bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacterial Detection via Surface‐Enhanced Raman Spectroscopy (SERS)
Authors: Sengupta Raghuvir N. , D’Apuzzo Fausto , Barcelo Steven
Published: April 2020

Surface‐Enhanced Raman Scattering (SERS) is a sensitive and label‐free detection technique that takes advantage of the large enhancements in Raman scattering of molecules adsorbed onto roughened or nanofabricated metallic surfaces. Since its discovery in the 1970s, SERS has been harnessed for sensing a wide variety of molecules, including drugs and biological molecules, and more recent work suggests that it can be a powerful method for bacterial identification. Here we provide a comprehensive review of bacterial detection via SERS, highlighting a key observation in the literature that bacteria, when deprived of nutrients, undergo a stress response that correlates with the release of biomolecules that can be detected by SERS. Such SERS spectra are consistent with sensing of purine‐based metabolites that presumably arise from rapid RNA degradation in bacteria in the absence of nutrients. Intriguingly, the SERS spectra from different bacteria appear to be unique, suggesting that this method can be used for “fingerprinting” bacteria. We subsequently describe new results to better understand bacteria SERS by employing unique SERS substrates designed at HP Inc as well as HP’s state‐of‐the‐art inkjet dispensing technologies for sample exposure. Our results provide evidence for time‐dependent release of purine metabolites by nutrient‐deprived bacteria which we suggest may be used as an additional parameter to further classify different types of bacteria. The ability to merge inkjet dispense with SERS (ID‐SERS) creates the possibility of using SERS for bacterial detection, quantification, and antibiotic susceptibility testing which, in turn, may have significant implications in healthcare and the life sciences.
Transcriptomic landscape of lenalidomide‐treated NK cells: Functional enhancement by down‐regulation of CD161
Authors: Sim JeongMin , Ahn JuWon , Park YoungJoon , Kim JongIl , Kang SuJung , Choi Seo-Hyeon , Lee SeLin , Hwang Min , Cho JooYeon , Lim Jaejoon , Oh Soo-Yeon , Kwack KyuBum
Published: April 2020

Lenalidomide is a potential immunomodulator that enhances the anti‐tumor effect of natural killer (NK) cells. However, the underlying molecular mechanism of anti‐tumor effects and long‐term activating effect on NK cells are unclear. Here, we report that lenalidomide in the long stimulation with interleukin‐2 (IL‐2) enhances the proliferation and specific anti‐tumor function of NK cells by modulating transcriptomic patterns, including downregulation of KLRB1 (CD161). In conclusion, the results suggest that lenalidomide may be more applicable in the field of cancer immunotherapy by exhibiting specific cytotoxicity ability enhancement of NK cells against cancer cells as well as long‐term activating effect on NK cells.

Support or Funding Information

The research fund was supported by Ministry of Science, Technology and Information, Republic of Korea (Grant no. 2019R1F1A1054920)
MOTS‐c inhibits high‐fat diet‐induced muscle wasting by suppressing myostatin expression via the PTEN/AKT/FOXO1 signaling pathway
Authors: Kumagai Hiroshi , Coelho Ana Raquel , Wan Junxiang , Huang Amy , Mehta Hemal H , Yen Kelvin , Zempo Hirofumi , Fuku Noriyuki , Oliveira Paulo J , Cohen Pinchas , Kim Su-Jeong
Published: April 2020

Insulin resistance is thought to contribute to the development of muscle wasting. MOTS‐c, a novel mitochondrial‐derived peptide, improves insulin resistance in skeletal muscle, but its effects on muscle wasting have yet to be explored. Here, we used a diet‐induced obese mouse model to examine the effect of MOTS‐c administration on high fat diet‐induced muscle wasting. Three weeks of MOTS‐c administration by intraperitoneal (IP) injection in high‐fat diet fed mice resulted in higher total skeletal muscle mass of soleus, gastrocnemius, and plantaris compared to its high‐fat diet fed control group. Myostatin mRNA expression levels were significantly decreased in the MOTS‐c treated group compared to the high‐fat diet fed controls in the gastrocnemius muscle. Furthermore, myostatin mRNA expression negatively correlated with the total skeletal muscle mass of soleus, gastrocnemius, and plantaris. To examine the detailed mechanism of MOTS‐c action, we injected high‐fat diet fed mice with MOTS‐c for eight weeks and detected significantly lower plasma myostatin levels than high‐fat diet fed controls, as well as mRNA expression of myostatin and atrogin‐1 in the skeletal muscle. We further examined the upstream signaling pathways of myostatin regulation in the skeletal muscle and showed that MOTS‐c treatment inhibited PTEN activity, leading to elevated AKT phosphorylation and inhibition of FOXO1 transcription activity, resulting in myostatin and atrogin‐1 suppression. These results suggest that potent MOTS‐c analogues could potentially treat conditions such as high‐fat diet‐induced skeletal muscle wasting, as well as other muscle wasting phenotypes including sarcopenia, and that circulating levels of myostatin may serve as a pharmacodynamic biomarker of MOTS‐c activity.
Treadmill exercise alleviates brain iron dyshomeostasis accelerating neuronal amyloid‐β production, neuronal cell death and cognitive impairment in transgenic mice model of Alzheimer’s disease
Authors: Choi Dong-Hun , Kwon Ki-Chun , Hwang Dong-Ju , Um Hyun-Seob , Koo Jung-Hoon , Kim Eung-Jun , Yeom Dong-Cheol , Cho Joon-Yong
Published: April 2020

