bluuebunny/crossref_metadata_2025 huggingface dataset:
The Lecture is Not Dead: Combined Peer Assisted Flipped Class, an Alternative to Classical Lecture in Basic Medical Education
Authors: Waly Nermien , Shawky Noha Osama , El-Bab Mohamed Fath
Published: April 2020

PURPOSE

Student engagement at lectures is declining especially at those of of basic sciences. The main method of Physiology teaching, in our settings, is the lecture. It has been increasingly challenging to employ new educational methodologies in teaching physiology for several economical and technical reasons. Our objective was to enhance lecture attendance and increasing student engagement in physiology lectures.

METHODS

combined peer assisted learning (PAL) and flipped classroom (FC) strategies (PAFC) was employed in the teaching of neurophysiology course for second year medical students. Course Material and reading assignments were provided ahead of class on instructor and departmental websites. Twenty Students participated in lectures preparation and presentation under instructor’s supervision. Class members participation was conducted via pre‐class reading assignments, in class discussions and instructor guided questions. Method evaluation was conducted using both course surveys, quizzes, end of course evaluations and lecture attendance.

RESULTS

lecture attendance showed about 30 % increase compared to anatomy class conducted in classical lecture format. Course surveys revealed a 77% of students were satisfied with the new methodology while 23% preferred the traditional method. All PAFC participants expressed their positive feedback when interviewed after the course had ended and their comments was documented. End of course evaluations showed that class results were Comparable to anatomy course taught in traditional method (same class and conducted at the same time).

CONCLUSION

In conclusion, PAFC can be beneficial in teaching basic science courses for medical students. It can enhance lecture attendance and student satisfaction. Also, it can provide an affordable alternative method to improve medical education in institutes with limited resources.
The Compatibility of Varied Rib Cartilage Shapes with the Inferior Rim of the Orbit: A Gross Anatomical Study
Authors: Hobeika Natalie A. , Bolden Alexys M. , Burkett Jesse T. , Zdilla Matthew J. , Lambert H. Wayne
Published: April 2020

Recently, reports have documented the reconstruction of the infraorbital rim with costal cartilage grafts. In order to determine the costal cartilage with the shape that is most compatible to that of the infraorbital rim, a geometric morphometric analysis was performed. Cadaveric costal cartilages 5–7 and infraorbital rims were skeletonized and photographed. Photographs were utilized to perform shape analysis. The report highlights the pros and cons of utilizing costal cartilage from each level and side of the body. This information will help improve pre‐operative decision making in the reconstruction of the infraorbital rim.

Support or Funding Information

WVU INTRO (Initiation to Research Opportunities) Summer Research program; WV Research Challenge Fund [HEPC.dsr.17.06]
Does creative writing improve scientific writing and learning?
Authors: Gillen Christopher M. , Kerkhoff Andrew J. , Lynn David H. , McFarlane Hewlet G. , Niemiec Andrew J. , Petersen Sarah C. , Reach Anna Duke
Published: April 2020

Although creative and scientific writing differ in form, objectives, conventions, and language, integrating creative writing into science pedagogy is a promising and increasingly prevalent strategy. For example, recent pedagogical research describes the value of reading and writing poetry in chemistry, neuroscience, pharmacology, nursing, and general physics courses and curricula. At Kenyon College, we incorporate creative writing into the training of biological scientists using diverse approaches at all levels of the curriculum. In several upper‐level Biology courses, students complete “News and Views” assignments in which they write two essays based upon a research article. One essay is targeted towards a general audience; the other is written for scientists. We have found that in‐class instruction, such as reading and discussing writing that effectively engages general readers, is needed for students to write well for general audiences. Members of the English and Biology departments have collaborated to develop and teach a Science Writing course that combines literary analysis of writing about science with a writing workshop that values accurate exposition, creative presentation, and connections to topics beyond science. In the Neuroscience department, a major assignment of the senior seminar is to produce an issue of a magazine for general readers modeled on Scientific American. Students select the topics, write and edit the essays, and contribute figures and relevant artwork. Scientists at Kenyon, in collaboration with the Kenyon Review and with creative writers at Kenyon and beyond, have developed and twice taught the Kenyon Review Young Science Writers Workshop. In the two‐week summer workshop, high school students read poems, stories, essays, and research articles about science and write in a variety of creative genres. Finally, Kenyon students have founded Lyceum, a creative science community that publishes a semesterly journal of science‐inspired poetry, artwork, and essays. We observe that many science students enjoy and benefit from opportunities to write creatively about science. Moreover, students report that to write effectively and creatively about science requires a deeper understanding of underlying scientific concepts than more typical assessments like exams. Thus, we hypothesize that including creative writing in science courses and curricula improves: 1) students’ understanding of scientific concepts, 2) students’ communication of science to scientific and general audiences, and 3) retention and success of students in STEM fields.
Proteolytic Activation of ENaC Mediates Fructose‐Induced Salt‐Sensitive Hypertension
Authors: Shukla Rashi , Hong Nancy , Henderson Ryan , Gonzalez-Vicente Agustin , Hopfer Ulrich , Garvin Jeffrey
Published: April 2020

