bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell Cycle‐Dependent Cleaving of Holliday Junctions via Yen1 in S. cerevisiae
Authors: Hadi Hafridha , Fischhaber Paula 
Published: April 2020

Double‐strand breaks (DSBs) in DNA can be harmful if left unrepaired. Eukaryotes use repair mechanisms, some of which involve intact chromosomes that template repair via an intertwined intermediate known as a Holliday Junction (HJ). In S. cerevisiae, recruitment of the endonuclease enzymes that cleave HJs back into individual chromosomes is not well understood. Yen1 resolves HJs but it’s unclear whether it localizes to anaphase bridges, the final intertwined regions during chromosomal separation. Yen1 appears in anaphase which occurs during a small time frame within the cell cycle, making it difficult to observe in an asynchronous cell population. Further, Yen1 is present in lower abundance relative to other proteins, therefore, microscopy experiments with fluorescently labeled Yen1 will suffer from low signal‐to‐noise. Our aim is to clone a yeast strain with a bright version of red fluorescent protein under the strong galactose (GAL1) promoter so that Yen1‐yEMRFP will be in higher abundance, thereby allowing us to track the position and timing Yen1 during HJ resolution. To construct this strain, we used adaptamer‐mediated Polymerase Chain Reaction (PCR), DNA digestions and ligations to prepare two DNA plasmids. Plasmid One contains a gene for yeast Enhanced Monomeric Red Fluorescent Protein (yEMRFP), a partial URA3 auxotrophic selection marker gene, and an upstream flanker sequence of the target yeast chromosome to direct integration. Plasmid Two contains the other half of the partial URA3 selection marker, the galactose promoter, and the downstream flanker sequence to target integration. Candidate clones were screened by PCR and DNA sequencing which showed that the candidate plasmids had the desired sequences and are ready to be integrated into the yeast chromosome. These DNA plasmids will allow cloning of a specialized yeast strain that will produce an improved signal‐to‐noise resolution to elucidate Yen1’s role at anaphase bridges.Support or Funding InformationThis research was funded by NIH grant, SC1GM127204.
Investigating SLX4‐Dependent Recruitment of a DNA Repair Protein to DNA Double Strand Breaks in Dividing Cells
Authors: Hakimian Jonathan , Cureg Gyana , Fischhaber Paula 
Published: April 2020

Cancer is a disease that functions as a consequence of abnormal cell growth, generally caused by DNA damage that triggers abnormal cell function. One form of DNA damage is the double‐strand break (DSB) in which both strands of a chromosome are severed in near proximity. In normal cells, several forms of DSB repair, including single strand annealing (SSA) and synthesis dependent strand annealing (SDSA), fix the double strand break (DSB), restoring an intact chromosome, albeit sometimes with an altered DNA sequence. These DNA repair processes require the sequential action of numerous proteins that enable their many steps. In S. cerevisiae these include the Rad1‐Rad10 endonuclease complex, which cleaves nonhomologous DNA flaps in SSA and SDSA as one of the final steps.We have evidence that the Slx4 protein is partially required for the recruitment of Rad1‐Rad10 to DSB sites when cells are dividing (in S, G2 or M phases). Those experiments did not show the basis for the “partial” nature of the recruitment, but our hypothesis is that the Slx4‐dependency is tied to a specific phase of the cell cycle, possibly S phase. A plausible explanation is that when Slx4 exercises its checkpoint signal dampening role in S phase, which promotes homologous recombination, it indirectly recruits Rad1‐Rad10 to double‐strand break repair sites. Since, following DNA damage, checkpoint signal dampening is triggered (in part) by phosphorylation of Slx4 in S phase and the specific phosphorylation sites for this are known, we will test our hypothesis by creating mutant strains which lack these sites and repeating the earlier experiments using synchronized cell cultures. We began creating the mutant strains by cloning DNA plasmids containing the slx4‐7MUT gene, our focus, in which wild‐type SLX4 has been mutated to alanine at the seven phosphorylation sites activating checkpoint signal dampening. Candidate clones were screened by PCR and DNA sequencing. The successful candidate plasmids were transformed into a panel of W303 background yeast strains and integration of the mutant cassettes was confirmed by PCR and DNA sequencing screens. The strains were then crossed to strains bearing the fluorescence microscopy assay markers we will use in the fluorescence microscopy assay being employed. We have carried out preliminary fluorescence microscopy assays in which cultures of the progeny of these crosses were synchronized at the G1/S boundary and released back into the cell cycle that show acceptable arrest but the limited release back into the cell cycle that was observed requires further optimization. Using this separation‐of‐function mutant strain will allow us to compare recruitment of Rad1‐Rad10 in SLX4 wild‐type strains with Slx4 checkpoint signal dampening mutants. Understanding the process of Rad1‐Rad10 recruitment may aid in the design and development of a more effective means of cancer treatment.
CKDNET: a program for fighting with chronic kidney disease in the Northeast Thailand
Authors: Cha’on Ubon , Pongskul Cholatip , Thinkhamrop Bandit , Limwattananon Chulaporn , Sharma Amod , Anutrakulchai Sirirat 
Published: April 2020

