bluuebunny/crossref_metadata_2025 huggingface dataset:
Incorporation of Tumor Spheroids into an Ex‐vivo Tissue Culture Model for Investigating Cancer Cell–Microvascular Interactions
Authors: Lampejo Arinola O. , Lightsey Suzanne E. , Sharma Blanka R. , Murfee Walter L.
Published: May 2022

A challenge in cancer research is the lack of biomimetic models that incorporate cancer cells, blood vessels, lymphatic vessels, and immune cells. This challenge highlights the gap between an in vivo system and common in vitro assays, which do not recapitulate the tumor microenvironment. To bridge this gap, our lab has introduced the rat mesentery culture model, which provides a complex and physiologically relevant culture system that enables the real‐time tracking of multiple cell types during angiogenesis and lymphangiogenesis. The objective of this study was to demonstrate the feasibility of culturing transplanted tumor spheroids on rat mesenteric tissues for investigating cancer cell‐angiogenesis interactions. Two lung carcinoma cell lines, A549 and H1299, were labeled with DiI and suspended in a hanging drop culture system consisting of MEM + 10% FBS for 4 days to form fluorescent spheroids. These spheroids were then seeded onto excised rat mesentery tissues (approximately 20 spheroids per tissue). Tissues with transplanted spheroids were cultured for 3 days. To identify blood vessels during various culture timepoints, tissues were labeled with FITC‐conjugated lectin and imaged on Day 2 and Day 3. On Day 3, tissues were labeled for PECAM, NG2, and CD11b to identify endothelial cells, pericytes, and macrophages respectively. Labeling a subset of tissues for Calcien AM enabled the detection of live cells. Observations confirmed that tumor spheroids remained viable and intact during culture. The apparent increase in individual cells along the periphery of the spheroids suggests radial cell migration into the tissue. Spheroids were observed in the same regions of angiogenic microvascular networks characterized by increased sprouting and vessel density. Angiogenesis was also confirmed by comparing time‐lapse lectin labeling of vessels. For some spheroids, directed sprouting towards spheroids was also observed. The presence of lymphatic vessels (identified by PECAM labeling morphology), NG2+ pericytes, and CD11b+ macrophages confirmed a multi‐cellular environment. These results support the feasibility of co‐culturing tumor spheroids and rat mesenteric tissues as well as a new approach for exploring how cancer cells interact with a surrounding microvasculature and their local environment.
An Exercise Mimetic Blunts Influenza‐Induced Mortality in Middle Aged and Obese Mice
Authors: Nunan Emily L. , Baker Hallie , Gammill Blake , Butcher Joshua T.
Published: May 2022

Viral infection is well known for its ability to significantly compromise cardiovascular health, and elevate morbidity and mortality in healthy (and unhealthy) patients. Susceptibility to the consequences of viral infection are significantly elevated in the obese and aged populations. Even modest amounts of exercise are protective against influenza, influenza severity and mortality, and additionally enhance immunization efficacy in both human and rodent models. However, exercise is contra‐indicated in those with viral infection, due to potential contagiousness and a weakened immune state. Further, a significant patient population is unable to exercise at a rate that confers cardiovascular benefit (ex: the aged, obese, those constrained by a pandemic). Thus, it would be inherently valuable to determine if an exercise mimetic can protect and preserve function, independent of increases in activity or exercise. To address this question, our lab manipulates the muscle myokine myostatin (GDF‐8) in obese and aged mouse models. Myostatin is a potent negative regulator of skeletal muscle growth that is upregulated in human and animal models of obesity, influenza, and downregulated following regular exercise. Our hypothesis is that targeting myostatin in a rodent model of influenza will prevent mortality via a mechanism that mimics the benefits of exercise.

Influenza infection was induced via intranasal administration of Influenza A/PR/8/34 in WT and obese (db/db) mice with and without myostatin deletion. Importantly, middle aged mice (aged 40‐60 wks) were used to appropriately mimic a susceptible human phenotype. Assessed variables included weight, survival, coat score and inflammatory factors in the lung.

Myostatin deletion significantly improved survival in mice compared to infected controls, with lean controls experiencing ~27% mortality and obese controls experiencing ~50% mortality. No mortality was observed in obese and lean groups with myostatin deleted. All groups experienced similar weight loss, demonstrating effective influenza infection. Lung inflammatory factors showed significantly elevated IL‐2, MCP‐1, and IL1b in infected lean myostatin KO mice, as well as NOX1 and NOX2, compared to infected controls. Taken together, our data suggests that myostatin may be an effective target for improving outcomes in influenza infection.
Functional analysis of the nitrogenase protective protein CowN
Authors: Willard Dustin
Published: May 2022

Nitrogenase is the only enzyme that can catalyze ATP‐dependent nitrogen fixation in bacteria, an important biological process that reduces atmospheric nitrogen gas into ammonia. The bacterium Gluconacetobacter diazotrophicus expresses nitrogenase and is agriculturally relevant since it provides ammonia to commercial crops such as sugarcane and pineapple. The environmental gas carbon monoxide tightly inhibits nitrogenase. To prevent this inhibition, diazotrophs have evolved a small protein, CowN, which interacts with nitroganse and lowers the affinity of CO binding. In doing so, CowN allows nitrogenase to stay active despite the presence of CO Although we know that CowN protects nitrogenase from CO, we do not know how it achieves its protective effect. Both CowN’s mechanism and structure remain undetermined. This poster presents a structure‐function analysis of CowN. The C‐terminus of CowN contains a conserved Cys (C90 in G. diazotrophicus) and a semi‐conserved second S‐containing residue, either Cys or Met. Due to this conservation, we hypothesize that the C‐terminus of CowN is crucial to its function. To test this hypothesis, we made a series of C‐terminal mutations, expecting that mutations to C90 would abolish activity. Surprisingly, we discovered that the conserved Cys is not required for CowN function. A C90S mutant was almost fully active, suggesting that either a thiol or a hydroxyl group at the CowN C‐terminus is sufficient for activity. In contrast, mutations to the second S‐containing residue, M91 in G. diazotrophcius, and to both C90 and M91 fully abolish CowN activity and destabilize the structure. Together, the results confirm our hypothesis that CowN’s C‐terminus is required for activity, however, they also reveal that the presence of the conserved Cys is not necessary for function. Future work will aim to solve the CowN structure and to elucidate the role of residue C90.
Amyloid‐Beta Precursor Protein: Essential to Lung Capillary Barrier Defense During Acute Infection
Authors: Voth Sarah B. , Choi Chung‐Sik , Morrow K. A. , Gwin Meredith S. , Knighten Jennifer , Francis C. M. , Lin Mike T. , Gautam Samir , Frank Dara W. , Audia Jonathan P. , Stevens Troy , Sharma Lokesh , Cruz Charles D.
Published: May 2022

