bluuebunny/crossref_metadata_2025 huggingface dataset:
SPARC: Neural elements mediating side effects during cervical vagus nerve stimulation in the pig
Authors: Nicolai Evan Neil , Settell Megan L. , Knudsen Bruce E. , Pelot Nicole A. , Grill Warren M. , Williams Justin C. , Ludwig Kip A.
Published: April 2020

Vagus nerve stimulation (VNS) is FDA‐approved and CE‐marked for a diverse set of conditions including epilepsy, depression, heart failure, and others. Despite widespread adoption and perceived efficacy, a post‐hoc analysis of patients treated for heart failure using VNS revealed that only 12% of patients showed stimulation evoked heart rate changes one‐year post‐implant. This finding is not unexpected given that A‐type nerve fibers have the lowest activation thresholds, and they innervate neck muscles, causing contractions that patients will not tolerate. Therefore, muscle responses at lower amplitudes may have prevented application of higher amplitudes required to achieve cardiac effects in clinical trials of VNS for heart failure. Understanding therapy‐limiting side effects to VNS and finding ways to mitigate them are critical for optimizing VNS for clinical therapies.

We developed a swine model of VNS which closely approximates the size and fascicular organization of the cervical vagus nerve observed in humans. The clinical LivaNova stimulation electrode was used as advised by a practicing neurosurgeon. We hypothesized that neck muscle responses are mediated by somatic branches of the vagus nerve as opposed to direct electrical activation of the muscle fibers. Further, we hypothesized that stimulation evoked responses depend on the location of the stimulation electrode on the vagus nerve. We measured compound nerve action potentials using intrafascicular electrodes placed within the vagus to identify activation of different nerve fiber types by conduction velocity. The downstream effects of nerve fiber activation, i.e. changes in heart rate and neck muscle responses (EMG), were measured using an arterial catheter and intramuscular electrodes, respectively. We then used a neuromuscular junction blocking agent, as well as systematic transection of somatic vagus nerve branches, to specifically identify the pathways causing neck muscle contractions.

Two pathways contributed to VNS‐evoked neck muscle responses: activation of nerve fibers in the recurrent laryngeal branch of the cervical vagus nerve at low amplitudes and in the superior laryngeal branch at higher amplitudes. Lesser activation of the superior laryngeal branch could be achieved by moving the stimulation electrode caudally down the length of the vagus. This work represents a step towards mitigating intolerable side effects of VNS, thereby allowing maximization of intended target engagement and therapeutic benefit.

Support or Funding Information

The Defense Advanced Research Projects Agency (DARPA) Biological Technologies Office (BTO) Targeted Neuroplasticity Training Program under the auspices of Doug Weber and Tristan McClure‐Begley through the Space and Naval Warfare Systems Command (SPAWAR) Systems Center with (SSC) Pacific grants no. N66001‐17‐2‐4010, and the NIH SPARC Program Award OT2 OD025340.
Probing the Nature of the Hydrophobic Cleft of Perilipin 5
Authors: Cool Lillian , Tansey John T.
Published: April 2020

Numerous conditions that currently threaten human health are related to aberrant lipid storage. These conditions include type II diabetes mellitus, non‐alcoholic fatty liver disease, obesity and heart disease. Nearly all cells of the body are able to store neutral lipids but this storage is not without consequence. Neutral lipids are stored in the cell in specific organelles termed lipid storage droplets. These organelles consist of a hydrophobic core surrounded by a phospholipid monolayer studded with several proteins that regulate the formation and breakdown of the droplet. Perilipin 5 is a lipid droplet protein which has been demonstrated to play key roles in lipid storage and mobilization in oxidative tissues including cardiac muscle and liver tissue in the fasted state. Perilipin 5 is a member of the perilipin protein family, a family encoded by five highly related genes. A different family member, perilipin 3, has had its structure solved. The C‐terminus of perilipin 3 consists of a four‐helix bundle domain and a mixed alpha‐beta structure. At the interface of these two units is a hydrophobic cleft. We have aligned the carboxy terminal region of perilipin 5 with perilipin 3 and have used threading and molecular modeling to generate a theoretical model of the region in perilipin 5. This structure retains the four‐helix bundle, mixed alpha‐beta domain and the hydrophobic cleft of perilipin 3, albeit with a potentially different amino acid sequence. The perilipin 5 structure differs in that the cleft contains a short insertion which may affect this pocket. Ongoing studies seek to generate mutations to this cleft to probe the function of this structural feature in perilipin 5.
Understanding the Conformational Changes of Kinesin‐5
Authors: Robinson Kingston , Griffin Jessica , DeVry Jenelle , Chaney Joseph
Published: April 2020

Human Kinesin‐5 (Eg5), an anticancer drug target, is key to the assembly of the bipolar spindle during mitosis. Its promise as a clinical target comes from the fact that it is involved only in metaphase and not interphase like some of the leading clinical drugs; however, the overall mechanism of inhibition needs further exploration. Inspection of the crystal structures of Kinesin‐1 dimers reveal the addition of strands to the central β‐sheet is found in one head and absent in the other; this previously unrecognized structural asymmetry between the two heads may suggest a regulatory mechanism involving the N‐terminal Neck‐Linker (NL) and the C‐Terminal Cover‐Neck. These structural changes in the neck‐linker and cover‐neck of Kinesin‐5 have not been reported. Thus, the importance of establishing this conformational switch in Kinesin‐5 requires additional experiments for understanding and molecular validation. But first, we must understand how changes in the neck‐linker modulate kinesin function. We have inserted three residues (+DAL) into the middle of the neck‐linker of dimeric Kinesin‐5 (1‐513) and KHC (1‐560). Our results indicate these neck‐linker mutants can allosterically regulate kinesin ATPase activity. Our future goal is to test the response to inhibition by current anticancer inhibitors such as S‐trityl‐L‐cysteine.

