bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Teaching Methodology and Course Duration on Student Performance at Different Assessment Types across Different Cognitive Levels as defined by Bloom’s Taxonomy
Authors: Sunba Khaleel , Rogers Kem
Published: April 2020

The University of Western Ontario currently offers a third year systemic human anatomy course (ANATCELL 3319) in face to face (F2F) and online sections during the fall/winter intake (F/W). While F/W sections share the same lecture and assessment materials, F2F students attend weekly one‐hour cadaveric labs while online students attend weekly interactive video conference labs. During the summer intake, the course is offered in the online section only, with labs given twice a week. The assessments consist of multichoice (MCQ) and short answer questions. Our previous publication
Sunba & Rogers (2018) showed that F/W F2F scored higher in their MCQ assessments than F/W online students at different Bloom’s Taxonomy levels. In addition, F/W online students scored higher than summer online students in the shared MCQ.

In the current study, we further compared the performance of students in MCQ against their performance in short answer questions, in total and at different Bloom’s Taxonomy levels within/against the assessment type(s). For F/W sections (F2F students n=142; online students n=172), there were 300 MCQ (knowledge (n= 149), comprehension (n= 120), application (n= 21), and analysis (n= 10)) and 118 short answer questions (knowledge (n= 88), comprehension (n= 18.5), application (n= 10.5), and analysis (n= 1)). Both groups scored higher in their MCQ than short answer questions, in total and at most cognitive levels (p ≤ 0.05). F/W F2F students scored higher than F/W online in both assessment types and at most levels. The variance across levels differed based on the assessment type. For example, All F/W students scored the highest in comprehension MCQ but the lowest in short answer comprehension questions. This indicates that a change of assessment type influences the way in which students answer.

With summer online students (n=44), we compared 169 MCQ (knowledge (n=79), comprehension (n=74), application (n=9) and analysis (n=7)) with 59 short answer questions (knowledge (n= 34), comprehension (n=12), application (10) and analysis (n=3)). Students scored higher (p ≤ 0.05) in their MCQ total, comprehension MCQ, and application MCQ than the adjacent levels of the short answers. Unlike their F/W counterparts, students scored lowest in their MCQ comprehension (p<0.001) in relation to the other levels.

Our findings indicate that teaching F2F is likely to improve student performance in terms of both overall grades and cognitive ability. In addition, students are likely to score higher in MCQ than short answer questions which is indicative of a reliance on cues within MCQ (especially comprehension) to help them overcome their lack of information. Finally, the performance of summer students, particularly in relation to comprehension‐type questions which rely on memorisation, indicates that the spacing of course material may help in achieving higher grades.

Support or Funding Information

Dr. Kem Roger
High‐throughput Targeted Lipidomics Analysis of Dihydroceramide Desaturase‐1 (DES1) Knockout Mice
Authors: Pearson Mackenzie Jo , Kapil Santosh J. , Ubhi Baljit J. , Summers Scott
Published: April 2020

Retention time scheduling for MRM transitions during targeted assays enables more compounds to be quantified with higher quality results. In this assay, MRMs to quantitate over 1150 lipid molecular species were combined into a single assay. The selected amide column chemistry provides reproducible isomer separation based on lipid class. This Liquid Chromatography separation strategy, is then coupled with the time scheduled targeted assay. To validate this method, we selected DES1 knockout mice to measure lipid changes in liver and adipose tissues. The MRM™ Pro Builder tool (SCIEX) was then used to optimize window width and dwell weight to enhance sensitivity and coverage of method. This method provides extensive, reproducible lipid coverage in complex biological samples like tissue, cells, and plasma.

Amide column chemistry was chosen for lipid class separation and minimize isomeric interference. The target list of lipids is comprehensive, covering most major lipid classes and categories, and MRMs were selected to cover lipids containing fatty acids with 14 to 22 carbons and 0 to 6 double bonds. The method is customizable, so new lipid categories, classes and molecular species can be added to the MRMs list. This method provides quantitative measurement of over 1150 different lipid molecular species in a rapid, highly reproducible manner. The sMRM Pro Builder template which was developed to streamline the method optimization process, enables assigning the retention time, optimize dwell weight and set window size per MRM to enhanced coverage and sensitivity of the method. This optimization improved results quality especially on low abundant lipids.

Liver and eWAT tissues were harvested from dihydroceramide desaturase‐1 (DES1) knockout mice. DES1 is the enzyme responsible for inserting the 4,5‐trans‐double bond into the sphingolipid backbone causing the dihydroceramide conversion to ceramide. While both of these classes of lipids are lower in abundance in the chosen tissues, this method shows significant changes in these lipid classes in a quantitative manner. However, lipids from another 17 classes were not changing.

Lipid standards from 19 different classes, which are either heavy isotopic labeled lipids or odd chain lipids, served as internal standard. This method provided extensive lipid class coverages including, CE, CER, DCER, HCER, LCER, TAG, DAG, MAG, LPC, PC, LPE, PE, LPG, PG, LPI, PI, LPS and PS.
GONADAL HORMONES MODULATE HIPPOCAMPAL NUCLEAR DEK EXPRESSION IN ADULT MICE AND RATS
Authors: Greene Allie , Balmer Nikolaus , Estrada Christina , Vinnedge Lisa Privette , Solomon Matia
Published: April 2020