Rationale

Pneumonia is a leading cause of acute respiratory distress syndrome (ARDS) and sepsis. Despite resolution of active infection, pro‐inflammatory mediators and cytotoxins elicited during the acute phase promote secondary tissue injury, impede recovery, and contribute to poor patient prognoses. Lung infection with Pseudomonas aeruginosa, Klebsiella pneumoniae, and influenza elicit pathogenic amyloids that propagate injury and likely contribute to this inflammatory milieu. However, clinical trials targeting amyloid‐beta (Aβ) have consistently failed. Lung endothelial‐derived amyloids have been implicated as innate antimicrobial effectors yet the contribution of Aβ to host defense of the lung capillary barrier remains poorly resolved. We hypothesized that lung endothelial Aβ contributes to 1) the attenuation of active infection, and 2) endothelial barrier resilience to infection.

Methods

To test our hypotheses, CRISPR/Cas9 was targeted to Aβ precursor protein (APP) in rat pulmonary microvascular endothelial cells (PMVECs), and the deletion was confirmed via PCR, sequencing, and immunoblotting. The contribution of APP expression to cell proliferation, self‐replication, wound closure, angiogenesis, and barrier integrity (transwell permeability, bacterial dissemination, ECIS) both at baseline and during virulent infection was assessed in both WT and APP‐/‐ PMVECs. Amyloid content was monitored with immunoblotting and Thioflavin T staining. Antimicrobicity was measured with kinetic bactericidal assays, agglutination, plating, and SYTO 9/propidium iodide.

Results

APP‐/‐ cells were ~49% less effective in reducing the bacterial load of an active infection as compared to APP expressing PMVECs. Immunodepletion of Aβ from WT endothelial cell supernatant reduced bactericidal efficacy in a comparable manner. APP‐/‐ cells also exhibited impaired proliferation (WT: 0.59 days, APP‐/‐ 0.91 days), a ~2.2‐fold reduction in replication capacity, poor wound closure and barrier formation, and little resilience to infection with loss of barrier integrity in roughly half the time of infected WT PMVECs. Moreover, conditioning of naïve cells with supernatant derived from WT but not APP‐/‐ PMVECs significantly increased proliferation and angiogenic capacity.

Conclusions

Lung endothelial APP is an important contributor to host defense. Both APP and its Aβ variant(s) appear to constrain infective bacterial burden whereas APP products appear to function as mitogens/morphogens within the microvascular milieu, likely contributing to tissue repair and recovery post‐insult.
A Novel Description of Breast Anatomy Indicates the Need for New Surgical Approaches
Authors: Lindvall Tyler J. , Suarez‐Venot Ana , Rodriguez‐Sosa Jose R. , Plochocki Jeffrey H. , Grossman Aryeh , Voegele Georgina M. , Valdez Dominik , Georgi Justin A. , Hall Margret
Published: May 2022

Mastectomy remains a major treatment for breast cancers and prophylaxis for carriers of the BRCA genes, yet the amount of tissue removed is variable across surgical approaches. The anatomical and surgical literatures provide conflicting and incomplete descriptions of breast gross anatomy. In order to clarify this anatomy, we dissected 6 male and 15 female human cadaveric specimens. We found that the mammary glandular tissue is constrained to a membrane‐bound, central structure referred to as the corpus mammae in the surgical literature, and not dispersed throughout the breast as described in the anatomical literature, consistent with other epidermally derived glands of the body. The major fasciae of the human ventral body wall, including the superficial fatty Camper’s fascia and the deeper membranous Scarpa’s fascia, both contribute to the structure of the breast. Based on these findings, we propose that during embryological development the mammary gland grows dorsally from the integument, pushing Camper’s and Scarpa’s fasciae ahead of it. When the mammary gland reaches the rigid thoracic wall, the gland grows laterally, pulling along Camper’s fascia to create the fat of the breast. Ventrally Scarpa’s fascia becomes a double layer that creates the shape of the breast and dorsally Scarpa’s forms a circummammary ligament that 1) stabilizes the breast against the thoracic wall and 2) is continuous with Scarpa’s fascia on the rest of the ventral body wall at the inframammary fold. The suspensory ligaments of the breast represent the typical, albeit thickened, septations between compartmentalized fat lobules found consistently throughout the human body, and do not attach to the skin. Instead, these ligaments attach to Scarpa’s fascia. We propose replacing surgical approaches that remove the entire breast with more targeted procedures focused on removing the gland only. Since the glandular material is constrained to the corpus mammae, in low‐grade cancers and prophylaxis surgeons can utilize fascial planes to remove only the gland while protecting the rest of the breast. Such a procedure could allow the patient to maintain most of the breast, thereby potentially reducing physical and emotional trauma.
Anomalous Origin of the Left Vertebral Artery from the Aortic Arch: A Case Report
Authors: Highhouse Kendall , Choobchian Paoula , Dixon Jared , Perozich Garrett , Raciti Catherine , Barremkala Malli
Published: May 2022

