bluuebunny/crossref_metadata_2025 huggingface dataset:
Simplified, open‐source analysis of DNA‐binding proteins
Authors: Gates Evan , Lin Jason , Dandpat Shiba , Balassy Zsombor , Simpsom Trey , Groen Bas , Driessen Rosalie , Llauró Aida
Published: May 2022

The structure, regulation, and maintenance of DNA is critical to cellular function. Errors in these underlying mechanisms play a major role in diseases such as cancer. As a result, insights into the molecular details are needed to further our understanding, improve biological models, and develop novel therapeutics. Single‐molecule techniques have emerged as useful tools for uncovering the activity and function of DNA‐binding proteins in real time. Furthermore, the data from single‐molecule techniques allows for an accurate quantification of many molecular properties, such as binding location, kinetics, etc. For many researchers, however, developing an analysis pipeline to quantify single‐molecule data is challenging due to the required time, resources, and expertise. To address these obstacles, we developed an automated, open‐source image processing and data quantification toolbox. We show that our toolbox allows for easy, rapid, and intuitive processing of single‐molecule data. Using systems like the gene editing CRISPR/Cas9 system, we demonstrate that our toolbox 1) reports DNA binding specificity at the base‐pair level, 2) measures binding constants (kon/koff) from the direct visualization of individual complexes interacting with DNA, and 3) allows for the accurate quantification of enzymatic processivity and protein diffusion. Overall, we show that our simplified, open‐source data analysis platform allows for automated processing of single‐molecule data and rapid characterization of virtually any DNA‐binding protein or polymerase. This toolbox is accessible to all researchers and will enhance efforts to understand a wide range of normal and pathological DNA processes.
A New Method for Quantifying 3D Skeletal Morphology in Living Subjects Using Low‐Dose Stereo Radiographs
Authors: Lawrence Austin B. , Boeyer Melanie E. , Middleton Kevin M.
Published: May 2022

Accurate and repeatable assessments of three‐dimensional (3D) skeletal morphology are critical to interpretations of vertebrate functional anatomy. In skeletal specimens, these goals can be accomplished using in‐silico models derived from 3D laser scan, photogrammetry, or computed tomography (CT) data. Assessments of skeletal morphology in living subjects, especially humans, are more challenging and are often either cost‐prohibitive (e.g., magnetic resonance) or expose subjects to high doses of ionizing radiation (e.g., CT). Here, we present a new, open‐source method for assessing 3D skeletal morphology using ultra‐low dose biplanar radiographs. Biplanar radiographs were obtained using an EOS Imaging System (EOS Imaging, Inc., Paris, France). A custom‐built calibration frame with radiopaque markers was scanned using this system on multiple days in multiple positions inside the scanning space. We quantified the 3D coordinates of markers by placing landmarks at the center of each marker in both anterior‐posterior and lateral radiographs. Landmark data for each scan were collected independently by two observers to determine inter‐observer error. 3D coordinates of landmarks in each scan differed by <1 mm between observers, suggesting strong repeatability in assessing morphology of living subjects. To assess distortion in EOS images, we constructed a second calibration device, a flat plate with radiopaque markers at the corners of three squares with 10 cm, 20 cm, and 30 cm sides, respectively, surrounding an origin point. The calibration plate was scanned with the origin point at the center of the scan area and the sides of the squares parallel to the detectors. We identified distortion at the edges of EOS images which could significantly impact measurements on skeletal features near the margins of the scan area (i.e., hips, pelvis, lower limbs) and developed a method to mathematically correct for resulting distortion in landmark data. Finally, we demonstrate this method using measurements of iliac blade and spinous process orientation in a sample of living human participants. This method will be applicable to studies of multiple human cohorts, including symptomatic and asymptomatic groups, as well as to other biplanar radiograph platforms and study organisms.
Chronic Ethanol Exposure of Basal Cells causes Barrier Dysfunction of Differentiated Mucocilliary Monolayers
Authors: Easley Kristen F. , Mehta Ashish J. , Staitieh Bashar S. , Koval Michael
Published: May 2022

Alcohol use disorder affects over 14 million adults and causes 88,000 deaths each year in the United States. Chronic alcohol use significantly increases people’s risk of developing lung infections and acute respiratory distress syndrome (ARDS). This increased sensitivity to injury is a condition known as alcoholic lung syndrome, and it is caused by dysfunction of the lung immune system and alveolar epithelial barrier. However, little is known about how alcohol impacts epithelial cells in the conducting airway, i.e. the trachea and bronchi, which is the first line of defense against infectious pathogens in the lungs. We hypothesize that in vitro ethanol exposure decreases barrier function of human primary bronchial epithelial cells. To test this, we dosed cells with ethanol at various timepoints: 1) during both the basal‐cell expansion and differentiation stages 2) the first 14 days of differentiation on Transwell permeable supports at air liquid interface (ALI); 3) beginning on day 14 of differentiation and continuing for 10 days. We treated cells with 10mM, 60mM or 100mM ethanol almost daily and measured barrier function by transepithelial electrical resistance (TER). We found that ethanol caused a dose‐dependent decrease in barrier function, but only in the group treated during both the expansion and differentiation stages. This suggests that chronically dosing basal cells with ethanol is required for barrier dysfunction of a fully differentiated cell monolayer. Additionally, we cultured primary airway epithelial cells isolated from healthy and alcoholic patients on Transwell permeable supports and differentiated them in vitro to determine if in vivo exposure caused long‐term changes to tight junction protein levels. It is known that the transmembrane protein junctional adhesion molecule A (JAM‐A) regulates barrier function by controlling the paracellular flow of small molecules as part of the tight junction barrier. Our results show in response to alcohol exposure, cells have lower JAM‐A protein expression compared to healthy cells. Together, these results indicate that chronic alcohol exposure detrimentally alters the conducting airway epithelial cell barrier potentially through the loss of JAM‐A. Future directions include determining if other tight junction proteins are altered by alcohol and whether the alcohol‐induced reduction in JAM‐A is sufficient to cause barrier dysfunction in airway cells.
C. elegansAttraction Learning in casy‐1Mutants
Authors: Gilpin Megan E. , Crowe Kelly E.
Published: May 2022

