bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of COVID‐19 on the Mental Health of College Students
Authors: Marushka Viktoriya , Hobbs Pamela
Published: May 2022

COVID‐19 has impacted the world greatly, leading researchers to be interested in the virus’s relation to mental health among various settings. Repeated isolation due to quarantining and virtual school created a change in academic approach for students across college campuses and is thought to have negatively influenced their mental health, leading us to hypothesize that college students will have decreased levels of motivation and increased percentages of depression over an academic year. This study anonymously surveyed students from Lee University on their mental health over the course of their COVID‐19‐influenced college experience regarding their motivation, sleep patterns, and physical activity. Results showed that about half of the students felt their academic motivation was affected by the pandemic. Additionally, almost half of the students scored in the moderate to severe range of depression, with a weak correlation to the number of hours slept or the inclusion of physical activity daily. Participants also shared insights into whether they believed they looked after their mental health well during the school year, noting their attitudes and ways in which community and activity helped maintain a balanced mental health status. Findings conclude that although most students said that their academic year was not affected by COVID‐19 in a drastic way on Lee University’s campus, students still agreed that their college experience and outlook on mental health was influenced by the pandemic positively and negatively. These outcomes can provide small universities a better understanding of student experiences during COVID‐19 to proactively meet the needs of students moving forward.
Influenza A Virus Causes Shedding of the Alveolar Epithelial Glycocalyx through Activation of Sheddases
Authors: Schaaf Kaitlyn R. , Buggs Charity J. , Putz Nathan D. , Langouet‐Astrie Christophe J. , Jetter Christopher S. , Nigretti Nicolas M. , Sucre Jennifer M. S. , Schmidt Eric P. , Bastarache Julie A. , Shaver Ciara M.
Published: May 2022

Objective

The healthy alveolar epithelium is protected by a heparan sulfate rich, glycosaminoglycan layer called the epithelial glycocalyx. Our group found that the epithelial glycocalyx is shed in patients with acute respiratory distress syndrome (ARDS). In murine models of LPS‐ or bleomycin‐induced acute lung injury, sheddases (membrane‐bound enzymes that cleave extracellular potions of transmembrane proteins) are upregulated and associated with glycocalyx shedding and increased lung permeability. ARDS is commonly caused by viral infections including influenza A (IAV). In murine models, IAV causes massive and persistent glycocalyx shedding into the airspace but the mechanisms by which this occurs are unknown. The objective of this work is to determine the molecular processes underlying IAV‐induced shedding of the glycocalyx.

Hypothesis

We hypothesize that IAV causes glycocalyx shedding through induction of host sheddases.

Methods

We examined the literature and curated a list of sheddases associated with IAV with potential to cleave the glycocalyx (MMP‐7, ‐2, ‐9 and their inhibitors TIMP‐1 and ‐2). C57BL/6 mice were infected intranasally with A/PR/8/34 (H1N1) at 30,000 PFU/mouse and bronchoalveolar lavage and lung tissue were collected at day 1, 3, and 7 post infection. Sheddase expression was assessed by RT‐qPCR and RNAscope was used to localize lung sheddase expression in infected and uninfected lungs. MLE‐12 mouse lung epithelial cells were infected with viable or heat‐inactivated (56C for 30 min) A/PR/8/34 (H1N1) at a MOI of 1 and sheddase expression measured by RT‐qPCR.

Results

Mice infected with IAV develop significant lung inflammation (increased BAL inflammatory cells), lung permeability (increased BAL protein), and increased glycocalyx shedding. MMP‐7 is upregulated in infected vs. uninfected lungs at day 1 and 3 post infection, then returns to baseline levels by day 7. MMP‐7 is only expressed in cells that are directly infected by IAV. Expression of the MMP‐7 inhibitor TIMP‐1 is similar to uninfected lungs on day 1, but increases 50‐fold on day 3. In contrast, MMP‐2 and MMP‐9, as well as their inhibitor TIMP‐2 are not upregulated in the first 7 days after IAV infection. Preliminary studies in lung epithelial cells suggest that heat‐inactivated IAV fails to upregulate MMP‐7.

Conclusions

Together, these data suggest that localized IAV infection increases MMP‐7 in a murine model of IAV infection, but has no effect on several other sheddases. This suggests that MMP‐7 may modulate IAV‐induced glycocalyx shedding. Future studies will explore the mechanisms of IAV induced glycocalyx shedding which could provide molecular targets for clinical intervention in IAV‐ARDS pathogenesis.
Dapagliflozin Stimulates Smooth Muscle Cell Voltage‐Gated K+ Channels KV7 to Induce Vasodilation in Resistance Mesenteric Arteries
Authors: Hasan Ahasanul , Hasan Raquibul
Published: May 2022

