bluuebunny/crossref_metadata_2025 huggingface dataset:
Respiratory Response‐Based Intermittent Hypoxia Treatment Leads to Diaphragmatic Long Term Facilitation in Adult Rats
Authors: Silverstein Aaron L. , Yoo Jae H. , Devine Caroline , Alilain Warren J.
Published: May 2022

Spinal cord injury (SCI) most commonly occurs at the cervical level and can lead to severe respiratory dysfunction such as paresis of the diaphragm, the primary mammalian inspiratory muscle. Intermittent hypoxia (IH) treatment (tx) can be applied to induce a prolonged increase in breathing motor output, known as long term facilitation (LTF), which is achievable following SCI in humans. However, this does not potentiate following multiple days of tx, indicating a need for optimization of existing IH protocols. Many seminal experiments demonstrating LTF in uninjured animals employ phrenic nerve recordings to measure respiratory response to moderate IH protocols consisting of 3, 5‐minute‐long periods of hypoxia (~11% O2) interspersed with periods of normoxia (~20.9% O2). This tx increases respiratory motor drive, stimulating serotonin (5‐HT) and growth factor‐dependent plasticity within the cervical spinal cord. Importantly, many studies have shown that the pattern and severity of IH dramatically affect LTF expression. Indeed, fundamental literature indicates that the details of 5‐HT‐based or other stimulation protocol are crucial for induction of plasticity across multiple phenomena, species, and preparations. In these studies, real‐time verification of the stimulus’ effect is key. Since LTF is a neuroplastic paradigm which depends on sequelae of effective stimulation of respiratory drive, we constructed an IH protocol which adapts to the animal’s real‐time respiratory response during treatment. We hypothesized that novel, respiratory response‐based (RRB) IH tx would induce a greater degree of diaphragmatic (dia) LTF than would standard fixed duration (FD) tx in uninjured adult rats. To develop RRB treatment, we first derived an initial setpoint from previous EMG/IH experiments in rats which reflected the magnitude of diaEMG amplitude increase expressed during hypoxic exposure. We then utilized this setpoint during each of three IH cycles such that the animal would qualify for progression from hypoxic to normoxic exposure by expressing a sufficiently robust diaEMG amplitude increase with respect to output measured in previous normoxic conditions. To evaluate our hypothesis, we conducted diaphragm EMG recordings in isoflurane‐anesthetized Sprague‐Dawley retired breeder rats before, during, and after three cycles of either moderate RRB or FD tx administered by nose cone. Preliminary data demonstrated that RRB treatment (n=2) resulted in LTF reaching 113.2% and 130.8% of baseline diaphragm amplitude for the two subjects at least 1‐hr. post‐IH. Further elucidation of appropriate hypoxia‐induced diaphragm amplitude setpoints is a necessary next step, followed by completion of greater numbers of both RRB and FD treatments to achieve adequate power for statistical comparison before applying RRB to the rat C2 hemisection injury model to explore the treatment’s utility in restoring breathing motor function once lost.
Multi‐omics analysis reveals regulatory mechanisms in chronic cyclosporine A‐induced nephrotoxicity studied a rat model
Authors: Demirci Hasan , Yilmaz Duygu E. , Popovic Suncica , Ellison David H. , Mutig Kerim , Bachmann Sebastian
Published: May 2022

Chronic calcineurin inhibitor (CNI) nephrotoxicity is a major drawback in current immunosuppressive regimens. In the chronic setting, arteriolar hyalinosis, decreased glomerular filtration rate, interstitial fibrosis and tubular dedifferentiation are the major adverse side effects. Since CsA is still widely used in transplant recipients, particularly in posttransplant diabetes, mechanistic insights into nephrotoxic mechanisms are required. To identify regulatory mechanisms in CsA nephrotoxicity we used quantitative transcriptomic, proteomic and phosphoproteomic methods. We tested the hypothesis that tubulointerstitial pathomechanisms play a predominant role in chronic CNI nephropathy. Whole transciptome RNA‐seq as well as global proteomic and phosphoproteomic methodologies were performed on kidney extracts from normal Wistar rats receiving CsA (25mg/kg b.w./day) or vehicle for 3 weeks. Differentially expressed genes and proteins as well as their phosphorylation status were obtained. Products of interest were tested using Western blot, qPCR, and immunohistochemistry. CsA treatment stimulated genome‐wide alterations in rat kidney according to RNA‐seq data. We identified 342 transcripts upregulated (padj<0.05) which included Ribosome (padj<0,0001) and Oxidative phosphorylation (padj<0,0001) pathways, whereas 331 transcripts were downregulated, with enrichment in genes critical for amino acid metabolism. Data were controlled by the established upregulation of renin and downregulation of calbindin (pdaj<0,05) in global proteomics. KEGG pathway and GO analysis from proteomics largely corresponded to the RNA‐seq results. Upregulated proteins were further related to ECM‐receptor interaction and focal adhesion pathways (padj<0,05). Phosphoproteomics demonstrated functional phosphorylation of components from unfolded protein response pathways, indicating an activation of the integrated stress response upon CsA. Products were verified at the tissue‐ and cell level. In sum, using integrated ‐omics analysis in CsA nephrotoxicity proved to be a powerful approach. Chronic CsA treatment was associated with enhanced energy metabolism and activation of the unfolded protein‐response pathways. A tubulointerstitial focus has been demonstrated. Potential biomarker candidates have been obtained.
Electrochemical DNA Biosensor to Detect Glycopeptidolipids of Nontuberculous Mycobacteria
Authors: Poch Dylan , Bonham Andrew J.
Published: May 2022

