bluuebunny/crossref_metadata_2025 huggingface dataset:
What You See is What You Understand: How Changes in Dissection Techniques Have Informed Our Understanding of What We Perceive in Anatomy
Authors: Nemetz Laurice D.
Published: April 2020

Dissection can reveal or obscure aspects in our understanding of the human body. Destroying one part of the connected form in order to reveal a particular perception of the body system is, by nature, part of dissection technique. However, our understanding of the body has continued to change based on techniques from fresh tissue to shellacked, embalmed, semi‐fixed, plastinated and more. This raises the question of how much of our perception of the human body at any one point is based on technique and approach or our desire to see and name in a defined manner. Does one influence the other? Anatomists during the Renaissance, for example, worked with fresh tissue, and artists portrayed the musculoskeletal system, as well as the importance of connection and fascia in their movement‐filled illustrations. Modern techniques from decellurization to plastination have created newer fascial models, reversing the thought process of figure/ground perception of our previously muscle‐focused atlases. For example, the interstitium was recently named a new organ, based on fresh tissue samples that allowed the ability to see 3D fluid‐filled spaces in what was previously flattened in preserved tissue. While anatomy remains a key component of medical school training, it is widely documented that time spent in a physical dissection lab is decreasing. With less time in lab, are key components of three‐dimensional learning being lost, or simply not “seen” based on preserved tissue cadavers? Three‐dimensional computer models are stunning in the process to bring learning to areas that have low access to donation programs, but the computer scans are still based on a limited number of forms and may choose to obliterate certain tissues. Additionally, the loss of touch and fine motor skill that is practiced in cadaver lab is difficult to reproduce in a virtual model. Through a survey of anatomical illustrations, engravings, drawings and photography, it is evident that this raises questions on our past anatomical history to the importance of how our anatomy models of the future (from virtual reality to synthetic cadavers) may once again influence how we perceive the body, particularly in relationship to fascia.
Efficacy of In‐Class Drawing Activities on Clinical Anatomy of the Vagus Nerve
Authors: Mansour Yusra , Kulesza Randy J.
Published: April 2020

Introduction

In class drawing has been shown to enhance learning and promote long term retention in anatomy and medical education. There has, however, been little research into the effect of instructor‐guided interactive drawing sessions on clinical anatomy compared to textbook reading as well as the effect of drawing on technology (i.e. tablets, ipads) compared to drawing on paper.

Aim

We set out to investigate the effect of student’s preferred learning style (i.e. visual, non‐visual) on learning after instructor guided interactive drawing sessions using anatomy of the vagus nerve and compare this to more traditional resources such as lecture and textbooks.

Methods

In order to explore the effects of interactive drawing sessions and use of technology, we recruited novice first year medical students. Students were asked to complete the Index of Learning Styles questionnaire to determine preferred learning style and categorize students as either visual or non‐visual learners. Students were further sorted into 1 of 4 groups including a drawing on paper group, drawing on technology group, lecture group and a reading group and participated in a learning session on the anatomy of the vagus nerve. Learning sessions involved an anatomy pre‐test and a post‐test to assess learning. Both pre and post‐test included functional, structural, clinical and dissection‐based questions. Long‐term retention will be assessed 6 months after the primary learning session with another anatomy post‐test. Students were also asked to participate in a survey asking their opinion on the effectiveness of their session. Data collection and statistical analysis will be completed by Feb 2020. The protocol was approved by the Institutional Review Board (protocol # 26‐167).

Results

Preliminary results of pre‐ to post‐test analysis suggest an increase in post‐test scores in each of the four groups, with a greater improvement in non‐reading groups compared to that of the reading group. Students in the drawing and lecture groups performed better than those in the reading group on clinical case‐based questions. On the post‐session survey, non‐readers preferred their particular learning sessions, were more likely to recommend more sessions be added to the curriculum and felt their learning session taught them anatomy better than reading a textbook.

Conclusion

We interpret these results to suggest that instructor guided drawing and lecture sessions lead to enhanced learning and clinical comprehension as well as increased student satisfaction compared to reading.
HMGB1‐induced vascular hyperpermeability requires β‐catenin phosphorylation
Authors: Guo Xiaohua
Published: April 2020

Endothelial hyperpermeability is a hallmark of acute lung injury in response to sepsis. The imbalance between adherence junction (AJ) mediated cell‐cell adherence forces and stress fiber driven contractile forces contributes to increased endothelial permeability. Here, we spotlight the effects of β‐catenin Y654 andY142 phosphorylation on HMGB1‐mediated endothelial barrier leakage. Our results showed that phospho‐deficiencies at both β‐catenin Y654 and Y142 ameliorated pulmonary vascular dysfunction in male C57 mice receiving a cecal ligation and puncture operation. In vitro analysis indicated that high mobility group box‐1 protein (HMGB1) triggered β‐catenin Y654 and Y142 phosphorylation, causing β‐catenin translocation and adherence junction (AJ) disruptions as well as cytoskeleton rearrangement. In addition, β‐catenin Y654 dephosphorylation attenuated HMGB1‐mediated dissociation of VE‐cadherin/β‐catenin and, hence, partially prevented endothelial hyperpermeability. β‐catenin Y142 dephosphorylation abolished HMGB1‐induced uncoupling of β‐catenin and α‐catenin, suppressed cytoskeletal reassembly and, hence, alleviated endothelial hyperpermeability. Further investigation demonstrated that RAGE and Src were required for β‐catenin Y654 phosphorylation in response to HMGB1, while FAK was responsible for HMGB1‐triggered β‐catenin Y142 phosphorylation. In sum, this study revealed the role of β‐catenin Y654 and Y142 phosphorylation in HMGB1‐mediated endothelial hyperpermeability through dysregulation between adherence and contractile forces. This result advances understanding of the mechanisms underlying pulmonary vascular hyperpermeability in sepsis.

