bluuebunny/crossref_metadata_2025 huggingface dataset:
Cortical and trabecular bone microstructural parameters and dietary stable isotopes throughout ontogeny
Authors: Beresheim Amy Catherine , Singh Atul K. , Grynpas Marc D.
Published: April 2020

Introduction

Recent research has revealed correlations between δ15N, an indicator of dietary protein sources, and measures of intracortical and endosteal bone remodeling in adults. Given that these isotopes are used to infer the prevalence and duration of breastfeeding in past populations, it may be possible to specifically target bone microstructural variation associated with early life nutrition. It is critical to develop a more robust understanding of skeletal responses to weaning and infant diet, as they ultimately influence final body size proportions, skeletal fragility, and osteoporosis risk.

Materials and Methods

The sample is 27 mid‐sternal rib bone sections, each representing one child (0–16 years of age) from the Wesleyan Methodist cemetery of Prospect Hill, Newmarket, ON (1824–1879 AD). All specimens were scanned using the SkyScan 1174 Micro‐CT system at the Lunenfeld‐Tanenbaum Research Institute, Toronto, ON. Quantitative parameters were derived using SkyScan CT‐analyser software. Carbon and nitrogen stable isotope ratios from bone collagen were previously reported (Katzenberg and Pfeiffer1995).

Spearman’s rank order correlation coefficients were calculated to explore relationships between each bone microstructural variable and age‐at‐death. Partial corrections, controlling for age, were used to examine the relationships between bone microstructural variables and δ13C and δ15N.

Results

Cortical bone variables relating to cross‐sectional size increase from birth to late adolescence. With age, bone volume fraction (BV/TV), trabecular number (Tb.N) and connectivity density (Conn.Dn) decrease, while trabecular spacing (Tb.Sp) increases. Relationships between bone microstructural variables and δ13C or δ15N values are not straightforward.

Discussion and Conclusion

Ontogenetic patterns of microstructural variation in the rib are similar to those of other bones undergoing endochondral ossification. Linkage to characteristics of infant and childhood dietary protein may be mitigated by causes of death.
Polysynaptic Neurotracing of Autonomic Nuclei Involved in Liver and Kidney Function
Authors: Williams Paul Allen , Rahmouni Kamal
Published: April 2020

Central nervous system (CNS) autonomic network plays an important role in the control of hepatic and renal functions with important implications in obesity and related hypertension. Previous studies employing pseudorabies viruses (PRV) have provided insight into the polysynaptic connections between various CNS nuclei and liver or kidney. However, whether the same autonomic network is involved in the control of hepatic and renal function remain unknown. Therefore, we used two groups of animals that received injections of PRV that express a green fluorescent protein (PRV‐GFP) into the left lobe of the liver or the two kidneys to compare labeled nuclei. The animals were sacrificed 5, 6, or 7 days after PRV injections, perfused, and the brains extracted. The brains were sectioned at 50 μm thickness, stained for GFP, and imaged with confocal microscopy. Sections were matched to the mouse atlas (Franklin & Paxinos, 3rd ed, 2008) and nuclei identified. Consistent with published work, we identified several nuclei that project to both the liver and kidneys although they may represent different orders (expression appearing on different days). In the brainstem, the nucleus of the solitary tract and area postrema expressed GFP after both liver and kidneys infections while the dorsal vagal nucleus expressed GFP after inoculation of the liver, but less after the kidneys. A few nuclei were observed in the midbrain after liver and kidneys infections, yet the ventral tegmental area projections observed previously from the liver were also observed from the kidneys which has not been previously reported. GFP staining was noted in several hypothalamic nuclei such as the paraventricular nucleus, arcuate nucleus, lateral hypothalamus, and dorsomedial hypothalamus after PRV infections of both organs. Projections from the cortical region and other areas such as the motor cortex, bed nucleus of the striatum, septal nuclei, and preoptic nuclei were also observed. In addition, we identified several nuclei such as the subfornical organ, nucleus accumbens, and insular cortex that project to the liver or kidneys which have not been previously reported and a few nuclei that were specific to the liver or kidneys. Our study supports the existence of an overlap in the projections that sub‐serve the liver and kidneys. The relevance of the newly identified autonomic projections to hepatic and renal functions is under investigation.
Academic Predictors of MCAT performance for Post‐baccalaureate Pre‐medical program students
Authors: Dobrydneva Yuliya , Ganjoo Rohini , Schwartz Lisa
Published: April 2020

Career changers, i.e. students who did not major in the traditional pre‐medicine fields like chemistry and biology, presently constitute a large percentage of medical school applicants. Post‐baccalaureate pre‐medical programs (PBPMP) provide prerequisite coursework for non‐life science majors who aspire to become physicians. Curriculum includes physics, general and organic chemistry, biology and biochemistry. Students entering these programs generally do not have significant exposure to natural sciences, and are considered “scientifically naïve”. Therefore, post‐baccalaureate programs are searching for criteria for admission process to help identify applicants with potential to succeed in PBPMP and matriculate into medical schools.

