bluuebunny/crossref_metadata_2025 huggingface dataset:
Multimodal communication modulates swimming behavior in sailfin molly (Poecilla Latipinna)
Authors: Hendrix Ashley , Bristow McKenna , Huertas Mar
Published: April 2020

Information received from the external environment modulates reproductive behavior in vertebrates and determines when and with whom to mate. If one of these signals is not received, then the selected behavior may not occur. It is known that chemical and visual signals play a role in reproductive behavior in fish, but whether they work together is not fully understood. We hypothesize that both visual and chemical cues are necessary in reproductive behavior of the sailfin molly, and therefore we expect the fish to respond to multimodal cues in order to complete the reproductive cycle. Our objective was to analyze attractive behavior of sailfin males and females to chemical, visual and chemical + visual cues from the opposite sex. A two‐choice maze constructed of acrylic was split into two sections with each side being 106cm (L) × 30.5cm (w). At the bottom of the maze is an open mixing area, which also doubled as the starting point for each trial. Plastic mesh was placed in each half of the maze to create turbulence and even flow throughout the maze. Water was pumped from the top of the maze using a hose and exited the maze downstream through four drain holes. Previously done dye tests confirmed that the water flow was even throughout each half of the maze and exited through the drain holes without mixing at the exit point for each trial. A small clear box with holes was placed in the maze and used for the chemical + visual signal, a black box with holes was used for the chemical only signals, and a separate clear box with no holes was used for the visual only signal. The fish of the opposite sex being tested was placed in the box located in the maze about 3/4 upstream. At the start of each trial a fish was placed in the mixing area for 10 minutes in order to acclimate. It was then given access to both sides of the maze for 10 minutes which established side preference. Cues were placed on the least preferred side of the maze in order to avoid bias and the fish behavior was observed for another 10 minutes for each cue. A Wilcox rank test was used to determine the preference for each cue. Behavioral responses showed a significant visual and a visual+chemical preference for both sexes. Visual cues had higher index of preference than visual+chemical cues, which suggest a deterrent component in sailfin odors. Chemical cues alone did not elicit swimming behavior. Although it was demonstrated in previous studies that male chemical cues induce release sex steroid release in female sailfin molly. This study shows that multimodal cues are critical for the reproductive behavior of sailfin molly and opens a new avenue of research in communication of live‐bearing species.
The Effect of a Histone Deacetylase Inhibitor on PD‐L1, HLA‐ABC, HLA‐E, and HLA‐G on Human Breast Cancer Cell Lines
Authors: Toufexis Alec George , Reddy Nikhil Cheruku , O’Donnell Robert W.
Published: April 2020

Increased expression of human leukocyte antigen (HLA) allows tumor cells to be more easily detected by the immune system. In previous research, epigenetic modifiers including the histone deacetylase inhibitor 3 (HDAC3), RGFP966, has been shown to decrease PD‐L1 expression. PD‐L1 expression leads to inhibition of T cell cytotoxicity, and decreasing it therefore can enhance the immune response. We hope to elucidate whether or not HLA‐ABC expression is similarly decreased upon exposure to the HDAC3 inhibitor which would be detrimental to immune detection. Two breast cancer cell lines that are known to express HLA‐ABC are MCF‐7 and MDA‐MB‐231. Our initial results show that HLA‐ABC is expressed on the MDA‐MB‐231 and MCF‐7 cell lines, while PD‐L1 is only expressed by MDA‐MB‐231 cell line as demonstrated by flow cytometry. However, in subsequent experiments testing the effect of the drug RGFP966, there were inconsistent results regarding the change in expression of HLA‐ABC which requires further investigation. In addition, we also wish to look at other HLA proteins, such as HLA‐E and HLA‐G, of which expression which has been shown to be detrimental to immune detection. Future experiments are planned to optimize the doses of RGFP966 to maximize HLA expression in these breast cancer cell lines while limiting the expression of PD‐L1 and HLA‐E and HLA‐G.

Support or Funding Information

Geneseo Foundation

Carl Savino ‘53 and Olga Gerych‐Savino ‘53 Endowment

David Wolf, DO
The Size of the Inferior Horn of the Lateral Ventricle is Negatively Correlated with the Thickness of the Entorhinal Cortex
Authors: Ritz Brianna Kathryn , Zdilla Matthew
Published: April 2020

The hippocampus and entorhinal cortex are implicated in the progression dementia. With structural changes in the parenchyma of the hippocampus and entorhinal cortex, will the inferolateral aspect of the lateral ventricle expand to fill the void? This study sought to determine the relationship between the volume of the lateral ventricle / inferolateral aspect of the lateral ventricle and the volume of the hippocampus and entorhinal cortex, as well as the thickness of the entorhinal cortex. The methods included analysis of Freesurfer data collected from 2100 hemispheres from 1050 brain MRIs. Imaging included scans from 733 unremarkable individuals, 82 pre‐dementia individuals, and 235 individuals with dementia. Pearson’s correlation coefficients were calculated to assess correlation. The inferolateral aspect of the lateral ventricle was significantly negatively correlated with hippocampus volume, in general; although, the correlation was small. The strongest statistically significant correlations existed between the inferolateral ventricular volume and the entorhinal thickness. Analysis of the total population revealed a moderate negative correlation (r=−0.51, p<0.0001), as did analysis of the pre‐dementia and dementia populations (r=−0.45, p<0.000 and r=−0.51, p<0.0001, respectively). However, in the unremarkable population, the correlation was very small albeit significant (r=−0.26, p<0.001). Therefore, the inferolateral ventricular size increases as entorhinal thickness decreases, in general, but especially in pre‐dementia and dementia populations. The results of this study suggest a relationship between the inferior horn of the lateral ventricle and key regions of the brain that are implicated in dementia and, therefore, have clinical value in the assessment of dementia.