This study presents and describes the anatomical variation of the left vertebral artery arising from the aortic arch in two female cadavers, discovered during dissection in an anatomical laboratory setting. This anomaly was initially discovered during a routine cadaveric dissection of the neck of a 64‐year‐old female cadaver. This finding prompted an evaluation of the remaining twenty‐five cadavers in the lab, leading to the discovery of the same variation in an 83‐year ‐old female. In both cadavers, the left vertebral artery originated from the aortic arch in between the origins of the left common carotid artery and the left subclavian artery. This is a variation, as the vertebral artery is typically known to branch off of the first part of the subclavian artery on both the right and left side. Following this discovery of the anomaly in the laboratory, a literature review was conducted to understand the prevalence of this variation in the general population as well as its clinical implications.
Perceptions of Students toward Flipped Learning in the STEM Courses at a Two‐Year and a Four‐Year Institution
Authors: Daughrity Sheyenne , Dickey Paige , Bracey Georgia , Fickas Julie , Bartels Lynn , Locke Sharon , Gopalan Chaya
Published: May 2022

Flipped teaching (FT) is shown to actively engage the learner in comparison to passive learning experience of traditional teaching (TT) by shifting lecture outside the classroom to allow in‐class time on activities at higher levels of Bloom's taxonomy. The impact of FT on STEM students in a two‐year institution (2‐YI) and a four‐year institution (4‐YI), male and female students, and Freshmen to Senior years were examined in this study. Twelve STEM faculty, six per cohort, from a 4‐YI and six per cohort from a 2‐YI were selected to receive faculty development and to implement FT in their classrooms. During the first semester of FT, students at the 4‐YI responded more favorably than students at the 2‐YI (p=0.0004), and female students rated FT greater than male students (p=0.04). In the second semester of implementation, all students equally favored FT. Furthermore, Juniors and Seniors were more likely to report a better experience than Freshmen and Sophomores in the subsequent semester (p<0.05). These results suggest that students in the 4‐YI, especially Juniors and Seniors, have a more positive and receptive attitude toward FT than the students at the 2‐YI.
Forster Resonance Energy Transfer in an Intrinsically Disordered Peptide
Authors: Martinez Roman J. , Martin Joshua P.
Published: May 2022

We report Forster Resonance Energy Transfer (FRET) studies of a 2‐cyanophenylalanine‐tryptophan donor‐acceptor pair within an intrinsically disordered peptide (IDP) region of an olfactory marker protein. In the olfactory IDPs (OFP) examined, tryptophan occupies the N‐terminus and 2‐cyanophenylalanine (2‐PheCN) is substituted in place of phenylalanine (Phe) at two different distances from that terminus; in OFP Long the 2‐PheCN is at the C‐terminus, whereas in OFP Short the nitrile derivatized amino acid is only two residues away from the tryptophan. While Phe is native to OFP, 2‐PheCN exhibits a larger fluorescence quantum yield providing better spectroscopic selectivity. Further, addition of the nitrile group to phenylalanine has been reported to change the peptide structure only minimally, thus resulting in an unperturbed IDP structure. As such, OFP Long and Short allow for comparison of energy transfer efficiency between the donor and acceptor fluorophores at two distances. Additionally, examining OFP Long and Short in solvents that either promote or inhibit secondary structures provide model systems in which spectroscopic techniques are used to determine structural perturbations induced by environmental changes. Intriguing trends observed in the various solvents provide a site‐specific view of an IDP structure and dynamics, which can be used to extrapolate applications of their manifestation in numerous human disease states.
Elevated Glucose Negatively Regulates Nkx6.1 Protein Level in the Pancreatic Beta Cell
Authors: Wieland Kristopher L. , Elison Weston , Tessem Jeffery S.
Published: May 2022

Millions of people suffer from T2D. A key characteristic of T2D is damage to the pancreatic beta cell. This damage affects the beta cell’s ability to sense glucose, produce insulin, and release insulin in response to elevated blood glucose levels. Homeobox protein Nkx6.1 is a beta cell transcription factor essential for beta cell differentiation, proliferation, and insulin secretion. To test the effect of hyperglycemia on the beta cell, INS‐1 832/13 beta cells were cultured under hyperglycemic conditions (20mM glucose) for 24 and 48 hours. This treatment significantly increased cellular ROS levels and impairs beta cell insulin secretion. Similarly, 20mM culture resulted in decreased nuclear localization of Nkx6.1, decreased overall Nkx6.1 protein levels, and decreased Nkx6.1 mRNA levels. We demonstrate that culturing beta cells in 20mM glucose increase Nkx6.1 ubiquitination and appears to impair new Nkx6.1 translation. Our data suggest that beta cells cultured in 20mM glucose result in increased ROS levels that result in Nkx6.1 relocalization and degradation, thus changing the ability of the beta cell to properly secrete insulin.
Hypoglycemic activity of CDCA (chenodeoxycholic acid) and GW4064 in type I and type II diabetic mouse model systems
Authors: Cho Young
Published: May 2022