Brain iron increases with age and brain iron dyshomeostasis is proving increasingly likely to be involved in the pathology of Alzheimer’s disease (AD) and Parkinson’s disease (PD), possibly via promoting oxidative damage. There is therefore an urgent need to research into improving role iron play in AD. Sustained exercise may be ways to slow the rate of overload of iron, by perhaps alleviating oxidative stress due to the Fenton reaction. Yet, the exact mechanisms of the effects of exercise on iron toxicity are not well understood. Here, we explored whether treadmill exercise impacts the AD‐related mechanism(s) of brain iron status in vivo, using APP‐C105 AD‐model mice. We observed iron‐induced disruptions of amyloid precursor protein (APP) processing, neuronal apoptotic signaling, oxidative stress, and cognitive impairment in APP‐C105 AD‐model mice. Further, brain iron dyshomeostasis associated with Aβ‐induced neuronal cell death possibly via APP misprocessing, and oxidative stress and cognitive decline was blocked by treadmill exercise, suggesting effect led to mitigated Aβ‐induced neuronal cell death and cognitive decline by promoting non‐amyloidogenic pathway possibly via enhanced furin, concomitant with inhibiting oxidative stress possibly via down‐regulating brain iron dyshomeostasis. Together, we show evidences to suggest that treadmill exercise may be ways to inhibit Aβ‐induced neuronal cell death by up‐regulating non‐amyloidogenic pathway through enhanced furin, concomitant with down‐regulating iron‐mediated oxidative stress.
Autologous Haematopoietic Stem Cell Transplantation Followed by Low‐dose Cyclophosphamide Ameliorates the Disease Course and Influences the Microglia Phenotype in Rats With Experimental Allergic Encephalomyelitis
Authors: Kowara Michał Krzysztof , Kasarełło Kaja , Seta Martyna , Sulejczak Dorota , Wrzesień Robert , Snarski Emilian , Cudnoch-Jędrzejewska Agnieszka
Published: April 2020

Experimental allergic encephalomyelitis (EAE) is the animal model of multiple sclerosis (MS), the human autoimmune disease, which leads to neurodegeneration. Immune response is developed by autoreactive lymphocytes infiltrating the CNS, but further maintained by resident immune cells, such as microglia. Microglia, until recently was recognized as pro‐inflammatory cells. Lately, it was discovered, that microglia can also differentiate into an anti‐inflammatory phenotype involved in the regression of inflammatory reaction and regenerative processes. Therapy based on the autologous haematopoietic stem cell transplantation (HSCT) was recently approved for treatment of highly‐active or progressive MS. In some regimens the additional conditioning is used after the HSCT to eliminate the autoreactive cells, which may persist after the first dose of chemotherapy.

In our experiment, we aimed to investigate the effect of HSCT preceded by high‐dose of cyclophosphamide (CP) on the disease course in rats with evoked EAE, and if the additional, low‐dose of CP following the high‐dose of CP and HSCT may improve the observed effect.

Rats with evoked EAE (day 0) were treated with (i) high‐dose (125mg/kg) CP (day +6), followed by HSCT (day +7), or (ii) high‐dose (125mg/kg) CP, followed by HSCT, followed by low‐dose (20mg/kg) CP (day +10, +11). Control animals were (iii) healthy non‐treated and (iv) EAE non‐treated. Clinical symptoms of EAE (0–5 scale) were observed during the disease course and after the resolution of symptoms rats were euthanized. Spinal cords were collected for immunofluorohistochemical analysis to exmine the microglia phenotype in the CNS.

Results showed that the applied therapy in the EAE rats, including only the high‐dose CP followed by HSCT did neither improve clinical symptoms observed in the rats nor influenced the microglia phenotype in comparison to the EAE non‐treated animals. However, the additional low‐dose CP after HSCT resulted in the significant reduction of clinical symptoms and increase in the number of anti‐inflammatory microglia in the CNS. There was no effect observed of applied therapies on the numer of pro‐inflammatory microglia.

Our results demonstrated, that the appliction of additional low‐dose CP after HSCT led to the increase in the number of anti‐inflammatory microglia in the CNS. This shift into the anti‐inflammatory phenotype might be engaged in the reduction of clinical symptoms in the EAE rats and accentuates the role of microglia in MS pathomechanism.

Support or Funding Information

Supported by grant no. 2018/02/X/NZ5/01487 financed by the National Science Centre
Sigma‐1 Receptor May Participate in the Regulation of Epigenetic Modifications Related to Neuropathic Pain
Authors: Goguadze Nino , Wu Hsiang-En , Su Tsung-Ping
Published: April 2020

Nerve injury causes abnormal hyperactivity of primary sensory nerves and causes changes in the expression of pro‐ and antinociceptive genes in the DRG.

The ER chaperone sigma‐1 receptor (Sig‐1R) was demonstrated to play a role in neuropathic pain and is known to exist in the dorsal root ganglia (DRG), notably, at the nuclear envelope. According to the published data, in Spared Nerve Injury (SNI) model of neuropathic pain, Sig‐1R knockout mice did not develop cold allodynia and showed significantly less mechanical allodynia. However, the molecular mechanisms whereby Sig‐1Rs modulate neuropathic pain at the DRG are largely unknown.

Recently, it was shown that the inhibition of histone methyltransferase at the DRG attenuates the neuropathic pain by correcting the dysfunctional potassium channels. Inasmuch as Sig‐1Rs exist at the nuclear envelope, we hypothesized that Sig‐1Rs may participate in the regulation of epigenetic modifications related to neuropathic pain.

Immunoblotting of extracted histones from Sig‐1R knockout DRGs, when compared to wild type DRGs, showed a decrease in the level of H3K9me2, whereas total H3 methylation/acetylation, H3K9ac, H3K9me3, H3K27me3 and H3K4me3 histone marks were unchanged. However, the protein level or mRNA expression of euchromatic histone‐lysine N‐methyltransferase‐2 (G9a) which is an enzyme responsible for H3K9me2 modification was not altered in wild type and Sig‐1R knockout mice. Those results suggest that the Sig‐1R plays a role on the maintenance of the level of H3K9me2 downstream of the G9a.