Support or Funding Information

Funding: WV Research Challenge Fund [HEPC.dsr.17.06] and NASA West Virginia Space Grant Consortium, Training Grant #NNX15AI01H
Variation in the morphology of the extensor pollicis longus and extensor digitorum muscles: A case report
Authors: John Jithin , Bukhsh Omar A. , Skowronski Christian , Ngo Han G. , Silverman Brittany R. , Barremkala Malli
Published: April 2020

This case report aims to highlight the variation in the morphology of the extensor pollicis longus (EPL) and the extensor digitorum (ED) muscles. EPL originates from the posterolateral surface of the ulna, and the ED originates from the lateral epicondyle of the humerus. Both of these muscles are innervated by the radial nerve. EPL’s primary function serves to extend the pollex, while the ED serves to aid in the extension of the metacarpal phalangeal, proximal interphalangeal, and distal interphalangeal joints of digits 2 through 5. During routine anatomical dissections of 48 upper limbs from 24 cadavers (n=24) at Oakland University William Beaumont School of Medicine, one adult female (75‐years‐old) was observed to have an EPL with an accessory tendon attaching on the 2nd metacarpal‐phalangeal joint and an accessory tendon of the ED inserting on the distal phalanx of the pollex unilaterally on the left side. The EPL is known to be one of the most anatomically consistent structures with its tendon, only inserting into the pollex (Kim, Lee, & Baek). This variation of the EPL was described as extensor pollicis et indicis accessorius, which was found in 1.2% of the cadavers (n=431) in Yoashida’s study. Although there are a few isolated patient cases describing the similar ED variation, such as in Hanrahan et al., very few results have been found regarding the prevalence of the accessory ED muscle belly in the general population. It is likely that the ED tendon inserting into the pollex weakly assists with the extension function. Additional variations of the extensor indicis muscle providing an accessory tendon to the 3rd digit and extensor digiti minimi muscle providing an accessory tendon to the 4th digit were noticed bilaterally on the same cadaver. The knowledge of these variations will be vital in proper surgical planning for tendon transfers, grafts, and transplantations.
Regulation of SPRTN Metalloprotease in DNA‐Protein Crosslink Repair
Authors: Perry Megan , Kollala Sai , Halbkat Halle , Su Grace , Kreiling Natasha , Ghosal Gargi
Published: April 2020

DNA‐protein crosslinks (DPCs) are generated when proteins become covalently trapped on the DNA upon exposure to agents such as endogenous reactive aldehydes or exogenous chemotherapy drugs. Failure to repair DPCs causes cells to acquire oncogenic mutations; conversely, specific crosslinking of replication enzymes can target highly proliferative cancer cells and cause fork collapse and cell death. Despite the potency of DPC‐inducing chemotherapies, cancer cells often become resistant to these treatments likely due to robust DNA damage repair pathways. Previously, we and others identified SPRTN metalloprotease as a critical DNA damage response protein. SPRTN cleaves proteins cross‐linked to DNA and facilitates DPC repair, but little is known about the molecular mechanisms that regulate SPRTN protease activity. SPRTN is mono‐ubiquitinated, and we are currently investigating the functional significance of SPRTN ubiquitination. Using mass spectrometry and co‐immunoprecipitation, we have identified USP11 as an interacting partner of SPRTN. USP11 shRNA knockdown cells are hypersensitive to DPC‐inducing agents and have delayed repair of DPCs. Continuing work is focused on the molecular function of USP11 in DPC repair. Our studies provide insight into the regulatory processes underlying the DPC repair pathway and identify repair enzymes that can be therapeutically targeted in cancer cells to prevent chemoresistance to standard DPC‐inducing therapies.

Support or Funding Information

This work is supported by K22, NCI Award CA188181‐01A1 to Gargi Ghosal and University of Nebraska Presidential Graduate Fellowship to Megan Perry.
Novel Salinomycin Analogs Show Improved Selectivity Towards Breast Cancer Stem Cells
Authors: Urbaniak Alicja , Reed Megan R. , Antoszczak Michał , Sulik Michał , Huczyñski Adam , Eoff Robert L. , MacNicol Melanie C. , MacNicol Angus M. , Chambers Timothy C.
Published: April 2020

Breast cancer tumor metastasis is the leading cause of mortality among women. Cancer stem cells (CSCs) are the tumor‐initiating cells which drive initiation, progression, metastasis, and tumor reoccurrence. Their resistance to conventional chemo‐ and radiotherapies and their ability to survive such treatment enables tumor reestablishment. Thus, therapeutic strategies targeting CSCs hold great potential for novel advances in cancer treatment. Salinomycin (SAL) is a naturally occurring polyether ionophore antibiotic with anti‐cancer activity towards various types of tumor cells, but its toxicity towards normal cells limits broad application. A library of 21 novel SAL analogs was screened to identify compounds with improved selectivity especially against breast cancer stem cells. SAL analogs were either single modified with ester or amide in the C1 position or double‐modified C20‐oxo derivatives. Eight single‐ and two double‐modified derivatives were more potent (IC50 range of 1.13 ± 0.19 to 3.93 ± 0.39 μM) towards the breast cancer cell line MDA‐MB‐231 compared to SAL (IC50 of 5.81 ± 1.50). These derivatives induced DNA fragmentation suggestive of apoptotic cell death. Further, most of these derivatives had improved selectivity toward MDA‐MB‐231 cells compared to SAL when also tested against normal breast epithelial MCF10A cells. In addition, a butyl ester derivative of SAL, chosen based on increased potency and selectivity versus SAL, significantly reduced the colony‐forming efficiency of MDA‐MB‐231 cells in association with selective loss of the CD44+/CD24−/low stem‐cell‐like subpopulation. Select derivatives were next screened against the NCI‐60 Human Tumor Cell Line Panel. A double‐modified analog of C20‐oxo‐SAL with hydroxamic acid was found to be more potent than SAL towards all 6 breast cancer cell lines in the panel as well as other tumor types especially leukemia. Studies are underway to test the analogs in three dimensional culture using breast cancer organoids derived from both cell lines and patients. The present findings highlight the therapeutic potential of SAL analogs towards breast cancer and breast cancer stem cells and support further research and clinical development of these compounds.