In many regions, nontuberculous Mycobacteria (NTM) pulmonary infections have outnumbered tuberculosis infections over the past 30 years, yet NTM infections are frequently misdiagnosed. One critical challenge is that the current “gold standard” for NTM diagnosis involves a weeks‐to‐month long microbiological culture‐based method. To provide a more efficient diagnostic method, we have developed a novel electrochemical DNA‐based (E‐DNA) biosensor to detect NTM. Our E‐DNA biosensor targets a mycobacterial glycopeptidolipid (GPL) found on the exterior cell wall of NTM, which demonstrates high exclusivity for NTM. To create the E‐DNA biosensor, we successfully generated a GPL‐specific single‐stranded DNA aptamer through a modified SELEX (systematic evolution of ligands by exponential enrichment) process followed by high throughput sequencing to quantify potential aptamer sequences. The putative aptamers were then synthesized and screened via fluorescence, with the best candidate aptamer displaying a dissociation constant in the low nanomolar range (KDapp = 0.73 nM). Computational analysis was used to predict secondary and tertiary structure of the aptamer, allowing optimization of that aptamer sequence to find the minimum binding domain and engineer in a two‐state folding mechanism. The aptamer will then be synthesized with a 5’ thiol linkage to facilitate attachment to a gold electrode along with an internal redox reporter (methylene blue). This will enable an electrochemical interrogation method wherein changes in the peak current in square wave voltametric analysis correlate to a conformational rearrangement of the aptamer upon binding to GPL. In future studies, this E‐DNA biosensor will be verified against NTM‐positive patient samples. Ultimately, this approach should provide a rapid, point‐of‐care diagnostic method that eliminates the current lengthy culturing requirements to shorten the duration between the onset of symptoms and correctly targeted treatment of patients infected with NTM.
ClC‐K2 Cl‐ Channel exhibits discrete actions on acid‐base transport of A‐ and B‐Type Intercalated Cells of the Collecting Duct
Authors: Pyrshev Kyrylo , Stavniichuk Anna , Tomilin Viktor N. , Zaika Oleg , Pochynyuk Oleh
Published: May 2022

The collecting duct is a highly adaptive terminal part of the nephron, which is essential for maintaining systemic homeostasis. Principal and intercalated cells perform different physiological tasks and exhibit distinctive morphology. However, acid‐secreting A‐ and base secreting B‐type of intercalated cells cannot be easily separated in functional studies despite virtually mirror opposite localization of their transport acid‐base systems.

Here, we used BCECF‐sensitive intracellular pH (pHi) measurements in split‐opened collecting ducts followed by immunofluorescent microscopy in WT and intercalated cell‐specific ClC‐K2‐/‐ mice to demonstrate that ClC‐K2 inhibition enables to distinguish signals from A‐ and B‐ types of intercalated cells.

We show that ClC‐K2 Cl‐ channel is expressed on the basolateral side of intercalated cells, where it governs Cl‐‐dependent H+/HCO3‐ transport. ClC‐K2 blocker, NPPB, caused acidification or alkalization in different subpopulations of intercalated cells in WT but not ClC‐K2 ‐/‐ mice. Immunofluorescent assessment of the same collecting ducts revealed that NPPB increased pHi in AE1‐positive A‐type and decreased pHi in pendrin‐positive B‐type of intercalated cells. Induction of metabolic acidosis led to a significantly augmented abundance and H+ secretion in A‐type and decreased proton transport in B‐type of intercalated cells, whereas metabolic alkalosis caused the opposite changes in intercalated cell function, but did not substantially change their relative abundance.

Concluding, we show that inhibition of ClC‐K2 can be employed to discriminate between A‐ and B‐type of intercalated cells in split‐opened collecting duct preparations. We further demonstrate that this method can be used to independently monitor changes in the functional status and abundance of A‐ and B‐types in response to systemic acid/base stimuli.
Development of an Electrochemical, DNA‐Based Biosensor to the Cancer Biomarker ENOX2
Authors: Quansah Mary , Bonham Andrew J.
Published: May 2022

One challenge to the identification of cancerous malignancies is the ability to detect cancer‐specific antigens in a rapid, reliable, and accessible manner. Human Ecto‐NOX Disulfide‐Thiol Exchanger 2 (ENOX2) is a cell‐surface metalocatalyst frequently expressed on virtually all major human cancer types and is critical for cell maturation. Importantly, ENOX2 regularly sheds into the sera, making it an attractive target for diagnostic blood testing. As a strategy for diagnostics focused on utilizing these shed ENOX‐2 particles, we here demonstrate an electrochemical DNA‐based (E‐DNA) biosensor directed against ENOX‐2 in solution. To do so, we first exploited a modified SELEX (systematic evolution of ligands by exponential enrichment) protocol to generate aptamers against ENOX2 recombinantly expressed in E. coli. A pool of candidate aptamers was identified by high‐throughput sequencing, and gel mobility shift assays were used to characterize the best candidate aptamers and classified those with the highest affinity for ENOX2. The leading aptamer sequence was used as the core of our E‐DNA biosensor, wherein changes in DNA conformation upon target binding result in changed dynamics of an appended redox reporter molecule, ultimately generating a rapid and quantitative change in the current of the electrochemical response when interrogated via voltametric methods such as square‐wave voltammetry. Our sensor displays robust response to ENOX2 in solution, and may allow accelerated, dose‐responsive readout for the presence of ENOX2 in both buffer and blood serum.
Developing RNA‐Seq and ChIP‐Seq Pipelines on an Oxford Nanopore Technologies Platform to Study Notch1 Transcriptional Dynamics
Authors: Klier Nikola , White Brandon
Published: May 2022