It is known that H3K9me2 preferentially marks chromatin at the nuclear periphery that interestingly is where Sig‐1Rs are located. Thus, we performed peptide pulldown experiments using unmodified and modified histone peptides to pull down Sig‐1Rs and found that indeed the H3K9me2 peptide can bind the Sig‐1R. When taken together, those results suggest that the Sig‐1R relates to the maintenance of the H3K9me2 level in the DRG and that the Sig‐1R may affect neuropathic pain by regulating the level of potassium channels by the epigenetic modification on histone 3.

Support or Funding Information

IRP/NIDA/NIH
Transcriptional Regulation of the Proteasome: Implications for Cancer Therapy
Authors: Radhakrishnan Senthil , Vangala Janakiram
Published: April 2020

While proteasome inhibitors are being effectively used in the clinic to treat multiple myeloma and mantle cell lymphoma, the reasons for why this strategy is not generally applicable to other tumor types is poorly understood. A detailed characterization of the cellular events triggered in response to these agents could offer vital clues to target the protein degradation pathway(s) more effectively in a broad range of tumors.

Our previous studies have established the transcription factor Nrf1 as a key player in the stress‐response pathway invoked by inhibition of cellular proteasome. Nrf1, by its ability to induce de novo synthesis of proteasome subunit genes in response to proteasome inhibitors, promotes the recovery of proteasome activity, thus mitigating proteotoxic stress and enhancing cellular survival. From a therapeutic stand‐point, this suggests that disabling the Nrf1‐mediated proteasome recovery pathway could potentiate the action of proteasome inhibitor treatments. However, effective means to curb the Nrf1 pathway remain scarce. Here, using an in silico chemical biology approach, we uncovered a class of therapeutic agents that can effectively inhibit the Nrf1 pathway. Mechanistic dissection of this phenomenon provided further novel insight into the way by which Nrf1 maintains its transcriptional activity. Using triple negative breast cancer (TNBC) cell lines and patient‐derived xenograft (PDX) models, we further validated our hypothesis that attenuating the Nrf1 pathway could increase the efficacy of proteasome inhibitor therapeutics. Thus, our study sheds light on the unique biology of Nrf1 along with validating this pathway as a therapeutic target in TNBC. This could serve as a motivation for similar future efforts where Nrf1 pathway inhibitors can be tested as adjuvants for proteasome inhibitor treatments in other cancer types.

Support or Funding Information

NIH grant R01GM132396
Cytotoxic Effects of Digested Edible Insects on Intestinal Epithelia
Authors: Scheers Nathalie , Dobermann Darja
Published: April 2020

Insects have been proposed as a substitute for livestock in future foods as a source of protein and micronutrients. In an attempt to compare iron bioaccessibility between different insect species to human intestinal epithelia, we discovered unexpected cytotoxic effects using standard procedures for estimating iron bioaccessibility. Pre‐confluent and confluent, differentiated epithelia of human intestinal Caco‐2 cells were exposed to simulated gastrointestinal digests of three edible insect species (meal worm, desert locust and black cricket) at varying concentrations. Cell survival and metabolic assays indicated cell death already at low levels of insect digests. Proteomic analyses revealed increased levels of cellular stress markers. Taken together, the data indicate that the investigated insects are cytotoxic to cultured human intestinal cell epithelia.

Support or Funding Information

This work was supported by the UK Biotechnology and Biological Sciences Research Council Nottingham‐Rothamsted Doctoral Training Partnership [grant number BB/J014508/1] and by the Swedish Research council FORMAS (grant # 2014‐417).
Effects of Dexamethasone on HDAC4 Expression and Cellular Localization in C2C12 Cell‐Derived Myotubes: Implications for Skeletal Muscle Atrophy
Authors: Alves Paula Ketilly Nascimento , de Oliveira André Cruz , Moriscot Anselmo Sigari
Published: April 2020

Skeletal muscle can rapidly decrease its mass and force to various conditions such as denervation, mechanical unload, kidney disease, sepsis and hormonal stimuli such as corticoids. Although it is well known that such conditions can trigger a set of genes related to atrophy (mainly Atrogin‐1 and MuRF1), the specific cellular processes that control atrophy under different conditions are not well understood. Accordingly, histone deacetylases (HDACs) can be important modulators of skeletal muscle atrophy and it has been shown that HDAC4 can trigger Atrogin‐1, and MuRF‐1 expression in denervation model. Nonetheless, it is not known whether HDCA4 is modulated in models involving hormonal stimulation such as corticoids. Thus, our objective was to investigate HDAC4 expression and localization in C2C12 cell‐derived myotubes submitted to dexamethasone treatment. We examined HDAC4 localization, gene expression and protein content on a in vivo, and in vitro models. C2C12 cells, treated with dexamethasone (10 nM) for 12h, 24h and 48h and the results are expressed by means ± SD, and the statistical analysis was performed using Student’s t test (p ≤ 0.05). Initially we confirmed the responsiveness of HDCA4 to atrophy mediated mechanical unload. As expected, we found a marked increase in HDCA4 mRNA expression (~3 fold, p<0.05) after 7 days of hind limb immobilization. Other marker of atrophy was also induced (Atrogin‐1 1.7 fold; p <0.05). In C2C12 cell‐derived myotubes, we confirmed decreased in myotube diameter induced by dexamethasone (~25% p<0.05). In addition, we observed increased HDAC4 mRNA levels after 24 h of dexamethasone treatment (~1.8 fold, p<0.05). Atrogin‐1 mRNA level, as expected, was also elevated (~1.6 fold, p<0.05). Concerning cell location, our preliminary findings suggest a nuclear accumulation of HDAC4 in myotubes treated with dexamethasone. As time course and dose response experiments are ongoing, overall, these results provide further insight into molecular mechanisms involving HDAC4 responses in skeletal muscle. This project was approved by the Ethics Committee on the Use of Animals by the University of São Paulo (Protocol No. 2034200718).