Support or Funding Information

The present study was funded by grants (to AMM and TCC) from the Arkansas Breast Cancer Research Program. Testing was performed by the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute,
http://dtp.cancer.gov.
Brain connectivity and activity during Alzheimer’s disease progression in a mouse model by manganese‐‐enhanced MRI in living brain correlated with post‐mortem histopathology.
Authors: Bearer Elaine L. , Medina Christopher S. , Uselman Taylor , Jacobs Russell E.
Published: April 2020

Alzheimer’s disease (AD) arises from inherited mutations in genes associated with plaque formation: amyloid precursor protein (APP), tau, and presenilin, or sporadically without these mutations. Studies of mice expressing these familial Alzheimer’s disease (FAD)‐associated mutations has led to new insights into the biological processes in sporadic AD. We study a mouse model in which expression of the FAD protein, APPSwInd, can be suppressed after plaque formation. This mouse thus corresponds to sporadic Alzheimer’s disease, where plaques are present in the absence of mutated protein. We use manganese‐enhanced magnetic resonance imaging (MEMRI), an emerging technology, coupled with histopathology of serial sections to investigate how plaques affect connectivity, neural activity degeneration throughout the whole brain as the mouse ages. MEMRI can be used in two ways: to determine axonal transport dynamics and to map neural activity in living mice. Stereotactic intracranial injection of Mn2+ traces transport in hippocampus occurring over 24h in the awake behaving mouse. Systemic Mn2+ enters active neurons in the brain of freely moving mice over 24h and is imaged retrospectively. Amyloid precursor protein (APP) is the precursor to A□eta plaques. The cytoplasmic domain of APP mediates cargo‐motor attachments for axonal transport. Our results show that in APP‐KO mice, axonal transport on Mn2+ is decreased demonstrating the APP plays a role in Mn2+ transport. In old transgenic mice APPSwInd, with both over‐expression of mutated protein and plaques, axonal transport is altered, as detected by time‐lapse manganese‐enhanced magnetic resonance imaging (MEMRI) of the brain in living mice. Time‐lapse MR images were captured before and successive time points after stereotactic injection of Mn2+ (3–5nL) into CA3 of the hippocampus. Images of multiple individuals from the two groups (with plaques and no expression, or without plaques) were aligned and processed with our automated computational pipeline, and voxel‐wise statistical parametric mapping (SPM) performed. Whole brain neural activity was decreased in 3yr old mice with plaque and expressing APPSwInd compared to non‐expressing littermates, particularly in the hippocampus. Brains were harvested after MEMRI for biochemistry, or fixed, embedded and serially sectioned for histopathology. Histopathology detected mouse p‐tau–positive dystrophic neurites, decreases in the volume of septal nuclei, decrease in dentate gyrus width and cell death. Thus, plaque alone depresses transport in the important hippocampal to basal forebrain memory circuit, and decreases neural activity in the aging mouse.

Support or Funding Information

Supported by NINDS RO1 NS062184 and The Harvey Family Endowment.
Amelioration of Podocytopathy by Smpd1 Gene Deletion in Podocyte‐Specific Asah1 Gene Knockout Mice
Authors: Li Guangbi , Huang Dandan , Camus Sarah , Li Ningjun , Li Pin-Lan
Published: April 2020

Podocyte‐specific deletion of Asah1 (AC gene code in mouse) gene has been found to produce podocytopathy and nephrotic syndrome in mice (Asah1fl/fl/PodoCre mice). However, the mechanism underlying the pathogenesis of podocytopathy in these mice remains unknown. To test whether ceramide accumulation contributes to podocyte injury in Asah1fl/fl/PodoCre mice, Asah1fl/fl/PodoCre mice were mated with Smpd1−/− mice (Smpd1 is mouse gene coding acid sphingomyelinase), which produced mice lacking both Smpd1 and Asah1 gene in podocytes, namely, Smpd1−/−/Asah1fl/fl/PodoCre mice. Using LC‐MS/MS, ceramide accumulation was detected in isolated glomeruli of Asah1fl/fl/PodoCre mice, and Smpd1 gene knockout blocked glomerular ceramide accumulation Asah1fl/fl/PodoCre mice. Correspondingly, severe proteinuria and albuminuria in Asah1fl/fl/PodoCre mice were ameliorated by Smpd1 gene deletion. By immunofluorescence staining, we found reduced glomerular podocin and increased glomerular desmin in Asah1fl/fl/PodoCre mice, which was confirmed using Western blot analysis in isolated glomeruli from these mice. All these podocyte injuries in Asah1fl/fl/PodoCre mice were substantially blocked by cross breeding with Smpd1 gene knockout mice. Under transmission electron microscope, Asah1fl/fl/PodoCre mice were found to have foot process effacement and microvillus formation in podocytes, which were also reversed by deletion of Smpd1 gene. These results suggest that AC deficiency induces ceramide accumulation in mouse glomeruli which is critical to the development of podocytopathy and nephrotic syndrome. Blockade of ceramide production via ASM may attenuate ceramide accumulation and consequent podocytopathy due to AC gene deficiency.