This study analyzed academic metrics of career changer pre‐medical students to predict their success on mandatory Medical College Admission Test (MCAT). Data analysis correlated MCAT scores with gender, high school Scholastic Aptitude Test (SAT) scores, undergraduate major or field of study, and undergraduate Grade Point Average (GPA). Undergraduate GPA is not correlated with performance on MCAT. Analysis yielded positive correlations between MCAT scores and SAT scores, and between MCAT and undergraduate non‐science major/field of study. The results provide insight into academic metrics that could predict future success on MCAT and matriculation into medical school. While positive correlations between certain sub‐scores on the SAT and MCAT were found, other holistic non‐quantitative characteristics should be considered for medical school admission.
Chemical Inhibition of the Replication Stress Response Causes DNA Damage and Delayed Regeneration in S. mediterranea
Authors: Nguyen Lillian , Ishaq Ariana , Sheppard Taylor , Velasquez Elizabeth , Ahmed Youssef , Montes Patricia , Arroyo Jimena , Sawyer Nathaniel , Tirgar Reyhaneh , Shamoon Ushna , Akpati Lois , Nam Edward
Published: April 2020

S. mediterranea is a planarian with extensive regenerative ability which requires substantial cell division. However, DNA replication stress occurs every cell division and must be resolved. From yeast to man, an evolutionarily conserved DNA replication stress response (RSR) is essential for life. The RSR is a signal transduction pathway regulated by Ataxia telangiectasia and Rad3‐related kinase (ATR) and Checkpoint 1 kinase (CHK1) to respond to replication stress. However, the RSR has not been established in planarian nor do we know if it functions in regeneration. We hypothesize that ATR may be essential for regeneration. Preliminary results show reduced regenerative ability in the presence of hydroxyurea (HU), a replication stress inducing agent. However, low concentrations (2mM) did not attenuate regeneration, indicating that ATR and the RSR may be present and able to overcome replication stress. To test this, we treated amputated tails with HU, ATR inhibitor (ATRi), and a combination of both. In 2mM HU, tails regenerated similarly to control. We observed only a one day delay in regeneration with ATRi alone. However, ATRi and 2mM HU in combination prevented planarian regeneration completely. In addition, replication stress was confirmed through comet assays, which quantified levels of DNA damage within each planarian experimental group. The ATRi and 2mM HU combination group exhibited the highest average comet tail moment. These synergistic effect suggests that ATR is significant in the response to replication stress during regeneration. Additionally, inhibition of CHK1, another kinase in the RSR pathway, exhibited similar tail moment values with ATRi. Future experiments will allow us to support the presence of CHK1 and its important role in resolving replication stress. Further, determining the importance of RSR genes in planarian will elucidate the conserved components and allow us to understand how ATR and the RSR contributes to the incredible regenerative abilities in planarian.

Support or Funding Information

UST Committee for Student ResearchAlvarado Sanchez LabJim Jenkin for S. mediterraneaDepartment of Education HSI‐STEM grantUST Biology Smith ChairUST Biology Department
Cyclin C Regulated Transcriptional Response to Stress
Authors: Stieg David
Published: April 2020

When cells are exposed to extracellular stress, there is a coordinated shift in cellular and molecular actions that determine the fate of the cell. An important aspect of this response involves transcriptional reprogramming to either mitigate the effects of the stress or commit to a cell death pathway. This altered transcriptome causes disruption of normal cellular functions, such as growth and proliferation. It also promotes the transcription of genes involved in stress response and cell death. Our previous findings from RNA sequencing (RNA‐seq) studies has shown that the conserved transcription factor, cyclin C, regulates the stress responsive transcriptome in mouse embryonic fibroblast (MEF) cells. Cyclin C is a member of the Cdk8‐kinase module (CKM) which, depending on the locus, can function to either induce or repress transcription. The genes induced by cyclin C prior to stress include those that promote growth and proliferation. In contrast, the genes repressed by cyclin C prior to stress include those that promote autophagy and apoptosis. After exposure to stress, the role of cyclin C is disrupted on each group of genes. This transcriptional reprogramming event results in the repression of the growth and proliferation genes, and induction of the autophagy and apoptosis genes. In the present study, chromatin immunoprecipitation (ChIP) studies show that cyclin C is associated with the promoter region of genes identified through RNA‐seq analysis. ChIP was used to determine promoter occupancy by cyclin C and transcriptional changes occurring from exposure to either oxidative stress or Torin1 to induce apoptosis and autophagy, respectively. Stressed cells show significantly reduced, or complete loss of cyclin C occupancy at specific genes. Cyclin C is also recruited to the promoter of other genes following exposure to stress to induce transcription. The genes identified in this study are involved in the cellular responses upon exposure to stress; specifically, the repression of growth and proliferation and the induction of autophagy and apoptosis. These findings demonstrate a direct role for cyclin C in the transcriptional changes occurring following exposure to stress, and subsequently contribution to cell fate determination.
The Herbal Medicine Extract Rutin Reduces Beta Amyloid‐induced Depolarization of Glutamatergic Neurons
Authors: Lin Yenshou , Kuo Li-Chen , Chuang Jen-Hua
Published: April 2020