Support or Funding Information

FAPESP; CAPES; CNPq
Systems Thinking: An approach for departmental transformation in the life sciences
Authors: Offerdahl Erika G. , Gangera Gita , Bronson Claire , Byers Steve , Davis William , DeMarais Alyce , Fitzhugh Ginger , Goedhart Christine , Linder Nalani , Liston Carrie , McFarland Jenny , Otto Joann , Pape-Lindstrom Pamela , Pollock Carol , Reiness Gary , Stavrianeas Stasinos , Wenderoth Mary Pat
Published: April 2020

The Partnership for Undergraduate Life Sciences Education (PULSE) is a national organization that supports department‐level efforts to transform life sciences programs to align with the recommendations of Vision and Change (V&C). With support from the NSF, a team of PULSE fellows from the Pacific Northwest region created a program consisting of (1) a three‐day, team‐based workshop on how to apply systems‐thinking approaches to organizational change and (2) a mentored execution of a department‐level transformation plan. Over the past five years, faculty teams have participated from over 40% (63/148) of institutions in the NW region, including 27 community colleges, 15 liberal arts, and 11 masters granting, 9 doctoral granting and one professional‐degree granting institutions.

Reported here are the short and long term outcomes of the first 45 institutional teams from years 1–3 of the project. A mixed‐methods approach was applied to (a) understand the ways in which NW PULSE supported departmental transformation, (b) identify strategies used by department and the relative efficacy of those strategies, and (c) determine emergent practices to inform departmental transformation efforts nationwide. Pre‐ and post‐surveys were administered prior to and immediately following the workshop and 6–7 months later. Long‐term effects of program participation were measured by a longitudinal survey. Fifty‐seven percent of the 138 individual participants responded to the survey, representing 87% (39 of 45) of institutions. An outlier sampling approach was subsequently used to interview faculty and administrators at 12 institutions to identify factors that contribute to departmental/institutional transformation.

Results from the evaluation provide insight into potential “better practices” for supporting biology education reform. First, our data support the recommendation for systematic and inclusive engagement of faculty, especially faculty that represent the composition of the department and those that have decision‐making power or influence in the department. Further, while a critical mass of faculty is needed for transformation, our data suggest that effective transformation need not involve all department faculty members. Second, our data underscore the importance of providing a range of resources and support. Not surprisingly, there is no “one‐size‐fits‐all” approach because each institution has its own unique context. Finally, our data demonstrate the efficacy of applying systems thinking to department transformation efforts. These data are consistent with prior literature encouraging multiple levers across levels within the organization to catalyze change.
Simulations of Combination Therapy in Hepatocellular Carcinoma Using a Quantitative Systems Pharmacology Model
Authors: Sove Richard J. , Jafarnejad Mohammad , Wang Hanwen , Yarchoan Mark , Popel Aleksander S.
Published: April 2020

Hepatocellular carcinoma (HCC) is a major health concern and despite efforts to screen at‐risk individuals, diagnosis often occurs at late stages when patients are no longer candidates for potentially curative therapies (i.e. surgical resection, ablation, or transplantation). For patients with advanced HCC, tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors may provide clinical benefit, but overall survival remains low. One strategy that has shown promise in on‐going clinical trials is to administer a combination of TKIs and immune checkpoint inhibitors. Despite the increased number of patients that respond to combination therapy compared to individual therapies, there is still a large fraction of patients who remain unresponsive. Therefore, to improve therapy outcomes for patients with HCC, there are two broad questions which need to be addressed. First, through which mechanisms are the therapies interacting in HCC, and secondly, why are they effective in only a subset of patients?

To address these questions, we developed a mechanistic quantitative systems pharmacology (QSP) modeling platform for immuno‐oncology (IO) that can be used to predict the progression of HCC in response to molecular and immune therapies. This model integrates cancer growth, immune response, and intracellular signaling in four compartments (tumor, tumor‐draining lymph node, central and peripheral), and considers the interactions and transport of various immune cells (e.g. effector T cells, T regulatory cells and antigen presenting cells) as well as the growth of tumor cells which are partially dictated by interactions with immune cells in the tumor compartment. Additionally, the model includes the binding kinetics of immune checkpoint proteins. Further, pharmacokinetic models are used to model the uptake, transport and clearance of immune checkpoint inhibitors, and the antibodies in the model by bind the immune checkpoint proteins. In this work, the model was applied to an on‐going clinical trial (ClinicalTrials.gov Identifier: NCT03299946) of the combination of TKI, cabozantinib and immune checkpoint inhibitor, nivolumab.

Overall, this work presents a modular platform for the development of QSP models in IO that integrate features at the molecular, cellular, tumor and whole patient scales. This framework can then be used to predict therapeutic response to the treatment of HCC, and is the first to predict the outcome of combination therapy for liver cancer. Additionally, it enables the analysis and interpretation of the complex interactions in HCC that may ultimately lead to the understanding of the drug interactions in cancer and enable the prediction of patient response to therapies, facilitating planning for patient cohorts and even individual patients.