Proteolytic cleavage of the amiloride‐sensitive epithelial sodium channel (ENaC) by serine proteases such as trypsin leads to channel activation. In nephrotic syndrome, glomerular injury allows filtration of such enzymes causing ENaC‐dependent Na retention and hypertension(PMID: 23216619). Dietary fructose also causes Na retention and salt‐sensitive hypertension at least, in part, via altered hormonal signaling and metabolism in the proximal nephron. Proximal tubules normally secrete proteases into the forming urine but whether they are a source of proteases that cleave and activate ENaC in fructose‐induced salt‐sensitive hypertension is unknown. We hypothesized that dietary fructose increases expression and release of proteases from the proximal nephron, and that these enzymes activate ENaC thereby causing Na retention and salt‐sensitive hypertension. To test this hypothesis we first measured proximal nephron protease expression and urinary protease excretion in rats on either a high‐salt diet containing 4% NaCl without fructose (high salt) or one containing high salt plus 20% fructose in the drinking water (high salt/fructose) for 7 days. High salt/fructose treatment increased proximal nephron expression of trypsin I, an enzyme known to cleave and activate ENaC, by 70% (p < 0.01) compared to high salt alone. Urinary excretion of trypsin I was 8.4±1.3 arbitrary units/μg protein in rats fed high salt while it was 20.3±4.6 arbitrary units/μg protein in those on the high salt/fructose diet, 142% greater (p < 0.02). There was no difference in total urinary protein excretion between groups. Finally, we examined the effect of high salt/fructose and high salt plus 20% glucose (high salt/glucose) on blood pressure before and after oral amiloride. After 7 days, the systolic blood pressure of rats on high salt/fructose was 142±6 mm Hg while it was only 121±6 mm Hg in those on high salt/glucose (p < 0.05). After 10 days, the systolic blood pressure of rats on high salt/fructose was 148±6 mm Hg while it was only 124±5 in those on high salt/glucose (p < 0.02). Amiloride treatment for 24 hrs reduced systolic blood pressure in rats on the high salt/fructose diet from 148±6 to 134±5 mm Hg but did not decrease it in the high salt/glucose group. We conclude that proteolytic cleavage of ENaC contributes to fructose‐induced salt‐sensitive hypertension and that the source of the protease(s) is likely the proximal tubule rather than glomerular filtration.

Support or Funding Information

NLHBI HL128053
Molecular Landscape of Canine Osteosarcoma
Authors: Das Sunetra , Idate Rupa , Fowles Jared S. , Lana Susan E. , Regan Daniel P. , Gustafson Daniel L. , Duval Dawn L.
Published: April 2020

Osteosarcoma affects about 2.8% of dogs with spontaneous cancer and its occurrence is significantly higher than observed in humans. The prognosis of this disease is relatively poor, where the survival rate of ~45% of dogs with bone cancer is one year. With the recent advancement in characterizing mutational landscapes of various cancer types, personalized medicine has become increasingly important for cancer treatment. The purpose of this study is to characterize the mutation and expression profile of canine osteosarcoma. Using whole exome capture (Agilent Sure Select Canine V2) we sequenced 26 primary tumors and matched normal samples, with coupled treatment and outcome data. These samples were previously analyzed for gene expression using Affymetrix Canine 2.0 microarrays.

The BWA tool was used to map Illumina reads to the CanFam3.1 genome and short variants were called and annotated using Mutect2 and SnpEff tools, respectively. Additionally, copy number variations were called and annotated using Sequenza and GISTIC tools. The total number of somatic variants identified across 26 samples ranged from 148 to 3,785 The protein coding mutations per Megabase ranged from 0.25 to 11.92. Among the protein coding somatic variants identified as SNVs and INDELS, an average of 80% were missense mutations (range 64% to 92%). The top recurrent mutations were observed in TP53 (82% of the samples), SETD2 (22%). We have also identified 56 known cancer genes with copy number variations (amplification or deletion) in at least 58% of the samples. When compared to pediatric osteosarcoma, there was an overlap of 38 genes (eg: TFEB, MYC) that were recurrently amplified and 69 genes (eg: RB1, MSH3) were recurrently deleted in both canine and human osteosarcoma.

Using the microarray data, we have identified 585 differentially expressed genes between normal bone and osteosarcoma samples. Using GeneSet Expression Analysis (GSEA) we observed down‐regulation of myogenesis genes and up‐regulation of extracellular matrix genes in tumor samples. Additionally, we have quantified T‐cell and macrophage infiltration in the tumors via immunohistochemistry. Across 20 samples, the median percentage of tumor area with CD3+ and MAC387 positive cells was 0.14% and 0.21%, respectively. There was no significant correlation between disease‐free interval (DFI) and median dichotomized T‐cell or macrophage infiltration. However, Kaplan‐Meier analysis revealed that dogs with TP53 missense mutations had significantly longer DFIs than those with TP53 wildtype/null mutations. Further, correlation of gene expression and DFI can provide us with prognostic biomarkers for osteosarcoma in dogs.

Support or Funding Information

Anschutz Foundation and Morris Animal Foundation
The effect of Ananixanthone in TGF‐ ß1 signaling
Authors: Yeh Yu-Hsuan , Chen Jih-Jung , Chen Chun-Lin
Published: April 2020

Transforming growth factor beta 1(TGF‐ß1) is a famous known growth factor which promotes malignancy and enhances tumor metastasis by inducing epithelial‐mesenchymal transition (EMT). Ananixanthone(GMP15), a derivative of xanthone, is extracted from Calophyllum teysmannii and has been reported to show lower toxicity compared to xanthone. Previous study has showed that xanthone repressed TGF‐ ß1‐mediated EMT, however there is no study about GMP15. Hence, in this study, we determine to study the effect of GMP15 in TGF‐ ß1 signaling. We found that GMP15 suppressed TGF‐β1‐induced PAI‐1 reporter gene activation and Smad2/3 phosphorylation in various type of cell. Also, GMP15 repressed TGF‐ ß1‐mediated expression of N‐cadherin and Fibronectin as well as abolished repression of E‐cadherin in HepG2 cell, thus blocking TGF‐ ß1‐mediated EMT. Our preliminary finding showed that GMP15 might be a potential inhibitor of TGF‐ ß1 signaling. Finding the underlying mechanism of GMP15 in TGF‐ ß1 signaling might help provide a new choice of cancer treatment.
Development of a micro‐plastination protocol for morphological research
Authors: Ottone Nicolas Ernesto , Baptista Carlos , Fuentes Ramon , del Sol Mariano
Published: April 2020