C. eleganshave a simple nervous system with approximately 300 neurons and exhibit well‐documented learning‐specific behaviors. Several genetic mutations, such as in the casy‐1 genes, have been shown to disrupt these learning behaviors in C. elegans. The casy‐1 gene encodes a calcium‐binding protein that is important for synapse regulation, and has shown deficits in learning behaviors. While casy‐1mutant C. eleganshave specifically shown deficits in aversion learning, their attraction learning is not well characterized. Here, we examined the effects of casy‐1 mutation on attraction learning in C. elegans. To do so, we have quantified C. elegans locomotor behaviors such as reversal frequency, run length, and pause frequency in wild‐type and casy‐1 mutant C. elegansin the context of a salt chemotaxis learning assay. This work helps to elucidate the effect of casy‐1on learning behaviors, which is particularly relevant as this gene is an ortholog of mammalian calsyntenins.
Lectin Biomarker Development in a Mouse Model of GNE Myopathy
Authors: Bradley Kara E. , Crowe Kelly E.
Published: May 2022

GNE Myopathy (GNEM) is a rare autosomal recessive disease that causes progressive muscle wasting. GNEM occurs due to mutations in the GNE gene that encodes a key enzyme of the biosynthetic pathway of sialic acid, a terminal glycan on extracellular chains of muscle cells. Mutations in the GNE gene cause a significant reduction in the production of sialic acid, which is associated with muscle weakness and wasting. In order to assess the cellular effects of sialic acid‐producing therapies in clinical studies, a robust biomarker is needed to quantify the levels of sialic acid in muscle. Several lectins have shown robust and replicable changes after sialic acid perturbations in human cells. However, the ideal lectin biomarker for GNEM would also need to reflect relevant sialic acid changes in mouse models of GNEM, which are used to test the efficacy of gene therapies during pre‐clinical studies. Here, we compare lectin staining in skeletal muscle of wild type mice and the GNED207VTgGne‐/‐ mouse model of GNEM. Validation of a biomarker in this mouse model can facilitate pre‐clinical studies of GNEM gene therapy in mice.
CD4+T Cells cause increased glucose, mitochondrial dysfunction, and hypertension in a Novel Pregnant Rodent Model of Gestational Diabetes Mellitus
Authors: Deer Evangeline , Williams Jan Michael , Amaral Lorena M. , Fitzgerald Sarah , Herrock Owen , Turner Ty , Campbell Nathan , LaMarca Babbette
Published: May 2022

Hypertensive (HTN) disorders of pregnancy complicate approximately 10% of all pregnancies worldwide and contribute to maternal and fetal morbidity and mortality. HTN increases risks for greater pathophysiology in gestational diabetes mellitus (GDM) pregnant women, thereby supporting a need for controlling maternal glucose and blood pressure. GDM is a major medical complication characterized by hyperglycemia and/or β‐cell dysfunction and associated with increased inflammatory cytokines, oxidative stress, and activated CD4+ T cells. Streptozotocin (STZ) is utilized in nonpregnant rats to induce β‐cell destruction causing features of diabetes. However, STZ is not ideal for pregnancy and leads to unsuccessful pregnancy outcomes, and, therefore other ways to establish animal models of GDM must be pursued. Previously, we have shown CD4+T cells from a rat model of preeclampsia causes HTN and mitochondrial (mt) dysfunction/ROS compared to normal pregnant (NP) rats. Therefore, we hypothesize CD4+ T cells cause pancreatic β‐islet cell destruction/dysfunction via mt dysfunction/ROS leading to HTN during pregnancy. To examine our hypothesis, we adoptively transferred CD4+ T cells from STZ Dahl diabetic rats to cause features of GDM in pregnant Sprague Dawley (SD) rats. Circulating CD4+ T cells were isolated from STZ induced diabetic Dahl virgin female rats and injected into pregnant SD rats on gestational day (GD) 12. On GD18 carotid catheters were inserted. On GD19, blood pressure (MAP) and tissues were collected and glucose levels were measured after 2h fasting in STZ CD4+ T cell recipients (GDM) and NP controls. Mt respiration and mtROS was measured in isolated mitochondria using an Oxygraph 2K and by production of H2O2 using a fluorescent microplate reader, respectively. A student’s t‐test was used for statistical analysis. On GD19, MAP increased to 106±4 mmHg (n=5, p<0.05) in GMD pregnant rats compared to control NP rats 90±2 mmHg (n=4). Blood glucose levels were elevated in GDM rats (138 ± 6 mg/dl, n=5, p<0.05) compared to NP controls (86 ± 7 mg/dl, n=4). Placental state 3 (27±3 vs 50.3±3 pmol/sec/mg, p<0.05) respiration rates, indicative of ATP production, was reduced in GDM rats (n=5) cells compared to NP controls (n=4). Placental mtROS was significantly increased in GDM rats (266 ± 34 % gated, n=5, p<0.05) compared to NP rats (100 ± 48 % gated, n=4). Collectively, the data indicate adoptive transfer of STZ CD4+ T cells causes increased circulating glucose, placental mt dysfunction and mtROS and HTN during pregnancy. These data demonstrate the importance of CD4+T cells in mechanisms causing the pathophysiology of GDM, and also introduces a potential novel animal model of GDM.
Metabolic Syndrome and SIRT1 Mutation Impair Ca2+Channels in Coronary Smooth Muscle Cells of Ossabaw Miniature Swine
Authors: Thamba Aish , Reed John , Strobel John , Byrd James , Alloosh Mouhamad , Coutts Alex , Sturek Michael
Published: May 2022

