bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Prolonged High‐Altitude Exposure on Heart Rate Response to Central Sleep Apnea
Authors: Berthelsen Lindsey , Steele Andrew , Vanden Berg Emily , Bird Jordan , Thrall Scott , Wilson Richard , Jendzjowsky Nicholas , Day Trevor , Steinback Craig
Published: May 2022

Central sleep apnea (CSA) is common at high‐altitude (>2500m) and is characterized by a cyclical pattern of hyperventilation and transient arterial hypocapnia, triggering apnea. We have demonstrated that voluntary wakeful apnea at altitude promotes a greater degree of bradycardia and cardiac arrhythmias than at sea level. However, whether bradyarrhythmias are observed during CSA at altitude has not been investigated. We investigated the duration dependent effect of altitude on heart rate changes during periods of apnea overnight. We hypothesized that overnight apnea would result in a greater magnitude of bradycardia with prolonged hypoxic exposure.

Central sleep apnea (Apnea‐hypopnea index; AHI) was assessed overnight at low altitude (1045m), early (Night 2‐3) and late (Night 9‐10) at high altitude (N=14; 6 female). Heart rate (HR) and rhythm (electrocardiogram; ECG lead II) was collected alongside sleep data (RESMED Apnealink) to quantify sleep apnea events and desaturations. HR data were averaged over 10s prior to and 10s following apneas. Delta HR during apnea (BL HR – Nadir HR apnea) was determined to characterize bradycardia. Differences in heart rate prior to, during, and post apnea were compared using a repeated‐measures ANOVA design, with a Holm‐Sidak post‐hoc analysis where main effect of condition was significant. The magnitude of bradycardia during apneas between early and late time points were compared using a paired t‐test. Additional analyses of sex differences were performed using a two‐way mixed model ANOVA.

AHI was augmented at high altitude (main effect p=0.012) but not different between early and late time points (p=0.193). The incidence of central apneic events was greater late at high‐altitude compared to low altitude (p=0.0045). Overnight oxygen saturation was decreased at high altitude (main effect of time, p<0.01) but was elevated late (84 ± 3%) compared to early (82 ± 4%) at altitude (p=0.038). Average HR overnight was similar between early (74 ± 11bpm) and late (69 ± 10bpm) time points (p=0.134). HR dropped during apneas at early (‐6.2±4.6 bpm; p=0.018) and late (‐6.4±2.8 bpm; p<0.001) timepoints. Female participants had a lower AHI at high altitude (p=0.006) but tended to have a greater bradycardia (‐7.7± 5.6bpm early, ‐7.6±2.9bpm late) compared to men (‐3.9±0.68bpm early; ‐4.5±1.4bpm late; main effect of sex, p=0.052).

We demonstrate that CSA at altitude is associated with bradycardia that does not appear to become greater with prolonged exposure. It appears females may be less susceptible to CSA but have a greater bradycardic response during apneas compared to males.
Ketones Improve the Mitochondrial Coupling Efficiency of Brown Adipocytes
Authors: Moazzami Zahra , Crawford Peter , Ruan Hai‐Bin
Published: May 2022

Brown adipose tissue (BAT) thermogenesis is an energy‐demanding response, and this capacity can be leveraged to dissipate excess energy in obesity and its associated metabolic complications. Therefore, understanding the regulatory mechanisms of BAT mitochondrial uncoupling and thermogenesis is crucial. Ketone bodies are endogenous metabolites mainly found during starvation and carbohydrate restriction; however, their regulatory role on BAT thermogenesis is elusive. In this study, we aim to investigate the potential metabolic reprogramming induced by ketones on BAT. We hypothesize that ketones are alternative fuels for BAT during energy/carbohydrate shortage and decrease mitochondrial uncoupling to counterbalance the systemic energy deficit. In mice, ketogenic diet reduces core body temperature by 0.4˚C. Using Seahorse respirometry in differentiated brown adipocytes, we found that ketones improve mitochondrial coupling efficiency (ATP‐linked respiration/ basal respiration) by 30%, implying an attempt to decrease thermogenesis. The gene Oxct1, encoding succinyl‐CoA:3‐ketoacid CoA transferase that is required for ketolysis, is abundantly expressed by brown adipocytes. BAT‐specific Oxct1 knockout renders mice more resistant to cold‐induced decrease in core body temperature during fasting or ketogenic diet, via mechanisms independent of UCP1. Together, our data suggest that ketones potentially communicate hunger to brown adipocytes, thus promoting mitochondrial coupling efficiency for energy conservation.
A Directed Evolution Experiment to Improve Protein Splicing of Two Inteins From Haloquadratum walsbyi
Authors: D'Angelo Anthony C. , Yalala Vaishnavi R. , Mills Kenneth V.
Published: May 2022

We are interested in the protein splicing of the inteins from the extreme halophile Haloquadratum walsbyi. Protein splicing is a post translational process by which an intein excises itself and ligates the two flanking polypeptides, the exteins. Two inteins interrupt the cell division control (cdc21) protein in H. walsbyi. One intein splices only at salt concentrations above 2 M, whereas the other will splice at lower salt concentrations. We have designed a directed evolution experiment coupling the growth of E. coli to successful splicing of each intein from the protein that confers resistance to the antibiotic kanamycin. Preliminary results suggest that each intein can promote splicing of the kanamycin resistance protein, and we are currently testing a pool of intein mutants for enhanced splicing activity.
A Shared Anchor on Primary Sigma Factor SigA by the WhiB‐Like Proteins
Authors: Guiza Beltran Daisy C. , Horova Magdalena , Wong Huey‐Xian , Zhang Zhang
Published: May 2022

