processes by altering serotonin system gene expression. This in turn may affect brain development, including the hippocampus, a region with dense serotonergic innervations and important in stress-regulation. The aim of this study was to test whether greater DNA methylation in specific CpG sites at the serotonin transporter promoter in peripheral cells is associated with childhood trauma, depression, and smaller hippocampal volume. We were particularly interested in those CpG sites whose state of methylation in peripheral cells had previously been associated with in vivo measures of brain serotonin synthesis. Thirty-three adults with Major Depressive Disorder (MDD) (23 females) and 36 matched healthy controls (21 females) were included in the study. Depressive symptoms, childhood trauma, and high-resolution structural MRI for hippocampal volume were assessed. Site-specific serotonin transporter methylation was assessed using pyrosequencing. Childhood trauma, being male, and smaller hippocampal volume were independently associated with greater peripheral serotonin transporter methylation. Greater serotonin transporter methylation in the depressed group was observed only in SSRI-treated patients. These results suggest that serotonin transporter methylation may be involved in physiological gene-environment interaction in the development of stress-related brain alterations. The results provide some indications that site-specific serotonin transporter methylation may be a biomarker for serotonin-associated stress-related psychopathology.