Background

Normal metabolic health and sirtuin1 (SIRT1) by its deacetylase activity maintain voltage‐gated Ca2+ channel (VGCC) function that promotes gene expression to maintain contractile coronary smooth muscle (CSM) phenotype. MetS impairs VGCC function and sarcoplasmic reticulum (SR) Ca2+ release, contributing to dedifferentiation of CSM to proliferative and osteogenic phenotypes.

Hypothesis

MetS and impaired SIRT1 will impair VGCC and SR Ca2+ release channels in CSM.

Methods

Using CRISPR/Cas9 methodology a point mutation (SIRT1L100P) was made in Ossabaw miniature swine to mimic the naturally occurring mutation in humans and decrease SIRT1 activity, thereby resulting in hyperacetylation (Ac) of Ca2+transporters and impaired function. Four groups of pigs were used to analyze genotype and diet interactions: wild type lean, SIRT1 lean, wild type MetS, SIRT1 MetS. Pigs were age 4 months at the start and fed normal chow (lean) or atherogenic diet (MetS) for 7 months. CSM cells were enzymatically dispersed and Ca2+ measured with fura‐2. Depolarization with 80 mM K+ assessed Ca2+ influx through VGCC and the peak Ca2+ response to 5 mM caffeine to open SR Ca2+ release channels assessed the caffeine‐sensitive SR Ca2+ store.

Results

Two‐way ANOVA showed SIRT1 mutation (p=0.02) and MetS diet (p<0.0001) significantly decreased VGCC function independently, but not additively or synergistically. Interaction of SIRT1 genotype and MetS diet was significant (p<0.0001). No significant effect of genotype or diet was observed on SR calcium store release.

Conclusion

SIRT1L100P mutation is likely to contribute to coronary atherosclerosis and SIRT1 activators may be effective therapies.
Lectin Staining for the Detection of In Vitro Sialic Acid Alterations
Authors: Roth Mackenzie , Crowe Kelly E.
Published: May 2022

GNE Myopathy (GNEM) is a rare disease caused by a mutation in the UDP‐N‐acetylglucosamine 2‐epimerase/N‐acetylmannosamine kinase (GNE) gene, which catalyzes sialic acid synthesis. GNEM results in decreased sialic acid and loss of strength in the muscles due to an unclear mechanism. Currently, there is no available treatment for GNEM. However, gene therapy approaches involving delivering a corrected copy of the GNE gene into GNEM patient muscle are currently being developed. A robust biomarker is needed to assess the efficacy of such a therapy. Here, we assess the ability of a panel of lectins, which are glycan‐binding proteins, to reflect changes in sialic acid levels in vitro that occur when sialic acid is enzymatically removed via sialidase or when its production is impaired due to mutations in the GNEgene. This work probes the ability of these lectins to reflect changes in sialic acid levels, which can inform biomarker development for GNE myopathy therapies.
Exercise Training Upregulates SK+ Channel Function in the Hypothalamic Paraventricular Nucleus (PVN) of Animals
Authors: Miodonski Gregory , Bruning Jessica , Johnson Christian , Gu Mingjun , Shan Zhiying , Chen Qing‐Hui
Published: May 2022

Increased sympathetic outflow is a hallmark of cardiovascular disease (CVD). Previously, our lab demonstrated that small conductance Ca2+ activated K channels (SK+) expressed in the PVN play a crucial role in regulating sympathetic outflow, and SK+ currents were significantly decreased in rats with salt‐sensitive hypertension (SSH). Since SK+ channels among autonomic PVN neurons play a crucial role in regulating sympathetic outflow in SSH, antihypertensive therapies targeted at upregulating SK+ channel expression in the PVN should be pursued. Exercise is a powerful, non‐pharmacological intervention to treat CVD, however, the interaction between SK+ channels among the hypothalamic PVN and exercise is unknown. The objective is to determine the role of exercise in regulating SK+ channels function and sympathetic outflow as well as arterial blood pressure (ABP) in animals. We hypothesize that exercise upregulates SK+ channel function in the PVN and attenuates sympathetic outflow. To test this hypothesis, Sprague Dawley (SD) rats are divided into sedentary (SED) and exercise (EXT) groups based on their willingness to run on a motorized treadmill. Exercise intensity is determined through a maximum exercise capacity test and rats run at 70% of their exercise capacity speed for 30 minutes 5 days/week for 8‐10 weeks. Then rats are anesthetized and renal sympathetic nerve activity (RSNA) and ABP are recorded following bilateral PVN injections of apamin, a selective SK+ channel blocker (0.25mM, 60nL/side). We found that the RSNA response to PVN apamin trended toward significance in EXT rats compared with SED rats (342.1 ± 164.2 % baseline, n=6 vs 120.9 ± 25.1 % baseline, n=3; p = 0.0598). The corresponding ABP response to apamin was not significantly different in EXT rats compared with SED rats (16.5 ± 5.7 mmHg, n=6 vs 24.6 ± 7.2, n=3; p = 0.1039). Collectively, these results indicate exercise activates PVN SK+ channels function and reduces sympathetic outflow. The improvement of channel function or increase in expression may contribute to the beneficial effects of exercise on reducing sympathetic outflow in patients with CVD, including hypertension.
Novel Functional FOXC1 Variants in Familial and Sporadic Atrial Septal Defect
Authors: Chen Huan‐Xin , Hou Hai‐Tao , Wang Xiu‐Li , Yang Qin , He Guo‐Wei
Published: May 2022