The antidiabetic drug dapagliflozin is a sodium‐glucose cotransporter‐2 (SGLT2) inhibitor that, in addition to glucose reduction, lowers systemic blood pressure and adverse cardiovascular events. Here, we sought to investigate if dapagliflozin dilates resistance mesenteric arteries that control peripheral vascular resistance and blood pressure. We performed pressurized arterial myography on freshly isolated, ex‐vivo preparation of resistance‐size mesenteric arteries from Sprague Dawley rats to examine if dapagliflozin can regulate arterial contractility. To assess the role of endothelial signaling, endothelium was denuded by slow passage of air bubbles through the vessel lumen. We also used different pharmacological modulators of major endothelium‐ and smooth muscle‐specific vasodilatory pathways. Our pressure myography data showed that dapagliflozin application produced a concentration‐dependent vasodilation in phenylephrine (PE)‐constricted rat mesenteric arteries. Non‐selective inhibition of KV channels and selective inhibition of smooth muscle cell voltage‐gated K+ channels KV7 significantly abolished dapagliflozin‐induced vasorelaxation. Inhibition of other major KV isoforms such as KV1.3, KV1.5 channels as well as large‐conductance Ca2+‐activated K+ (BKCa) channels, ATP‐sensitive (KATP) channels did not abolish vasodilation. Dapagliflozin‐evoked mesenteric artery vasodilation remained unaltered upon pharmacological inhibition of endothelial cell to smooth muscle signaling axis involving nitric oxide (NO), soluble guanylyl cyclase (sGC) and protein kinase G (PKG), as well as the inhibition of endothelium‐derived prostacyclin (PGI2). Consistent with pharmacological inhibition, endothelium denudation had no effect on dapagliflozin‐induced vasodilation. Overall, our data suggests that dapagliflozin selectively stimulates arterial smooth muscle cells KV7 channels, leading to vasodilation in resistance mesenteric arteries. These findings are significant as it uncovers a direct vasodilatory action of dapagliflozin in resistance mesenteric arteries, which may lower systemic blood pressure.
Production of recombinant Mtb membrane efflux pump for structural and functional studies to reveal mechanisms of drug resistance
Authors: Georgieva Elka R. , Ahmad Akram B. , Adetuyi Oluwatosin , Majeed Saman
Published: May 2022

We investigate the molecular mechanisms of the Mycobacterium tuberculosis (Mtb) EfpA membrane exporter. EfpA is a ca. 56 kDa monomeric integral membrane protein, which belongs to the Major Facilitator Superfamily transporters, and the QacA subfamily. It has fourteen predicted transmembrane segments/helices (TMs) with both N‐and C‐termini located in the intracellular space. EfpA exports anti‐Mtb drugs (e.g., isoniazid, fluoroquinolones, rifampicin, tetracycline, clofazimine) out of the cell coupled to the antiport of H+. Thus, EfpA supports a major mechanism of Mtb drug resistance. However, its structure and structure‐function relationship are currently poorly understood. What is more, to the best of our knowledge, the protein was not produced recombinantly in quantities needed for large‐scale in vitro investigations. To overcome this deficiency, we cloned the gene encoding EfpA and expressed the protein in E. coli host. Two fusion constructs of EfpA with respectively His8‐FLAG tag and His8‐maltose binding protein (MBP) tags were produced. These tags were designed to facilitate protein purification utilizing double affinity chromatography. We used SDS‐PAGE and western blotting to identify the protein localization in E. coli cellular fractions. We found that although these fusion constructs were expressed to a minor extent in the E. coli plasma membrane and can be partially purified, the proteins were mostly deposited in inclusion bodies, similarly to what was observed previously for other Mtb membrane proteins. We purified to a high degree and refolded these proteins from inclusion bodies. Currently, we are working on producing large quantities of highly‐pure EfpA protein for functional and structural studies. We further used Swiss Model program and the structures of the distant homologues NorC transporter and peptide transporter DtpA solved in outward‐ and inward‐facing states, respectively, to predict the structure of EfpA in the two major functionally‐relevant conformations. After that, we designed two double cysteine EfpA mutants E141C/F398C and F170C/Q401C, which report on large conformational rearrangements upon transition from outward‐ to inward‐facing conformations, for spin labeling and electron paramagnetic resonance (EPR) study. These mutants were generated via site‐directed mutagenesis on the background of cysteine‐free EfpA construct and are being produced in E. coli. We will further discuss results from EfpA‐drug binding and EPR experiments. Our studies have the potential to significantly advance the detailed characterization of EfpA functional mechanism.
Beneficial effects of AT1 antagonists treatment, losartan, on cardiovascular and cognitive function on aged male Dahl salt sensitive rats
Authors: Rocha dos Santos Carla , Grigorova Yulia , McDevitt Ross , Zernetkina Valentina , Wei Wen , Juhasz Ondrej , Lakatta Edward G. , Fedorova Olga V.
Published: May 2022

Introduction and objective

Cardiovascular diseases and hypertension are the major contributors to the pathogenesis of vascular dementia. The Dahl salt‐sensitive (DSS) rat model is characterized by age‐associated cardiovascular remodeling and the development of central arterial stiffness (CAS) in the presence of renin‐angiotensin system disbalance, accompanied by cognitive decline. We hypothesized, that the anti‐hypertensive treatment, antagonist for angiotensin II receptors losartan (LOS), via reducing CAS, will improve cardiovascular parameters and cognitive function in aged DSS male rats.

Methods

Male DSS rats (n=30) were kept on a normal salt diet (0.5% NaCl) for the duration of the study. At 6 months of age, rats were given either LOS in drinking water (30mg/kg/day, n=14) or control treatment (n=16) for 6 months. Measurements, including systolic and diastolic BP (SBP, DBP) and pulse wave velocity (PWV), a measure of CAS (by echocardiology), were taken after 6 months of LOS treatment. Rats were tested in an operant chambers (OCh) attention challenge to assess attention and impulsivity; elevated plus maze (EPM) was performed to estimate anxiety‐like behavior. Statistical analyses were performed using 1‐way ANOVA, t‐test and Pearson correlation. Data are presented as mean ± SEM.