Support or Funding Information

BUILD Technicians supported under grant #RL5GM118966 and BUILD undergraduate students under grant #TL4GM118968. An Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant #5 P20 GM103424‐15 and 3 P20 GM103424‐15S1. The Louisiana Cancer Research Consortium (LCRC) and National Center for Research Resources RCMI program Grant #8 G12MD007595 and the Louisiana Biomedical Research Network Grant #8P20GM103424. The National Institute of Minority Health and Health Disparities of the National Institutes of Health under award #G12MD007595. Students are supported by GRAD Star Scholar Mini Grants through Center for Undergraduate Research and Graduate Opportunity (CURGO).
Genetically‐Encoded ATP Sensors for Fluorescence Lifetime Measurements
Authors: Tantama Mathew
Published: April 2020

Fluorescent protein‐based genetically‐encoded sensors of adenosine triphosphate (ATP) have proven valuable for the detection and imaging of both intracellular and extracellular ATP. Current ATP sensors employ either a Förster‐type resonance energy transfer (FRET) fluorescent protein pair or circularly‐permuted fluorescent proteins to report changes in ATP levels. Steady‐state fluorescence spectroscopy and imaging has been the predominant mode of measurement, but both single‐channel intensity and two‐channel ratiometric signals are subject measurements artifacts from a variety of confounding factors such as variance in sensor expression levels and wavelength‐dependent scattering. Recently, we demonstrated that time‐resolved measurements of fluorescence lifetime offer a robust and quantitative alternative mode of detection that relies only on an intrinsic photophysical parameter. Here, we characterize the fluorescence lifetime responses of a panel of the currently available ATP sensors that span different protein architectures. We find that ATP sensors can provide large fluorescence lifetime dynamic ranges, with some sensors exhibiting nearly 1 ns changes in response to ATP. Thus, our study demonstrates that time‐resolved measurements expand the application space of genetically‐encoded fluorescent protein ATP sensors and will facilitate multicolor, multiplexed quantitation of live‐cell energy metabolism and purinergic signaling dynamics.
Assessing Sialic Acid Uptake in an In Vitro Model of GNE Myopathy
Authors: Goines Briana N. , Sattler Kristina M. , Crowe Kelly E.
Published: April 2020

Mutations in the GNE gene leads to GNE myopathy (GNEM), a rare autosomal disease that causes skeletal muscle weakness. The GNE gene encodes an enzyme for sialic acid (SA) biosynthesis, causing a reduction of SA when biallelic GNE mutations are present. Currently, gene therapies are in development to replace the mutated GNE gene and restore SA production. Determining that SA can be used as a biomarker would allow researchers to quantify the success of the gene therapy. Our lab is attempting to take a panel of SA‐binding proteins called lectins that are known to show a binding difference in muscle cells from GNEM patients as compared to cells from their unaffected family members and determine which lectin best reflects SA changes in GNEM patients. Preliminary data does not show the expected difference in SA levels between cells derived from GNEM patients and their unaffected family members. As this could be from SA scavenging from serum in the media, we have cultured these GNEM patient‐derived myotubes in serum‐free media prior to fixation to assess SA scavenging from serum in this model. Here, we show the extent to which SA scavenging from serum affects cell surface sialylation in GNEM patient cells in vitro.

Support or Funding Information

This work was supported by funds from the Beta Beta Beta Research Scholarship Foundation Fund.
Ketone Bodies Attenuate Wasting in Diverse Models of Atrophy
Authors: Koutnik Andrew Paul , Poff Angela M. , Ward Nathan P. , Deblasi Janine M. , Soliven Maricel A. , Romero Matthew A. , Roberson Paul A. , Fox Carl , Roberts Michael D. , D’Agostino Dominic P.
Published: April 2020

Background

Cancer anorexia cachexia syndrome (CACS) is a distinct atrophy disease negatively influencing multiple aspects of clinical care and patient quality of life. Although it directly causes 20% of all cancer‐related deaths, there are currently no model systems that encompass the entire multifaceted syndrome, nor are there any effective therapeutic treatments.

Methods

A novel model of systemic metastasis was evaluated for the comprehensive CACS (metastasis, skeletal muscle and adipose tissue wasting, inflammation, anorexia, anemia, elevated protein breakdown, hypoalbuminemia, and metabolic derangement) in both males and females. Ex vivo skeletal muscle analysis was utilized to determine ubiquitin proteasome degradation pathway activation. A novel ketone diester (R/S 1,3‐butanediol acetoacetate diester) was assessed in multifaceted catabolic environments to determine anti‐atrophy efficacy.

Results

Here, we show that the VM‐M3 mouse model of systemic metastasis demonstrates a novel, immunocompetent, logistically feasible, repeatable phenotype with progressive tumor growth, spontaneous metastatic spread, and the full multifaceted CACS with sex dimorphisms across tissue wasting. We also demonstrate that the ubiquitin proteasome degradation pathway was significantly upregulated in association with reduced IGF‐1/insulin and increased FOXO3a activation, but not TNF‐α‐induced NF‐κB activation, driving skeletal muscle atrophy. Additionally, we show that R/S 1,3‐butanediol acetoacetate diester administration shifted systemic metabolism, attenuated tumor burden indices, reduced tissue catabolism, and mitigated comorbid symptoms in both CACS and cancer‐independent atrophy environments.