Objective

Atrial septal defect (ASD) is one of the most common forms of congenital heart disease. Genetic defects play important roles in the pathogenesis of ASD but are complex and unclear. This study tries to discover the genetic pathogenesis in familial and sporadic ASD patients.

Methods

In Stage I, in a family with 30 members whole exome sequencing was performed in 4 ASD patients and the identified genetic variants were screened in the rest of the family members. In Stage II, 335 unrelated sporadic and isolated ASD patients were enrolled to test whether the genetic variants were involved in these patients. In Stage III, biological functions of the variants were elucidated at the cellular level and the CRISPR/Cas9 was used to construct variant‐specific mutant mice in order to observe the abnormality of the heart structure.

Results

A novel heterozygous missense variant, FOXC1 c.518G>A (p.R173H) was identified in the ASD family by whole exome sequencing. Subsequently, other 4 heterozygous variants in FOXC1, including 2 novel missense variants: c.556‐558delAAG (p.K186del) and c.559G>A (p.D187N) were identified in unrelated 335 sporadic ASD patients. In dual‐luciferase reporter assay, the transcriptional activity of R173H decreased to almost 30% of the wild‐type. In the Foxc1 R173H site‐specific mutant mice, the atrial septum became very thin in tissue slices of the heart that was similar to the echocardiographic finding in the above family members.

Conclusion

Variants in FOXC1 likely cause cardiac anomaly, particularly ASD. These findings provide a new insight into genetic mechanisms and counseling of familial and sporadic ASD.
The Cognitive Load of Learning Anatomy in a Virtual World
Authors: Touliopoulos Evangelea , Ives Rowan , Chan Sachi , Lohit Simran , Reis Isabella R. , Sinha Sakshi , Nehru Amit , DeYoung Veronica , Mitchell Josh P. , Brewer‐Deluce Danielle , Sonnadara Ranil , Wainman Bruce C.
Published: May 2022

Introduction

Cognitive load refers to the amount of working memory that is being used in a task, like memorizing the anatomical landmarks on distinct boney specimens. Critically, cognitive load may be compromised when the load imposed by the environment and the content to be learned together exceeds a student’s capacity. Previous research shows that stereoscopic materials delivered in virtual reality (VR) can be more mentally taxing compared to desktop (i.e., two dimensional) delivery but may be similar to that encountered in real life. There is no data on the cognitive load of autostereoscopic displays. Given the increased reliance upon digital media for teaching in learning in anatomy classrooms, it is prudent to better understand the cognitive load imposed on learners across a variety of modalities employed.

Methods

Cognitive load will be compared across three different learning modalities: immersive virtual reality (VR, displayed on the Oculus Quest 2TM), autostereoscopic (displayed on the AlioscopyTM screen), and an identical printed, physical model. During a four‐minute learning phase, undergraduate students, with no prior formal anatomy education, will learn 10 anatomical landmarks on a displayed bony model (calcaneus, zygomatic bone, or hemipelvis) in each of the three modalities. A Stroop test will be administered as a secondary task throughout the learning phase to evaluate cognitive load. Stroop test reaction time, and accuracy of the participants' responses to the Stroop test will be recorded. After the learning phase, an untimed, recognition‐based test will be administered wherein participants will be asked to recall the ten landmarks learned with the aid of a 3D‐printed bone identical to the one used in the learning phase. Performance will be evaluated based on landmarks correctly identified and the results will be correlated with cognitive load measured in each learning modality.

Results

We hypothesize that the cognitive load will be highest for VR when compared to the cognitive load on the AlioscopyTM and physical model modalities which would manifest as lower reaction times and/or accuracy on the Stroop test. Further, we hypothesize that cognitive load will inversely correlate with the recognition test performance.