Support or Funding Information

This study was supported by NIH grants DK54927 and DK102539.
Carotid body‐specific shRNA knockdown of PKCɛ blunts TRPV1‐dependent asthmatic bronchoconstriction
Authors: Wilson Richard J.A. , Roy Arijit , Jendzjowsky Nicholas
Published: April 2020

Rationale

Aggravated airway bronchoconstriction and mucus production in response to allergen and/or bradykinin (i.e., airway hyperresponsiveness) is a primary defining symptom of asthma. Recently, we discovered a critical role for the carotid bodies: inhaled allergen causes an increase in arterial plasma concentrations of asthmakines which excite the carotid bodies via a transient receptor potential vanilloid 1 channel (TRPV1) dependent mechanism. Elevated carotid body excitation in turn increases efferent vagal activity, resulting in heightened bronchoconstriction. While our data demonstrate that the carotid bodies are strongly excited by multiple Th‐2 cytokines, in the ovalbumin primed and sensitized Brown Norway rat model of asthma, the primary asthmakine is likely lysophosphatidic acid (LPA). However, the cellular link between LPA receptors and TRPV1 has not been identified. Here we show that the effect of LPA on the carotid bodies is likely mediated by PKCɛ‐dependent phosphorylation of TRPV1.

Methods

Lentivirus vectors containing shRNA to knockdown PKCɛ or scrambled sequence were injected bilaterally into the carotid bodies of asthmatic rats (n=7, each). The responses of carotid bodies to LPA (5μM), and their subsequent responses to TRPV1 blockade (AMG9810, 10μM) were tested using the en bloc perfused carotid body preparation. The effects of carotid body‐specific PKCɛ knockdown on changes in lung mechanics in response to inhaled bradykinin were tested using the Flexivent system.

Results

Carotid bodies from asthmatic rats injected with PKCɛ knockdown sequence mounted smaller responses to LPA than those injected with scrambled sequence. No difference in LPA response was detected between groups following TRPV1 blockade, suggesting the primary target for PKCɛ is TRPV1. Asthmatic rats with carotid bodies injected with PKCɛ knockdown sequence had reduced lung resistance in response to bradykinin at 1, 10 and 20 minutes. Similar results were obtained using a PKCɛ inhibitory peptide.

Conclusion

We show for the first time that carotid body‐mediated bronchoconstriction in asthmatic rats occurs through LPA receptor activation of PKCɛ and subsequent TRPV1‐mediated carotid sinus nerve excitation. Targeting carotid body LPA receptors, PKCɛ and/or TRPV1 may provide a new therapeutic target to reduce asthmatic airway hyperresponsiveness.

Support or Funding Information

CIHR, Alberta Innovates, Alberta Lung Association and the Parker B. Francis Foundation.
Self‐Reported Student Assessments as Predictors of Gross Anatomy Success
Authors: Graham John H. , Barizo McTheophilus , Coates Darren , Rustin Emily , Weicht Jessica , Wilkinson Elizabeth
Published: April 2020

This study sought to identify factors that contribute to success in an interprofessional, graduate‐level, cadaveric anatomy courses, composed of physical therapy (DPT) and occupational therapy (MOT) students. One hypothesis predicted that team‐based learning (TBL) would improve student outcomes. A second hypothesis predicted that students classified by the M4 (highly capable and confident) category, of the Hersey and Blanchard Situational Leadership Theory, would be more successful in the anatomy course. The course utilized two different types of cadavers – traditionally‐embalmed (TEC) and soft‐embalmed (SEC). Analysis was conducted on self‐reported factors, from graduate level DPT and MOT students, which could underlie success in a cadaveric gross anatomy course. The participants consisted of 34 MOT and 33 DPT first‐year cohort students, who completed an 18 question Qualtrics self‐reported survey consisting of Likert scales, categorical choices, and open‐ended responses, regarding their overall performance in a cadaveric gross anatomy lab and lecture course. Based on the participants’ responses to the survey, it was found that the mean lab grade for students who dissected the TEC cadaver was only marginally lower than students who dissected the SEC cadaver. The mean lab grade for students with three group members was slightly lower than the mean grade of students with four group members (87.80 vs. 88.82). These results indicated that TBL in the lab was not a significant factor, as there was little difference in average lab grade when comparing three‐ and four‐person teams. Participants responded that their average skill level before participation in dissection was 1.4 and after was 3.61, which was found to be significantly different (p < 0.001). Studying up to eight hours per week appeared to be the optimal time that yielded successful results. Variability in reported time spent studying and grades suggests that quality needs to be taken into account. Results from other survey questions did not support the hypothesis that students fitting the M4 category of Hersey and Blanchard’s Situational Leadership Theory would be more successful in the anatomy course. Overall, success appeared to be most dependent on a cumulative result from hands‐on experience, learning style, and quality of study.
Probiotic‐generated product protects against alcoholic liver disease through increasing intestinal AhR‐IL22‐Reg3 and Nrf2 signaling
Authors: Gu Zelin , Liu Yunhuan , Li Fengyuan , Jiang Mengwei , Zhang Lihua , McClain Craig , Feng Wenke
Published: April 2020

Background

Probiotics are known to modulate intestinal barrier integrity against alcohol‐induced endotoxin leakiness and bacterial translocation. Our previous studies showed that Lactobacillus rhamnosus GG (LGG) and its culture supernatant protected against alcoholic liver disease (ALD) through increasing intestinal mucus and epithelial tight junction protein expression resulting in a reduction of circulation LPS level. The precise mechanisms underlying this protective effect are still not clear. Recent studies demonstrated the critical role of intestinal aryl hydrocarbon receptor (AhR) in the regulation of RORγt+ innate lymphoid cell (ILC3) function to produce IL22, which modulates intestinal microbiota homeostasis and barrier function through increasing the expression of epithelial antimicrobial peptide regenerating islet‐derived protein 3γ (Reg3γ) and Reg3β.