Conclusions

Our findings suggest the ketone diester attenuates multifactorial CACS skeletal muscle atrophy and inflammation‐induced tissue catabolism, demonstrating anti‐catabolic effects of ketones bodies in multifactorial atrophy.

Support or Funding Information

This work was supported by Disruptive Nutrition (Grant #: 61431150), Florida High Tech Corridor (Grant #: FHT 18‐15), Donner Foundation (Grant #: 61431151), and Office of Naval Research (Grant #: N00014‐18‐1‐2701). Additional funding for muscle molecular analysis were provided by discretionary laboratory funds of MDR.
Exogenous Root Uptake of Glycine Betaine Mitigates Improved Tolerance to Salinity Stress in Avicenna germinans under Ambient and Elevated CO2 Conditions
Authors: Maurer Rosa , Tapia Meghan E. , Shor Audrey C.
Published: April 2020

In recent years, climate change has caused unpredictable variations in sea levels and an increase in atmospheric CO2 levels. Increasing temperatures and subsequent rates of evaporation have been demonstrated to cause increased coastal salinity in certain areas, which induces abiotic stress, in mangroves systems. The osmolyte, glycine betaine, has been shown in numerous studies to increase the tolerance of plants to environmental stressors, including salt stress. A handful of studies have demonstrated that increased CO2 concentrations confer increased salt tolerance and photosynthetic productivity of different mangrove species. Avicenna germinans, commonly referred to as the black mangrove, is native to Pacific and Atlantic coasts, and produces glycine betaine endogenously. This study sought to characterize the response of A. germinans exposed to increasing saline conditions, grown under normal and high CO2 concentrations. Elucidation of the role glycine betaine accumulation plays in tolerance to abiotic stress was investigated. A. germinans seedlings were subjected to increasing levels of salt stress (15–90 ppt) and treated with a dose response of glycine betaine (0–50 μM). Half of the seedlings were grown under ambient (400 ppm) CO2 conditions, and the other half were grown under elevated (800 ppm) CO2 conditions. Increased salinity tolerance and higher photosynthetic rates were observed among seedlings exposed to elevated CO2 conditions, as indicated by increased survival rates and qualitative health observations. This research may provide insights into potential consequences posed by climate change, as well as characterizing the glycine betaine signaling pathway in this mangrove species. Looking into the future, the knowledge obtained through this study may potentially contribute to our predictions of mangrove adaptability and therefore conferred protection of our coastlines in an ever‐changing world.
Using Edpuzzle as a Method to Improve Student Engagement and Performance in the Introductory Biochemistry Laboratory
Authors: Shelby Shameka J. , Fralish Zachary
Published: April 2020

Various engagement tools have been commonly used within biochemistry courses to increase student engagement and maximize student exposure to material within the time constraints of a class session. These time constraints also extend to the laboratory component of these courses which often leads to increased levels of anxiety for students. One method that has been used successfully in the past to increase student understanding and performance during laboratory experiments is the utilization of pre‐laboratory video lectures. While the use of pre‐laboratory videos has been shown to be beneficial for students, instructors often have no efficient way of assessing student understanding of the video content prior to the laboratory session. Edpuzzle is a free online service that allows for instructors to upload videos and incorporate questions into videos at various points within the video to assess student understanding. In an effort to increase student performance in an introductory biochemistry laboratory, edpuzzle was adapted to engage students with the material in preparation for the laboratory session. Prior to each laboratory session, students viewed an edpuzzle video and answered questions to assess their understanding of the experimental details and concepts that would be covered in the lab. Surveys were conducted to determine student satisfaction, learning outcomes, and level of anxiety associated with the laboratory as a result of using edpuzzle as a laboratory preparation tool. Students reported high satisfaction rates, increased confidence during laboratory experiments, and decreased anxiety about the laboratory overall. The results suggest that students viewed the edpuzzle application as a valuable component of their laboratory preparation routine. Due to the flexibility of edpuzzle, it can be adapted for use in other chemistry or biology laboratories at various levels. In addition, edpuzzle can also be used as a supplementary tool for expanding lecture content.

Support or Funding Information

Florida Southern College Department of Chemistry, Biochemistry, & Physics
EGFR Activation is Suppressed by High Doses of Folic Acid in HPV Negative Cervical Cancer Cells
Authors: Zito Christina , Tsai Cheng-Hsiu
Published: April 2020