Currently, two of the major clinical hallmarks of Alzheimer’s disease (AD) are senile plaques formed by beta‐amyloid (Aβ) deposits and neurofibrillary tangles caused by tau protein hyperphosphorylation. Extracellular Aβ accumulation could cause neuronal death and this eventually leads to the individual developing cognitive deficits and memory impairment. Aβ accumulation seems to mediate an increase of glutamate concentration by which excess glutamates bind NMDA receptors and/or AMPA receptors at the synaptic and/or extrasynaptic cleft manner and subsequently trigger excitotoxicity. Memantine, the only clinical drug on the basis of synthetic NMDA receptor antagonist, developed to treat mild to severe AD patients. Therefore, we aim to develop more new drugs which could alleviate the Aβ‐induced pathophysiology. We previously established a drug screening model that employed glutamatergic neurons that were cultivated from mouse brain cortex with the help of bis‐(1,3‐dibutylbarbituric acid)‐trimethine oxonol (DiBAC4(3)), a fluorescent dye able to sense depolarization. We found rutin, a pure compound extracted from Ginkgo biloba, is able to attenuate Aβ‐induced abnormal depolarization in these glutamatergic neurons. Utilizing receptor agonists, we revealed that rutin mediates the inhibition of NMDA receptor, but not AMPA receptor to exert such an effect. Next the molecular mechanism(s) behind rutin and Aβ‐induced abnormal depolarization, as well as various other parameters such as electrophysiology, are at present under investigation. Our findings, when taken together, clearly show that the pure compound rutin is able to reduce Aβ‐induced depolarization of these neurons. Although research exploring the detail mechanisms of rutin is ongoing, this drug may have important benefits and great potential with respect to AD therapy.
Rap1 exacerbates myocardial ischemia/reperfusion injury through activation of NFκB signaling pathway and NLRP3 inflammasome
Authors: Cai Yin , Xie Xiang , Pan Qixian , Zhi Qiqi , Woolford Michael G. , Xia Zhengyuan
Published: April 2020

Background

Ischemic heart disease is a leading cause of morbidity and mortality worldwide. Activation of nuclear factor kappa B (NFκB)‐mediated inflammation has been demonstrated in various models of experimental myocardial ischemia/reperfusion injury (I/RI). Repressor activator protein 1 (Rap1), an established telomere‐associated protein, is a novel modulator of NFκB‐mediated inflammatory response. However, it is unknown whether Rap1 modulates NFκB signaling in cardiomyocytes to contribute to myocardial I/RI. Therefore, the present study aimed to explore the potential pro‐inflammatory role of Rap1 in myocardial I/RI and to determine the underlying molecular mechanism.

Methods and Results

In a mouse model of myocardial I/RI (30 minutes of left descending coronary artery ligation followed by 24 hours reperfusion), Rap1 deficiency significantly reduced myocardial infarct size and improved cardiac systolic/diastolic function (all p < 0.05, Rap1−/− I/R vs. Rap1+/+ I/R). This was associated with a reduction in NFκB activity [reduced phosphorylation, degradation of IκBα and phosphorylation of p65] and suppressed production of NFκB‐mediated pro‐inflammatory cytokines, including tumor necrosis factor α (TNFα), interleukin (IL)6 and IL1β in the post‐ischemic myocardium (all p < 0.05, Rap1−/− I/R vs. Rap1+/+ I/R). In cultured cardiac origin H9C2 cardiomyocytes, Rap1 knockdown did not significantly influence the pro‐inflammatory response in hypoxia/reoxygenation or in lipopolysaccharide‐stimulated H9C2 cells, but deletion of Rap1 significantly attenuated macrophage infiltration into the post‐ischemic myocardium of mice (p < 0.05, Rap1−/− I/R vs. Rap1+/+ I/R). Also, knockdown of Rap1 significantly suppressed NLRP3 inflammasome formation in THP‐1 macrophages in response to LPS and ATP (p < 0.05, Rap1KD vs. Rap1WT). When post‐hypoxic H9C2 cardiomyocytes were co‐cultured with mock‐ or Rap1‐siRNA transfected THP‐1 macrophages, knockdown of Rap1 in macrophages significantly attenuated macrophage‐mediated/exacerbated cardiomyocyte injury (p < 0.05, Rap1KD vs. Rap1WT).

Conclusions

These data collectively suggest that Rap1 may exacerbate myocardial I/RI through enhancing activation of NFκB signaling pathway and NLRP3 inflammasome formation, which may yield a novel therapeutic target for myocardial I/RI.

Support or Funding Information

The work was supported by the National Natural Science Foundation of China (No. 81800245) and General Research Fund (17117217M, 17123718M Research Grants Council of Hong Kong).
STAT3 decoy oligodeoxynucleotides improve end‐organ tissue injury and survival in septic mice
Authors: Matsuda Naoyuki , Imbaby Samar , Hattori Kohshi , Hattori Yuichi
Published: April 2020

STAT3 serves as a key transcription factor in both immunity and inflammatory pathways. We examined whether in vivo introduction of STAT3 decoy oligodeoxynucleotides (ODNs) is beneficial in reducing septic end‐organ injury. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in mice. STAT3 was rapidly activated in major end‐organ tissues following CLP, which was accompanied by activation of the upstream kinase JAK2. Transfection of STAT3 decoy ODNs significantly improved histopathological changes in lung, liver, kidney, and heart tissues. STAT3 decoy ODN transfection downregulated pro‐inflammatory cytokine/chemokine overproduction and reduced the increases in tissue and serum levels of HMGB1 in CLP mice. These beneficial effects of STAT3 decoy ODNs led to s significant survival advantage in mice after CLP. Our results indicate a detrimental role of STAT3 in the sepsis pathophysiology and the potential usefulness of STAT3 decoy ODNs for sepsis therapy.