Alzheimer’s Disease (AD) is currently the 6th leading cause of death in the United States, and is the most common cause of dementia. Women are more likely than men to be diagnosed with AD. The cause of this discrepancy is not known; however, the loss of circulating estrogen after menopause is hypothesized to be a leading factor for women’s increased risk of AD. Estrogen is considered neuroprotective; it can ameliorate the effects of brain injury, contribute to DNA repair in mitochondria, and maintain synapse number and spine density in the hippocampus. Our laboratory is investigating the role of an estrogen receptor alpha (ERα) target gene, DEK, as a novel player in Alzheimer’s disease and age‐related dementias. DEK is a nuclear chromatin phosphoprotein that is expressed throughout the brain, and is prominently expressed in the hippocampus, a brain region important for learning and memory. Preliminary data indicate that DEK loss in vitro and in vivo results in AD and dementia‐like phenotypes, such as cell death, increased total tau and phosphorylated tau expression, DNA damage, and decreased hippocampal microtubule associated protein (MAP2) expression. These findings suggest a neuroprotective role of DEK. Given that DEK is an ERα target gene, we postulated that DEK expression in the brain would be modulated by circulating levels of estrogen. We specifically hypothesized that loss of estrogens would decrease DEK expression in the hippocampus. To study this, we used a surgical rodent model of menopause, ovariectomy (OVX), to remove the main source of circulating estrogen in female rats. OVX was performed in 3‐month‐old Sprague Dawley rats. Cell counts and optical density analysis were done using NIH Image J. We found that following two months ovariectomy, DEK expression was decreased in the hippocampus of adult female rats. Besides estrogen, the gonadal hormones testosterone and progesterone can exhibit neuroprotective effects as well. Therefore, we included males in our next experiment in order to determine if gonadal hormone depletion in males would also affect DEK expression. Similar to rats, gonadectomy decreased hippocampal DEK expression in adult male and female mice. Interestingly, OVX significantly decreased DEK expression relative to castration in the CA1 and CA3 regions of the hippocampus. These findings suggest that the impact of gonadal hormone loss on DEK expression is evident across species. We do not yet know the effect of specific gonadal hormonal administration (e.g., E2 or testosterone) on central DEK expression in castrated males and OVX females; therefore, future studies will explore this. Our results suggest that biological factors such as circulating hormones can affect the expression of DEK, whose deficiency may be linked with neurodegeneration and brain aging in dementia and AD.

Support or Funding Information

L.I.F.E. Foundation
Accelerating SWATH® Acquisition for Protein Quantitation – Up to 100 Samples per Day
Authors: Arnold Randy J. , Hunter Christie , Morrice Nick , Demianova Zuzana
Published: April 2020

The combination of microflow LC with SWATH Acquisition for large scale quantitative proteomics studies is becoming increasingly more widespread, due to the improved robustness and throughput obtained relative to the traditional nanoflow LC approach. High quality quantitative datasets have been generated using a standard 1 hour gradient, demonstrating large numbers of proteins quantified routinely.

The impact of gradient length on protein identification results using data dependent acquisition (DDA) was also explored previously and summarized here. Next, an exploration into the impact of gradient length on proteins quantified using data independent acquisition (DIA) was undertaken to provide researchers expanded workflow options with microflow SWATH acquisition. Using microflow liquid chromatography gradients as short as five minutes (total run time <15 mins), SWATH acquisition parameters were first optimized then used on multiple TripleTOF® 6600 systems to study impact of shortened separations on number of proteins quantified.

Testing on multiple instruments with different complex matrices, it was observed that the number of proteins quantified using SWATH acquisition for the fastest gradients was over 1300 proteins for multiple complex matrices from 1 μg protein load. When combined with larger ion libraries, over 2100 proteins were identified and quantified in a 5 min gradient from a complex HEK digest (1 μg). The combination of microflow LC with the high MS/MS acquisition rates of the TripleTOF® 6600 system enables high‐throughput yet comprehensive analysis for proteomics samples, at rates approaching 100 samples per day.
Evaluation of Adherence to Dietary Guideline for Patients with Type 2 Diabetes Mellitus According to Age
Authors: Jang Haeun , Park Kyong
Published: April 2020

Background

This study aimed to compare adherence to dietary guidelines between elderly and non‐elderly patients with type 2 diabetes mellitus (T2DM) in Korea.

Methods

Data of 4577 patients with T2DM were collected from the 1998–2015 Korea National Health and Nutrition Examination Surveys. The patients were classified into two groups based on age: non‐elderly T2DM group comprising patients aged 30–64 years and elderly T2DM group comprising patients aged ≥65 years. Adherence to dietary guidelines was assessed using the Korean Diabetes Association‐Korean Ministry of Health and Welfare (KDA‐KMHW) index, comprising six components of dietary guidelines for T2DM patients. Multivariate generalized linear regression analysis was performed to analyze the KDA‐KMHW index scores.

Results

The adherence levels to the individual components of the KDA‐KMHW index were mostly lower in non‐elderly T2DM patients (P < 0.001) than elderly group, except for moderate carbohydrate consumption. The total KDA‐KMHW index score was significantly lower in non‐elderly T2DM group than in the elderly T2DM group (P < 0.001).

Conclusions

The study results suggest the need for developing patient‐specific education programs that focus on adherence to dietary guidelines, particularly for non‐elderly patients, to adequately intervene with the difficulties experienced in T2DM dietary management.

