bluuebunny/crossref_metadata_2025 huggingface dataset:
A Three‐Pronged Synergistic Approach to Increase the Biofeedstock Viability of Chlorella vulgaris
Authors: Slone Danielle Renee , Smythers Amanda Louise , Kolling Derrick
Published: April 2020

Over the last several decades, the global temperature has increased at an alarming rate, increasing the need for efficient renewable energy sources.
1 Microalgae are a promising source of biodiesel due to their high rate of lipid accumulation, but are not efficient enough to produce biofuel on a scale matching current fuel needs.
2

Chlorella vulgaris is a fast‐growing unicellular green microalga with a high percent of lipids per biomass, making it a promising candidate for biodiesel production. Previous research has used two approaches to increase the accumulation of lipids and photosynthetic activity: the addition of exogenous glycerol or the addition of excess Mn2+. When used separately, the addition of glycerol increases neutral lipid production in C. vulgaris while the addition of Mn2+ increases photosynthetic capacity.
3 Glycerol was found to be directly incorporated into the triacylglycerol (TAG) backbone, which allowed for an increased flux toward TAG synthesis without significantly changing the energy exerted. Manganese was shown to increase the rate of electron transfer through photosystem II, increasing the overall photosynthetic output. While it was assumed that combining these conditions would further optimize the cultures for lipid production, this was not the case: when used simultaneously, they decrease TAG production. To improve lipid productivity, a three‐pronged synergistic approach was taken combining Mn2+‐replete conditions, nitrogen deprivation, and exogenous glycerol. Nitrogen was deprived from the cells, because under nitrogen deplete conditions cells shift away from protein synthesis and towards energy storage compounds that can be used to produce TAGs. Cultures were then monitored for turbidity, chlorophyll content, lipid productivity, biomass, and TAG content. Cells grown using this three‐pronged approach showed an increase in the amount of TAGs produced over time as the glycerol concentrations were increased. This novel approach can be used to increase lipid synthesis to increase biofuel feasibility.

Support or Funding Information

#NNX15AI01H.

D.S. was supported from the Marshall University Department of Chemistry alumni donors to the Marshall University Department of Chemistry’s Summer Undergraduate Research Fellowship.

D.R.J.K. and A.L.S. were supported by the National Science Foundation under Cooperative Agreement No. OIA‐1458952. A.L.S. was also funded by the NASA West Virginia Space Grant Consortium, Grant #NNX15AK74A and Training Grant
Stiffness Induces NAFLD‐like Metabolic Dysfunction in Primary Hepatocytes
Authors: Kidambi Srivatsan
Published: April 2020

Background

Non‐alcoholic fatty liver disease (NAFLD), the liver manifestation of the metabolic syndrome linked to obesity and insulin resistance, affects 25% of the general population both in western and developing countries. The development and progression of NAFLD is mediated by increase in lipid accumulation and triglycerides levels, excessive oxidative stress and inflammation that in turn exacerbates the diseased condition and impairs metabolic parameters. NAFLD progression is also characterized by liver stiffness due to extensive remodeling of the extracellular matrix. The role of matrix stiffness as related to subtle but pivotal changes in hepatocytes physiology and metabolic dysfunction is under explored. The overall goal of our study is the development and implementation of a platform that enables the convergence of engineered cell microenvironments with the phenotypic and functional analysis of hepatocytes. Using our innovative biomimetic liver fibrosis model that allows modulation of substrate stiffness, we investigated the role of liver matrix stiffness in modulating hepatocytes metabolic function in fibrotic‐like microenvironment.

Materials and Methods

Primary hepatocytes were cultured on our novel polymer film coated polydimethylsiloxane (PDMS) gels with 2 kPa, 9 kPa 25 kPa and 55 kPa elastic modulus mimicking healthy, early fibrotic, fibrotic and extremely fibrotic substrates. A wide range of functional/metabolic analysis were performed to determine: lipogenic and oxidation genes, glutathione expression, dynamics by Western Blot; Respirometry by Seahorse XFe24 Analyzer.

Results

We demonstrated that stiffness impedes hepatic urea and albumin production, expression of drug transporter gene and epithelial cell phenotype marker, hepatocyte nuclear factor 4 alpha (HNF4a). We also demonstrated that hepatocytes cultured on NAFLD‐like stiffness showed an induction of lipogenic genes along with lowered‐oxidation genes expression, mitochondrial respiration, and glycolytic capacity, 2) increased ROS production, and 3) disruption of the mitochondrial fusion process and dynamics. We observed a significant increase in oxidized glutathione (GSSG) and reduced glutathione (GSH) in hepatocytes cultured on NAFLD‐like stiffness compared to healthy liver stiffness. Similar effect was observed in hepatocytes isolated from NAFLD rat models indicating correlation to physiological conditions. These data suggest a plausible mechanism that increased stiffness modulates hepatocyte function causing liver functional failure.

Conclusions

Together, all these data demonstrates the plausible role of stiffness in regulating hepatocytes function and contribute to metabolic dysregulation in NAFLD. Understanding the impact of stiffness on hepatocytes biology will provide significantly more nuanced data to aid drug development for NAFLD.