Farnesoid X receptor (FXR) is an orphan nuclear receptor that plays roles in diverse metabolic pathways including blood glucose. Previously we tested a signal transduction pathway of the phosphoinositide 3‐kinase (PI3K) pathway in in vitro model. In HepG2 cells and Caco‐2 cells, FXR in the presence of GW4064 (synthetic FXR agonist) and CDCA (chenodeoxycholic acid, physiological FXR agonist) enhanced activity of PI3K in FXR:PI3K immunocomplex that led to increase in glucose (GLC) uptake and glycogen synthase (GS) activity. However, positive effects on PI3K were reversed in the presence of wortmannin. We also confirmed that in vivo effects of FXR were comparable to those of insulin in mouse models. GW4064 reversed hyperglycemia in Db/Db mice in vivo. In addition, we found the comparable hypoglycemic effects of FXR in the C57BL/6 mice which were treated with streptozotocin that resulted in type I diabetic hyperglycemia. Time course and levels of antihyperglycemic effects of GW4064 were comparable to those of insulin. Accordingly, we report insulin‐mimetic action of FXR both in vitro and in vivomodel systems.
Impact of Dietary Empagliflozin on Renal Cortex Metabolome and Lipidome in Mice
Authors: Bangarbale Surabhi , Bansal Shivani , Jayatilake Meth B. , Levi Moshe , Shepard Blythe D. , Ecelbarger Carolyn M.
Published: May 2022

Sodium glucose cotransporter, type 2 inhibitors (SGLT2i) have been demonstrated to be protective of kidney by unclear mechanisms. The aim of the current study was to determine how treatment with a common SGLT2i, Empagliflozin affected kidney cortical metabolome and lipidome in high‐fat‐fed, male TallyHo/Jng (TH) mice. TH mice are prone to obesity and insulin resistance progressing to type 2 diabetes (T2D). Adult mice (n = 7 or 8/group) were fed a control high‐milk‐fat diet (60% by weight) or this same diet supplemented with empagliflozin (0.01%) for 8‐weeks, then euthanized and kidney cortex dissected and rapidly frozen. Metabolomics (targeted and untargeted) and lipidomics (targeted) were conducted on separate sections of cortex in our core facility by liquid chromatography‐followed by tandem mass‐spectroscopy. Data pre‐processing included signal drift and inter‐batch correction, as well as, removal of species with high coefficient of variability on pooled or reference analysis. Metabolites and lipids passing quality control (QC) included 207 and 467 unique species, respectively, which were further analyzed by MetaboAnalyst 5.0 and lipids, additionally by LipidSig, both publically available software platforms. Volcano plotting of metabolites and lipids revealed oppositely skewed patterns in that metabolites tended to be relatively decreased and lipid species increased with Empagliflozin. Seventy‐four metabolites met our threshold of changed > 20% change and/or p < 0.05 and were analyzed for pathway enrichment. The top three pathways down regulated by Empagliflozin were urea cycle, spermine/spermidine biosynthesis, and aspartate metabolism. Analysis of metabolites of glycolysis/oxidative phosphorylation revealed a general 20‐45% reduction in several species including phosphoenolpyruvate (PEP), succinate, and malic acid. In contrast, in general, several lipid species were increased with hierarchical clustering showing greatest effect on phosphatidylglycerol (16:1/16:1), 40% increase, p < 0.02, lysophosphatidylcholine (LPC, 20:4) 38% increase p < 0.03, and 10 additional phosphatidylcholine (PC) species. Overall, these analyses suggest a greater abundance of lipid, relative to carbohydrate and protein‐based species, in the kidney cortex in response to chronic Empagliflozin treatment. It is unclear whether these differences are due to intracellular metabolic alterations or transport reduction in substrate (glucose and peptides) for metabolic pathways. In addition, we found no evidence of elevated gluconeogenesis with Empagliflozin observing a significant reduction in one key metabolite in this particular pathway, PEP. We may speculate oxidative phosphorylation and ATP generation is maintained relatively normal by greater oxidation of lipids (as opposed to proteins and carbohydrates) in the kidney proximal tubule of Empagliflozin‐treated mice.
Exploring the Distribution and Orientation of Cell Proliferation as Drivers of Mouse Facial Development
Authors: Dauter Andreas C. , Green Rebecca M. , Lo Vercio Lucas D. , Barretto Elizabeth C. , Robertson Samuel , Mahika Anandita , Vidal Garcia Marta , Forkert Nils D. , Hallgrimsson Benedikt
Published: May 2022

During the development of the face, tissues move, change shape, and fuse in tightly orchestrated patterns to create all the parts of a normal face. These shape changes are driven by factors such as cell signaling, migration, proliferation, and apoptosis. However, the contributions of each of these drivers to morphogenesis are poorly studied. Here, we explore differential cell proliferation as a driver of mouse facial morphogenesis. We quantify patterns in both the spatial distribution and orientation of proliferation in the developing face in 3D over a critical period of murine facial development (E9.5‐E11.5). We use immunostaining with light sheet microscopy (LSM) to capture total and proliferating nuclei. To compare proliferative density in facial tissues, we segment these images using a novel convolutional neural network. We then generate atlases of average proliferation at each half‐day age point within our range and use these to identify relationships between morphology and cell proliferation. We show that regions with more dense proliferation tend to undergo more intensive shape changes. We then simulate outgrowth of the maxillary process using a cell simulation engine, PhysiCell, to demonstrate that differential proliferation is necessary to maintain expected morphology in growing tissues. In addition to differential proliferation, localized orientation of cell division could also affect morphology. In plants and some animal tissues, including murine limb buds, preferentially oriented cell proliferation drives shape change by causing tissue elongation in specific directions. We explore the orientation and distribution of cell proliferation using LSM: we inject pregnant dams with a synthetic nucleotide, EdU, 5 minutes before harvest to mark the daughter cells of proliferative events occurring in the interim. We then compare the angles of the proliferative axes for each pair of daughters relative to the primary direction of tissue growth. Preliminary results suggest that cell proliferation in the maxillary and nasal processes is oriented preferentially towards the axis of tissue growth. These results suggest that both the distribution and orientation of cell proliferation play a role in murine facial morphogenesis. Understanding the mechanisms underlying morphogenesis is important to guide future research that could lead to earlier and more robust diagnosis and treatment of syndromes and facial abnormalities.
Deletion of Prorenin Receptor in the Rostral Ventrolateral Medulla Results in Biphasic and Sex‐Dependent Pressor Responses in Deoxycorticosterone Acetate‐salt Hypertension
Authors: Nakagawa Pablo , Brozoski Daniel , Mathieu Natalia M. , Lu Ko‐Ting , Gomez Javier , Reho John , Fang Shi , Segar Jeffrey L. , Grobe Justin L. , Sigmund Curt D.
Published: May 2022