Support or Funding Information

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (grant number: NRF‐2017R1A1A3A04069759).
Differential Tissue‐Specific Effects of Fructokinase Blockade in Sugar‐Induced Metabolic Syndrome
Authors: Lanaspa Miguel , Andres-Hernando Ana , Johnson Richard J.
Published: April 2020

The intake of fructose and the fructose‐containing sugars, sucrose and high fructose corn syrup, are strongly associated with the development of obesity and diabetes. Here we tested the hypothesis that the metabolism of fructose is the key feature whereby these added sweeteners cause metabolic syndrome. To this end, metabolic syndrome was induced by administering fructose‐glucose (10 or 30%) solutions in the drinking water. Of interest, we found that glucose enhances fructose intake and transport and that the blockade of fructose metabolism in fructokinase deficient (KHK‐A/C KO) mice is sufficient to fully prevent sugar‐induced metabolic syndrome. Our data indicate that this protection is not only due to reduced fructose metabolism but also to decreased preference and intake of sugar. Furthermore, and by using tissue specific KHK‐A/C KO mice, we determined that the reduction in sugar preference was mainly driven by the deficiency of KHK in the jejunum. However, gut‐specific KHK‐A/C KO mice showed enhanced risk for metabolic syndrome when equal intake of fructose was consumed due to increased fructose delivery to the liver. In contrast, liver specific KHK‐A/C KO mice continued to show a preference for sugar despite being protected from metabolic syndrome. In conclusion, these studies document a pivotal role for fructose metabolism in the mechanism by which added sweeteners cause metabolic syndrome. Intestinal fructose metabolism plays a key role in sugar intake whereas liver fructose metabolism drives fatty liver, obesity and diabetes indicating that metabolic syndrome is a hepatocyte‐centered condition.

Support or Funding Information

This work has been supported by NIH Grants 1RO1DK105364‐01A1 (to M.A.L. and R.J.J.) and 1R01DK108859 (to M.A.L.)
A Quantitative Proteomic Approach for Elucidating the Effects of Antidiabetic Drugs on Coronary Artery Endothelial Cells
Authors: Rodríguez Cindy Manríquez , Ulloa Aldo Moreno
Published: April 2020

Individuals with type 2 Diabetes Mellitus (DMT2) possess an elevated risk of mortality due to cardiovascular disease (CVD). Although there is available a repertoire of antidiabetic drugs (ADD) to treat the disease, mortality risk reduction remains an elusive goal despite improvement in glucose metabolism by drug therapy. Moreover, some drugs further increase the CVD risk in subjects with T2DM, while others appear to partially reduce the risk. Hence, new approaches and thinking are needed to explore this phenomenon beyond the classical drug’s antihyperglycemic effects and principal organs involved in glucose metabolism. In this study, we explored the effects of selected ADD (with epidemiological CVD evidence in the clinic) on Human Coronary Artery Endothelial Cells (HCAEC) —involved in the development of CVD— using a quantitative SWATH‐based proteomic and comprehensive bioinformatic approach. To create a template or network of pathways linked to cell damage in HCAEC, we generated a list of proteins modulated (Up and down‐regulated) by the treatment with the pro‐inflammatory cytokine TNF‐a and performed a Functional Enrichment Analysis against REACTOME and KEEG Databases using ClueGO software. We further expanded our network by utilizing reported data of endothelial cells treated with cytokine IL‐1B. ADD treatment consisted of cells incubated with physiological concentrations of either metformin (100 μM), rosiglitazone (1 μM), empagliflozin (1 μM), saxagliptin (1 μM), and vehicle (DMSO) for 48 h. The list of proteins modulated by the ADD was analyzed by ClueGO using as a template the pathway network of cytokines. Our results hint at anti‐inflammatory actions of all ADD tested, but at different levels within the immune system network. Rosiglitazone, followed by saxagliptin, modulated more proteins linked to anti‐inflammatory actions compared to the rest of the drugs. Neutrophil degranulation, signaling by interleukins, and interferon signaling were modulated by rosiglitazone and saxagliptin, while the rest of the drugs modulated less or none of these specific pathways. Also, ADD down‐modulated pathways associated with the metabolism of proteins and angiogenesis. In conclusion, our study provides insight into the pathways on HCAEC modulated by drugs used to treat subjects with T2DM. Further, our results contribute to the understanding of molecular mechanisms, that could explain the clinical effects noted with some ADD for which there is no clear mechanistic data beyond their classical antihyperglycemic effects.

Support or Funding Information

Research funded by an Internal Project to AMU and a Graduate Scholarship from the Mexican National Council for Science and Technology (CONACyT) to CMM
Transcutaneous Electrical Stimulation of the Vague Nerve X Depressive Symptoms in Patients with Metabolic Syndrome
Authors: Fernandes Daniella Marques , de Moraes Tércio Lemos , Costa Fernando Oliveira , Cabral Dannielly Gomes , de Albuquerque Natália Cristina Mesquita , Coelho Fernando Morgadinho Santos , Consolim-Colombo Fernanda Marciano
Published: April 2020

Currently, some techniques such as nerve stimulation have been used as a treatment to reduce inflammation levels in the organism. Inflammation is present in both metabolic syndrome and depression. In brain stimulation through the vagus nerve, the main neurotransmitter acetylcholine modulates the actions of various peptides, hypothalamic incretins and cholecystokinin, consequently modulating the actions of the Autonomic Nervous System. According to the World Health Organization, depression occurs in people of all genders and ages, affects 350 million people worldwide, is among the leading causes of disability. This study aimed to verify the impact of transcutaneous electrical stimulation of the vagus nerve on depressive symptoms in patients with metabolic syndrome. This is the analysis of patients with metabolic syndrome who underwent transcutaneous electrical stimulation in the left ear once a week for 30 minutes for a period of 8 weeks. Pre‐ and post‐Transcutaneous Electrical Stimulation analyzes were performed through interviews with the application of three psychometric depression assessment scales of Beck I (IDB), Hamilton (HAM‐D) and Montgomery‐Asberg (MADRS). Nine patients with Metabolic Syndrome from the city of São Paulo, Brazil, were interviewed. Of the 09 respondents, the study showed in the IDB Scale there are 06 (66.6%) with depression, HAM‐D 08 (88.8%) and MADRS all 08 (88.8%) with depression before Transcutaneous Electrical Stimulation. The results showed a significance level of 55.5% for depression scores after Transcutaneous Electrical Stimulation. A high prevalence of depressive symptoms was observed in the study sample. The research allowed us to reflect on the Transcutaneous Electrical Stimulation of the vagus nerve as an alternative treatment for individuals with Metabolic Syndrome and depressive symptoms aiming to prevent or mitigate the effects of these pathologies.