Results

following 6 months of treatment, at 12‐mo of age, LOS treated rats had lower BP and PWV vs. control (Table 1). Performance on the OCh indicated that LOS rats had more correct responses vs. control rats (Fig. 1). The LOS group exhibited lower anxiety levels, because they spent less time on the closed shoulder in EPM (Table 1) and demonstrate 1.5‐fold increase of time in the open shoulder (p < 0.01). PWV and SBP was negatively correlated with time on open shoulder, total distance traveled and total correct answers at 12‐mo of age (Fig.2).

Conclusion

Aged male DSS rats show beneficial effects of LOS on cardiovascular and cognitive function. The CAS and BP improvement by LOS was associated with improved attention and lower anxiety levels. The mechanistic basis of LOS effects on cognition via cerebrovascular and brain changes will be further investigated.
Bilateral extra‐renal pelvises and calyxes with additional renal blood vessel variations identified in a single prosected medical school donor body
Authors: McGarvey Terence W. , Nemeth Denise V. , Chalk Robert M.
Published: May 2022

While there are relatively commonly observed, reported anatomical variations associated with the posterior abdominal wall, especially with the kidneys, we report here over a dozen variations in one prosected medical school donor body (Caucasian, male, age 87, cause of death: sepsis). These variations could contribute significantly to negative outcomes, especially in surgical procedures. During a routine dissection, we found bilateral extra‐renal pelvises. In the right kidney, there were two extra‐renal major calyxes, and five extra‐renal minor calyxes (two extra‐renal minor calyxes from the superior major calyxes, and three extra‐renal minor calyxes from inferior major calyxes). In the left kidney, there were four extra‐renal major calyxes and two extra‐renal minor calyxes off the most inferior major calyx. In addition, we found four venous variations and four arterial anomalies including one in a major artery. Namely, there were bilateral accessory renal veins found at the hilum that drained into the posterior aspect of the inferior vena cava. There was an additional accessory left renal vein that drained into the left renal vein, at the junction of the left testicular and left suprarenal veins. There was also a left lumbar vein that drained into one of the accessory left renal veins. Regarding the arterial supply to the kidneys, there were two polar right renal arteries directly off the abdominal aorta in addition to two long hilar right renal arteries, and three left hilar renal arteries that branched individually from the abdominal aorta. Finally, there was a long left common iliac artery compared to the right common iliac artery. Anatomical variations within patients can be of no significance or can turn a routine surgical procedure into a hazardous process if unrecognized at the time of intervention. Further documentation of such anatomical variations in prosected donor bodies can further inform surgeons for the need for imaging prior to various surgical procedures.
Strategically Substituted Agmatine Analogs Reduce Neuropathic Pain and Show Improved Pharmacokinetics Compared to Agmatine
Authors: Clements Benjamin M. , Peterson Cristina D. , Kitto Kelley F. , Wilcox George L. , Fairbanks Carolyn A.
Published: May 2022

Chronic pain remains a serious issue, affecting millions in the United States. Opioids serve as the most powerful therapeutic option but show limited efficacy in neuropathic pain and consequential side‐effects, such as tolerance, addiction, and respiratory depression. Development of novel drugs to treat pain, though, continues to stagnate. Additionally, in conditions in which opioids are required, adjuvants to reduce opioid‐induced neuroplasticity is critical. Agmatine has been shown to reduce neuropathic pain behavior and opioid tolerance, but use remains limited due to potentially reduced CNS penetration due to its hydrophilicity. Therefore, we have developed a series of agmatine‐based compounds that exhibit greater lipophilicity while exhibiting equivalent pharmacological responses (Figure 1). The goal of this study was to assess differences in pharmacokinetics and dynamics of 4 of these strategically substituted agmatines (SSAs). Our hypothesis is that these compounds will have an increased plasma half‐life and CNS distribution compared to agmatine, with equivalent reductions in neuropathic pain and opioid tolerance.

Analysis of the pharmacokinetics in rat plasma was done using serial sampling via jugular catheter. All in vivo experiments were reviewed and approved by the University of Minnesota IACUC and the ACURO of the Department of Defense. After intravenous administration, the SSAs exhibit 2‐phase distribution and elimination and have an increased elimination half‐life over agmatine (9 minutes), with SSA3 and SSA4 showing the longest (Table 1). This increased half‐life aligns with the increased lipophilicity of these compounds. Additionally, SSA2 and SSA4 show large volumes of distribution, suggesting increased CNS distribution.