Notch signaling plays a crucial role in cell development and proliferation. The Notch receptor is an inducible transcription factor found on the cell surface which is cleaved resulting in the release of the intracellular domain (NICD). The NICD enters the nucleus and complexes with CBF1 and Mastermind (MAM) to activate target genes. CBF1 recognizes the binding motif RTGRGAR (R=G or A), and this is referred to an NRE (Notch Response Element). Recent studies have shown that a paired CBF1 binding site is also important for target gene activation. While individual genes regulated by SPS are known, the extent to which an SPS vs individual NREs are responsible for Notch‐mediated transcription on a genomic level remains an active area of research. It is hypothesized that the majority of Notch‐mediated transcription occurs as a result of a dimeric Notch complex in the promoters or enhancers of transcribed genes. Recently, high‐throughput techniques have allowed the study of Notch complex assembly and their effects on transcription. Here, we use an Oxford Nanopore Platform was used.

To identify genes regulated by Notch transcriptional complexes, RNA‐Seq was performed. Total RNA was isolated from SUPT1 cells treated with 5 µM of the Notch inhibitor DAPT for 24 hours, or DMSO as a control. Poly‐A RNA was purified using an oligo‐dT column and double‐stranded cDNA generated. PCR adapters were nonspecifically ligated to cDNA and amplified to increase library yield. Sequencing was performed using an Oxford Nanopore Minion, and results were mapped to the human reference genome to identify genes significantly downregulated when Notch was inhibited. To identify NREs and SPS in the regulatory elements of these genes, ChIP‐Seq was performed for Notch. ChIP was performed in DAPT and DMSO treated cells, and ChIP DNA used to prepare a sequencing library as above. Sequenced fragments were aligned to a known regulatory elements dataset, and genes with significant Notch binding identified.

RNA‐Seq data shows significant downregulation of known Notch targets genes, such as Hes1, Hey1, and Hes4. Sequenced mRNA fragments, however, averaged 1kb suggesting size selectivity during the purification or amplification steps of library preparation. Additionally, underrepresentation of other Notch known targets such as Hes5 may suggest that computational stringency may have to be adjusted. We are currently pursuing ChIP‐Seq experiments that will be presented at the conference to identify the locations of SPS in the genome. Identified SPS in regulatory elements will then be correlated with the genes they activate, and the downstream effects on transcription analyzed. It is expected that a large number of genes that are inactivated under Notch inhibition conditions will exhibit a SPS in a corresponding regulatory element.
Decline of gross anatomy in medical education. I. Independent learning and need for assessment of effects of curricular change on medical practice
Authors: Zill Sasha N.
Published: May 2022

The importance of Gross Anatomy in medical education has declined in recent years as the result of curricular reforms at many schools. The goals of our ongoing observational study are 1) to adapt Anatomy instruction to the compression of content and reduction in duration of anatomy instruction, 2) to evaluate the effectiveness of pedagogical methods in conveying information essential to understanding body structure and 3) to attempt to reduce student stress. Our approach to pedagogical reorganization is to base anatomy instruction upon core clinical conditions. These conditions were not presented as clinical problems, as many students had limited prior training in medical terminology, but focused upon clinical symptoms, allowing for direct correlation of structure and function. Overall methodology has been multi‐modal. Content was significantly reduced: for example, knowledge of muscle muscle origins and insertions is no longer required, only muscle actions and innervations. Performance has been evaluated using a long‐term data base of student scores on written and regional practical examinations. Results of written examinations were relatively constant during the period prior to the introduction of curricular changes but practical examinations could show large anomalous variability in some years (including data utilized in a previously published study of the effects of independent learning). To increase the accuracy of assessment we have developed a set of questions that were asked repetitively in successive years on written examinations. Current data on exam performance (including repeat questions) indicate that the pedagogical changes were effective during the initial period of curricular change and a survey of students indicated that these changes effectively decreased stress. However, performance in Head and Neck anatomy declined in 2020‐2021 as curricular compression and increased reliance upon independent learning has intensified. We are currently assessing whether the decline was associated with the curricular change or the unique circumstances associated with the recent viral pandemic. We also discuss the need for assessment of the effects of these changes on medical practice. The results to date suggest that training in gross anatomy can be modified to a compressed duration but do not support reliance upon independent learning as a pedagogical method.
Tyrosine Hydroxylase‐Positive Neurons in the Hypothalamic Paraventricular Nucleus Modulate Blood Glucose Levels in Mice
Authors: Souza Lucas A. C. , Gayban Ariana J. , Feng Earley Yumei
Published: May 2022

