bluuebunny/crossref_metadata_2025 huggingface dataset:
“The Physician's Statement”: A novel test item strategy for medical education
Authors: Wright Bruce E. , Averill David B. , Janssen Herbert F.
Published: June 2021

We introduce a novel test item strategy that requires higher‐order thought processes. In this format, students first receive a clinical vignette. Then a physician makes a higher‐order statement presumably based on the data contained within a clinical vignette. This statement may propose a diagnosis, a treatment, the physiological basis for a clinical condition, etc. Students then analyze the vignette and this physician's statement, in a two‐question format. In the first question, students confirm whether the statement is: a) correct, b) incorrect, c) logical but requires more information to adequately support, d) illogical but requires more information to definitively refute, or e) unrelated to available data; only guessing. In the second question, students suggest how to support or refute the statement by choosing either: a) no additional information is required, b) or c) as two options that could provide support for the statement, or d) or e) as two options to refute the statement. Requiring students to analyze a physician's third‐order statement and then justify their decisions takes this assessment format a level beyond that of standard scenario style questions. This format also requires students to apply basic science information to a clinical case in a manner that does not appear contrived and which promotes classroom discussions. This format may be equally useful in clinical training.
Investigation of the Virtual Physiology of Exercise Laboratory
Authors: Dobson John
Published: June 2021

The Virtual Physiology of Exercise Laboratory (VPEL) replicates the test construction, data collection and analysis phases of several common “hands on” exercise physiology lab (HOEPL) experiences. The VPEL includes four modules: 1) Cardiovascular, 2) VO2 max, 3) Lactate and Ventilatory Thresholds and 4) Respiratory Exchange Ratio. This investigation sought to compare student learning from both HOEPL and VPEL activities. Students from the spring 2009 Integrated Fitness Programming course were randomly assigned to investigational group 1 or 2. Group 1 completed a HOEPL version of a VO2 max lab activity, whereas group 2 completed the VPEL VO2 Max Module. Both groups then took the same exam to assess their understanding of VO2 max lab concepts. Group 2 then completed the HOEPL version of a lactate and ventilatory thresholds lab activity, whereas group 1 completed the VPEL Lactate and Ventilatory Thresholds Module. Both groups then took the same exam to evaluate their understanding of lactate and ventilatory threshold lab concepts. Mean VO2 max exam scores were 86.39[plusmn]4.13 and 85.64[plusmn]6.72, and mean lactate and ventilatory thresholds exam scores were 85.50[plusmn]8.05 and 86.15[plusmn]6.45, for groups 1 and 2, respectively. There were no statistical differences between the two groups on either exam. This study provides evidence that students learned equally well from the HOEPL and VPEL activities.
Using General Models, Summary Sheets and Learning Paragraphs to Integrate Learning in Physiology
Authors: Wenderoth Mary Pat
Published: June 2021

To help students develop an integrative approach to learning physiology, three learning activities are routinely used in my courses for Biology majors. First, General Models (GM; Modell 2000) are incorporated into each class. GMs help students realize that the same basic principles govern many physiological processes, for example movement of blood through the circulatory system, air through the respiratory tree and oxygen across the alveoli are each explained by Flux. On exams, students are required to note which GM they used to solve the problem. Students who correctly identified the GM gave more correct explanations than those who had not identified the GM. Second, students create a weekly summary sheet that requires them to synthesize and integrate their understanding of the week's material in a pictorial format. Third, students submit a weekly learning paragraph that address connections made between material discussed in class and their lives outside the classroom. Students found the last two learning tools helped them see the relevance and better integrate material. The Revised‐Study Process Questionnaire (R‐SPQ; Biggs et al. 2001) was used to determine if these learning strategies caused students to use a deep or superficial study approach. Seniors in the 400 level course using these three learning tools showed a significant increase in their deep approach to studying compared to age matched colleagues.
Integrative learning of physiology and other biomedical sciences organized around primary care cases with healthy patients
Authors: Hansen Penelope Ann , Peters Sharon , Wells Mary
Published: June 2021

Many health professional school curriculum planners struggle with the problem of using clinical cases for teaching and learning normal physiology and other biomedical sciences. Such cases traditionally involve patients who are ill and are seen by their physicians for diagnosis and treatment of diseases. This tends to focus learning on pathophysiology and management of disease rather than on healthy structure and function, resulting in normal physiology, anatomy, and biochemistry receiving short shrift. An alternative approach is to use clinical scenarios, called “presenting features” cases, in which healthy individuals see their family physicians for preventive care, check‐ups, medical certificates, education, or advice. In this way, physiology is linked with health promotion. After students have learned about normal structure and function, cases can be elaborated to encompass interruptions in health due to patients' deleterious behaviors or to external circumstances, creating a natural continuum from normal physiology to pathophysiology. Here disease prevention is emphasized and biomedical sciences are naturally integrated with social sciences, epidemiology, and other public health topics. This development of cases is founded on the constructivism theory of learning, and authentically reflects the reality of primary health care.
Synthetic education in pathophysiology. Role of etiopathogenetic clusters.
Authors: Kovac Zdenko
Published: June 2021

In order to make pathophysiology teaching/learning, both, teacher‐workable and student‐friendly, we developed educational model with four steps. 1. Exposition of problem gives short presentation of “raw data” derived from patient records, selected publications with experimental data, etc. 2. Repetition of relevant knowledge is a multiple choice test, whose statements is related to the exposition and referred teaching materials. 3. Algorhythmic workout of the pathogenesis is task in which students, out of given 25–30 units of etiopathogenesis, build‐up the cause‐consequence sequence of events, with positive and negative feedback loops, and parallel and contextual events. 4. Feedback integration of the problem deals with additional relations, systematization and quantitative aspects of the same problem.

