bluuebunny/crossref_metadata_2025 huggingface dataset:
A Cadaveric Analysis of Morphological Variations of the Anterior Belly of the Digastric Muscle
Authors: Anderson Hana , Tucker Richard P.
Published: April 2020

The digastric is a suprahyoid muscle characterized by two distinct muscle bellies, anterior and posterior, linked by an intermediate tendon. The anterior belly of the digastric muscle (ABDM) displays a wide array of anatomical variations and there is rich literature documenting its variations, going back as early as the 18th century. A wide range of prevalence of ABDM variations is found in the literature, ranging from as low as ~5% to as high as ~70% of the examined cadavers in each study. Morphologies of ABDM variations are also fairly complex, including fusion of the right and left ABDM, addition of a unilateral or bilateral accessory muscle, and composites of multiple accessory muscles. Reflecting the common embryological origin in the first pharyngeal arch and shared innervation of the mandibular branch of the trigeminal nerve, ABDM variations frequently also involve the mylohyoid muscle.

In the current study, we sought to improve our understanding of ABDM variations in the ethnically diverse whole‐body donor population in Northern California. Specific aims were: (1) To determine the frequency of ABDM variations in cadavers donated to the UC Davis Body Donation Program, (2) To classify the observed ABDM variations in this population using the nomenclatures proposed by Zlabek (1933) and Yamada (1935), and (3) To investigate the innervation and arterial supply to ABDM accessory muscles.

Forty‐eight cadavers (male 23, female 25, average age 75) were examined over a two‐year period and ABDM variations were identified in 15 donors (31.2%). These variations were classified as: (1) Atavistic (Atavique) type – 2 cadavers, (2) Origin (Antérieur) type – 5 cadavers, (3) Insertion (Postérieur) type – 5 cadavers, (4) Mixed (Mixte) type – 1 cadaver, and (5) Composite (Composé) type – 2 cadavers. Innervation and arterial supply were determined for one of the atavistic types and one of the origin types. It was found that innervation was from the nerve to mylohyoid and arterial supply was from the submental artery originating from the facial artery, which fit to the typical innervation and blood supply to the ABDM.

In summary, on average we found ABDM variations in 1 in 3 individuals in the local Northern California population. Knowledge of a high prevalence of ABDM variations in the general population as reported here would be valuable information when an operation or examination is performed in the submental region.
Protease Activities of Proteins Secreted by Fusarium sp. in Aerobic and Anaerobic Conditions
Authors: Mercado Benjamin , Bayman Paul
Published: April 2020

A broad spectrum of infections has been associated with opportunistic pathogens in the genus Fusarium, ranging from the superficially and locally invasive to disseminated infections. Proteases secreted by filamentous fungi like Fusarium help the pathogen colonize the mucosa and penetrate tissues during dissemination, causing disease. When Fusarium infections are not treated correctly, the host can develop an abscess, necrosis or ischemia resulting in severe damage to the infected tissue or even death. Fungal infection may occur in low oxygen in the lungs and there are study cases of immunocompromised patient dying after a disseminated fungal infection developing necrosis and abscess. There are no studies on growth of Fusarium in infected tissues with low oxygen. The main aim of our study is to determine and compare the activities of proteases secreted by Fusarium in aerobic and anaerobic conditions. A strain of Fusarium was grown in media culture enriched with soybean grains for 3 weeks in aerobics and anaerobic conditions. Extracts from cultures were filtered and tested at pH ranging from 8–11 at 40° 50° 60° and 70° C to determine protease activity against azocasein as measured by absorbance at 436 nm. Results showed higher activity of proteases at 70° C for enzymes secreted in both aerobic and anaerobic conditions. Protease activity was highest at pH 9 for enzymes secreted in aerobic conditions but was highest at pH 10 for enzymes secreted in anaerobic conditions. These results confirm the secretion of proteases by filamentous fungi in anaerobic conditions. The differences in pH maxima could suggest that a different protein profile is secreted under low oxygen availability. Also, the secretion of proteases in anaerobic conditions by Fusarium could suggest an active role in the process of tissue damage during anaerobic infections.

Support or Funding Information

G‐RISE program: 5R25GM061151‐18.
Studying KLF6 and PEPD‐gene Promoter by Chromatin Immunoprecipitation Assay
Authors: Lanaghan Zeljka Miletic , Eni-Aganga Ireti , Balasubramaniam Muthukumar , Dash Chandravanu , Pandhare Jui
Published: April 2020

Prolidase (PEPD) is a ubiquitously expressed cytosolic dipeptidase that has the unique ability to cleave proline or hydroxyproline on the C‐terminus of imidodipeptides and imidotripeptides. The reaction is vital for collagen degradation. Since collagen is the most abundant protein in the body, our objective is to get a better understanding of the enzymes involved in its turnover is crucial. In silico analysis on prolidase promoter showed that putative transcription factor of interest is KLF6. To test the hypothesis that the expression of PEPD gene may be regulated by the binding of the transcription factor KLF6 to candidate binding sites we used chromatin immunoprecipitation (ChIP) assay. HEK293T cells were transfected with FLAG‐hKLF6 plasmid and the cellular lysates were obtained to measure the KLF6 protein expression by immunoblotting. After that, purified DNA from the ChIP assays were used in end‐point PCR with primers targeting candidate KLF6‐binding sites in PEPD promoter. The primers used were expected to yield a 615 bp amplicon. PCR products were resolved on 1% agarose/TAE gel. The second round of PCR was performed in which endpoint PCR products from the first round of PCR were used as a template for nested PCR with primers designed to amplify 164 bp. Nevertheless, our results showed no detectable PEPD promoter‐specific signals in the PCR analysis of the ChIP products. However, due to the inherent caveats in the experimental design, choice of reagents, and analysis method, it would be premature to conclude at this time point that PEPD expression is not regulated by KLF6. Accordingly, the design of future experiments will effectively address the limitations of this current work and will help reveal the identity of transcription factors regulating PEPD expression.