Tyrosine hydroxylase (TH)‐positive neurons in the paraventricular nucleus of the hypothalamus (PVN), termed THPVN neurons, have emerged as key metabolic regulators. Recent studies showed that THPVN neurons are mostly Gamma‐aminobutyric acid (GABA)ergic and/or dopaminergic. Previous results from our laboratory indicate that THPVN neurons project to brainstem autonomic regulatory nuclei, such as nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM). However, it is not known if THPVN neurons play a role in blood glucose regulation. We hypothesize that THPVN neurons are key regulators of blood glucose. To test our hypothesis, we used adeno‐associated virus serotype 2 (AAV2)‐mediated Cre‐recombinase driven by a rat TH promoter (AAV2‐TH‐Cre), in combination with chemogenetic approach (Designer Receptors Exclusively Activated by Designer Drugs, DREADD) to activate or inhibit THPVN neurons and continuously monitored blood glucose levels using telemetry system. hM3Dq‐LoxP (excitatory‐DREADD) and hM4Di‐LoxP (inhibitory‐DREADD) mice, 10‐14 weeks old, were bilaterally microinjected with AAV2‐TH‐Cre into the PVN. One month following AAV injection, mice were implanted with telemetry glucose transmitters (HD‐XG, DSI) to monitor blood glucose (BG) levels. To activate or inhibit THPVN neuronal activity, Clozapine‐N‐oxide (CNO, 1 mg/Kg) or vehicle were administered (i.p.) and BG was monitored by telemetry. To examine the functional role of THPVN neurons at baseline and avoid the influence of feeding on blood glucose levels, mice were fasted for 2 hours and fasting was maintained throughout the experiment. We found that activation of THPVN neurons by CNO in hM3Dq‐LoxP mice did not alter baseline blood glucose (n=5); on the other hand, inhibition of THPVN neurons by CNO in hM4Di‐LoxP mice increased baseline blood glucose levels compared with vehicle only in male mice (Male: ΔBG: 31.6 ± 8.6 mg/dL vs. 11.0 ± 6.7 mg/dL, n=4, p=0.03; Female: ΔBG: 11.0 ± 7.4 mg/dL vs. 7.3 ± 5.9 mg/dL, n=4, p=0.16). To study the functional role of THPVN neurons on whole body glucose handling, mice were fasted overnight and simultaneously injected with glucose (1 g/Kg) and either vehicle or CNO. We found that activation of THPVN neurons by CNO improved glucose clearance after glucose overload when compared with vehicle (AUC: 3133.0±109.0 vs. 6021.0±102.0, p<0.0001, n=3) in hM3Dq‐LoxP mice. In summary, our data suggest that THPVN neurons negatively regulate blood glucose possibly by their GABAergic input to downstream neural circuits. We conclude that THPVN neurons play a functional role in the regulation of glucose homeostasis; while the mechanisms remain to be determined.
Design and characterization of a FGF1‐FGF21 chimeric protein with increased stability and enhanced antidiabetic activities
Authors: Phan Phuc , Kumar Thallapuranam K. S.
Published: May 2022

Fibroblast growth factor (FGF) family is consisted of 22 proteins that regulate a wide variety of biological functions such as wound healing, tissue repairs, and angiogenesis. FGF family follows three main modes of action: paracrine, intracrine or endocrine. One member of the paracrine FGFs is FGF1, which works locally through interaction with heparin or heparin sulfate (HS). With its mitogenic and cell survival activities, FGF1 participates in many developmental processes, cell growth and differentiation, tissue repairs, and tumor growth and invasion. A representative of endocrine FGFs is FGF21, which is often used in long range physiological processes, but does not require HS. FGF21 engages in many functions across various organs like liver, pancreas, adipose tissues. One of FGF21’s abilities is the regulation of glucose uptake in adipocytes through upregulating the transcription of glucose transporter‐1. Thus, FGF21 is a potential treatment for metabolic diseases such as Type‐2 Diabetes. However, FGF21 has been reported to possess an inherently unstable core with low thermal stability and receptor affinity. Herein, we designed a structure‐based chimera protein by replacing the core of FGF21 with a thermally stable paracrine FGF1 (sFGF1). Using techniques such as fluorescence spectroscopy, and circular dichroism, we show that the chimera protein sFGF1‐FGF21 adopts a β‐trefoil core. Intrinsic fluorescence spectra of the chimera protein shows an emission maximum centered at 307 nm indicating that the emission of the conserved tryptophan is quenched similar to wildtype FGF1. Differential calorimetry data suggests that chimera sFGF1‐FGF21 is significantly more stable than wild type FGF21. The findings of this study provide valuable clues for the rational design of FGF21‐based therapeutic against hyperglycemia and obesity.
Maternal opioids decrease mu‐opioid receptor expression in the neonatal preBötzinger Complex
Authors: McDonald Emilee A. , Naidoo Robyn , Albarrán David , Beyeler Sarah A. , Huxtable Adrianne G.
Published: May 2022