Cervical cancer is one of the leading causes of death due to cancer in women. Human Papilloma Virus infection is the primary risk factor for developing cervical cancer, however very little is known about HPV negative cervical cancers. Folate fortification of flour for prevention of neural tube defects has been mandatory in North America since 1998; and folate supplementation is recommended during pregnancy. Folate fortification is not mandated in all countries, however, due to concerns about possible adverse effects on cancer incidence or prognosis. There is evidence that folate intake can reduce risk of developing tumors but other contradictory studies indicate that supraphysiologic doses of folate can actually lead to disease progression. Folate receptor alpha is known to be expressed in many cancer cell lines, including HPV positive cervical cancer. There is some evidence that folic acid can promote methylation of the EGFR, leading to a decrease in EGFR expression, and that EGFR might be active in some HPV positive cervical cancers. The purpose of this study is to determine if EGFR plays a role in HPV negative cervical cancer cell growth, and the effects of folic acid on EGFR protein expression and activation. Two HPV negative cervical cancer cell lines were used for this study: C33A and DoTc2‐4510. High doses of folic acid caused a decrease in both C33A and DoTc cell proliferation. Interestingly, qPCR and western blotting analysis indicate that only the DoTc2‐4510 cells expressed EGFR. The DoTc2‐4510 cells were sensitive to the EGFR inhibitor, gefinitib, with an IC50 of 0.01 uM. There was no change in EGFR expression upon folic acid treatment, but interestingly high doses of folic acid did inhibit EGFR activation. Taken together this data indicates that EGFR may be a target for a subset of HPV negative cervical cancers, and that high doses of folic acid may help prevent EGFR positive HPV negative cervical cancer cell growth.
Compensatory Enzyme Expression In Response To Chronic Ethanol Intake And Partial Hepatectomy Disrupts Zone‐Specific Organization Of Lobular Metabolism In The Rat Liver
Authors: Barnhart Benjamin , Chopra Chetna , Achanta Sirisha , Hoek Jan B. , Vadigepalli Rajanikanth
Published: April 2020

Regulation of liver metabolism is heterogeneous across lobular zones. In response to the acute metabolic challenge of partial hepatectomy (PHx), hepatocytes enact a global transcriptional program both to compensate for and replace lost tissue mass. Chronic ethanol intake has also been shown to rewire transcriptional programs in hepatocytes, with suppressive effects on hepatocyte proliferation and blunted regeneration post PHx. However, it is not clear how zone‐specific metabolism transiently adjusts to ensure PHx‐induced regeneration, or how zone‐specific metabolism is adapted due to chronic insult by ethanol intake.

Male Sprague‐Dawley rats were fed a Lieber‐DeCarli liquid diet containing ethanol or carbohydrate for 5–6 weeks prior to 70% PHx in which left lateral and medial lobes were surgically removed. Liver tissue samples were collected at the time of surgery and after 24 hours of recovery. Laser‐capture microdissection was used to collect regions of tissue from eight distinct lobular zones between portal triads and central veins, and high throughput qPCR was used for transcriptional analysis.

We show here that regrowth after PHx involves multiple patterns of compensatory gene expression of enzymes from most major metabolic pathways. Genes whose expression is normally restricted to a particular zone responded to PHx in a wide range of patterns, e.g., by increasing expression with no change of zone (e.g., Arg1), expanding expression to nearby zones (e.g., Glul), or transient uniform expression across the whole lobule (e.g., Ldha). We tested whether chronic ethanol intake dysregulates zonation patterns of gene expression by exaggerating, dampening, redistributing, or eliminating preexisting lobular heterogeneity. After PHx, chronic ethanol intake dysregulated zonation of expression patterns in a variety of ways, including a reversal of healthy adaptations (e.g., Pck1). Other ethanol‐induced patterns of expression seen were suppression of PHx‐induced expression (e.g., Ldha) and redistribution of expression (e.g., Arg1).

The dysregulation of zonated metabolic gene expression patterns in chronic‐ethanol‐fed rats suggests that zone‐specific compensatory mechanisms may contribute to the ethanol‐impaired liver regeneration post‐PHx. Overall, this work shows the transient changes in zone‐specific transcriptional regulation in response to PHx, and identifies genes whose zone‐specific expression is affected early in ethanol‐induced disease progression. These heterogeneous changes within the hepatic lobule could be a marker of ethanol‐induced adaptation preceding more severe impairment.

Support or Funding Information

Supported by NIH NIAAA R01 AA018873, T32 AA007463, and NIBIB U01 EB023224.
Skeletal muscle‐specific PHD2 deficiency improved running endurance in mice
Authors: Kubota Takahiro , Nunomiya Aki , Osana Shion , Takada Hiroaki , Nagatomi Ryoichi
Published: April 2020

Introduction

Hypoxic training is one of the methods supposed to improve the performance of endurance athletes. Under a normoxic condition, prolyl hydroxylase domain containing protein 2 (PHD2) leads hypoxia inducible factor (HIF) to proteasomal degradation. Under hypoxia, PHD2 becomes inactive and let HIF be stabilized. HIF, then, promotes the expression of downstream genes, resulting in enhanced glycolysis, suppressed electron transport system, promotion of red blood cell production and angiogenesis. In PHD2 systemic knock‐out mice, hypoxic responses are elicited even under normoxia due to stabilized HIF. Interestingly, an enhanced endurance training effect was confirmed in PHD2 systemic knock‐out mice (Nunomiya et al. 2017). In PHD2 systemic knock‐out mice, calcineurin dependent increase in the proportion of type I fibers in skeletal muscles has been confirmed. (Shin et al. 2016). Whether the shift in fiber type, and more importantly the training effect is dependent on the hypoxic response within the muscle fibers or on the systemic hypoxic response has not been elucidated. The purpose of this study was to examine the effect of hypoxic response in skeletal muscle on the endurance training effect.

Method

In order to delete PHD2 specifically in skeletal muscle, doxycycline was administered for 18 days to PHD2flox/flox/Acta1rtTA/tetO‐Cre mice. The running time on a rodent treadmill until exhaustion was measured 8–10 days after the final administration of doxycycline before training and after 4 weeks of treadmill training, Endurance testing protocol: treadmill speed was set at 10 m/min for the initial 30 min, then after the speed was increased by 2 m/min every 15 min with no inclination. Training protocol: training was performed 5days per week for 4 weeks. Mice ran for 30 min per day. The running speed was 18m/min with 5 degrees uphill inclination.