WhiB‐like (Wbl) proteins are a unique group of iron‐sulfur cluster ([4Fe4S])‐containing transcription factors exclusive to actinobacteria. They play crucial roles in the virulence, survival, and propagation of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. Seven Wbl proteins (WhiB1‐WhiB7) are found in Mtbwith diverse regulatory roles. All Mtb Wbl proteins except for WhiB5 have been shown to interact with the conserved region 4 of the primary sigma factor (SigAr4) in RNA polymerase holoenzyme. Our recent structural and biochemical analyses indicate that two Wbl proteins, WhiB1 and WhiB7, bind to the same site on SigAr4 unexpectedly through tight hydrophobic interactions involving several conserved aromatic residues of the Wbl proteins and His516 of SigAr4. Substitution of these aromatic residues in either WhiB1 or WhiB7 at the molecular interface abolishes their binding to SigAr4. Here, we hypothesize that all Mtb Wbl proteins share a similar molecular interface when in complex with SigAr4, based on the primary sequence analysis and structural modeling. Using the site‐directed mutagenesis and the in vitro protein‐protein interaction assays, we confirm that all Mtb Wbl proteins, including WhiB5, bind to the same site on SigA centered on His516, and the conserved aromatic residues within the Fe‐S cluster binding pocket are required for SigA binding. The results from this study will offer insights into how the Wbl proteins are utilized in Mtb to orchestra gene expression in response to environmental cues in the host.
Mimicking a Model: A Novel Approach to Pelvis and Perineum Dissection
Authors: McManus Lauren , Reynolds Amberly
Published: May 2022

Pelvic dissections are difficult for students to visualize and complete given the standard dissection approach and limited time provided in standard anatomy curricula at U.S. medical schools. The purpose of this project is to propose a novel approach to dissecting the pelvis with aims of increasing student understanding of pelvic anatomy. The proposed novel technique allows for ease of viewing by making the donor more mobile and more model‐like, like what is seen with common anatomical models used at medical schools.

The technique begins with transverse sectioning of the body into, an upper, mid, and lower section with the mid‐section containing the pelvic region. This section allows the dissector to utilize the pelvic bowl in a standing position, show cross‐sectional anatomy of the thigh muscles, and provide focus on three different dissection pathways. The pathways created include, neurovascular, organ, and muscle specific.

On a preliminary dissection, this method provided ease at dissecting this region, a structurally similar view to live bodies and models, and additional pathways for learning. This dissection was done by a first‐year medical student six months following typical anatomical dissection. The technique is beginning to redefine in situ study of the pelvis and perineum by providing an alternative, more focused way to utilize donors for this region. Models tend to provide the “perfect” anatomy, which can be useful in understanding the typical relationships, however, it is important for students to understand this region and the relationships found with each structure to help better prepare them for future patients.

Future studies to enhance this technique will determine how the pathways can be better utilized. In having students perform this technique alongside the typical techniques, a comparison can be made with a larger population. By having subjective feedback, it can show how students interacted with the dissection, as well as how it was utilized throughout this region of study.
Biochemical analysis of human ERK2 mutants reveals important residues in MEK1‐ERK2 binding and phosphorylation
Authors: Ye Ming , Hotan Quynn , Glatz Gwendolyn , Finkelstein Maxwell T. , Hayashi Scout , Miller Emma P. , Lau Benjamin , Li Kevin , Chen Han , LeBlanc Julia , Zaidi Hussain , Fera Daniela
Published: May 2022

The Ras‐Raf‐MEK‐ERK (MAPK) pathway is a signal transduction cascade used to regulate cellular processes including cell cycle progression and proliferation. Aberrant activation of this pathway is implicated in cancer development, and treatment with Raf, MEK and ERK inhibitors often leads to adaptive resistance. Combination therapies have been shown to offer benefits; however, the MEK‐ERK interface remains poorly understood, hindering structure‐guided approaches to the design of potent MAPK pathway inhibitors targeting this complex.

To identify important residues for the MEK1‐ERK2 interaction, we performed site‐directed mutagenesis on ERK2. We used circular dichroism to assess the secondary structure of the ERK2 mutants. We also used biolayer interferometry binding experiments coupled with phosphorylation assays to evaluate the impact of the mutated residues on the formation of the MEK1‐ERK2 complex and activity.

Circular dichroism showed no differences in secondary structure for any of our ERK2 mutants. Of all the mutations generated, the L234D mutation in ERK abrogated binding and phosphorylation by MEK1 the most. Other mutants showed some reductions in binding or activity but require further analysis. Of note, L234 is located on an ERK2 α‐helix adjacent to the phosphorylation lip, consistent with MEK1 binding this face during phosphorylation. Our results suggest that this α‐helix may play critical roles in the MEK1‐ERK2 complex. Studying the impact of additional mutations in this and additional regions will develop our understanding of the MEK‐ERK interface and inform the design of allosteric inhibitors that can modulate MEK‐ERK complex formation.
The Role of Polyphenols on Blue Light‐Induced Retinal Pigment Cell Damage
Authors: Bel Nicholas J. , Gupta Sanjoy K. , Khaper Neelam
Published: May 2022