An understudied population in the opioid crisis are infants exposed to in utero opioids experiencing severe respiratory deficits. Our recent work demonstrated maternal opioids destabilized neonatal breathing, increased apneas, and blunted opioid‐induced respiratory frequency depression; however, the mechanism(s) underlying these neonatal breathing deficits remains unclear. We hypothesized deficits in neonatal breathing after maternal opioids are due to downregulation of mu‐opioid receptors on key cell types (e.g. neurons and astrocytes), essential for respiratory rhythm generation and modulation in the preBötzinger Complex. While opioid receptor expression is downregulated in the whole brain after maternal opioids, it remains unclear if opioid receptor expression changes in the preBötzinger Complex after in utero opioids to induce neonatal breathing deficits. To assess the typical developmental changes in opioid receptors in the preBötzinger Complex, mu‐opioid receptor expression was first assessed using immunohistochemistry and confocal microscopy on neonatal neurons and astrocytes in the preBötzinger Complex at three ages: the day of birth (postnatal day 0, P0) when neonates must begin robust rhythmic breathing, an early neonatal age (postnatal day 4, P4), and during a critical developmental window (postnatal day 11, P11). At all ages, mu‐opioid receptors were expressed on both neurons and astrocytes in the preBötzinger Complex; however, mu‐opioid receptor expression decreased from P0 (n=6) to P4 (n=6) and was lowest at P11 (n=6). Preliminary data after maternal opioids suggest mu‐opioid receptor expression was lower in preBötzinger Complexes at P0 (n=2) and P4 (n=2), but returned to control levels by P11 (n=2). These results are further supported by in vitro brainstem‐spinal cord recordings of the cervical rootlets (C4) from neonates after maternal opioids, demonstrating decreased opioid sensitivity of isolated respiratory control circuitry after maternal opioids (n=2). Results from these studies will contribute to our understanding of the mechanisms contributing to impaired neonatal breathing after maternal opioids and may lead to better treatments for infants suffering breathing deficits after maternal opioids.
Investigating the Mechanistic Strategies of the Two‐component FMN‐dependent Alkanesulfonate Monooxygenase Systems
Authors: Somai Shruti , Ellis Holly R.
Published: May 2022

The two‐component alkanesulfonate monooxygenase systems, consisting of a flavin reductase (SsuE and MsuE) and an alkanesulfonate monooxygenase (SsuD and MsuD), enable bacterial organisms to utilize a broad range of alkanesulfonates when sulfur is limiting. The flavin reductases supply reduced flavin to their partner monooxygenase for the desulfonation of sulfonated compounds through the formation of a flavin oxygenating intermediate. Commonly, flavin monooxygenases have been proposed to utilize a C4a‐(hydro)peroxyflavin as the oxygenating intermediate. However, unlike other flavin monooxygenase enzymes, this intermediate has not been spectrally observed in the alkanesulfonate monooxygenase enzymes. Recent studies reported the formation of a flavin‐N5‐oxide which forms as an intermediate during turnover or as the final product in some flavin monooxygenases. It is hypothesized that the alkanesulfonate monooxygenases could employ a flavin‐N5‐adduct based on amino acid sequence alignments and structural similarities with enzymes employing similar intermediates.

SsuD and MsuD share comparable structural properties but have different specificities for alkanesulfonate substrates. Although the substrate preference is critical for catalytic competence, SsuD and MsuD likely utilize similar catalytic steps for desulfonation. Previous studies identified various polar and nonpolar amino acid residues that were critical for the formation and stabilization of the flavin‐N5‐oxide. The conserved nonpolar amino acids are proposed to control the interaction of the flavin‐N5 with molecular oxygen, whereas the polar amino acids are involved in stabilizing the superoxide anion involved in the formation of the flavin‐N5 oxygenating intermediate. Some of these amino acids were also found to be conserved in the alkanesulfonate monooxygenases and their role was further evaluated through site‐directed mutagenesis. The V108T and T109A SsuD variants had similar activity to wild‐type SsuD; however, the N108L SsuD variant had no measurable activity. These results support the role of Asn in stabilizing the proposed flavin‐N5 oxygenating intermediate. Since a flavin‐N5‐oxide has been identified as an intermediate or as the final product in some two‐component monooxygenases, HPLC and mass spectrometric analyses were performed to determine if the alkanesulfonate monooxygenases form the flavin‐N5‐oxide. To provide evidence for kinetic steps and identify flavin‐N5 reaction intermediates, stopped‐flow kinetic experiments were performed with wild‐type and variants of SsuD and MsuD. The findings from this study provide insight into the mechanistic features of the alkanesulfonate monooxygenases as well as their role in the overall global sulfur cycle.
Enriching Anatomy Learning with Virtual Reality Clinical Scenarios
Authors: Hasan Farah Z. , McBride Jennifer , Mitchell Josh P. , Anwarzi Deewa , Sonnadara Ranil R. , Wainman Bruce C.
Published: May 2022

Introduction

The use of virtual reality (VR) to simply display anatomical specimens typically fails to exploit the potential of VR to enhance the learning environment. For example, anatomy education normally occurs within the confines of a laboratory which is devoid of relevant context, while VR is uniquely capable of providing a clinically‐relevant space that may enhance the anatomy learning experience. However, it is also possible that an enriched VR environment may inhibit learning, perhaps as a result of increased cognitive load. In general, well‐designed educational materials create schemas and organize the acquired knowledge in a way that promotes deeper learning, retention, and easy retrieval in other settings. Efforts must be made, in instructional design, to reduce extraneous and increase germane cognitive load, but how exactly this is done in VR remains an open question.

Objective

The purpose of this study is to evaluate the educational efficacy of a VR module which includes a clinical scenario presented in an immersive, contextually‐relevant virtual environment.

Hypothesis

Although learning in a VR enriched environment with a clinical scenario can increase extraneous cognitive load, we hypothesize that it will ultimately facilitate memorization of anatomical structures, when compared with using 3D‐printed physical models or an interactive, 2D environment.

Methods

Participants with no prior knowledge of pelvic anatomy will be randomly assigned to one of three groups to learn human pelvic anatomy in an identical module presented in VR, an interactive 2D computer‐based module, or physical environment consisting of 3D‐printed models. Prior to the learning phase, participants will complete the Mental Rotation Test (MRT), as well as the Titmus Fly and Titmus Circles tests to assess spatial visualization ability and stereoscopic vision, respectively. They will then complete a pre‐test assessment where they will be asked to identify anatomical structures labeled on a 3D‐printed model. Participants will then be given 10 minutes to learn and memorize pelvic anatomy using their assigned anatomy modality. This will be followed by a post‐test assessment with another set of labeled structures to identify. Finally, participants will complete a survey to share feedback on the learning experience and will complete the Simulation Task Load Index (SIM‐TLX) questionnaire to assess cognitive load.