Conclusion

The results of this study will allow educators and students to make informed decisions when deciding which learning modalities should be used for anatomy education or any other education that requires nominative learning on complex objects. Understanding which modalities minimize cognitive load and improve learning will help improve outcomes and allow for more efficient anatomical education.
Mental Stress Pressor Response and Post‐Stress Aortic Wave Reflection
Authors: Vyas Aditi P. , Thivierge Grant S. , Toorongian Colleen A. , Larson Robert A. , Durocher John J.
Published: May 2022

Reactivity to mental stress has been linked to cardiovascular risk, and is shown to negatively influence aortic wave reflection for up to an hour after acute mental arithmetic. It has been postulated that sympathetic catecholamine release during the stressful task drives the sustained elevation in wave reflection. In the present study we sought to determine how muscle sympathetic nerve activity (MSNA) and mean arterial pressure (MAP) reactivity influence post mental stress aortic augmentation index (AIx). Twenty‐seven volunteers (25±1 years) with resting blood pressure ≥120/80 mmHg participated in the present study. Baseline pulse wave analysis and pulse wave velocity recordings were assessed on participants after 10 minutes of supine rest. Participants were then instrumented for measurements of MSNA (microneurography), beat‐to‐beat blood pressure (finger plethysmography), and heart rate (3‐lead ECG). Subsequently MSNA, blood pressure, and heart rate were measured during a 5‐minute baseline, 5‐minute mental stress task (serial subtraction), and 5‐minute recovery. Finally, aortic wave reflection (i.e. AIx) was reassessed 10 minutes after completion of mental stress. Statistical analyses included paired t‐tests to compare MSNA, MAP, heart rate (HR), and perceived stress during baseline and mental stress. We used standard multiple regression with change in AIx as the dependent variable and changes in MSNA, MAP, and perceived stress as the independent variables. Means were considered significantly different when p<0.05. The mental stress task significantly increased HR (Δ15±2 beats/minute), MAP (Δ14±1 mmHg), and perceived stress (Δ1.9±0.1 a.u.), while MSNA was not significantly increased. There was however a range of changes in MSNA from ‐13 to +20 bursts/minute. The change in MAP during mental stress was a significant predictor (ꞵ=0.47; p=0.03) of the change in AIx (post‐stress vs. baseline). Changes in MSNA and perceived stress were not predictors of mental stress‐related changes in AIx. Our results indicate that the aortic wave reflection responses to mental stress appear to be linked to the pressor response, but not sympathetic or perceived stress responses. This is novel preliminary data that suggests there may not be a direct link between sympathetic activation and aortic wave reflection following cognitive stress.
LHX2 Regulates Shh Expression in the Limb Independent of the ZPA Regulatory Sequence
Authors: Whitley Kristen L. , Britton Jessica C. , Pira Charmaine U. , Oberg Kerby C.
Published: May 2022

During limb development, proximal‐distal patterning is controlled by fibroblast growth factors (FGFs) secreted from the apical ectodermal ridge (AER), while anterior‐posterior expansion is coordinated by sonic hedgehog (SHH) released from the zone of polarizing activity (ZPA). Both FGFs and SHH regulate each other through an autoregulatory loop. The transcription factor LIM homeodomain 2 (LHX2) is an intermediate of the Fgf‐to‐Shh portion of the loop. In the absence of Lhx2 (and its redundant family member, Lhx9), Shh expression is markedly reduced. However, the mechanism by which LHX2 mediates FGF‐regulation of Shh is unclear. Shh is regulated by a limb‐specific enhancer called the ZPA Regulatory Sequence (ZRS). Using in silicoanalysis, our lab has identified two putative LHX2 binding sites within the ZRS. We hypothesized that LHX2 regulates Shh expression directly via the ZRS. To determine if LHX2 binding sites were necessary for ZRS activity, we mutated both LHX2 binding sites within a control tk‐ZRS‐eGFP reporter construct using site‐directed mutagenesis. We electroporated these constructs into the presumptive limb buds of chicken embryos (Hamburger‐Hamilton stage 14) and assessed for changes in ZRS activity in the limb buds 48 hours post transfection via fluorescence microscopy. We found that the mutation of both LHX2 binding sites within the ZRS resulted in activity similar to our control. These results suggest that LHX2 may not bind directly to the ZRS in its regulation of Shh. Instead, LHX2 may use another pathway to regulate Shhexpression. Further experiments are needed to confirm this connection. Investigating the mechanism by which LHX2 regulates Shh will provide insight into how LHX2 contributes to coordinated patterned‐limb development.
Intermittent Cocaine Self‐Administration Infusions Increases Subthreshold Activity in Putative Dopaminergic Neurons of The Ventral Tegmental Area
Authors: Calo Cristhian G. , Velez Omaris , Bosque Karl , Consuegra Daisy , Vazquez Rafael , Jimenez‐Rivera Carlos A.
Published: May 2022

Intermittent Access (IntA) cocaine self‐administration is a protocol suggested to better simulate human addictive behavior due to the intermittent pattern of drug administration. IntA is also known to produce incentive salience and psychomotor sensitization. It is documented that IntA produces a neurochemical sensitization of the mesolimbic dopamine (DA) system by increasing both release and uptake of DA. The ventral tegmental area (VTA) DA neurons display a prominent mixed cation current conductance known as the hyperpolarization‐activated cyclic nucleotide current, or Ih. Neural processes such as resting membrane potential, firing frequency modulation, and synaptic integration are influenced by the Ih. Previous results from our laboratory demonstrated that Ih amplitude and membrane capacitance of putative VTA DA neurons are significantly reduced after cocaine sensitization. This Ihand capacitance reduction resulted in an increased temporal summation, mean depolarization and excitatory postsynaptic potential (EPSP) amplitude, all related to an enhanced neuronal excitability state. It is not known how IntA alters the intrinsic properties of VTA DA cells. Since the cocaine sensitization model involves experimenter administered drug injections (non‐contingent) it is important to determine if electrophysiological changes in VTA DA cells are present when drugs are self‐administered (contingent). In the present study we explored if synaptic integration, membrane capacitance and cell activity alterations are present after exposure to cocaine IntA. Our hypothesis is that IntA enhances synaptic integration and neuronal excitability of VTA DA cells. Whole‐cell patch‐clamp technique in rat brain slices was used to inject a 33‐Hz train of 5 αEPSCs (α = 5 ms; Imax = 50) into the soma of putative VTA DA neurons when clamped at ‐70 mV and analyze the effects of cocaine IntA, and passive cocaine infusions (yoked controls) on synaptic integration. Increasing depolarizing current injections were used to evaluate how neurons respond to a depolarizing stimulus. Our results demonstrate that an IntA protocol, but not passive cocaine infusions, produces a significant increase in the number of APs (P<0.05). Temporal summation was increased at depolarized potentials in the IntA group and Yoked controls (P<0.0001). These results suggests that neuronal activity regulation is dependent on associative learning to drug cues. The findings also suggest that enhanced synaptic integration could possibly be a cocaine‐induced pharmacological effect.
Mathematical Modeling of Protracted HCMV Replication using Genome Substrates and Protein Temporal Profiles
Authors: Monti Christopher E. , Mokry Rebekah , Schumacher Megan , Dash Ranjan , Terhune Scott
Published: May 2022