Support or Funding Information

Supported by National Institutes of Health (NIH) Grants DA037779 and MD007586 (to Jui Pandhare) and DA024558, DA30896, DA033892, DA021471, AI22960 and MD007586 (to Chandravanu Dash). Ireti Eni‐Aganga is supported by RISE Grant. The work is also supported by the RCMI Grant G12MD007586, the Vanderbilt CTSA Grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 Grant MD007593 from NIMHD/NIH, and the Tennessee Center for AIDS Research (P30 AI110527).
Kv3 channels Modulate Epithelial Mesenchymal Transition through Akt pathway
Authors: Sim Hun Ju , Lee So Yeong
Published: April 2020

Voltage‐gated potassium (Kv) channels, especially Kv3.1 and Kv3.4, have been suggested that they are closely related to cell migration and invasion. This process is associated with epithelial mesenchymal transition (EMT) which is important to cancer metastasis.

In the present study, we used HeLa and canine breast cancer cells, CHMp, to investigate the involvement of Kv3 channels in the EMT processes. We found that blood depressing substance‐II (BDS), which is a specific Kv3 subfamily blocker, decreased the protein expressions of vimentin and N‐cadherin, mesenchymal cell markers. In addition, BDS inhibited cancer cell migration. Blocking Kv3 subfamily by BDS down‐regulated of phospho‐Akt S473 and up‐regulated of PTEN. These results suggest that blockade of Kv3 channels using BDS induced reversed EMT through Akt pathway in HeLa and CHMp cells. Further study is needed to explain the detailed mechanisms of controlling the relationship between Kv3 channels and EMT.

Support or Funding Information

This research was supported by the Bio and Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF‐2016M3A9B6026771).
In‐frame Genetic Disruption of SPECC1L Microtubule‐Interaction Domain Causes Embryonic Tissue Movement and Fusion Defects
Authors: Saadi Irfan , Wenger Luke , Hall Everett , Goering Jeremy , Isai Dona Greta , Umar Zaid , Moedritzer Michael , Paroya Shahnawaz , Czirok Andras
Published: April 2020

Movement and fusion events are critical during embryonic development; defects in these events lead to common birth anomalies. For example, failure of neural tube, secondary palate and ventral body wall closure leads to exencephaly, cleft palate, and omphalocele, respectively. Autosomal‐dominant SPECC1L mutations have been identified in patients with syndromic malformations, including hypertelorism, cleft palate and omphalocele. These mutations cluster in the second coiled‐coil (CCD2) and calponin homology (CHD) domains, which interact with microtubule and actin, respectively. To study SPECC1L function in mice, we first generated genomic deletions that resulted in out‐of‐frame truncations. Homozygous mutants for these truncations, including those lacking CCD2 and CHD, died shortly after birth without cleft palate or omphalocele. We then generated a series of targeted in‐frame deletions in CCD2, homozygotes for which also exhibited perinatal lethality but now with ~50% exencephaly, ~50% cleft palate, and ~50% omphalocele. While the omphalocele phenotype segregated equally with the other two phenotypes, exencephaly and cleft palate were never observed in the same embryo, suggesting antagonistic tissue mechanics between neural tube and palate closure. Consistently, we found that the oral cavity was narrower in mutant embryos with exencephaly, allowing for the palatal shelves to close eventually. We have shown previously that SPECC1L deficiency leads to impaired cell speed and directionality. We now show that subcellular SPECC1L‐ΔCCD2 protein distribution was abnormal, accumulating asymmetrically and associating almost exclusively with filamentous actin. Thus, loss of SPECC1L interaction with microtubules – while maintaining actin association – was more detrimental to cell and tissue movement than loss of SPECC1L protein entirely. Taken together, our results showed that in‐frame perturbations of CCD2 in SPECC1L protein specifically affect embryonic closure of several tissues, suggesting that human SPECC1L CCD2 missense mutations are gain‐of‐function. Our data also revealed a novel role for SPECC1L cross‐linking of actin and microtubule cytoskeletons in embryonic tissue movement and fusion events.

Support or Funding Information

This project was supported in part by the National Institutes of Health grants DE026172 (I.S.). Core support and pilot funding was also provided by National Institutes of Health Center of Biomedical Research Excellence (COBRE) grant (P20 GM104936), Kansas IDeA Network for Biomedical Research Excellence (INBRE) grant (P20 GM103418), and Kansas Intellectual and Developmental Disabilities Research Center (IDDRC) grant (P30 HD 002528).
Over‐expression of BICD1 gene as a marker for clinically predicting Temozolomide resistance in Glioblastomas
Authors: Huang Shang-Pen , Chan Yung-Chieh , Chang Yu-Chan , Hsiao Michael
Published: April 2020