Support or Funding Information

This work was supported by National Natural Science Foundation of China (81670076, 31871183); Guangdong Natural Science Foundation (2017A030313462); General Program from Natural Science Foundation of China (81370226, 81170297); The Team project of Natural Science Foundation of Guangdong, China (S2013030013217); Grant of NSFC Guangdong Joint Foundation of China (U1601225); Key Scientific and Technological Program of Guangzhou City (201607020016); Special Funds for the Cultivation of Guangdong College Students’ Scientific and Technological Innovation [pdjh2017a0095].
Effect of temperature fluctuations on larval function of the Baja California chorus frog, Pseudacris hypochondriaca
Authors: Ramirez Marie , Manzanares Samantha , Mueller Casey A.
Published: April 2020

Environmental temperature has an extensive impact on the phenotypes of all animals.

Temperature fluctuations, often experienced naturally across daily or longer cycles, may cause profound effects on physiological phenotypes of animals, particularly during development and in ectotherms like amphibians. This study tested this hypothesis by evaluating the effects of temperature fluctuations during the larval development of the eurythermal Baja California chorus frog (Pseudacris hypochondriaca), a species native to southern California and northern Baja California. Eggs were collected from ponds in San Marcos, California within 24 hours of fertilization and separated into four temperature treatments: 15°C constant, and fluctuating treatments over 24 hours of 12.5°C – 17.5°C, 10°C – 20°C, and 15°C – 25°C. Oxygen consumption rate (V̇o2), critical thermal maximum (CTMAX), and swim speed were measured for tadpoles at 15°C during hindlimb toe development. Relative to mass‐specific V̇o2 in 15°C constant tadpoles, mass‐specific V̇o2 measured at 15°C showed a slight increase in 12.5°C – 17.5°C tadpoles, a larger increase in 10°C – 20°C tadpoles, but no difference in 15°C – 25°C tadpoles. Relative to CTMAX in 15°C constant tadpoles, CTMAX was similar in 12.5°C – 17.5°C tadpoles, slightly higher in 10°C – 20°C tadpoles, and greatly increased in 15°C – 25°C tadpoles. Swim speed measured at 15°C was similar across all temperature treatments. Overall, greater fluctuations had stronger effects on larval V̇o2 and CTMAX, and tadpoles tend to acclimate to higher temperatures during daily cycles. Acclimation in thermal tolerance and metabolic rates of this eurythermal amphibian indicates the animals will likely show plasticity in some traits in response to fluctuating temperatures. Future studies will examine these traits across a range of acute measurement temperatures to fully understand the functional implications of fluctuating temperatures.
Invasive Physiological Measurements in Patients with Peripheral Artery Disease: Willingness and Barriers to Participation
Authors: Muller Matthew D. , Levenberg Kate , Luck J. Carter , Miller Amanda J. , Maman Stephan R. , Aziz Faisal , Radtka John F. , Proctor David N.
Published: April 2020

Invasive physiological measurements such as microneurography, arterial and venous catheterization, and central neuraxial anesthesia are necessary to investigate mechanisms of cardiovascular control in humans. To date, these procedures have not been utilized in patients with peripheral artery disease (PAD). The purpose of this project was to quantify the willingness, motivating factors, and barriers to participating in invasive physiological experiments in adults with and without PAD. Data were gathered through focus groups (n = 19 participants age 55–79) and questionnaires (n = 2543 mailed, 438 adults age 18–85 responded). Of the people who returned the questionnaire, 90% were willing to participate in ultrasound assessment of vascular function and 76% were willing to undergo a venous blood draw, whereas only 40% were willing to participate in microneurography, 30% were willing to undergo arterial or venous drug infusions, 12% were willing to receive spinal or epidural anesthesia, and 14% were willing to undergo a muscle biopsy. PAD patients (n=45) were more willing than those without PAD (n=360) to participate in drug infusion studies (48% vs 18%, P<0.001) and trials of investigational drugs (44% vs 21%, P<0.001). The participants also provided feedback on our recruitment materials and made suggestions to increase enrollment in our ongoing randomized controlled trial. In the past three years, some of these strategies have been implemented. Based on our focus groups and questionnaire data, we speculate that engaging PAD patients in all aspects of the research process will lead to better recruitment and retention as compared to the “standard of care” (i.e., spending large amounts of money on radio and television advertising, printed brochures, searching electronic medical records, and direct mailing). However, this remains to be directly tested in future studies.