This four‐step matrix‐like approach clearly outlines etiopathogenetic pathways and networks of interconnected elements within the hierarchy of the system. Along with data analysis, students are asked to design a synthetic view of the problem. They use both exact and fuzzy logic in such integration. Some interconnecting nodes (e.g., processes, structural alterations, concentration abnormalities, etc) form clusters, with multiple entries and outputs. Focusing to such clustering points seems to be a reliable approach to master complexities in medicine and bridging the basic and clinical knowledge.
Blood pressure and heart rate changes in simulated human apnea diving
Authors: Valic Zoran , Valic Maja , Giaconi JoAnn A , Peros Kristina
Published: June 2021

Physiology exercise employing simulated human breath‐hold diving is used at University of Split School of Medicine to integrate knowledge in cardiovascular and respiratory physiology gained during lectures and seminars. We improved on otherwise widely used model, with emphasis on importance of continuous recordings of physiological signals. Pulsatile arterial pressure, heart rate and arterial oxygen saturation were continuously measured and digitalized signals stored for later analysis. All apneas lasted for 30 seconds, which produced no change in arterial oxygen saturation. Responses in arterial pressure and heart rate to breath‐hold in air, breath‐hold in water, breathing in air and cold water with snorkel, and applying warm and cold stimulus to forehead were analyzed. We describe our experience with organizing the exercise, and explain how application of modern technology can improve scientific investigation. Several points for discussion are accentuated in relation to possible medical problems (middle ear and sinus problems, regulation of breathing, death of most experienced divers/shallow water blackout) occurring during apnea diving.

This study was supported by Croatian Ministry of Science, Education and Sports.
A physiological model of factors that determine skeletal muscle hemoglobin oxygen saturation
Authors: Klabunde Richard Edwin
Published: June 2021

A mathematical model for hemoglobin oxygen saturation (StO2) in skeletal muscle was derived from Poiseuille's equation, the Fick principle, and from published data on skeletal muscle hemodynamics and oxygen consumption (VO2). The model shows that StO2 is about 85% under resting conditions of blood flow, arterial oxygen content and VO2. A 40% reduction in flow caused either by reducing perfusion pressure or increasing vascular resistance reduces StO2 from 85 to 75%. Flow reductions greater than 40% cause large, disproportionate decreases in StO2. At any given flow, a change in oxygen consumption (VO2) causes a reciprocal change in StO2. If VO2 decreases as flow decreases, which occurs in low flow states, a paradoxical increase in StO2 may result. At a given perfusion pressure, increasing hematocrit (Hct) from 40 to 60% has no effect on StO2; however, increasing the Hct to 80% reduces StO2 because blood viscosity increases more than blood O2 carrying capacity. Reducing Hct from 40 to 20% decreases StO2 because O2 carrying capacity decreases more than blood viscosity. The predictions of the model closely parallel clinical observations of thenar muscle StO2 when monitored in normal subjects and trauma patients. Therefore, the model helps to explain how changes in patient status before and during resuscitation alter StO2.
Gender Differences in Thinking about Ethical, Experimental and Quantitative (TEEQ) Biology related to Functional Diversity of the Vasopressin and Oxytocin Receptor Superfamily
Authors: Dutta Debalina , Pelaez Nancy
Published: June 2021

TEEQ Biology targets a large lecture course on organismal biology for biology majors. TEEQ aims to improve undergraduate students' understanding of bioscience research and thereby foster development necessary for dynamic careers. A 2008 AAMC‐HHMI report says future physicians need a scientific foundation including the ability to critically evaluate conclusions from a scientific study (Competency E1.6). TEEQ engages students in reviewing scientific research by using the Calibrated Peer Review (CPR) program to structure writing assignments followed by anonymous peer‐ and self‐ review. For this study, evolutionary and functional diversity of the vasopressin/oxytocin receptor family was examined in vertebrates. For a CPR assignment, students found another example of research where traits were used to hypothesize evolutionary relationships, and they evaluated a scientific conclusion suggesting a relationship tree. Results show gender differences and multicultural influences on students' selection of topics, suggesting that CPR fosters innovative learning in a manner allied to student interests. CPR assignments can be shared by faculty who want to interact with students in ways that lead to better engagement and retention of students in the major. NSF #0837229
The functional and comparative anatomy of the forelimb muscles in the sun bear (Helarctos malayanus)
Authors: Adrian Brent , Barton Michael , Tang Samuel Y. , Fisher Rebecca E.
Published: June 2021