In models of low renin hypertension such as the deoxycorticosterone acetate (DOCA)‐salt model, the local generation of angiotensin II in the brain mediates the increase in blood pressure. The prorenin receptor (PRR) might play a potential role in the local activation of prorenin in the brain. We have new evidence that prorenin and PRR are co‐expressed in the proximity to the rostral ventrolateral region of the medulla oblongata (RVL). Therefore, we hypothesized that selective ablation of PRR targeting the RVL attenuates responses to DOCA‐salt in mice. PRR ablation was targeted to the RVL by stereotactic microinjections of adeno‐associated virus (AAV) expressing Cre recombinase in PRR‐flox mice (PRRRVL‐KO). AAV mCherry was used as a control virus (WT). Pressor response to L‐glutamate in the injection site served as confirmatory stereotactic target hit. During the first week of DOCA‐salt treatment, RVL‐targeted ablation of PRR resulted in lower pressor responses to DOCA‐salt in females (WT=115±3 vs KO=104±4 mmHg; p<0.05; n=8), but not males. However, after week 2 on DOCA‐salt treatment, female PRRRVL‐KO unexpectedly exhibited an exaggerated increase in systolic blood pressure (WT=149±3 vs KO=163±3 mmHg; p=0.004; n=8) and this was accompanied by a trending increase in urinary norepinephrine (WT=177±28 vs KO=270±38 ng/ml; p=0.07; n=7‐8). Next, female mice were challenged with an intraperitoneal hypertonic saline injection equivalent to 10% of their body weight followed by 4 hours of urine collection at baseline and on DOCA‐salt treatment. Urinary sodium excretion in female PRRRVL‐KO was significantly lower when compared to WT in both conditions (p<0.05). Finally, using nuclear magnetic resonance/bioimpedance spectroscopy we assessed body fluid compartmentalization. We observed no difference in total body fluid among groups at any time point, but there was a significant mobilization of extracellular fluid to the intracellular compartment in PRRRVL‐KO. Our data indicate that the role of PRR in the RVL is sex‐dependent and biphasic. That is, PRR contributes to the pressor response during the initial stage of DOCA‐salt hypertension in females, presumably by facilitating the generation of angiotensin peptides in the RVL, while it plays a protective role by promoting renal sodium excretion and preventing blood pressure elevation during the maintenance stage of DOCA‐salt hypertension. This study also suggests that distinct PRR expressing cell populations within the RVL might elicit diverging physiological functions.
Sirt6 activation blocks LPS induced expression of inflammatory cytokines in cardiac cells
Authors: Gupta Madhu , Hund Samantha , Pillai Vinod , Samant Sadhana , Gupta Mahesh P.
Published: May 2022

Sepsis induced myocardial dysfunction is a common complication that has been linked to an increased mortality. The activation of NFkB pathway and excessive production of pro‐inflammatory cytokines is considered to be one of the most important signaling pathway involved in the pathogenesis of sepsis. Earlier studies have shown that Sirt6, a member of class III of histone deacetylases, attenuates hyper‐activation of NFkB signaling that contributes to inflammation and the aging process. In the present study we tested the hypothesis whether pharmacological activation of Sirt6 can prevent LPS induced myocardial inflammation. Primary cultures of adult rat cardiac fibroblasts were treated with UBCS039, an allosteric activator of Sirt6, for 48 hours. The results showed a dose‐dependent increase in steady‐state levels of Sirt6 as well as deacetylation of its target H3K9 by Western analysis. A similar effect of UBCS039 treatment was observed in H9C2 cardiac myocytes. We also tested effects of UBCS039 on LPS induced NFkB activation and cytokine production in these cells. Our data showed that pretreatment of cardiac fibroblasts with UBCS039 for 48 hrs significantly reduced LPS (100ng/ml)‐induced phosphorylated levels of NFkB (pNFkB). By real‐time PCR analysis we found that LPS led to increased expression (4‐9 fold) of mRNA of IL‐6, IL‐1b, TLR4 and CD‐38 in cardiac fibroblasts within 6h of treatment. This effect of LPS in cardiac fibroblasts was completely blocked when cells were pretreated with UBCS039. These data thus indicated that pharmacological activation of Sirt6 is capable of blocking LPS induced inflammatory response in cardiac cells, and has the potential to alleviate sepsis induced myocardial dysfunction and related health consequences.
Limb Morphology in the Macaroni Penguin (Eudyptes chrysolophus) Exhibits Adaptations for Both Sub‐aqueous Flying and Locomotion on Land
Authors: Hall Margret , Brantley Michaela B. , Long Michelle Y. , Hirsh Morgan L. , Rodriguez‐Sosa Jose R. , Lee Andrew H. , Simons Erin L. , Valdez Dominik , Georgi Justin A.
Published: May 2022