Support or Funding Information

This project was supported by NIH R21 AG054940 (to Dr. Proctor). This project was also supported, in part, by National Center for Advancing Translational Sciences Grants UL1 TR002014, UL1 TR‐000127 and KL2 TR‐000126 and also under a grant with the Pennsylvania Department of Health using Tobacco CURE funds (to Dr. Muller).
Integrin‐targeting ferritin for drug delivery and tumor imaging
Authors: Chen Yan-Jun , Wang Hsu-Yuan , Huang Feng-Ting , Huang Chiun-Wei
Published: April 2020

Various potent chemotherapeutic drugs have been developed over decades. Doxorubicin, the lead compound of the anthracycline family, has been applied clinically in a wide range of malignancies including leukemia, lymphomas, myeloma, breast cancer, lung cancer and sarcoma etc. Despite its profound therapeutic efficacy, this chemotherapeutic drug causes numerous side toxicity effects. One main side effect is cardiotoxicity. To eliminate the side effect of this conventional chemotherapeutic drug, targeted delivery of chemotherapeutic drugs to tumor cells is being investigated. This concept can be achieved using nano‐scaled drug‐containing particles, which are called nanoparticles. Many promising nanoparticles with various materials have been developed, such as liposomes, micelles, and carbon nanotubes. However, biocompatibility and metabolism issues related to these nanoparticles have limited their clinical applications. Hence, various nanoparticles have been fabricated to improve above disadvantages. Ferritin, consisting of 24 subunits, could self‐assemble into a shell‐like sphere and which make ferritin a suitable drug‐carrier. Moreover, with precisely assembled nanoscale shape, high safety and biocompatibility, ferritin has the potential for in vivo imaging and therapeutic purpose.

Integrin α2β1 is a major collagen receptor that highly expresses on various tumor cells such as prostate, lung, and colorectal cancer cells. In this study, we have constructed integrin‐targeted ferritin which expresses integrin α2β1‐targeting peptide on the ferritin surface to specifically target cell lines with high‐expression of integrin α2β1. Our results indicated that the integrin‐targeted ferritin showed higher doxorubicin loading efficiency and yield than unmodified ferritin. Moreover, flow cytometry analysis showed that integrin‐targeted ferritin had better binding ability to U‐87 MG and PC3, cell lines with high integrin α2β1 expression, than unmodified ferritin. Furthermore, in cell cytotoxicity assay, doxorubicin encapsulated within integrin‐targeted ferritin showed higher cytotoxicity effects than doxorubicin encapsulated within unmodified ferritin and free doxorubicin in U‐87 MG and PC3 cells. All of our results support that integrin‐targeted ferritin has the potential to be a novel anticancer nanomedicine.

Support or Funding Information

This study was supported by Chang Gung Memorial Hospital, Taoyuan, Taipei, TAIWAN and Department of Biochemical Science and Technology, National Taiwan University, Taipei, TAIWAN
Exogenous Lactate Intake Can Induce Muscle Synthesis in Rats
Authors: Kyun Sunghwan , Yoo Choongsung , Jang Inkwon , Kim Jisu , Lim Kiwon
Published: April 2020

Purpose

Lactate had been recognized as fatigue substance during high intensity exercise. However, the lactate paradigm has changed in the last decades, and it was reconsidered as a potential factors of energy resource and muscle adaptation. Nevertheless, the effect of administration of exogenous lactate on muscle has not been elucidated, and it has been no molecular signaling study of the muscle synthesis over time after ingestion. Therefore, we aimed to investigate the effect of lactate on muscle synthesis on skeletal muscle, according to the different time intervals within 2 hours.

Methods

Male rats were randomly assigned into one of following four timings that were sacrificed time after oral administration of sodium lactate (2 g/kg); 0 min (n = 8), 30 min (n = 8), 60 min (n = 8) and 120 min (n = 8). After administration lactate, rats were sacrificed and immediately obtained blood and skeletal muscle (plantaris). To determine the effect of lactate on muscle synthesis (Akt, mTOR) and degradation (MuRF1, atrogin‐1), we measured mRNA expression in plantaris by RT‐PCR, and analyzed blood parameters of lactate, insulin, IGF1, glycerol and glucose.

Results

The results showed that, despite no significant differences in the hormone receptors for androgen and IGF1 associated with synthesis, Akt remained 1.5~2 fold higher after 30minutes, and mTOR was lower than the 30‐minute expression level but showed a high trend at 60 and 120 minutes. In muscle degradation factors, MuRF1 remained elevated after 30 minutes, but atrogin‐1 had no change. Lactate intake increased plasma lactate by about 3.2 mmol / L at 60 minutes and decreased insulin and IGF 1 after 30 minutes. Furthermore, plasma glycerol was increased at 120 min, but glucose has not changed within 2 hours.

Conclusion

It is demonstrated in the present study that lactate administration contributes to increase muscle synthesis factors at 30 minutes, and it remained at high levels within 2 hours. Moreover, the reduction of insulin and IGF1, and the upregulation lactate confirmed that the administration of lactate might be similar to the blood response of the human body during low or moderate exercise. Therefore, our results suggest that lactate intake as a supplement has the potential to increase muscle synthesis and have a positive effect on muscle injury and recovery after exercise. Despite these results, because our study is acute intake and synergies with exercise are not identified, further research is required to investigate the long‐term administration of lactate and the effects of exercise on muscle synthesis.

Support or Funding Information

This work was supported by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea(NRF‐2019S1A5A8033825)
Loss of von Hippel‐Lindau protein in renal collecting duct cells is associated with massive changes in gene expression and induces a mesenchymal phenotype
Authors: Edemir Bayram , Chernyakov Dmitry , Fischer Annika , Becker Franziska , Gallwitz Lisa , Bornkessel Nicola , Groß Alexander
Published: April 2020

Introduction

Clear‐cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer. It is characterized by a loss of function mutation of the von‐Hippel‐Lindau tumor suppressor (VHL). The proximal tubule is traditionally the source of ccRCC but there is also evidence that a subset con also originate from collecting duct. Alongside the renal nephron, cells are exposed to increasing concentrations of NaCl and urea. In the collecting duct, osmolality can reach up to 1200 mosmol/kg, generating a hyperosmolar environment. It has previously been shown, that VHL deficient mice express a phenotype of polyuria and reduced aquaporin‐2 expression, which is predicted to be caused by a hypoosmolar environment in the nephron. This implicates a physiological function of VHL in the collecting duct segment. Most research however, that discusses the effects of VHL deletion has been conducted with renal cancer cell lines such as 786‐0, Caki or RENCA, which harbor various other specific mutations. Within this study we analyzed the impact of VHL deletion, using “healthy” renal collecting duct cells.