Rationale

Clinical manifestations of human cytomegalovirus (HCMV) infection, a major cause of morbidity and mortality in the immunocompromised, are associated with the lytic replication cycle. Lytic replication occurs over 96 hrs and is marked by viral DNA (vDNA) synthesis and expression of viral proteins, the latter of which can be generalized into distinct temporal classifications. Given the complexity of the lytic replication cycle, mathematical modeling can be used to probe this elaborate system. Recently, mathematical modeling has been used to study the early lytic replication cycle and the transition between lytic and latent cycles. To date, there appears to be few modeling studies focusing on the late phases of the lytic replication cycle.

Method

vDNA replication kinetics were probed using qPCR assays with absolute quantification over varying multiplicities of infection (MOIs) in fibroblasts. An empiric model of vDNA replication was generated and parameterized using these data and then compared to qPCR data obtained post hoc. The vDNA model was used to drive a model of late viral infection that incorporated protein temporal class expression and culminated in virion production. This model was parameterized using data from immunoblots of representative proteins from the late temporal profile and viral titering data at three different MOIs. Predictions from this model were then compared to post hoc high‐temporal‐resolution, fluorescence‐based concentration measurements of one of the representative proteins over a 96‐hr replication cycle.

Results

A MOI‐dependent model of vDNA and late viral replication was developed from the experimental data. The model of late viral replication is mechanistic. It predicts feedback inhibition of late viral protein production and an increase in late viral protein degradation following an increase in vDNA. Both the vDNA and late replication models yielded 3 primary predictions: (1) presence of saturation kinetics at high MOIs, (2) inefficient replication at low MOIs, and (3) a range of MOIs in which vDNA replication and virus production are maximized. Saturation kinetics at high MOIs likely represents physical limitations of cellular machinery. Inefficient replication at low MOIs likely indicates a minimum MOI required to sustain infection. Identification of a maximal range of MOIs likely represents a set of conditions most favorable to HCMV lytic replication where extraneous viral material is minimized, while both the number of cells and the amount of viral material delivered to each cell is maximized.

Conclusion

A mathematical model of HCMV vDNA synthesis and late protein expression was generated and predicts a range of MOIs where maximal replication occurs, which can be extended to predict protein dynamics during infection.
Insights into the Binding of the Dengue Virus Nonstructural 5 (NS5) Protein to Stem Loop A (SLA)
Authors: Obi Juliet , Fields James , McQueen Linfah , Deredge Daniel
Published: May 2022

Dengue virus (DENV) is the most prevalent mosquito‐borne virus that infects humans, and it continues to pose a public health threat to over 2.5 billion people globally. There are no clinically approved antivirals available to treat dengue infection to date, and available vaccines are limited to seropositive individuals which calls for intensified research efforts in therapeutic development. The non‐structural 5 (NS5) protein is the largest protein encoded by flaviviruses and is responsible for viral RNA genome replication. NS5 has an N‐terminal methyltransferase (MTase) domain responsible for 5’ RNA capping, and a C‐terminal RNA‐dependent‐RNA‐polymerase (RdRp) domain responsible for de novo RNA synthesis. These two domains are connected by a flexible linker which allows NS5 to adopt multiple conformations in performing its multiple replicative functions. The dual enzymatic activity of NS5 and its essential role in dengue viral RNA replication, makes it an attractive target for the development of antiviral for the treatment of dengue infection. However, there is limited understanding on the structural basis for the interactions of NS5 to its different RNA partners during the viral replication cycle. Studies have shown that dengue NS5 discriminates between viral and host RNA by binding to RNA elements as the 5’‐untranslated regions (5’‐UTR) including stem loop A (SLA) to promote viral RNA synthesis. In this study, we characterized the binding of NS5 from dengue virus serotype 2 (DENV2) with stem loop A (SLA) at the 5’‐UTR using surface plasmon resonance (SPR) studies and determined their binding stoichiometry using analytical ultracentrifugation (AUC) studies. Hydrogen‐deuterium exchange coupled to mass spectrometry (HDX‐MS) studies of DENV2 NS5 alone and in complex with SLA reveal large conformational fluctuations consistent with an EX1 kinetic regime of HDX‐MS at the interface between the MTase and RdRp domains and identify regions of interactions between DENV2 NS5 and SLA. These experiments were repeated in the context of the individual MTase or RdRp domains to gain insight on how they coordinate binding to SLA compared to full‐length NS5. The results from our study will advance our understanding of how dengue NS5 binds SLA and its other RNA interaction partners.
The Effects of Vaping on Human Esophageal Keratinocytes
Authors: Lehman Heather
Published: May 2022