Glioblastoma multiforme (GBM) is a highly malignant tumor with a median survival of only 14 months, for which surgical resection, radiotherapy, and chemotherapy are the major clinical treatments. Temozolomide (TMZ) is currently the only FDA approved effective chemotherapy drug for treatment of GBM; however, cancer cells often develop drug resistance after TMZ treatment and eventually lead to treatment failure. Here, we demonstrated that Bicaudal D Homolog 1 (BICD1) gene would be a potential biomarker for predicting TMZ resistance in GBM patients. In our study, we found that T98G GBM cell is highly resistant to TMZ (IC50>200μM), while U87 GBM cell is highly sensitive to TMZ (IC50<100μM) by MTT assay. After treated with TMZ, gene expression of BICD1 was significantly upregulated in T98G cells and downregulated in U87. The available clinical datasets of the Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) indicated that over‐expression of BICD1 correlates with a poor outcome of GBM patients. Moreover, the TMZ sensitivity was recruited in T98G and SF126 GBM cells after their BICD1 genes were knocked down. Thus, over‐expression of BICD1 plays an important role in TMZ resistance when treating GBM.
Sex‐differences in AMPK activity and kidney function parameters post uninephrectomy
Authors: Saitta Biagio , Li Hui , Pham Jessica , Mancino Valeria , Sepehr Saman , Hallows Kenneth R. , Pastor-Soler Nuria M.
Published: April 2020

Background

Living donor (LD) kidney transplant is the preferred therapy for end‐stage kidney disease. LDs have generally excellent outcomes, yet in the first year post‐uninephrectomy (UNX) there is an increased risk of hypertension and microalbuminuria. In some chronic kidney disease (CKD) models activity of the metabolic sensor AMP‐activated kinase (AMPK) is lower compared to healthy kidneys. We hypothesized that low AMPK activity has a detrimental effect on kidney function post‐UNX when combined with a high‐Na diet in both male and female mice. Our findings could lead to protective interventions for LDs with AMPK activation.

Methods

We used adult male and female mice with double‐floxed AMPK alpha subunits (AMPKfl) and ± tamoxifen‐driven (Tam) expression of CAG‐Cre recombinase (Cre+ vs. Cre‐AMPKfl). Mice underwent UNX 5–6 wks post‐Tam (vs. vehicle corn oil injection). We also performed control Sham surgeries. Post surgery (Sham or UNX), mice were placed on a high‐Na (HNa) diet. Euthanasia was performed at 22 weeks. We measured GFR, urine albumin and plasma complete blood count electrolytes pre‐ and post intervention. Explant and remnant kidneys were examined by immunoblot and qPCR.

Results

Cre+AMPKfl males and females kidneys had significant AMPK knockdown (KD) compared to Cre‐AMPKfl mice after Tam. There was a significant decrease in AMPK protein expression in mice receiving Tam compared to vehicle. AMPK‐KD females had significant albuminuria compared to AMPK‐sufficient females, although we did not detect such change in males. In both males and females, AMPK‐KD mice was associated with worse anemia as compared with AMPK‐sufficient mice. Interestingly, GFR increased after UNX+HNa in AMPK‐sufficient females, yet this increase was not evident in the AMPK‐KD female mice. GFR changes were not observed in males. In male mice, UNX+HNa had a detrimental effect on BUN that was independent of AMPK status.

Conclusions

AMPK KD in female mice worsens kidney injury in a model of kidney donation. Male mice did not achieve the same degree of AMPK KD as females, which may explain the lack of AMPK dependence of kidney injury post‐UNX+HNa in males. These findings implicate AMPK as an important target for potential pharmacological interventions to prevent CKD in men and women LDs.

Support or Funding Information

Keck School of Medicine of USC Dean’s Pilot. University Kidney Research Organization (UKRO).
Mechanosensitive Smad5 Enhances MiR‐487a Processing to Regulate Vascular Endothelial Cell Proliferation in Response to Disturbed Flow
Authors: Chiu Jeng-Jiann , Wang Wei-Li , Wei Shu-Yi , Shih Yu-Tung
Published: April 2020

MicroRNAs (miRs) and bone morphogenetic protein receptor‐specific Smads are mechano‐responsive molecules that play vital roles in modulating endothelial cell (EC) functions in the face of blood flow. However, the roles of interplay between these molecules in modulating EC functions under different flows remain unclear. We elucidated the roles of interplay between miRs and Smad5 in modulating EC proliferation in response to different flows. Microarray and quantitative RT‐PCR showed that disturbed flow with oscillatory shear stress (OS, 0.5±4 dynes/cm2) up‐regulates EC miR‐487a in comparison to static controls and pulsatile shear stress (12±4 dynes/cm2). MiR‐487a was up‐regulated in ECs in the inner curvature (OS region) of rat aortic arch and diseased human coronary arteries. MiR‐487a expression was promoted by nuclear phospho‐Smad5, which bound to primary‐miR‐487a to facilitate miR‐487a processing. Algorithm prediction, luciferase reporter, and argonaute 2‐immunoprecipitation assays showed that miR‐487a binds to 3’UTR of CREB binding protein (CBP) and p53. Knockdown or overexpression of miR‐487a decreased and increased, respectively, phospho‐Rb and cyclin A expressions through CBP and p53. BrdU incorporation assay showed that miR‐487a enhances EC proliferation under OSS in vitro and in disturbed flow regions of experimentally‐stenosed rat abdominal aorta in vivo. In summary, disturbed flow with OSS induces EC expression of miR‐487a through its enhanced processing by activated‐Smad5. MiR‐487 inhibits its direct targets CBP and p53 to induce EC cycle progression and proliferation. Our findings suggest that EC miR‐487 is an important molecular target for intervention against disturbed flow‐associated vascular diseases resulting from atherosclerosis.
Inter‐ventricular difference in the excitation‐contraction coupling and myofilament proteins of rat cardiomyocytes
Authors: Jeon Young Keul , Kwon Jae Won , Jang Ji Hyun , Yin Ming Zhe , Zhang Yin Hua , Kim Sung Joon
Published: April 2020