Materials and methods

We created a VHL deficient mpkCCD cell line by using CRISPR/Cas9. Single clones with functional VHL deletion were selected. The knockout was confirmed by western blotting. Cell morphology studies were performed, using the immune fluorescence method. The gene expression profiling was performed by Next Generation Sequencing analysis and results were validated with real time PCR. Proliferation and migration assays were performed for functional characterization of the cells.

Results

We successfully generated single clones harboring dysfunctional VHL. This was associated with stable Hif1a expression. VHL deficient mpkCCD cells undergo drastic morphological and functional changes. We observed the partial loss of tight and adherent junctions. Deletion of VHL is also associated with higher migration and lower proliferation potential compared to wildtype cells. The expression of Aqp2, a specific marker of the collection duct epithelial cells, was massively reduced. RNA‐seq analysis showed massive changes in global gene expression pattern and cancer associated pathways were affected by VHL deletion. Correlation with the Humans Pathology Atlas showed that VHL loss induces an unfavorable gene expression pattern.

Conclusions

Since the mpkCCD cells only lack active VHL, we are able to conclude a direct link between the loss of function of VHL and the cancerous transformation of epithelial cells. Therefore, our results might be useful for further research and novel treatment strategies in the future.

Support or Funding Information

German Research Fundation
Racial Differences in Cardiometabolic Complications in Polycystic Ovary Syndrome
Authors: Stockton Savannah Marie , Alexander Nathan , Fernandez Edgar Torres , Huffman Alexandra , Reckelhoff Jane , Romero Damian , Cardozo Licy Yanes
Published: April 2020

Background

Polycystic Ovary Syndrome (PCOS), the most common endocrine disorder in reproductive‐age women, is characterized by androgen excess and ovarian dysfunction. PCOS women have an increased prevalence of components of the metabolic syndrome, such as increased blood pressure (BP), insulin resistance and obesity. Whether African American women with PCOS have worse cardiometabolic profile than Caucasian remains unknown.

Aims

To determine the prevalence of components of the metabolic syndrome in patients of different ethnicity with PCOS in the state of Mississippi.

Methods

University of Mississippi Medical Center (UMMC) de‐identified patient data from 2013–2017 was obtained from electronic medical records (EMR) using UMMC’s Research Data Warehouse. Cardiometabolic risk factors were analyzed and compared between African American and Caucasian PCOS women. Data is expressed as mean. SEM and statistical analysis was performed using SPSS.

Results

During the 2013 to 2017 period, EMR of 1,200 PCOS patients were obtained and analyzed from a total of 784,666 patients that were evaluated at UMMC. Compared to Caucasians, African American women with PCOS have significantly higher mean arterial BP (97.06±0.52 vs 94.23±0.48 mmHg p<0.001), hemoglobin A1C (6.03±0.08 vs 5.63±0.07 % p<0.001) and Body Mass Index (39.30±0.43 vs 36.05±0.39 p<0.001). About 44% of patients with PCOS have a BMI more than 40 (morbid obesity).

Conclusions

Morbid obesity is highly prevalent in patients with PCOS in the Mississippian population cared for at UMMC. African American women with PCOS have a significantly higher prevalence of cardiometabolic risk factors than Caucasians. Lifestyle modifications should be implemented to ameliorate the prevalence of cardiometabolic risk factors in patients with PCOS.

Support or Funding Information

Funding for this research is supported by NIH R01 HL135089 (JFR), P20 GM12334(LLY, ETF, JFR, DGR) and PO1HL51971 (JFR).
SPARC: Determine the topology and function of DRG neurons innervating mouse colon and rectum.
Authors: Feng Bin , Guo Tiantian , Bian Zichao , Chen Longtu , Zheng Guoan
Published: April 2020

Visceral pain is the major complaint of patients with irritable bowel syndrome (IBS) that affects ~15% of the U.S. population, and satisfactory management of IBS pain remains an unmet clinical challenge. Neuromodulation targeting afferent somata in dorsal root ganglions (DRG) has emerged as a new and effective therapy for many types of chronic pain. To advance neuromodulation for treating IBS‐related visceral pain, new knowledge is needed regarding the topological distribution and neural encoding functions of lumbosacral DRG neurons that innervate distal colon and rectum (colorectum). Here, we implemented GCaMP6f to conduct high‐throughput optical recordings of colorectal afferent activities in lumbosacral DRG. By establishing this optical approach with single‐spike resolution, the current study characterizes the neural encoding from hundreds of colorectal afferents in the DRG, which are sparsely distributed and difficult to study with conventional methods. Using a mouse ex vivo preparation with distal colorectum and L5‐S1 DRG in continuity, we recorded 791 colorectal afferents’ responses to graded colorectal distension (15, 30, 40, 60 mmHg) and/or luminal shear flow (20–30 mL/min), then functionally classified them into four mechanosensitive classes, and determined the topological distribution of their somata in the DRG. Of the 791 colorectal afferents, 90.8% were in the L6 DRG, 8.3% in the S1 DRG, and only 0.9% in the L5 DRG. L6 afferents had all four classes: 29% mucosal, 18.4% muscular‐mucosal, 34% low‐threshold (LT) muscular and 18.2% high‐threshold (HT) muscular afferents. S1 afferents only had three classes: 19.7% mucosal, 34.8% LT muscular and 45.5% HT muscular afferents. All seven L5 afferents were HT muscular. In L6 DRG, somata of HT muscular afferents, i.e., putative colorectal mechano‐nociceptors were clustered in the caudal region whereas somata of the other classes did not cluster in specific regions. Outcomes of this study can directly inform the design and improvement of next‐generation neuromodulation devices that likely implement spatially selective DRG modulation to alleviate visceral pain in IBS patients.