Conclusion

The impact of contextually‐relevant enriched virtual environments and clinical scenarios for VR‐based anatomy education have yet to be explored. The findings from this study will provide valuable insight to inform the design of future VR learning tools that not only reduce or limit factors which can impair learning, but also suggest factors within the learning environment which have the potential to improve anatomy learning.
Evidence of Conformational Changes in the Structure of Y‐Family Human Polymerase Kappa
Authors: Schneider Paula L. C. , Sampoli Benitez Benedetta
Published: May 2022

Y‐family DNA polymerases, unlike replicative DNA polymerases, are enzymes characterized by their ability to bypass DNA lesions and continue extension of damaged DNA via Translesion Synthesis (TLS). One such polymerase, human polymerase kappa (Pol κ), can efficiently bypass minor groove adducts, most notably N2‐furfuryl‐deoxy‐guanosine (N2‐ffdG), but it is inhibited by major groove adducts such as O6‐methylated‐deoxy‐guanosine (O6‐Met‐dG). Pol k is composed of five domains including its novel N‐clasp domain, as well as the thumb, palm, fingers, and little finger (polymerase associated domain or PAD) domains which are found across Y‐family polymerases. The specificity that Pol κ demonstrates is thought to be related to Y‐family polymerases having smaller finger domains when compared to replicative DNA polymerases thus limiting major groove contact, while Pol κ’s PAD and catalytic core move to open the minor groove side. It is believed that Pol κ and its Escherichia colianalogue, DinB (DNA polymerase IV), undergo one of two conformational changes, open or closed, in their active site loops when in contact with undamaged DNA or minor groove adducts. Using molecular dynamics (MD) simulations, we examined conformational changes and utilized RMSD analysis to observe activity within Pol κ’s domains across three different Pol κ ternary structures in the presence of different templating lesions and their correct incoming nucleotides. These structures include, one with a minor groove N2‐furfuryl‐deoxy‐guanosine adduct (FDG for short), one with a major groove O6‐methylated‐deoxy‐guanosine adduct, and finally one with a mutation of a residue in Pol κ’s PAD (R507K) with an FDG lesion on the DNA templating strand. All systems were solvated using CHARMMgui, minimized, equilibrated, and run for 110 ns with NAMD, and observed in VMD. Our findings support that Pol κ exists in two conformations, which depend on the kind of lesion on the DNA and/or the incoming nucleotide. In particular, the movement is concentrated in the N‐clasp and fingers domains. Some residues which seem important for this transition are N52, M135, N464, and R507. Interestingly, kinetics studies have already identified some of these same residues as important for extension. Our MD studies give an atomistic explanation of why such residues are crucial for the correct functioning of this protein.
Protein‐protein interactions play significant roles in molecular motors
Authors: Li Lin
Published: May 2022

Dynein is a molecular motor for cargo transportation and force generation in cells. Dysfunction of dynein is associated with many diseases, such as ciliopathies, lissencephaly and other neurodegeneration disorders. Understanding the functions of dynein is crucial for developing new treatments of such diseases. Electrostatic interactions play important roles in proteins including dyneins. A lot of efforts have been made to study the electrostatic interactions in biological systems. However, it is extremely challenging to accurately calculate the electrostatic interactions in large biological systems such as dynein. I will introduce a novel multi‐scale simulation approach which is used to study dynein’s motion along microtubules. The electrostatic binding funnel around microtubule is observed, which drags the dynein to the binding pocket. The electrostatic forces on dynein residues form a torsion which reorients the dynein when it is in an un‐native orientation. Furthermore, the electrostatic component of the binding energy of dynein and microtubule strongly affects the velocity and run length of the dynein. These results reveal the mechanisms of dynein’s motility and functions along microtubule, which shed light on treatments of molecular motor related diseases.
Acute Manipulation of Outer Membrane Phospholipid Composition Directly Alters Mitochondrial Dynamics and Ultrastructure
Authors: Pemberton Joshua , Kim Yeun Ju , Ferrer Elizabeth , Joshi Vijay , Fischer Tara , Korzeniowski Marek , Youle Richard , Heuser John , Balla Tamas
Published: May 2022