Support or Funding Information

NIH R01AA027189‐01A1; Nebraska Research Initiative; NIH 1P20GM113126

Project Summary

Figure 1
High Fat Feeding Primes Mice to Binge Alcohol‐induced Liver Injury: Critical Role of Cathelicidin as a Chemoattractant
Authors: Li Fengyuan , Liu Yunhuan , Gu Zelin , Jiang Mengwei , Zhang Lihua , McClain Craig J. , Feng Wenke
Published: April 2020

Background

Alcohol consumption and obesity are known synergistic risk factors of steatohepatitis. Combination of high fat diet (HFD) and acute alcohol synergistically induced acute liver inflammation via enhanced hepatic neutrophil infiltration. CRAMP (Cathelicidin‐related antimicrobial peptide), the murine ortholog of LL‐37, which is the only known member of human cathelicidin antimicrobial peptide family, possess both anti‐ and pro‐inflammatory activity. Our previous studies showed that CRMAP deficiency exacerbated binge‐on‐chronic alcohol‐induced liver injury. Here we aimed to investigate the role of CRAMP in alcohol‐induced liver injury in HFD fed mice, and to explore the underlying mechanism.

Methods

CRAMP KO mice (Camp−/−) and wild type (WT) mice were fed control diet (CD) or HFD for 10 weeks, a bolus of alcohol was gavaged 9 hours before sample harvesting.

Results

Ten‐week HFD + acute alcohol markedly induced liver steatosis and injury in WT mice, reflected by significantly increased liver macrosteatosis and serum levels of AST and ALT. Immunofluorescent assay showed a significantly stronger staining of Ly6G in the livers of mice treated with HFD + acute alcohol compared to CD feeding + acute alcohol, indicating an increased neutrophil infiltration. Interestingly, HFD+acute alcohol treatment significantly increased hepatic protein levels of CRAMP, which co‐localized with Ly6G‐positive neutrophils. In addition, hepatic mRNA expression of chemokines, Mcp‐1 and Cxcl‐2, was markedly increased. Depletion of Camp gene (Camp−/−) markedly reduced HFD + acute alcohol‐induced liver steatosis, injury and neutrophil infiltration, along with reduced expression of Mcp‐1. Importantly, feeding Camp−/− mice with HFD + acute alcohol significantly reduced the mRNA expression level of Cxcr‐2, a known CRAMP receptor. In vitro study showed that synthetic CRAMP peptide increased neutrophil migration, which was dependent on Cxcr‐2 expression upon treatment by fatty acid and ethanol. In addition, significantly decreased mRNA levels of hepatic Tgf‐β and Col‐1a1 were found in Camp−/− mice compared to WT mice, suggesting that CRAMP may play a role in hepatic stellate cells activation.

Conclusions

HFD + acute alcohol treatment synergistically induces liver injury through systemic upregulation of chemoattractant CRAMP and subsequent enhancement of hepatic neutrophil infiltration. CRAMP deficiency protects against acute alcohol‐induced steatohepatitis in the HFD primed mice.

Support or Funding Information

Supported by grants from NIH

Schematic hypothesis of CRAMP‐mediated neutrophils infiltration in HFD and alcohol‐induced liver injury in mice

Figure 1
3D Printing and the Organ Crisis
Authors: Cevallos Manuel , Butler Jessica , Stabile Diana
Published: April 2020

According to the United Network for Organ Sharing (UNOS) published in January 2019, there are 124,206 men, women, and children currently in need of a lifesaving organ. With barely 26,448 transplants performed annually and only 12,740 people registered as donors, this, unfortunately, equates to 8,000 individuals dying per year in the United States alone. Nevertheless, can advances in bio‐engineering alleviate this upward trending crisis?

Researchers utilizing three‐dimensional (3D) bio‐printing are currently able to mimic the structure of natural tissues. Recently published in The Journal of Thoracic and Cardiovascular Surgery (2016), Lei Wand et al. reported successful 3D‐printed titanium ribs implanted into a patient following an extensive lung cancer resection. Further research is being conducted regarding more complex tissues on animal models to test the viability of the 3D printed tissues before implanting into human patients. In 2019, Jae‐Hyun Park et al. published in Science Reported, a 3D‐bioprinted artificial trachea with autologous epithelial cells and chondrocytes in rabbits are feasible. 3D printing has successfully been used for skin grafts, tracheal splints, artificial ears, knee meniscus, corneas, and bone, but not complex organs.

Though challenging to replicate synthetically, researchers are still working on angiogenesis as one of significant limitation in the process of transplantation. This presentation reviews the current status of 3D printed organ replication for life‐saving transplants.

3D‐Bioprinting Organs

Figure 1
Role of the Epithelial Glycocalyx in Maintaining the Alveolar‐Capillary Barrier During Acute Lung Injury
Authors: Putz Nathan D. , Habegger Luke E. , Shaver Ciara M. , Ware Lorraine B. , Schmidt Eric P. , Bastarache Julie A.
Published: April 2020

Rationale

Disruption of the alveolar‐capillary barrier is a signature feature of acute lung injury. Previous studies have shown the importance of the glycocalyx in maintaining the integrity and homeostasis of the lung endothelium during acute lung injury. However, the role of the glycocalyx on lung epithelial barriers during acute lung injury has not been fully explored. We hypothesize that the lung epithelial glycocalyx is important in maintaining lung epithelial tight junctions in the setting of acute lung injury.

Methods

Cultured mouse lung epithelial cells (MLE12) were treated with functional or heat‐inactivated heparinase I/III. Additionally, some MLE12 cells were treated with LPS after pretreatment with functional heparinase I/III. Glycocalyx shedding was measured by immunohistochemistry for heparan sulfate (HS). To assess paracellular permeability, electrical cell‐impedance sensing (ECIS) was used to measure the resistance across the epithelial monolayer. To determine whether loss of the glycocalyx affects epithelial tight junctions we measured gene and protein expression of ZO‐1, by PCR, western blot, and immunofluorescence.