The sun bear (Helarctos malayanus) is the smallest extant member of the Ursidae, inhabiting the tropical mainland forests of Southeast Asia and the islands of Sumatra and Borneo. This study provides morphological data for the forelimb of Helarctos, whose anatomy has been scarcely documented. The left and right forelimbs of a 16 year old female sun bear from the Reid Park Zoo (Tucson, AZ) were dissected and photodocumented. In addition, muscle origins and insertions were recorded and muscle maps produced, providing a valuable resource for those analyzing the postcranial remains of fossil ursids. The forelimb musculature was compared to that of other carnivores and analyzed for functional adaptations. Dissections revealed common ursid features, such as powerful flexors of the digits, elbow and carpal joints, as well as traits particular to the sun bear. For example, m. flexor carpi radialis has an extensive insertion onto the scapholunatum and metacarpals II and III, assisting in adduction of the carpal joint. Mm. flexor digitorum brevis manus and palmaris brevis were also observed, which may contribute to increased grasping ability of the manus. Taken together, these findings are likely adaptations to the climbing and feeding behaviors which are central to the arboreal lifestyle of Helarctos.
What does modern human variation in MT1 abduction angle and arch height reveal about hominid pedal morphology?
Authors: Kramer Patricia Ann
Published: June 2021

Although modern humans exhibit many different morphologies, anthropological wisdom dictates that an effective bipedal foot is one with an adducted first metatarsal (MT) and medial longitudinal arch (MLA). The recently announced pedal morphology of Ardipithecus calls into question this assumption, prompting the question of what variation among modern human feet might reveal about the function of extinct hominid feet.

Using anterior‐posterior and lateral radiographs of 50 osteological adults (aged 15–78 years, 25 women), arch height and the angle between MTs (MT12, MT23, MT34, MT45, MT15) were measured. MT12 of STW 575 and MT angles of OH 8 were measured on photographs/drawings of reconstructions.

In the modern sample, MT12 ranged from 3–15° (mean = 9°, σ = 3°) and feet with more abducted 1st MT tended to have more abducted 5th MT (p = 0.051) and higher arches (p=0.018). MT12 measured 20° on STW 575 and 10° on OH 8.

For MT12, STW 575 is > 3σ from the human mean, while OH 8 exhibits the human mean value. If the fossil reconstructions are accurate representations of weight‐bearing position, then the 1st MT of STW 575 was more abducted than is typically seen in modern humans, but what, if any, functional consequences accrue from this difference are less clear. As with other morphological features, effective bipedal feet can come in many forms.
Base composition changes indicate biased gene conversion is a major factor in the evolution of the Fam53A gene
Authors: Bergemann Andrew David , Reidenberg Joy S. , Laitman Jeffrey T. , Skrabanek Lucy , Genecin Isabel
Published: June 2021

We have conducted a phylogenetic analysis of the Fam53A gene throughout vertebrate evolution. Our studies reveal extreme differences in the rates of change between lineages. In particular, rates of change are much higher in placental mammals (Eutheria) than in other groups. The faster evolutionary rates of change in Eutheria correlate with a transition of the Fam53A‐containing isochore towards a much higher guanosine and cytosine (G+C) content. Changes in isochore G+C content are now thought to reflect changes in regional meiotic recombination rates, and to result from base composition biases in the recombination machinery. We have investigated the recent base composition changes for the Fam53A gene in two eutherian groups, rodents and primates, in which we have dense taxon sampling. Our initial goal in comparing these two groups was to compare the evolution of Fam53A in a clade in which the gene is located close to the telomeres (primates) to one in which the telomeric location has been lost (rodents). While we found that chromosomal location did not correlate with evolutionary changes, our studies indicate that the mapping of historical sequence changes may allow the mapping of ancient recombination hot spots. Sequences within the primate Fam53A genes display localized, transient, increases in G+C content, as would be expected from the known biology of recombination hot spots.
Anatomy of the facial musculature in two diurnal anthropoid primates: exploring the relationship between facial mobility and the muscles of facial expression
Authors: Kandath Rachael E , Muldoon Kathleen M , Dobson Seth D
Published: June 2021

Recent anatomical research suggests a pattern of uniformity in the muscles of facial expression across several primate species. This pattern appears to conflict with the observed diversity in facial mobility (the number of facial movements an individual can produce) among primate species. Exploring the potential disparity between uniformity of facial expression musculature and diversity of facial movements requires further expansion of the existing anatomical data on primate facial muscles. In this study, we examined the facial musculature of 2 species of diurnal anthropoid primates: Macaca mulatta (high facial mobility) and the previously unexamined Callithrix jacchus (low facial mobility). Using the ‘facial mask’ dissection technique, we determined the number (presence or absence) and degree of differentiation of the facial muscles in each species. We identified at least 18 highly differentiated muscles in Macaca as compared to 6 muscles with less differentiation in Callithrix. Our results indicate some degree of correspondence between facial expression musculature and facial mobility, and as such have implications for the role of these two variables in the evolution of primate social communication. This study was funded by the Claire Garber Goodman Fund to REK.

Grant Funding Source: Claire Garber Goodman Fund (Anthropology Department)
Auditory Functional Analysis: Lessons From the Primate Auditory Ossicles
Authors: Carter Yasmin , Silcox Mary T.
Published: June 2021

Soon after the auditory structures were first described, mechanical engineering theory was applied in order to understand the movements of the various components and the forces generated during this process, with the aim to accurately postulate the actual hearing of an individual or species. Many acoustic hypotheses are limited by their assumption of perfect conditions or ideal movements and rely on the ossicular chain functioning as a mechanical lever unit, however, changes in the angles between these bones have not been considered and until such studies are conducted, the movements of the ossicular chain under sound pressure cannot be accurately modelled. To address this issue, a landmark analysis was conducted on ultra‐high resolution computer tomography (UhrCT) scans of twenty‐six primate auditory ossicle chains. The study allowed not only visualisation but quantification of unexpected angles between bones and articulations which may answer questions regarding ossicle flexibility and relative motions. The morphometry of stand‐out specimens including the articulation angle of V.v. variegata, the relatively large chain of Daubentonia, the bulbous and shortened chain of A. calabarensis and the mediolaterally constricted bodies of T. bancanus suggest that many of the foundational mechanical theories of the auditory system will need to be revised to include more variables than they currently accept.