Support or Funding Information

Without funding.
Mutation‐Dependent Capacity of the ER Proteostasis Network to Quality Control Disease‐Causing Collagen Misfolding
Authors: Bikovtseva Agata , Doan Ngoc-Duc , DiChiara Andrew S. , Del Rosario Amanda , Shoulders Matthew D.
Published: April 2020

Osteogenesis imperfecta (OI) is an inherited disease most commonly caused by autosomal dominant mutations in collagen type‐I. OI‐causing mutations are found in both the triple helical region and the C‐propeptide region of collagen‐I. Mutations in the triple helical region cause disruption to triple helical folding, stability, and structure. Mutations in the C‐propeptide region, the C‐terminal nucleation domain of collagen‐I, disrupt the initial assembly steps in collagen folding. Currently, the mechanism by which the cellular protein folding machinery recognizes and processes these types of misfolded variants of collagen‐I is not well‐understood. Here, we report how the endoplasmic reticulum (ER) differentially processes normal and misfolding, OI‐causing collagen‐I variants. We employed state‐of‐the‐art, quantitative mass spectrometry‐based proteomic methods to determine the full complement of proteostasis network components that differentially engage wild‐type (wt) and several OI‐causing mutants of collagen‐I. We identified >75 putative proteins that might play a role in collagen‐I proteostasis, in addition to the currently known collagen‐I interactors. Interestingly, we found that while there is no significant difference in the interactomes of wild‐type (wt) and triple‐helical mutant collagen‐I variants, mutant C‐propeptide collagen‐I is well‐recognized by a number of components of the ER proteostasis network. These results indicate that the cell can specifically recognize collagen C‐propeptide misfolding, but has limited capacity to address triple‐helix domain misfolding. Our findings contribute to an understanding of how the proteostasis network reacts to different types of collagen folding defects and maintains proper collagen folding and secretion.
Effects of Central Intervention of Tumor Necrosis Factor‐α Converting Enzyme (TACE) on Neuroinflammation and Cardiac Function in Heart Failure
Authors: Bell Balyssa , Yu Yang , Felder Robert , Wei Shun-Guang
Published: April 2020

Central nervous system inflammation has been implicated in the neurohumoral activation and disease progression in experimental animal models of heart failure (HF). The pro‐inflammatory cytokine tumor necrosis factor‐α (TNF‐α) increases in cardiovascular/autonomic brain regions including the paraventricular nucleus of the hypothalamus (PVN) and subfornical organ (SFO), and contributes significantly to the augmented sympathetic nerve activity in HF. TNF‐α is initially synthesized as a membrane bound protein that is proteolytically cleaved by TNF‐α converting enzyme (TACE), also known as a disintegrin and metalloprotease 17 (ADAM17), to become the fully functional inflammatory mediator. Our previous study demonstrated that TACE has elevated expression in the PVN and SFO and mediates TNF‐α‐induced sympathetic excitation and hemodynamic responses in HF rats. However, whether inhibition of TACE in the brain has a protective effect on central inflammation and cardiac function in HF remains unclear. We hypothesized that inhibition of TACE activity in the brain reduces central inflammation and improves left ventricular (LV) function during HF. Adult male Sprague‐Dawley rats were studied 4–5 weeks after induction of HF by coronary artery ligation. TACE activity was chronically inhibited specifically in the brain by infusion of the TACE inhibitor TAPI‐1 (30μg/day) via an osmotic minipump connected to an intracerebroventricular (ICV) cannula. Treatment with TAPI‐1 or vehicle began immediately following the myocardial infarction. At the end of experiments, LV function was assessed by echocardiography and tissues were collected for molecular analysis. In HF rats‐treated with TAPI‐1, the LV ejection fraction (26.8±0.9% vs 34.8±6.4%) was improved, although this did not reach statistical significance vs. HF treated with vehicle. Wet lung weight to body weight ratio also showed a trend towards being improved in TAPI‐1 treated animals (6.3±0.3mg/g vs. 4.8±0.4mg/g, vehicle vs. TAPI‐1, p=0.32). Additionally, TAPI‐1 treatment significantly decreased mRNA levels of proinflammatory mediators including TNF‐α, IL‐1β, and TACE in the SFO (TNF‐α: 0.28±0.10, p<0.05; IL1‐β: 0.25±0.11, p>0.05; TACE: 0.68±0.14, p<0.05, fold change vs. vehicle) and resulted in a trend toward reduced levels of these measurements in the PVN (TNF‐α: 0.52±0.13, p=0.24; IL1‐β: 0.58±0.11, p=0.21; TACE: 0.87±0.11, p=0.43, fold change vs. vehicle). TNF‐α expression was also reduced in both the superior cervical (0.35±0.07, p<0.05, fold change vs. vehicle) and stellate ganglia (0.44±0.14, p<0.05 fold change vs. vehicle). Taken together, these data suggest that central inhibition of TACE activity diminishes the inflammatory state in the brain and improve cardiac function in HF.
Effect of sulfated galactans from red seaweed Gracilaria fisheri on extracellular matrix production in human dermal fibroblast
Authors: Pratoomthai Benjamart , Gangnonngiw Warachin , Songtavisin Thanitsara , Wongprasert Kanokpan
Published: April 2020