The increased exposure of eyes to blue light is a growing concern in our everyday lives. Blue light is responsible for cell damage, cell death, and oxidative stress all of which can lead to vision loss. Resveratrol and pterostilbene are polyphenols found in the skin of various fruits including grapes and blueberries and have been known for antioxidant properties. The aim of this study was to investigate the protective role of resveratrol and pterostilbene on cell death, cell proliferation, and oxidative stress in Human Retinal Pigment Epithelial (ARPE‐19) cells exposed to blue light. ARPE‐19 cells were pre‐incubated with either; 50 or 100uM of resveratrol or 10 or 50uM of pterostilbene for 4 hours followed by exposure to blue light (475nm) for 12 hours. Subsequent to the blue light exposure, cellular viability, oxidative stress, and death were measured in each treatment condition. Blue light exposure resulted in a decrease in cell viability, an increase in caspase 3/7 activation, and cell death. This was accompanied by an increase in protein expression of catalase, and manganese superoxide dismutase (MnSOD) and an increase in 4‐Hydroxynoneal (4HNE) protein adduct formation. In conclusion, resveratrol mitigated the damaging effects of blue light on ARPE‐19 cells by attenuating cell death and oxidative stress. This in‐vitro study is the first step into understanding how polyphenols can possibly be used to reduce the oxidative damage caused by blue light in the eyes and prevent unnecessary vision loss.
Upregulation of GCLC is Responsible for SFN‐induced Tumor Cell Proliferation
Authors: Jang Chan Ho , Lee Jinkyung , Kwon Min Jeong , Kim Yoonsu , Chae Ji Hyeong , Jeong Yoon Ah , Kwon Eun Jeong , Oh Jisun , Kim Jong‐Sang
Published: May 2022

Sulforaphane (SFN), a sulfur‐containing compound that belongs to the isothiocyanate class of dietary phytochemicals found in cruciferous vegetables, has been known to cause a hormetic effect on cancer cell growth. We previously reported that SFN at low doses stimulated proliferation of HCT116 human colorectal carcinoma cells through Nrf2 activation in vitro and in vivo. Recently, we discovered that brusatol, a nuclear factor erythroid 2‐related factor 2 (Nrf2) inhibitor, significantly suppressed the SFN‐induced proliferation of HCT116 cells. Therefore, we hypothesized that SFN‐induced proliferation of HCT116 cells is mediated by an Nrf2 downstream gene(s). Transcriptomic profiling revealed a set of differentially expressed genes (DEGs) involved in glutathione (GSH) metabolism as compared between wildtype (WT) and Nrf2 knockout (KO) HCT116 cells treated with or without SFN. In particular, we found that glutamate‐cysteine ligase catalytic subunit (GCLC) was highly elevated in SFN‐treated WT HCT116 cells among the DEGs, but not in SFN‐treated Nrf2 KO HCT116 cells. To confirm if upregulation of γ‐glutamate‐cysteine ligase (γ‐GCL) is associated with SFN‐induced proliferation of HCT116 cells, inhibiting γ‐GCL by buthionine sulfoximine (BSO, a selective γ‐GCL inhibitor) and silencing GCLC by short hairpin RNA (shRNA) were carried out. The results showed that SFN‐induced proliferation of HCT116 cells was significantly reduced by γ‐GCL inhibition and abolished by GCLC knockdown. Furthermore, we confirmed that BSO suppressed SFN‐induced tumor growth of WT HCT116 xenograft mice and the tumor growth was not stimulated in both Nrf2 KO and GCLC shRNA HCT16 xenograft mice by SFN at a low dose. Collectively, these findings indicate that upregulation of GCLC‐dependent GSH biosynthesis by SFN promotes proliferation of HCT116 cells and thus GCL could be a potential target for chemotherapy.
Utilization of CYP3A‐Specific Inhibitors to Study Cytochrome P450 CYP3A7 Activity in Neonatal Human Liver Microsomes
Authors: Work Hannah M. , Kandel Sylvie E. , Lampe Jed N.
Published: May 2022

CYP3A7 is a member of the cytochrome P450 (CYP) 3A enzyme sub‐family that accounts for ~50% of the total CYP content in neonate and fetal livers. The amount of other CYP3A enzymes, CYP3A4 and CYP3A5, expressed in neonates is low but highly variable, and therefore, the activity of individual enzyme isoforms is difficult to differentiate. Recent studies report that CYP3cide, clobetasol propionate, and azamulin are specific inhibitors for CYP3A4, CYP3A5, and CYP3A4/5, respectively. However, the inhibitory effects towards CYP3A7 were not investigated for the latter two compounds, and the use of these compounds to study CYP3A7 activity has not been reported/investigated. Thus, we sought to characterize these compounds further and utilize them to isolate and study the activity of CYP3A7 in neonatal human liver microsomes. We have examined three substrate probes—dibenzylfluorescein, luciferin‐PPXE, and midazolam—to determine the IC50 values and mechanism‐based inactivation parameters (Ki, Kinact) of the purported isoform specific inhibitors. We discovered that azamulin was unable to selectively inhibit any of the CYP3As, but CYP3cide and clobetasol propionate exhibited IC50values for CYP3A4 and CYP3A5 two to three orders of magnitude lower than CYP3A7. CYP3cide and clobetasol propionate were further tested in mixtures of CYP3A4, CYP3A5, and CYP3A7 to mimic the neonatal human liver microsome environment and ascertain their effectiveness in isolating CYP3A7 activity. We were able to inhibit CYP3A4 without disturbing CYP3A7 activity using CYP3cide at concentrations below 5 μM. The majority of CYP3A5 activity was inhibited using low clobetasol propionate concentrations, but using higher concentrations also inhibited CYP3A7 activity due to its lower selectivity. These findings provide valuable insight regarding testing conditions to investigate the metabolism of new drug candidates and help determine drug safety in neonates.
Sympathetic Responses to Isometric Handgrip Exercise in Women following Preeclamptic Pregnancies
Authors: Orr Harlan R. , Adler Tessa E. , Leone Cheryl , Paidas Michael J. , Stachenfeld Nina S. , Usselman Charlotte W.
Published: May 2022