Introduction

Ultrathin‐sheet plastination technique has been used to study microscopic details in anatomy. The aim of this study was to develop an ultra‐thin sheet plastination protocol in an induced model of osteoarthritis in rat humeral joint.

Material and method

Rat humeral joints, with osteoatrhitis, were used for this research. Sixteen weeks after osteoatrhitis induction, the animals were euthanized and injected with red epoxy resin through toracic aortha. Humeral joints and surrounded tissue were fixed in 10 % formalin. Samples were submitted to the plastination technique. The first step was dehydration during ten days, with acetone (100 %) at −25 °C. Later, for degreased, samples were inmerse in methylene chloride at room temperature during one week. Forced impregnation, the most important step in plastination, was peformed inside a stove with vacuum chamber. The plastinated blocks were cutted with a low speed band saw, with a diamond saw blade. Slices were placed in curing chambers to achieve polymerization and final transparentation.

Results

We obtained slices of humeral joint of 230 μm thick, in which articular cartilage characteristic osteoarthritis morphological changes, including neovascularization, were observed.

Discussion

Ultra‐thin sheet Plastination is generally not used as a methodology for the identification of morphological changes caused by pathologies. However, we have applied this technique to identify the changes caused by osteoarthritis in the humeral joint of rats, as tjhe invasion of blood vessels in subchondral bone.

Conclusion

Ultra‐thin sheet plastination technique was a methodology that can be used for the identification, measurement and comparison of morphological modifications caused by various pathologies, including osteoarthritis.
Horseshoe Kidney: A Vascular Survey With Surgical Significance
Authors: Ford Stephen , Beger Aaron
Published: April 2020

During routine cadaveric dissection in a medical school anatomy lab, a 77‐year‐old male was found to have complete fusion of the inferior poles of the kidneys by an isthmus, forming a u‐shaped horseshoe kidney. Although reported rates of horseshoe kidney are as frequent as 1 in 300 with a 2:1 predominance in males, this is the first case that has been discovered in LMU’s anatomy lab. This anomaly, in many patients, goes unnoticed due to its asymptomatic presentation and, if identified, is typically an incidental finding from ancillary testing. In this case, the superior poles of the kidneys were located in approximately normal anatomical positioning with classic right kidney displaced inferiorly in relation to the left kidney; however, the right kidney’s hilum was displaced more inferiorly than was expected. Both kidneys were elongated, extending down to the level of L3–L4 where the isthmus passes inferior to the inferior mesenteric artery. An impression was observed on the anterior surface of the isthmus due to its relationship to the inferior mesenteric artery. Vasculature was documented in this report which showed several unique features. Multiple inferior polar arterial branches were seen branching from the right common iliac artery but were not observed on the contralateral side. Both testicular veins were observed coalescing into the left and right renal veins respectively. The complete vasculature has been documented in this report for future research and evaluation prior to kidney transplantation or resection.
Pressure‐Induced Constriction of the Middle Cerebral Artery is Abolished in TrpC6 Knockout Mice
Authors: Nemeth Zoltan , Ryan Michael J. , Granger Joey P. , Drummond Heather A.
Published: April 2020

Pressure‐induced constriction (PIC) is an inherent response of small arteries and arterioles in which increases in intraluminal pressure evoke vasoconstriction. It is a critical mechanism of blood flow autoregulation in the kidney and brain. Degenerin (Deg) and transient receptor potential (Trp) protein families have been implicated in transduction of PIC because of evolutionary links to mechanosensing in the nematode and fly. We hypothesize that Deg and Trp proteins associate to form a multi‐meric mechanosensing complex in vascular smooth muscle cells (VSMCs), in which Deg and Trp proteins play different, yet equally critical, roles as sensors and amplifiers, in PIC molecular signaling. Our laboratory has provided direct evidence that the Deg protein bENaC acts as a sensor by mediating mechanoreceptor currents in isolated VSMCs. While TrpC6 has been suggested to mediate PIC, direct supporting evidence is lacking. Therefore, the aim of this study was to determine the importance of TrpC6 in PIC signaling using a mouse model lacking TrpC6. To address this aim, we evaluated graded pressure (20–90 mm Hg), depolarization‐ (20–80 mM KCl) and adrenergic receptor‐(phenylephrine, PE 10−7–10−4 M) mediated constriction of isolated middle cerebral artery (MCA) segments from 9 weeks old male wild‐type (+/+, n=7) and homozygous null (−/−, n=9) TrpC6 mice (Jackson Laboratories). Isolated MCA segments were cannulated and pressurized with physiological salt solution using pressure myography (Living Systems). The data were analyzed preliminarily using independent 2‐tailed t‐test. Normalized MCA vasoconstrictor responses to KCl (80 mM, 45.5±1.1% vs 40.9±1.2%, p=0.2) and PE (10−4 M, 37.4±1.7% vs 38.7.0±1.2%, p=0.8) were similar in TrpC6−/− and TrpC6+/+ mice, respectively. In contrast, PIC responses were totally abolished in TrpC6 −/− mice; at 90 mmHg calculated myogenic tone was 0.8±0.5% vs 10.7±1.7%, p=0.0002, in TrpC6−/− and TrpC6+/+ mice, respectively. Additionally, there were no changes in mechanical properties of circumferential wall strain (0.2±0.02 vs. 0.2±0.02, p=0.8) and stress (2.7±0.1 vs. 3.1±0.4, p=0.4), at 90 mmHg; or morphological properties wall thickness (13.4±0.8μm vs. 14.3±1.5μm, p=0.6) and wall‐to‐lumen ratio (0.3±0.02 vs. 0.3±0.04, p=0.7), at 50 mmHg between TrpC6−/− and TrpC6+/+ mice. Although these results suggest that TrpC6 is required for the PIC response, they do not identify the role of TrpC6. Studies in the nematode suggest that Deg and Trp channels play different, yet equally critical roles in mechano‐signaling; Deg channels mediate mechanoreceptor currents, while Trp channels mediate post‐transduction signal amplification. Future studies will determine the role of TrpC6 in isolated VSMCs and cerebral blood flow autoregulation. This research was supported by P20GM104357, P20121334, P01HL051971, R01HL1136684.