Results

MLE12 cells in culture express a robust glycocalyx that is completely shed following treatment with active heparinase I/III at 24 hours. Active heparinase I/III did not cause significant change in monolayer resistance from baseline compared to inactive heparinase I/III by ECIS (Active −142±39.2 v Inactive −118.9±57.9 ohms, p=0.75). Active heparinase I/III did not decrease protein expression of ZO‐1 compared to inactive heparinase I/III (Active
1.55±0.33 v Inactive 1.8±0.25 fold change ZO‐1/Actin, p=0.61). MLE12 cells treated with activated heparinase I/III and LPS had a significantly greater decrease in resistance from baseline compared to inactive heparinase and LPS (Figure
, Active + LPS −145±8.8 vs Inactive + LPS − 7.7±10.8 ohms, p=0.0022)

Conclusion

Treating MLE12 cells with functional heparinase I/III degrades the epithelial glycocalyx. Loss of the glycocalyx alone was not sufficient to cause permeability defects nor alter the expression of tight junction proteins. However, glycocalyx shedding did increase the susceptibility of MLE12 cells to LPS‐induced injury. In clinical situations, the shedding of the glycocalyx in ALI is often accompanied by compounding factors such as infection, inflammation, or mechanical stress. The current findings suggest that damage to the epithelial glycocalyx may play a key role in maintaining the alveolar epithelial barrier in acute lung injury.

Support or Funding Information

Supported by a research grant from Department of Defense

MLE12 cells treated with active heparinase + LPS had a significantly greater decrease in resistance compared to baseline than cells treated with inactive heparinase + LPS p=0.0022.

Figure 1
Activated Retinal Microglia Secrete Exosomes Containing Galectin‐3
Authors: Eom Annette Yunsung , Bondarenko Vladimir , Roque Rouel S
Published: April 2020

Purpose

Numerous reports support a potential role for activated microglial cells in the mechanisms of degenerative retinal and brain diseases. Although the exact mechanism remains unknown, our study and those of others describe the transport and release of cytotoxic substances by activated microglia during the initial stages of neuronal cell death. Among these substances, galectin‐3 has emerged as an important player in inflammatory conditions and neurodegenerative diseases by mediating and/or modulating microglial activation. Galectin‐3, a member of an evolutionary conserved family of carbohydrate‐binding lectins, lacks a signal peptide and is most likely secreted via a non‐classical exocytic pathway involving extracellular vesicles (exosomes and microparticles).

Methods

To investigate the role of galectin‐3‐derived retinal microglia in degenerative retinal diseases, activated microglia were isolated from 4–5 week old mice retinas with a targeted mutation of the c‐mer proto‐oncogene tyrosine kinase gene (Mertk−/−) and characterized for expression of microglia specific markers. Extracellular vesicles were purified from 48h conditioned media of cultured microglia and characterized for expression of galectin‐3 and Alix, an exosome marker, using immunocytochemistry and Western blots.

Results

Retinal microglia from Mertk−/− mice displayed an activated phenotype characterized by expression of CX3Cr1, CD11b, and Griffonia simplicifolia isolectin B4 but not of the Müller glial marker glial fibrillary acidic protein (GFAP) nor the vascular endothelial cell marker PECAM‐1. Intense galectin‐3 immunofluorescence was observed throughout the cell body and processes of microglial cells; producing a ~30 kDa galectin‐3 reactive band in the cytosolic component in Western blots. Abundant amounts of extracellular vesicles (~100–200 nm in size) secreted by the cells in 48h conditioned medium displayed a ~30 kDa galectin‐3‐reactive band and an Alix‐reactive band, consistent with exosomes, in Western blots.

Conclusions

Our study shows that activated microglia isolated from early Mertk−/− mice with retinal degeneration expressed high levels of galectin‐3. Moreover, galectin‐3 co‐localized with Alix‐expressing exosomes secreted by cultured microglia. The increased production and release of exosomal galectin‐3 by activated retinal microglia support a potential role for galectin‐3 and microglial cells in the mechanisms of degenerative retinal diseases.
Development of a new technique of embalming and preservation until almost one year at room temperature and dry storage: histological and microbiological analysis
Authors: Masuko Telma Sumie , Dos Santos Andrade Lucas , Ottone Nicolas Ernesto , Baptista Carlos Augusto Camargo , Mimica Marcelo Jenne , Barros Mirna Duarte
Published: April 2020

Introduction

In recent years, a large number of fixation and preservation liquids have been proposed in the search for alternative methods of body preservation. Two processes occur postmortem: putrefaction with bacterial action on body tissues and autolysis with decomposition of the body by endogenous substances. Thus, we intend to present a new formula that allows the body to be preserved with its morphological and mobility characteristics similar to the fresh cadaver, but ensuring its fixation and conservation over time, maintaining at room temperature.

Material and Method

We use a donated 69‐years‐old female body, was embalmed using a new solution based on Baptista et al. (1986) with embalming pump. The formula used was: alcohol 1000 ml; phenol 1000 g; glycerin 1000 ml; sodium nitrate 1000 g; formaldehyde 2000 ml; distilled water 14000 ml. Arterial accesses and complementary injections of formaldehyde were performed in muscles limbs and cranial cavity. The cadaver was maintained at room temperature, ranging from 20.1 to 32.6 °C (68.18–90.68 °F) and the humidity (41–94) in body bag covered with blackout sheet. Samples of tissues (skin and subcutaneous tissue, rectus abdominis muscle, quadrate lobe of liver, transverse colon and brain) were collected after 130, 150, 185, 360 and 512 days of embalming and submitted to histological staining (HE). For the microbiological analysis we used samples after 185 and 360 days and seeded on agar plates (blood, chocolate, and Sabouraud) and placed in liquid medium (TSB) and incubated for 72 hours (35–37 °C).