Fibroblasts are derived from mesenchymal stem cells that play an important role in produce the extracellular matrix (ECM). ECM creates a tissue microenvironment for regulation of cell signaling. The loss of ECM plasticity is associated with several pathologies, especially those involving chronic inflammation, therefore, the ECM represents a potential therapeutic target for certain conditions. Sulfated galactans was extracted from red seaweed Gracilaria fisheri that previous studies have been reported about its activities on anti‐oxidant, anti‐tumour, anti‐melanogenesis, and anti‐bacteria. Therefore, the aim of this study was to investigate the effect of potential of sulfated galactans from red seaweed G. fisheri on enhancing the ECM production. Cytotoxicity was tested following determination of ECM‐related gene and protein expression on human dermal fibroblast (HDF) by quantitative RT‐PCR and enzyme‐linked immunosorbent assay (ELISA), respectively. The result showed that sulfated galactans not effects to survival rate of HDF. Additionally, we found that sulfated galactans was able to modulate ECM production by regulating the collagen, elastin, hyaluronic acid, MMP‐1, and TIMP production. The present results demonstrating the effects of a natural multi‐component compound on ECM composition, highlighted the possibility of pharmacologically modulating ECM molecules. The recovery and the maintenance of ECM homeostasis might be considered as a potential therapeutic goal to ameliorate pathological conditions.

Support or Funding Information

This work was financially supported by Navamindradhiraj University Research Fund.
Molecular Hydrogen Manages Mitochondrial Proteostatic Stress and Induces Cellular Response
Authors: Hasegawa Tomoya , Ito Mikako , Hasegawa Satoru , Takeda Kouki , Nishiwaki Hiroshi , Negishi Shuto , Ohno Kinji
Published: April 2020

Molecular hydrogen (H2) has an anti‐oxidant effect on hundreds of animal models. However, the exact molecular mechanisms of molecular hydrogen still remains elusive. Mitochondrial unfolded protein response (mtUPR) is a known stress response to perturbed mitochondrial proteostasis caused by oxidative stress or mitochondrial dysfunction.

We examined the effects of molecular hydrogen on mtUPR by examining mtUPR‐related protein expression in vivo and in vitro. Mice drinking hydrogen water showed higher expression of the HSP60 chaperone protein, which is an mtUPR marker, as well as other mtUPR‐related proteins including protein kinase R (PKR), phosphorylated eukaryotic translation initiation factor 2a (eIf2a), and activating transcription factor 5 (ATF5). Incubation of cultured cells in 10% hydrogen gas increased expression of HSP60 and some mtUPR‐related proteins in four human cell lines (A549, HeLa, HT1080, and PC3 cells, which are called “responders” hereafter), but not in three other human cell lines (HCT116, HepG2, and SH‐SY5Y cells, which are called “non‐responders”). Flowcytometric analysis disclosed that higher mitochondrial mass, higher superoxide production, higher mitochondrial membrane potential in “responders” compared to “non‐responders’. Similarly, measurement of the oxygen consumption rate revealed high spare capacity of the electron transport complex (ETC) activity in “responders”. Indeed, the gene set enrichment analysis (GSEA) of gene expression profiles in gene expression omnibus (GEO) of two groups of cell lines demonstrated that ETC‐related pathways were upregulated in “responders”. In addition, quantification of DNA synthesis by BrdU assay revealed that incubation of cultured cells in 10% hydrogen gas for 48 h induced cell proliferation, and rescued proliferation blockage by rotenone in “responders”.

We report that hydrogen induces mtUPR and cellular proliferation in cell lines with higher mitochondrial ETC activity. Molecular hydrogen ameliorates a variety of diseases including Parkinson’s disease, myocardiac or cerebral infarction, and inflammatory diseases. Molecular hydrogen is thus likely to manage mitochondrial stress through activation of mtUPR, and exerts its effects on these oxidative stress‐related diseases.

Support or Funding Information

This research was supported by grants‐in‐aids from MEXT, MHLW, and AMED of Japan.
A Novel Interaction Reveals LC8 as a Potential Hijacking Point for a Respiratory Virus
Authors: Rodriguez Joaquin , Howe Jesse James , Donner Brianna , Barbar Elisar
Published: April 2020

Lower respiratory tract infections (LRTIs) are a major cause of death among immunocompromised patients. Human parainfluenza viruses (hPIVs) are non‐segmented, negative‐stranded RNA viruses that frequently give rise to these infections. In order to transcribe genes or replicate their genomes, these viruses encode three major genes: a nucleocapsid (N), a phosphoprotein (P) and a large RNA‐dependent RNA polymerase (L). P is indispensable for transcription and replication as it positions L at the replication/transcriptional start site and recruits accessory proteins. Phosphoprotein structure has been characterized in related viruses such as measles and mumps, where P has been shown to be an oligomer, featuring an intrinsically disordered region as well as a flexible multimerization domain. However, P has not been well studied in the hPIVs and its oligomerization state and dynamics remain unclear. In this study, we show that the phosphoprotein of hPIV type 3 (hPIV3‐P) interacts with host ‘hub’ protein LC8 resulting in structural changes. LC8 is known to bind over a hundred different proteins in eukaryotic cells, facilitating a functional structure. LC8 preferentially binds to intrinsically disordered regions within binding partners which ultimately results in self association, support, oligomerization or other structural rearrangements required for proper function.