Result

In PHD2flox/flox/Acta1rtTA/tetO‐Cre+/− mice, a significant reduction in PHD2 expression in gastrocnemius, tibialis anterior muscle, and soleus was confirmed. Skeletal muscle specific PHD2 knocked out mice had significantly increased endurance time compared to control mice before training. But there was no significant difference in the training effect between skeletal muscle‐specific PHD2‐deficient mice and control mice. There was no significant difference in training effect between skeletal muscle‐specific PHD2‐deficient mice and control mice.

Conclusion

Hypoxic response in the skeletal muscle resulted in a higher endurance capacity without training. There was no additive training effect in the mice the mice with hypoxic response. It was suggested that systemic hypoxic response may be responsible for the additive endurance training effect confirmed in the previous study.
Understanding Sarcospan Protein Function by Exposure of Mice to Beta‐Adrenergic Stress Conditions
Authors: Wacker Amanda L. , Elsheikh Salma , Valera Isela C. , Hwang Hyun Seok , Parvatiyar Michelle S.
Published: April 2020

Introduction

Sarcospan (SSPN) is a core component of the dystrophin‐glycoprotein complex (DCG) located at the sarcolemma of muscle fibers. SSPN has been shown to play a major role in membrane stabilization of the heart and other striated muscles, however many of its additional functions remain unknown. Previous studies have shown that SSPN deletion diminishes cardiac β‐adrenergic responsiveness, however it elicits increases in cardiac mass (JAHA, 2015 Dec 23;4(12). Blunted β‐adrenergic responsiveness in SSPN‐null hearts appears to be part of a complex phenotypic presentation that occurs downstream of alterations in sympathetic drive, and a propensity toward metabolic syndrome and insulin resistance.

Methods

Here, we document the whole‐body effects of ß‐adrenergic stimulation in three‐month‐old WT and SSPN‐null male and female mice after treatment with an isoproterenol (ISO) dosing regimen (8 mg/kg of body weight/day for 5 days) with tissues collected 2 weeks after treatment and examined for alterations in histology, gene and protein expression. After the treatment regimen, morphometric measurements were recorded and cardiac function assessed by echocardiography.

Results

After ISO treatment significant differences were found in sympathetic and cholinergic receptor expression in SSPN‐null cardiac and white adipose tissue (WAT) that were distinct from WT. The response of SSPN‐null mice to ISO treatment displayed distinct sex‐differences. After ISO treatment, WT and SSPN‐null male mice exhibited similar levels of WAT lipolysis, evidenced by loss of body and reduced size of epidydymal fat pads. However, female SSPN‐null mice were protected from ISO treatment induced loss in body weight. Furthermore, WT females had reduced spleen weights after ISO treatment, indicative of normal immune cell migration to other tissues, whereas spleen weights were unchanged in SSPN‐null females after ISO treatment suggesting suppressed immune responsiveness.

Discussion

The goal of this study is to better understand how the cardiac function of SSPN‐null mice appears largely unaltered by β‐adrenergic stimulation, whereas other tissues display overall normal responsiveness to ISO treatment. Since lipolysis is induced through activation of β‐adrenergic receptors, we will examine associated markers in untreated and β‐adrenergic‐stimulated WT and SSPN‐null mice of both sexes.

Support or Funding Information

Research Support: Florida State University, FSU CRC FYAP Award, and AHA 16SDG29120002.
Molecular Mechanism and Functional Role of Hypoxia‐induced Reactive Oxygen Species in Pulmonary Arterial Endothelial Cells
Authors: Wang Harrison , Song Tengyao , Wang Yong-Xiao , Vincent Peter , Zheng Yun-Min
Published: April 2020

Hypoxia‐induced (Group 3) pulmonary hypertension (PH) is a common devastating lung disease. Pulmonary artery endothelial cells (PAECs) are a major contributor in hypoxic PH due to possible roles of reactive oxygen species (ROS). However, the production up‐and‐down, molecular mechanisms and functional roles of hypoxia‐induced ROS in PAECs are not well established. In this study, we first used Amplex UltraRed reagent to assess hydrogen peroxide (H2O2) generation. The result indicated that hypoxic exposure resulted in a significant increase in Amplex UltraRed‐derived fluorescence (i.e., H2O2 production) in human PAECs. To complement this result, we employed lucigenin as a probe to detect superoxide (O2−) production. Our assays showed that hypoxia largely increased O2− production. Hypoxia also enhanced H2O2 production in isolated mitochondria from PAECs. In support, using the genetically encoded H2O2 sensor HyPer, we revealed that hypoxia caused a large increase in HyPer‐derived fluorescence (ROS production) in PAECs, which was blocked by the mitochondrial inhibitor rotenone or myxothiazol. Hypoxia largely increased proliferation of PAECs, determined using Ki67 staining and direct cell number accounting. The hypoxic cell proliferation was blocked by rotenone or myxothiazol. Like rotenone and myxothiazol, lentiviral shRNA‐mediated knockdown of Rieske iron‐sulfur protein nicotine (RISP), known to be a primary molecule in mitochondrial ROS generation, inhibited the hypoxic proliferation as well. Hypoxic exposure evoked a significant increase in migration of PAECs, evaluated by wound healing assay. Pharmacological and genetic inhibition of mitochondrial ROS generation with myxothiazol and RISP knockdown both diminished the hypoxic cell migration. Taken together, we conclude that hypoxia significantly increases intracellular ROS generation in human PAECs, the hypoxia‐induced intracellular ROS generation primarily occur in mitochondria, and RISP serves as an essential molecule in controlling the hypoxic mitochondrial ROS generation. Equally Importantly, and RISP‐dependent mitochondrial proliferation that is likely to be ROS‐dependent. Conceivably, hypoxia remarkably causes proliferation and migration of human PAECs, which is predominantly mediated by RISP‐dependent mitochondrial ROS, thereby making an essential contribution to hypoxic pulmonary hypertension.
Durable Changes to Airway Mucus Secretion Following a Single Cholinergic Challenge
Authors: Collins Emily , Liao Yan-Shin , Guevara Maria Valentina , Schurmann Veronica , Atanasova Kalina , Reznikov Leah R.
Published: April 2020