Grant Funding Source: NSERC discovery grant to MTS and the Canadian Research Chairs program for YC
Positional variation in the molecular regulation of mammary gland development
Authors: Veltmaat Jacqueline M.
Published: June 2021

Our lab aims to unravel the molecular mechanisms of early mammogenesis, using the five pairs of mammary glands in mouse embryos as a model. We found that loss of Gli3 results in induction of only mammary rudiment pairs #1, #2 and #4, but not #3 and #5. Moreover, the #2 and #4 pairs each have distinct morphogenetic defects, while pair #1 is morphologically normal. Our in vivo approach to rescue all defects by activating a pathway downstream of Gli3 resulted in induction of pair #3 only, but not of pair #5, nor did it restore morphogenesis of pairs #2 and #4. Analysis of the latter two pairs is ongoing. We conclude that all five pairs of mammary rudiments in mice differ in their molecular regulation of induction and early morphogenesis, despite their identical functional outcome. This contrasts with other multiples of the same structures, e.g. somites, limbs or hair follicles, which are generally thought to share the same inductive signals, while unique specification signals establish their anatomical and functional differences such as those between fore‐ and hind limbs. The variation in molecular inductive mechanisms of mammary glands may explain the differences in numbers and positions of mammary glands among mammalian species. It also increases the chances of survival of the species when a gene function is altered.

Funding provided by CBCRP:10FB‐0116 and A*STAR.

Grant Funding Source: A*STAR
Pathophysiological changes associated with sustained delivery of DHT and TP by means of Hydroxyapatite (HA) delivery system
Authors: Benghuzzi Ham , Tucci Michelle
Published: June 2021

The objective of this study was to investigate the effect of various dosages of testosterone propionate (TP) and dihydrotestosterone (DHT) delivered in a sustained manner by means of hydroxyapatite lysine delivery system (HADS), on seminal vesicles as well as other vital organs. Adult male Sprague Dawley rats were randomly divided into eight equal groups (n=16). Rats in group I‐ III were implanted subcutaneously with HADS containing 200, 120, or 50 mg of TP. Rats in group IV‐IV were implanted subcutaneously with HADS containing 200, 120 or 50 mg of DHT. Group VII animals were implanted with empty HADS, and group VIII served as unimplanted controls. At the end of 1, 2, 3, and 6 months post‐implantation, four animals from each group were sacrificed and the seminal vesicles (SV), adrenal, heart, liver, and kidney were collected and evaluated. The results demonstrated the following: 1) SV weights were directly proportional to the dose of TP and DHT. 2) SV weights from animals implanted with 200mg TP were 3 fold greater than control. 3) No significant changes were observed in the weights of the spleen, kidney, liver and heart tissues. 4) In contrast to the other vital organs, the mean weight of the adrenal gland was slightly affected by high dosage of TP delivery compared to DHT and controls groups. 5) Cross sections of the seminal vesicles demonstrated that there was a decrease in the secretory epithelial cell height as well as an increase in papillary folding in all experimental groups. The results of this study suggest that TP and DHT are key factors in maintenance of secretory epithelium of the seminal vesicles.
VEGF up‐regulates pro‐inflammatory factors in the cervix of mice
Authors: Schwabe John , Boggess Bethany , Nguyen Bao‐Tran , Gosai Puja G. , Tashakkori Parisa , Mowa Nonde K , Mowa Chishimba N
Published: June 2021

Cervical remodeling (CR) entails a physiological inflammatory response that can be induced by inflammatory agents, such as lipopolysaccharide (LPS), and inflammation is closely associated with vascular remodeling. Recently, we showed that LPS‐induced inflammation up‐regulates mRNA levels of vascular endothelial growth factor (VEGF) and its receptors, KDR and Flt‐1. Here, we sought to investigate whether VEGF can modulate inflammation by characterizing the expression pattern of pro‐inflammatory factors in VEGF‐treated mice using real time PCR. Our data revealed that VEGF up‐regulates a group of pro‐inflammatory factors in the cervix, including IL‐6. Collectively, our studies show a possible existence of a positive feed forward loop between inflammatory and vascular factors in the remodeling cervix.

Grant Funding Source: Appalachian State University
Mechanisms of transcription repression by HIV‐1 Vpr binding protein
Authors: An Woojin
Published: June 2021

In the human body, genomic DNA is compacted into each cell's nucleus as a highly‐organized structure called chromatin. Histones are the major protein components to establish this extensive packaging of DNA within the limited volume of the nucleus. Regulation of transcription processes within this chromatin context requires histone modifying activities that counterbalance this repressive nature of chromatin. HIV‐1 Vpr binding protein (VprBP) was originally identified on the basis of its ability to interact with HIV‐1 Vpr protein and implicated in DNA replication and cell cycle regulation, but its precise role remains unclear. Here, we show that VprBP suppresses p53 transactivation by recognizing unmodified histone H3tail via its Lis homology domain. RNAi‐complemented ChIP and binding assays demonstrate that HDAC1‐mediated deacetylation of H3 tail is prerequisite to tether VprBP at p53 target gene promoters. Furthermore, suppression of VprBP expression induces transcription of p53 target genes, and causes an increase in DNA damage‐induced cell death. Consistent with these results, VprBP is over‐expressed in human cancer cells, and this increased level of VprBP is tightly correlated with reduced apoptosis. Our results thus reveal a previously unknown role for VprBP in DNA repair pathway and illustrate the power of combined biochemical and cellular approaches for mechanistic analyses.
Role of the H4K20me1/H3K9me1 trans‐tail histone code in transcriptional regulation
Authors: Congdon Lauren Marie , Sims Jennifer K , Rice Judd C
Published: June 2021

