bluuebunny/crossref_metadata_2025 huggingface dataset:
Antioxidant Effect on Myofibroblast Differentiation
Authors: Palmer Tiffany Robin , Vaughan Melville B.
Published: June 2021

Antioxidants have been shown to decrease various fibrotic cells in culture, however little is known about their effects on dermal fibrotic cells. In this study we will investigate the effects of several different antioxidants on dermal myofibroblast differentiation. This will be accomplished by measuring the percent of myofibroblasts and cell number in differing treatment groups. The antioxidants chosen for our study include: carcinine, epigallocatechin gallate, and lycopene. Immortalized human fibroblasts treated with TGF‐β are plated at a concentration of 0.2 x 105 cells per coverslip. After incubating 72 hours, the coverslips are fixed, stained with immunofluorescence, and photographed. The preliminary results suggest that myofibroblast presence is somewhat reduced by carcinine; however this antioxidant had little effect on the cell number. Experiments with epigallocatechin gallate and lycopene are underway. The antioxidants that affect myofibroblast differentiation in the coverslips will then be tested in stress‐relaxed collagen lattices. These studies may be useful to develop future therapies for Dupuytren's contracture and other fibrotic tissue pathologies. Funded by a UCO student RCSA Award.

Grant Funding Source: UCO RCSA Award
The characteristics of rabbit and rat mesenchymal stromal cell growth and attachment to mesh used in hernia repair
Authors: Fagan Diana L. , Lydic Melissa , Marie Hazel , Fredette Paul , Heffner Jeremy , Dorion Heath
Published: June 2021

The goal of this project is to create an optimal environment for wound healing. Preliminary results from a rabbit model suggest that the addition of mesenchymal stromal cells (MSC) to a repaired incision improves the biomechanical properties of the wound. To increase sample size and reproducibility, these studies will be repeated in an inbred rat model. A review of the literature shows a wide variety of culture conditions used for rat MSCs. This study compares proliferation of rabbit and rat MSCs when grown in ten different growth medias. The Cell Titer 96 ® Cell Proliferation Assay and direct cell counts were used to measure the proliferation of MSC's. Rabbit and rat MSC growth was optimal in alpha MEM + 20% FCS or Hams F12 + 10% FCS. To prepare for the extension of these studies into the rat model, four mesh materials commonly used in hernia repair were tested to determine the best vehicle for the addition of MSCs. MSCs were incubated with the mesh for 30 min at 37oC. Non‐adherent cells were removed and the remaining cell number determined by incubation with Cell Titer 96 ® solution and comparison to an MSC standard curve. Optimal adherence was seen with Bard ® CollaMend (38% of added cells). Future studies will use Bard ® CollaMend as a vehicle when adding MSCs to a repaired abdominal incision. Supported by St. Elizabeth Health Center and Youngstown State University.
Upregulation proliferation and senescence markers in hyperplasia induced in mouse skin by sulfur mustard exposure
Authors: Chang Yoke‐Chen , Wang James D. , Soriano Melannie , Gordon Marion K. , Gerecke Donald R.
Published: June 2021

Chemical vesicants such as sulfur mustard (SM) can produce severe tissue damage. A time‐course study using mouse ear skin exposed to a low dose (0.08 mg) of SM resulted in excessive keratinocyte hyperplasia about three days after exposure. The abnormal cell proliferation was reminiscent of that observed in several types of cancer and potential oncogenic hyperproliferative signal molecules were examined to determine if the two systems were comparable. Immunohistochemical, Western blotting and RT‐PCR studies were performed and markers of skin injury and repair were examined for various time‐points. The protein, p16‐INK4a, which triggers either growth arrest or apoptosis was hyperexpressed in the hyperplasia areas of injured skin. The p16‐INK4a co‐expressed with laminin γ2, a polypeptide that promotes epithelial sheet migration over a wound bed. The endoplasmic reticulum stress‐regulated gene, GADD153/CHOP had a similar expression pattern to that of p16‐INK4a. In contrast, both keratin 5, an intermediate filament protein of basal epithelial cells and Ki67, a marker of cells in the growth stage of the cell cycle had a much more restricted distribution. Understanding the basic mechanism of action of vesicant‐induced skin injury and repair should help to identify new targets of potential medical countermeasures against chemical vesicants. This work is supported by ES005022, EY09056, and AR055073.

Grant Funding Source: ES005022, EY09056, and AR055073.
Oxygen concentration modulates in vitro differentiation of porcine muscle derived stem cells
Authors: Loughna Paul Thomas , Redshaw Zoe
Published: June 2021