BackgroundChronic kidney disease (CKD) in the Northeast Thailand (22.2%) was in the highest range of the world, therefore, Chronic Kidney Disease Prevention in the Northeast of Thailand (CKDNET) program was established to prevent and reduce CKD in this area, https://ckd.kku.ac.th/.MethodsCKDNET program attempt to cover all general population, high risk groups and all stages of CKD patients with several strategies including early screening, effective CKD registry, CKD comprehensive care models in community and hospital levels, as well as cost effectiveness analysis.ResultsPreliminary results from CKD screening in general population of two rural sub‐districts shows that the prevalence of CKD is approximately 27%. Ten million individuals are registered in CKD hospital registry with hundred thousands of CKD cases. The CKD guideline that suits for local situation has been formulated and CKD care trainings were developed for physicians and nurses. Moreover, our multidisciplinary intervention and self‐management supports were effective to improve glomerular filtration rate and blood pressure, and quality of life among CKD patients.ConclusionCKDNET program is the holistic approach to deal with the different aspects of CKD which in turn to attenuate the CKD burden in the Northeast Thailand.Support or Funding InformationFaculty of Medicine, Khon Kaen University, Thailand
The Role of Direct Effects of Drugs of Abuse Binding to the hDAT Molecule in their Diverse Behavioral Phenotypes
Authors: Bergman Shana , Razavi Asghar , Khelashvili George , Weinstein Harel 
Published: April 2020

Substrates, inhibitors, and various classes of drugs of abuse that target the human dopamine transporter (hDAT) elicit a variety of responses measurable in vitro and in vivo. Classification of such ligands in terms of their chemical and/or pharmacological properties has failed to produce sufficient mechanistic explanations, or hypotheses, to guide a satisfactory understanding of the nature of this diversity in the effects of hDAT ligands. With the objective of advancing the current understanding of these drugs and their mechanisms of action, we set out to investigate the role that the response of the hDAT molecule to the binding of these ligands may have in their known diverse behavioral effects. We use structurally and pharmacologically diverse ligands of hDAT (e.g., typical inhibitors, atypical inhibitors, substrates and releasers) in extensive all‐atom molecular dynamics (MD) simulations to reveal the structural and dynamic effects that characterize the response of the transporter molecule to each of them and to enable a classification of hDAT molecular responses. MD trajectories describing the time‐dependent dynamic behavior of each complex are analyzed to quantify the molecular responses of hDAT, and machine learning algorithms serve to classify each response and reveal specific dynamic features underlying the molecular mechanism of hDAT’s response to each ligand. This approach offers mechanistic insight into the role of the ligand‐determined molecular response of hDAT in determining the diversity of effects expressed in the functional outcomes.Support or Funding InformationSB is supported by T32 “Genetic and environmental influences on addiction” (DA03980), research is supported by NIH Grant R01 DA041510 (to H.W.)
Novel FRET‐based biosensors for monitoring Presenilin/γ‐secretase activity in live cells
Authors: Maesako Masato , Sekula Nicole M. , Anderson Lauren C. , Berezovska Oksana 
Published: April 2020