We previously discovered a novel trans‐tail histone code in which monomethylated histone H3 lysine 9 (H3K9me1) is enriched in genomic regions containing monomethylated histone H4 lysine 20 (H4K20me1). Based on this, we hypothesized that the PR‐Set7 H4K20 monomethyltransferase recruits an H3K9 monomethyltransferase to establish this histone code to regulate the expression of specific genes. A survey of known H3K9 methyltransferases revealed that only PRDM2/Riz1 binds PR‐Set7 in vivo. To further understand the role of this histone code in transcription, gene expression profiling was used to identify a panel of target genes regulated by PR‐Set7. In all cases, the absence of PR‐Set7 and/or H4K20me1 resulted in a >200% increase in their transcription indicating a repressive role for this histone modification. Although both H3K9me1 and H4K20me1 were enriched at these genes, depletion of Riz1 had no effect on H4K20me1 levels and had varying effects on transcription. Collectively, these findings indicate that the repression of a specific set of human genes is conferred by H4K20me1 and suggests that H3K9me1 may be dispensable for gene regulation.
Interactions between the U2 snRNP and SAGA reveal new links between co‐transcriptional RNA splicing and chromatin modification
Authors: Johnson Tracy L
Published: June 2021

Recent studies have shown that mRNA splicing can occur co‐transcriptionally. The next key challenge is to identify the mechanisms by which specific factors couple the two reactions. We have discovered that Gcn5, which encodes the HAT activity of the SAGA, and other components of SAGA that support its catalytic activity, have functional interactions with the genes encoding the heterodimeric U2 snRNP proteins Msl1 and Lea1. ChIP analysis has revealed that Gcn5, and particularly its HAT activity, is required for co‐transcriptional recruitment of Msl1 and Lea1 to pre‐mRNA. Consistent with a role for Gcn5 in co‐transcriptional splicing, Gcn5 protein and Gcn5‐dependent histone acetylation are enriched in intron‐containing genes. These results have led us to examine how the activity of the SAGA complex affects ATP‐dependent rearrangements of the U2 snRNP that are critical for branchpoint recognition. A genetic analysis has revealed surprising interactions between the DExH/D box protein Prp5, which hydrolyzes ATP to remodel the U2 snRNP and facilitate branchpoint recognition, and components of SAGA. A lethal mutation in PRP5 that eliminates ATP‐binding is suppressed when components of the SAGA complex are deleted, thereby for the first time linking SAGA with a catalytic activity that drives spliceosomal rearrangements.

(Supported by NSF CAREER grant MCB 0448010 and NRSA predoctoral fellowship 5F31 GM078745)
Role of Gβγ in regulation of class II histone deacetylases
Authors: Budayeva Hanna , Hamm Heidi E , Spiegelberg Bryan D
Published: June 2021

Canonically, G protein‐coupled receptors (GPCRs) participate in biological information flow via their actions at the cell periphery. Recent data, however, have demonstrated the presence of nuclear GPCR‐initiated pathways that have direct access to the genome. We discovered that liberated Gβγ regulates DNA processing through physical interaction with histone deacetylases (HDACs), allowing a fine‐tuning of gene expression upon activation of specific GPCRs. Our work has focused on understanding the mechanisms by which binding to Gβγ inhibits the biological function of class II HDACs. We have found that the interaction occurs, at least in part, within the nucleus. Importantly, we present data that Gβγ interacts directly with the regulatory N‐terminus of HDAC5, which also contains the transcription factor binding domain. We propose a hypothesis that Gβγ inhibits HDAC5 by perturbing protein‐protein interactions that are necessary for full functioning of the deacetylase activity. To test this hypothesis, we are developing an in vitro equilibrium binding assay that will allow us to quantitatively characterize Gβγ/HDAC5 complex formation. As proof of concept, we are using this assay to characterize the known interaction between the N‐terminus and the transcription factor MEF2A. We present data characterizing additional interactions of the N‐terminus of HDAC5 with other regulatory proteins.
H2B ubiquitylation regulates nucleosome stability at replication forks
Authors: Lin Chia‐Yeh , Wu Meng‐Ying , Tseng Shin‐I , Hsu Guoo‐Shyng W. , Kao Cheng‐Fu
Published: June 2021