Support or Funding Information

Grants‐in‐Aid for Scientific Research from Japan Society for Promotion of Science (17K08586, 19H03757)
Evaluation of Periarticular Pathology Surrounding Knee Joint Tissues in an Osteoarthritis Rat Model
Authors: Sriwatananukulkit Orada , Likitnukul Sutharinee , Desclaux Scarlett , Srikuea Ratchakrit , Tawonsawatruk Tulyapruek , Hemstapat Ruedee
Published: April 2020

Osteoarthritis (OA) is a multifactorial disease associated with pathological changes in a wide variety of musculoskeletal tissues, including articular cartilage degeneration, synovitis, ligament tears as well as muscle wasting. Cartilage is known as a predominant pathology, however the association between the pathogenesis of OA around the knee joint tissues and pain is limited. Infrapatellar fat pad (IFP) fibrosis and muscle atrophy are the main features of chronic OA phenomenon that cause joint pain, stiffness and impairment of skeletal muscle, which contributing to locomotor restriction in OA patients. Despite a high prevalence of knee OA, there is still a lack of comprehensive information explaining the interplaying mechanisms among these OA features. Although various animal models of OA have been characterized, none of them can truly represent all aspects of the OA features. Monoiodoacetate (MIA)‐induced OA is one of the most common animal model used in the OA research, in which intra‐articular injection of MIA can lead to articular cartilage destruction and development of pain‐like behaviour. This study aimed to characterize and determine the suitable time point, at which MIA could significantly induce the development of IFP fibrosis and hind limb muscle atrophy in this OA rat model. IFP and soleus muscle were collected for histological analysis at 4 and 8 weeks post‐OA induction. In addition, pain‐related behavior was also evaluated using hind limb weight distribution test. Semi‐quantitative analysis of Masson’s trichrome staining revealed that the percentage of IFP fibrosis area was not significantly different at 4 weeks compared to 8 weeks post‐OA induction. In addition, there was a significant (p<0.05) reduction of soleus muscle fiber cross‐sectional area at 4 and 8 weeks of OA‐induced groups compared to their relative controls; however, the muscle fiber atrophy was more pronounced at 4 weeks compared to 8 weeks. Moreover, there was a significant reduction in the mean values of percent weight borne on the injured hind limb for the OA‐induced group compared to the saline group throughout the study period (p<0.05). Interestingly, pain response significantly reversed at 8 weeks compared to 4 weeks (p<0.001), suggesting that this reversal in pain response could be the contributing factor on alleviation of soleus muscle atrophy under chronic OA pain. These results suggested that at 4 weeks post‐MIA injection is sufficient and suitable period to investigate OA‐related pain, IFP fibrosis and muscle atrophy. Taken together, a MIA‐induced OA rat can be used as a representative model in translational research to understand IFP fibrosis and hind limb muscle atrophy in OA as an integrated musculoskeletal disease.
Anti‐obesity Effects of Ecklonia stolonifera Extract in 3T3‐L1 Cells
Authors: Jin Heegu , Lee Kippeum , Oh Jae-Hoon , Chei Sungwoo , Oh Hyun-Ji , Lee Boo-Yong
Published: April 2020

Obesity is caused by excessive fat accumulation when energy intake exceeds energy expenditure. Recent studies have shown that dietary phytochemicals can suppress adipogenesis and promote lipolysis. Ecklonia stolonifera, which belongs to the family Laminariaceae, is a perennial brown marine alga that is rich in phlorotannins. Many studies have been reported that Ecklonia stolonifera has antioxidative, hepatoprotective, and anti‐inflammatory activities. However, it is not known yet whether Ecklonia stolonifera has an anti‐obesity property and improves lipid metabolism. In our study, the anti‐obesity effects of Ecklonia stolonifera Extract (ESE) were investigated in 3T3‐L1 cells. ESE reduced the expression levels of adipogenesis‐related genes such as CCAAT/enhancer‐binding protein alpha (C/EBPα) and fatty acid‐binding protein 4 (FABP4). In addition, ESE reduced the expression levels of lipogenesis‐related genes such as lysophosphatidic acid acyltransferase theta (LPAATθ), diacylglycerol acyltransferase 1 (DGAT1) and sterol regulatory element‐binding protein 1 (SREBP1). Moreover, ESE increased the expression of adipose triglyceride lipase (ATGL) and hormone‐sensitive lipase (HSL), which are related to lipolysis pathway. Lastly, ESE promoted lipolysis via protein kinase A (PKA) mediated pathway. In conclusion, ESE modulates adipocyte differentiation and lipid metabolism by reducing lipogenesis and stimulating lipolysis. These results suggest that ESE may have therapeutic implications for the development of new anti‐obesity agent.
EP1 Receptor Antagonism as a Therapeutic Intervention Strategy in Renal Fibrosis
Authors: Kresse Jean-Claude , Tingskov Stine Julie , Jensen Michael Schou , Mutsaers Henricus A. M. , Praetorius Helle , Nørregaard Rikke
Published: April 2020

Renal fibrosis is a hallmark of Chronic Kidney Disease (CKD), which affects 10–16% of the world’s adult population. It has previously been shown that the cyclooxygenase/prostaglandin (COX/PG) system affects the progression of renal fibrosis. The downstream elements of this system, the eicosanoid prostaglandin (EP) receptors, EP1–4, have various physiological roles in health and disease. We therefore evaluated the effect of antagonizing one of these downstream receptors for prostaglandin, the EP1 receptor, on renal fibrosis.

This was accomplished with a translational approach by utilizing three models of renal fibrosis; Transforming growth factor β (TGF‐β) treated Madin‐Darby Canine Kidney (MDCK) cells, mice that underwent seven days of unilateral ureteral obstruction (UUO), as well as TGF‐β treated Human Precision Cut Kidney Slices (HPCKS). We evaluated the extent of fibrosis in these models on the gene and protein level using QPCR as well as Western blotting and immunohistochemical analysis. Moreover, we determined the corresponding effects of the EP1 agonist 17‐phenyl trinor prostaglandin E2 (17‐phenyl PGE2) and the EP1 antagonist SC‐19220 on the intracellular Ca2+ concentration in MDCK cells measured by Flou‐4‐AM.