Support or Funding Information

Supported by NIH SPARC grant U01 NS113873 (BF and GZ)
Sex Differences in the Cerebrovascular Response to a Metabolic Stimulus
Authors: Pearson Andrew G. , Miller Kathleen B. , Howery Anna J. , Corkery Adam T. , Barnes Jill N.
Published: April 2020

Background

Blood pressure control and regulation of cerebral blood flow are influenced by biological sex. We have previously demonstrated sex differences in cerebrovascular reactivity to hypercapnia in young adults. However, sex differences in the cerebrovascular response to a metabolic stimulus are unknown. As prostaglandins play a role in regulating cerebrovascular reactivity, evaluating cerebrovascular responses to a metabolic stimulus with cyclooxygenase (COX) inhibition may reveal important differences between males and females. Therefore, the purpose of this study was to investigate sex differences in the cerebrovascular response to a metabolic stimulus with and without COX inhibition in young adults.

Methods

Forty‐three young adults, 21 males (age = 28 ± 5 y) and 22 females (age = 25 ± 5 y), completed two levels of difficulty of the Stroop Color Word Test and the average response was calculated. Beat‐to‐beat mean arterial pressure (MAP) and middle cerebral artery velocity (MCAv) were measured at baseline and in response to each level of the Stroop test before and after administration of the COX inhibitor indomethacin. The peak MAP, MCAv, pulse pressure (PP), and cerebral pulsatility index (PI) responses were calculated as the highest 3‐beat average during each Stroop test.

Results

At rest, MCAv was higher (Females: 65 ± 2, Males: 55 ± 2 cm/s, p < 0.01) and PI was lower (Females: 0.70 ± 0.02, Males: 0.78 ± 0.02, p < 0.05) in females compared to males, with no differences in MAP or PP. There were no differences in the percent change or peak MCAv, MAP, PP, or PI between males and females in response to the metabolic stimulus. COX inhibition reduced MCAv (Females: −28 ± 2%, Males: −28 ± 3%, p < 0.01 compared to baseline) and increased PI (Females: 17 ± 2%, Males: 20 ± 4%, p < 0.01 compared to baseline), with no change in MAP or PP. During COX inhibition, MCAv at rest was higher (Females: 46 ± 2, Males: 38 ± 1 cm/s, p < 0.01) and PI was lower (Females: 0.81 ± 0.03, Males: 0.93 ± 0.03, p < 0.05) in females compared to males, with no differences in MAP or PP. During COX inhibition, the percent change in MCAv, MAP, and PI in response to the metabolic stimulus did not differ between groups (p > 0.05); however, the percent change in PP during the metabolic stimulus was greater in males compared to females (Males: 16 ± 2%, Females: 9 ± 1%, p < 0.01).

Conclusion

Young females had greater MCAv and lower PI at rest and this difference persisted following COX inhibition. Prior to COX inhibition, there were no differences between males and females in response to the metabolic stimulus. After COX inhibition, young males demonstrated a greater change in PP in response to the metabolic stimulus. These results suggest that COX inhibition reveals sex differences in the PP response to a metabolic stimulus.

Support or Funding Information

Funding

This research was funded by NIH grant HL118154.
Maternal Nicotine Exposure During Lactation Alters Craniofacial Development
Authors: mohi amr , Kishinchand Rajiv , Durham Emily , Cray James
Published: April 2020

Maternal exposure to nicotine has been linked to increased incidence of craniofacial abnormalities in infants. One of the craniofacial anomalies is craniosynostosis, which results from the premature fusion of the calvarial sutures. In the US, incidence of craniosynostosis is 1:1800–2500 births and often requires neurosurgical intervention to allow for normal brain growth. As the use of nicotine is increasing among young population specially through vape/electronic cigarettes use, we feel the need to investigate the effects of maternal nicotine on craniofacial development. We aim to determine the most critical period along pregnancy and lactation where maternal nicotine exposure could cause craniofacial anomalies and how does nicotine affect cranial sutures and other craniofacial structures. We compared the widths of coronal, sagittal, and posterior interfrontal sutures in offspring after maternal nicotine exposure during pregnancy only, lactation only, and pregnancy + lactation. Using Micro‐CT scans for cephalometric measurements, our preliminary data shows that maternal exposure to nicotine during lactation affects coronal suture development evidenced by decrease suture width and incidence of fusion in PN day 15 mice. We plan future experiments using histomorphometry and immunohistochemistry to better understand how sutural tissue and cells are affected by maternal nicotine exposure.