Methods

We isolated a product (LGGp) from LGG and examined its role in the modulation of ALD. C57B6 mice were subjected the Lieber DeCarli liquid diet containing 5% alcohol for 10 days, and one bolus of alcohol (5 g/kg) was gavaged on the last day 9 hours before tissue collecting. Control mice were fed isocaloric maltose dextrin. LGGp was supplemented on day 7 once a day for three days.

Results

This three‐day administration of LGGp reversed alcohol‐induced hepatic steatosis and injury, demonstrated by reduced Oil red O staining of liver section, decreased serum ALT and AST levels and reduced hepatic apoptosis. The protective effects were associated with decreased circulating LPS concentration and hepatic bacterial load, indicating an increased gut barrier function. Further analysis showed that LGGp activated AhR luciferase activity and increased Cyp1a1 (AhR target) enzymatic activity. LGGp treatment increased intestinal mRNA expression of IL22, Cyp1a1, Reg3g and Reg3b, which play a critical role in modulating intestinal immune response to bacteria invasion and translocation. In addition, LGGp treatment significantly increased intestinal epithelial nuclear factor erythroid 2‐related factor 2 (Nrf2) protein expression, which were associated with increased protein expression of epithelial tight junctions ZO‐1, occludin, claudin‐1. Furthermore, LGGp inhibited LPS‐induced pro‐inflammatory factors TNFα and IL1β in mRNA and protein levels in Raw264.7 macrophage cells.

Conclusions

Our results demonstrate that LGGp treatment protects against ALD through activation of intestinal AhR‐mediated upregulation of IL22 and Reg3 and through Nrf2‐mediated upregulation of intestinal tight junction proteins that lead to the decreased LPS release and bacterial translocation and reversal of ALD.

Support or Funding Information

Supported by grants from NIH.
Effects of Vacuolar H+‐ATPase Inhibition and the a4 Isoforms on Secretion and Activation of Cathepsin D in MDA‐MB231 Breast Cancer Cells
Authors: Swickard Jamie Nicole , Henry Abhijeet , Hinton Ayana
Published: April 2020

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are important for acid‐base balance in the blood and sperm maturation. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231 and may contribute to its metastatic phenotype. V‐ATPases are hypothesized to create a localized low pH environment that promotes tumor cell invasion by activating secreted cathepsins. Studies indicate that cathepsins secreted by cancer cells are involved in metastasis by cleaving extracellular matrix proteins and activating other proteases, which facilitates tumor invasion. We are interested in a connection between cell surface V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. Previous data from our lab indicates that inhibition of V‐ATPases by a general inhibitor showed a trend of reducing the activity of secreted cathepsin B, but not cathepsin L. However, that trend, was not statistically significant. Cathepsin D is another protease that is secreted by cancer cells including metastatic breast cancer. For this study we looked at the activity of cathepsin D following inhibition of V‐ATPases. Our preliminary data indicates that inhibition of V‐ATPases reduces the activity of secreted cathepsin D. To investigate the role of V‐ATPases in the activation of cathepsins further we are using CRISPR/Cas9 to knockout the gene for the a4 isoform of the V‐ATPase “a” subunit. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. We hypothesize that the a4 isoform knockout cells will have reduced activation of secreted cathepsin D while not effecting activation of cathepsin L. Separate abstracts submitted by our lab are focused on the a1, a2 and a3 isoform knockouts.

Support or Funding Information

Anderson Summer Research Assistantship
MMP‐12 knockdown during acute and chronic phases promotes post‐stroke neurological recovery
Authors: Nalamolu Koteswara Rao , Challa Siva Reddy , Fornal Casimir A. , Martin Ryan C. , Olson Elsa A. , Ujjainwala Ammar L. , Klopfenstein Jeffrey D. , Pinson David M. , Wang David Z. , Kalyanasundaram Ramaswamy , Vemuganti Raghu , Veeravalli Krishna Kumar
Published: April 2020

Matrix metalloproteinases (MMPs) are known modulators of ischemic brain damage. We recently showed that ischemic stroke in adult rodents induces MMP‐12 in brain during both acute and chronic phases of ischemic stroke. We also showed that MMP‐12 is detrimental during acute phase as it promotes BBB disruption leading to brain damage. In the present study, we evaluated the significance of elevated MMP‐12 during the chronic phase after focal ischemia on neurological recovery. Adult male Sprague‐Dawley rats were subjected to a 2h transient middle cerebral artery occlusion (MCAO). These rats were administered with either a single dose (at 10–15 min of reperfusion) or 3 doses (at 10–15 min, 7 days and 14 days of reperfusion) of plasmids expressing either MMP‐12 shRNA or a scrambled control shRNA (1 mg/Kg; i.v.; n = 9–11/group) in nanoparticle formulation. To assess reflexes, balance, sensory and motor functions, rats were subjected to modified neurological severity scoring, adhesive removal test, beam walk test and rotarod test at 1, 7, 14 and 21 days after reperfusion. The cohort of rats treated with a single dose of MMP‐12 shRNA showed significantly better functional recovery as compared to control shRNA cohort. Longer‐term suppression of MMP‐12 also resulted in better functional recovery than control shRNA cohort. Thus, these studies indicate that induction of MMP‐12 during both acute and chronic phases after focal ischemia contributes to neurological deficits and hinders recovery.