Results

This method resulted in soft and flexible cadavers as can be seen at elbow joint (Figs.
1a, 1b) with almost natural colors (Fig.
2). Histological analysis indicated the preservation of tissue and cellular structure in the different samples examined, even after 512 days post‐embalming: skin and subcutaneous tissue (Fig.
3); muscle (Fig.
4); liver (Fig.
5) and brain (Fig
6). The cultures were negative for growth of microorganisms in both analysis (after 185 and 360 days).

Discussion

The cadaver preservation technique presented has low cost and easy execution, being effective in maintaining the morphological characteristics of the anatomical specimen for an extended period of time.

Conclusion

It presents as an alternative to anatomical teaching and research centers that have to deal with the toxicity of fixation products and the costs of obtaining and maintaining anatomical specimens.

Demonstration of the cadaver elbow joint flexibility after 512 days post embalming;

Figure 1

Aspect of the coloration of the tissues of the abdominal organs, 360 days post embalming.

Figure 2

Histological sections after 512 days of fixation of skin and subcutaneous tissue, muscle, liver and brain, respectively. HE. 200x.

Figure 3
Preliminary biochemical characterization of Flavin Dependent Monooxygenase DszA
Authors: Sanchez Misael , Tavassoli Alireza , Palfey Bruce , Vey Jessica
Published: April 2020

Sulfur dioxide production through fossil fuel combustion is a major contributor to atmospheric pollution. This molecule has been known to cause respiratory illness in humans and land deterioration through acid rain formation. Fortunately, the biodesulfurization pathway (BDS) in R. erythropolis and other bacterial species have shown promise in becoming a useful tool for the removal of sulfur from crude oil. However, improvements are still necessary for this pathway to become competitive and industrially applicable. Our current objectives are to rationally design the BDS pathway in R. erythropolis, starting with enzymes DszC and DszA. The project goal is to decrease substrate specificity in the pathway, allowing it to work on a larger selection of organosulfur compounds found in fossil fuels. Here we exhibit preliminary biochemical characterization of the second enzyme in the BDS pathway, DszA, a flavin dependent monooxygenase. Stopped‐flow spectrophotometric studies are being used to monitor the reduced flavin dissociation constant(Kd) as well as its rate of oxidation in the presence or absence of DszA and its native substrate, DBTO2. A 20.3 ± 0.451 μM Kd value was determined for the reduced form of flavin. As expected, flavin oxidation increases with oxygen concentration in the presence of DszA. The rate of flavin oxidation also increases in the presence of both DszA and DBTO2 compared to DszA alone, suggesting that the oxidation we observe is enzymatically mediated. Contrary to previous studies, which suggested the production of a flavin‐N5‐oxide intermediate during catalysis, we have not observed the formation of flavin‐N5‐oxide. This intermediate should be detectable through stopped‐flow experiments. Our work towards confirming our enzyme’s activity and ability to generate either the N5‐oxide or the better‐understood C4a‐(hydro)peroxyflavin intermediates is ongoing.

Support or Funding Information

California State University Program for Education in Biotechnology

NIH Grant 5SC3GM122652
Exercise Improves the Fasting Regulation of Skeletal Muscle Protein Synthesis in Tumor Bearing Mice
Authors: Halle Jessica , Fix Dennis , Counts Brittany , Carson James
Published: April 2020

Introduction

Cancer is a debilitating disease that is often accompanied by decreased physical activity and chronic energetic stress that disrupts muscle proteostasis. Skeletal muscle protein turnover is highly sensitive to changes in feeding and activity. Raptor (Ser 792) and TSC1/2 (Ser 1387) phosphorylation can inhibit mTORC1 activation and suppress anabolic signaling, which has been implicated in cancer‐induced skeletal muscle wasting.

Purpose

To examine the effect of the cancer environment on the fasting regulation of Raptor and TSC1/2 in male ApcMin/+ mice and determine if increased physical activity could alter this response.

Methods

Male C57BL/6 (B6, N=24) and ApcMin/+ (MIN, N=31) mice were either sacrificed under ad libitum conditions (B6‐fed, M‐fed), fasted for 12hrs (B6‐fast, M‐fast), or fasted for 12hrs following 4wks of voluntary wheel running (B6+W, M+W). TSC1/2 serine 1387 and Raptor serine 792 were measured in the gastrocnemius muscle as phosphorylation to total ratio by western blot. Protein synthesis was measured by puromycin incorporation.

Results

All MIN mice exhibited body weight loss (p<0.001) and reduced gastrocnemius mass (p<0.001) when compared to all B6 mice. Raptor phosphorylation (pRaptor) was induced in M‐fast compared to M‐fed (p=0.019), which was not changed by fasting in B6 mice (p=0.414). TSC1/2 phosphorylation was induced in M‐fast compared to M‐fed (p=0.001) and in B6‐fast to B6‐fed (p=0.039). Puromycin was trending to be reduced in M‐fast compared to MIN‐fed (p=0.070), but there was no change in B6‐fast to B6‐fed (p=0.323). Raptor phosphorylation was not different in M‐fast compared to M+W (p=0.302), however pRaptor was reduced in B6+W compared B6‐fast (p=0.028). TSC1/2 phosphorylation (p=0.001) and puromycin (p=0.016) were induced in MIN+W compared to M‐fast, with no changes in the B6‐fast to B6+W (p=0.360, p=0.196; respectively).