Presenilin (PS)/γ‐secretase is responsible for the proteolytic processing of wide variety of membrane associated proteins that include APP and Notch. Therefore, a change in PS/γ‐secretase activity is linked to essential biological events as well as to the progression of many diseases. However, not much is known about how PS/γ‐secretase activity is spatiotemporally regulated in live cells, mainly because shortcomings of the existing assays prevent monitoring dynamic changes in the activity of PS/γ‐secretase in its normal physiological environment. Here we present successful development and validation of the Förster resonance energy transfer (FRET)‐based biosensors, so called the APP C99 Y‐T and the Notch1 N100 Y‐T biosensors, that enable quantitative monitoring of endogenous PS/γ‐secretase activity in live cells longitudinally on a cell‐by‐cell basis. Using these novel FRET biosensors, we uncovered dynamic spatiotemporal pattern of PS/γ‐secretase activity that is heterogeneously regulated among live neurons.Support or Funding InformationNIH: AG 44486 (OB), AG 15379 (OB) and BrightFocus Foundation: A2019056F (MM)
Amino Acid Complexed Zinc Has Higher Bioavailability Than Hydroxy Acid Complexed Zinc
Authors: Crabtree Amber , Shay Sheila , Moore Christie , Gladson Santhi , West James 
Published: April 2020

Poor zinc absorption causes zinc deficiency, leading to skin, gut, and immune pathologies, and psychiatric changes, and are potentially life threatening in infants and children. This can be caused by dietary deficiencies, but also inherited mutations of the ZIP4 gene.ObjectiveAlthough zinc complexed with organic molecules have previously been shown to have better uptake, we hypothesized that bonding the zinc to the amino group would be less liable to breakdown in the gut than zinc complexed to hydroxy acids, leading to higher bioavailability.MethodsWe treated wild‐type and mice with inducible universal knockout of one copy of the ZIP4 gene with reduced zinc diet (15 ppm) ad libitum, either in the form of amino acid complexed (CSK18271) or hydroxy acid complex (CSK18273) zinc in an otherwise nutritionally complete diet. Weight was measured at weekly intervals, and mice placed in metabolic phenotyping chambers for a week near the end of the trial.ResultsWe found that ZIP4+/− mice fed CSK18271 had weight gain comparable to wild‐type mice; wild‐type mice fed CSK18273 had lower weight gain, indicating bioavailability was lower even with intact zinc transporter. Zip4+/− mice fed CSK18273 had the worst overall weight gain. Metabolic phenotyping chambers found an increase in respiratory exchange rate (RER) and water intake in the Zip4+/− mice, indicating reduced kidney function in the heterozygous knockout mice, which was compensated for functionally by CSK18271 diet.ConclusionsZinc complexed to the amine group has higher bioavailability than zinc complexed to the hydroxyl group in amino acids, likely because of increased resistance to degradation in the gut. This results in improved weight gain in both wild‐type and Zip4+/− animals.Support or Funding InformationThe work was funded by Vanderbilt University Medical Center internal funds.
Effects of white adipose tissue denervation on cardiovascular and renal parameters in Wistar rats treated with high‐fat diet
Authors: Nishi Erika E. , Ferreira Giovanna R. , Garcia Michele L. , Campos Ruy R. , Bergamaschi Cassia M. T. 
Published: April 2020