Histone modifications are important determinants of gene expression, replication, DNA repair and cell cycle control. Ubiquitylated lysine 123 on H2B (H2Bub) is shown to contribute to efficient reassembly of nucleosomes during RNA polymerase II (Pol‐II)‐mediated transcription elongation in yeast. Here, we demonstrate that absence of H2Bub (htb1‐K123R mutant) causes a slowdown G1/S transition and a prolonged S phase as the initiation of nascent DNA synthesis is defective. Significantly, the cell cycle control of H2Bub is independent of H3K4 and K79 methylations, the two putative downstream signals of H2Bub. Moreover, the altered S phase progression is not a result of checkpoint activation but caused by distorted nucleosome stability at the replication fork. Genetic analysis suggests that H2Bub functions as an intrinsic cell cycle regulator in parallel with replication factors, Sgs1 and Mrc1. Deletion of histone chaperones Asf1 deteriorates the growth of htb1‐K123R cell under replication stresses but the synergistic phenotypes are not observed when combined with the deletions of Cac1, a histone chaperone assembles newly synthesized histone onto replicated DNA. Thus, these findings suggest that H2Bub contributes to nucleosome stability at replication fork independent of Asf1.
p300 mediates DNA looping between far upstream enhancer and core promoter of human iNOS gene
Authors: Guo Zhong , Shao Lifang , Geller David
Published: June 2021

Our previous study identified a cytokine‐induced inducible enhancer located between ‐5 and ‐6 kb in the human iNOS (hiNOS) upstream promoter. The exact mechanism for how the distal enhancer to regulate hiNOS gene is not clear. In this study, we apply 3C assay and ChIP loop assay to determine whether cytokines have any effect on chromatin structure of the hiNOS locus. Our results show that 5kb DNA looping is induced by the cytokine mixture (CM) of TNFα + IL‐1β + IFNγ. The distal enhancer and TATA box in the hiNOS core promoter are bridged by p300, a ubiquitous transcription coactivator. The binding site at enhancer for which transcription factor involves this DNA loop in iNOS transcription is still under investigation.
Mammalian Tethered Catalysis: a novel in vivo approach to identify and characterize histone post‐translational modification binding proteins
Authors: Tuzon Creighton T , Spektor Tanya M , Rice Judd C
Published: June 2021

Post‐translational modifications (PTMs) of nucleosomes are critical for genome architecture and function. Remarkably, more than 70 different sites for histone PTMs have been reported; however, the identification of histone PTM binding proteins (PTMBPs) and the mechanistic understanding of how various PTMs translate in to biological outputs is less understood. We developed mammalian tethered catalysis (MTeC) as an in vivo approach to identify novel histone PTMBPs. To validate this approach, we first used MTeC to identify H3K9 PTMBPs in an unbiased manner. Surprisingly, we isolated G9a as an H3K9me1 binding protein in vivo. Given this selectivity, we then tested the binding specificity of methyl‐specific histone PTMBPs to two different histones – H3 and H4. To our surprise, only one of the three histone PTMBPs assayed, HP1β, exhibited specificity for H3K9 methylation status in vivo. Interestingly, both tandem tudor‐domain proteins, JMJD2A and 53BP1, did not demonstrate binding specificity for H4K20 methylation status. Instead, we discovered that JMJD2A and 53BP1 interacts with all three methylated states of H4K20 in vivo, unlike previously reported in vitro assays. Thus, the utility of MTeC extends beyond the impartial identification of histone PTMBPs and allows an objective evaluation of the binding specificity of histone PTMBPs in vivo.
The β Subunit Gate Loop Mediates Antitermination Modification of RNA Polymerase
Authors: Sevostiyanova Anastasia , Belogurov Georgiy , Artsimovitch Irina
Published: June 2021

Synthesis of long RNA chains by multisubunit RNA polymerases (RNAPs) requires accessory proteins that help RNAP bypass numerous roadblocks it encounters along the way. These “antitermination” proteins switch the elongating RNAP into a processive state, but the molecular mechanism of this phenomena remains unknown. Bacterial antiterminator RfaH is an excellent model to address this key mechanistic question because its structure, the mechanism of recruitment and its binding site on RNAP have been determined. RfaH increases expression of the distal genes in operons that have ops DNA elements. We found that RfaH binds simultaneously to the regions of β and β′ subunits that constitutes “jaws” of RNAP, locking it in a closed conformation. Deletion of a conserved β gate loop (GL) has little effect on intrinsic properties of RNAP but completely abolishes its response to RfaH and NusG, a general paralog of RfaH. qRT‐PCR analysis of ops‐containing operon expression driven by wild‐type or ΔGL RNAP in the presence or absence of RfaH confirmed in vitro observations. Finally, we showed also that GL deletion does not compromise stimulation of Rho‐dependent termination by NusG, function that is thought to be independent from AP effect of NusG and based on physical tethering of Rho to the elongation complex. We propose that NusG‐like proteins restrict conformational mobility of RNAP, thereby locking it in a processive state.
Ubiquitin Carboxy Hydrolase‐L3 (UCH‐L3): A Molecular Sensor of Protein Stability?
Authors: Romo‐Fewell Octavio , Navarro Mario , Love John J.
Published: June 2021