Requiring both wing‐propelled swimming and rugged overland locomotion, Macaroni penguins possess highly specialized morphology in both limbs. We dissected both the wing and hindlimb of one female and one male macaroni penguin. The distal wing exhibits very reduced musculature, and only retains very small brachialis and dorsal ulno‐metacarpal muscles. The elbow and metacarpal joints exhibit significantly decreased mobility compared to other birds. Proximal extrinsic wing muscles extend long tendons into the distal wing to control the dorso‐ventrally flattened flipper. Two sesamoid bones at the elbow joint provide a main attachment point for the proximal musculature, and probably contribute to the stiffening of the wing during underwater propulsion. The enlarged and extended sternum houses an extensive pectoralis muscle, responsible for the downstroke, which comprises nearly the entire ventral body wall. Supracoracoideus, found anatomically deep to pectoralis major and responsible for the upstroke, weighed about half of the pectoralis muscle, unlike most birds, where supracoracoideus weighs only about one‐fifth. An enlarged supracoracoideus increases the strength of the upstroke against viscous water. The tendons of latissimus dorsi cranialis and caudalis pass through a fibrous trochlea extending from the triceps complex to attach to the humerus, and possibly increases wing forces when moving the wing caudally. Unlike the sparse wing musculature, extensive leg musculature comprises about half of the dorsal body wall. The sartorius muscle, the most cranial hindlimb muscle, lies cranial to the caudal edge of the scapula, and cranial to the caudal margin of the sternum. Despite this, penguins exhibit restricted mobility during locomotion on land including apparent reduced range of motion in the knee joint, which contributes to their signature waddle. In addition, both Macaroni penguin specimens possess a unique muscle we have named the adductor tibialis. This muscle extends from the tibia of one hindlimb, across the midline where it slightly connects to the sternum, and attaches to the tibia of the contralateral limb, effectively tying the hindlimbs together. This permanent adduction probably helps create a streamlined figure while swimming to create faster speeds. In contrast to the knee, the penguin can greatly extend and flex the tarsus during locomotion. The considerable extensor system in the tarsus helps the penguin cross different terrains while walking, as well as give them the ability to jump.
Comparison Of Dynamic Calcium Signaling Profiles On Activation Of TRPV4 Channel And T‐Type Calcium Channel In Pulmonary Microvasculature
Authors: Sen Pallavi , Choi Chung‐Sik , Taylor Mark
Published: May 2022

Background

Acute Lung Injury (ALI) is characterized by inflammation, barrier disruption and edema within lung microvasculature. A dysregulation in intercellular signaling of calcium (Ca2+) within pulmonary microvascular endothelium potentially damages lung endothelial barrier integrity. Recently, two plasma membrane ion channels, the vanilloid transient receptor potential IV (TRPV4) and the T‐type voltage gated calcium channels (TTCC) have been implicated as potential conduits for spatially and temporally targeted Ca2+ signals within lung endothelium. Ca2+ entry via TRPV4 causes barrier disruption and Ca2+ entry via TTCC regulates P‐selectin expression, but their underlying patterns of specific Ca2+ signaling events are unknown. Therefore, in this study we aim to determine whether these channels elicit distinct Ca2+ signaling events that might contribute to their different functions in lung endothelium.

Method

Pulmonary microvascular endothelial cells (PMVECs) grown in 35mm glass‐bottomed dishes were loaded with fluorescent Ca2+ indicator Cal‐520 AM and recorded for signals via spinning disk confocal microscopy. After baseline recording, cells were subjected to GSK1016790 (TRPV4 activator) and then to GSK2798754 (TRPV4 inhibitor). Another set of cells were treated with high K+ solution (TTCC activator) and then with mibefradil (TTCC inhibitor). Lung slices were mounted on Sylgard inserts, and loaded with Cal‐520 AM. Two different slices were recorded for baseline activities and were respectively subjected to same drug treatments as PMVECs. Time‐lapse image sequences recorded after each addition were analyzed for amplitude and frequency of events using ImageJ and customized signal detection software LC_Pro.

Results

In PMVECs with TRPV4 channel activation, the amplitude and frequency of events increased 1.5‐fold when compared to baseline, whereas the TTCC activation showed an increase by 1.3‐fold. In lung slices, the TRPV4 channel activation exhibited an increase by 1.4‐fold compared to baseline, as opposed to an increase of only 1.1‐fold with TTCC activation. The addition of respective inhibitors brought the signals back to near baseline levels.

Conclusion

Our findings suggest both TRPV4 channel and TTCC activation caused an increase in Ca2+ dynamics, with the TRPV4 channel activation showing a greater increase in event amplitude and frequency than TTCCs, thereby indicating they both contribute to distinct signaling events in PMVECs and lung slices. Further studies will focus on how these different signatures contribute to the differential functional roles of the channels.
Effect of Pre‐Conditioning with Copolymer‐Based Cell Membrane Stabilizer Poloxamer 188 on Adult Mouse Coronary Artery Endothelial Layer Integrity after Prolonged Hypoxia/Reoxygenation
Authors: Maknojia Insha H. , Hampton Matthew J. , Li Zhu , Barajas Matthew B. , Riess Matthias L.
Published: May 2022

Prolonged ischemia followed by reperfusion can cause significant tissue damage and cell death. This ischemia/reperfusion (IR) injury can be alleviated by administration of Copolymer‐based Cell Membrane Stabilizers (CCMS), like the tri‐block Poloxamer (P)188, thought to be able to seal gaps in cell membranes and/or tight junctions and, thus, repair leaks attributable to severe cellular stress. We have previously tested post‐conditioning, i.e. the administration of P188 upon reperfusion/reoxygenation, to attenuate IR injury in various models. Here, we hypothesized that pre‐conditioning, i.e. its administration before simulated ischemia (hypoxia), has a protective effect on endothelial cell layer integrity as assessed by Trans‐Endothelial Electrical Resistance (TEER).

Confluent adult mouse coronary artery endothelial cell (ECs) layers were pre‐treated with 0, 30, 100, 300, 1,000, or 2,000 µM P188 during plating of cells before simulated IR with a 24‐hour hypoxic followed by a 2‐hour reoxygenation (HR) period. EC monolayer barrier integrity was assessed by TEER.

In contrast to mere post‐conditioning, pre‐conditioning dose‐dependently attenuated HR injury compared to control/normoxia (Fig 1) even after prolonged HR for 24+2 hrs.