We have identified two conserved potential LC8 binding sites within hPIV3‐P using the LC8Pred algorithm. We recombinantly expressed and purified a construct featuring the coiled coil domain of the protein and the LC8 binding site in closest proximity to it. LC8 binding was confirmed via pulldown assays, isothermal titration calorimetry (ITC) and size exclusion chromatography (SEC). Further, we characterized the stability of the coiled coil domain using circular dichroism spectroscopy (CD) and the oligomerization state of the protein bound and unbound to LC8 using analytical ultracentrifugation (AUC). Our results show a well‐defined novel interaction that might be indispensable for phosphoprotein activity and overall viral viability. This adds to the evidence that LC8 is a common target for viral hijacking due to its structural properties.

Support or Funding Information

This work is funded by NSF MCB 1617019
Carbon tetrachloride‐induced hepatotoxicity in weaning and adult mice
Authors: Son Seung Won , Choi Hun Ji , Jung Young-Suk
Published: April 2020

Age‐related differences in drug‐induced liver injury (DILI) have been reported, but DILI in children has been not fully understood as compared to that in adults. In the present study, male ICR mice at two different ages, 2 (weaning) and 8 (adult) week‐old, were intraperitoneally injected with CCl4 (15.4 mg/kg), and the time‐dependent liver damage and cellular events were compared. Significant elevation of serum ALT/AST levels and hepatic centrilobular necrosis were induced by CCl4 in weaning mice, while mild liver injury was observed in adult mice. CCl4‐treated weaning mice showed higher pro‐apoptotic proteins (Bax, cleaved caspase‐3, ‐7, and ‐9), activated MAPKs (p‐JNK and p‐Erk), and ER stress indicators (ATF6 and CHOP), but lower anti‐apoptotic Bcl‐2 in their livers than those in the adult mouse livers. Weaning mice showed higher basal level of hepatic GSH due to their higher GCL and lower CDO enzyme levels. However, after CCl4 injection, hepatic GSH in weaning mice was dramatically decreased, while that in adult mice was not changed. Hepatic malondialdehyde, 4‐hydroxynonenal, nitrotyrosine‐protein adducts, and oxidized proteins resulting from oxidative stress were highly detected in CCl4‐administered weaning mice compared to the corresponding adult mice. The higher levels of CYP2E1 and 3A in the weaning mouse livers appear to be the primary reason of their higher hepatic susceptibility, and the lower GST‐π and GR in these mice also could contribute to the liver damage.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) funded by the Korea government (MSIT) (NRF‐2018M3A7B4071233 and NRF‐2019R1I1A3A01058584).
NILCO signaling is a novel biomarker in thyroid cancer
Authors: Erkasap Nilufer , Badak Bartu , Ozyurt Rumeysa , Ozkurt Mete , Guzel Derya , Erkasap Serdar , Ihtiyar Enver , Dundar Emine , Arik Deniz
Published: April 2020

The pathways and therapeutic targets play a role in potential molecular mechanisms of thyroid cancer therapy have been studied in recent years. One of these pathways is Notch signaling. Recent studies on cancer have been reported to be Notch regulators of leptin and IL‐1. In addition, a new signaling crosstalk between Notch, interleukin‐1 (IL‐1) and leptin [Notch, IL‐1 and leptin crosstalk outcome (NILCO)] is important in proliferation, migration and angiogenesis. However, it is not clear whether activation of this signaling crosstalk is related to thyroid cancer and diverse clinicopathological factors. The aim of this study was to demonstrate the expression changes of Notch1, Leptin, Leptin receptor ObRb, IL‐1α, IL‐1β, IL1R axis in human thyroid cancer tissues. Notch1, Leptin, ObRb, IL‐1α, IL‐1β, IL1R mRNA expressions were analyzed by real‐time PCR in human tissue of normal and thyroid tumors from 27 patients. In this study, we show that leptin, IL‐1α, IL1R are over‐expressed in human thyroid carcinoma tissues compared with normal thyroid tissues. In addition to, mean plasma leptin, T3 and T4 concentration was significantly higher in thyroid cancer patients compared with healthy people. These results indicate that NILCO could be critical in the development of thyroid cancer and this is a useful clinical biomarker in thyroid carcinoma. The present study was approved by the Ethics Committee of Eskisehir Osmangazi University for Clinical Research (80558721/66) and was performed in accordance with the ethical standards of Helsinki Declaration.
Therapeutic Effects of a Small Molecule Agonist of the Relaxin Receptor in Mice with Unilateral Ureteral Obstruction
Authors: Agoulnik Alexander I. , Ng Hooi Hooi , Soula Mariluz , Rivas Bryan , Marugan Juan J.
Published: April 2020

Diseases such as diabetes and hypertension often lead to chronic kidney failure. The peptide hormone relaxin has been shown to have therapeutic effects in various organs. In the present study, we tested the hypothesis that ML290, a small molecule agonist of the human relaxin receptor (RXFP1), is able to target the kidney to remodel the extracellular matrix and reduce apoptosis in the unilateral ureteral obstruction (UUO) mice. UUO was performed on the left kidney of humanized RXFP1 mice, where the right kidney served as contralateral controls. Mice were randomly allocated to receive either vehicle or ML290 (30 mg/kg) via daily intraperitoneal injection, and kidneys were collected for apoptosis, gene and protein analyses. UUO significantly increased pro‐apoptotic activity in both vehicle‐ and ML290‐treated mice when compared to their contralateral control kidneys. Specifically, Bax expression and Erk1/2 activity were upregulated, accompanied by an increase of TUNEL‐positive cells in the UUO kidneys. Additionally, UUO induced marked increase in myofibroblast differentiation and aberrant remodeling on the extracellular matrix. ML290 suppressed these processes through a reduction in pro‐apoptotic, fibroblastic and inflammatory markers in the UUO kidneys. Finally, the potent effects of ML290 to remodel the extracellular matrix were demonstrated by its ability to reduce collagen gene expression in the UUO kidneys. Our data indicate that daily administration of ML290 has renal protective effects in the UUO mouse model, specifically through its anti‐apoptotic and extracellular matrix remodeling properties.