The regenerative properties of skeletal muscle are attributed to the resident myogenic progenitor cells known as satellite cells. They are reportedly heterogeneous in nature, with regard to their proliferation, fusion and transplantation abilities. Most in vitro studies to date have examined the differentiation of rodent satellite cells at atmospheric oxygen (O2) concentration. Our work focuses on the isolation of satellite cells from the pig, comparing differentiation at atmospheric (21%) with physiological (5%) O2 levels. Cells derived from two distinct skeletal muscles, the diaphragm (D) and hind limb semi‐membranosus (SM) muscles differ in their capacity to undergo myogenesis and adipogenesis in vitro. D cells exhibited greater myogenic ability compared with those from SM, while the converse was true for adipogenesis. The myogenic ability of cells isolated from D as measured by expression of desmin, myosin heavy chain and myotube number was significantly enhanced at 5% compared to 21% O2. The myotubes formed were significantly larger, with greater nuclei number at the lower O2 concentration. In contrast, when encouraged towards an adipogenic fate, formation of adipocytes in SM cells was reduced at the lower O2 level. These data demonstrate that stem cells derived from metabolically distinct muscles exhibit different lineage potentials, which can be influenced by O2 concentration.
Efficient myogenic commitment of hESCs and iPSC‐derived cells on biomimetic materials replicating myoblast topography
Authors: Hwang Nathaniel Suk‐yeon , Langer Robert S
Published: June 2021

Recent development in stem cell technology has demonstrated human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) as possible cells for cell‐based therapy and studies of particular differentiation program. However, spontaneous and heterogeneous differentiation of these cells may limit the potential use. Here, we describe the efficient generation of mesenchymal cell population from both hESCs and iPSCs having non‐turmoigenic potentianl and their commitment into myoblast. In skeletal development, increased cell‐cell contact plays critical initial steps for myogenic commitment. We demonstrate that iPSCs and hESCs‐derived cells can undergo efficient myogenic commitment by topographical cues present in their environment. We have created substrates from biomimetic materials that can replicate the mico‐ and nanoscale topography of fully differentiated skeletal myoblast. When hESCs and iPSCs‐derived mesenchymal cells were cultured on biomimetic pattern, mesenchymal cells followed the underying myoblast pattern. Furthermore, gene expression and cell fusion index showed enhanced myogenic commitment on these substrates. These results demonstrates that myogenic potential of hESCs and iPSCs‐derived cells are highly dependent on the micro‐ and nanoscale topographical cues provide by skeletal myoblast.

Grant Funding Source: NIDCR (NIH)
Dose‐response relationship of human CD34+ cells in an athymic rat model of myocardial infarction
Authors: Brooks Ben , McKee Jeff , Rice Deborah , Baumgartner Bernhard , Motlagh Delara , Amrani David
Published: June 2021

The athymic rat model of myocardial infarction is routinely used to evaluate the efficacy of cell‐based, proangiogenic therapeutics. Most studies employ a range of cells (104 to 2X106 per rat) with little to no justification as to why a particular dose was used and whether the dose is clinically relevant based on allometric scaling. Adding to the uncertainty of dose is which route of administration will yield the greatest therapeutic benefit. It's been shown that the intravenous administration of 104, 106 or 2X106 human CD34+ cells has positive, dose‐dependent effects on histopathological and functional endpoints in an athymic rat model of myocardial infarction (Schuster et al 2004). The purpose of this work was to evaluate if a similar dose‐response could be demonstrated with human CD34+ cells transplanted by direct intramyocardial injection. Athymic (HSD:RH‐FOXN1RNU) rats underwent left anterior descending artery ligation followed by direct intramyocardial injection of saline, 105, or 106 human CD34+ cells. Echocardiography was performed before ligation and at 4 and 32 days post‐ligation. The intramyocardial transplantation of human CD34+ cells improved cardiac performance as demonstrated by improvements in left ventricular fractional shortening and left anterior wall motion as compared with saline‐treated animals but no dose‐response was observed. In conclusion, these data suggest that the dose‐response relationship of human CD34+ cells may be shifted to the left when transplanted into the athymic rat by direct intramyocardial injection as compared to the intravenous route. Baxter Healthcare Corporation provided all research support.
Student interaction with a novel computer interface in a gross anatomy lab
Authors: Goldman Evan Michael
Published: June 2021

Results. Philadelphia University retrofitted its on‐campus gross anatomy lab with a customized computer system and interface. The student use of the technology has revolutionized the way the course material is learned. The computers provide a host of interactive tools to instantly correlate the anatomy of the cadaver with radiographs, 3‐dimensional rotatable views, animations, photographs, and illustrations. Instant access is also available to the course lecture notes, study guides, muscle reference sheets, and online resources. The computer interface developed for the lab computers provides intuitive and reliable access to these materials.

Conclusion. The goal of this project was to create an intuitive user‐friendly interface that students would freely choose to integrate into their dissection and studying routines. As evidenced by student feedback and instructors observations, students receive tremendous benefits from the computer access. Students are continually observed referencing online atlases, dissectors, and lecture notes. When asked questions there is greater prevalence of students providing correct answers as compared to previous years. Instructors also receive fewer basic questions from students and more questions indicative of a greater comprehension of the anatomy.
Evaluation by medical students of a new computer‐based dissection manual
Authors: Kraszpulska Barbara , Gainor Robert
Published: June 2021