These results suggest that CCMS may indeed elicit its cardioprotective effect, in part, through protection of the EC layer. However, more research is needed to determine optimal dosage and timing for CCMS administration.
Effects of Mitragynine and its Active Metabolites on the Reinforcing Effects of Remifentanil and Cocaine in Rats Self‐Administering Remifentanil
Authors: Hiranita Takato , Zuarth Gonzalez Julio , Patel Rohan C. , Mazpule‐Carrigan Christian C. , Martins Rocha Joelma , Gamez Jimenez Lea R. , Ho Nicholas P. , Patel Avi , Obeng Samuel , Leon Francisco , Mottinelli Marco , Mukhopadhyay Sushobhan , McCurdy Christopher R. , McMahon Lance R.
Published: May 2022

Kratom is being developed as a pharmacotherapy to mitigate withdrawal symptoms from opioids; however, the presence of active metabolites of the primary kratom alkaloid mitragynine (MG) at mu‐opioid receptors [(MORs), 7‐hydoxymitragynine (7‐OH‐MG) and mitragynine pseudoindoxyl (MG‐P)] may limit effectiveness due to their abuse potential. Yue et al (2018) indicated specificity of the antagonizing effects of MG for the reinforcing effects of the MOR agonist heroin relative to the non‐opioid methamphetamine. Using an intravenous drug self‐administration assay, the present study compared abuse potential of MG and its metabolites, and assessed specificity of MG to antagonize the reinforcing effects of the MOR agonist remifentanil and the non‐opioid cocaine in rats. In rats self‐administering remifentanil (0.1‐3.2 µg/kg/injection), reference MOR agonists (methadone and buprenorphine) and the dopamine uptake inhibitor cocaine maintained self‐administration responding above extinction (no injection) levels whereas antagonists at MOR (naltrexone and naloxone) did not. Self‐administration responding above extinction was maintained by 7‐OH‐MG (1.0‐10 µg/kg/injection) and MGP (0.32 µg/kg/injection), but not MG (up to 3.2 mg/kg/injection) when substituted for remifentanil. Self administration of remifentanil, cocaine, 7‐OH‐MG, and MG‐P was decreased by naltrexone (3.2 mg/kg, i.p.). Self administration of remifentanil was also decreased by MG, 7‐OH‐MG, and MG‐P (ED50 values: 34.5, 4.35, and 2.66 mg/kg, respectively). Self administration of cocaine was also decreased by MG, 7‐OH‐MG, and MG‐P (ED50 values: 59.2, 5.12, and 5.17 mg/kg, respectively). Despite the abuse potential of active metabolites of MG, these results suggest less abuse potential of MG than that of its metabolites in rats. The lack of obvious reinforcing effects of MG also suggests if any low contribution of 7‐OH‐MG and MGP to the in vivo MOR activity of MG in rats. Though MG was least potent to decrease the reinforcing effects of remifentanil and cocaine, the comparable ED50 values per compound suggest a lack of specificity of the antagonizing effects of MG and its metabolites in rats with opioid history. These results also indicate that drug history could affect the antagonizing effects of MG on the reinforcing effects of stimulants.
Effects of a ketogenic diet on recognition memory and locomotor activity
Authors: Warren Cali E. , Saito Erin , Miller Gavin , du Randt Johannes D. , Cannon Mio E. , Bikman Benjamin T.
Published: May 2022

Ketones are generally considered neuroprotective and may have therapeutic potential in conditions such as Alzheimer’s and other diseases in which cognition is impaired. The novel object recognition (NOR) assay is a widely‐used test of non‐spatial memory that relies, not on food reward or survival instincts, but on rodents’ innate predisposition to explore in the absence of external stimuli. Here we investigated whether a ketogenic diet (KD) enhances behavioral recognition memory. Adult‐to‐middle‐aged male and female mice were placed on a ketogenic diet supplemented with an exogenous ketone ester for eight weeks and memory was assessed in a two‐object novel object recognition test. We observed trends toward significant improvements in recognition memory (p=0.12) and velocity (p=0.066), and a significant increase in total distance traveled (p=0.047) in the KD mice compared to controls. These data suggest a KD induces beneficial locomotor adaptations, but cognitively, may be more relevant in conditions associated with more severe cognitive deficits.
Targeting ADAM‐17 to Alleviate Cytokine Storm Associated with COVID‐19
Authors: Mattos Pereira Vitoria , Pradhanang Suyasha , Thakar Amit , Mansell John , Nair Sreejayan
Published: May 2022

Background

Acute respiratory distress syndrome (ARDS) is the primary cause of mortality in critically ill subjects infected with the novel coronavirus (severe acute respiratory syndrome‐coronavirus‐2, or SARS‐CoV‐2). Given the elevated levels of circulating proinflammatory cytokines such as tumor necrosis factors (TNF)‐⍺ and interleukin (IL)‐6, it has been postulated that a “cytokine storm” is responsible for the COVID‐19 ARDS. A critical step in cytokine signaling is the shedding of membrane‐anchored cytokines and their receptors.

Objective

To determine the role of the metalloprotease/shreddase ADAM‐17 (A disinterring and metalloproteinase 17) in COVID‐19‐ mediated cytokine release from lung epithelial cells. We hypothesized pharmacological inhibition of ADAM‐17 attenuated COVID‐19‐induced release of TNF‐α in lung epithelial cells.

Materials and Methods

Human primary bronchial/tracheal epithelial cells (ATCC®, PS‐300‐010) were cultured to sub‐confluence and challenged with COVID‐19 Spike Coronavirus pseudovirus (SARS‐COV‐2 pseudo‐virus, MyBioSource), and phorbol‐12‐myristate‐13‐acetate (PMA, Sigma), which was used as a positive control in the presence or absence of the ADAM‐17 inhibitor TNF‐α Protease Inhibitor‐0 (TAPI‐0, Sigma). TNF‐α levels were determined in the cell media at different time points (2‐12h) following the challenge using an ELISA assay. Data are represented as mean ± SEM, and statistical significance was determined by using the Student’s t‐test.