Support or Funding Information

This research was supported by the U.S National Institutes of Health (NIH) Grant R01DK110167 (to A. I. Agoulnik). H. H. Ng received the American Heart Association Postdoctoral Fellowship Grant 19POST34380255, and M. Soula was supported by NIH National Institute of General Medical Sciences (NIGMS) T34GM083688 fellowship.
Cytokines Modulate the Expression of Calcitonin‐Gene‐Related‐Peptide and Substance P in Primary Rat DRG Neuron‐glia Co‐cultures
Authors: Grodzki Ana Cristina G. , Kurland Janice S. , Gorin Fredric A. , Lein Pamela J.
Published: April 2020

In the peripheral nervous system, dorsal root ganglion (DRG) neurons are the primary sensory neuron responsible for the transmission of temperature, touch, proprioception and pain. Recently, there has been increased interest in the role of neuro‐immune interactions in peripherally‐mediated neuropathic pain. Activated immune cells that infiltrate DRG release a mixture of inflammatory mediators that play a role in the development and maintenance of pain. However, the relationship between immune cells, pro‐inflammatory mediators and sensory neurons in the development and maintenance of pain are not completely understood. In this study, we investigated the effects of the cytokines interferon gamma (IFNγ) and tumor necrosis factor‐alpha (TNFα), and the chemokine fractalkine on the expression of calcitonin‐gene‐related‐peptide (CGRP) and substance P (SP) in DRG neuron‐glia co‐cultures. IFNγ decreased CGRP and substance P expression while TNFα increased CGRP expression, preferentially in small and medium size neurons. IFNγ and TNFα, both also increased nitric oxide (NO) production in a concentration‐dependent manner, at concentrations that affected CGRP and SP expression. However, inhibitors of nitric oxide synthase (L‐NAME) and of IKKB/NFkB (TPCA‐1) did not alter TNFα effects on CGRP expression. The data from this study demonstrates that cytokines and chemokines differentially modulate the expression of CGRP and SP in DRG neurons. Understanding the regulation of these two key inflammatory neuropeptides may provide novel insights regarding the development of new and effective therapeutics for neuropathic pain.

Support or Funding Information

This work was supported by NIH (grant # R21 AG065908).
The Acute Effects of Preworkout Supplement Ingestion and Whole‐Body Resistance Exercise on Clinical Safety Markers in Resistance‐Trained Males
Authors: Koozehchian Majid S. , Hosseini Seyyed Vahid , Eynavi Hafez
Published: April 2020

PURPOSE

To examine the acute effects of ingesting a multi‐ingredient preworkout supplement (PWS) and conducting a whole‐body resistance exercise on clinical safety markers before and following exercise.

METHODS

We recruited eight apparently healthy resistance‐trained males with at least six months immediate prior history of resistance training (19±1.1 yr, 173±2.6 cm, 66±2.9 kg, and 13.6±3% body fat) to participate in a randomized, double‐blind, crossover, and placebo‐controlled manner. We instructed participants to maintain their current diet and training regimens throughout the study. Supplements were a 6.5 g maltodextrin placebo (PL) and a commercially available PWS containing 1.6 g beta‐alanine, 1.0 g creatine nitrate, 1.0 g arginine alpha‐ketoglutarate, 321 mg explosive energy blended (including 160 mg caffeine anhydrous, l‐tyrosine, Tea Cor, tetramethyluric acid), 250 mg ascorbic acid, 30 mg niacinamide, 500 mcg vitamin B6, 150 mcg folate, and 20 mcg vitamin B12. In the first and second sessions, which were interspersed with a seven‐day washout period, participants ingested their assigned supplement 30 min before exercise. The exercise including a whole‐body resistance exercise initiated 30‐min after supplement ingestion. Blood samples were taken before, immediately after, and an hour following exercise. The measured blood markers included alkaline phosphatase (ALP), aspartate transaminase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine, and lactate. Repeated‐measures ANOVA and Bonferroni tests were used to analyze data. Results are expressed as mean±SD.

RESULTS

Analysis of data demonstrated no significant between‐group differences for any biomarkers associated with liver enzymes such as ALP, AST, and ALT; kidney enzymes, including BUN and creatinine function; and lactate levels (all makers, p>0.05).