Support or Funding Information

P20GM104357, P20121334, P01HL051971, R01HL1136684.
The Sigma1 Receptor Antagonist CM304 Potentiates the Antinociceptive but not the Discriminative Stimulus Effects of the Cannabinoid Receptor Agonist THC in Rats
Authors: Obeng Samuel , Patel Avi , Burns Mallory , Intagliata Sebastiano , Mottinelli Marco , Reeves Morgan E. , Behnke Mira , Restrepo Luis F. , Ho Nicholas P. , Gamez Jimenez Lea R. , Williamson Morgan R. , McCurdy Christopher R. , McMahon Lance R. , Hiranita Takato
Published: April 2020

Opioid overdose is one of the leading causes of death in the United States in people under 50 years, which has resulted in a decline in life expectancy in the United States. Thus, there is a need for an alternative to prescription mu‐opioid agonists for pain treatment. Delta (9)‐tetrahydrocannabinol (THC), a cannabinoid (CB) agonist, has been shown to produce antinociceptive effects albeit less effectively than mu‐opioid receptor agonists. Sigma1 receptor (σ1R) antagonists in combination with THC may provide a viable and safe pharmacological alternative to prescription opioids for pain treatment. The present study compared the pharmacological effects of the σ1R antagonist CM304 alone and in combination with THC in Sprague Dawley rats. Hotplate latency was determined at 52°C followed by rectal temperature measurement and the drugs were administered intravenously (i.v.) and in cumulative doses every 5 min. THC dose‐dependently increased maximum possible effects (MPEs) up to 84% which were reversed by 10 mg/kg rimonabant (cannabinoid CB1 receptor antagonist). CM304 alone up to a dose of 10 mg/kg did not produce significant antinociceptive or hypothermic effects, but CM304 (1.0 and 3.2 mg/kg, i.v.) dose‐dependently left‐shifted the dose‐effect functions of the antinociceptive (7.3‐fold at 3.2 mg/kg CM304) and hypothermic effects of THC. In rats discriminating 3.2 mg/kg THC i.p. from vehicle under a fixed ratio (FR) 10 schedule of food delivery, the cannabinoid CB1 receptor agonist CP55,940 fully substituted for THC, whereas CM304 i.p. produced a maximum of 27 % THC‐lever responding at 17.8 mg/kg. CM304, at doses of 32 and 56 mg/kg, decreased response rate to 24 and 9 %, respectively. No dose of BD1063 (σ1R antagonist), rimonabant, SR144528 (cannabinoid CB2 receptor antagonist), morphine (μ‐opioid receptor agonist), or naltrexone (opioid antagonist) produced greater than 30% THC‐lever responding. Pretreatment with rimonabant (1.78 mg/kg) produced a rightward shift in the dose effect curve of THC (4.5‐fold) and CP55940 (4.5‐fold) while pretreatment with CM304, BD1063, SR144528, and naltrexone had no significant effect on the dose effect curve of THC. The present results may support the development of a σ1R antagonist as an adjunct to cannabinoids for treatment of acute pain without enhancing an undesirable side effect of THC.

Support or Funding Information

This work was supported by National Institute on Drug Abuse grants DA23205 and DA48353.
Methocinnamox Produces Sustained Antagonism of the Antinociceptive Effects of Morphine and Persistent Decreases in [3H]DAMGO Binding to μ Opioid Receptors in Rat Cortex
Authors: Gerak Lisa R. , Gould Georgianna G. , Daws Lynette C. , France Charles P.
Published: April 2020