Support or Funding Information

This work was supported by the NIH Grant 1R01NS102573‐01A1 to KKV
Impact of Aging and G Protein‐Coupled Estrogen Receptor Deletion in Arterial Stiffening and Cardiac Function in Male and Female Mice
Authors: Ogola Benard Ojwang , Zimmerman Margaret A. , Harris Nicholas R. , Kilanowski-Doroh Isabella , Groban Leanne , Lindsey Sarah
Published: April 2020

Arterial stiffening assessed by pulse wave velocity (PWV) independently predicts cardiovascular events and is exacerbated in aging men and women. Our previous studies demonstrated that G protein‐coupled estrogen receptor (GPER) plays a protective role in the heart and vasculature. The current study evaluated sex differences and the impact of GPER and aging on arterial stiffening and cardiac function. We hypothesized that genetic deletion of GPER induces sex differences in arterial stiffness and cardiac function during aging. Male and female wildtype (wt) and global GPER knockout (ko) mice were used between 4–6 months (adult) and 12–13 months (middle‐aged; n=6–17 per group). Echocardiography was performed under isoflurane anesthesia in short axis view to assess cardiac function, and local PWV was obtained within the carotid artery. Data was subjected to 3‐way ANOVA and correlation analysis. PWV was significantly higher in adult male ko versus wt mice (1.60 ± 0.07 m/s vs. 1.21 ± 0.05 m/s, P<0.001) but was not altered by GPER deletion in female mice. Aging significantly increased PWV (P<0.0001) in both males and females. PWV did not correlate with ejection fraction (P=0.69) or heart rate (P=0.89) but inversely correlated with early left ventricular filling deceleration time (Edec time; P=0.0005, r= −0.37) and left ventricular ejection time (LVET; P=0.044, r= −0.22) and positively correlated with relative wall thickness (RWT; P=0.008, r= 0.281). In conclusion, GPER deletion impacted PWV in adult male mice, while aging increased PWV in all groups. Future studies will investigate whether other pathological conditions such as hypertension reveal a phenotype of vascular stiffness in female GPER ko mice. The inverse relationship between PWV and Doppler‐derived indices of left ventricular distensibility underscores the important relationship between arterial and cardiac stiffness.

Support or Funding Information

NIH Grant # R01HL133619 to SHL
Annotation of Contig53 on Chromosome 3L of Drosophila takahashii
Authors: Garcia Janette , Fung Siaumin
Published: April 2020

DNA wraps around histones to form chromatin in the nucleus of eukaryotic cells. DNA densely packed histones, heterochromatin, is associated with silencing or transcription inactive genes. While, the loosely packed chromatin, euchromatin, is associated with actively transcribed genes. The F element, dot chromosome in Drosophila melanogaster is composed of mostly heterochromatin, yet has active transcribed genes. The goal of our research is to elucidate the regulatory motif within the dot chromosome by comparing the dot chromosome genes with gene located in the euchromatin, D element. In this study, Contig 53 in D element from Drosophila takahashii is characterized with 7 features. Through the creation of a gene model or outline of where exons and introns begin and end, instances of where regulatory motifs occurs can be observed. The Genome Browser allows for visualization of the predicted genes and isoforms. BLAST allows for the alignment of genes between the two species. FlyBase contains genetic and molecular data for Drosophila melanogaster. By annotating and comparing different contigs or gene segments of the two species and the divergence between two different species D. melanogaster and D. takahashii can be visualized as well as any silencing of genes that has occurred through evolution. Our research shows that although many of the jim isoforms vary slightly, data can be correlated with RNA seq, Gene scan predications, and TopHat predictions. Of 11 total isoforms of D. melanogaster, seven share almost 90% similarity to D. takahashii. This shows that through evolution, contig 53 of D. takahashii and D. melanogaster have only diverged marginally.

Support or Funding Information

This project was supported in part by grants from the Department of Education (Award #P031S150199), from Southern California Edison, and through Vanguard University’s SURP Program generously supported by the Office of the Provost and the Institute for Faculty Development.”

Special thanks to the Genomics Education Partnership (GEP)
Elimination of Wnt Secretion from Hepatic Stellate Cells Impairs Hepatocyte Proliferation After Partial Hepatectomy
Authors: Hu Shikai , Ko Sungjin , Kim Min-wook , Preziosi Morgan , Bell Aaron , Monga Satdarshan
Published: April 2020