CONCLUSION

The acute ingestion of the PWS examined in the current study indicated no adverse effects on liver enzymes, kidney enzymes, and lactate levels, with respective responses being similar to treatment with a PL treatment. These findings agree with other studies testing similar ingredients, and more extended studies appear to be safe to perform on performance‐related outcomes.
Activation of apoptotic signaling by DAPK1 suppresses neural remodeling and functional recovery in stroke
Authors: Hong Yonggeun , Won Jinyoung , Choi Jeonghyun , Kim Joo-Heon
Published: April 2020

Death‐associated protein kinase 1 (DAPK1), a key player in multiple types of cell death, is activated in ischemic stroke. We examined whether suppression of DAPK1 contributes to neuronal regeneration and, thereby, improves functional outcomes. Animals were subjected to middle cerebral artery occlusion (MCAo) and were administered an oxazolone‐based DAPK1 inhibitor. Animals with ischemic stroke had neurological damage and altered behavioral outcomes. However, DAPK1 inhibitor treatment reduced the expansion of the ischemic infarct and improved functional recovery. Inhibition of DAPK1 attenuated the structural abnormalities of the cortical pyramidal neurons and progressively improved neurological deficits. Additionally, while the levels of anti‐apoptotic proteins were upregulated, those of pro‐apoptotic and autophagy‐related proteins were down‐regulated in the ischemic brain by DAPK1 inhibition. Likewise, brain infarct volume and neurological deficit scores were reduced in DAPK1 KO mice. Activation of DAPK1 was exclusively associated with brain injury. These findings suggest that modulation of apoptosis by DAPK1 suppression could be used to treat ischemic stroke.

Support or Funding Information

National Research Foundation (NRF‐2013R1A2A2A01067169, NRF‐2017R1D1A1B03029565) Korea, Creative Research Program (2016–2017) of Inje University, Korea.
Stearoyl‐CoA desaturase 1 determines pancreatic β‐cell fate through regulation of DNA methylation pattern
Authors: Dobosz Aneta Maria , Janikiewicz Justyna , Borkowska Anna Maria , Kwiatek Wojciech Maria , Dobrzyn Agnieszka
Published: April 2020

Study objective

Stearoyl CoA desaturase 1 (SCD1) is a pivotal enzyme involved in saturated fatty acids metabolism that exhibits a protective role against lipotoxicity in pancreatic β‐cells. It has been shown that SCD1 deficiency contributes to impaired insulin secretion, β‐cell failure and development of type 2 diabetes (T2D). In the present study we investigated the involvement of SCD1 in regulation of DNA methylation and expression of transcription factors (TFs) responsible for maintenance of mature β‐cells function and identity.

Methods

The experiments were carried out ex vivo on pancreatic islets isolated from wild type (WT) and SCD1 knock‐out (SCD1 KO) mice, and in vitro on INS‐1E pancreatic β‐cell line, where lipotoxicity was induced by palmitic acid treatment. Morphological and functional characteristics of islets was evaluated by ELISA assays, immunofluorescence labeling and transmission electron microscopy techniques. DNA methylation and gene expression levels were assessed by bisulfite sequencing and real‐time quantitative PCR analyses, respectively.

Results

Our data demonstrated that pancreatic islets of SCD1 KO mice are characterized by different microarchitecture, secretory granules content and functional abnormalities manifested by diminished insulin secretion and greater proinsulin secretion in comparison to WT islets. On both ex vivo and in vitro models we observed significantly reduced expression of TFs crucial to maintenance of β‐cell identity such as Pdx1, MafA, Nkx6.1 and Neurod1. Interestingly, transcriptional repression of Pdx1 and MafA was related to excessive methylation of promoter regions of those TFs. In addition, we showed that downregulation of SCD1 activity/gene expression affects global DNA methylation pattern and changes methyl groups distribution within chromosomes in pancreatic β‐cells. DNA hypomethylation which occurred in SCD1‐deficient β‐cells was followed by decreased expression of DNA methyltransferase 1 (Dnmt1). We indicated that activation of AMP‐activated protein kinase (AMPK) axis influences post‐translational regulation of Dnmt1, thereby mediating the effect of SCD1 inhibition on global DNA methylation profile in β‐cells.

Conclusions

Together, our findings suggest that SCD1 is a gatekeeper that preserves epigenetic inheritance of DNA methylation pattern in β‐cells. Thus, SCD1 protects β‐cells from lipid‐derived loss of identity in methylation‐dependent manner through regulation of expression of TFs responsible for maintenance of β‐cell identity. Our results provide additional mechanistic insights toward understanding the role of SCD1 in T2D pathogenesis.

Support or Funding Information

This work was supported by grants from the National Science Center, Poland, grants no UMO‐2017/27/N/NZ3/01987 (AMD) and UMO‐2015/19/D/NZ4/03705 (JJ), and by The National Centre for Research and Development, grant no STRATEGMED 3/305813/2/NCBR/2017 (AD).
Host‐parasite Interaction Model Using Bead‐Grown Biofilms
Authors: Abu-Hassan Diala Walid , Alkawareek Mahmoud Yousef , Hamed Rania “Ahmad Azzam” , Banat Fahmy , Al-Muhtaseb Saja
Published: April 2020

Infectious diseases are widely spread and can significantly affect people’s lives and cause social and economic damages. Co‐culture systems have been used to investigate different types of cell‐cell interactions just like those that occur in infections between microorganisms and host cells. These techniques have many applications in biology and medicine. Although various co‐culture models using different approaches were published, more modelling strategies are being tested.

In this project we present a new co‐culture model based on using agar/glass beads to grow biofilms of different types of bacteria which then can be used to infect cultures of human cells. Biofilms of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Streptococcus intermedius, and Streptococcus oralis were successfully grown using this model. Beads containing bacterial biofilms were co‐cultured with PANC1 epithelial cell line to simulate its infection. This co‐culture model proved to be a facile method to study bacteria‐human cell interaction which also can be used to evaluate the efficacy and toxicity of different drug treatments.

Support or Funding Information

Scientific Research Support Fund, Ministry of Higher Education and Scientific Research, Al Jubaiha, Amman, Jordan 11941.