Conclusions

Our results provide evidence that the cancer environment disrupts fasting regulation of mTORC1 in skeletal muscle. Interestingly, TSC1/2 phosphorylation was sensitive to fasting independent of the tumor environment, while wheel activity induced protein synthesis independent of Raptor phosphorylation. Therefore, further study is warranted to determine physical activities regulation of fasting skeletal muscle protein synthesis in tumor bearing mice.

Support or Funding Information

Acknowledgements

NCI R01‐CA121249
Redox and light signals in the partitioning of photosynthetic electrons
Authors: Puthiyaveetil Sujith , Ibrahim Iskander M.
Published: April 2020

The light‐driven electron transport reactions of photosynthesis convert the free energy of sunlight into usable chemical energy. Plants contain two primary electron transport pathways: the linear electron transport from water to NADP+, producing NADPH and ATP, and the cyclic electron flow around photosystem I, generating just ATP. Recent research from our laboratory reveals that the sensory and gene regulatory function of a Chloroplast Sensor Kinase (CSK) is central to the partitioning of electrons into the linear and cyclic pathways. A conserved iron‐sulfur cluster enables CSK to sense the redox state of the electron carrier plastoquinone. CSK thus places the expression of multiple chloroplast genes under the regulatory control of plastoquinone. Through the phytochrome photoreceptor, light provides a further fail‐safe input into chloroplast gene regulation. The combined action of redox and light signals brings about stoichiometric changes in electron transport complexes with important implications for the flow of photosynthetic electrons.

Support or Funding Information

Purdue University, Showalter Trust, and AgSEED

The CSK‐based genetic switch in chloroplasts

Figure 1
In Defense of Intrinsic Efficacy: The Didactic Value of a Modern Implementation of Furchgott’s Views on Agonism
Authors: Iniguez-Lluhi Jorge Alberto
Published: April 2020

Mastery of fundamental pharmacology concepts is a defining characteristic of basic science graduate pharmacology programs. The acquisition of these skills, however, is challenging for most trainees and a solid understanding of the meaning and use of appropriate pharmacology terms such as efficacy, potency, receptor reserve, allostery and biased agonism is often not fully realized.

In an effort to overcome these challenges, I have developed a consistent stepwise approach to the introduction of concepts that involves a modern and expanded implementation of Furchgott’s classical views of agonism. Although largely superseded by the Black and Leff operational model of agonism, Furchgott’s approach has distinct didactic advantages by clearly separating properties of the drug/receptor complex (intrinsic efficacy) from the specific properties of the system that interprets the stimulus generated by the receptor species (sensitivity). This approach lends itself readily to useful didactic analogies that can be implemented and illustrated in the teaching environment and are easier to conceptualize by the learners. To illustrate the behavior of this model, an easy to use simulation tool has been developed in the widely used Excel platform that allows learners to manipulate individual parameters such as affinity, intrinsic efficacy, number of receptors and sensitivity of the system to explore their influence on the shape and position of concentration response and ligand occupancy curves. Furthermore, this formalism is readily expanded in a stepwise manner to introduce progressively more advanced concepts such as interactions between two different ligands, as well as concepts of constitutive activity and allostery. These functionalities are revealed progressively in the simulation tool such that nearly all forms of major scenarios can be explored, including concepts such as receptor reserve, competitive antagonism, inverse agonism, and allosteric effects on both affinity and/or efficacy.

The power of this approach is also evidenced by the fact that it is easy to illustrate to the learners not only how different mechanisms can yield similar functional outcomes but also to allow them to explore and identify experiments that can most clearly distinguish between them. This overall didactic approach has proven effective in diverse environments ranging from Medical Student instruction, Doctoral and Masters Pharmacology programs, Doctor in Pharmacy as well as advanced undergraduate courses.

Pharmacodynamics simulation tool depicting the functional interaction between an orthosteric and allosteric ligand on two linked responses.

Figure 1
Stereotactic Radiosurgery Effect on Ion Channel Activity: Implications in Metastasis and Cancer Pain Relief
Authors: Azorin-Vega Erika Patricia , Picones Arturo , Moranchel Mario , Oros Rigoberto
Published: April 2020

Influence of aberrantly expression ofvoltage‐gated sodium channels (VGSCs) in metabolic regulation, cell cycledistribution, proliferation, radioresistance and metastasis confirm their role incarcinogenesis. Recently VGSCs have been proposed as prognostic markers andantimetastatic targets with diagnostic and therapeutic potential
[1]. Increasedsodium levels observed in cancer tissue, due to the persistent currents thatVGSCs develop under hypoxic conditions, have been correlated to tumor growth, invasiveness and metastasis
[2]. Quality of life of bone metastasis patientssignificantly improves after radiotherapy (10 fractions of 3Gy or 1 fraction of8 Gy) due to the control of tumor spreading and the sustained pain relief
[3,
4]. Mechanism trough which radiotherapy controls tumor spread and achieves painrelief is poorly understood. In the present work the role of VGSCs in tumorcontrol and pain relief mechanisms after irradiation were analyzed by invasionassays and whole‐cell patch clamp. Monolayers of NaV1.7 expressing and wildtype CHO cells were irradiated in a LINAC. SL 15 Phillips (8X8 cm2 field, 6MV xrays, gantry 180°) before evaluating the clonal and invasive capacity of cellsand the function of NaV1.7. Resuls show that NaV1.7 expression reduces CHOcells plating efficiency and confers radioresistance. Irradiation reduces sodiumcurrent density and increased the voltage to hyperpolarize. Apparently, irradiationdelayed NaV1.7 activation and significantly abolished invasiveness induced byradiotherapy.