The opioid crisis continues in the US despite the availability of pharmacotherapies that are effective in some patients. A novel μ opioid receptor antagonist, methocinnamox (MCAM), has a long duration of action in blocking the effects of opioids, which could provide a substantial advantage over currently available medications, particularly for overdose. MCAM has been shown to attenuate the behavioral effects of μ, but not k, opioid receptor agonists, and this antagonism, which can last for several weeks, is thought to be due to pseudoirreversible binding of MCAM to μ opioid receptors. In the current study, a warm water tail withdrawal procedure was used to examine sustained antagonism by MCAM in one group of 8 rats. This study showed that 10 mg/kg MCAM attenuated the antinociceptive effects of morphine (1.78–17.8 mg/kg) 5 and 13 days after MCAM administration with antagonism beginning to wane 21 days after MCAM. In separate groups of 8 rats, that same dose of MCAM decreased [3H]DAMGO binding to μ opioid receptors in rat cortical homogenates measured 1, 5 and 21 days after MCAM injection, indicating that MCAM occupied μ opioid receptors at all time points tested. For example, maximal binding (Bmax) was 28.51 ± 3.33 and 30.96 ± 1.73 fmol/mg protein 5 and 21 days, respectively, after vehicle administration, and 16.03 ± 4.15 and 12.50 ± 1.27 fmol/mg protein 5 and 21 days, respectively, after administration of 10 mg/kg MCAM. Binding affinity (KD) was not markedly changed after MCAM administration. Thus, MCAM produces a sustained decrease in [3H]DAMGO binding to μ opioid receptors in rat cortex that parallels antagonism of the antinociceptive effects of μ opioid receptor agonists. This finding supports the notion that MCAM blocks the behavioral effects of μ opioid receptor agonists by binding pseudoirreversibly to these receptors. The unique binding profile of MCAM to μ opioid receptors could increase the usefulness of MCAM as a treatment for opioid abuse and overdose.

Support or Funding Information

Supported by NIH (R01DA005018 and R01048417) and the Welch Foundation (AQ‐0039).
N‐Acetylcysteine Attenuates Aldosterone induced Vasoconstriction in Individuals with Type 2 Diabetes
Authors: Finsen Stine Louise Hoyer , Mortensen Stefan
Published: April 2020

Introduction

Individuals with type 2 diabetes (T2D) have a high risk of developing cardiovascular disease. Chronic elevation of plasma aldosterone could contribute to endothelial dysfunction within this population by ROS dependent mechanisms. The antioxidant N‐acetylcysteine (NAC) is known for its free radical scavenging properties and NAC may potentiate the activity of nitric oxide (NO) directly by increasing NO‐bioavailability. A shift in the balance between vasoconstrictor and vasodilator responses to aldosterone could be involved in the detrimental actions of aldosterone, and NAC could revoke this effect. The purpose of this study was to investigate if infusion of NAC would improve the vascular response to infused aldosterone in individuals with T2D.

Methods

In 13 subjects with T2D and 14 control subjects (matched on age and activity level), we measured leg hemodynamics during 10 min of incremental doses of infused aldosterone (0.2 and 5 ng/min/L leg volume) into the femoral artery, with and without co‐infusion of NAC (125 mg/kg/hour for 20 min; 25 mg/kg/hour (maintenance dose)). Leg blood flow (LBF; ultrasound Doppler) and arterial blood pressure was measured, and femoral arterial and venous blood samples were collected. Data was analyzed with one‐way repeated measure ANOVA (within groups) and two‐way ANOVA (between groups).

Results

LBF and leg vascular conductance (LVC) decreased during infusion of aldosterone at the high dose (p<0.05) in the individuals with T2D, whereas co‐infusion of NAC attenuated this response. In the control group, there was no change in LBF and LVC during aldosterone infusion independently of NAC infusion. In both groups, venous aldosterone levels increased during aldosterone infusion (p<0.05), and there was no difference between groups in aldosterone concentrations at baseline or during infusions.

Conclusion

These results indicate that T2D is associated with a vasoconstrictor response to physiological levels of infused aldosterone and an increase in NO‐bioavailability attenuates this effect.

Support or Funding Information

Supported by the Independent Research Fund, Denmark and Region of Southern Denmark
Variation of CD36 genotype in Adolescent Females
Authors: Primeaux Stefany D. , Schreiber Allyson L. , Denstel Kara D. , Martin Corby K. , Hsia Daniel , Staiano Amanda E.
Published: April 2020

The pervasiveness of obesity in children and adolescents aged 5 to 19 years has increased by more than 10‐fold over the past 40 years. This profound increase in childhood obesity has become a major public health problem that is predicted to result in multiple obesity‐related comorbidities in adulthood. Therefore, a better understanding of both genetic and environmental factors that contribute to excess weight in adolescents is paramount. Obesity disproportionately affects ethnic minorities, indicating a racial disparity, and a higher percentage of adolescent African‐American females have obesity compared to non‐Hispanic, white adolescent females. Studies suggest that overconsumption of highly palatable, energy dense, high fat foods contributes to the current rates of obesity. Recent clinical studies have linked a single nucleotide polymorphism (SNP), rs1761667 of CD36, to higher rates of obesity and altered fat perception in adults and children. The goal of the current study was to investigate the intersection of genotype and phenotype on the risk for obesity in adolescent African‐American and white females with the AA genotype by investigating differences in dietary intake, food cravings, and body composition. CD36 genotype was determined by an allelic discrimination assay in a subset of adolescent girls (10–16yo; n=142) enrolled in the Translational Investigation of Growth and Everyday Routines in Kids (TIGER Kids) Study. Anthropometrics, 24‐h diet recall, food craving inventory and body composition assessments were conducted for each participant. Expression of the AA genotype was higher in white females (27.7%) compared to African‐American females (15.3%), while expression of the GG genotype was higher in African‐American (44.1%) compared to white (26.5%) females. Preliminary results indicated that BMI percentile and rates of obesity were higher in African‐American females across all genotypes. However, white females with the AA genotype consumed significantly more daily calories, including daily fat (g), carbohydrates (g), and sugar (g) than African‐American females. No differences in daily food intake were detected between African‐American and white females in the AG or GG genotype. Furthermore, no differences were noted in the Food Craving Inventory or in body composition based on race or genotype. Results from this study suggest that the AA genotype of rs1761667 of CD36 in African‐American and white female adolescents may influence overall food intake and macronutrient selection.