Women who have had preeclamptic (PE) pregnancies are at increased risk for developing cardiovascular diseases later in life. One potential mechanism that may mediate this increased risk is sympathetic dysregulation. Indeed, there is evidence for elevated muscle sympathetic nerve activity (MSNA) both during and following PE pregnancies. Moreover, previously PE (PPE) women may demonstrate impaired MSNA responses during stress, although this has only been examined to a limited extent. Therefore, we tested the hypothesis that MSNA responses to isometric hand grip exercise (HG) would be greater in PPE women (n=6, age 31±6 years, BMI 29±5 kg/m2, 17±4 months postpartum) compared with women who had a healthy pregnancy (HP; n=8, age 29±2, BMI 25±4 kg/m2, 14±4 months postpartum). MSNA (peroneal nerve microneurography), mean arterial pressure (MAP; finger photoplethysmography), and total peripheral resistance (TPR; MAP/cardiac output), were assessed at baseline, during a 2‐min HG protocol, and during 2‐min post‐exercise circulatory occlusion (PECO). As previously reported, baseline MSNA was higher in PPE than HP (22±7 vs 13±5 bursts/min, P=0.01). Baseline MAP was not different between PPE and HP (94±11 vs 89±9 mmHg, P=0.23), nor was TPR (13±3 vs 13±2 mmHg/L/min, P=0.63). During HG, MSNA was greater in PPE than HP (24±8 vs 16±6 bursts/min, P=0.03), whereas MAP was not different between PPE and HP (95±12 vs 95±11 mmHg, P=0.99), nor was TPR (13±3 vs 14±2 mmHg/L/min, P=0.39). During PECO, no differences were observed between PPE and HP women in MSNA (26±8 vs 18±6 bursts/min, P=0.08), MAP (93±12 vs 96±12 mmHg, P=0.70), or TPR (12±3 vs 15±2 mmHg/L/min, P=0.18). These findings indicate that PPE women demonstrate exaggerated MSNA during isometric HG exercise relative to HP women, although the role of the metaboreflex in mediating this effect, as assessed using PECO, remains unclear. Importantly, these data also demonstrate that young PPE women were able to buffer the deleterious cardiovascular outcomes of elevated MSNA such that MAP and TPR outcomes were similar to age‐matched HP women during all conditions.
The Association between High Intake of Omega‐3 Fat Food, Perceived Stress and Mental Distress Levels
Authors: Kumarasivam Tharsana , Moser Lindsey , Dempsey Gerard , Colabelli Michael , Rosenberg Lexi , Welch Megan , Begdache Lina
Published: May 2022

The connection between stress and eating patterns has been extensively studied; however, the reverse association between dietary factors and perceived stress is still a gap in the literature. In particular, the effect of omega‐3 fatty acids on perceived stress is little known. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are omega‐3 fatty acids that play major roles in the nervous system. Therefore, the aim of this study was to assess the relationship between intake of omega‐3 fatty acids rich food, perceived stress and mental distress. It is hypothesized that a diet high in omega‐3 will have a negative correlation with mental distress and perceived stress. An anonymous online survey, consisting of the validated Food‐Mood Questionnaire, Kessler‐6 and Perceived Stress Scale, was distributed to participants 18 years and older through various means, including email and social media platforms. A total of 1591 individuals completed the survey. Data was analyzed using Spearman’s Rho correlation and Principal Component Analysis, in SPSS Version 25.0. Our results indicated that a diet high in omega‐3 fatty acids, specifically flax seeds and nuts (p <0.01), fish oil supplements (p <.05) and fatty fish such as salmon (p <.01) negatively correlated with perceived stress. Additionally, our results suggest that flax seeds and nuts (p <0.01) as well as fish (p <0.01) negatively correlated with mental distress. Collectively, the results of this study suggest that a diet high in omega 3 fatty acids may reduce perceived stress and improve mental wellbeing.
Immune Cell Regulation by Macrophage Derived Small Extracellular Vesicles in Pain
Authors: Pande Richa , Jean Toussaint Renee , Muir Roshell , Haddad Elias E. , Ajit Seena
Published: May 2022

Background

Development of chronic pain is related to aberrant neuroimmune signaling. Small extracellular vesicles (sEVs) are 30‐150nm size particles of endosomal origin. They play an important role in neuroimmune crosstalk facilitating transfer of biomolecular cargo between cells. Our studies show that sEVs from RAW 264.7 macrophage cells can be therapeutic or prophylactic in mouse model of inflammatory pain. We investigated the mechanistic basis of how sEVs from lipopolysaccharide (LPS) stimulated (Exo+) and unstimulated (Exo‐) cells impact primary glial cells, dendritic cells (DCs) and adaptive immune cells such as CD4+T cells. We hypothesize that macrophage derived sEVs can potentiate T cell activation directly, or indirectly by the upregulation of major histocompatibility complex‐II (MHC‐II) and co‐stimulatory molecules on DCs, attenuating pain hypersensitivity. We will also test if sEVs confer protection to glial cells in the context of inflammation.