Support or Funding Information

This study was performed as part of the activities of the “Laboratorio Nacional de Investigación y Desarrollo de Radiofármacos‐CONACyT”. Grant 293334.

NaV1.7 expression reduces CHO cells plating efficiency and confers radioresistance

Figure 1

NaV1.7 expression (red line) confers radioresistance to CHO cells (black line)

Figure 2
Vascular Remodeling with Reduced Renal Mass
Authors: Adam Ryan J. , Marks Amanda , Kriegel Alison J.
Published: April 2020

Maintenance of vascular anatomy is critical for the kidneys to perform their chief function of blood filtration. It is then no surprise that compromises in kidney vascular anatomy and function are associated with chronic kidney disease (CKD), which afflicts >15% of the US population. Rat renal remnant models of CKD, including 1/3 and 5/6 nephrectomies (1/3NX, 5/6NX, respectively), recapitulate many elements of human CKD. We have observed from prior studies that remnant kidneys of 5/6NX rats spontaneously undergo remarkable hypertrophic remodeling, and by 10 weeks post NX are, on average, larger than Sham NX, intact kidneys. In contrast, the 1/3NX remnant kidney (2/3 of mass excised with contralateral kidney intact) atrophies over time. These disparate observations of whole‐organ remodeling must occur concomitantly with commensurate differences in vascular remodeling. Careful examination of differences in vascular remodeling in these two CKD models may uncover important molecular pathways and mechanisms underlying alterations in renal function. The first step toward realization of this goal, and the primary aim of this study, was to anatomically characterize the vasculature of the remnant kidneys from 1/3NX and 5/6NX rats.

Methods

1/3NX, 5/6NX, and Sham surgeries were performed on 10 week old male Sprague Dawley rats. Gross vascular anatomy was assessed 10 weeks post NX using two approaches. The first was in‐vivo assessment of major renal vessels with using Doppler ultrasound imaging of the short axis. The second was morphometric analysis of microfil kidney vascular cast photos.

Results

Doppler imaging revealed a reduction in renal blood flow through large vessels in the 1/3NX remnant kidney. The 5/6NX rats demonstrated pronounced vascular remodeling, with both increased diameter of larger vessels and turbulent flow. Quantitative analysis of microfil photos indicates that 1/3NX remnant kidneys have, on average, reduced vessel‐area fraction (FAV, area of photo occupied by vessels/area of tissue) compared to Sham (0.96 ± 0.2 vs. 0.53 ± 0.15; mean ± SEM), and that 5/6NX kidneys have reduced FAV compared to 1/3NX remnant kidneys (0.24 ± 0.06). Sham animal microfil casts extended to the capsule, while the mean distance from the most superficial casted vessels was greater in 1/3NX remnant kidney (0.20 ± 0.13 mm) and much greater in 5/6NX kidneys (1.57 ± 0.32 mm).

Conclusion

These observations demonstrate pronounced anatomical differences between 1/3, 5/6, and Sham NX rat kidney vasculature, including apparent differences in peripheral vessel density. These anatomical differences were accompanied by functional differences in blood flow. Thus, these rat models of CKD appear to recapitulate elements of the vessel rarefaction/injury of human CKD, which is associated with hypoxia, fibrosis, tubular damage, and functional loss. Improved understanding of these anatomical changes and underlying molecular pathways may reveal novel treatment approaches.

Support or Funding Information

1R01HL128332‐01A1

Figure 1
Investigating the CYP450‐Catalyzed Metabolism of Cannabidiol and Its Implications for Drug Safety
Authors: Murray Jessica L. , Nardone-White Dasean , Jackson Klarissa D.
Published: April 2020

Cannabidiol (CBD) is a naturally occurring phytocannabinoid derived from Cannabis sativa. It was recently FDA‐approved as Epidiolex® for the treatment of seizures associated with Lennox‐Gastaut or Dravet syndrome in patients aged two and older. CBD’s rise in popularity among consumer products and supplements has led to an increase in safety events and concerns for toxicity. Epidiolex® is known to cause dose‐dependent hepatotoxicity, and monitoring of serum transaminases and bilirubin is recommended for six months following treatment initiation. Cannabidiol is reported to undergo hepatic metabolism by cytochromes P450 (CYP) 2C19 and CYP3A4, and UDP‐glucuronosyltransferases (UGT) 1A7, UGT1A9, and UGT2B7, respectively. However, the relative enzyme contributions have not been described. The objectives of this project were to determine the specific roles of P450 and UGT enzymes in CBD metabolism with respect to formation of the active metabolite 7‐hydroxy‐CBD (7‐OH‐CBD) and glucuronides, and to investigate the effect of CYP2C19 activity on formation of 7‐OH‐CBD in human liver microsomes. Cannabidiol (1 μM) was incubated with pooled human liver microsomes (0.2 mg/mL protein) in the presence and absence of enzyme cofactors NADPH and UDPGA over time (0–60 min). Cannabidiol and its 7‐hydroxy metabolite were detected and quantified using LC‐MS/MS. The results from substrate depletion studies indicated that P450 enzymes were faster than UGT enzymes in metabolizing CBD (depletion rate constant = 0.12 min−1 with NADPH versus 0.05 min−1 with UDPGA). Formation of 7‐OH‐CBD increased rapidly in incubations supplemented with NADPH, followed by a gradual reduction in metabolite generation. Based on substrate depletion data in the presence of UDPGA, UGTs appeared to play a slightly lesser role in CBD metabolism. To determine the influence of CYP2C19 activity on CBD metabolism, a sample of individual CYP2C19‐genotyped human liver microsomes with poor, rapid, and ultrarapid metabolizing phenotypes were incubated with CBD (1 μM) for 0–30 min. Interestingly, CYP2C19 genotype appeared to influence CBD metabolite generation, with CYP2C19 poor metabolizers producing significantly less 7‐OH‐CBD compared to rapid and ultrarapid metabolizers. Future work will investigate the metabolism, transport, and toxicity of cannabidiol in human hepatocytes.