Support or Funding Information

Supported by NIH grant U01CA212007.
Bacterial Nanocellulose Membrane As Bolus in Radiotherapy: “Proof of Concept”
Authors: Chiozzini Giulia Cristina , Mendes Guilherme Paulão , Claro Amanda Maria , Amaral Creusa Sayuri Tahara , da Silva Barud Hernane , Amaral André Capaldo
Published: April 2020

Bolus characterize materials with an electromagnetic radiation attenuation coefficient similar to biological tissue and which are positioned on the skin of patients during radiotherapy treatment. Such devices are responsible for the superficialization of the maximum dose of high energy photons and electrons, restricting its penetration and improving the coverage of the treatment isodose. The development of new materials, mainly from sustainable biotechnological routes, will contribute to increasing efficiency and expanding the use of these technologies. The objective of this research was to develop the “proof of concept” regarding the use of the bacterial nanocellulose membrane (BNCm) as a bolus for radiotherapy procedures. For this purpose, 1.4 cm thick BNCm were produced from static cultivation of the K. rhaeticus, purified and subjected to the physical‐chemical characterization process by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT‐IR). The samples were then subjected to the determination of the radiological density (RD), by computed tomography, and the radiation attenuation potential (%), after being irradiated with 6MV photons in a linear accelerator using ionization and phantom chamber at doses of 100 and 50 UM. The results were compared with data from the analyzes in a commercial bolus (CB) with same thickness. The physical‐chemical analysis demonstrated the constitution of a pure, highly hydrated, homogeneous and nanostructured network of cellulose fibers. The mean RD of BNCm was 1.1 and 5 Hounsfield units respectively, demonstrating high water (0HU) equivalence from BNCm. The values corresponding to the radiation attenuation at the dose of 100 MU were 5.1 ± 0.1% and 4.6 ± 0.2%, for CB and BNCm respectively. The values for the dose of 50 MU were 3.8 ± 0.1% and 3.0 ± 0.2%. In both doses, the attenuation of BNCm was similar to the CB. It stands out the superiority of the BNCm moldability and the possibility to change its composition to improve the radiation attenuation. The results support the concept of use the BNCm as an effective biotechnological device, align to the sustainability idea, as a bolus for radiotherapy treatment.

Support or Funding Information

I.P.F.B. Indústria de Produtos Farm. e Biotecnológicos Ltda.
Finding molecular targets to reverse nerve degeneration associated with diabetic peripheral neuropathy (DPN)
Authors: Voithofer Gabrielle Elizabeth , Clark Jessica
Published: April 2020

One of the most common comorbidities associated with diabetes mellitus is diabetic peripheral neuropathy (DPN), which is the progressive degeneration of peripheral nerves. The manner in which DPN develops and the underlying mechanisms of peripheral nerve degeneration are currently unknown. Our lab has characterized a glucose‐induced model of DPN in zebrafish (Danio rerio) in which we observe defasciculation/degeneration of motor axons, decreased myelination, and a decrease in ensheathment by perineurial glia. Currently, we are interested in characterizing the molecular pathways involved in the degeneration we observe. To accomplish this, we are testing dexamethasone, a FDA‐characterized small molecule drug, in an effort to decrease the degree of nerve degeneration present after DPN onset. Beginning 5 days post fertilization (dpf) experimental larvae were immersed in 120 mM
d‐glucose for 7 seven days to induce DPN. At 10 dpf, larvae were additionally incubated with 0.5% dimethyl sulfoxide (DMSO) and a 1:1000 dilution of dexamethasone. As a control, a group of larvae were exposed to the same conditions without the addition of the drug treatment. Tg(nkx2.2a:meGFP) fish were used to allow visualization of perineurial glia 12 dpf using EVOS fluorescent microscopy. The percentage of motor axons ensheathed by perineurial glia was measured and quantified using FIJI. An increase in motor axons ensheathed by perineurial glia was apparent, furthermore the number of perineurial cells surrounding the motor axons appeared to increase as well. This increase in perineurial glia is meaningful because these cells form the blood‐nerve‐barrier, have phagocytic capabilities, and have also been shown to provide guidance cues for axonal regrowth. Future work will focus on characterizing the pathways affected by dexamethasone to uncover the molecular mechanisms potentially responsible for facilitating nerve regeneration after DPN onset.

Support or Funding Information

Support was provided by the Henson School of Science and Technology at Salisbury University.
Rosmarinic Acid‐Induced Apoptosis and Cell Cycle Arrest in Triple‐Negative Breast Cancer Cells
Authors: Messeha Samia Shoukry , Zarmouh Najla Omar , Asiri Abrar Mohamed , Soliman Karam Farah
Published: April 2020

Rosmarinic acid (RA) is one of the potent polyphenolic compounds in rosemary (Rosmarinus officinalis) extract. The compound has shown various bioactive properties, including, anti‐inflammatory, immunomodulatory, anti‐mutagenic, hepatoprotective, and neuroprotective, as well as having antioxidant and anti‐cancer activity. This study evaluated the effects and mechanisms of RA in two triple‐negative breast cancer (TNBC) cell lines; MDA‐MB‐231(MM‐231) and MDA‐MB‐468 (MM‐468); which were derived from a Caucasian and an African American woman, respectively. In this study, RA significantly and similarly inhibited cell viability and proliferation in both cell lines in a dose‐ and time‐dependent manner. The results obtained show that RA differentially affected the two cell lines in cell cycle arrest‐related apoptosis and in the alteration of the expression of many apoptosis‐involved genes. In MM‐231 cells, RA arrested the cells in the G0/G1 phase. In contrast, the data suggest that RA causes S‐phase arrest in MM‐468 cells and leads to a 2‐fold greater apoptotic effect than in MM‐231 cells. In MM‐231 cells, RA significantly upregulated the mRNA expression of three genes; harakiri (HRK), tumor necrosis factor receptor superfamily 25 (TNFRSF25), and BCL2 interacting protein 3 (BNIP3). In contrast, in MM‐468 cell line, the compound induced a significant transcription activation in five genes, including (TNF), baculoviral IAP repeat‐containing 3 (BIRC3), receptor‐interacting serine/threonine‐protein kinase 2 (RIPK2), BNIP3, and growth arrest and DNA damage‐inducible 45 alpha (GADD45A). Furthermore, RA repressed the expression of TNF receptor superfamily 11B (TNFRSF11B) in MM‐231 cells in comparison to the ligand TNF superfamily member 10 (TNFSF10) in MM‐468 cells. In conclusion, the data suggest that the polyphenol RA may have therapeutic potential for TNBC. However, RA should be further evaluated for in‐vivo studies and in non‐cancer cells.