Sympathetic nervous system and renin‐angiotensin‐aldosterone system overactivation plays a major role in cardiovascular changes in obesity. The white adipose tissue (WAT) is an essential endocrine organ that synthesizes several biologically active compounds that regulate metabolic homeostasis and contains both sensorial and sympathetic innervation. This study aimed to investigate the effects of retroperitoneal white adipose tissue (rWAT) denervation (DNX) on cardiovascular and renal parameters in obese rats. Animals were treated with a high‐fat diet (HFD) for 8 weeks to induce obesity and underwent surgical DNX procedure at 6 weeks. At the end of the experimental protocol, animals treated with HFD were significantly overweight, whereas the HFD+DNX group had a slightly decrease in body weight. The HFD group presented a discrete increase in baseline mean arterial pressure, which was normalized after DNX. Moreover, DNX improved bradycardic response gain when cardiac baroreflex control was evaluated in the HFD group. No changes were found in renal parameters. However, it was found a significant decrease in circulating angiotensinogen and leptin after rWAT DNX. Therefore, these findings suggest that rWAT innervation has influence on cardiovascular changes found in obesity.Support or Funding InformationCAPES, CNPq and FAPESP
Flip it or not for histology teaching in dental school?
Authors: Sun Xinxin , Leavis Paul 
Published: April 2020

Over the years, many novel electronic teaching tools and approaches have complemented and supplanted traditional educational offerings in histology such as lectures. The teaching method frequently used in our dental school histology class is the “flipped classroom” model. Prior to formal classroom sessions, students preview course materials from videos and the syllabus. During formal classroom sessions, students apply the knowledge that they acquired before classes by answering quiz questions using the interactive electronic tool, Poll Everywhere. The instructors will see the live responses from students and review important concepts in histology. The flipped classroom has changed the traditional teacher‐centered lecture‐based teaching method and is designed to encourage self‐motivated learning. However, the attendance of the flipped classroom is very low, even though the interactive class is not recorded.To find a better strategy for teaching histology in dental school, we compared the effectiveness of “flipped classroom” with a non‐flipped interactive learning class. In the non‐flipped classroom, dental students are not required to watch videos before class. Instead, the instructor provides a brief introduction of day’s topic in class, then students will practice their understanding of freshly learned knowledge by discussing and answering quiz questions using the same interactive tool, Poll Everywhere. Since, in this model, the interactive classes are recorded, students may choose to forgo attending the scheduled interactive classes.In this study, we used the flipped class model for D22 students and the non‐flipped interactive approach for D23 students. The lecture content, quiz questions, instructor and exam questions were identical for the two classes. We compared the normalized exam performances of students who attended class in the two models.The research results showed that dental students who were exposed to the non‐flipped interactive class have significantly higher attendance and better normalized exam performance than those who received the flipped class. In the flipped classroom model, dental students exhibit a strong tendency to use the pre‐class videos and syllabus only, which is basically a traditional lecture‐based teaching method without scheduled time restrictions. In contrast, the non‐flipped interactive class model encourages students to attend the classes, even if they missed reviewing pre‐class materials (for example, syllabus). Once the students come to the classroom, they will benefit from the interactive learning that follows the instructor’s outline of the topic.This study will provide insights for choosing histology teaching strategies for dental students.Support or Funding InformationDepartment of Medical Education, Tufts University, School of Medicine; Dental Research Administration, Tufts University School of Dental Medicine
Endurance Exercise Training Does Not Alter Key Receptors Within the Renin‐Angiotensin System in Skeletal Muscle
Authors: Nguyen Branden , Deminice Rafael , Yoshihara Toshinori , Lawrence Jensen , Hyatt Hayden , Ozdemir Mustafa , Powers Scott 
Published: April 2020