Support or Funding Information

This study was supported by research grants from the National Institute of Dental and Craniofacial Research[R03DE026192 (JC), F31DE026684 (ED)], National Institutes of Health National Center for Advancing Translational Sciences [UL1 TR000062]Plastic Surgery Foundation [Pilot Award 512114].
Store‐Operated Calcium Entry Upregulated IL6 Expression in Glomerular Mesangial Cells through NF‐κB Pathway
Authors: Chaudhari Sarika , Shotorbani Parisa Yazdizadeh , Wei Celine , Davis Mark E. , Ma Rong
Published: April 2020

Background

IL6 is a pleotropic cytokine and functions as a pro‐ as well as anti‐inflammatory mechanism depending on the cell type or the specific pathological environment. It is unclear if a particular signaling pathway confers IL6 with this property. Immunological and inflammatory mechanisms play a significant role in the development of diabetic nephropathy (DN). Locally produced cytokines/chemokines contributes to the early changes and histological impairment in DN. Glomerular mesangial cell (MC) is a major cell type in glomerulus to produce cytokines/chemokines in response to diabetes and a major contributor to mesangial expansion in DN. We have previously demonstrated that the Orai‐1 mediated store‐operated calcium entry (SOCE) suppressed ECM protein production by MCs. The aim of this study was to determine whether and how SOCE in MCs regulated IL6 production by MCs.

Methods

Orai1, the channel protein mediating SOCE in MCs was knocked down using the targeted nanoparticle‐siRNA delivery system in wild type C57BLKS/J mice at the age of 16 weeks. Immunohistochemistry was performed on the paraffin embedded kidney sections to examine glomerular IL6 expression. In cultured human MCs, the expression of IL6 was examined in culture media and whole cell lysates using ELISA and Western blot analysis, respectively in the presence of normal glucose (5 mM D‐glucose + 20 mM L‐glucose) with/without an activator (thapsigargin at 1 μM) or inhibitor (GSK‐7975A 10 μM) of SOCE for 15 hrs. To determine the role of NF‐κB in SOCE effects on IL6 production, human MCs were treated with an inhibitor of NF‐κB, helenaline or siRNA against p65 subunit of NF‐κB. Furthermore, the effect of SOCE on the nuclear translocation of p65 was examined by assessing abundance of nuclear p65 protein in response to activation and inhibition of SOCE.

Results

The IL6 expression level was reduced in the glomeruli of the mice treated with nanoparticle/Orai1 siRNA for 2 weeks compared to that in the control mice. In cultured human MCs, thapsigargin significantly increased IL‐6 level in supernatant media and in whole cell lysates of MCs, and this effect was attenuated by GSK 7975‐A, a selective inhibitor of SOCE. However, tunicamycin, an ER stress inducer did not alter the amount of IL6 as thapsigargin did. Treatment with helenaline, an inhibitor of NF‐κB pathway, or knockdown of p65, a subunit of NF‐κB significantly blunted the thapsigargin‐induced increase in IL6 protein abundance. Moreover, thapsigargin stimulated the nuclear translocation of p65 in human MCs.

Conclusion

Orai1‐mediated SOCE positively regulates IL6 production by MCs through activation of NF‐κB pathway.

Support or Funding Information

Research Support: NIH/NIDDK (1RO1 DK115424); AHA Southwest Affiliate.
Identifying the 20‐HETE Binding Site on the 20‐HETE Receptor (GPR75)
Authors: Pascale Jonathan V. , Adebesin Adeniyi , Falck John R. , Schwartzman Michal , Garcia Victor
Published: April 2020

The G‐protein coupled receptor GPR75 and its ligand the cytochrome P450 (CYP)‐derived vasoactive eicosanoid 20‐hydroxyeicosatetraenoic acid (20‐HETE), are implicated in the activation of pro‐inflammatory and hypertensive signaling cascades including decreased nitric oxide (NO) production and increased angiotensin converting enzyme (ACE) protein levels. Little is known as to how and where 20‐HETE interacts with GPR75. Computational modeling of the putative ligand binding pocket identified a site located across the fifth and sixth transmembrane on GPR75 (SiteScore: 1.182 and Dscore: 1.303). An independent analysis modeling the ligand‐receptor interaction between 20‐HETE and GPR75 places 20‐HETE in close proximity to the putative binding site location (Docking Score: −3.229, Glide Emodel: −29.045). Using the PRESTO‐TANGO methodology as a measure of receptor activation through the quantification of a ligand‐dependent β‐arrestin recruitment, 20‐HETE (1 nM) increased β‐arrestin recruitment by 3.48‐fold (± 0.63; p<0.05) compared to vehicle treated HTLA cells transfected with the WT‐GPR75‐TANGO construct. Recruitment of β‐arrestin was not observed with other structurally similar lipids including arachidonic acid, arachidonoyl ethanolamide and 12(S)‐HETE. Moreover, co‐treatment of WT GPR75‐TANGO cells with equimolar concentrations of 20‐HETE and the 20‐HETE antagonist N‐disodium succinate‐20‐hydroxyeicosa‐6(Z),15(Z)‐diencarboxamide (AAA) prevented the 20‐HETE‐mediated increase in GPR75 receptor activation, i.e., β‐arrestin recruitment [3.45 ± 0.19 vs. 0.89 ± 0.15 fold, 20‐HETE (1 nM) vs. 20‐HETE (1 nM) + AAA (1 nM), respectively; p<0.05]. Analysis of the 20‐HETE‐GPR75 interaction revealed several potentially key amino acids involved in hydrogen bond interactions with 20‐HETE including Ser205, Thr212 and Ser209. To assess the significance of these amino acids with respect to 20‐HETE‐mediated activation of GPR75, we generated site‐directed mutants of each residue. Cells transfected with the Ser205Ala and Ser219Ala mutant GPR75‐TANGO constructs exhibited similar increases in 20‐HETE receptor activation when treated with 1nM 20‐HETE (3.73 ± 0.79‐fold and 3.01 ± 0.57‐fold increases in luminescence, respectively, compared to vehicle treated controls; p<0.05). Interestingly, Thr212Asn GPR75‐TANGO transfected cells showed no significant changes to receptor activation when exposed to 20‐HETE (1nM) (1.00 ± 0.29 vs. 0.84 ± 0.18‐fold, vehicle vs. 20‐HETE). Taken together, this study identifies Thr212 as a critical residue of the 20‐HETE receptor binding site located across GPR75’s 5th and 6th transmembranes. Further studies are necessary to uncover other essential residues and their role in 20‐HETE binding and receptor activation.