Methods

Primary mouse DCs, microglia and astrocytes were incubated with 1µg Exo‐/+sEVs for 24 hours. Splenocyte derived CD4+ T cells were treated with 0.1ug Exo‐/+ sEVs for 72 hours. Flow cytometry and qPCR was used for analysis.

Results

Characterization of RAW sEVs showed the presence of sEV markers CD81 and Alix. sEV treatment of DCs resulted in an increase in CD80+/CD86+ MHCIIhiCD11c+/hi DCs. Treatment of CD4+T cells with sEVs increased Tgf‐βexpression. Pretreatment with Exo+ sEV increased Tfgf‐β expression in glial cells upon LPS stimulation. An increase in PPAR (Peroxisome proliferator activated receptor) expression was observed upon sEV treatment of glial cells.

Conclusion

Upregulation of MHC‐II and costimulatory molecules on DCs as well as PPAR upregulation in glial cells upon sEVs treatment indicates that sEVs from RAW 264.7 cells can modulate immune response. These results indicate sEVs modulate innate cells such as microglia and DCs and adaptive immune cells such as CD4+ T cells may contribute to therapeutic effect in attenuating chronic pain.
Cranial Dysmorphology in the Palatine, Vomer, and Pterygoid Plates of Fgfr2mice
Authors: Pennings Amanda N. , Ogg Miette L. , McNulty Margaret A. , Holmes Megan A. , Mussell Jason C. , DeLeon Valerie B.
Published: May 2022

The fibroblast growth factor and receptor system (FGF/FGFR) regulates cell proliferation and differentiation for various tissue types in the body – including osseous tissue. Mutations in the FGFR2 gene are associated with craniosynostosis, leading to alterations of the shape of bones in the head and face. We have previously demonstrated premature fusion of the spheno‐septal synchondrosis in Fgfr2C342Y/+ mice, a model for Crouzon syndrome. Here, we further describe the specific morphology of individual bones as a result of the Fgfr2C342Y/+ mutation.

We used microCT scans of mutant and wild‐type littermates at two developmental stages (adult and postnatal day 14) to create 3D virtual reconstructions of the skull in the program 3DSlicer. After transforming each scan to a uniform, orthogonal orientation, we collected coordinate data for 38 fixed craniometric landmarks to estimate the shape of the cranium. We used the geomorphpackage in R to perform a Procrustes superimposition and principal components analysis (PCA). We used the resulting shape coordinates in a two‐way Procrustes ANOVA to test the effects of Age and Genotype. In addition, we isolated key cranial elements (palatine, vomer, and pterygoid plates of the sphenoid) to compare between genotype groups at the P14 stage using a mesh‐to‐mesh comparison. This allowed us to test our expectation that fusion of the spheno‐septal synchondrosis has a greater impact on the vomer than on the palatines or pterygoid plates.

Our morphometric analysis and two‐way Procrustes ANOVA confirmed previous reports of statistically significant cranial shape differences between wild‐type and Fgfr2 mutants at both timepoints. Vomer shape was significantly altered in the mutant P14 cranium when compared to other key cranial elements (palatine, and sphenoidal pterygoid plates), suggesting that fusion of the spheno‐septal synchondrosis has a greater impact on the vomer compared to the other bones in the skull. Our mesh‐to‐mesh comparison showed that the vomer is both dorso‐ventrally and rostro‐caudally compressed in mutants relative to wild‐type mice at this early stage. Additionally, the palato‐sphenoid suture appears prematurely fused in the mutants.

This study provides insight into the skeletal phenotypic mechanisms that characterize diseases involving mutations of the FGFR2 gene, such as Crouzon syndrome. The dysmorphology of individual bones affect other osseous structures due to morphological integration within the skull. Elucidating the underlying mechanisms of the phenotypic expression of FGFR2 syndromes supports further translational research.
NCLX expression regulation by mild stress conditions
Authors: Cabral‐Costa João Victor , de Lima‐Júnior José Carlos , de Miranda Ramos Vitor , Kowaltowski Alicia J.
Published: May 2022

Mitochondrial matrix Ca2+ efflux is largely mediated by the mitochondrial Na+/Ca2+ exchanger (NCLX), directly coupling mitochondrial Ca2+ and Na+ handling. NCLX has been shown to play a central role in many pathophysiological processes, including colorectal tumor metastasis, and Parkinson’s and Alzheimer diseases. Indeed, sporadic Alzheimer´s disease patients had decreased frontal cortex NCLX levels, an effect recapitulated in mouse models. However, transcriptional mechanisms specifying its functional states has not been clearly defined. Here, we aimed to investigate how NCLX expression is regulated. NCLX mRNA was consistently found to be increased after mild stressful events, including scrambled siRNA transfection and control adenoviral vector transduction. We then hypothesized that NCLX gene expression was controlled by a stress‐sensitive pathway. Indeed, serum‐deprived astrocytes and mouse embryonic fibroblasts (MEFs) showed an increase in NCLX mRNA levels, an effect that was even further modulated by incubation with thapsigargin or H2O2, indicating expression sensitivity to a wide variety of stress signals. Here, we took advantage of a genome‐wide approach using available epigenomics datasets, and transcriptomic data to pinpoint transcriptional factors that orchestrate NCLX gene expression upon homeostatic conditions in astrocytes. We identified promoter‐distal enhancer‐like elements in forebrain mouse and primary astrocytes using chromatin immunoprecipitation high‐throughput sequencing (ChIP‐seq) data marked by histone acetylation of lysine 27 (H3K27ac). We also used a public ATAC‐seq to define a coincident region with accessible chromatin. Still, transcription factor binding sites uncovered through sequence‐based single site analysis of the NCLX gene indicated several putative transcription factors, including many components of the NF‐κB family. In parallel, public RNA‐seq databases were mined for patterns that were correlated with transcription factor known effects, before proceeding with binding confirmation through ChIP‐PCR. In summary, we were able to build a foundation to test the modulation of NCLX expression. This proof‐of‐concept of NCLX stress‐induced mechanism may be further explored as a tool for targeting cellular Na+/Ca2+signaling, mitochondrial function, and metabolism.
Interaction of Zika Virus with a Mosquito Axl‐like Receptor
Authors: Donahue Jacob , Miller Catherine
Published: May 2022