Support or Funding Information

U54 RCMI
Determination of hMATE1 Turnover Rate Using Quantitative Western Analysis
Authors: zhang xiaohong , Wright Stephen H
Published: April 2020

Human MATE1 (Multidrug And Toxin Extrusion 1, hMATE1) is an electroneutral H+/organic cation (OC) exchanger mainly expressed in the apical membrane of renal proximal tubule cells. It plays a critical role in the final step of organic cation excretion of the kidney, and these OCs comprise about 40% of prescribed drugs. Understanding the transport turnover rate (TOR) has important consequences for investigation of the transport mechanism, and is critical for interpreting in vitro‐in vivo extrapolation (IVIVE) required for physiologically‐based pharmacokinetic (PBPK) modeling to predict the influence of transport on pharmacokinetics. In the present study, we purified MATE1 protein from CHO cells stably expressing V‐5 epitope tagged hMATE1 by cell surface biotinylation, and the amount of MATE1 protein was obtained by quantitative western analysis using standard curves based on the V‐5‐containing Multiple tag fusion protein (GenScript). Cell surface protein represented approximately 25% of total MATE1 protein expressed by the cell. The number of cell surface transporters was 29.2 fmol cm−2. (~50,000 transporters per cell). The kinetics of MATE1‐mediated uptake of 1‐methyl‐4‐phenylpyridinium (MPP), atenolol, and metformin were performed at pH 7.4 (room temperature) in these CHO cells following an ammonia pulse to produce an outwardly‐directed H+ gradient. Apparent Kt and Jmax values (±SE; n=4) were: MPP, 79.6 ± 10.9 μM and 284 ± 65 pmol cm−2 min−1; atenolol, 352 ± 67 μM and 398 ± 121 pmol cm−2 min−1; metformin, 629 ± 60 μM and 1,561± 153 pmol cm−2 min−1. TOR values (±SE, n=4) calculated using the number of transporters and Jmax values were: MPP, 180 ± 41 s−1; atenolol, 253 ± 77 s−1; and metformin, 993 ± 97 s−1. These values are consistent with TOR values determined for a variety of exchangers (NHEs), cotransporters (SGLTs, Lac permease) and uniporters (GLUTs, ENTs).

Conclusion

TORs of hMATE1 for a kinetically diverse set of substrates (MPP, atenolol, and metformin) determined using a quantitative Western blot technique, ranged from 100 to 1000 s−1, similar to TOR values of other Solute Carrier transport proteins. (supported by NIH grant 1R01GM129777)
Effect of Medium Chain Triglycerides Consumption on Running Performance in Long‐Distance Careers Recreational Runners
Authors: Castro-Sánchez Francisco Humberto , Ruiz-Ulloa Amayrani Vanessa , Ochoa-Acosta Dora Alicia , Urías-Lugo Diana Angelina , de Jesús Vergara-Jiménez Marcela
Published: April 2020

Objective

The purpose of the study was to determine the effect of medium chain triglycerides (MCT) consumption on running performance in long‐distance careers recreational runners.

Methodology

A double‐blind placebo controlled crossover study was conducted with 10 recreationally runners. Participants underwent two 21‐kilometers training sessions in random sequence, once with a commercial hydrating sport drink consumption as placebo and once with the same sport drink plus 30g of MCT that were divided and given in four doses of the same amount once before start and the rest at kilometers number 5, 10 and 15. Due to the physical requirements of the tests, a rest period of 45 days was indicated between both evaluations. Anthropometric measurements through ISAK methodology and dietary assessment by 24 hours dietary recalls and food frequency questionnaires were evaluated before both training sessions to analyze that there were no changes in these variables. Participants were encouraged to not change their training sessions or modify their diets.

Results

50% of participants finished both training sessions. No differences in weight and body composition or dietary intake were observed during the rest period. Gastrointestinal upsets were reported by 66.6% of runners with MCT consumption and there no reports when they consumed placebo. There were no differences in running time when participants consumed MCT, in fact time was a bit higher when compared to placebo (132.6±22.1 vs 128.4±28.1 minutes; P=0.8). No difference were observed when partial times were compared.