Cholinergic signaling in the airway elicits robust airway smooth muscle contraction and mucus secretion. In our previous work, we showed that re‐exposure of the airways to a cholinergic agonist 48 hours post initial challenge exaggerated airway narrowing. Here we test the hypothesis that re‐exposure to a cholinergic agonist exaggerates mucus secretion. To test this hypothesis, we challenged male and female pig airways with the synthetic cholinergic agonist bethanechol. Forty‐eight hours later, we re‐exposed the airways and assessed mucus secretion ex vivo using jacalin lectins. We found that re‐exposure mildly increased mucus secretion. The increase in mucus secretion was not associated with an impairment or alteration in mucociliary transport. We also stained tracheal cross sections with antibodies directed against mucin5B and mucin5AC, the major secreted glycoproteins that comprise mucus. Our ongoing studies suggest that cholinergic challenge had no major effect on the amounts of mucin5B or mucin5AC. Critical next steps include measuring the amount of mucins in the lung lavage fluid, as well as examining key components of the cholinergic signaling cascade.

Support or Funding Information

HL119560, OD023854
Cardiorespiratory Fitness, Hippocampal Subfield Volumes, and Pattern Separation Task Performance in Older Adults
Authors: Kern Kathryn L. , Nauer Wehr Rachel K. , Storer Thomas W. , Schon Karin
Published: April 2020

Cognitive aging is associated with reduced integrity of the medial temporal lobe memory system. A key facet of episodic memory that demonstrates behavioral impairment with aging is pattern separation (PS), or the disambiguation of similar stimuli during neural encoding. PS is supported by the dentate gyrus (DG), a subfield of the hippocampus that demonstrates striking plasticity in response to exercise. Recent work from our laboratory has demonstrated that increasing cardiorespiratory fitness (CRF) through exercise training is associated with increased left anterior DG/CA3 volume and improved PS task performance in young adults. However, how these variables interact in cognitive aging is unknown, yet critical, given that aging is associated with impairments in hippocampal plasticity and PS task performance. Based on our findings in young adults, we predicted that CRF would positively correlate with DG/CA3 volume and PS task performance in older adults. Secondly, given the suggested role of the DG in PS task performance in humans, we predicted that DG/CA3 volume would significantly correlate with PS task performance. Sixty‐four participants aged 55–85 years underwent a submaximal treadmill test to estimate maximal oxygen uptake (VO2MAX), which operationally defined CRF, an MRI scan to examine volume of the hippocampal subfields, and a behavioral task designed to tax PS at parametrically varying levels of stimulus similarity (10, 30, 50%). There was a significant effect of similarity on PS task performance, with participants demonstrating lowest task accuracy in the condition theoretically placing the highest taxation on PS (50%). Left DG/CA3 body volume significantly predicted performance in the 50% condition. However, there was no relationship between CRF and PS task performance. Furthermore, while CRF did not predict DG/CA3 volume, it did significantly predict right and left subiculum volume, which may be unique to female participants between the ages of 60–80 years. Altogether, these findings provide support for a role of the DG in PS in older adults and suggest a relationship between CRF and hippocampal subfield volume that may be unique to the bilateral subiculum in female older adults.

Support or Funding Information

This work was supported by NIH grant R00 AG036845 (Schon), R21 AG049968 (Schon), and the BU Clinical and Translational Science Institute (1UL1TR001430).
Antioxidant and Anti‐inflammatory Activities of Methanolic Fraction from Native Mexican Avocado Seed Oil
Authors: Zavala-Guerrero Baruc , Hernández-García Alejandra , Torres-Martínez Rafael , Meléndez-Herrera Esperanza , Ríos-Chávez Patricia , Ochoa-Zarzosa Alejandra , López-Meza Joel E. , Saavedra-Molina Alfredo , Salgado-Garciglia Rafael
Published: April 2020