To test the impact of these compounds in vivo, we induced neuropathic pain by performing spared nerve injury (SNI). SNI drives robust tactile hypersensitivity measured using the von Frey assay in the ipsilateral hind‐paw, with the contralateral paw serving as a non‐injured control. Following intravenous administration, SSA2‐4 reduced SNI‐induced hypersensitivity at similar doses to agmatine, showing equivalent pharmacological activity. Additionally, after chronic morphine treatment alongside co‐treatment with SSA1 and SSA2, opioid reduction in the warm water (52.5°C) tail flick response was preserved, representing a prevention of opioid tolerance. In conclusion, these chemical alterations to agmatine are able to improve pharmacokinetic parameters while maintaining the pharmacological reduction in neuropathic pain and opioid tolerance, suggesting potential as novel therapeutics to replace opioids in pain treatment.
Obesity‐Induced Breathing Variability During Sleep Is Independent of Apneas and Sleep Fragmentation
Authors: Kim Lenise J. , Alexandre Chloe , Pho Huy , Latremoliere Alban , Polotsky Vsevolod Y. , Pham Luu V.
Published: May 2022

Introduction

Obesity is a major cause of sleep‐disordered breathing (SDB). Conventional metrics of SDB, such as the apnea‐hypopnea index, can be confounded by the effects of obesity on oxygenation and lack of standard definitions. Thus, the impact of SBD on sleep is often difficult to quantify. Sleep fragmentation is frequently observed in obese individuals, but whether it occurs independently of SDB remains unknown. Quantitative analysis of ventilation may delineate the effects of obesity on breathing patterns and sleep fragmentation. We aimed to examine the effects of obesity on respiratory patterns during sleep and the relationship between obesity‐related respiratory variability and sleep fragmentation.

Methods

Sleep recordings were performed in 15 male lean C57BL/6J mice and 17 male diet‐induced obese (DIO) mice on the same genetic background. We applied Poincaré analysis of minute ventilation (VE) during sleep to estimate the breathing variability. Apneas were scored as ≥90% reductions in airflow for two breath cycles or ≥0.7s regardless the presence of arousals. Arousals were defined as single waking events lasting less than 16s. Arousals were classified as respiratory when associated with ≥30% drops in VEfrom baseline.

Results

Breathing variability was significantly higher in DIO mice during NREM sleep, but not during REM sleep. Obesity was associated with an increased apnea severity and 45% greater sleep fragmentation. However, obesity‐induced breathing variability was not affected by apnea or arousal indexes. Respiratory arousals comprised 15% of the arousals in both strains.

Conclusions

Obesity increased respiratory variability during NREM sleep independently of apneas and macro‐sleep architecture. Obesity caused sleep fragmentation that was not fully explained by SDB severity. Our quantitative analysis of VE identified differences in breathing variability in obesity that were not captured by traditional SDB metrics, which may be applicable for human SDB.
Cadaveric Study of Anatomical Variations in the Limbic System
Authors: Morgan Rachel , Nation Haley
Published: May 2022

The limbic system is a collection of brain structures responsible for affective processing, spatial coding, and certain types of memory. A comprehensive investigation of numerous limbic system components is necessary to thoroughly understand anatomical and pathological differences in individuals with related disorders (e.g. Alzheimer’s disease, depression, etc.). This study examines anatomical variations of limbic system structures including the hippocampus, dentate gyrus, amygdala, and cingulate gyrus. We hypothesize variations in each of the limbic structures studied exist and that these variations will be correlated with one another. This cadaveric study includes the dissection of 47 formalin‐fixed brain hemispheres and measurements of pertinent structures. The dentate gyrus, hippocampus, and amygdala are given categorical results: ill‐defined, moderately defined, and well‐defined; additionally, quantitative measurements are taken for all structures. While previous research has detailed anatomical variations of some of these structures, none have investigated the existence of any relationships between them. Our results indicate variations in the classification of the dentate gyrus (36.2% well‐defined, 51.5% moderately defined, and 12.8% ill‐defined) and amygdala (57.4% well‐defined, 40.4% moderately defined, and 2% ill‐defined). Additionally, there were significant relationships between the length of the dentate gyrus and hippocampus (p=0.004), as well as the width of the amygdala and length of the dentate gyrus (p=0.019). The length of the hippocampus was shown to be dependent on age (p=0.006); increasing age is linked to a shorter hippocampus. These findings are consistent with previously published literature, and this is the first study to highlight the relationships between these variations. The knowledge of these interactions further the understanding of the limbic system and could be used to better understand clinical presentations in future studies.
The use of Hololens increases the engagement while reducing the cognitive load of senior medical students when overlaying medical imaging of body donors during the dissection
Authors: Noel Geoffroy , Xiao Isabella , Ilie Alexandru , Chaouachi Maher , O’Brien Jeremy , McWatt Sean
Published: May 2022

Augmented reality (AR) has recently been implemented in medicine as an integrative way to view virtual objects while simultaneously interacting with the physical environment. The technology offers many benefits for education in fields that require interactive and visual learning activities, such as human anatomy. However, AR is a novel and very advanced technology, so justifying its use in the field or classroom necessitates extensive study of its effects on mental processes. Accordingly, the purpose of this study was to evaluate cognitive markers of students’ engagement and cognitive load while they used AR technology to overlay donor‐specific diagnostic imaging (DSDI) onto the corresponding body donors in a fourth‐year medical elective course at McGill University. Each participnt (n = 12) used DSDI on a head‐mounted Microsoft HoloLens and DSDI on an Apple iPad to examine the underlying anatomy of their assigned body donor before beginning their dissection. Participants wore portable five‐lead electroencephalographic (EEG) devices to collect cognitive processing data. Engagement (engagement index; EI) and cognitive load (theta‐alpha ratio; TAR) were compared between HoloLens and iPad use conditions. Mean EI under the HoloLens condition (0.499 ± 0.038) was significantly higher than the mean EI under the iPad condition (0.297 ± 0.037; P = 0.002) while the mean TAR under the HoloLens condition (1.508 ± 0.047) was significantly lower than that collected during the iPad trial (1.813 ± 0.071; P= 0.012). Together, these results indicate that use of the HoloLens to superimpose radiographic images onto a human cadaver during dissection is significantly more engaging than examining the same images on a 2D iPad screen, and also imposes a lesser cognitive load for the same task.
Ex Vivo and In Vivo Studies of the Lysophospholipids Edelfosine and Mitelfosine to Develop Novel Anti‐Epileptic Therapies
Authors: Perez Daraishka M. , Ferrer Yancy , Delgado Yamixa , Ferchmin Pedro A.
Published: May 2022