Conclusion

30 g of MCT consumption divided in 4 doses does not improve velocity or running performance during 21‐kilometers training sessions in recreationally runners. MCT consumption was associated with gastrointestinal discomfort, thus possible could affect sport performance.
Abnormal Sarcoplasmic Reticulum/Mitochondria Ca2+ Microdomains Impairs Mitochondrial Ca2+ in Cardiomyocytes in Aging
Authors: Suarez Angelica E. , Cividini Federico , Suarez-Ramirez Jorge , Dillmann Wolfgang H.
Published: April 2020

Mitochondrial (Mito) metabolism is regulated by free calcium concentration ([Ca2+]m) in the mitochondrial matrix. Cardiac function is impaired in aging and is associated with Mito disfunction. [Ca2+]m homeostasis depends on the Mito calcium uniporter complex and functional sarcoplasmic reticulum (SR)/Mito calcium microdomains. We hypothesized that Mito dysfunction could be the result of aging‐induced maladaptive changes in protein levels of key proteins of the SR/Mito microdomain resulting in deficient Mito Ca2+ uptake. Therefore, the objective of this work was to determine the protein levels of key members of the SR/Mito microdomain and analyze Mito Ca2+ handling as well as spatial SR‐Mito interaction using the proximity ligation assay. We measured protein levels by Western blot of MFN2, GRP75 and SERCA2a, in hearts of young (YH; 2–3 months) and old (OH; 19–22 months) mice. In addition, we analyzed Mito Ca2+ uptake, release and [Ca2+]m using Mitycam and Mito‐pericam. Our results showed that the SR‐Mito Ca2+ microdomain tethering proteins MFN2 and GRP75 were decreased in the OH by 55% and 60% respectively. SERCA2a, the key mediator of SR Ca2+ loading was also decreased by 41% in the OH. SR‐Mito interaction was dramatically diminished in OH. Consequently, Mito Ca2+ uptake and release as well as [Ca2+]m were significantly decreased. In vivo cardiac AAV‐based expression MFN2 improved SR‐Mito interaction. Adenoviral MFN2 expression in cardiomyocytes increased [Ca2+]m.

In conclusion, SR/Mito interaction is impaired in the cardiomyocyte in aging. This results in abnormal Mito Ca2+ homeostasis and can be improved by expression of MFN2.

Support or Funding Information

Authors acknowledge funding from a Merit Grant from Veterans Affairs system I01BX003429. This work was supported by the National Institute on Aging, Grant T35 AG26757 (PI: Dilip V. Jeste, MD), and the Stein Institute for Research on Aging and the Center for Healthy Aging at the University of California, San Diego. Angelica Suarez was a 2018 UC San Diego MSTAR Student.
Exogenous Infusion of Epinephrine Augments Cardiac Output during Simulated Hemorrhage in Humans
Authors: Butterfield Brian P. , Shepherd John R. A. , Senefeld Jonathon W. , Wiggins Chad C. , Joyner Michael J. , Curry Timothy B. , Convertino Victor A. , Baker Sarah E.
Published: April 2020

Background

Lower body negative pressure (LBNP) simulates hemorrhage, evoking compensatory cardiovascular mechanisms of increased heart rate (HR) while stroke volume (SV) and cardiac output (CO) progressively decline, ultimately leading to presyncope. Previous work has shown that circulating levels of plasma epinephrine are elevated during LBNP; however, the role of early administration of epinephrine in compensatory cardiovascular mechanisms to simulated hemorrhage is not well understood.

Purpose

We hypothesized that during simulated hemorrhage, a low‐dose exogenous infusion of epinephrine in humans would: 1) augment HR response, 2) attenuate the decline of SV and CO, and 3) prolong time to tolerance.

Methods

Nine healthy subjects (5M/4F, 35±9yrs, BMI 25±1 kg·m−2) participated in LBNP on two separate study days. Protocols on each day consisted of a 10‐minute pre‐infusion baseline, 15‐minute infusion period (randomized to saline or epinephrine, dose = 0.1 μg·kg−1·min−1, delivered through a peripherally inserted central catheter), 10‐minute post‐infusion baseline, and 5 minute stages of progressive LBNP with stepwise increases of 10–15 mmHg negative pressure until tolerance (systolic blood pressure less than 80 mmHg, bradycardia, or voluntary termination due to presyncopal symptoms). Heart rate (HR; electrocardiogram) and blood pressure (BP; radial arterial catheter) were recorded continuously. Cardiovascular function (HR, SV, CO, and systemic vascular resistance (SVR)) was determined using arterial waveform analysis. Data were analyzed via percent tolerance using repeated measures ANOVA with student method for pairwise comparisons.

Results

Infusion of exogenous epinephrine lowered MAP (p<0.01) and diastolic blood pressure (DBP; p<0.001), but raised systolic blood pressure (SBP; p=0.019) throughout all stages of LBNP. Epinephrine compared to saline augmented mean HR, SV and CO to the penultimate stage of LBNP (p≤0.01 for all cardiac measures) and concurrently reduced SVR (p=0.002). However, time to tolerance was not significantly different between epinephrine and saline conditions (1415 ± 79 vs. 1475 ± 150s, respectively; p=0.69).

Discussion

Our findings suggest that during simulated hemorrhage exogenous epinephrine can augment cardiac function without increasing tolerance to progressive central hypovolemia. During the epinephrine infusion, benefits to cardiac function may have been limited by reduced vascular resistance.

Support or Funding Information

The United States Department of Defense W81XWH‐13‐2‐0038
Knockdown of Letm1 Protein Affects Mitochondrial Calcium Homeostasis and Cell Viability in Trypanosoma cruzi
Authors: dos SANTOS Guilherme Rodrigo , Leite Ana CR , Lander Noelia , Chiurillo Miguel A , Docampo Roberto , Vercesi Anibal E
Published: April 2020