Starting this year we offered an online tutorial that serves as a computer‐based dissection manual (CBDM) for medical students enrolled in the Human Structure course. The CBDM is an additional source to the mandatory Gray's Dissection Guide. The idea behind this multimedia approach is to provide a resource to guide students through the requisite steps of each laboratory, as well as to serve as a powerful tool for independent study and review. The first part of the CBDM (Back and Upper Limb) was evaluated by all enrolled students (54 females & 46 males) who completed a survey. The majority of them (82) had a BA/BS degree; some had a master (14) or a doctorate (4) degree. Most students (74) had no prior dissection experience. The vast majority of students (94) decided to use this new tool. The CBDM was used by 93% students who have never done dissection before and by 96% students who have done it previously. Among all CBDM users 97% found it very useful or somewhat useful. Most of the users (95%) wanted to use it additionally as a preparation for the practical exam. Interestingly, all students (including non‐users) concluded that the CBDM is a very helpful, user‐friendly resource, and suggested the development of additional interactive activities to the CBDM. Supported by the WSU, Neuroscience, Cell Biology & Physiology, grant fund 667162 under RC grant 666953.
Production and Assessment of Interactive Animations for Medical Education
Authors: Fredieu John R. , Watson Jacklyn , Hughart Cory , Almon Amanda , Nikiforova Tanya
Published: June 2021

Embryology is not given much attention in medical education, but has a large impact on clinical fields, including pediatrics, surgery, and primary care. The current lack of resources and learning time can be overcome by use of technologies that present the material in a clear, accurate, and interactive format. In this study, we investigated the impact of an interactive 3D animation module depicting facial development on medical student learning. The interactive animations were developed through a collaboration between medical school faculty, a medical student, and two biomedical artists. This production model is being examined as a basis for a collaborative curriculum between the CWRU School of Medicine and the Cleveland Institute of Art. For assessment of the impact of the animations on student learning, students were divided into two groups: those receiving the standard curriculum and those receiving the standard curriculum augmented with the interactive animations. Differences between the two groups were assessed using written pre‐ and post‐tests, as well as focus group surveys and discussions. Statistical analysis of the differences in test performance between the two groups showed that scores of the student group exposed to both the standard curriculum and the interactive animations tended to be increased over those exposed to the standard curriculum alone. Larger test group and focus group studies are currently underway.
Popularity versus effectiveness of two methods to teach gross anatomy: A preliminary study
Authors: Cochran Adam , Nichols Carol
Published: June 2021

During an 8 week anatomy course for Allied Health Sciences students taught in 2009, two distinct teaching modalities were used. The first was interactive lecture where the class as a whole was asked questions. The second was directed questioning where individual students were asked questions. Since both modalities involved engaging students by asking questions, the two strategies were hypothesized to be equally effective. At the conclusion of the course, performance on written exam questions related to a topic taught using interactive lecture was compared with exam questions related to a topic taught by directed questioning. Students performed 37.5% better (p < 0.05) on exam questions related to material taught using directed questioning. To determine if performance on those questions was statistically different between years regardless of teaching modality, Student's T‐Tests were performed on the two groups of secure questions from 2008 and 2009. Since no other differences were found, we conclude that directed questioning seems to result in improved exam performance perhaps because this modality encourages preparation prior to class as students know they will be called on individually to answer questions. However, based on student evaluations this method is less popular with students presumably due to the apprehension of being called on publicly. Research support from MCG School of Medicine.
Public domain (copyright free) resources in human gross anatomy
Authors: Albrecht Gene H.
Published: June 2021

Anatomy is the most visual of the basic medical sciences. Imagine teaching anatomy using words alone without illustrations that show structure, relationships, and function. The visual nature of anatomy is even more important as student learning evolves from books to electronic resources with their emphasis on graphic media. Recently published texts, atlases, and online sites help bridge the transition from text to screen but their use by anatomists wanting to develop and distribute teaching materials is severely limited by the fair use doctrine of U.S. copyright law. Older textbooks and atlases, however, contain thousands of images that can be used legally without restriction because their copyright has expired. Any publication from anywhere in the world published before 1923 is now in the public domain as are most U.S. works from 1923–1964 because their copyright was not renewed after 28 years. Examples include Poirier's c. 1900 5 volume text (3,487 illustrations), Rauber's c. 1920 6 volume text/atlas (2,230), Sobotta's 1922 3 volume atlas (774), Gray's 1924 text (1,283), Morris’ 1933 text (1,166), and Grant's 1962 atlas (665). The anatomical illustrations from these public domain works can be scanned, copied, edited, revised, combined, cropped, colorized, and otherwise used in any way in any number in teaching materials developed by anybody in any format with unlimited distribution.
The Clinical Relevance of Teaching the Medial line Theme of the Lower Limb to Future Healthcare Professionals
Authors: Benninger Brion
Published: June 2021

Objective

This project investigated the current common overview approaches to lower limb anatomy teaching and suggests an alternative technique. Introduction. The overview of the lower limb is often described as thigh, leg and foot regions, and subdivided into anterior, posterior, medial and lateral compartments. This method guarantees all structures of the lower limb are taught. Virtually all healthcare institutions have had to distill anatomy into accelerated courses.

Methods

Literature search was conducted regarding teaching techniques of the lower limb and morphology of 60 dissected embalmed cadavers was observed to assess patterns which correlate with common clinical conditions and techniques.

Results

Literature review revealed no specific teaching techniques. However, observations commonly revealed compartment and/or systems layout. Dissections revealed an overwhelming number of clinically important structures along the medial line of the lower limb. Reinforcement or stability structures, venous access, arterial supply, and nerves were emphasized greater along the medial line versus the lateral line.