Epilepsy is a chronic neurological disorder characterized by repeated seizures. Today, most anti‐epileptic therapies are a combination of anticonvulsant drugs that work on the excitability of neurons by inhibiting sodium and calcium channels, enhancing GABAergic transmission, inhibiting glutamate transmission or have several combined actions. However, these drugs have detrimental side effects, such as congenital disabilities and psychiatric comorbidities such as mood disorders, depression, and anxiety. In addition, ~35% of adults and ~15% of children epilepsy patients are resistant to these available therapies. Thus, it is critical to study new drugs against epileptic seizures that target neuronal mechanisms different from those used by classical anticonvulsants. Edelfosine (Ef) is a lysophospholipid that works as a phospholipase‐C‐β3 (PLCβ3) inhibitor. PLCβ3 is an effector of the G protein coupled to muscarinic acetylcholine receptors that produces inositol triphosphate and diacylglycerol increasing the release of calcium (Ca+2) from the calcium‐storing organelles. This cytoplasmic Ca2+ regulates several signaling cascades including opening of ion channels that leads neurons to excitotoxic death. Thus, Ef utilizes a neuroprotective mechanism against excitotoxicity that does not act directly on ion channels. This makes Ef a superior drug against muscarinic hyperstimulation. Unfortunately, Ef is not effective within the brain due to the blood‐brain barrier's (BBB) high selectivity, which does not allow its entry. Our project aims to deliver therapeutic concentrations of Ef to the brain to diminish neuronal excitotoxicity. Our first studies test the ex vivo effect of Ef in rat hippocampal slices after two different muscarinic hyperstimulation (excitotoxic insults). Our results showed that Ef could decrease brain excitotoxicity from both insults by inhibiting the PLCβ3 enzyme. This ex vivo preliminary data demonstrates for the first time in a brain model that exposure of hippocampal slices to Ef induced a significant recovery after a muscarinic hyperstimulation insult. For the next studies, we developed two non‐BBB‐targeted serum albumin (SA)‐based drug delivery system (DDS) loading Ef and the Ef analog, mitelfosine (Mf). We characterized the particle physical properties of these Ef‐SA DDS and Mf‐SA DDS using dynamic light scattering and circular dichroism. For both DDS, SA showed characteristic structural changes compared to the native SA, demonstrating the capacity of SA to load these two lysophospholipids. We also performed a short in vivo experiment for four days administrating these DDS to determine their safety in mice. From these in vivo preliminary results, we concluded that Ef‐SA DDS and Mf‐SA DDS showed no sign of toxicity and did not affect the weight, grooming or mobility. Future experiments will focus on the development of BBB‐targeted Ef‐SA DDS and Mf‐SA DDS and their in vivo effect in an epilepsy mouse model.
Anaerobic Bacterial Biofilms Inhibit Neutrophil Responses via the Release of Stable Small‐Molecule Effectors
Authors: Lockhart Joey , Allain Thibault , Buret Andre , Morck Douglas
Published: May 2022

Background and Objective

Fusobacterium and Porphyromonasare two genera of opportunistic pathogens that often aggregate in complex communities known as biofilms. Host innate immune cells are frequently frustrated when they encounter biofilm bacteria, resulting in ineffectual clearance and persistent infection. Anaerobic pathogens generate or accumulate organic molecules and use them as defense against environmental pressures such as oxidative stress or attack from host immune cells. Porphyromonasspecies utilize protoporphyrin IX (PPIX) to protect against neutrophil oxidative‐killing. The objective was to develop a model of mixed‐species biofilm generation and to evaluate bovine neutrophil responses to bioactive molecules released from either biofilm or planktonic bacteria. We hypothesized that biofilm‐conditioned growth medium would contain more PPIX than planktonic medium and therefore inhibit the functional responses of peripheral neutrophils.

Methods

Neutrophils were isolated from whole blood via hypotonic lysis and bacterial biofilms were generated using previously validated methods for the cultivation of mixed‐species biofilms. Spent media from biofilm and overnight planktonic cultures were collected, centrifuged, and filtered to remove viable bacteria. Supernatants were fractionated via <3kDa‐cutoff filtration and exposed directly to neutrophils. Neutrophil responses to the supernatants were assessed with a fluorescent microplate assay for reactive oxygen species (ROS) and chemotaxis was measured with a Transwell® transmigration assay. The specific mechanism of neutrophil activation was investigated by employing NF‐κB and NADPH oxidase inhibitors.

Results

Neutrophils exposed to planktonic supernatant showed significantly elevated oxidative and chemotactic responses compared to biofilm products from identical bacteria. Ultra‐filtration revealed that a <3kDa molecule is differentially expressed in the supernatants and allows biofilm‐bacteria to inhibit neutrophil responses. Intensive heat, nuclease, lipase, and protease treatment of the <3kDa fractions did not alter neutrophil functional responses. Preliminary experiments suggest that the accumulation of PPIX in the biofilm supernatant attenuates neutrophil chemotaxis and ROS production. In addition, chemical inhibition experiments indicated that neutrophils respond to planktonic supernatants independently of NF‐κB signaling.