The left (LV) and right ventricle (RV) have distinctive structural and functional characteristics; thinner free wall and different kinetics of contraction‐relaxation in RV. However, the precise comparison of the excitation‐contraction (E‐C) coupling mechanisms and the Ca2+ homeostasis in the isolated cardiomyocytes are lacking, especially in rats. Whole‐cell patch clamp study showed significantly shorter action potential duration (APD) in RV myocytes. Consistently, the amplitude of transient outward K+ current (Ito) was higher in RV, whereas the amplitude of L‐type Ca2+ current was not different. However, when measuring the [Ca2+]i and sarcomere length (SL) of intact myocytes by using IonOptics, the Ca2+ transient amplitudes and shapes were not significantly different. Nevertheless, the accompanied shortening of SL was smaller and the speed of relaxation appeared slower in RV than LV myocytes. Interestingly, the immunoblot analysis revealed lower expression of cardiac troponin complex proteins (cTnC, cTnI, cTnT) and nebulette in RV myocytes. The lower level of Ca2+‐binding cTnC, i.e. smaller Ca2+‐buffering capacity, might be responsible for, at least partly, the similar Ca2+ transient despite the shorter APD in RV myocytes. Introduction of the higher Ito and lower cTn into the mathematical computer simulation of rat ventricular myocytes showed changes similar with the experimental data of Ca2+ transient and of contraction from the RV myocytes. Taken together, in addition to the comprehensive physiological analysis, we firstly show the lower expression of cTn proteins in the RV myocytes, which gives a clue to explain the inter‐ventricular difference in the E‐C coupling kinetics.
Hormonal Regulation of Novel Glycogen Metabolic Proteins Hari Priya Vemana, Ehab M. Abo‐Ali, John L. Croft, Shraddha Bhutkar, Michael Woolford, Vikas V. DukhandeDepartment of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, NY, USA
Authors: Vemana Hari Priya , Abo-Ali Ehab M. , Croft John L. , Bhutkar Shradda , Woolford Michael , Dukhande Vikas V.
Published: April 2020

Lafora disease (LD) is a rare autosomal recessive disorder manifesting as a progressive myoclonic epilepsy. LD is characterized by the formation of Lafora bodies, insoluble deposits containing abnormal glycogen‐like material in many tissues. LD is caused by mutations in the genes encoding dual‐specificity phosphatase laforin (EPM2A) or the E3 ubiquitin ligase malin (EPM2B / NHLRC1). Several research studies on LD have shown that laforin functions as a phosphatase for glycogen. Malin forms a functional complex with laforin and either malin alone or the laforin‐malin complex is known to ubiquitinate multiple proteins involved in glycogen metabolism. It is well established that hormones such as insulin regulates glycogen metabolism but hormonal regulation of laforin and malin is not completely elucidated. In type‐2 diabetes, defects in insulin responsiveness leads to a state called insulin resistance. Our objective is to study the effect of hormones and nutrients on novel glycogen metabolic proteins laforin and malin in physiology and diabetes pathophysiology. We used liver cells in culture and a rodent model of diabetes using high‐fat diet and low dose streptozotocin (HF‐STZ). We measured gene expression, protein expression, protein posttranslational modifications, and functional activities of laforin and malin. Our preliminary data indicates that both diabetic conditions and insulin modulate the expression of LD proteins. Our ongoing studies are directed towards delineating the molecular mechanisms of these alterations. This study can serve to find novel potential targets for the pharmacotherapy of type‐2 diabetes.

Support or Funding Information

This study is supported by an award from National Institute of General Medical Sciences of the National Institute of Health, Award number: SC2GM125550.
Effects of Social Defeat on Hippocampal CA1 Neuronal and Sympathetic Nerve Activities in Conscious Rats
Authors: Yaguchi Kana , Ikegame Shizuka , Yoshimoto Misa , Miki Kenju
Published: April 2020

Social defeat stress has been linked to depression, autonomic disorders, and cardiovascular disorders. The hippocampus has been implicated in the pathogenesis of depression, and hippocampal neuronal activity is presumed to be involved in autonomic disorders induced by social defeat stress. Potential linkages among hippocampal neuronal activities, sympathetic nerve activities, and cardiovascular function have received less attention. In the present study, we measured time courses of changes in hippocampal CA1 neuronal activity (CA1NA), renal sympathetic neuronal activity (RSNA), and lumbar sympathetic nerve activity (LSNA), arterial pressure (AP), and heart rate (HR) in response to social defeat stress in rats. Wistar rats were chronically implanted with electrodes for the measurement of CA1NA, RSNA, LSNA; electroencephalography, cervical electromyography, and diaphragm electromyography were performed, and a catheter was used to measure AP. Social defeat was induced by introduction of a Long–Evans rat, which was heavier and more aggressive than a Wistar rat, into the Wistar rat’s home cage for 30 minutes. The intrusion of the Long–Evans rat sometimes resulted in fighting between the Long–Evans and Wistar rats. Following defeat, Wistar rats displayed a submissive posture and exhibited immobilization for several minutes. Fighting caused immediate reduction of CA1NA, which remained suppressed during the period of submissive posture and immobilized behavior. However, the AP, HR, RSNA, and LSNA increased in transient fashion during immobilized behavior. These findings showed that the defeat stress considerably influenced CA1NA, whereas it marginally influenced RSNA, LSNA, AP, and HR. Therefore, it is likely that social defeat stress suppressed hippocampal function in tonic fashion, which could not be measured on the basis of sympathetic nerve activity and cardiovascular function.