Although the first electronic cigarette‐like device was patented in the 1930s, the electronic nicotine delivery systems (ENDS) seen today did not infiltrate the US market until 2006. Since the 1960s, nicotine use had plummeted, but the invention and popularization of vapes caused its nationwide resurgence. Unlike smoking, however, little is known about the effects of vaping on the human body. Before individuals can weigh the risks associated with vaping, science must come to a consensus on what those risks are. Much of the vaping research today examines the effects on the brain or lungs; however, effects on the esophagus are not understood. Our study seeks to investigate the effects of different vape chemicals on human esophageal keratinocytes. In vitro experiments suggest that esophageal keratinocytes undergo phenotypic and physiological changes when exposed to various concentrations of vape liquid or nicotine. Additionally, when exposed to the vapor produced by an ENDS, esophageal cells undergo changes in morphology, cell viability, and motility capabilities.
Examining Flipped Teaching in an Anatomy Course at a Community College
Authors: Hackmann Elizabeth , Daughrity Sheyenne , Nguyen Kim‐Leiloni , Gopalan Chaya
Published: May 2022

Flipped teaching (FT) is a student‐centered instructional method, but its effectiveness at community colleges is poorly understood. A study was designed to compare the unflipped (UF; N=41) versus partial (PF; N=62) and full FT (FF; N=29) in an accelerated 8‐week and a regular 16‐week Introduction to Human Anatomy course. For the FT portion, students were expected to independently review the content before class and participate in the discussion and engage in learning activities during class, unlike students in the UF portion. Both UF and PF scores were more significant than the 8‐week FF class (p<0.05). When the FF courses, 8‐week and 16‐week (N=65), were compared, the final exam scores were higher in the 16‐week course (p<0.05). Male students scored less in the 8‐week FF course compared to 8‐week PF (p<0.001) and 16‐week FF (p<0.001). They also scored lower than female students in the 8‐week FF course (p<0.05). When the final exam scores of different modalities were compared between the above‐ and below‐median of each class, FF in an 8‐week course was found to be challenging by both above‐ (p<0.01) and below‐median groups (p<0.05). These results suggest FF is helpful in a regular 16‐week semester compared to the accelerated 8‐week course at the community college where male students, in particular, find it difficult.
Tau Mediated Coupling Interactions between Pol III Core DNA Synthesis and DnaB Helicase Unwinding
Authors: Welikala Malisha U. , Perera Himasha , Behrmann Megan , Trakselis Michael
Published: May 2022

DNA replication is a vital process ensuring the passing down of intact genetic information over generations. DNA replication in the model organism, Escherichia coli (E. coli), has been extensively studied, providing significant insights into the various enzyme mechanisms. However, while the prime functions of individual components of the replisome have been deciphered, the more complex interactions between the components is an active area of study to understand replisome coupling. At the core of the replisome, the DnaB helicase unwinds the double strand DNA which is followed by rapid synthesis of nascent strand by Polymerase III core (Pol III core). It is proposed that the Tau subunit of the clamp loader complex acts as the physical link between both DnaB and Pol III core, leading to a coupling phenomenon between unwinding and synthesis.

Using a reconstituted E. coli replisome in vitro, unwinding of DNA substrates with variable lengths and assemblies can be monitored through electrophoresis to better understand the kinetics of coupling. Mutations have been introduced in the exterior surface of DnaB, disrupting its charged interactions with the excluded strand, and promoting more constricted conformation that result in faster unwinding. The unwinding kinetics of wild‐type versus mutant DnaB enzymes on different length substrates are being examined. Further experiments include Pol III synthesis alone or with proposed coupling from the tau‐subunit and can simultaneously examine unwinding and synthesis. Mutations that affect DnaB conformation or disrupt Pol III‐tau interactions will be utilized to assess their relative influences in maintaining a coupled replisome. Conducting in vitro experiments will provide mechanistic insight into the nature of interactions between these replisome components in a coordinated system.

Coupling interactions are also being investigated in vivo using precise CRISPR‐Cas9 genome editing. Coupling mutations have been introduced in the dnaX gene encoding the tau‐subunit. These mutant strains exhibit a reduced growth rate when compared to wild‐type strains, indicating that disrupted coupling leads to reduced doubling times. These strains are being further characterized to determine the genomic and cellular consequences of helicase‐polymerase decoupling in vivo. Therefore, a combination of in vitro and in vivotechniques are being used to better understand the importance of coupling during DNA replication to maintain a stable genome.
Inhibition of Wnt/β‐catenin Signaling Pathway on Melanogenesis‐ Insights from Zebrafish (Danio rerio)
Authors: Silva Praneeth T. , Atukorallaya Devi S. A.
Published: May 2022

Introduction

Vertebrate color patterns are composed of specialized pigment‐producing cells, the chromatophores. Melanin is the main pigment type that colors the skin and skin appendages in mammals. Melanin is produced by specialized cells called melanocytes. Melanocytes have embryonic neural crest cells origin and their development and functions are ubiquitous across vertebrates, residing in dermal and epidermal skin layers. Studies of melanogenesis have provided experimentalists with a valuable system for understanding pattern formation in the developing embryo. In addition, studying these mechanisms may have important consequences for the clinical sciences. Alcohol is a widely abused teratogenic substance. Fetal alcohol spectrum disorder (FASD) is described as birth defects associated with prenatal alcohol exposure (PAE). PAE is likely one of the leading causes of birth defects but pathophysiology of FASD associated body pigmentation disorders are needed to be investigated further. Zebrafish (Danio rerio) (ZF) is identified as a good model to study FASD and melanocyte biology. Wnt signaling pathway is a conserved signal transduction pathway which is important in embryonic development and it regulates many aspects of melanogenesis. The current study was designed to investigate the effect of Alcohol‐Wnt signaling pathway activity on ZF melanogenesis.