Support or Funding Information

This study was supported by National Institute of Health grants HL39793 (M.L. Schwartzman), HL39793‐01S1 (V. Garcia) and the Robert A. Welch Foundation (I‐0011, J.R. Falck).
Does Clopidogrel Decrease the Efficacy of Carboxylesterase‐1 Prodrugs?
Authors: Laizure Steven C. , He Hui , Parker Roert B.
Published: April 2020

It has previously been reported that clopidogrel is a potent inhibitor of the carboxylesterase‐1 (CES1) mediated hydrolysis of oseltamivir to its active metabolite based on in vitro inhibition studies. However, clopidogrel undergoes extensive first‐pass hepatic metabolism resulting in 85% of the dose being metabolized to clopidogrel carboxylate prior to reaching the systemic circulation. Thus, despite its potency as an inhibitor of CES1 enzyme activity, it is unlikely to affect the formation of oseltamivir or any other CES1‐substrate prodrug due to the low level of systemic exposure. We studied the inhibition of clopidogrel and its major metabolite, clopidogrel carboxylate, on another CES1‐substrate prodrug used in the treatment of multiple sclerosis, dimethyl fumarate. Our finding corroborates the previous study demonstrating potent inhibition of CES1 enzyme activity by clopidogrel, but also demonstrated a similar potency in the inhibition of enzyme activity by its major metabolite, clopidogrel carboxylate. In human recombinant CES1 enzyme 50 μM of clopidogrel or clopidogrel carboxylate inhibited the CES1‐mediated hydrolysis of dimethyl fumarate to monomethyl fumarate by 90%. Given that the maximum plasma concentration of the clopidogrel carboxylate metabolite is over 1000‐fold greater than clopidogrel after normal therapeutic doses, the primary inhibition of CES1 enzyme activity will be due to clopidogrel carboxylate and not clopidogrel. Furthermore, the maximum concentration of the clopidogrel carboxylate metabolite reported after a 600 mg clopidogrel loading dose was 71.4 μM, which would be in excess of the concentration required to achieve a 90% reduction in CES1 enzyme activity. Therapeutic dosing with clopidogrel is likely to inhibit the formation of the active metabolite of CES1‐substrate prodrugs resulting in therapeutic failure.

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None
Perception of Pharmacology Education by Medical Sophomore students from 2000 to the present: impact of digital technology and educational policy
Authors: Arai Amy
Published: April 2020

Pharmacology is one of the disciplines that medical students find difficult to study. Reasons are that there are numerous drug names to remember with a variety of side effects; even within a given class, each drug may have unique advantages or disadvantages with regard to drug‐drug interaction, pharmacokinetics, and contraindications. To make things worse, a deep anxiety about the medical licensing step 1 exam induces students to learn pharmacology through simple rote memorization by relying predominantly on pharm cards and online visual‐mnemonics interfaces, and too little time is spent trying to understand drug action in the context of physiology and cellular biochemistry. Thus, the goal of Pharmacology education seems not to be achieved during the basic science years. Our SIU School of Medicine medical curriculum has introduced unique curriculum methods. This includes problem‐based learning modules (PBLM) and group discussions as major curricular activities in Year 1 and 2 basic science teaching, with optional attendance at resource sessions. In this study, AAMC Graduation Questionnaires and students’ comments on Pharmacology curriculum at SIU School of Medicine are analyzed for the academic years 2001 to 2019. One question addressed if the basic science curriculum provides a sufficient foundation for clinical clerkship. The percentage of ‘Good’ and ‘Fair’ remained similar (35–38%) across 18 years. However, ‘Disagree’ was notably reduced from 16% to less than 10% around 2003, whereas the national average remained about the same. The nature of students’ perceptions on Pharmacology Education and their performance will be discussed in relation to the incorporation of digital technologies like online interfaces, cloud and social media into the curriculum, as well as the introduction of innovative teaching methods.

Support or Funding Information

None
Childhood Cancer Drug Development: Why Alternative Designs for Drug Development are Required
Authors: Houghton Peter J.
Published: April 2020