Discussion

Anatomy courses have resulted in less student‐instructor contact time. Specific techniques would aid the efficiency of learning. This project demonstrates that emphasizing lower limb structures along the medial line would be a useful technique teaching future clinicians.
Death is only the Beginning: Cadaver Use at Indiana University
Authors: Husmann Polly
Published: June 2021

Cadaver shortages have plagued some areas of the country, yet other places have no problems fulfilling their cadaveric need. The state of Indiana has had few issues maintaining a supply of donated remains and has begun providing cadavers to other states in 2009. One possible reason for this abundance is that there is only one medical school the state is attempting to accommodate. However, it is one of the largest medical schools in the nation and thus requires many cadavers. Other possibilities include high levels of knowledge of the anatomical donation program or less restrictive criteria for donation, though none of these have been studied. This demographic study examines the origin and use of cadavers at the Indiana University School of Medicine (IUSM). De‐identified donor data from IUSM was examined including age at death, sex, cause of death, last location of residence, year of death, ethnicity, and final use of donation. Descriptive statistics were run to assess trends among demographic data and final use. Analysis revealed that most donors are Caucasian, die of chronic medical conditions, and tend to come from large cities. These data support the hypothesis that those with chronic diseases are more likely to donate their remains for research and education out of respect for and/or knowledge of health care. The study also discusses the uses of donated cadavers and the proportion of donations that are used for each purpose. Utilization of the donors typically begins within six months to one year of death and the largest numbers of cadavers are used for medical education. This information may be valuable for those deciding whether or not to donate. This study represents the first step in a larger project to compare these data with data from areas combating cadaver shortages to establish ways that these shortages may be resolved.

Grant Funding Source: None
The logistics of facilitating a dissection based anatomy curriculum in a distributed medical program
Authors: Fraser Jennifer , Mogerman Emma , Krebs Claudia
Published: June 2021

When the University of British Columbia (UBC) medical school began a distributed model for medical undergraduate education in 2004, the delivery of gross anatomy teaching needed to adapt to these new circumstances. The pedagogical decision to continue with a dissection – based anatomy curriculum, supplemented with selected prosections was made. Core gross anatomy teaching was front‐loaded into the curriculum during the first term when all students are based at the Vancouver site. Thereafter, all cadaveric material is shipped to the satellite sites so that students can continue with their anatomy education remotely. As the Body Donation Program at UBC's Vancouver campus is the only program of its kind in British Columbia, all cadavers for the distributed program are processed at UBC. The distributed medical education program has been operational for five years, and while successful, there have been some logistical and operational challenges:

The acquisition of cadaveric material.

The complexities of tracking and monitoring the anatomical material between all three sites.

The processing and maintenance of anatomical material.

The set up and monitoring of technical audio‐visual (AV) equipment in the lab.
Evaluating quality of medical students’ dissections in the gross anatomy laboratory
Authors: Kumar Neha , Hassinger James Peyton , Scoville Elizabeth A , Christensen Kevin , Nyauncho Clive , Lachman Nirusha , Pawlina Wojciech
Published: June 2021

Gross anatomy laboratory dissection experience continues to be an integral part of the majority of gross anatomy courses in the United States. Student‐dissected specimens, typically used for practical exams administered during the course, vary in quality. In most anatomy courses the quality of cadaveric dissection is not counted toward the final grade, therefore students are not overly concerned with completing their daily laboratory assignments. Often gross anatomy teaching faculty is challenged to find good examples of dissected structures to be used for testing purposes, frequently spending additional dissecting time to secure adequate material for examinations. Dissection at Mayo Medical School is performed as a collaborative effort of a four‐member dissection team. A new method was implemented to evaluate dissection and provide the students with weekly formative feedback regarding the quality of their dissection. Team performance was graded and constituted 5% of the total grade for the course. A ten‐point evaluation tool was used that incorporated both objective and subjective measures for determining whether students had met learning objectives. Implementation of dissection evaluation resulted in the improvement of dissection quality. The dissection evaluation proved to be a valuable assessment tool in our anatomy curriculum and one that students found beneficial.

Grant Funding Source: Department of Anatomy
Using histology as a predictor of academic success in a post‐baccalaureate program
Authors: Barritt Laura C , Jergenson Margaret A , Norton Neil S , Ayers Frank J
Published: June 2021

Numerous schools across the country offer post‐baccalaureate programs to assist individuals who have completed a bachelor's degree and wish to pursue a career change. Many of these programs focus on preparing individuals for careers in the health professions. While the purpose of these programs varies, they are frequently designed to improve an individual's academic credentials and satisfy existing deficiencies in prerequisite core courses. Four years ago the School of Dentistry at Creighton University established a Pre‐dental Post‐ Baccalaureate program specifically designed to strengthen the academic abilities of individuals from educationally, economically, and socially disadvantaged backgrounds. As part of the program, in the summer prior to their matriculation into dental school, the students are required to take a week long, intensive course in histology which focuses on the microscopic analysis of cell structure and the four basic tissues that comprise the organ systems. During the week, students are given the same lectures and laboratory sessions that they would receive during the regular dental school histology course. At the end of the course students are required to take a lecture and practical exam. We have observed that a student's performance in the Histology Post‐Baccalaureate course serves as a good predictor of future academic success in dental school.
Assessment of Biomedical Students Performance in Human Anatomy
Authors: Afifi Nahla M. , Bargal Hala W. , Thani Amna Al
Published: June 2021