Support or Funding Information

JSPS Grant
Isolating Group A Streptococcus Infected Phagosomes
Authors: Young Kyrlia , Johnson Mitchell , Yuen Isabelle , Okumura Cheryl Y.M.
Published: April 2020

The number of Group A Streptococcus (GAS) infections has dramatically risen in the last several decades and is an increasing public health concern worldwide. Normally, the body’s innate immune system responds to pathogens by phagocytosing and degrading the bacteria within the phagolysosome by a number of different molecular processes, including the oxidative burst response and lysosomal acidification. However, it has been shown by our lab and others that GAS has a number of different mechanisms to subvert immune defenses, including evasion of digestion in the phagolysosome. Because it is unusual for pathogens to reside in phagolysosomes, we want to develop a proteomic profile of GAS‐infected phagosomes. Because isolation of phagosomes is technically difficult, we set out to develop a method to isolate and purify GAS‐infected phagosomes using magnetic particles. We coupled magnetic particles to live GAS and demonstrated that we can effectively isolate viable coupled bacteria. Following infection of THP‐1 macrophages, we employ a magnetic separation protocol that yields a high concentration of GAS‐infected phagosomes largely absent of other organellar contamination. With these magnetically‐isolated GAS phagolysosomes, we aim to conduct a proteomics survey using mass spectrometry to characterize GAS phagosome modification to elucidate its mechanisms of survival. Our work will help identify novel therapeutic targets and strategies to treat GAS infections to enhance normal immune processes aimed at combating GAS infections, and thus limiting the use of antibiotics.

Support or Funding Information

American Heart Association (17GRNT3410851), Occidental College Undergraduate Research Center
Roles of nNOS and eNOS in rat heart ; comparison between the left and right ventricle myocytes
Authors: Kwon Jaewon
Published: April 2020

Right ventricle (RV) has physiological as well as anatomical differences compared with left ventricle (LV). There are constitutive NO synthase isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS) in myocardium, regulating the calcium dynamics and contractility. Here we compared the roles of nNOS and eNOS in the RV and LV myocytes from S‐D rats. After isolating the myocytes using Langendorff perfusion system, their sarcomere shortening and [Ca2+]i were simultaneously measured using the Ionoptix system while paced by 2 Hz field stimulation. nNOS‐specific inhibitor, S‐methyl‐L‐thiocitrulline (SMTC) and the non‐specific NOS inhibitor, L‐NG‐Nitroarginine methyl ester (L‐NAME) were used to pharmacologically dissect the roles of NOS isoforms.

According to the immunoblot assay, the protein amounts of nNOS and its phosphorylated form were not different between RV and LV. Although the amount of eNOS was lower in RV than LV, its phosphorylated form was similar. RV myocytes showed slower contraction (longer TPeak) and slower relaxation (longer Trelax) than LV myocytes. The treatment with SMTC or L‐NAME shortened TPeak and Trelax of RV myocytes, abolishing the kinetic differences of ΔSL. It was notable that the Trelax of LV myocytes became slightly longer by SMTC. Paradoxically, the length of sarcomere shortening (ΔSL) was increased while the amplitude of calcium transient (Δ[Ca2+]i) was decreased by SMTC in RV myocytes. However, SMTC did not affect the ΔSL and Δ[Ca2+]i of LV myocytes. Our study suggest that the slower contraction and relaxation of RV myocytes might be partly due to differential responses to the nNOS‐dependent intracellular signaling. The opposite direction of changes in the amplitudes of ΔSL and Δ[Ca2+]i by SMTC suggest that the nNOS in RV myocytes is responsible for negative inotropy despite the positive effects on the calcium transient, which is not evident in LV myocytes.
Afferent Nerve Fibers of the Skin ‐ Influence of salt intake
Authors: Ditting Tilmann , Rodionova Kristina , Reeh Peter , Eberhard Miriam , Veelken Roland Albert
Published: April 2020

Sodium can be accumulated without commensurate water retention in the skin (non‐osmotic sodium storage). Macrophages play a pivotal role in this context and their depletion can induce salt‐sensitive hypertension (shin). On the other hand renal peptidergic nerves are also involved the development of ssHTN. Since the skin is densely innervated by peptidergic sensory nerves we hypothesized that high salt diet might enhance the release of neuropeptides from these nerve fibers.

In a cross‐over design, two groups of rats (n=4, each) were fed either low salt diet (LS; 0.2%) with free access to tab water for 14 days or high salt diet (HS; 8%) with free access to 0.9% saline as drinking water. After 14 days a skin sample (3×3mm) of the groin area was excised, and the diet was switched for another 14 days. Then a contralateral skin sample was taken. Tissue analyzed in an organ‐bath and CGRP content in the supernatant was measured with ELISA. After two baseline measurements within 5 min, the tissue was superfused with hypertonic saline (4.5%) for 5 min, and three further samples of the supernatant were taken every 5 min.

Baseline CGRP release was similar with both diets (LS 11.9±1.5 vs HS 13.6±1.5ng/g skin). Maximum release was higher with HS diet (LS 17.5±1.8 vs HS 29.8±1.3ng/g skin; P<0.05). After diet switch the results were similar: baseline LS 9.4±1.2 vs HS 10.1±, however, in rats that were on HS diet before max release tended to be higher with LS diet, Nevertheless with HS diet the release was higher again (LS 19.7±2.1 vs HS 29.3±1.7; P<0.05).