Prolonged skeletal muscle inactivity results in muscle fiber atrophy and weakness. Interestingly, endurance exercise training promotes a protective phenotype in skeletal muscle fibers that resists inactivity‐induced muscle wasting. The mechanism(s) responsible for this exercise‐induced preconditioning of muscle remain unknown and are explored in these experiments. In this regard, evidence reveals that activation of the classical arm of the renin‐angiotensin system (RAS) contributes to inactivity‐induced muscle wasting. Specifically, activation of the classical RAS pathway via the angiotensin II type I receptor (AT1R) is associated with activation of proteases and muscle fiber atrophy. Additionally, a non‐classical RAS axis exists that acts antagonistically to the classical RAS axis. Therefore, activation of the non‐classical RAS pathway via the Mas receptor (MasR) inhibits signaling through the classical RAS pathway. Indeed, pharmacological studies reveal that blockade of the AT1R and/or activation of the non‐classical RAS axis protects skeletal muscle against inactivity‐induced muscle atrophy. Studies also reveal that endurance exercise training decreases the abundance of AT1R and increases the quantity of MasR in blood vessels. If similar changes occur in skeletal muscles, these changes in receptor abundance could shift the RAS signaling to favor the non‐classical RAS axis and reduce activation of classical RAS signaling in muscle fibers during prolonged inactivity. These experiments test the hypothesis that endurance exercise training decreases the abundance of AT1R and increases the abundance of MasR in skeletal muscle fibers. To test this postulate, adult rats performed 10 days of endurance exercise training (60 min/day @ ~70% VO2Max). At the completion of the training protocol, the abundance of both AT1R and MasR were determined in isolated single muscle fibers. Moreover, a sensitive angiotensin II (Ang‐II) binding assay was employed to confirm exercise‐induced changes in AT1R abundance in muscle. The results reveal that endurance exercise training does not alter the protein abundance of either the AT1R or MasR in the soleus, plantaris, or diaphragm muscle fibers. Moreover, exercise training did not change the Ang II‐binding affinity within skeletal muscles. Collectively, these results do not support the hypothesis that endurance exercise training results in a decrease in the abundance of AT1R and an increase in MasR in skeletal muscle fibers. It follows that exercise‐induced changes in the abundance of RAS receptors does not appear to be the mechanism responsible for exercise‐induced protection of muscle fibers against inactivity‐induced muscle atrophy.Support or Funding InformationNIH AR064189 (SKP).
GAME BOARD IMPROVES THE ENDOCRINE SYSTEM ANATOMY TEACHING AND LEARNING PROCESS
Authors: de Paula Uchôa Francisco Ewerton , Nunes Bruna Marques Barreto , Forte Guilherme Aguiar , Filho Cézar Nilton Rabelo Lemos , Cerqueira Gilberto Santos , Morano Domingos Antonio Clemente Maria Silvio 
Published: April 2020

Board games are important tools of active methodology in the anatomy teaching‐learning process, especially when traditional learning is to be diminished. Several studies demonstrate that board games are effective in promoting learning. At the present time, in our University, the traditional methodology is predominant, and building an appropriate and qualified learning environment is a big challenge nowadays. Based on these assumptions, the aim of this paper was to investigate whether the endocrine anatomy board game promotes meaningful learning. A descriptive exploratory study with quantitative approach was conducted with 31 students of the discipline of anatomy. For learning assessment, a tested and validated board game of endocrine anatomy was applied. For statistical analysis, the normality of the data was verified and the Mann‐Whitney test and the Chi‐Square test were used, and those with p <0.05 were considered significant. It was observed that there is a statistically significant difference between the ages of the participants, and females had an average age of 20.38±1.03 when compared to male students 18,25±0.30. Regarding the perception of learning, there was a statistically significant difference in learning after the board game (p <0.05). Regarding the positive aspects of using the game, it was found that 23.5% reported that it helps to fix the content, 19.6% that provides integration between students, 18.9% that facilitates learning anatomy, 15.5% that raises students self‐esteem and 15.16% promotes better interpersonal relationships. The parameters of Reasoning, Safety, Learning, Evaluation Effectiveness, Speed and Cognition were statistically significant when compared to traditional teaching. It was found that the board game anatomy of the endocrine system is an important tool in the construction of anatomical knowledge, promoting dynamic, playful and meaningful learning, when compared with the traditional methodology.Support or Funding InformationCNPQ and FUNCAP