We found, that as a whole, treatment with the EP1 receptor antagonist SC‐19220 successfully ameliorated renal fibrosis in all three models. Treatment with the EP1 receptor antagonist SC‐19220 (25mg/kg per day) diminished fibrosis in mice that underwent seven days of UUO. UUO‐induced protein expression of the fibrosis markers fibronectin (FN), α Smooth Muscle Actin (αSMA) and collagen measured by western blot, qPCR and immunohistochemical staining, was lowered by SC‐19220 treatment when compared to SHAM operated mice. Adding to this, treatment of HPCKS with SC‐19220 reduced TGF‐β‐mediated fibrosis as shown by decreased collagen 1A1, FN and αSMA gene expression as well as reduced staining intensity of Sirius Red and Masson’s Trichrome stains. In MDCK cells, fibrosis induced by TGF‐β was significantly reduced when treated with SC‐19220. This was evident by lower FN protein levels and possibly mediated by hampering of TGF‐β signalling. The specificity of SC‐19220 as an antagonist of the 17‐phenyl PGE2‐induced [Ca2+]i response was determined with standard concentration response experiments.

Thus, this study shows that the EP1 receptor is a promising target for the treatment of renal fibrosis. However, further investigations into the mechanism underlying the anti‐fibrotic effect of EP1 receptor antagonism are necessary. It seems though, that intracellular calcium regulation is profoundly altered by the EP1 receptor and that this may alter fibrosis dynamics.

Support or Funding Information

Funding for the project was provided by the Danish Research Council.
Alterations in Heart Rate Fragmentation After Myocardial Infarction in Rats: A Novel Approach in Heart Rate Variability Analysis
Authors: Omoto Ana Carolina Mieko , Lataro Renata Maria , da Silva Thaís Marques , Salgado Helio Cesar , Silva Luiz Eduardo Virgilio , Fazan Rubens
Published: April 2020

Heart rate variability (HRV) has been largely studied by linear and nonlinear approaches, in a variety of clinical scenarios and animal models of diseases. Recently, a new pattern of ultra‐rapid heart rate variation has been proposed and named heart rate fragmentation (HRF). This approach showed that aging and coronary disease, increase the fragmentation of heart rate fluctuations. Since HRF was only evaluated in humans, we aimed to investigate this new HRV index in a rat model of myocardial infarction (MI). For this purpose, Wistar rats (250 a 300g) were submitted to MI by permanent ligation of the left anterior descending coronary artery, or sham‐operation (control animals). At the 4th or 12th weeks after MI, or sham‐operation, animals were instrumented with a polyethylene arterial catheter for arterial pressure recordings which were carried out in freely moving rats. At the end of the protocol, cardiac function was evaluated by echocardiography using a 21 MHZ transducer (VisualSonics‐VEVO2100®). For HRF analysis, the pulse interval (PI) series was transformed into a sequence of symbols named "−1”, “0” or “1", when the difference between successive values (transitions) was negative, zero, or positive, respectively. Next, sequences of 4 consecutive symbols were classified according to the numbers of transitions. The percentage of inflection points (PIP) of PI series were also quantified. As expected, cardiac function was found reduced in rats subjected to MI. Ejection fraction was smaller 4 (28±3 vs 68±2%) and 12 weeks after MI (38±3 vs 70±3%). Similarly, fractional shortening was also smaller 4 (13±2 vs 41±2%) and 12 weeks after MI (20±2 vs 41±3%). On the other hand, PIP values were increased in rats subjected to MI at the 4th (74±2 vs 69±1%) and 12th weeks after surgery (70±2 vs 63±1%), revealing a more fragmented pattern compared to control counterparts. We also found a significant correlation between cardiac functional parameters (ejection fraction and fractional shortening) and HRF (PIP) at both 4 and 12 weeks after MI. These findings reveal that in rats, cardiac ischemia also increases HRF, reinforcing the importance of this new HRV approach as a promising tool to explore physiological and pathophysiological conditions at clinical and experimental levels.

Support or Funding Information

Financial support: CNPq (870308/1997‐1) and FAPESP (2013/20549‐7).
PPP1R2, a Protein Phosphatase‐1 Regulatory Subunit, Balances Aurora A and PP1 Activities to Maintain Centrosome Number
Authors: Bresch Alan-Michael Jay , Sperry Ann
Published: April 2020