Support or Funding Information

This work is supported by the UNC‐Chapel Hill Royster Society of Fellows and the UNC Eshelman School of Pharmacy Faculty Start‐Up Funds.

Oxidative Metabolism of Cannabidiol

Figure 1
Short bowel upregulates oxalate and citrate transporters and increases CaOx crystal deposition in the rat hyperoxaluric kidney
Authors: Ma Ming-Chieh , Tseng Yi-Shiou
Published: April 2020

Short bowel (SB) increases the risk of kidney stones. However, the underlying mechanism related to intrarenal changes is unclear. Here we examine SB may affect oxalate and citrate handlings on calcium oxalate (CaOx) crystal formation in the hyperoxaluric kidney. SB was induced by small intestine resection in male Wistar rats. Sham‐operation was performed the same surgery without resection. After 7 days of recovery, rats were then fed with 0.75% ethylene glycol (EG and SB+EG groups) for 28 days as a hyperoxaluric induction. The corresponding controls were fed with distilled water (control and SB groups). Blood and 24 h of urine were collected for analysis. Renal CaOx deposition was examined by Pizzolato’s staining. Oxalate transporters Slc26a2 and Slc26a6, and citrate transporter sodium‐dependent dicarboxylate cotransporter NaDC‐1 at their mRNA levels were examined (n=6 in each group). After 7 days of SB, hypocalciuria and hypocitraturia were found when compared with those in sham‐operated control rats. These were associated with hypocalcemia and low renal NaDC‐1 expression after 28 days. SB aggravated EG‐mediated CaOx crystal nephropathy by fostering hyperoxaluria, degree of supersaturation, and CaOx crystal deposition. Renal expression of Slc26a6 but not Slc26a2 was increased in SB+EG group when compared to those in EG or SB group. SB lowered NaDC‐1 expression, but it was reversed in SB+EG group. Intestinal expression of these transporters was similar among groups. We conclude that SB enhances CaOx crystal formation in the hyperoxaluric kidney. More oxalate and less citrate secretion due to Slc26a6 upregulation and insufficient NaDC‐1 expression, respectively, possibly contribute to the aggravated effect of SB on crystal formation.

Support or Funding Information

This research was supported by grants of the Far Eastern Memorial Hospital (FEMH‐2018‐C‐046, Tseng YS and Ma MC) and the Ministry of Science and Technology (MOST108‐2314‐B‐030‐004‐MY3, Ma MC), Taiwan.

Hyperoxaluria and hypocitraturia in the short bowl (SB)‐treated hyperoxlauric rats.

More prominent hyperoxaluria (A) and oxalate transporter Slc26a6 upregulation (B) was seen in the SB+EG group. Hypocitraturia were found in the SB group (C), this associated with citrate transporter NaDC‐1 downregulation (D). SB increased CaOx formation in the hyperoxaluric kidney (E). N=6 in each group. *P<0.05, EG vs. C group; #P<0.05, SB+EG vs. EG group; @P<0.05, SB+EG vs. SB group

Figure 1
Deduced Roles of Cardiac 14‐3‐3 Protein Interactome in Heart Metabolism, Protein Synthesis and Proteostasis
Authors: Qu Jia-Hua , Tarasov Kirill V. , Chakir Khalid , Tarasova Yelena S. , Lakatta Edward G.
Published: April 2020

The 14‐3‐3 protein family is known to interact with many proteins in non‐cardiac cell types to regulate multiple signaling pathways, particularly those relating to energy and protein homeostasis, and the 14‐3‐3 network is a therapeutic target of critical metabolic and proteostatic signalings in cancer and neurological diseases. Although the heart is critically sensitive to nutrient and energy alterations, and multiple signaling pathways coordinate to maintain the cardiac cell homeostasis, neither the structure of cardiac 14‐3‐3 protein interactome nor potential functional roles of 14‐3‐3 protein‐protein interactions (PPIs) has been explored in heart. Here, we evaluated both RNA expression and protein abundance of 14‐3‐3 isoforms in mouse left ventricle, followed by co‐immunoprecipitation of 14‐3‐3 proteins and mass spectrometry in left ventricle. We identified 594 proteins comprising the cardiac 14‐3‐3 interactome. Immunofluorescence assays indicated that 14‐3‐3 proteins localize in mitochondria and ribosome. Multiple bioinformatic analyses indicated that more than half of proteins bound to 14‐3‐3 are related to mitochondria, and the deduced functions of the mitochondrial 14‐3‐3 network are to regulate cardiac ATP production via interactions with mitochondrial inner membrane proteins, especially those in mitochondrial complex I and V, and to regulate cardiac fatty acid metabolism via interactions with enzymes within mitochondrial matrix. 14‐3‐3 protein binding to ribosomal proteins may triage energy production to coordinate protein synthesis and protein quality control. In summary, we establish the comprehensive landscape of cardiac 14‐3‐3 PPIs, dissecting its structure, deducing its function to have a major role to regulate cardiac metabolic homeostasis and proteostasis. Because 14‐3‐3 binds to proteins that are crucial to cardiac functions and heart health, the cardiac 14‐3‐3 interactome may be a potential therapeutic target in cardiovascular metabolic and proteostatic disease states, as it already is in cancer therapy.