Human anatomy forms the foundation for all medical sciences; thus its place in the school curriculum deserves careful attention. The Biomedical Sciences Program at Qatar University is the first international program obtained 5 years accreditation by NAACLS at 2008, Educators at the program put much emphasis on the content of the curriculum and how biomedical students should be assessed. As so we tried to evaluate the performance of students to different formats of examination. We analyzed the test scores of 414 students from fall 2004 to spring 2009 who completed their Human Anatomy course. The parameters analyzed statistically were short essay questions (SEQs), multiple choice questions (MCQs), and practical exams. For all assessments, mean scores ± SD were calculated and compared using ANOVA test. Correlation between scores for MCQs and SEQs in each year for the entire cohort was determined. Preliminary results showed that student performed better in practical exams than in MCQ and SEQ in the five years of the study. The mean and high scores were consistently higher in MCQ than SEQ tests. The percentage of total grades indicated an increase in B grade in the last two years 2008 &2009 and the worst result was in year 2007 as the percentage of failed students was 7%. MCQs grades showed better performance in year 2004, 2006 and 2009 by the number of students getting (A) and (B) grades with mainly (A) in year 2009 in practical exam. In conclusion, our results indicate that the performance of students improved after changing the admission criteria in correlation to the accreditation and the clusters of various modes of assessment may give all students better opportunities to demonstrate their knowledge and competence.
Inhibition of Notch is Directly Correlated with VPAC Repression in HT‐29 Colon Carcinoma Cells
Authors: Bakke Danika Sue , Benton Keith , Hermann Rebecca , Dorsam Glenn , Bakke Danika
Published: June 2021

In 2005, 72,007 men and 69,398 women were diagnosed with colon cancer in the United States alone. Although the cause of cancer is not well defined, colon cancer can arise from upregulation of the Notch signaling pathway, which enhances intestinal stem cell and progenitor cell proliferation. A vital step in activating Notch signaling is its cleavage by gamma‐secretase. It has been shown that vasoactive intestinal peptide receptor 1 (VPAC1), a pro‐proliferative receptor in solid‐tumors like colon cancer, contains multiple putative binding sites for Notch in the gene locus. However, the effect of Notch on VPAC1 signaling is not yet determined. We hypothesized that Notch would activate VPAC1 expression. The gamma‐secretase inhibitor N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycinet‐butyl ester (DAPT) was added to cultures of HT‐29 colon cancer cells, followed by determination of VPAC1 steady state mRNA levels measured after 24 hours via SYBR green qPCR. The data showed a decrease in VPAC1 due to the addition of DAPT, supporting the idea that Notch is a transcriptional activator of VPAC1 expression in HT‐29 cells. These results positively impact human health by guiding the development for new colon cancer therapies directed at inhibiting VPAC1 signaling. Funding: NIH‐K011K01DK064828 & COBRE 2P20RR05566.
Neuropeptides and Colon Cancer; Another Notch in the Complexity of Tumorigenesis
Authors: Light Naomi G , Benton Keith D , Hermann Rebecca J , Failing Jarrett , Dorsam Glenn P
Published: June 2021

In 2009 an estimated 50,000 deaths in the United States will be attributed to colon cancer. Dysregulation of tumor suppressors and oncogenes are a major cause of uncontrolled cellular proliferation, leading to tumerogenesis. The proto‐oncogene, Notch, is associated with colon cancer. It has been previously shown that cleavage of membrane tethered Notch releases the intracellular domain fragment (NICD) which can enter the nucleus. Manifestation of tumerogenesis is caused by elevated NICD entering the nucleus and activating gene transcription. A proliferative G‐protein coupled receptor, vasoactive intestinal peptide receptor 1 (VPAC1), is highly expressed in colon cancer cells and contains numerous Notch putative consensus sequences within its promoter. We hypothesize that the reason why colon cancer cells, HT‐29, have elevated VPAC1 expression levels is due to enhanced Notch signaling. To this end, Dibenzazepine, a γ_secretase inhibitor which prevents the cleavage of Notch was utilized to determine whether VPAC1 expression was altered. By SYBR green qPCR, VPAC1 mRNA was found downregulated by Dibenzazepine in a concentration dependent manner over 48 hours. This observation is the first to link VPAC1 regulation to Notch signaling and therefore further elucidation of this mechanism will allow for greater insight into the etiology of colon cancer.