Results

Treatment with both PMA (0.1 µM) and SARS‐COV‐2 pseudovirus (1x108pseudoviral particles), resulted in a robust increase of TNF‐α in the culture media 8 h post‐stimulation (3243.75± 55.3 and 1278.6 ± 357 pg/mL respectively, p <0.05, n = 4). Furthermore, treatment with TAPI‐0 resulted in a concentration‐dependent inhibition of the pseudovirus‐induced TNF‐α release by cultured bronchial epithelial cells, with a complete inhibition observed at 100 µM of TAPI‐0.

Conclusion

Targeting ADAM‐17 is a potential strategy to mitigated the cytokine storm associated with COVID‐19‐ARDS.
Cryo‐EM Structural Determination of Met18, a Scaffold Protein for Iron‐Sulfur Protein Maturation
Authors: Vasquez Sheena , Brignole Edward J. , Marquez Melissa D. , Perlstein Deborah L. , Drennan Catherine L.
Published: May 2022

Iron‐sulfur ([Fe‐S]) clusters are essential cofactors for sustaining life. Because [Fe‐S] clusters play a role in a wide range of cellular processes, defects in their biosynthesis, trafficking, and incorporation into target proteins lead to human diseases. The molecular details of how iron‐sulfur clusters are assembled and transferred to their target proteins are not well understood. Therefore, the goal of this work is to structurally and biophysically characterize proteins involved in the Cytosolic Iron‐Sulfur Cluster Assembly (CIA) pathway. One of these proteins, Met18, is in the CIA targeting complex, and this complex is believed to receive a mature [4Fe‐4S] cluster and transfer it to target proteins downstream. To better understand its function, we determined a 3.3 Å resolution cryo‐EM structure of Met18. In the absence of other CIA targeting complex proteins and target proteins, we observed that Met18 forms a hexamer with its N‐terminus exposed and its C‐terminus buried. Conserved sequences within Met18 assist in forming the hexamer. Upon the addition of Cia2, a CIA targeting complex protein, the hexamer appears to be broken up. We hypothesize that the hexamer is a storage form of Met18 that protects itself from ubiquitination and proteosomal degradation. Here we present the cryo‐EM structure determination of the Met18 hexamer.
Cardiovascular Reactivity During a Fatiguing Exercise in Women: The Influence of Menopause Duration and Age
Authors: Anselmo Miguel F. , Stokes William , Lee Emma , Tahsin Chowdhury T. , Panigrahy Nisha , Glazos Aline , Reilly Cavan , Noven Marnie V. , Carter Jason R. , Keller‐Ross Manda L.
Published: May 2022

Introduction

The risk of hypertension (HTN) and cardiovascular disease (CVD) dramatically increases following menopause, and premature and early menopause (<45yr) substantially increase this risk. Greater blood pressure (BP) reactivity to stressors such as exercise can predict future risk of developing HTN. Thus, we aimed to determine if the time since menopause completion contribute to greater BP reactivity during a fatiguing hand grip (HG). We hypothesized that women who have been postmenopausal for a longer duration would exhibit greater cardiovascular reactivity to a fatiguing HG exercise.

Methods

Women who experienced early and typical‐age (>46yr) menopause completed two visits. Visit 1: Informed consent, medical and health questionnaires. Visit 2: Participants rested in a supine position. Three maximum voluntary contractions (MVC) of their left hand were completed followed by a baseline rest (BL) of 10 minutes. Following BL, a HG test at 30% of MVC was performed until time to task failure. Non‐invasive beat‐to‐beat BP and heart rate (HR) were recorded during BL and HG test. Systolic (SAP), diastolic (DAP), mean arterial (MAP), and HR were analyzed as BL averages and in 20‐30s intervals at start, 25%, 50%, 75%, and task failure. Repeated‐measures ANOVA and independent T‐tests were used for the statistical analysis; results are presented as mean±SD.

Results

Eighteen postmenopausal females completed the study and were divided into two groups: 1) postmenopause ≤10yrs (PM≤10; 8±2 yrs; body mass index (BMI): 25±3 kg/m2, n=8) and 2) postmenopause ≥11 (PM≥11; 15±5 yrs; BMI: 25±5 kg/m2, n=10). The PM≥11 group was slightly older (64±3yrs) than the PM≤10 (60±4yrs, p=0.005). BL SAP was not different between PM≤10 (133±14mmHg) and PM≥11 (128.75±14mmHg; p=0.95). DAP (85±9 vs 81±10mmHg; p=0.68) and MAP (100±10 vs 96±9mmHG; p=0.40), were not different between PM≤10 and PM≥11, respectively. During the fatiguing contraction, SAP increases were exaggerated in PM≥11 compared with PM≤10 (Δ63±25 vs Δ40±16mmHg; group × time, p=0.03). Similarly, MAP increased more in PM≥11 compared with PM≤10 (Δ49±16 vs Δ31±10mmHg; group × time, p=0.04), but not DAP (group × time, p=0.1) or HR (group × time, p=0.08). When co‐varying for age, however, the interactions were no longer significant (p>0.05, for all). In addition, peak increases in SAP (p=0.04), MAP (p=0.01), and HR (p=0.02) were greater in PM≥11 compared with PM≤10, but these observations were no longer significant when co‐varying for age (p>0.05, for all).

Conclusion

Novel findings from this study suggest that although the duration of menopause influences BP reactivity during a moderate intensity fatiguing contraction, this greater increase may be explained by older age. This elevated response to submaximal exercise may contribute to the increased risk of HTN and CVD in postmenopausal women.