Wnt‐dependent β‐catenin activation contributes to hepatocyte proliferation following 2/3 partial hepatectomy (PH) through regulation of Cyclin‐D1 (Ccnd1) expression, as we have previously shown using β‐catenin or Wnt co‐receptors, low‐density lipoprotein receptor‐related protein 5–6, conditional knockouts (KO). However, given the redundancy between the 19 mammalian Wnt proteins and various cell sources for Wnt secretion, the precise mechanisms of Wnt signaling in liver regeneration (LR) are not completely understood. Recently, using endothelial cell (EC)‐specific deletion of Wntless (Wls), a cargo receptor required for Wnt secretion, we have reported that sinusoidal ECs secrete Wnt2 and Wnt9b to activate β‐catenin in hepatocytes and enhance Ccnd1 expression, which is indispensable for normal and optimal proliferation of hepatocytes during LR after PH. Alternatively, conditional deletion of Wls in hepatocytes or cholangiocytes did not impact LR, whereas Wls deletion in macrophages only marginally affected LR post‐PH. Here, to investigate the roles for hepatic stellate cell (HSC)‐secreted Wnts in LR, we characterize the HSC‐Wls‐KO, generated by interbreeding Wls‐floxed and Lecithin Retinol Acyl Transferase (LRAT)‐driven Cre (Lrat‐Cre) transgenic mice. HSC‐Wls‐KO mice showed no effect on liver weight or β‐catenin‐dependent pericentral gene expression at baseline. However, hepatocyte‐specific CCND1 protein normally evident in midzonal hepatocytes, was diminished in baseline KO liver as also evident by WB, whereas Ccnd1 expression was unaltered as seen by RNA‐seq and qPCR, suggesting posttranslational regulation. No difference in CCND1 protein was seen in the non‐parenchymal cells of the KO livers when compared to WT. After PH, HSC‐Wls‐KO showed modest decrease in gene expression of Ccnd1 at only 24 hour (h) post‐PH as compared to WT but comparable levels between the two groups were seen at 40h and 72h. While similiar PCNA+ staining in hepatocytes was observed at 40–72h post‐PH in HSC‐Wls‐KO and WT, HSC‐Wls‐KO displayed significantly diminished BrdU+ or pHH3+ hepatocytes at 40h, which rebounded by 72h, suggesting delayed regeneration due to the impaired hepatocyte replication.

Conclusion

At baseline, Wnt ligands secreted by HSCs are essential for CCND1 protein expression in midzonal hepatocytes but not for pericentral zonation. Following PH, HSCs‐derived Wnts also contribute to normal Ccnd1 expression and hepatocyte mitosis.

Support or Funding Information

This work was supported by NIH grants 1R01DK62277, 1R01DK100287, R01CA204586 and Endowed Chair for Experimental Pathology to S.P.M., and by NIH grant 1P30DK120531‐01 to Pittsburgh Liver Research Center (PLRC) and by PLRC Pilot & Feasibility grant PF 2019‐05 to S.K.
Mechanistic Insights into pH‐sensing GPCRs
Authors: Rowe Jacob , Kapolka Nick , Taghon Geoffrey , Morgan William , Isom Daniel
Published: April 2020

Many aspects of protein function, cell biology, human physiology, and disease are influenced by pH changes. However, our mechanistic understanding of pH effects in most of these areas is limited. This is especially true in the case of cell signaling, for which very few pH‐sensing receptors and signaling cascades have been identified. To address this issue, we are studying how pH regulates G protein‐coupled receptors (GPCRs), the largest and most therapeutically targeted family of cell‐surface receptors in humans. Unlike most cellular proteins, GPCRs are regularly exposed to a wide range of dynamic pH values (from pH 7.4 on the cell surface, to pH 5.0 in endosomes) and can be chronically exposed to acidic microenvironments caused by cancer and processes such as inflammation. While there are more than 800 GPCR family members, only three receptors (i.e. GPR4, GPR65, and GPR68) are known to be activated directly by pH. In this study, we used structural informatics and cell‐based signaling assays to predict and validate residues responsible for pH sensing by these receptors. As part of these efforts, we engineered and interrogated 44 GPR4, GPR65, and GPR68 point mutations in a library of 440 cell strains that covered all possible GPCR‐Gα subunit signaling combinations. This comprehensive strain library included 14 mutations predicted by our structural informatics software, pHinder, and the full set of 30 mutations for these receptors available in the literature. Our results for the known mutants agree with previous reports and expand our understanding of the relationship between Gα‐signaling specificity and pH sensing. More importantly, several of our newly predicted mutations appear to have a greater influence on the pH‐sensing capabilities of these receptors than any of the previously reported GPR4, GPR65, and GPR68 mutations to date. Most of the pH‐sensitive residues predicted by pHinder were found within the protein core, a region that has been largely overlooked when considering pH‐sensing properties of GPCRs. Our cell‐based experiments confirmed that these residues are critical for pH sensing and provide a more thorough knowledge‐base for understanding GPCR pH sensing. We anticipate that the outcome of these studies will enable us to more comprehensively quantify the extent of pH sensing across the GPCRome and rationally engineer new pH‐sensing GPCRs for use in industry, synthetic biology, and medicine.

Support or Funding Information

R35GM119518R01NS103906R03TR002908
The influence of high fat, high cholesterol diet on expression of myocardial remodelling biomarkers in the mouse model of atherosclerosis ApoE−/−
Authors: Kowara Michał Krzysztof , Czarzasta Katarzyna , Jędrzejewski Michał , Koperski Łukasz , Segiet Agnieszka , Cudnoch-Jędrzejewska Agnieszka Barbara
Published: April 2020

Introduction

Despite the significant development of cardiology, little is still known about the molecular basis of myocardial remodelling under conditions of high fat, high cholesterol diet, which is known to be proatherogenic.

Purpose

The aim of the project was to investigate whether a diet with high amount of fat and cholesterol contributes to changes in the expression of proteins regulating the structure of extracellular matrix in the heart muscle.

Methods

The study was conducted upon 20 ApoE−/− mice (Charles Rivers Laboratories, Inc.), which from the 10th week of age for the next 14 weeks were fed a standard diet (5% fat, 19.5% casein, Animalab) (n = 10 – control group) or high fat, high cholesterol diet (21% fat, 0,15% cholesterol, 19.5% casein; Animalab) (n = 10 – experimental group). At the end of the experiment, mice were euthanized to collect the heart for immunohistochemical analysis. In myocardium, levels of matrix metalloproteinase 1 (MMP‐1), matrix metalloproteinase 2 (MMP‐2), matrix metalloproteinase 9 (MMP‐9) and pro‐inflammatory cytokines ‐ interleukin 1β (IL‐1β), interleukin 6 (IL‐6) and interleukin 17 (IL‐17) were determined. In addition, the levels of type I collagen, type III collagen and tissue metalloproteinase inhibitor type 1 (TIMP‐1) were also examined in the myocardium. Consent was obtained from The Local Animal Research Ethics Committee (20/2015).