Conclusions

Experiments reveal a minimal neutrophil response to molecules released from biofilm bacteria and may explain the inadequate host response to biofilm‐mediated infections. The phenotypic changes in the biofilm mode of growth allow for the generation and accumulation of PPIX, providing protection from host immune clearance mechanisms. Understanding the mechanism of immune avoidance is a crucial step to reduce the persistent inflammation and negative clinical outcomes associated with chronic biofilm‐mediated disease.
An siRNA library screen for endothelial PAR1‐specific deubiquitinases regulating p38 MAPK inflammatory signaling
Authors: Patwardhan Anand , Cheng Norton , Trejo JoAnn
Published: May 2022

Endothelial dysfunction is induced by inflammatory mediators that signal through GPCRs and a hallmark of inflammation. Protease‐activated receptor‐1 (PAR1), a GPCR for thrombin, induces endothelial dysfunction via multiple signal transduction pathways including p38 mitogen‐activated protein kinase (MAPK). We showed that thrombin‐stimulates K63‐linked ubiquitination of PAR1 that drives recruitment of transforming growth factor‐β‐activated kinase‐1 binding protein‐2 (TAB2), an adaptor protein that binds TAB1, which triggers p38 activation on endosomes and promotes endothelial barrier disruption. However, the regulatory processes that control thrombin‐stimulated PAR1 ubiquitin‐dependent p38 inflammatory signaling are not known. The objective of this study was to identify specific deubiquitinases (DUBs) that counter thrombin‐induced ubiquitin‐mediated p38 signaling by conducting an unbiased genome‐wide siRNA library screen targeting all 96 human DUB genes in human cultured endothelial cells. We hypothesize that a PAR1‐specific DUB is essential for controlling proper dynamics of thrombin‐mediated p38 signaling and might be altered in endothelial dysfunction. To systematically identify DUBs, endothelial cells were transfected with pools of 4 siRNAs targeting each of the 96 DUBs and thrombin‐stimulated p38 phosphorylation was assessed by immunoblotting. In parallel, a genome‐wide DUB siRNA library screen was conducted in HeLa cells. We identified 8 different DUBs that preferentially cleave K63‐linked ubiquitin and regulate thrombin‐p38 signaling in both endothelial and HeLa cells. A time‐course analysis of thrombin‐stimulated p38 signaling confirmed that depletion of each of the 8 DUBs alone caused an increase in the magnitude and/or duration of p38 signaling. Among the candidate DUBs, USP34, USP47 and CYLD exhibited the highest expression in both endothelial and HeLa cells detected by RT‐qPCR and were further examined. Using individual siRNAs targeting each of the 3 candidate DUBs, we validated that knockdown of either USP34, USP47 or CYLD alone was sufficient to markedly enhance thrombin‐induced p38 signaling in endothelial cells. New data examining the function of CYLD on thrombin‐induced inflammatory responses will be presented. While ubiquitin‐dependent signaling is widespread, our knowledge of the processes that control ubiquitin‐mediated GPCR signaling is limited, especially related to the function of GPCR‐specific DUBs. In this study, we report the first genome‐wide DUB siRNA library screen on GPCR signaling and identified a subset of DUBs that specifically regulate PAR1‐p38 inflammatory signaling that may serve as important therapeutic targets for vascular inflammation.
Circulating hematopoietic stem cells (cHSC) in elite athletes: an antidoping perspective
Authors: Donati Francesco , Bonelli Cristina , Biasini Giorgia M. , Concetti Livia , de la Torre Xavier , Botre Francesco
Published: May 2022

Hematopoietic stem cells (HSC) are multipotent progenitor cells, resident primarily in human bone marrow. Their main function is the continuous renewal of all hematopoietic cells. A small fraction of HSC is also circulating in peripheral blood (cHSC) where they can be possibly detected thanks to the application of the new very sensitive flow cytometry methodologies.

In recent times it has been debated whether the utilization of several different types of stem cells can be abused by athletes for doping purposes. Moreover, there is growing knowledge that the dynamic and turnover of circulating HSC may reflect changes affecting the physiologic processes at the erythropoietic level; also, cHSC and other circulating hematopoietic progenitors (cHP) resulted to give different responses in athletes engaged in endurance and maximal exercise sports. If it is true that the use of stem cells (mainly mesenchymal stem cells) to allow a rapid recover from an injury is not considered a doping practice, it is also true that the 2022 WADA list of prohibited substances and methods forbids “the use of normal or genetically modified cells” as well as “any form of intravascular manipulation of the blood or blood components by physical or chemical means”. In this context, HSC and hematopoietic erythroid progenitors may be a potential threat and the possibility of their abuse needs to be exploited in sport antidoping.