Support or Funding Information

USDA 3092‐51000‐056‐04A
Upregulation of Prox‐1, podoplanin and LYVE‐1 in retinal endothelial cells under hyperglycemic conditions
Authors: Eshaq Randa , Warar Ravdeep , Harris Norman
Published: April 2020

Background

Diabetic retinopathy (DR) is a leading cause of blindness among working age adults worldwide. It is marked by changes in the retinal microvasculature including an increase in permeability, vascular blockage, angiogenesis, and alterations in the expression of endothelial cell surface proteins, all of which could be due to retinal endothelial cell dysfunction. Prospero‐related homeobox 1 (Prox‐1) is a transcription factor and a master gene regulator that could repress or induce various genes. Moreover, Prox‐1 has been implicated in regulating genes that are important for the functions of endothelial cells, such as platelet endothelial cell adhesion molecule‐1 (PECAM‐1) and vascular endothelial‐cadherin (VE‐cadherin). We have previously reported a significant decrease in retinal PECAM‐1 levels under hyperglycemic conditions, which could possibly contribute to the development of DR. However, limited studies to date have investigated the hyperglycemia‐induced phenotypic changes occurring to retinal endothelial cells, and how it contributes to DR.

Methods and materials

For our in vitro studies, primary rat retinal microvascular endothelial cells (RRMECs) were grown in normal glucose (NG, 5 mM glucose) or high glucose (HG, 25 mM) media for 1 hour, 24 hours, or six days. Additionally, RRMECs were grown in mannitol, NaCl, L‐glucose, or dextran (25 mM) as osmolar controls. For our animal studies, age‐matched male Wistar rats were injected with either vehicle (control), or streptozotocin (STZ) (diabetic, 30 mg/kg/day) for three consecutive days in a model of type I diabetes. Retinas and plasma were collected eight weeks post‐STZ injection for analysis. Levels of Prox‐1, PECAM‐1, podoplanin, lymphatic vessel endothelial receptor 1 (LYVE‐1), VE‐cadherin, nuclear factor of activated T cells 5 (NFAT‐5) were assessed using western blotting, ELISA, immunofluorescence, and immunoprecipitation assays.

Results

Prox‐1 was expressed in RRMECs under normoglycemic conditions, with its expression significantly increased under hyperglycemic conditions. Interestingly, podoplanin and LYVE‐1, both considered specific lymphatic markers, were expressed in RRMECs under normoglycemic conditions, with their expression significantly increased under hyperglycemic conditions. On the other hand, PECAM‐1 and VE‐cadherin expression was significantly decreased in RRMECs grown under hyperglycemic conditions.

Conclusion

These results are showing, to our knowledge for the first time, the expression of the lymphatic markers Prox‐1, podoplanin, and LYVE‐1 in retinal endothelial cells, and a hyperglycemia‐induced modulation in their expression. Taken together, these data strongly suggest the possible role of Prox‐1 increased expression in the hyperglycemia‐induced downregulation of PECAM‐1 and VE‐cadherin, and the upregulation of podoplanin and LYVE‐1 in the retinal vasculature.

Support or Funding Information

NIH EY025632
Tobacco Smoke Extract Causes Mitochondrial Dysfunction Associated with Myotube Atrophy
Authors: Fitzgerald Liam F. , Thome Trace , Burke Sarah K. , Salyers Zachary R. , Ryan Terence E. , Hepple Russell T.
Published: April 2020

INTRODUCTION

Whereas the link between chronic tobacco smoke (TS) exposure and development of disease (e.g., chronic obstructive pulmonary disease; COPD) is well established, how chronic TS exposure relates to the mitochondrial dysfunction and muscle atrophy seen in COPD remains unclear.

METHODS

We exposed C2C12 myotubes to TS extract (TSE) on day 5 post‐differentiation to determine the impact of TS on mitochondrial function (respiration, reactive oxygen species [ROS] emission and sensitivity to mitochondrial permeability transition [MPT]), and myotube atrophy. Briefly, for respiration experiments myotubes were treated with 0.02% TSE or vehicle (DMSO) for 24 h, and subsequently washed and assessed for maximal uncoupled respiration in an oxygraph using carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone (FCCP; 250 nM – 1.5 μM). For ROS experiments, following 24 h TSE or DMSO treatment myotubes were incubated with the superoxide indicator MitoSox (500 nM) for 15 min and imaged for MitoSox positive area. For MPT experiments, after 24 h TSE or DMSO treatment myotubes were fixed in 4% PFA and incubated with anti‐Apoptosis Inducing Factor (AIF; a protein released from mitochondria during MPT) and NucBlue (to stain nuclei). Images (n=5 per well, 8 wells per treatment) were analysed using Image J to quantify the fraction of AIF co‐localizing with nuclei using the Manders coefficient. For myotube atrophy experiments, 24 h following TSE or DMSO treatment myotubes were fixed, labelled with anti‐sarcomeric myosin, and subsequently imaged to assess myotube area using automated batch processing to avoid human bias. Two‐tailed Students t‐tests were used to evaluate differences between DMSO vs. CSE with an alpha level of 0.05. Data are presented as mean±SD with exact p‐values.