Funding: NIH‐K01 1K01DK064828 and COBRE 2P20RR05566.
Can neuropeptide receptors really cause leukemogenesis
Authors: Failing Jarrett , Dorsam Sheri , Hermann Rebecca , Batra Sandeep , Dorsam Glenn
Published: June 2021

Leukemia is cancer of the blood that results in 50,000 deaths annually in the US. A majority of leukemias have mutations/deletions in either Notch or Ikaros genes, which encode transcriptional regulators that recognize the same DNA sequence. Notch/Ikaros binding sites are found >10 times in the promoter regions (5kb) of vasoactive intestinal peptide receptor 1 (VPAC1) and VPAC2. These neuropeptide G‐protein coupled receptors transmit growth regulating signals when bound to their ligand. Lymphocytes are normally in a quiescent state with a high VPAC1:VPAC2 ratio. However, upon a mitogenic signal (i.e. T cell activation) the anti‐proliferative VPAC1 “shield” is lost and the VIP receptor ratio becomes equalized. We hypothesized that leukemia cells (hyperproliferative phenotype) would have a greater skewing in favor of the anti‐apoptotic VPAC2 receptor. To this end, total RNA was isolated from human leukemia tumors and cell lines, and VIP receptor mRNA levels were measured by SYBR green qPCR. In support of our hypothesis, T cell leukemia revealed a dramatic receptor reversal, downregulating VPAC1 and upregulating VPAC2. Similarly, B cell leukemia also showed VIP receptor dysregulation. We concluded that a shift from an anti‐proliferative to an anti‐apoptotic phenotype is driven by VIP receptor expression changes. This work was supported by COBRE (2P20RR015566) and NIH/NIDDK Career Award (KO11K01DK064828).
Nicotinic Acid Degradation in Bordetella bronchiseptica: Mechanistic Studies of the Novel Enzyme NicF
Authors: Kincaid Virginia Anne
Published: June 2021

The mechanism of aerobic nicotinic acid degradation in bacteria has not been well characterized. Based on studies in Pseudomonas putida, it is predicted that maleamic acid is converted to maleic acid by the protein product of peg.1774 in Bordetella bronchiseptica (Bor). This gene (nicF) was cloned using several vectors to enable expression of a C‐terminal His6‐tagged or N‐terminal maltose‐binding protein fusion recombinant enzyme in E. coli. Results of purification by HisTrap nickel affinity chromatography and subsequent kinetic activity using a linked assay with glutamate dehydrogenase will be presented. Catalytic proficiency, defined as (kcat/KM)/kuncat, of NicF will be determined by comparing the enzyme's catalytic efficiency with the rate constant of uncatalyzed maleamic acid hydrolysis, measured by 1H NMR.
Facile chemoenzymatic platform for protein tyrosine‐sulfate production.
Authors: Lu Lu Yi , Chen Bo Han , Wu Jennifer Yun‐Shin , Liu Tzu An , Chen Da Huang , Wang Chen Chu , Lin Tsu Hsiang , Hsu Ching Chen , Yang Yuh Shyong
Published: June 2021

Protein tyrosine sulfation is a key post‐translational modification that mediates various critical functions, such as HIV entry, inflammation, coagulation, and sterility. Tyrosine O‐sulfation is catalyzed by membrane‐associated tyrosylprotein sulfotransferase (TPST, EC 2.8.2.20), which is responsible for the sulfation of secreted and transmembrane proteins. The sulfated proteins are generally attained from tissue extraction, chemical synthesis, and enzymatic catalysis. Enzymatic catalysis provides direct, specific and easy method for the identification and preparation of sulfated proteins. Currently, TPST is mainly acquired from nature tissues or cell cultures. In this report, the constant source of active and homogeneous TPST at large quantity was achieved through prokaryotic expression. Furthermore, a continuous and cheap 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) regenerating system was utilized to provide the activated sulfate for protein sulfation. This methodology rendered the increase in catalytic efficiency of TPST up to 10–100 folds than previous studies. Moreover, we applied this platform in vivo to produce target protein concurrently with sulfation on the specific tyrosine residue throughout bacterial cultivation. This platform will be beneficial to synthesize sulfated proteins and investigate the functions of protein sulfation. support: 98‐2627‐B‐009‐009
Premature translation termination products are substrates for the rapidly degraded polypeptide pathway
Authors: Lacsina Joshua Rene , Liu Xiongfei , Nicchitta Christopher V.
Published: June 2021

In mammalian cells, nearly one‐third of all newly synthesized polypeptides are rapidly degraded (t1/2 = 10 min), however it is unclear what mechanisms govern access to the rapidly degraded polypeptide (RDP) pathway. The objective of this study is to determine if the products of premature translational termination are RDPs. To elicit premature termination, cells were treated with the aminoacyl‐tRNA analog, puromycin. Puromycin‐treated cells were labeled with [35S]‐Met, and puromycin‐labeled translation products were recovered by immunoprecipitation with an anti‐puromycin antibody. [35S]‐Met pulse‐chase studies demonstrated that peptidyl‐puromycins are polyubiquitinated and rapidly degraded in a proteasome‐dependent manner; proteasomal inhibition led to a 2.3‐fold increase in radiolabeled peptidyl‐puromycins. These results demonstrate that premature translation termination products can serve as substrates for the RDP pathway. Our findings suggest that the nature of the translation termination event (premature vs. stop codon‐mediated) influences the stability of the nascent polypeptide. Supported by NIH grants F31GM086127, R01GM077382, and T32GM007171.
Careers in Regulatory Affairs
Authors: Kwong Joan C
Published: June 2021