Centrosomes are the primary microtubule organizing centers in the cell and nucleate spindle microtubules at mitosis. A balance of kinase and phosphatase activity controls centrosome number and mitotic spindle function. PPP1R2 (R2) is a negative regulator of protein phosphatase 1 (PP1) and an activator of Aurora A Kinase (AURKA). Both PP1 and AURKA play critical roles in centrosome regulation, however R2’s role at the centrosome has not been examined. Given that R2 interacts with PP1 and AURKA, critical regulators of the centrosome, we hypothesized that disruption of R2 function would impact centrosome behavior and mitosis. We overexpressed R2 in cultured cells and then measured centrosome number and nuclear size. R2 overexpression caused an increase in centrosome number compared to controls. R2 cooverexpression with either AURKA or PP1 returned centrosome number to normal levels. Since inhibition of PP1 by R2 is dependent on phosphorylation of R2, we tested the role of R2 phosphorylation on centrosome number. We transfected cells with R2 phosphorylation site mutants and measured centrosome number as well as nuclear size. Phosphomimetic R2 increased the percentage of cells with supernumerary centrosomes while cells transfected with phospho‐null R2 exhibited normal centrosome number. Overexpression of R2 and its phosphomimetic caused significantly larger nuclei than controls suggesting that R2 overexpression may interfere with cytokinesis. We also examined the localization of these proteins in dividing cells by indirect immunofluorescence and discovered that R2, its phosphorylated isoform pR2, AURKA, and PP1 were all localized to the midbody. Together these findings suggest that R2, AURKA and PP1 regulate cytokinesis. To test the role of specific protein domains of R2 in recruitment of PP1 to the midbody, we overexpressed R2 deletion mutants lacking either the N or C terminus of R2 and measured PP1 localization during cytokinesis. Overexpression of R2 truncation mutants resulted in a dramatic reduction of PP1 at the midbody supporting an important role for R2 in directing PP1 to the midbody during cytokinesis. We conclude that R2 balances opposing activities of AURKA and PP1 to regulate centrosome number through control of cytokinesis.
Beyond Binding Kinetics: Stoichiometry via SPR can identify novel binding modes, assess ligand quality and inform functional design in biopharmaceutical discovery.
Authors: DiGiammarino Enrico L. , Izeradjene Kamel , Harding Fiona , Akamatsu Yoshiko , Xu Hattie , Chervin Adam , Stone Jennifer , Wang Ningyan , Durning Sean , Pefaur Noah , Durkin Travis , Urick Andrew , Harlan John
Published: April 2020

Biological therapeutics have seen a dramatic increase in molecular complexity and biological mechanisms. Antibody drug conjugates (ADCs) and multi‐specific formats are filling therapeutic pipelines and fundamentally changing traditional platform approaches to biologics discovery. These new entities pose unique challenges for characterization; as with traditional mAbs they are generally triaged through the discovery process by a series of functional assays including ELISA, FACS and binding kinetics however, we have implemented an additional and often underutilized SPR measure to characterize the function and quality of these entities: binding stoichiometry. Here we will discuss the special requirements for quality stoichiometry measures and show how stoichiometry can be used to 1) identify novel binding modes for a mAb that may have implications to ADC function, 2) identify signs of molecular instability, 3) aid in engineering functional biotherapeutic molecules and 4) assess simultaneous binding of analytes in multi‐specific formats. These examples will show an important measure that can be used to identify liabilities and to demonstrate function of biotherapeutic candidates.

Support or Funding Information

All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
Muscle Wasting in Cancer Involves Suppression of Ribosomal Production and Increased Expression of the Ribophagy Receptor NUFIP1
Authors: Kim Hyo Gun , Huot Joshua , Pin Fabrizio , Bonetto Andrea , Nader Gustavo A.
Published: April 2020

Muscle wasting/cachexia is a major contributor of mortality and morbidity in cancer and is frequently accompanied by functional and metabolic muscle abnormalities. Muscle mass is determined by the balance between protein synthesis and degradation, yet very little is known about the mechanisms controlling the translational capacity (i.e., ribosome biogenesis) of skeletal muscle in cancer cachexia. Muscle ribosome content is mainly regulated by transcription of the ribosomal (r)DNA genes and also by degradation of ribosomes via ribophagy. In this study, we investigated whether muscle wasting in a previously described model for the study of ovarian cancer cachexia is associated with reduced rDNA transcription and ribosomal mass. Nod SCID gamma mice were injected with 1 × 107 ES‐2 human high‐grade serous ovarian cancer cells and observed for fourteen days. Gastrocnemius muscle was collected for qPCR and western blot analysis. Fourteen days of tumor bearing resulted in a 24% (P < 0.0001) loss in gastrocnemius muscle weight. Muscle wasting was associated with ~50% (P = 0.0003) reduction in ribosomal capacity and a significant suppression in rDNA transcription rates (35%, P = 0.0086). This was accounted by a significant reduction in specific rDNA loci. Factors associated with the regulation of rDNA transcription (i.e., Pol1rb, UBF, RRN3, TAF1C, PAF53) were significantly higher than control (1.5 to 4‐fold, P < 0.05). Muscle wasting was also associated with a 4‐fold increased expression (P < 0.0001) of the ribophagy receptor NUFIP1. Our findings indicate that in cancer cachexia, the anabolic capacity of skeletal muscle is diminished due to reduced ribosome production and likely increased ribosome degradation via ribophagy.

Support or Funding Information

Supported by NIH grant R56AR‐073385 to GAN, The Pennsylvania State University, and The Huck Institute of the Life Sciences.
Electronic Hookah Vaping: Inflammatory Biomarkers and Biomarkers of Exposure after Acute Exposure
Authors: Dobrin Daniel , Cheng Chiao-Wei , Means Angelica , Brecht Mary-Lynn , Rezk-Hanna Mary
Published: April 2020