High sodium diet sensitizes neuropeptide release from peptidergic sensory nerves in the skin. Hence peptidergic afferent nerves might be an integrated body‐wide system involved in sodium handling in very different target areas like skin and kidney. Putative peptidergic mechanisms (vasoregulation, chemotaxis) remain to be determined.
Comparison of Anti‐diabetic Activities of Selected Phenolic Acids in Relation to Antioxidant Activities
Authors: Yu Myeongnam , Lee Hana , Kim Hyun-Joo , Yang Jinwoo , Lee Junsoo
Published: April 2020

Dietary phenolic acids are antioxidants with diverse physiological functions that are beneficial for human health. Phenolic acids show anti‐mutagenesis and anti‐microbial activities and play a role in immune regulation in humans. The objective of this research work was to compare antioxidant and anti‐diabetic activities of selected 11 phenolic acids. 1,1‐Diphenyl‐2‐picrylhydrazyl (DPPH), 2,2‐azino‐bis‐(3‐ethylbenzothiazoline‐6‐sulphonic acid) (ABTS) radical scavenging capacities, reducing power, α‐glucosidase inhibitory activity and glucose uptake in HepG2 cells were evaluated. The relation of antioxidant activities with anti‐diabetic activities was also investigated. Gallic acid showed significantly higher on DPPH and ABTS radical scavenging activities and reducing power followed by caffeic acid and sinapic acid. Caffeic acid among samples showed the highest α‐glucosidase inhibitory activity followed by gallic acid and coumaric acid. Glucose uptake was the highest in gallic acid and coumaric acid. Compared to the positive control (metformin), all the phenolic acids showed equivalent or more potent effects on the glucose uptake in HepG2 cells. However, low relationship was found between antioxidant capacity and anti‐diabetic activity. These results suggested that phenolic acids can be used as functional food ingredients or blood glucose regulators.
Defective membrane insertion of mutant Na,K‐ATPase, a cause of fatal epilepsy
Authors: Howard Rebecca , Ygberg Sofia , Andersson Magnus , Akkuratov Evgeny , Jans Daniel , Brismar Hjalmar , Lindstrand Anna , Aperia Anita
Published: April 2020

Given its critical role in establishing ionic gradients in neurons and other excitable cells, functional disruption of the Na/K‐ATPase is expected to substantially compromise neurological homeostasis. However, to date only a few examples of spontaneous Na/K‐ATPase mutations have been identified in human patients. Here we describe a de novo Trp‐Arg mutation in the ATP1A1 gene, encoding the Na/K‐ATPase catalytic α1 subunit, in an infant who died at 10 months from therapy‐resistant epilepsy. Located in a conserved transmembrane helical region of the α1 subunit, the mutation was associated primarily with neurological symptoms and a low capacity to retain magnesium. Confocal imaging of the mutant Na/K‐ATPase α1 subunit expressed in rat hippocampal neurons revealed decreased but detectable membrane expression. Expressed in Xenopus oocytes, the mutant Na/K‐ATPase catalytic α1 subunit was associated with nonselective, ouabain‐resistant leak currents, consistent with a sizable, continuous hydration defect in the transmembrane domain. Indeed, molecular dynamics simulations in both POPC and mixed‐lipid bilayers showed accumulation of water at the protein periphery, particularly in the vicinity of crystallographic Na‐binding sites. These results provide evidence, from clinical diagnosis to atomistic simulation, for a mechanism of ion‐gradient disruption that could account for leak currents now observed in multiple pathological Na/K‐ATPase variants. Our finding of a pathological interaction between the mutated ion transporter and the membrane lipids has implications for development of new treatment strategy in many cases of genetic epilepsy.

Support or Funding Information

Swedish Research Council to AA and to HB
Effect of MED28 on glucose metabolism in human non‐small cell lung cancer (NSCLC) cells
Authors: Lin Yi-Chang , Cheng An-Chin , Lee Tzu-Yi , Lee Ming-Fen
Published: April 2020

Cancer cells can undergo “Warburg effect” for energy production to support cell growth by increasing glucose uptake and glycolysis. Aberrant Wnt/β‐catenin signaling in cancer may not only increase cell proliferation but also upregulate glycolytic capacity. Our laboratory has previously reported that MED28, a Mediator subunit for transcriptional activation, modulates metalloproteinase 2 (MMP2)‐dependent migration in human non‐small cell lung cancer (NSCLC) cells. We have also found that MED28 mediates Wnt/β‐catenin signaling in human colorectal cancer cells. The objective of the current study is to investigate if MED28 also plays a role in glucose metabolism in NSCLC besides MMP2‐dependent migration. Suppression of MED28 in A549 human NSCLC cells decreased the expression levels of Akt/Wnt/β‐catenin signaling‐related molecules including p‐Akt (Ser473), p‐GSK3β (Ser9), β‐catenin, and c‐Myc as shown by RNAi technology and protein immunoblotting. Meanwhile, siMED28 downregulated glycolysis by reducing the expression of relevant proteins such as glucose transporter 1 and hexokinase 2. Our data indicate a role of MED28 in regulating glucose metabolism, partially through Akt/Wnt/β‐catenin signaling, in human NSCLC cells. The underlying mechanism is currently under investigation.