Support or Funding Information

Intramural Research Program, National Institute on Aging, National Institutes of Health

Conduction of 14‐3‐3 interactome

Figure 1

Deduced roles of 14‐3‐3 protein‐protein interactions in metabolism and maintenance of cardiac homeostasis

Figure 2
Investigating the Effects of Epoxy Fatty Acids against LPS and Pesticide Induced Toxicity
Authors: Atone Jogen , Hwang Sung Hee , Hammock Bruce D.
Published: April 2020

Neuroinflammation is a hallmark of many neurodegenerative diseases including Parkinson’s Disease (PD) and Alzheimer’s Disease (AD), and mitigating neuroinflammation could avert the progression of these disorders. While it is commonly known that inflammation is propagated by COX metabolites of arachidonic acid (ARA) called prostaglandins, it is less known that metabolites of ARA produced by CYP450 called epoxyeicosatrienoic acids (EETs) have been shown to have an anti‐inflammatory and anti‐apoptotic effect in many disease models. Previous studies have demonstrated that increasing the biological availability of EETs by inhibiting soluble epoxide hydrolases (sEH) reduces MPTP or paraquat induced neuroinflammation and prevents the loss of dopaminergic neurons caused by these toxicants. However, the mechanisms underlying such effect is still unknown. In this study, we have treated primary rat astrocyte mixtures with LPS to determine how EETs can affect regulation of growth factors such as its BDNF, GDNF, and VEGF‐A/B. In addition, primary microglia culture was treated with LPS to determine whether microglial activation can be mitigated through the addition of EETs. Finally, N27 dopaminergic neurons were treated with a pesticide rotenone, and the effect of EETs on the survival of dopaminergic neurons were assessed. We have found that EETs and/or sEH inhibition can affect all cells in ways that are be neuroprotective against neuroinflammatory disorders such as Parkinson’s Disease, with reduced inlammatory marker, increased growth factor signaling, and increased neural viability. Future studies may investigate how inhibition of sEH can be used to combat PD in human patients.

Support or Funding Information

This work was supported by the National Institute of Environmental Health Sciences (NIEHS) Grant R01 ES002710, NIEHS Superfund Research Program P42 ES004699, and NIEHS T32 ES007059 (J.A).

EETs and sEH inhibitors cause reduction in rotenone induced loss of N27 cell viability.

Figure 1

EETs and sEH inhibitor along with LPS stimulate growth factor signaling and reduce LPS induced IL‐1b induction.

Figure 2
Optimizing the Fracture Repair Mouse Model by Using Different Sizes of Femoral Defects
Authors: Chang Chih-Hsiang , Hu Chih-Chien , Chang Yuhan , Sia Kee-Chin , Ueng Steve W. N. , Chen Mei-Feng
Published: April 2020

Although mouse models are crucial in orthopedic studies, no study has explored the bone healing process in mice through continuous imaging. Thus, we optimized a mouse model for femoral defect by continually monitoring the bone healing process of different‐sized femoral defects. We observe bone healing images and quantify the trabecular bone value up to 1 month after surgery. In the 0.3‐mm group, significant bone bridge formation and spongy bone trabeculae were found on the day 14 after the defect operation. The bone healed on day 21 with the formation of external callus and internal callus structures and transformed to a remodeled compact bone on day 28. In the 1‐mm group, bone bridge formation occurred on day 21 after surgery. Moreover, bone healing was observed on day 28 with the formation of external callus and internal callus structures. Mice in the 1.5‐mm group were prone to fracture during feeding after bone defect surgery. The fluorescent staining results of osterix and cathepsin K also revealed that the bone formation process in the 0.3‐mm group was faster than that in the 1‐mm group. This study optimized a bone healing mouse model that can be applied for developing new strategies for accelerating bone healing.

Support or Funding Information

During the study period, the authors received funding support from the Ministry of Science and Technology, Taiwan (grant numbers MOST 107‐2314‐B‐182A‐036‐MY3 and MOST 108‐2320‐B‐182A‐020 ‐MY3), and the Chang Gung Memorial Hospital (grant numbers CRRPG3H1311, CRRPG3H1312, CRRPG3H1313, CMRPG3F2011, CMRPG3F2012, CMRPG3F2013). The funders had no role in study design, data collection, data interpretation and analysis, decision to publish, or preparation of the manuscript.

Continuous observation of the bone healing process in the 0.3‐mm bone defect group of the mouse femoral bone.

Images of 3D micro‐computed tomography (CT) angiography as well as hematoxylin and eosin (H&E)‐stained and Safranin O (SFO)‐stained histopathological slides. Immunofluorescence staining of osterix and cathepsin K indicates osteoblast and osteoclast signals, respectively.

Figure 1