Mitochondria undergo coordinated rounds of fusion and fission that are critical for maintaining the functional integrity of this essential organelle. While a growing number of proteins have been identified as important regulators of mitochondrial dynamics, the direct role of membrane lipid composition during the fusion and fission processes is poorly understood. To address these shortcomings, we devised a protein‐engineering platform that allows for the acute remodeling of structural phospholipids within the outer mitochondrial membrane (OMM) of intact cells. Specifically, we modified a bacterial phospholipase C (Bacillus cereus (Bc)PI‐PLC) to initiate the rapid hydrolysis of phosphatidylinositol (PI) and locally generate diacylglycerol (DAG); an important intracellular signaling molecule and metabolic precursor that is used in diverse lipid biosynthetic pathways. Spatial restriction of enzyme activity was achieved using a chemically inducible system consisting of a rapamycin‐dependent dimerization module (FKBP‐BcPI‐PLC) along with an OMM targeting sequence tagged with the FKBP‐rapamycin binding domain (OMM‐FRB). Using these unique molecular tools, we show that recruitment of FKBP‐BcPI‐PLC to the OMM not only causes the expected local accumulation of DAG, but also initiates the rapid and uniform fragmentation of the mitochondrial network. Mitochondrial fission induced by FKBP‐BcPI‐PLC is accompanied by profound swelling of the mitochondrial matrix along with vesiculation of the inner mitochondrial membrane (IMM) and a general loss of cristae, which all occur within minutes of tethering FKBP‐BcPI‐PLC to the OMM. Expression of dominant‐negative constructs targeting essential GTPases known to regulate OMM fission suggest that both dynamin‐related protein 1 (Drp1) and dynamin 2 (Dnm2) work together to drive efficient BcPI‐PLC‐induced mitochondrial division. However, results using a validated Drp1 knockout cell line show that the loss of Drp1 alone is sufficient to prevent the mitochondrial fragmentation initiated by FKBP‐BcPI‐PLC recruitment, indicating that Drp1 likely functions upstream or independent of Dnm2 in this context. Interestingly, unlike the induced OMM fission, removal of Drp1 from cells does not prevent the matrix swelling or OMM constrictions observed in response to acute generation of DAG within the OMM. Ongoing experiments are now focused on characterizing new methods to sequentially metabolize the DAG generated within the OMM as well as investigate how local lipid composition influences the binding and oligomerization of membrane‐shaping proteins that may function in concert with Drp1 to regulate mitochondrial remodeling. Overall, these studies establish a direct relationship between lipid metabolism within the OMM and clinically relevant morphological changes that are known to manifest in mitochondrial‐associated diseases.
Microbial metabolite mitigates arsenic induced oxidative stress, inflammation, and barrier dysfunction in gut epithelia
Authors: Ghosh Sweta , Banerjee Mayukh , Bodduluri Haribabu , Jala Venkatakrishna Rao
Published: May 2022

The intestinal barrier acts as a physical and immunological defense against luminal microorganisms, viruses, food antigens, and environmental factors. Intestinal inflammation and the pathogenesis of inflammatory bowel diseases (IBD) are closely associated with loss of gut barrier integrity and increased immunological responses. Environmental heavy metal toxins like arsenic (iAs3+) effectively regulates several cellular processes involved in cellular proliferation and homeostasis. Recently, it was shown that gut microbiota is required for full protection against acute arsenic toxicity using mouse models. In current study, we investigated the potential role of microbial metabolites such as urolithins to provide protection against arsenic‐induced gut barrier damage and inflammation. Urolithin A (UroA), a polyphenol derived microbial metabolite has been reported to provide protection against several diseases caused by inflammation and oxidative stress. Previously, our group demonstrated that microbial metabolite reversed the colitis induced defects in epithelial barrier function. In this study, we for the first time reveled the potential of UroA to mitigate arsenite induced toxicity and loss of gut barrier integrity. UroA showed a time‐ and concentration‐ dependent protection against iAs3+‐induced apoptosis and oxidative stress. Moreover, UroA treatment protected against iAs3+‐induced gut epithelial tight junction protein disruption/down regulation. Importantly, treatment with microbial metabolite attenuated arsenic induced barrier dysfunction in pre‐clinical human 3D small intestinal organoid tissue model. Taken together, our study indicates that beneficial microbial metabolites such as UroA intervention is a putative preventive/therapeutic approach against environmental iAs3+ related adverse activities on gut barrier.
Aortic Stiffness is Negatively Associated with Microvascular Glycocalyx Thickness and Microvascular Perfusion among Older Adults: Role of Carotid Pressure Pulsatility
Authors: Gimblet Colin , Armstrong Matthew , Nuckols Virginia , Stroud Amy K. , Guerrero Mariah , Pierce Gary
Published: May 2022

Introduction

The glycocalyx layer is essential to the maintenance of a healthy microvascular endothelium and diminishes with advancing age. Similarly, the aorta stiffens with aging and increases the risk of cardiovascular disease and target organ damage, possibly through pressure pulsatility‐associated microvascular damage. However, whether aortic stiffness and/or pressure pulsatility are associated with microvascular endothelial glycocalyx structure/function among older adults is unknown. Therefore, the objective of this analysis was to investigate the potential relation between aortic stiffness and pressure pulsatility with glycocalyx thickness and microvascular perfusion in older adults. We hypothesized that increased aortic stiffness and pressure pulsatility would be associated with less glycocalyx thickness and microvascular perfusion in older adults.

Methods

Sublingual microvessels were assessed using intravital microscopy in older adults (n=24; age 61 ± 6y, 46% women). Perfused boundary region (PBR) and red blood cell (RBC) filling %, indirect indices of glycocalyx thickness (larger PBR=smaller thickness) and microvascular perfusion respectively, were determined in vessels between 5‐25 µm RBC column width. Aortic stiffness was quantified by carotid‐femoral pulse wave velocity (cfPWV) and carotid pressure pulsatility as carotid pulse pressure (PP)/mean arterial pressure (MAP).

Results

Adjusting for age, sex and MAP, cfPWV was associated with larger PBR 5‐25 (β: 0.08; p=0.001) and lower RBC filling % (β: ‐0.02, p=0.002). Higher carotid pressure pulsatility was associated with lower RBC filling % (β: ‐0.12; p=0.03), but this relation was attenuated after adjusting for sex (p=0.11). Upon stratification by sex, higher carotid pressure pulsatility was associated with larger PBR 5‐25 (r=0.66, P=0.03) and lower RBC filling % (r=‐0.73, P=0.01) in women, but not men (P=0.94 and P=0.77 respectively).