Support or Funding Information

This work was supported by the grants MOST 106‐2320‐B‐039‐062‐MY3 and CMU108‐MF‐13 to M‐F Lee.
Immune Modulatory Effects of Caryophyllene Oxide in Alcohol Related Behaviors
Authors: Oppong-Damoah Aboagyewaah , Abney Sarah E. , Gannon Brenda M. , Blough Bruce E. , Uchakin Peter N. , Murnane Kevin S.
Published: April 2020

Alcohol‐use disorder (AUD) is the third leading cause of preventable death in the United States following tobacco and obesity, and current treatments for AUD yield disappointing remission rates. Chronic alcohol intake has been established to negatively impact the body’s immune system, and the endocannabinoid system has been shown to modulate the abuse‐related effects of ethanol. In this study, and we investigated the effects of 30 mg/kg caryophyllene oxide (
BCPO), in two widely accepted animal models of AUD: two‐bottle choice
ethanol consumption and ethanol‐induced conditioned place preference (CPP). Finally, we used multiplex bead‐based flow cytometry to evaluate the effect of BCPO on plasma level of cytokines following these behavioral assays. BCPO significantly decreased ethanol intake and preference without changing total fluid intake in mice that consumed high amounts of ethanol. In mice that consumed low amounts of ethanol, BCPO did not alter ethanol intake, preference, or total fluid intake. BCPO significantly attenuated the expression of an ethanol‐induced CPP.
Following the CPP assay, we observed a significant decrease in circulating levels of IFNγ, TNFα, MCP1, IL12p70, IL1b, and IL10 which was reversed by treatment with BCPO. Our findings support the current knowledge that excessive consumption of alcohol may lead to immune suppression and predispose individuals to a number of infections as well as neoplastic diseases. This study expands and supports the use of immunoregulatory molecules as a viable target for the treatment of AUD.
HlyA cause platelet activation and neutrophil/ monocyte interaction during urosepsis
Authors: Johnsen Nanna , Christensen Mette G. , Hamilton Aimi D.M. , Praetorius Helle
Published: April 2020

The urinary tract is frequently a primary infection site in patients with severe sepsis or septic shock
1. Urinary tract infections are most commonly caused by E.coli sub‐strains that produce several virulence factors
4–
8, of which α‐haemolysin (HlyA) is the most abundant
9. Our previous data demonstrate that the biological effects of HlyA are intimately associated with extracellular ATP signalling secondarily to ATP release from cells through the HlyA‐pore. Our previous data support that HlyA is largely responsible for the septic symptoms observed in response to bacteraemia with uropathogenic E.coli. ATP and its degradation products are aggressive activators of immune cells
10 and platelets
11–
13, and prime candidates for promoting the intravascular coagulation observed during septic shock.

We tested the effect of antagonising platelet P2Y receptors (P2Y12 and P2Y1) during sepsis with uropathogenic E.coli(ARD6, O6:K13:H1) in anaesthetised mice. In aggreement with the literature, infusion of the P2Y12 antagonist cangrelor (8.6 or 86mg/hour) did not change survival or any tested sepsis parameters. However, the P2Y1 inhibitor MRS2500 reduced platelet activation by pore‐forming bacterial toxins (70–80%) in vitro, measured as surface exposure of fibronectin, DC63 and P‐selectin. In vivo, infusion of MRS2500 (624μg/hour) increased the survival in mice exposed to uropathogenic E. coli. Moreover, infusion of MRS2500 partially prevented the 60% fall in platelet count otherwise seen in response to sepsis in control mice.

Interestingly, targeting of P2Y2 receptors, which is highly expressed on neutrophils and monocytes
14 but not in platelets, showed that lack of P2Y2 receptors entirely prevented the fall in circulating platelets in response to sepsis. Moreover, P2Y2 receptor‐deficient mice die much earlier in response to intravenous application of uropathogenic E. coli compared to controls. By flow cytometry, we are able to detect increased complex formation between thrombocytes (CD42) and neutrophils (Ly6‐G) /monocyte (Ly6‐G/CD11B). These data could potentially suggest that complex formation between thrombocytes and neutrophils and/or monocytes are important for the observed reduction in free thrombocyte numbers during sepsis and that the complexes could dampen the septic reaction to the circulating bacteria and improve survival.

Support or Funding Information

This project is funded by

The Independent Research Foundation ‐ Denmark

The Augustinus Foundation
Efficacy of the curcumin to ameliorate ovalbumin induced atopic dermatitis and progression of atopic march in mice
Authors: SHARMA SUKRITI , NAURA AMARJIT SINGH
Published: April 2020

Atopic dermatitis (AD) is a chronic pruritic skin inflammatory condition which arises due to skin barrier disruption and immune dysregulation. Curcumin has been widely exploited in the treatment of various skin‐related pathologies due to its pharmacologic effects. The present study was designed to investigate the beneficial effect of curcumin in the amelioration of ovalbumin (OVA)‐induced atopic dermatitis and ensuing atopic march. AD was induced in female Balb/c mice by epicutaneous application of OVA at the back skin site for 1 week followed by a resting period of two‐weeks and same protocol was repeated thrice. Curcumin treatment was given either intraperitoneally or topically in two separate groups of animals. Curcumin was administered intraperitoneally at different doses during the last sensitization phase for a period of 7 days. Topical application of curcumin at a dose of 10 mg/kg was done for a period of 14 days during the second resting period. Skin tissues were examined for histologic changes, Th2 cytokines and the oxidative stress markers. Systemic curcumin administration ameliorated the onset of AD‐like lesions and significantly improved the dermal destruction in mice upon OVA exposure. Curcumin reduced the levels of various inflammatory mediators such as Th2 promoting cytokines (TSLP and IL‐33), Th2 cytokines (IL‐4, IL‐5, IL‐13 and IL‐31) and also downregulated the activation/expression of Stat‐6 and GATA‐3. Next, when curcumin preparation was applied topically, it shows a remarkable efficacy in restoration of the histo‐architecture of the skin suggesting that the phytochemical based ointment(s) can offer a newer treatment option for AD and associated atopic march. Finally, curcumin blocked the OVA initiated progression of AD towards asthma as evident by the reduced number of BALF inflammatory cells and suppressed expression of Th2 cytokines in the lung tissue upon aerosolized exposure of the allergen to the AD mice. Overall, our findings demonstrate for the first time that curcumin has the efficacy to ameliorate AD and halt the progression of associated atopic march.