Viral infections continue to be the bane of human medicine, often with minimal options for treatment. Of particular concern are arboviruses, or viruses transmitted by an arthropod vector, such as a mosquito. Recently, Zika virus (ZIKV), a mosquito borne arbovirus, has emerged as a major threat to public health in many parts of the world. It is unknown what protein(s) ZIKV uses to bind to and infect cells within its mosquito vector. The receptor used by ZIKV to bind to and infect human cells has been identified as the tyrosine kinase receptor family Axl. A homolog of the Axl gene is not present within the mosquito genome. However, genes for the Down‐Syndrome Cell Adhesion Molecule family members (DSCAMs) found within multiple mosquito species share key functional modular ectodomains with the human Axl gene. Here, we extracted total RNA from Culex and Aedes species of mosquitos, reverse transcribed the RNA into cDNA and then cloned the mosquito DSCAM cDNA with the highest homology to human Axl into a eukaryotic expression vector. The vector will then be transfected into a mammalian cell line void of human DSCAM and human Axl expression to determine if the mosquito DSCAM molecule is expressed as a surface protein. Finally, we will determine if ZIKV is capable of binding to the mammalian cell line that now expresses the mosquito DSCAM protein. This project will allow us to learn if ZIKV is capable of binding to the mosquito DSCAM protein. In turn, this information could lead to the possibility that the mosquito DSCAM protein allows infection by ZIKV as well. Ultimately, this research could assist vaccine development for Zika, or lead to treatments that could block ZIKA from infecting its mosquito vector.
Differential Effects of the Fgfr2c C342Y Mutation on Developing Cranial Cartilage
Authors: Durham Emily L. , Kawasaki Mizuho , Pitirri M K. , Perrine Susan M. M. , Kawasaki Kazuhiko , Richtsmeier Joan T.
Published: May 2022

Crouzon syndrome is an autosomal dominant condition characterized by craniofacial anomalies in the absence of major hand and foot abnormalities. Although premature closure of the coronal suture is the focus of most studies of Crouzon syndrome, cartilage elements have also been described as anomalous. The Fgfr2cC342Y/+ Crouzon syndrome mouse model carries a cysteine to tyrosine substitution at amino acid position 342 (Cys342Tyr; C342Y) in Fgfr2, equivalent to one of the FGFR2 mutations commonly associated with Crouzon syndrome. This mutation results in constitutive activation of the receptor and is associated with up‐regulation of osteogenic differentiation and consequent skull deformities. Development of the bony skull of these mice is known to be affected, but is preceded by development of the chondrocranium, a cartilaginous skull whose elements either disappear, remain as cartilage, or ossify endochondrally. In conjunction with identified disparities in the size and shape of the chondrocranium between Fgfr2cC342Y/+ mice and unaffected Fgfr2c+/+ littermates throughout development, we analyzed the morphology and proliferative activity of chondrocytes of the nasal septum (mostly remains as cartilage), braincase floor (ossifies endochondrally), and lateral walls (disappears) of the chondrocranium at embryonic day 14.5 (E14.5) and E15.5 to determine the effects of the mutation on cartilage cells. Consistent with a larger chondrocranium, Fgfr2cC342Y/+ mice had relatively more chondrocytes in the nasal septum (p≤0.01) and lateral wall (p≤0.001) at E14.5. At E15.5 there were more chondrocytes in the lateral wall (p≤0.001) of Fgfr2c+/+ chondrocrania, but the cells of the lateral wall in Fgfr2cC342Y/+ mice were larger (p≤0.001) than in control (Fgfr2c+/+) mice at both timepoints. There were no differences between genotypes in either chondrocyte number or size in the braincase floor, however more proliferative activity was identified in Fgfr2c+/+ braincase floors at E14.5 (p≤0.001). No differences in proliferation were identified between genotypes in either the nasal septum or the lateral walls. Our data indicate that regions of the chondrocranium are differentially affected by the Fgfr2c C342Y mutation, potentially based on the fate of each cartilage. Disruption to the tightly regulated developmental processes of the chondrocranium including cell proliferation and hypertrophy can have significant and lasting effects on craniofacial development through direct effects on cartilage formation and the indirect effects of chondrocranial morphology on development of bone.
Application of Physiological Autonomic Activity Tracking GUI and Computational Algorithms for Measuring Persistent Vagal Nerve Dysfunction After Recovery from SARS‐CoV‐2 Infection
Authors: Wilson Damen A. , Nowak Thomas , Gupta Anita , McKinnon Michael , Clements Isaac , Everett Thomas , Powley Terry , Ward Matthew
Published: May 2022