Electronic (e‐) hookah vaping has increased in popularity, especially among youth and young adults. Contributing to e‐hookah’s popularity is the unsubstantiated belief that smoke is detoxified as it passes through the water‐filled basin, rendering e‐hookah as a safer alternative to combustible tobacco smoking. With combustible tobacco smoking, vascular inflammation is a key component in the development and progression of atherosclerosis. However, the comparable effects of e‐hookah vaping have not been examined to date. In light of recent outbreaks of vaping‐associated deaths and related‐injuries, it is critical that the use of vaping devices is subjected to scientific scrutiny. Accordingly, the purpose of this work was to elucidate acute effects of e‐hookah vaping on a comprehensive set of systemic vascular inflammatory biomarkers and biomarkers exposure. In 23 young healthy subjects (age 26±1 years, BMI 24.1±0.6 kg·m2, mean±SE) who regularly smoke hookah but do not smoke cigarettes, we measured heart rate, blood pressure, systemic vascular inflammatory biomarkers, including, C□ reactive protein (hsCRP), interleukin‐6 (IL‐6), fibrinogen, and tumor necrosis factor alpha (TNFα), and biomarkers of exposure, including plasma nicotine and exhaled carbon monoxide (CO) before and after a typical 30‐minute session of e‐hookah bowl vaping and, for control comparison, before and after sham smoking (n=8). All biomarkers were measured before and ≤10 minutes after the vaping session. Our results show that after e‐hookah vaping, heart rate and blood pressure increased significantly (Δ heart rate: +9±2 beats.min−1; Δ systolic blood pressure: +16±1 mm Hg, Δ diastolic blood pressure: +10±1 mmHg, p<0.001 as compared with baseline). While nicotine levels increased after vaping (+5.34±1.24 ng/ml, p=0.002), exhaled carbon monoxide levels did not change (p=ns). Notably, plasma indices of systemic vascular inflammation increased significantly (all p<0.05, except for IL‐6: p=ns). All indices were unchanged after sham smoking. Though e‐hookah users endorse marketing claims that these products are safer alternatives to combustible tobacco smoking, our data herein show that despite the absence of combustion, acute exposure to e‐hookah among young healthy adults does not elude adverse cardiovascular effects and, in fact, leads to a transient increase in vascular systemic inflammation. Our data help inform the public about potential cardiovascular disease risk of vaping and guide further studies to examine the chronic long‐term vascular effects of exposure to e‐hookah vaping.

Support or Funding Information

This work was supported by the National Institutes of Health, National Heart, Lung, and Blood Institute 1R21HL145002‐01 and the University of California, Los Angeles Clinical and Translational Science Institute grant UL1TR000124.
Molecular basis for the exploitation of nuclear mRNA export by influenza A virus
Authors: Ren Yi , Zhang Ke , Xie Yihu , Morris Andrew , Wang Zhen , Muñoz-Moreno Raquel , Wang Juan , Zhang Liang , Esparza Matthew , García-Sastre Adolfo , Schey Kevin , Fontoura Beatriz
Published: April 2020

Influenza A virus replicates in the cell nucleus and has evolved at least two mechanisms to exploit the host nuclear mRNA export pathway. First, we show that a key influenza virulence factor NS1 blocks nuclear export of host mRNAs. We identify the direct target of NS1 as the mRNA export receptor complex, NXF1‐NXT1, which is the principal receptor mediating docking and translocation of mRNAs through the nuclear pore complex via interactions with nucleoporins. We determined the crystal structure of NS1 in complex with NXF1‐NXT1 at 3.8 Å resolution. The structure reveals that NS1 prevents binding of NXF1‐NXT1 to nucleoporins, thereby inhibiting mRNA export through the nuclear pore complex into the cytoplasm for translation. Second, packaging of influenza virus genome has been suggested to require the mRNA export factor UAP56 through interaction with the influenza NP protein, but the mechanism remains elusive. Our biochemical characterization of the NP‐UAP56 interaction suggests that UAP56 primes NP for engaging with viral RNA. Together, our studies uncover the molecular basis of how influenza A virus targets nuclear mRNA export to inhibit host gene expression and promote viral proliferation.

Support or Funding Information

NIH R01 GM113874, R01 AI125524, and R35 GM133743
The chondrocranial key: Development of the sphenoid bone in primates
Authors: Mano Nanami , Wood Brody , Oladipupo Lanre , Reynolds Rebecca , Vinyard Christopher , Cray James , DeLeon Valerie , Smith Tim
Published: April 2020

It has been hypothesized that the human sphenoid bone is uniquely truncated, which in turn contributes to a reduction of forward midfacial growth. If so, the perinatal fusion of the intrasphenoidal synchondrosis (ISS) in humans may contribute to midfacial reduction. However, there is a lack of detailed knowledge on sphenoid development of non‐human primates. In this study, orientation and direction of growth of basicranial interface with the midface of late prenatal and early postnatal sphenoid development was examined in ontogenetic samples of primates including three species of monkeys (n = 25) and four species of lemurs and bushbabies (strepsirrhines; n= 28). Micro‐computed tomographic (CT) and histological methods were used to track cross‐sectional age changes in the sphenoid bone. In monkeys (Saguinus spp.), histological findings indicated the number of proliferating chondrocytes is reduced across age leading to a reduction in absolute anteroposterior length of the proliferating zone. Preliminary data from micro‐CT reconstructions suggest that absolute length of the ISS decreases more rapidly in monkeys than in strepsirrhines. Measurements of presphenoid (PS) and basisphenoid (BS) length indicate that in strepsirrhines, these bones grow similarly to one another as cranial length increases. In contrast, in monkeys, the PS increases at a faster pace (i.e. higher linear regression line slopes) than the BS. However, unlike humans, the monkeys studied have prolonged postnatal patency of the ISS. Thus, the reduced midfacial projection in humans and these monkeys cannot be explained by the timing of ISS fusion alone. Dichotomous growth patterns of the cranial base and midface among primates suggest different patterns of regional integration of the cranium. In particular, our results suggest that in monkeys, integration of the midface is stronger with the anterior compared to the posterior portion of the sphenoid bone. In strepsirrhines by comparison, growth of the midface progressively deviates from that of the sphenoid bone over time.