RESULTS

Treatment of C2C12’s with TSE reduced maximal uncoupled respiration (TSE: 312.0±24.0 vs. DMSO: 415.1±45.5 pmoles/sec/μg, p=0.002) and increased MitoSox positive area (TSE: 8,607.0±2,963.1 vs. DMSO: 2,254.7±1,034.7 μm2, p=0.0001); the latter suggesting TSE increases ROS. AIF: nuclear co‐localization was greater in C2C12’s treated with TSE compared with vehicle (Manders coefficient in TSE: 0.66±0.08 vs. DMSO: 0.46±0.05, p=0.0001), suggesting TSE causes MPT. We also observed significantly smaller myotube area in TSE‐treated cells compared with vehicle (TSE: 62,972.9±43,527.5 vs. DMSO: 85,284.9±28,136.1 μm2, p=0.048), consistent with TS inducing myotube atrophy.

DISCUSSION

Our experiments demonstrate that TSE exposure causes significant mitochondrial impairment, as evidenced by reduced respiratory capacity, increased ROS generation, and nuclear co‐localization with a mitochondrial protein released during MPT. Furthermore, TSE treatment caused significant myotube atrophy. These observations are consistent with chronic TS exposure playing an important role in the mitochondrial dysfunction and muscle atrophy seen with the TS‐related disease COPD.
Competing modes of assembly restrict amyloid formation by a neurodegenerative protein, TDP43
Authors: Wu Jianzheng , Halfmann Randal
Published: April 2020

The accumulation of aberrant deposits of specific protein(s) in the brain is a hallmark of many neurodegenerative diseases including amyotrophic lateral sclerosis (TDP43) and frontotemporal lobar degeneration (FUS and/or TDP43). These proteins exhibit “prion‐like” low complexity domains (LCDs), with the defining property of templating their own assembly into highly ordered aggregates known as amyloid. Structural biology investigations from other labs have revealed a multitude of TDP‐43 self‐assembly modes in vitro, ranging from cooperatively folding alpha‐helices to amyloid‐like beta‐sheets of varying stability. However, the extent to which these assembly modes occur in the intracellular environment, and their full relationship to physiological and/or pathological self‐assembly, have not yet been feasible to study.

To interrogate mechanistic features of amyloid nucleation by the TDP43 LCD in the cellular milieu, I employed a recently developed FRET‐based flow cytometry approach along with rational mutagenesis informed by existing structural models. I observed that the LCD transitions from monomers to small condensates with a prominent phase boundary. I further found that these condensates were qualitatively distinct from amyloid, and that amyloid occurred probabilistically and only in cells harboring pre‐existing amyloids of other pathogenic proteins.

Mutations designed to enhance or disrupt alpha‐helicity exhibited corresponding effects on non‐amyloid condensation, confirming the role of this assembly mode in non‐pathogenic aggregation. Remarkably, amyloid formation exhibited the opposite dependence, with strong alpha‐helix‐promoting mutations eliminating amyloid formation altogether. Mutations designed to probe the role of cooperative beta‐sheet formation revealed that amyloid‐like interactions either promote or inhibit amyloid formation depending on their stability. Importantly, I verified that the full‐length protein follows the same aggregation pathway, but does so at lower concentrations due to additional homotypic interactions mediated by non‐LCD domains. In summary, my data suggested the tendency of TDP43 toward amyloid is balanced by competing alternative modes of self‐assembly.

Support or Funding Information

NIH Director’s Early Independence Award DP5‐OD009152 (to RH), the University of Kansas Alzheimer’s Disease CenterStowers Institute for Medical Research
Treatment with Sacubitril‐Valsartan Reduces Muscle Sympathetic Nerve Activity and Improves Functional Capacity in Heart Failure Patients with Reduced Ejection Fraction
Authors: Bunsawat Kanokwan , Ratchford Stephen M. , Alpenglow Jeremy K. , Stehlik Josef , Smith Adam , Richardson Russell S. , Wray D. Walter
Published: April 2020

Background

Chronic sympathetic overactivity and reduced functional capacity, characteristics of heart failure with reduced ejection fraction (HFrEF), are associated with poor prognosis and an increased risk of mortality in this patient group. Sacubitril‐valsartan, a recently approved angiotensin receptor neprilysin inhibitor (ARNI), reduces the risk of death from cardiovascular causes or hospitalization for heart failure in patients with HFrEF, but the mechanisms underlying its benefits are not fully understood. This study tested the hypothesis that treatment with sacubitril‐valsartan reduces muscle sympathetic nerve system activity and improves functional capacity in patients with HFrEF.

Methods

In nine Class II and III patients with HFrEF (69±3 yrs; 28.6±1.2 kg/m2), measurements of muscle sympathetic nerve activity (MSNA), functional capacity (six‐minute walk test (6MWT) distance), and blood pressure (BP) were performed at baseline prior to being transitioned to sacubitril‐valsartan. The measurements were repeated after two months of treatment with sacubitril‐valsartan.

Results

Sacubitril‐valsartan treatment reduced MSNA burst frequency (baseline: 43±3 bursts/min; 2‐month: 36±3 bursts/min, p=0.05) and burst incidence (baseline: 68±5 bursts/min; 2‐month: 55±5 bursts/min, p=0.02), as well as improved 6MWT distance (baseline: 446±26 m; 2‐month: 500±30 m, p<0.001). The changes in MSNA burst frequency and burst incidence were not correlated with the change in 6MWT distance (r=0.13 and r=0.11, respectively; both p>0.05). No changes in BP were observed with the treatment (p>0.05).

Conclusion

Sacubitril‐valsartan reduced sympathetic nervous system activity and improved functional capacity in patients with HFrEF, providing new evidence for the potential favorable effect of this new drug class on autonomic and functional outcomes in this patient group.