Post‐Acute Sequelae of SARS‐CoV‐2 infection (PASC) is now recognized as a constellation of symptoms such as postural hypotension, anxiety, and “brain fog” in addition to autonomic nervous system dysfunction such as tachycardia and labile hypertension. Better quantification of these conditions, specifically, autonomic nervous system dysfunction, is desired for future diagnostics, treatment modalities, and gaining a further understanding of PASC. This research has led to application of a novel autonomic activity tracking algorithm and GUI suite from previous work, see Figure 1, to study the physiological autonomic activity in six patients who had recovered from acute SARS‐CoV‐2 infection a mean of six months prior but were still experiencing symptoms. By measuring skin sympathetic nervous activity (SKNA), heart rate variability, and the cutaneous electrogastrogram (EGG), all before and after a water meal challenge, the data was fed into a custom analysis pipeline, shown in Figure 1. Each of the PASC patients were compared to a mean response of 34 healthy controls, each undergoing a 20‐minute baseline recording and another 20‐minute recording after ingestion of an 8 oz water test meal. All six patients showed significantly abnormal heart rate variability on frequency domain analysis in predominantly the low frequency (LF) and very low frequency (VLF) bands, and less so in the high frequency (HF) band, suggesting sympathetic nerve dysfunction. Three patients showed a significant decrease in SKNA while two showed a significant increase. All patients showed an abnormal cutaneous EGG. As shown in Figure 2, the temporal responses of aSKNA, VLF, LF, HF, and EGG for the PASC patients revealed that on average they were statistically different (p<0.05) from the healthy controls’ responses respectively during 98.4%, 78.4%, 86.76%, 47.9%, and 86.1% of the 40‐minute time period in testing, 20 minutes of baseline and 20 minutes after ingestion. In conclusion, we are looking at the sympathetic, parasympathetic, and enteric nervous systems synced temporally for applications with classification and further stratification of PASC based on the temporal dynamics of their autonomic nervous system mediated coordination from digestion. This shows that SARS‐CoV02 infection appears to have a significant effect on sympathetic and parasympathetic autonomic nervous system function and may be responsible for the disturbances noted in PASC. This work provides the framework and example of use for further applications in autonomic disorder physiological response exploration and furthermore can be expanded to other areas of neuromodulation.
Ligand Specificity in Progesterone Receptor
Authors: Dube Namita , Okafor Denise C.
Published: May 2022

Progesterone receptor, nuclear receptor subfamily 3, group C, member 3 (PR, NR3C3) is a member of the steroid receptor subfamily of nuclear receptors (NR3), i.e. ligand‐regulated transcription factors. PR regulates multiple biological processes in response to binding of steroid‐derived hormones. PR is promiscuously activated by various steroid hormones, including estrogens, androgens, and corticosteroids. Currently, it is not understood how steroid hormones differentially modulate the ligand binding domain (LBD) of PR to achieve various transcriptional outcomes. Here, we use a computational approach to investigate how dynamics of PR are altered by ligand binding to achieve distinct transcriptional properties. Previous studies on the PR LBD revealed that residues in helices 3, 5 and 12 are functionally conserved in nuclear receptors and play important roles in stabilizing agonistic conformations. To probe the roles of these residues in discriminating between steroid hormones, we performed long MD simulations, using a library of 34 steroidal ligands with EC50 values ranging from inactive to 1E‐02 nM. To investigate ligand‐specific allosteric signaling, we investigated communication pathways between the ligand binding pocket and key regulatory surfaces on the PR LBD surface to determine the specificity with which ligands modulate allosteric coupling between two sites. Our weighted dynamic network analysis revealed that the active and inactive steroids modulated key regions of PR distinctly. Additional analysis of communication paths allowed us to further categorize agonists into three groups, consistent with their potency of activation. We used in silico mutagenesis to investigate roles for specific residues in achieving ligand‐selective PR signaling. Combined, the results of the study provide structural and dynamic insight into how PR achieves ligand specificity. Further, these studies can illuminate strategies for the design of novel PR ligands for therapeutical uses.
Physical Therapy Student Knowledge Retention from a Remotely Taught Anatomy Course
Authors: Nesbitt Allison , Briedwell Teresa
Published: May 2022

Introduction

A whole‐body donor anatomy course is a foundational learning experience for additional musculoskeletal courses for Doctor of Physical Therapy (DPT) students. The summer 2020 human anatomy course at the University of Missouri was taught remotely with daily asynchronous and synchronous components. The DPT students were evaluated with online multiple‐choice lecture and picture‐based lab practical exams. Due to the novel nature of the course, student knowledge retention and interpretation of three‐dimensional anatomy is unknown.

Aim

The purpose of this study was to evaluate student retention of anatomy knowledge from the remotely taught course, test if students can identify structures on anatomical donors in a laboratory setting that they previously identified on picture online exams and survey the students’ prior anatomy experiences.

Methods

A 90‐minute in‐person laboratory session was held seven months after the completion of the remote anatomy course for first year DPT students. Students’ previous anatomical course work and opinions of remote and lab learning were assessed with a survey. Prior to entering the anatomy lab, the students completed a multiple‐choice retention quiz of eight questions from the four‐unit exams that all students previously answered correctly. During the laboratory session, students worked in pairs to answer fifteen questions and identify some of the same structures from the retention quiz on the donors.

Results

Every student had taken at least one undergraduate course as a requirement for DPT school admission or as part of their major. All the students described benefits to a lab‐based anatomy course compared to a remote course and a majority emphasized the hands‐on and spatial learning in their comments. Most students (73.7%) also believed there were some advantages from the remote course, including having additional study time, cost savings, and more individualized learning. Fifty‐nine of the 60 students in the remote anatomy course took the retention quiz and the average score was 86.7% (range 62.5% ‐ 100%). Students performed well on the musculoskeletal questions on the lab identification quiz.