Support or Funding Information

NSF; grant numbers BCS‐1830894, BCS‐1830919, BCS‐0959438
Science Advocacy Serves Communities and Scientists
Authors: Kozlovich Shannon , Bushana Piryanka , Szlenk Christopher
Published: April 2020

Science is largely funded by taxpayer dollars, and because of this, scientists have a responsibility to ensure that their research is being effectively communicated back to taxpayers and the policy makers who determine the distribution of those funds. The importance and impact of effective science communication is compounded when research is used to inform legislative action. Science impacts policy, and policy can impact science. However, formal education of scientists does not usually include specific training on interacting with science policy. Immersive service learning modalities should be integrated into formal science training programs, especially for Masters and Ph.D. programs. These programs are training our future scientists and should be responsible for introducing a fundamental understanding of the interaction between science and policy to trainees. Currently, service learning opportunities for science policy, communication, and advocacy are being organized by student organizations. This project describes strategies for engaging trainees in science policy ‐ starting with simple and easy policy actions, and delving into more complex event planning and group organizing. We aim to distribute this as a model for the types of educational resources that should be adopted into the curricula of formal science training programs. We propose that formal service‐learning projects around science policy, communication, and advocacy can serve as opportunities to practice integrating scientific knowledge and expertise into dissemination of scientific advancements to the general public and policy makers. Similar trainings are integrated into many clinical health professional programs. After all, service‐learning facilitates learning that persists over a career and a lifetime. Whether advocating for evidence‐based policies, policies which impact the scientific enterprise or STEM education, practicing skills pertinent to science policy can help scientists gain comfort in translating their experiences and experiments into lasting change for the benefit and advancement of humanity and human health.

Support or Funding Information

This work was supported in part by the National Cancer Institute grant CA‐113710(fellowship support for S. Kozlovich), the Research!America Civic Engagement Grant, generous funding from the Associated Students of Washington State University HealthSciences (ASWSUHS), and the Washington State University Health Sciences campus Services and Activities fee committee.
MicroCT Scanner to Differentiate Histotechnological Processing of Bone Tissue Using Different Nucleic Acid‐Based Fixatives
Authors: Yodice Anthony Nicholas , DeOcampo Francis Garcia , Gill Shaheryar M. , Khwaja Zabi , Gagna Claude E. , Lambert W. Clark , Gupta Megha , Malhotra Alisha , Ahsan Mina
Published: April 2020

Micro‐computed tomography (μ‐CT) scanners permit researchers to characterize the three‐dimensional structure of bone. They allow for the visualization of many bone structural parameters to be quantified with an extremely high degree of precision. Immunohistochemistry (IHC) (colorimetric) and immunofluorescence (IF) allows for characterization of DNA and RNA in fixed tissue sections. μ‐CT scanners along with IHC and IF allows processed tissue sections to be examined by a variety of different fixatives, in order to characterize the effects of fixation on bone morphology (i.e., adult chicken and mice), and preservation of different types of intact, non‐denatured nucleic acids (e.g., right‐handed B‐DNA, left‐handed Z‐DNA). Fixation was performed using 10% Neutral Buffered Formalin (NBF), Formalin‐Alcohol‐Acetic Acid (FAA), Davidson’s fixative, and Carnoy’s solution for 72 hours at room temperature. Both decalcified and non‐decalcified bone tissues were processed. Ultra‐pure molecular biological grade fixatives and water were used in order to ensure the best histotechnological data. Tissues processed in FAA resulted in hardening of tissue, but good fixation of intact DNA. Tissues processed in 10% NBF resulted in softer tissue, but poor fixation of intact DNA and required antigen retrieval (AR). Tissues processed in Carnoy’s solution resulted in hardened tissue, but excellent DNA IHC and IF. Tissues processed in Davidson’s fixative resulted in hardened tissues, but good preservation of DNA. We will try to correlate structural bone differences observed with the μCT scanner, relative to DNA isolation and IHC and IF staining of bone tissue sections with anti‐B‐DNA and anti‐Z‐DNA monoclonal antibodies. These results will better enable researchers to histotechnologically process tissue while obtaining data on DNA structure and function.
Gamification of General Education Science Courses for Online Delivery
Authors: Clauson-Kozina Cheryl L. , Decius Pamela Painter
Published: April 2020

Gamification is an approach to learning that has students participate in gaming style activities while engaging with the course material. While this can be readily implemented in face‐to‐face modalities, it can be more difficult to envision in an online course delivery. Here we examine this approach in two general education science courses that have been developed for online students. Each of these courses seek to empower students and give them a sense of autonomy and control of their learning “path”. Much like a choose your own adventure book, the students navigate their own way through each module, enabling them to take as much or as little time with each activity, as is appropriate to their individual learning styles. Gamified courses presented here include an evolution course and a biochemistry course focused on cooking. Game elements were used to present new material to students or for them to review content that they had already learned. Approaches that were effective (or not) are presented along with a discussion of challenges and how they were addressed throughout the course design process. Overall, the gamification has been well‐received by students, resulting in improved student evaluations and a reduction in DFW numbers for redesigned courses.