The accurate assessment of protein stability, as well as exploring new means to enhance protein stability are of keen interest. To achieve this we are exploring the properties of ubiquitin carboxy hydrolase‐L3 (UCH‐L3), a protease that cleaves proteins N‐terminally fused to ubiquitin. Based on the hydrolysis rate we are exploring whether UCH‐L3 is capable of differentiating between proteins with different thermal stabilities. First, we developed a panel of mutants derived from streptococcal protein G that span a thermal stability range from 28–100 °C. The genes for these variants were fused N‐terminal to the gene for ubiquitin. To explore the activity of UCH‐L3 in vivo, E. coli (BL21) cells were cotransformed with plasmids coding for both the enzyme and the fusions in compatible plasmids. After protein induction the hydrolysis rates were estimated with SDS‐PAGE. For further characterization, ex vivo and in vitro assays were run with cell extracts and the purified enzyme and substrates. In addition, size exclusion chromatography and CD (Circular dicrhoism) studies were performed in order to determine the oligomerization state and the melting temperature (Tm) of the substrates. In the three formats tested in vivo, ex vivo and in vitro, the hydrolysis was similar and consistent. Hydrolysis of the different constructs by UCH‐L3 is correlated to the thermal stability of each particular test protein. Interestingly, the enzyme is capable of differentiating between substrates with only one amino acid change, even for a position far from the cleavage site. Founding has been provided by National Science Foundation, Consejo Nacional De Ciencia y Tecnologia, Mexico, and by the NIH/NIGMS SDSU MARC Program 5T34GM08303.
Estimating the activation barrier to each step of protein splicing for the non‐canonical P. abyssi PolII intein
Authors: York Daniel J. , Duffee Lauren R. , Dorval Deirdre M. , Reitter Julie N. , Mills Kenneth V.
Published: June 2021

Protein splicing is a post‐translational event by which an intervening polypeptide, called an intein, facilitates its own excision from the flanking polypeptides, called the exteins, and the ligation of the exteins. We are able to study the protein splicing of the Pyrococcus abyssi PolII intein in vitro, and can determine the rate of the overall splicing reaction as well as that of each step. The robust temperature‐dependence of this reaction allows us to estimate the activation barrier for individual steps of the reaction. This is especially interesting for the third step of the reaction, cyclization of Asn or Gln coupled to peptide bond cleavage. We will use these data to assess mechanistic models for the catalysis of side‐chain cyclization.

This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.
Hemoglobin induced oxidative stress is associated with both endogenous peroxidase activity and H2O2 generation from polyunsaturated fatty acids
Authors: Cheng Tsai‐Mu , Pan Ju‐Pin , Chen Hong‐Wei , Chang Yi‐Chun , Chang Chia‐Ching
Published: June 2021

Patients with increased hemolytic hemoglobin (Hb) have 10–20 times greater incidence of cardiovascular mortality than the general population. In the presence of an exogenous source of H2O2, Hb is essential and responsible for the oxidation of low‐density lipoproteins (LDL). However, the role of Hb peroxidase activity in LDL oxidation remains elusive. Our study indicated that Hb could induce the formation of H2O2 by reacting with LDL, linoleic acid, and cell membrane lipid extracts. In the presence of NaN3 (a peroxidase inhibitor), endogenous peroxidase activity and subsequent Hb‐induced LDL oxidation was significantly inhibited and H2O2 concentrations accumulated. Furthermore, Hb was shown to dramatically stimulate the expression of intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) in both a concentration‐ and time‐ dependent manner. Nevertheless, this stimulation was completely inhibited by a potent antioxidant, probucol. We found that 2, 2′azo‐bis‐(2‐amindinopropane) hydrochloride (AAPH) could also induce LDL oxidation and stimulate the expression of ICAM‐1 and VCAM‐1, but was not inhibited by NaN3. In conclusion, the present study suggests that the oxidative effect of Hb to produce atherogenic oxidized LDL and to induce the production of ICAM‐1 and VCAM‐1 are due to H2O2 formation as well as its endogenous peroxidase activity toward polyunsaturated fatty acids. The biochemical and physiologic significance of this study are discussed.
Size and Stability are Critical Elements in the Substrate Selectivity of Insulin‐Degrading Enzyme
Authors: Kalas Vasilios , Ralat Luis A , Zheng Zhongzhou , Sosnick Tobin R , Tang Wei‐Jen
Published: June 2021

Insulin‐degrading enzyme (IDE) is a zinc metalloprotease involved in the clearance of small bioactive peptides, such as insulin and amyloid‐beta. Given the considerable diversity in substrate tertiary structure, the criteria for substrate recognition and degradation by this protease have been difficult to determine. Here, we evaluate size and local stability as parameters in the substrate selectivity of IDE using opioid peptides and ubiquitin variants as model systems, respectively. Kinetic analyses of IDE, using opioid peptides ranging from 5 to 31 residues, demonstrate that opioid peptides containing 17 or more residues exhibit Km values of 50 μM or less and kcat values that do not exceed 40 s−1. In contrast, smaller opioid peptides exhibited Km values above 900 μM and kcat values above 600 s−1. Mutations at conserved substrate binding sites in IDE are currently being investigated to identify the molecular basis of substrate size discrimination. To probe local stability as a parameter in substrate selectivity, we mutated residues on each of the seven major structural elements of ubiquitin (Ub), a 76‐residue inhibitor of IDE, which destabilize Ub by 0.5–9 kcal/mol. Surprisingly, Ub variants which lose at least 1.6 kcal/mol in local stability are cleaved by IDE with kcat values above 0.5 sec−1. While the first cut site is identical in all digested variants, the cleavage pattern varies with increasing instability of Ub proteins. Together, these findings imply that changing a substrate's property modifies the enzyme's specificity and thus suggest a knowledge basis for the rational design of peptidomimetic modulators of IDE activity.
Mutational disruption of the nitric oxide synthase/calmodulin interaction causes increased formation of reactive oxygen species
Authors: Kellogg Dean L. , Masters Bettie Sue S. , Roman Linda J.
Published: June 2021