Results

ApoE−/− mice in the experimental group had significantly higher body weight compared to the ApoE−/− mice in the control group. ApoE−/− mice in the experimental group showed significantly higher levels of MMP‐9 in myocardium compared to control mice. In contrast, TIMP‐1 expression was significantly higher in the control group compared to the experimental group. The high fat, high cholesterol diet also contributed to significantly higher expression of type I collagen and type III collagen in myocardium in the ApoE−/− mice. We also showed that the mice in the experimental group had significantly lower myocardial IL‐6 levels compared to the mice in the control group.

Conclusion

High fat, high cholesterol diet significantly affected the level of proteins regulating extracellular matrix structure in the ApoE−/− mice. It suggests, that the diet influences myocardial remodeling. This subject is worth of further investigations due to potential clinical implications.

Support or Funding Information

This research was supported by the Medical University of Warsaw, Young Researcher’s Grant (1MA/PM1/17/17).
High‐precision and Scalable Fabrication of Biomimetic Culture Environments for Replicating the Extracellular Matrix In Vitro
Authors: Geisse Nicholas A. , Fisher Elliot C.F. , Gray Kevin , Ghazizadeh Hamed , Smith Alec S.T. , Kim Deok-Ho
Published: April 2020

In vivo, cell and tissue organization is driven by a complex interplay of cells and their environment, including the extracellular matrix (ECM). The ECM can provide varied cues such as structure, protein content, mechanical stiffness, stretch, and electrical stimulation to influence cell and tissue development and function. Traditional in vitro cell culture environments—typically composed of non‐dynamic hard and unstructured glass or plastic—fail to fulfill the roles of the ECM in development. Consequently, many in vitro cell models fall short in correctly reproducing critical in vivo phenotypes as evidenced when cultured cells lose type‐specific characteristics or express phenotypes indicative of an immature developmental stage. Considerable effort is directed at fabricating biomimetic culture environments that maintain or promote mature phenotypes. However, making biomimetic substrates typically involves costly or hard‐to‐reproduce techniques that are often incompatible with many standard assays. These challenges are further compounded when using high‐throughput assays. In this study, our objective is to develop a host of techniques for recapitulating certain aspects of the ECM in vitro. To replicate the shape and stiffness of the extracellular niche, we first developed novel nanopatterning techniques based on high‐precision photolithography methods that are highly reproducible, scalable, and amenable to integration with most industry‐standard endpoint assays, including high‐NA microscopy. Our data demonstrate that a variety of cell types‐‐including cardiac, skeletal, endothelial, neuronal, and cancer‐‐can cue off of patterns that mimic the fibrous structure of the native ECM. We then extended this biomimetic patterning technique to deformable elastomeres which permitted the addition of mechanical strain to recapitulate mechanical signals present in the ECM of specific tissues. Our data show that mechanical strain can orient skeletal muscle cells and tissues along the axis of stretch, resulting in a polarized orientation that is similar to what is seen in vivo but is not recapitulated in traditional culture environments. Last, we demonstrate that a combination of ECM patterning, mechanical stretch, and electrical pacing can reproduce the extracellular environment of the intact myocardium to facilitate studies of induced pluripotent stem cell‐derived cardiomyocytes. We conclude that our approach is a viable method for recreating specific aspects of the ECM that are critical for driving the development and maturation of cells in culture in a cell, instrument, and assay‐agnostic manner.

Support or Funding Information

National Institutes of Health Small Business Innovation Research Grant.
What influences research medical students?
Authors: Elizondo-Omaña Rodrigo Enrique , Muñoz-Leija Milton Alberto , Quiroga-Garza Alejandro , Salinas-Alvarez Yolanda , Gutierrez-de la O Jorge , Zarate-Garza Pablo Patricio , Martinez-Garza Javier Humberto , Guzman-Lopez Santos
Published: April 2020

Introduction

There are several influencing factors that aid in promoting student involvement in scientific research. Due to the steady decrease of medical researchers over time, identifying these factors would provide valuable data regarding research productivity and attitudes among medical students. Our objective is to determine the influencing factors in undergraduate students who were already interested in research.

Materials and methods

A cross‐sectional study was performed, applying a survey validated by the Delphi method on students involved in the anatomy research group established in our University. Our school has a 6‐year program, and the group was established 17 years ago. Informed consent was obtained from all participants. The survey was voluntary and anonymous,

Results

A total of 85.5% of the 90 surveyed students answered with a mean age of 20.5±2 years. Most students still belonging to their 2nd year, and just starting to get involved, without an active research project. Most had as a goal to contribute new knowledge (45.5%) as well as to the scientific community (42.9%). Their primary limitation is the lack of time. More than half of the students mentioned a professor/doctor was their primary motivation to get involved in research, and half also mentioned interest due to a peer already involved. Only 1.3% have considered a full career in research.

Conclusion

Professors are the primary source of motivation for student recruitment, preferably those that lead by example. These same students who get involved in research will also play an important part in leading by example their younger peers. Universities should focus on these two variables to resolve the rapid decrease or physician‐researchers, as well as nourishing the mentor‐mentee relationship.