In view of the above, we have preliminarily assessed the possibility of detecting circulating hematopoietic stem cells by flow cytofluorimetry. In details, we applied two flow cytofluorimetric protocols for the accurate and precise counting of circulating HSC, also discussing the potential applicability in a potential antidoping scenario. We concluded that i) flow cytofluorimetry is a pratical and rapid platform for the identification and the counting of cHSC and other erythroid progenitors in peripheral human whole blood, ii) the correlation between CD34+ circulating progenitors and standard haematological parameters may be an effective new tool to be exploited as in direct markers of blood doping and, iii) accurate baseline levels of HSC and erythroid progenitors need to be determined in order to identify abuses of HSC and/or conventional blood doping.
Exploring the Dynamics and Regulatory Functions of Nuclear Lipids
Authors: Niu Yang , Kim Yeun Ju , Pemberton Joshua , Balla Tamas
Published: May 2022

Phospholipid composition of biological membranes is critical for the control of numerous cellular processes in eukaryotic cells. Much less is known about the role of the nuclear phospholipids in the control of nuclear functions. Recently, biosensors with higher sensitivity and specificity, have been successfully used to follow the cellular distribution and dynamic changes of selected lipids in living cells. These included sensors for various phosphoinositides (PPIns), diacylglycerol (DAG), phosphatidic acid (PA), phosphatidylserine (PS) and cholesterol (Cho). To explore the importance of nuclear phospholipids in the control of different cellular processes, we set out to assess their presence and distribution in the nucleus both in steady state and in response to environmental challenges. For this, we created versions of the lipid sensors that allow probing lipid distribution and changes within the nucleus. Using these tools, we observed the enrichment of PS and DAG in the inner nuclear membrane (INM) in steady state and found that both PS and DAG showed characteristic changes in response to metabolic challenges, such as fatty acid loading or nutrient deprivation. These changes prompted us to seek nuclear proteins that respond to and are controlled by these lipid changes. In addition, we also created cell lines with stable and inducible expression of the biosensors to follow the dynamic changes in those lipids both within and outside of the nucleus during the cell cycle. Since such sensors may interfere with the biology regulated by these lipids, it was critical to allow for their controlled expression. These experiments are still in progress and we will share our newest data obtained by these ongoing efforts. We believe our data will shed important new light on the regulatory changes in phospholipids within the inner nuclear membrane and on their importance in the control of nuclear processes.
A Novel Cell Death Mechanism Involving the Sphingosine‐to‐Glycerophospholipid Pathway
Authors: Leak Logan , Dixon Scott
Published: May 2022

Apoptosis is the most well‐studied form of cell death, but there exist other cell death pathways that also have roles in normal physiology and disease. It is unclear what other forms of cell death remain to be discovered and how these pathways could inform our understanding of cell and molecular biology. Caspase‐independent lethal 56 (CIL56) is a compound that kills cells via a novel cell death mechanism. We conducted a genome‐wide CRISPR screen to identify novel regulators of this death pathway. From the screen, we identified trans‐2‐enoyl‐CoA reductase (TECR) as a key regulator of CIL56‐induced death. TECR is localized to the endoplasmic reticulum and is required for very long‐chain fatty acid (VLCFA) synthesis. When the gene encoding TECR is disrupted, cells become resistant to CIL56‐induced death. We find that VLCFA synthesis is dispensable for CIL56‐induced death. On the contrary, TECR has a non‐canonical role in the conversion of sphingosine to palmitate in the sphingosine‐to‐glycerophospholipid pathway. Through chemical complementation analyses, we find that this pathway is important for death induction by CIL56. It remains unclear how this pathway could be acting to execute cell death. It is known that palmitate produced in the endoplasmic reticulum can be incorporated into ceramide species, which have long been associated with cell death. Preliminary lipidomic analyses reveal a dramatic increase in palmitoyl‐ceramide upon CIL56 treatment. We are investigating the role of this species in CIL56‐induced cell death to further characterize the mechanism of death and its importance.
Self‐DNA induced inflammation impairs the normal function of myoepithelial cells of the lacrimal gland
Authors: Yang Menglu , Dartt Darlene
Published: May 2022

Sjögren's Syndrome (SS) is an autoimmune disease that mainly affects the lacrimal glands and salivary glands. Patients with SS suffer from severe dry eye which leads to corneal ulceration. The pathogenic nuclear acid triggered autoimmune response is playing a key role in autoimmune disease like SS. Previously our group observed a significantly higher expression of a DNA sensing mechanism Absent in Melanoma (AIM)2 inflammasome in two mice models of primary SS compared to the wildtype. AIM2 is a member of innate immune sensors that detects the presence of DNA in the cytoplasm, and then forms the AIM2 inflammasome to activate caspase‐1, which cleaves immature pro‐IL‐1ß to mature IL‐1ß that contribute to inflammation. In this study we investigate the effect of the AIM2 inflammasome DNA sensing pathway on primary myoepithelial cell (MEC) function of the lacrimal gland.

Primary MECs culture was acquired from female wildtype C57Bl/6 mice aged 4‐6 weeks. Genomic DNA (gDNA) extracted from MECs was used to resemble the self‐DNA. The MECs were treated with gDNA for 6 hours, with random double‐stranded DNA poly A:T as a positive control. Immunofluorescent (IF) staining result shows the accumulation of AIM2 signals and a speck of Apoptosis‐associated speck‐like protein containing a CARD (ASC), indicating the assembly of AIM2 inflammasome in both gDNA and poly AT treated group (Fig 1). The activation of caspase‐1 by gDNA treatment was confirmed using FAM‐FLICA assay.