In the US, the regulatory environment, through the Research to Accelerate Cures and Equity for Children Act (RACE for Children Act) requires FDA to develop a list of molecular targets and molecular targets of new drugs and biologics in development, that are determined to be substantially relevant to the growth and progression of pediatric cancer, and that may trigger the requirement for pediatric investigations. The intent is to engage Pharma in pediatric testing at an early stage in drug development. Under the RACE for Children Act, FDA may now require pediatric assessments when molecular targets under FDA review are substantially relevant to childhood cancer. This may require evaluation of up to 200 entities each year. However, it is clear that because a cancer has a particular ‘molecular driver’ it does not necessarily predict tumor response to a targeted agent. For example, some patients with low‐grade BRAF‐driven glioma have dramatic responses to MEK inhibitors, whereas others have some disease stabilization. Thus, factors other than the driver‐mutation appear important to tumor response. Consequently, to encompass clinical heterogeneity (genetic/epigenetic) that may determine response to treatment, a large number of models having similar molecular characteristics will have to be developed to allow adequate pre‐clinical evaluation of new targeted entities. Previously, the Pediatric Preclinical Testing Program (PPTP) conducted a retrospective analysis of over 2100 studies using 83 PDX and xenograft models and 67 drug entities undertaken using conventional testing (10 mice/group for solid tumor PDX models, 8 mice/group for leukemia PDX models). This analysis showed that use of one mouse per treatment group would accurately predict the group median response in 78% of experiments. Allowing a deviation of + or − one response category, the accuracy was 95% (Murphy B. et al, Cancer Res. 2016). Here we have prospectively tested the ‘single mouse’ experimental design to evaluate both small molecule and antibody‐drug conjugate drugs. We conclude that the ‘single mouse’ approach is highly accurate, and yields more information than traditional experimental approaches, particularly with respect to identifying biomarkers that associate with tumor response. Further, within the current resource constraints, this design allows up to 20 PDX models to be used for every one model used in the conventional testing design. Thus, the single mouse design potentially allows incorporation of disease heterogeneity that may be required for evaluating new agents under the RACE for Children Act.

Support or Funding Information

NO1‐CM42216, UO1CA199297, CA165995 from NIH, and CPRIT RP160716
Cervical Vagal Nerve Stimulation Restores Ventilatory Pattern Variability in an E. Coli Sepsis Rodent Model.
Authors: He Zehong , Nethery David , Campanaro Cara , Dick Thomas E. , Jacono Frank J. , Hsieh Yee-Hsee Hsee
Published: April 2020

Sepsis is a life‐threatening disease characterized by dysregulated systemic inflammation and organ failure. We have associated an increase in predictability of the ventilatory pattern variability (VPV) with progressive development of systemic inflammation leading to sepsis. Vagal nerve stimulation (VNS) is a proposed electro‐therapeuty that reduces circulating pro‐inflammatory cytokines, like interleukin‐1 beta (IL‐1β), that are associated with systemic inflammation. We hypothesize that the anti‐inflammatory effects of VNS will be reflected in reduced predictability of VPV. Urethane‐anaesthetized adult Sprague Dawley male rats (350–400g n=17) were instrumented for blood pressure (BP), heart rate (HR), and airflow (pneumotach). The left cervical vagus was dissected, isolated and prepared for electrical stimulation via nerve cuff. Each rat was subjected to a staircase protocol of monophasic electrical stimulus pulses (0.6 mA, 0.1 ms pulse width, 10 Hz) to identify the maximal current that was subthreshold for cardiac and respiratory responses. Four experimental cohorts were then established: 1) (+stim/+E. coli) (n=4), 2) (−stim/+E. coli) (n=5), 3) (+stim/−E. coli) (n=2), 4) (−stim/−E. coli) (n=6). In +stim groups, a stimulus train was initiated 10 min before and continued for 20 min after an E. coli solution (60 μg/kg rat; 50,000 cells suspended in normal saline) was injected intravenously (IV). The effects of stimulation on the systemic inflammatory response were determined by comparing serum cytokine levels prior to stimulation to those 90 min after inoculation and the effects on VPV was quantified from non‐linear complexity index (NLCI) prior to the blood draws. Cervical VNS reduced expression of IL‐1β from 102.33 ± 49 pg/ml to 69.50 ± 28 pg/ml and reduced NLCI (0.57 ± 0.04 bits to 0.48 ± 0.03 bits; p < 0.001). VPV became more predictable with the infusion of E. Coli and VNS in the presence of E. coli infusion reduced IL‐1β expression and decreased NLCI. These data suggest that VNS impacts both peripheral and central inflammation during E. Coli infusion.

Support or Funding Information

Supported by U01 EB021960, and VA I01BX004197
Uncovering the DNA binding properties of the GATA4 and TBX5 transcription factor complex
Authors: Rodríguez Emili Patricia Rosado , Ríos Jessica Rodríguez , Martínez José Rodríguez
Published: April 2020

Transcription factors (TFs) are essential master regulators of gene expression. These DNA binding proteins are fundamental for cellular differentiation during development. Eukaryotic TFs are notorious for not working alone and they often bind DNA as multimeric protein complexes to regulate gene expression. GATA4 and TBX5 are transcription factors that are central components of the gene regulatory network of human heart development and function. Recent studies have determined the binding specificity of these transcription factors as monomers. However, the DNA‐binding properties of the cooperative complex between GATA4 and TBX5 remain undetermined. Based on this, we want to know the intrinsic DNA binding preferences and the emergent properties that result from the cooperative complex formed by GATA4 and TBX5. We used a cell‐free protein expression system to produce GATA4 and TBX5, and we validated their monomeric and complex DNA‐binding activity through an Electrophoretic Mobility Shift Assay (EMSA). Subsequently, we successfully determined the in vitro DNA‐binding specificity of the GATA4:TBX5 complex using Systematic Evolution of Ligands by Exponential Enrichment (SELEX‐seq). For further studies, we will use this data to computationally predict genome‐wide recognition sites of this transcription factor complex. The findings of this study will help understand the DNA‐recognition rules of the GATA4:TBX5 complex and its potential roles in normal heart development.

Support or Funding Information

This research was supported by:NIH SC1GM127231NSF PR‐LSAMP #1400868