Career opportunities for those trained in chemistry and biology extend beyond academia and medicine to interdisciplinary roles such as pharmaceutical regulatory affairs (RA). RA operates at the interface of science, law, and business, applying scientific rationale along with statutory regulations and business demands to enable regulatory approval throughout drug development. RA is responsible for all interactions with health authorities from initial clinical trials through marketing applications and beyond. An RA professional influences and collaborates with diverse groups from R&D scientists to project teams to global regulators. Strong communication skills are essential to successfully providing appropriate clinical, nonclinical, and CMC (chemistry, manufacture, and controls) information to regulatory bodies. Creativity is required in the development and negotiation of regulatory strategy with both internal teams and regulators worldwide. Preparation for a career in RA can include bench top research experience, project development team membership, and formal education. The dynamic nature of drug development provides ample opportunity for continued learning and growth throughout a career in RA.
Careers in Science Publishing
Authors: Jabri Evelyn
Published: June 2021

How do scientists transition from the bench to the publishing desk? Working in a lab or a publishing office requires the same critical thinking, problem solving, project management and teamwork skills required in the lab. It is these skills, coupled with scientific breadth that will help you succeed numerous publishing jobs. Staff scientific editors have a Ph.D in the sciences. These editors may work with editorial board or within a small staff editorial group to evaluate manuscripts (or books) and manage peer review. They may also read and digest the scientific literature, commission new content such as reviews, attend scientific conferences, write news pieces on published articles, and contribute scientific features to a web site. News Editors have a scientific background (a M.S. or Ph.D.). News editors focus on writing and commissioning news pieces (such as highlights, commentaries, & perspectives). In the digital world, they are increasingly writing for the web and enhancing public understanding of science through blogs, Twitter, and Facebook. I will briefly discuss my career path from bench scientist to professional editor, describe the evolving publishing environment, and provide pointers on how to succeed in a publishing career
Activation of muscarinic receptor 2 stimulates proteasome function in cardiomyocytes
Authors: Ranek Mark John , Wang Xuejun
Published: June 2021

Ubiquitin‐proteasome system (UPS)‐mediated proteolysis plays a central role in protein quality control and degradation in the cell. UPS dysfunction was observed in various forms of heart disease and might play an important role in the development of congestive heart failure (CHF).

The parasympathetic nervous system (PNS) activity is decreased during CHF. As an initial step to investigate if and how PNS regulates the UPS in the heart, we employed neonatal rat ventricular myocytes (NRVM) culture. To better monitor UPS activity, a validated UPS surrogate substrate (a modified Green Fluorescent Protein, GFPu and GFPdgn) and a red fluorescent protein (RFP, not a specific substrate of the UPS) were co‐expressed in cultured NRVMs via adenoviral gene delivery.

We found that stimulation of the muscarinic 2 receptor with pilocarpine significantly reduced the GFPu/RFP ratio, which is reversed by inhibiting protein kinase G (PKG). We have further found that the phosphorylation status of the Rpt6 subunit of the 19S proteasome and the beta 5 subunit of the 20S proteasome was markedly altered by stimulating this receptor. These preliminary findings suggest that PNS may positively regulate cardiac proteasome through activating PKG. Supported by NIH grants HL072166; HL085629, and HL068936 and American Heart Association grant 0740025N.
CHIP E3 Ligase and USP2 Deubiquitinating Enzyme Antagonistically Control AIF‐mediated Programmed Cell Death
Authors: Yang Seung Wook , Oh Kyu Hee , Park Jung Mi , Jeon Young Joo , Chung Chin Ha
Published: June 2021

Under normal conditions, apoptosis inducing factor (AIF) in the mitochondrial membrane functions as an oxidoreductase that scavenges reactive oxygen species. Under certain stresses, such as exposure to N‐methyl‐N′‐nitro‐N′‐nitrosoguanidine (MNNG), AIF is truncated from the mitochondria and translocated to the nucleus, where it induces caspase‐independent programmed cell death, featured by chromatin condensation and large‐scale DNA breakage. Here we demonstrate that USP2 deubiquitinates AIF whereas CHIP functions as an ubiquitin E3 ligase of AIF, thus controlling the stability of AIF. Expression of USP2 resulted in acceleration of AIF‐mediated apoptotic process in HeLa cells, whereas that of CHIP attenuates it. Consistently, CHIP−/− mouse embryonic fibroblast (MEF) cells showed an increased sensitivity to MNNG. Collectively, these results indicate that CHIP and USP2 display antagonistic roles in the regulation of AIF‐mediated, caspase‐independent apoptosis.
Compartmentalization of eicosanoid substrates, signaling lipids, and enzymes into nanoparticles in human cerebrospinal fluid
Authors: Fonteh Alfred Nji , Oborina Elena , Harrington Michael
Published: June 2021

Objective

To find out whether lipid substrates and metabolizing enzymes are compartmentalized into discrete particles in human cerebrospinal fluid (CSF).

Methods

Lumbar CSF was fractionated by ultracentrifugation to obtain a 17,000 g pellet (P2), a 200,000 g pellet (P3), and supernatant fluid (S3). We used transmission electron microscopy (TEM) to examine the morphology of particles, and liquid chromatography tandem mass spectrometry (LCMS) to obtain lipid composition of all fractions. Colorimetric or fluorometric assays, and shotgun sequencing were used to measure enzyme activities and identify proteins, respectively.