Avocado fruit oil (Persea americana), mainly cultivar Hass, has excellent medicinal properties as antioxidant and anti‐inflammatory activities. However, these properties have not been evaluated in the seed oil of the native Mexican avocado (P. americana var. drymifolia). This study investigated the antioxidant and anti‐inflammatory activity of native Mexican avocado seed oil and the methanolic fraction. Seed oil was obtained with hexane by the Soxhlet method, which was extracted liquid‐liquid to obtain the methanolic fraction. In vitro antioxidant was examined with three radical scavenging methods (ABTS, DPPH and TAC) at 1 mg/mL, and IC50 was determined. Although the oil seed presented high percentage of antioxidant activity in a range of 90.5 to 91.38% with in vitro methods, the highest antioxidant activity was shown by methanolic fraction (99.7%) with ABTS method, presenting an IC50 of 0.035 mg/mL. Anti‐inflammatory activity of oil seed and methanolic fraction (0.1, 1 and 10 mg/kg of body weight) was estimated by measuring the paw edemathickness by carrageenan‐induced paw edema model in male Wistar albino rats (400 g), and compared to Ibuprofen (25 mg/kg of body weight), with topical and oral administration (50 μL) dissolved in DMSO 5%. Control animals received an equal volume of vehicle. Edema was induced with 1% carrageenan to all the groups after four hour of treatments, and paw thickness was checked at 1, 2, 3, 4, and 8 h. With topical administration of 10 mg/kg, the highest anti‐inflammatory effect was observed at 8 h, decreasing by 70.8% and 72.28% in paw thickness with oil seed and methanolic fraction, respectively. This anti‐inflammatory activity was 1.8 times more than Ibuprofen effect. At 8 h, the anti‐inflammatory activity with oral administration of 10 mg/kg of oil seed and methanolic fraction (by 28 days), was similar than with topical administration of oil seed (68.6%) and less than that methanolic fraction (46.69%), although these values were higher than Ibuprofen (39.61%). It shows a direct relationship between antioxidant and anti‐inflammatory activities of methanolic fraction from native Mexican avocado seed oil.

Support or Funding Information

Funding from CIC‐UMSNH (2.10 to RSG).
Dynamics of Acetyl Homeostasis in Bacillus subtilis
Authors: Best Brian , Reverdy Alicyn , Chai Yunrong
Published: April 2020

Bacillus subtilis biofilms are effective models for studying prokaryotic multicellular systems. These biofilms, multicellular bacterial communities encased in a protective polysaccharide and protein matrix, are associated with several bacterial and chronic infections but can also serve beneficial purposes in agriculture and wastewater treatment. Recent investigation into post‐translational protein modification in bacteria has elucidated important regulatory mechanisms for growth and biofilm formation. Protein acetylation has been observed to regulate bacterial multicellularity, and it is closely linked to cellular metabolism by using acetyl metabolites as donors of acetyl groups. The goal of this study is to examine the role of protein lysine acetylation as a regulatory mechanism for metabolism B. subtilis. We hypothesize that bacteria use acetylation as a buffering mechanism for acetyl metabolites to balance levels and prevent metabolite toxicity by increasing acetylation during times of high acetyl metabolite levels and deacetylating proteins to restore metabolites during times of low levels. Wild type bacteria as well as knockout mutants affecting protein acetylation and the carbon overflow pathway are grown in biofilm‐inducing minimal media, and cell and supernatant samples are collected at various ODs. Acetyl‐CoA, acetyl‐phosphate, and acetate levels over the course of growth are measured using fluorometric and colorimetric metabolite test level kits. Dynamics of global lysine acetylation levels are also measured by an anti‐acetyl lysine Western blot. Work so far has focused primarily on optimizing and troubleshooting metabolite assay kits by modifying growth and sample preparation conditions. Acetate dynamics have been measured for the wild type bacteria, showing a continuous secretion of acetate into the supernatant throughout exponential growth. This result is expected, as rapid growth should coincide with continuous utilization of the carbon overflow pathway. Initial Western blots show increased acetylation in the ΔackAΔacuCΔsrtN triple knockout mutant, which is expected due to a buildup of acetyl phosphate and inhibition of deacetylase activity. Future research will also focus on analyzing mutated genes in Δpta suppressor mutants to uncover genes related to acetyl‐CoA‐mediated cell death. Findings from this study will promote the understanding of the regulatory mechanisms behind biofilm formation and potentially uncover novel information about bacterial survival strategies and programmed cell death.

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This project was funded by grants from the National Science Foundation, the Northeastern University Honors Program, and the Northeastern University Office of Undergraduate Research and Fellowships.
Detection of Mycobacterial Mannose‐capped Lipoarabinomannan via Electrochemical Biosensor
Authors: Sodia Tyler , Poch Dylan , Honda Jennifer R. , Bonham Andrew J.
Published: April 2020

Mycobacterium tuberculosis (M.tb) is one of the world’s most prevalent bacterial pathogens. However, Nontuberculous Mycobacterium (NTM) are less recognized than M.tb while remaining a pressing public health risk. Often, NTM lung disease is confused with that of a tubercle infection leading to unreliable diagnosis. Additionally, the time spent to fully diagnose a mycobacterial disease is extensive and dependent on the species growth rate due to the necessity of culturing for identification. Thus, designing a sensitive, electrochemical DNA‐based (E‐DNA) biosensor as a diagnostic tool for NTM would dramatically decrease the timeline of diagnosis and therefore could improve patient outcomes. In this work, we pursued detection of the cell envelope of NTM, which includes many complex glycolipids and glycopeptidolipids—many of which are believed to have immunopathogenic mechanisms. As mannose‐capped lipoarabinomannan (ManLAM) is one of the most prevalent of the glycolipids, we adapted it as a biomarker for the sensitive detection of NTM. An aptamer specific to ManLAM was incorporated into our E‐DNA biosensor, and exhibits significant current change under square wave voltammetric interrogation.
Identification of a Small Protein Involved in the Regulation of the Stationary Phase Sigma Factor RpoS
Authors: Viala Stephane , McDowell Briana , Peterson Celeste
Published: April 2020