Conclusion

The physical therapy students from this remote anatomy course had slightly higher retention scores than most studies of health professions students’, particularly in topics that were repeated throughout their first‐year courses.

Significance

The study supports the importance of whole‐body donor anatomy lab learning for students and the importance of flexibility, and ample inclusive access to learning materials for students.
Assessing the Importance of Chloroplast and Mitochondrial Small Heat Shock Proteins in Arabidopsis thaliana
Authors: Suri‐Payer Fabian , Patel Parth , Vierling Elizabeth
Published: May 2022

Organisms have a dedicated class of proteins to safeguard their proteome – molecular chaperones. Many chaperones help proteins fold and unfold, maintain their structure, and even re‐fold denatured protein aggregates by using ATP. Small heat shock proteins (sHSPs) are ATP‐independent chaperones that are proposed to prevent irreversible aggregation of denaturing proteins and handing them off to the ATP‐dependent chaperones. sHSPs are especially diverse in plants, possibly due to the exposure of plants to environmental stress due to their sessile nature. Despite their widespread occurrence, little is known about the exact function and role of sHSPs, especially in chloroplasts and mitochondria. Both organelles are critical to plant growth, development and reproduction and are also sources of cellular stress via production of reactive oxygen species. Understanding the role of sHSPs in these organelles is vital to grasp how plants deal with stress and could allow for transferring this knowledge into crop plants to improve agricultural yield. The model plant Arabidopsis thaliana possesses four sHSPs localized to the chloroplasts and mitochondria. HSP23.5‐MI/CP and HSP23.6‐MI/CP are thought to dual‐localize to both organelles, HSP25.3‐CP localizes to chloroplasts and HSP26.5‐MII is targeted to mitochondria. These sHSPs are primarily expressed under heat stress conditions. We have developed single, double, triple and quadruple knockout mutants of these four organelle sHSPs and confirmed that they are protein nulls by immunoblotting with specific antisera. Under standard growth conditions these organelle sHSPs appear dispensable, as the knockout mutants grow similarly to wild type. Notably, the organelle sHSP mutant plants show only minimal growth defects when heat stressed, which is in stark contrast to a lethal phenotype shown by a null mutant of the ATP‐dependent chaperone HSP101. Further work is in progress to determine if chaperones or other proteins are altered in expression to compensate for absence of the sHSPs during stress. Additionally, the dual‐localization of HSPs 23.5‐MI/CP and 23.6‐MI/CP has yet to be fully established and also raises the question of crosstalk between chloroplast and mitochondrial sHSPs. To answer these questions a combination of immunoblotting and proteomics approach on isolated chloroplasts and mitochondria is being taken.
Development of Cancer‐on‐a‐chip Technology for the Study of Tumor Microenvironment and Metabolism
Authors: Ericson Daniel L. , Azukas Joshua , Strey Helmut H. , Rubenstein David
Published: May 2022

Cancer metabolism has become an area of intense interest. In the 1920s, Otto Warburg showed that cancer cells metabolize glucose to produce lactate and ATP in the presence of oxygen, i.e. aerobic glycolysis, also known as the Warburg Effect. It is now appreciated that cancer cells display a complex metabolic phenotype. Metabolic reprogramming by oncogenes and tumor suppressor genes has been linked to the altered metabolic function of cancer cells. However, the tumor microenvironment (TME), which has different nutrient gradients for glucose, oxygen, and amino acids, has a dramatic effect on cancer cell growth and proliferation. In vivo studies have demonstrated a heterogeneous metabolic phenotype that is difficult to characterize. In addition, the exchange of nutrients and metabolites between the TME, vasculature, and tumor cells has been difficult to fully recapitulate. As a tumor grows, nutrients diffuse from the blood vessels to the cancer cells, in a process partially described by A. Krogh in 1919 through a reaction‐diffusion differential equation. Krogh’s work showed that for typical tissues the oxygen decay radius is around 150μm and we find approximately the same ‘Krogh radius’ for high glucose consumption cancer cells (e.g. MDA‐MB‐231). Thus, around 150μm from a blood vessel exists an oxygen deprived environment that imposes tremendous competitive stresses on the cancer cells for survival. There is rising evidence that this selection pressure favors the transformation to metastasis and causes invasive tumor cell lines to exhibit high glycolytic rates and overall metabolic reprogramming. However, most studies focused on defining the metabolic function of tumor cells have been conducted in vitro, under conditions that do not represent the physiological conditions accurately.

Here, we present a cancer‐on‐a‐chip technology that produces a well‐defined 3D TME that can be exposed to precise nutrient stresses such as varying oxygen or other nutrient gradients. In addition, we can determine the metabolic phenotype of cancer cells with varying oncogenic changes and degrees of aggressiveness. Our technology should be able to address the question of the role of the TME on metabolic reprogramming and in particular glycolysis. Our design is based on growing tumor microenvironments inside 360µm ID capillary tubing that can be accessed by 360µm PEEK tubing. To allow/control perfusion through the microenvironment we embed a 70µm nylon fiber into a photocross‐linkable gelatin gel in which the cells are dispersed (MDA‐MB‐231/GFP). After exposing the gel to UV‐light for a few seconds, the nylon fiber is pulled, leaving a perfusable microchannel. Using this technique, we have successfully perfused cell‐laden hydrogels over weeks while observing cell‐growth in a fluorescence microscope.