Support or Funding Information

This project is funded in part by the National Institutes of Health (HL118313; D.W.W., R56AG057584; R.S.R., T32HL139451; R.S.R. (recipient) and K.B. (trainee)), the U.S. Department of Veterans Affairs (I01RX001311; D.W.W., I01RX001697; R.S.R., I01RX002323; R.S.R., I21RX001433; R.S.R., I01RX000182; R.S.R.), and the American Heart Association (18POST33960192; K.B.).
The Role of Mitochondrial‐derived Reactive Oxygen Species in Non‐Invasive Anterior Cruciate Ligament Injury
Authors: Kwon Oh Sung , Davi Steven M. , White McKenzie S. , Lepley Lindsey K.
Published: April 2020

BACKGROUND

Anterior cruciate ligament (ACL) injury results in protracted quadriceps atrophy, however the mechanisms that drive atrophic pathways remain undefined. Mounting evidence has revealed that mitochondrial dysfunction and redox disturbances are causal events in the initiation of muscle atrophy, yet the extent to which mitochondria damage play a key role in quadriceps muscle atrophy after ACL injury has yet to be explored.

PURPOSE

Using a pre‐clinical animal model of ACL injury, a time course study was performed to investigate the role of mitochondria function and Reactive Oxygen Species (ROS) after ACL injury.

METHODS

48 Long Evans rats (n=8 per group; 4m/4f) underwent non‐invasive rupture of the right ACL and were euthanized at 1, 3, 7, 14, 28, 56 days post‐injury. 8 rats (4m/4f) served as healthy controls (HC). Mitochondrial respiration was measured, by high‐resolution respirometry, in permeabilized muscle fibers from the right quadriceps. ROS production was determined using Amplex Red assays. ROS production was determined using Amplex Red assays. VL weight was normalized to total body mass for measuring muscle mass loss. One‐way ANOVAs with Bonferroni post‐hoc were used to determine differences between groups (P < 0.05).

RESULTS

Reductions in complex I + II state 3 respiration were observed at 7 and 56 days post‐injury (HC: 33.92 ± 4.26 pmol·s−1·mg−1; 7D: 15.95 ± 1.38 pmol·s−1·mg−1; 56D: 18.80 ± 2.15 pmol·s−1·mg−1; F = 5.99, P = 0.002 and P = 0.015 respectively). State 4 respiration did not differ between groups (P > 0.05). Respiratory Control Ratio (RCR), defined as respiration in state 3 divided by respiration in state 4, significantly decreased 7 through 56 days post‐injury (HC: 4.00 ± 0.13; 7D: 1.54 ± 0.16; 56D: 1.82 ± 0.20; F = 13.29, P = 0.001 and P = 0.001 respectively) along with increased mitochondrial ROS production 7 through 56 days post‐injury (HC: 10.16 ± 0.41 pmol·s−1·mg−1; 7D: 12.23 ± 0.54 pmol·s−1·mg−1; 56D: 26.83 ± 0.54 pmol·s−1·mg−1; F = 178.05, P = 0.001 and P = 0.001 respectively). VL atrophy was observed at 7 and 14 days post‐injury (HC: 3.52 ± 0.08 mg·g−1; 7D: 3.14 ± 0.07 mg·g−1; 14D: 3.18 ± 0.07 mg·g−1; F = 5.06, P = 0.013 and P = 0.044 respectively).

CONCLUSION

These results demonstrated that mitochondrial dysfunction and redox disturbances contribute to ACL injury‐induced quadriceps atrophy and that mitochondria are an important source of muscular ROS production after ACL injury.

Support or Funding Information

NIH grant K01AR071503 from NIAMS
MERS‐CoV infection is associated with downregulation of genes encoding Th1 and Th2 cytokines/chemokines and elevated inflammatory innate immune response in the lower respiratory tract
Authors: Alosaimi Bandar A.
Published: April 2020

MERS‐CoV, a highly pathogenic virus in humans, is associated with high morbidity and case fatality. Inflammatory responses have a significant impact on MERS‐CoV pathogenesis and disease outcome. However, CD4+ T‐cell induced immune responses during acute MERS‐CoV infection are barely detectable, with potent inhibition of effector T cells and downregulation of antigen presentation. The local pulmonary immune response, particularly the Th1 and Th2‐related immune response during acute severe MERS‐CoV infection is not fully understood. In this study, we offer the first insights into the pulmonary gene expression profile of Th1 and Th2‐related cytokines/chemokines (Th1 & Th2 responses) during acute MERS‐CoV infection using RT2 Profiler PCR Arrays. We also quantified the expression level of primary inflammatory cytokines/chemokines. Our results showed a downregulation of Th2, inadequate (partial) Th1 immune response and high expression levels of inflammatory cytokines IL‐1α and IL‐1β and the neutrophil chemoattractant chemokine IL‐8 (CXCL8) in the lower respiratory tract of MERS‐CoV infected patients. Moreover, we identified a high viral load in all included patients. We also observed a correlation between inflammatory cytokines, Th1, and Th2 downregulation and the case fatality rate. Th1 and Th2 response downregulation, high expression of inflammatory cytokines, and high viral load may contribute to lung inflammation, severe infection, the evolution of pneumonia and ARDS, and a higher case fatality rate. Further study of the molecular mechanisms underlying the Th1 and Th2 regulatory pathways will be vital for active vaccine development and the identification of novel therapeutic strategies.

Support or Funding Information

The authors thank the Research Center at King Fahad Medical City for funding this study (Grant No 017‐066).