The nitric oxide synthases (NOSs) catalyze the formation of nitric oxide (NO) from L‐arginine. The NOSs are bidomain enzymes, consisting of a heme‐containing oxygenase domain and a flavin‐containing reductase domain, bisected by a calmodulin (CaM) binding site. Occupancy of this site is required for NO formation. Recently, Xia et al. (2009, J. Biol. Chem., 284, 30708) published the structure of human iNOS CaM‐bound FMN domain, showing that the only interactions between the FMN domain and CaM involve R536 of iNOS, which makes a salt bridge with E47 of CaM and forms H‐bonds with N42 of CaM and S562, F565 and A564 in the FMN domain. Mutations of R530, the homologous residue in the murine iNOS reductase domain, and R753 in the nNOS reductase domain, yield enzymes active in electron transfer to cytochrome c and potassium ferricyanide. The rate of ferricyanide reduction is equivalent to that of wild‐type, but cytochrome c reduction is diminished by approximately 60%. NADPH oxidation in the absence of acceptor, however, is elevated by about 40% over that of wild‐type, indicating an increase in reduced oxygen species production by these mutants.

Supported by NIH GM052419 to BSSM and LJR.
Molecular chaperone HSP90α isoform 1 forms telomerase complex
Authors: Zhdanov Dmitry , Kovalenko Natalia , Orlova Elena
Published: June 2021

Telomerase is a specialized reverse transcriptase multiprotein complex responsible for synthesizing telomeric DNA repeats (TTAGGG) at the ends of eukaryotic chromosomes. One of six telomerase subunits is HSP90α that is thought to stabilize the complex. Two isoforms (IS1 and IS2) of HSP90α that are the result of HSP90AA1 gene alternative splicing are known. IS1 has 122 additional amino acid residues at the N‐terminus of the molecule. We examined HSP90α IS1 and IS2 expression and its correlation with the expression of telomerase catalytic subunit hTERT and telomerase activity to investigate which of HSP90α isoforms forms telomerase complex. Purified RNA from human colon and gastric adenocarcinoma was used for RT‐PCR analysis to examine the mRNA expression rate of HSP90α IS1 and IS2, hTERT and B2‐microglobulin (as positive control).

Telomerase activity was detected using TRAP analysis. HSP90α IS2 constitutively expressed at the same level both in tumor and normal tissues. HSP90α IS1 was detected in colon and gastric tumor samples (p = 0.046) and was not found in normal tissues. Expression of this isoform correlated with telomerase activity (r = 0.86) and hTERT expression (r = 0.78), but did not depend on adenocarcinoma differentiation and a stage of disease.

According to these data, we can suggest that HSP90α IS1 forms telomerase complex. It can be used as a target for production more specific telomerase inhibitors.
The functional architecture of a cationic pore that modulates citrate synthase quaternary structure and allostery
Authors: Brayer Gary D. , Maurus Robert , Stokell David , Donald Lynda J. , Nguyen Nham , Duckworth Harry W.
Published: June 2021

The active form of the allosteric Type II citrate synthase of E. coli is a hexamer, having as a large central feature an unusual cationic pore lined with 18 arginines. We have investigated this remarkable concentration of positive charge using a variety of techniques. The importance of the combined repulsive effect of these arginines is evident from the extreme conditions necessary to reestablish a dimer‐hexamer equilibrium with their substitution. Structural studies have determined three repulsive arginine based forces act to destabilize the hexamer state, including: interactions between two arginine rings that encircle the cationic pore; a novel arginine cassette structure formed between dimer units; and, repulsions between arginine side chains at the very center of the pore. Notably, kinetic studies suggest these repulsive pore interactions give the hexameric enzyme the energetic flexibility needed to carry out catalysis. Our results emphasize the finely tuned relationship between the dual dimer ‐ hexamer and R (active) ‐ T (inactive) allosteric equilibria present. Indeed it seems the allosteric NADH binding site is a mechanism designed to fine tune the cationic pore ‐ active site energetic relationship and thereby allow for an equilibrium wherein a T‐state enzyme can be induced without irretrievable inactivation of the enzyme. Supported by the Natural Sciences and Engineering Research Council of Canada.
Novel alpha‐glucosidase from Human Gut Microbiome‐ substrate specificity and switch.
Authors: Tan Kemin , Tesar Christine , Wilton Rosemarie , Keigher Laura , Babnigg Gyorgy , Joachimiak Andrzej
Published: June 2021

The human intestine harbors a large number of microbes forming a complex microbial community that greatly affects the physiology and pathology of the host. In the microbiomes, the enrichment in certain protein gene families appears to be widespread. They include emzymes involved in carbohydrate metabolism such as glycosyl hydrolases of dietary polysaccharides and glycoconjugates. Here we report the crystal structure of an alpha‐glucosidase from human gut bacterium Ruminococcus obeum and its substrate specificities. The first ever protein structure from this bacterium reveals a structural homolog to human intestinal maltase‐glucoamylase (MGAM) with a highly conserved catalytic domain and reduced auxiliary domains. The alpha‐glucosidase shows substrate preference for alpha(1–6) over alpha(1–4) glucosidic linkages and produces glucose from isomaltose as well as maltose. The preference can be switched by a single mutation at its active site, suggestive of utilization of a variety of polysaccharides by intestinal bacteria as energy resources and their complex interplay with their host.