We next determined the primary function of MECs which is contraction, under the effect of DNA‐induced cell response. A time lapse microscope was used to record a 2‐hour video of the MECs upon ATP (10‐5M) stimulation, as ATP is an important neurotransmitter released by the parasympathetic nerve end which is responsible for tear production. The first and last images will be used as a quantification study, and the reduction (%) of the area after the 2hrs period is calculated to represent the contractility. The area reduction in the gDNA as well as the positive control poly A:T treated group was significantly decreased from the non‐treated (NT) group. We next investigated the mechanism of the reduced contractility caused by DNA. One of the most important signals for muscle cell contraction is the Ca2+. We measured the change of intracellular Ca2+ concentration ([Ca2+]i) in MECs upon ATP stimulation but observed no difference between gDNA and NT group. We then determined the actin and cytoskeleton of the MECs upon DNA‐induced inflammation. The structure of ß‐tubulin which supports the cell structure, and F‐actin which enables the mobility, were investigated by IF. The result shows that the poly A:T treated group presents with higher ß‐tubulin but lower F‐actin signals compared to the control.

To conclude, self‐DNA induces inflammasome formation and caspase‐1 activation in MECs, which results in the impairment of contraction by altering the cytoskeletal proteins.
Protective Effect of Hydrogen Sulphide in Cardioplegia on Global Myocardium Ischemia‐reperfusion Injury
Authors: Zuo Ji‐Yang , Hou Hai‐Tao , Liu Zhi‐Gang , Yang Qin , He Guo‐Wei
Published: May 2022

Objective

The endogenous gaseous transmitter hydrogen sulfide (H2S) is an important regulator of the cardiovascular system and is reported to have cardioprotective activity against ischemia/reperfusion (I/R) injury. We investigated the cardioprotective effect of H2S added in cardioplegia against I/R injury in global cardioplegic arrest.

Methods

Isolated Wistar rat hearts were perfused in Langendorff apparatus and underwent 30 min perfusion, 120 min cold ischemia, and 90 min reperfusion. The rats were randomly divided into 5 groups: Control group, ischemic and reperfusion (I/R), St. Thomas cardioplegia (St. Thomas +I/R), St. Thomas plus H2S donor NaHS (NaHS+St.Thomas+I/R), St.Thomas plus NaHS and glibenclamide (an antagonist of KATP channel; NaHS+Glib+St.Thomas+I/R). The following indices were compared among the groups: the change of left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), heart rate, heart rhythm, and coronary blood flow. The difference in left ventricular developed pressure (LVDP), the maximum left ventricular pressure rise rate (+dp/dtmax), and the maximum left ventricular pressure drop rate (‐dp/dtmax) were also compared. Western blot was used to detect the changes of related myocardial enzymes among different groups.

Results

NaHS+St.Thomas+I/R treatment significantly improved cardiac function (LVSP, LVDP, +dp/dtmax and ‐dp/dtmax) compared to other groups (p<0.05), although no significant differences on coronary flow and heart rate were observed. After reperfusion, the frequency of ventricular arrhythmia in I/R group and St.Thomas+I/R group was significantly higher than that in NaHS+St.Thomas+I/R group. Further, the expression of cardiac Troponin T, troponin I, and creatine kinase in the NaHS+St.Thomas+I/R group was significantly lower than that in other groups (p<0.05).

Conclusion

Cardioplegia containing H2S confers significant protection against global I/R injury during cold cardioplegic arrest. This effect is associated with opening of the myocardial KATP channels. Therefore, H2S may be a useful adjunct to cardioplegic solutions in heart surgery.
Quantitative proteomics profiling of lysine 2‐hydroxyisobutyrylation in right atrial appendage from rheumatic heart valve disease patients
Authors: Hou Hai‐Tao , Yang Qin , He Guo‐Wei
Published: May 2022

Objectives

Lysine 2‐hydroxyisobutyrylation (Khib) is a novel acylation that regulates glycolysis. Little has been done in human diseases regarding the role of Khib. The present study was designed to reveal the role of Khib in the development of atrial fibrillation (AF) associated with heart valve disease.

Methods

Discarded right atrial appendage tissues during the cannulation procedure were collected from AF and sinus rhythm (SR) patients with rheumatic heart valve disease. The protein profiles and Khib sites were identified and qualified by Tandem Mass Tag labeling and liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) technology. Bioinformatic tools, including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and protein‐protein interaction, were also used to explore the biological function between AF and SR.

Results

A total of 6,523 matched spectrum, 3,234 peptides, 2,867 modified peptides, corresponding to 579 proteins and 2,900 sites from LC‐MS/MS analysis were identified. Of these identified proteins and sites, 480 proteins and 2,275 sites were quantifiable. The length of most peptides distributed between 7 and 22, which was consistent with the length of tryptic peptides. Differentially expressed Khib sites analysis showed that there were 35 and 48 Khib sites related to 124 and 67 proteins exhibited upregulation and downregulation in AF compared to SR. Subcellular location analysis of Khib ‐related proteins revealed that cytoplasm, nucleus, and extracellular accounted for the most proportion. Bioinformatic analysis demonstrated that proteins with Khib were significantly enriched in different biological approach, such as cysteine and methionine metabolism, cGMP‐PKG signaling pathway, and glucose‐responsive energy metabolism.

Conclusions

The present study for the first time revealed larger number of differentially expressed Khib sites between AF and SR in patients with rheumatic valve disease. The proteins related to these sites are involved in diverse cellular processes including energy metabolism. These results indicate that Khib modification might be a key factor in occurrence of AF in patients with rheumatic heart valve disease.