Calcium ion (Ca2+) is an important second messenger in the life cycle of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease. It is well established that mitochondria have a central role in intracellular Ca2+ homeostasis. While the mechanisms of mitochondrial Ca2+ release in T. cruzi are not completely understood, it has been proposed that Letm1 (Leucine Zipper and EF‐Hand Containing Transmembrane Protein 1), a protein localized to the inner mitochondrial membrane, mediates the extrusion of Ca2+ from mitochondria via a Ca2+/H+ exchange mechanism. Using the CRISPR/Cas9 system we generated TcLetm1 knockdown (TcLetm1‐KD) cell lines. By electroporation of the parasite in presence of pTREX‐Letm1 plasmid, we also obtained a parasite line overexpressing Letm1 protein (OE‐Letm1). Using control parasites for knockdown (transfected with a scrambled sgRNA ‐ SCR) or overexpressing Letm1 protein (pTREX‐n) we have performed cell biology and biochemical experiments to assess the function of Letm1 in T. cruzi. We found that, although overexpression of Letm1 has no effect in epimastigotes proliferation, TcLetm1‐KD epimastigotes have a significantly lower proliferation rate. The ongoing biochemical experiments suggest that the protein Letm1 is, indeed, involved in mitochondrial Ca2+ homeostasis. Using digitonin permeabilized cells, we observed that, while overexpression of Letm1 increases the rates of both net mitochondrial Ca2+ uptake and release, TcLetm1‐KD has the opposite effect. Additionally, we found that TcLetm1‐KD parasites showed an increase in basal mitochondrial calcium concentration. In cell respiration experiments, TcLetm1‐KD cells showed lower oxygen consumption rates than control cells and increased ADP:ATP ratio. Knockdown and overexpression of Letm1 were achieved in T. cruzi for the first time and these cells are being used to study the activity of TcLetm1 as a mitochondrial Ca2+/H+ exchanger.

Support or Funding Information

FAPESP, CNPq, and CAPES.
Effects of Environmental Acclimation versus Transport Stress on Barrier Recovery in a Pig Model of Intestinal Ischemia and Repair
Authors: Ziegler Amanda L. , Blikslager Anthony T.
Published: April 2020

The pig is a powerful model for intestinal barrier studies, and it is important to carefully plan animal care and handling for optimal study design as psychological and physiological stressors significantly impact mucosal barrier function. Here, we report the effects of a period of environmental acclimation versus acute transport stress on mucosal barrier repair after intestinal ischemic injury. Jejunal ischemia was induced for 30‐minutes in 8–10‐week‐old pigs which had been transported and allowed to acclimate to a biomedical research housing environment for 3‐days prior to injury or been transported immediately prior to injury. Jejunal mucosa was then allowed to recover ex vivo in Ussing chambers while transepithelial electrical resistance (TEER) and mannitol flux were measured, and epithelial integrity was assessed by histomorphometry. In uninjured mucosa, there was no difference in basal TEER (P=0.8801) or epithelial integrity on histology between groups (P=0.5686), however, acclimated pigs had increased flux as compared to transported pigs during the first hour of ex vivo incubation (P=0.0186). Ischemia‐injured mucosa of acclimated pigs had less robust TEER recovery ex vivo (P=0.0101) as compared to transported pigs, but an increased initial flux in this group was significantly reduced during recovery (P=0.0025). Ischemia induced greater epithelial loss in transported pigs as compared to acclimated pigs (68.5% versus 87.8% epithelial coverage, P<0.0001), yet both groups restored control levels of epithelial coverage after 120‐minutes ex vivo recovery. These results indicate that acute transport stress on the day of experimental intestinal injury modeling may increase mucosal susceptibility to epithelial loss, but also prime the tissue for a more robust barrier repair response. Brief environmental acclimation, on the other hand, appears to increase intestinal permeability to molecular flux in the absence of injury, while possibly having a protective effect on epithelial loss during injury. These are important considerations for appropriate study design when utilizing highly translational pig models for intestinal barrier research.

Support or Funding Information

NIH K01 OD 028207, NIH P30 DK 034987, NIH‐NICHD R01 HD095876, USDA‐NIFA VMCG‐0065, USDA‐NIFA 1007263.
Regulation of ENaC by aldosterone and potassium
Authors: Leite Dellova Deise C. A. , Saha Bidisha , Takagi Enzo , Shabbir Waheed , Pearce David
Published: April 2020

In the cortical collecting duct (CCD), aldosterone regulates the transcription of serum and glucocorticoid‐regulated kinase 1 (SGK1) and stimulates the activity of epithelial sodium channel (ENaC). In addition, an aldosterone‐independent mechanism contributes to regulation of ENaC. We have shown that K+ itself acts through the master kinase mTOR within its type 2 complex (mTORC2) to activate SGK1, which stimulates ENaC to enhance K+ excretion in principal cells. However, the potential interconnections of these mechanisms have not been determined. Here, we have explored the aldosterone effect in association with K+ effect on ENaC activity regulation in the distal nephron. mpkCCD cells were grown on type I collagen–coated filters and subjected to aldosterone treatment and changes in extracellular [K+]. Amiloride‐sensitive short‐circuit current (ISC), which reflects ENaC activity, was measured by voltohmmeter (Millipore Millicell ERS). Following ISC measurements, cells were harvested and processed for immunoblot analysis. Exposure of mpkCCD cells to 1 μM aldosterone for 3 h increased ISC about 1.9‐fold compared to serum and hormone restricted cells. Elevation of basolateral [K+] to 3, 5 and 7 mM, after aldosterone treatment, promoted an additional increase in ISC of 1.5, 1.6 and 1.7‐fold, respectively. SGK1 HM phosphorylation increased proportionately to the increase in ENaC activity. The effect of K+ on ENaC activity and SGK1 HM phosphorylation is rapid and mediated by basolateral Kir 4.1 K+ channel activity. Overall, our data suggest that the effects of aldosterone and elevation of basolateral [K+] on ENaC activity and SGK1 phosphorylation were additive. Therefore, it is possible that basolateral [K+] acts in concert with aldosterone to regulate ENaC activity in CCD cells. However, in this cell culture model the effects are not synergistic suggesting that they do not proceed through convergent signaling pathways.

Support or Funding Information

This work was supported by NIH R01‐DK56695, Manning Foundation and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.