Results

TEM revealed particles of different sizes (30–200 nM) and structures in P3 fractions. Organic extraction showed P3 fractions contained lipids. LCMS identified the major phospholipid classes in P3. Positive or negative parent ion scanning revealed enrichment of polyunsaturated fatty acids, phosphatidylinositol and sphingomyelin in P3 compared with S3 fractions. Shotgun sequencing and enzyme activity assay identified proteins and enzymes of the prostaglandin biosynthetic pathway in P3.

Conclusions

There are discrete membrane particles within CSF that are compositionally distinct from bulk CSF. Compartmentalization of enzymes on these particles can play important roles in brain function by controlling the metabolism of eicosanoid and other lipid signaling pathways.
Targeting of acyl‐CoA synthetase 3 to lipid droplets
Authors: Fuellekrug Joachim , Grossmann Regina , Rudolph Berenice , Becker Melanie , Thiele Christoph , Stremmel Wolfgang , Ehehalt Robert , Digel Margarete
Published: June 2021

Lipid droplets emerge as dynamic organelles highly relevant for lipid homeostasis and the pathophysiology of metabolic diseases. We are using fatty acyl‐CoA synthetases (ACS), especially ACSL3 as a paradigm to investigate the formation and growth of lipid droplets. Out of eleven mammalian long chain ACS, ACSL3 is the only family member localizing specifically to lipid droplets and the ER. The distribution of ACSL3 between these two organelles depends on the cell type and whether exogenous fatty acids are being supplied. The N‐terminal region of ACSL3 is sufficient to direct reporter proteins to lipid droplets which we analysed by confocal microscopy. Mutational analysis demonstrated that only amino acids 12 to 51 are essential for targeting. ACSL3 is tightly membrane associated by an amphipathic helix contained within the N‐terminal sorting signal. During lipid droplet biogenesis, ACSL3 moves from the bilayer membrane of the ER to the phospholipid monolayer of nascent lipid droplets as we demonstrated by live microscopy. Overexpression of ACSL3 results not only in increased cellular enzyme activity but also in the enhanced uptake of exogenous fatty acids. This suggests that ACSL3 is functionally relevant for the regulation of fatty acid uptake. Moreover, we speculate that the specific localization of ACSL3 is important for the local biosynthesis of phospholipids at the surface of the lipid droplets.
Regulation and physiological roles of the phospholipase B's encoded by PLB1‐3 in Saccharomyces cerevisiae
Authors: Surlow Beth , Bishop Andrew , Patton‐Vogt Jana
Published: June 2021

The phospholipase Bs (PLBs) encoded by PLB1, PLB2, and PLB3, are associated with the plasma membrane, cell wall, and periplasmic space of Saccharomyces cerevisiae. PLB activity catalyzes phospholipid deacylation and the concomitant production of intracellular and extracellular glycerophosphodiesters.. The extracellular glycerophosphodiesters produced, glycerophosphocholine and glycerophosphoinositol, are transported into the cell via the permease encoded by the GIT1 gene. Whereas GIT1 is transcriptionally regulated by phosphate levels, we report here that PLB1‐3 are not similarly regulated. Alterations in RAS/cAMP signaling, however, do affect expression of PLB1‐3; PLB1 and PLB3 message levels are increased upon upregulation of RAS/cAMP signaling, whereas PLB2 message levels are decreased. The mechanism of this regulation is under study. To probe the physiological roles of Plb1‐3‐mediated phospholipid turnover, we performed phenotypic analyses. A strain in which PLB1‐3 have been deleted (a plb1‐3Δ mutant) exhibits slower growth as compared to its wild type counterpart. In addition, a plb1‐3Δ mutant exhibits increased latrunculin A sensitivity as compared to wild type, suggesting a connection between cell‐surface phospholipase B activity and stability of the actin cytoskeleton. Current studies are aimed at determining the molecular mechanisms involved in producing the observed phenotypes.
Prohibitin interacts with phosphatidylinositol 3,4,5‐triphosphate (PIP3) and modulates insulin signaling
Authors: Ande Sudharsana Rao , Mishra Suresh
Published: June 2021

Mitochondrial protein prohibitin (PHB) is known to associate with the plasma membrane of various cell types. However, biological function of plasma membrane associated PHB is not known. Recently we have shown that PHB undergoes tyrosine phosphorylation in response to insulin and here we report that PHB interacts with phosphatidylinositol 3,4,5‐triphosphate (PIP3). Furthermore, we demonstrate that over expression of PHB attenuates insulin signaling downstream of phosphatidylinositol 3 (PI3) kinase. This effect was not observed with over expression of tyrosine phosphorylation site mutant‐PHB suggesting a role for tyrosine phosphorylation of PHB in this process. Interestingly phosphorylation of PHB by Akt attenuates its interaction with PIP3 and enhances insulin signaling. Thus, phosphorylation of PHB and its interaction with PIP3 may be a part of the regulatory mechanisms that is involved in the modulation of insulin signaling. We speculate that phosphorylation of PHB may serve as a general mechanism in the regulation of PI3 kinase signaling including growth factors and immune receptor signaling. This work was made possible by financial support from Manitoba Health Research Council (MHRC) and Winnipeg Health Sciences Centre Foundation to SM and MHRC postdoctoral fellowship to SRA.