Upon encountering stressful environments, E coli induces the sigma factor RpoS to activate stationary phase and provide general protection against stress. To better understand the regulation of RpoS, we carried out an overexpression screen with an RpoS’‐‘LacZ fusion. This screen identified an intergenic region that increased RpoS expression and significantly slowed down growth when overexpressed. A short open reading frame within this region was sub cloned and shown to also induce RpoS expression and affect growth. Thus RpoS may be regulated by a novel small protein. This work highlights the importance of small proteins that were not originally annotated in the E coli genome.
Bacterial Peptidoglycan and Leukocyte Reactivity in the Brain Following Ischaemic Stroke
Authors: Broughton Brad , Modarresi Mahnaz , Rupasinghe Samoda , Hollands Cameron , Kemp-Harper Barbara , Aguilar Mibel , Forsythe John
Published: April 2020

It has recently been reported that bacteria found in the brain of neurodegenerative diseases, such as Alzheimer’s and amyotrophic lateral sclerosis, may cause neuroinflammation. Whilst infiltrating immune cells into brain are a key contributor to the pathophysiology of ischaemic stroke, whether or not this is due to the presence of bacteria within the brain remains to be determined. Therefore, the aim of this study was to establish whether peptidoglycan positive bacteria located within the ischaemic hemisphere post‐stroke attract infiltrating immune cells. To test this aim, C57BL6 male mice (8–12 weeks old) were subjected to either photothrombotic stroke (n=16), 0.5 h middle cerebral artery occlusion (MCAO, n=12) or sham surgery (n=12). Functional tests assessing motor function were conducted pre‐surgery, 24 h and/or 72 h post‐surgery. Infarct volume was assessed at 24 or 72 h post‐stroke via thionin staining and infiltrating immune cells within the brain were identified by immunofluorescence. Photothrombosis or MCAO caused significant functional impairment, as determined by the hanging wire and cylinder tests, compared to sham (P<0.05). Photothrombotic stroke caused cortical infarct damage whereas MCAO induced hypothalamic, thalamic, hippocampal and cortical infarct damage. Peptidoglycan immunoreactivity was observed within the infarct core of both ischaemic stroke models at 24 and 72 h post‐surgery. Whilst infiltrating myeloperoxidase‐positive neutrophils were located in close proximity with peptidoglycan‐positive cells at 24 h post‐stroke, F4/80‐positive macrophages were co‐localised with peptidoglycan‐positive cells at 72 h post‐stroke. Collectively, these data suggest that bacteria located in the infarct core following cerebral ischaemia may trigger an immune response, and thus could be a therapeutic target for stroke patients.
Regulation of the chaperone‐mediated autophagy receptor LAMP2A by DYNC1LI2 in cystinosis
Authors: Rahman Farhana D. , Johnson Jennifer L. , Catz Sergio D.
Published: April 2020

The dynein motor protein complex is required for retrograde transport but the specific functions of the intermediate and light chains that form the cargo‐binding complex are not elucidated and the importance of individual subunits in the maintenance of cellular homeostasis is unknown. Cystinosis is a lysosomal storage disorder caused by the accumulation of the amino acid cystine due to genetic defects in the CTNS gene, which encodes cystinosin, the lysosomal cystine transporter. Increased levels of intra‐lysosomal cystine lead to cell malfunction, which is especially manifested in the kidney epithelial proximal tubule cells (PTCs). Many cellular defects in cystinosis are independent of lysosomal overload, suggesting that cystinosin regulates cellular processes independently of its role as a cystine transporter. We showed that cystinotic cells (Ctns−/−) are characterized by vesicular transport defects, increased endoplasmic reticulum (ER) stress and susceptibility to oxidative stress. Here, using a mRNA array and immunoblot assays, we show that the dynein subunit, intermediate chain 2 (DYNC1LI2) is downregulated in cystinotic cells. Reconstitution of the expression of DYNC1LI2 in Ctns−/− fibroblasts re‐established endolysosomal dynamics quantified by Total Internal Reflection Fluorescence microscopy (TIRFM). Rescue of vesicular trafficking by DYNC1LI2 correlated with decreased ER stress manifested as decreased levels of the chaperone Grp78 in cystinotic cells. Mitochondrial fragmentation in cystinotic fibroblasts was also reduced by DYNC1LI2 expression. Survival of cystinotic cells to oxidative stress insult was increased by DYNC1LI2 but not by its paralog DYNC1LI1, which also failed to reestablish endolysosomal trafficking, or to decrease ER stress levels and mitochondrial fragmentation. Cystinotic (CTNS‐KO) PTCs, generated using CRISPR‐Cas9, showed defective endolysosomal trafficking and decreased expression of the Rab‐GTPase Rab11 carrier of the chaperone‐mediated autophagy receptor LAMP2A and the endocytic receptor megalin, both mislocalized in cystinosis. Restoring DYNC1LI2 expression rescued the localization of LAMP2A and restored the expression of megalin at the plasma membrane of CTNS‐KO cells. Expression of GFP‐DYNC1LI2 failed to rescue phenotypes in cystinotic cells (Ctns−/−) when co‐expressed with dominant negative (DN) RAB7/RAB11 but not DN‐Rab5 and in correlation with this finding, TR‐FRET assays show that DYNC1LI2 associates with RILP, a RAB7 effector and Rab11‐FIP4, a Rab11 effector. The rescue of the increased ER stress phenotype by DYNC1LI2 in cystinotic cells was blocked by downregulation of LAMP2A expression. Our results suggest that DYNC1LI2 reconstitution improves cellular homeostasis in a LAMP2A‐dependent manner highlighting DYNC1LI2 as a possible target molecule to improve cellular function in cystinosis.

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