Materials and Methods

Wild‐type (WT) ZF embryos were treated with Wnt pathway inhibitor (LiCl) and combined chemical treatment (LiCl and 1% alcohol) at 10 hours post fertilization (hpf). The differential effects of above substances on early development, differentiation, patterning of melanocytes were investigated using cell biology and imaging techniques.

Results

Melanocytes of LiCl exposed samples showed defects in migration, arrangement and survival. Ectopic melanocytes and developmental aberrations of eye pigmentation were noticed in both chemical treatments. Significant variation of the melanocyte cell size, shape and body pigmentation patterning was observed among the LiCl and combined treated samples compared to the control.

Conclusion

Embryonic LiCl and alcohol exposures affect the development and patterning of the melanocytes in ZF. The molecular mechanisms underlying the above phenotypes will be investigated further to understand the FASD associated pigmentation defects.
Verifying the Insertion of the Fibulocalcaneus (Peroneocalcaneus) Internus Muscle – A Rare Variant Leg Muscle
Authors: Quinn Makaela , Kellum Chloe E. , Cook Ryan L. , Herrold Christina B. , Zdilla Matthew J. , Lambert H. Wayne
Published: May 2022

The fibulocalcaneus (peroneocalcaneus) internus (PCI) muscle is a rare variant leg muscle with a low prevalence of <1%‐3% in cadaveric dissection and imaging studies. The PCI arises from the medial aspect of the distal third of the fibula, descends posterior and lateral to the flexor hallucis longus (FHL) muscle, traverses the tarsal tunnel inferior to the sustentaculum tali of the calcaneus, and inserts into the plantar surface of the calcaneus. However, controversy exists concerning the exact location of the insertion site of the PCI muscle. The insertion of the PCI muscle was recently described as being located distal to the coronoid fossa, a small depression between the anterior tuberosity and the anterior apex of the sustentaculum tali of the calcaneus. However, historical descriptions described the PCI as inserting into either the sustentaculum tali itself or a small tubercle on the medical surface of the calcaneus distal to the sustentaculum tali. During routine dissection of a 53‐year‐old Caucasian male, a PCI muscle was identified, and it inserted into distal to the coronoid fossa on the plantar surface of the calcaneus. More specifically, it inserted into the plantar surface of the calcaneus in the space between the origin of the plantar talonavicular (spring) ligament and the plantar calcaneocuboid ligament. Knowledge of the PCI muscle is important due its involvement with various ankle pathologies, including predisposing individuals to tarsal tunnel syndrome, FHL tenosynovitis, and posterior ankle impingement and pain.
Aging and G Protein‐Coupled Estrogen Receptor Exacerbates Carotid Artery Structural Remodeling
Authors: Ogola Benard , Clark Gabrielle , Kilanowski‐Doroh Isabella , Visniauskas Bruna , Abshire Caleb , Diaz Zaidmara , Horton Alec , Kiley Jasmine , Zimmerman Margaret A. , Groban Leanne , Miller Kristin , Lindsey Sarah
Published: May 2022

Aging is a nonmodifiable risk factor for cardiovascular disease associated with arterial stiffening and cardiac dysfunction. We previously showed an important role for G protein‐coupled estrogen receptor (GPER) in modulating female protection from arterial stiffening. In this study, we hypothesized that aging would decrease vascular compliance in GPER knockout (ko) mice to a greater extent than wildtype (wt) mice. Adult (25 weeks) and middle‐aged (57 weeks) mice were subjected to high frequency ultrasound for pulse wave velocity (PWV), and structural remodeling in the carotid artery was assessed with biaxial pressure myography followed by constitutive modeling. Data was analyzed by 3‐way ANOVA and Sidak’s post hoc tests. As we previously showed, PWV was significantly lower in adult female wt mice versus their male counterparts (1.4 ± 0.05 vs. 1.1 ± 0.02 m/s; P=0.0004) but was not different in adult ko mice (1.6 ± 0.07 vs 1.4 ± 0.08, P=0.20). Interestingly, PWV increased with aging in female but not male wt mice, so that the sex difference was lost (1.7 ± 0.03 vs 1.7 ± 0.06, P=0.99). Despite the lower PWV, circumferential linearized material stiffness in passive conditions was elevated in adult female wt versus male wt (1.9 ± 0.2 vs. 1.4 ± 0.2 MPa; P=0.02), but this sex difference was lost in middle‐aged wt mice (1.3 ± 0.1 vs. 1.2 ± 0.1 MPa; P=0.87). Aging significantly increased the ratio of thick to thin collagen only in male wt and female ko groups. We found that females but not males experience an increase in PWV at middle‐age along with a decrease in circumferential stiffness. In contrast, female GPER mice already display a higher PWV as adults, indicating that GPER deletion in females is a model of accelerated vascular aging.