Conclusion

Higher aortic stiffness was negatively associated with microvascular glycocalyx thickness and microvascular perfusion among older adults. Additionally, carotid pressure pulsatility was negatively associated with glycocalyx thickness and microvascular perfusion in women only. The mechanisms that contribute to sex differences in the relation between pressure pulsatility and glycocalyx structure/function in older adults require further study.
Effects of hsa‐miR‐9‐3p and hsa‐miR‐9‐5p on Topoisomerase IIβ Expression in Human Leukemia K562 Cells with Acquired Resistance to Etoposide
Authors: Carvajal‐Moreno Jessika J. , Hernandez Victor A. , Yalowich Jack C. , Elton Terry S.
Published: May 2022

DNA topoisomerase IIα (TOP2α/170; 170kDa) and Topoisomerase IIβ (TOP2β/180; 180kDa) are important targets for DNA damage‐stabilizing anticancer drugs, whose clinical efficacy is often attenuated by chemoresistance. Our laboratory has characterized an etoposide‐resistant human leukemia K562 clonal subline, designated K/VP.5. These cells show a decrease in both TOP2α/170 and TOP2β/180 expression. We recently demonstrated that a microRNA (miRNA)‐mediated post transcriptional mechanism plays a role in drug resistance via reduced TOP2α/170 protein levels in K/VP.5 cells. We hypothesize that a miRNA mechanism is similarly responsible for decreased TOP2β/180 levels in K/VP.5 cells. Both miR‐9‐3p and miR‐9‐5p are overexpressed in K/VP.5 compared with K562 cells, demonstrated by miRNA sequencing, and validated by quantitative polymerase chain reaction (qPCR). The 3’‐untranslated region (UTR) of TOP2β/180 contains a well‐defined miRNA recognition element (MRE) for miR‐9‐5p and putative MREs for miR‐9‐3p. Co‐transfection of K562 cells with a luciferase reporter plasmid harboring TOP2β/180‐3’UTR plus miR‐9‐3p and/or miR‐9‐5p mimics resulted in a statistically significant decrease in luciferase expression. miR‐9‐5p MRE mutation prevented this decrease validating direct interaction between this miRNA and TOP2β/180 mRNA. Transfection of K562 cells with miR‐9‐3p or miR‐9‐5p mimics led to decreased TOP2β protein levels without a change in TOP2β/180 mRNA. Inversely, K/VP.5 cells transfected with miR‐9‐3p or miR‐9‐5p inhibitors led to increased TOP2β/180 protein and no change in TOP2β/180 mRNA. Taken together, these results strongly suggest that TOP2β/180 mRNA is translationally repressed by miR‐9‐3p and miR‐9‐5p, and that these miRNAs play a role in acquired resistance to etoposide via regulation of TOP2β/180. Further experiments are underway to characterize miR‐9‐3p and miR‐9‐5p effects on TOP2β/180‐specific cytotoxicity.
Investigating CTLA‐4 binding to generate novel small‐molecule inhibitor
Authors: Khoroshilov Olivia , Greig James , MacGillivray Brendan
Published: May 2022

The CTLA‐4 receptor may be found on the plasma membranes of T‐cells where it acts as a negative regulator of T‐cell activation. When CTLA‐4 receptors bind B7 ligands (CD80/86) found on antigen presenting cells, they activate signaling pathways which, in combination with signaling from T‐cell receptors bound to antigen‐carrying MHC proteins, decrease the inflammatory response. Regulatory T‐cells (Tregs) constitutively express CTLA‐4 on their membranes and have T‐cell receptors that recognize self‐antigens; this allows them to moderate the self‐tolerance of the immune system. Specifically, CTLA‐4 signaling in Tregs leads to the release of anti‐inflammatory cytokines which decrease the proliferation of helper and cytotoxic T‐cells and prevent monocytes, macrophage precursors, from differentiating into macrophages that would produce pro‐inflammatory cytokines. Unfortunately, it is frequently the case that Tregs recognizing cancerous cells accumulate within tumors. This leads to the inability of the body to form an immune reaction against the cancer. For this reason, cancer therapies which inhibit CTLA‐4 signaling are being developed. Current strategies to limit CTLA‐4 signaling have been successful in treating multiple forms of cancer but primarily rely on reducing CTLA‐4 interactions with B7 through monoclonal antibody therapy targeting the extracellular domain of CTLA‐4. Monoclonal antibodies are not particularly convenient as they must be administered through IV infusion and some formulations carry the risk of inducing serum‐sickness, in which the immune system develops a reaction against the foreign anti‐CTLA4 antibodies. However, instead of inhibiting CTLA‐4 binding to B7 with an antibody, it may be possible to inhibit the intracellular signaling of CTLA‐4 that occurs after this binding with a small molecule that is easier to administer. One of the main proteins that binds to and is activated by stimulated CTLA4 receptors is the phosphatase SHP2. SHP2 uses an SH2 domain at its N‐terminus to bind CTLA‐4 through the YVKM motif of the latter. Binding causes a conformational change in the SH2 domain which makes the phosphatase active site on an adjacent domain of SHP2 more accessible. This conformational change also causes SHP2 to unbind from CTLA‐4 so that a new protein can take its place to be activated. Activated SHP2 dephosphorylates many cytoplasmic proteins at their tyrosine residues leading to Treg activation and thus decreased inflammatory response.