Support or Funding Information

Financial support from Department of Biotechnology, Government of India [BT/PR17968/MED/122/33/2016]; University Grants Commission, New Delhi, India [UGC‐SAP] and [UGC‐BSR‐SRF]
Nonselective Inhibition of PDE4 Induces Gastroparesis in Mice
Authors: McDonough Will , Aragon Ileana , Rich Justin , Murphy James M. , Saleh Lina Abou , Boyd Abigail , Koloteva Anna , Richter Wito
Published: April 2020

Purpose

Non/PAN‐selective inhibitors of Type 4 phosphodiesterases (PDE4s), a group of isoenzymes that hydrolyze the second messenger cAMP, exert promising therapeutic benefits, such as anti‐inflammatory as well as cognition‐ and memory‐enhancing effects. However, these drugs also induce several side effects, in particular emesis and nausea, curbing their clinical use. The PDE4 family consists of four subtypes/genes (PDE4A to D). As each plays unique and non‐overlapping roles in the body, targeting individual PDE4 subtypes is a promising approach to separate the therapeutic benefits from the side effects of current PDE4 inhibitors. To this end, this study aimed to identify the specific PDE4s involved in emesis and nausea. While mice are anatomically unable to vomit, PDE4 inhibition in mice induces gastroparesis, an abnormal retention of food in the stomach, which is a well‐known cause of nausea and vomiting in humans and was thus used as a correlate.

Methods

Gastroparesis in knock‐out mice deficient in individual PDE4 subtypes and in mice treated with PAN‐PDE4 inhibitors was compared to their respective controls. We used a long term model, in which food accumulated in the stomach of mice fed ad libitum for 3 days, as well as an acute model, in which fasted mice were given a FITC‐labeled food bolus by oral gavage, euthanized 30 min later, and FITC/food‐retention in the stomach was then measured.

Results

Treatment of wildtype mice with PAN‐PDE4 inhibitors increased stomach sizes to twice that of controls without changing their food intake or the weight of their intestines, suggesting a specific effect of PDE4 inhibition in restricting gastric emptying. Structurally distinct PDE4 inhibitors all induced gastroparesis, indicating it is a class effect. Conversely, YM976, an inhibitor which does not cross the blood‐brain‐barrier, did not induce gastroparesis suggesting that PDE4 inhibitor‐induced gastroparesis results from altered neuronal regulation, rather than from PDE4 inhibition in the stomach/periphery. Ablation of PDE4A, B, C or D in the respective KO mice did not induce, nor did it protect the animals from PAN‐PDE4 inhibitor‐induced gastroparesis, implying that it results from the concurrent inhibition of multiple PDE4s.

Conclusions

Selective ablation of individual PDE4 subtypes does not induce gastroparesis in mice. Thus, potentially, any of the four PDE4 subtypes may be targeted individually for therapeutic benefits without inducing nausea or emesis. In addition, PDE4 inhibitors that do not cross the blood‐brain barrier may also be free of these side effects.

Support or Funding Information

Supported by grants from the NIH (HL76125, HL141473, HL066299).
Serine/theonine kinase 4 is a potential biomarker for assessment of β‐catenin‐mediated colon cancer prognosis
Authors: Hsu Tai-I , Lu Pei-Jung , Hsiao Micheal
Published: April 2020

Colon cancer has highly incident rate in world, and the surgical therapy can cure early stage colorectal cancer patients. But the most difficult is to predict patients’ disease prognosis. STK4 is a one of the Hippo pathway component and was shown to have important role to control the organ size and cell to cell contact inhibition ability and was considered a potential tumor suppressor. However, little is known about the relationship between the expression of STK4 and patient survival. The aim of this study was to investigate prognostic value of STK4 in colon cancer. STK4 expression in normal and tumor tissues from 140 colon cancer patients was examined by immunohistochemistry. The relationship between STK4 expression and clinicopathological factors as well as patient survival was analyzed. Human colon cancer cell lines (H3347and DLD‐1) were used to analyze the effect of STK4 knockdown and overexpression on invasion/migration in vitro. IHC analysis showed STK4 was significantly downregulated in tumor tissues compared to their corresponding non‐tumor part (P< 0.01) and was significantly associated with tumor size (P = 0.003), distal metastasis (P = 0.03), disease recurrence and poor survival (P< 0.005). Univariate and multivariate analysis indicated loss of STK4 expression predicted significantly poor outcome. Knockdown of STK4 promoted, whereas overexpression of STK4 inhibited invasion and migration of human colon cancer cells in vitro. STK4 is a prognosis maker to predict patient survival in colorectal cancer. Loss of STK4 expressed in tissue is correlated with poor prognosis and survival.

Support or Funding Information

This research was supported by Academia Sinica [AS‐SUMMIT‐108].