bluuebunny/crossref_metadata_2025 huggingface dataset:
Performance in Medical Gross Anatomy as a Predictor of USMLE Step 1 Scores
Authors: Fletcher Donald , Musick David
Published: June 2021

With a mission to accept disadvantaged students at our medical school, we need reliable predictors of USMLE Step 1 performance. In this study, we compiled averages for 464 students in the Gross Anatomy course taken in the fall semester over 7 years (2004–2010). We examined the predictive validity of these averages by correlating them with scores of initial takers of the Step 1 exam (Pearson's coefficient). Correlation between the final course average (60% written exams and 40% lab practicals) and Step 1 score was 0.603 (p ≤ .01), explaining 36% of the variance in students’ scores. Correlation between the average performance on the four written exams and Step 1 score was stronger (0.642) and accounted for 41% variance in students’ scores. These correlations were higher than the correlation between these students’ MCAT and Step 1 scores (0.518). These findings are consistent with conclusions of similar studies from other medical schools and validate the use of student performance in Gross Anatomy as a reliable predictor of Step 1 scores at our medical school. These predictions can be used to counsel and provide additional assistance to at‐risk students in terms of their Step 1 preparations.
Human structure in 7 weeks: one approach to providing foundational anatomical competency in an era of compressed medical school curricula
Authors: Halliday Nancy Louise , O'Donghue Daniel , Thompson Britta
Published: June 2021

As part of a new integrated systems‐based pre‐clinical curriculum, the University of Oklahoma College of Medicine reduced its gross anatomy course from 200 to 120 contact hours in 2010. Along with the reduction in contact hours, content from embryology, histology and radiology were integrated into the course. To reflect the newly integrated content, the course title changed from Gross Anatomy to Human Structure. This paradigm shift in clinical anatomical education is becoming common in medical school curricula in the USA and internationally. Faced with the challenge of providing medical students with sufficient anatomical training, we developed an immersive, multimodal curriculum designed to maximize learner outcomes. The new curriculum includes innovations such as student presentations of anatomically based clinical cases to peers and faculty, meeting with families of anatomical donors, and group learning activities. We instituted multiple weekly summative and formative assessments to provide students ongoing measures of their learning. Our purpose in this presentation is to provide a descriptive overview of the approaches recently applied to our course. We will include an assessment of the results of course effectiveness based on student evaluations, and student performance results based on Step 1 of the USMLE.
The integration of osteology, radiology, and surface anatomy into a clinical skills 101 course for first‐year medical students
Authors: Giffin Bruce Francis , Lowrie D.J. , Pettigrew David , Gunderson Anne
Published: June 2021

Clinical Skills 101 is a new course with the goal of imparting patient centered skills fundamental to promoting health and treating disease. Teaching the physical examination requires knowledge of surface anatomy, and understanding the variation of normal visible and palpable anatomy enables the physician to determine when pathology may be responsible for abnormalities in surface anatomy or underlying structures. Since the skeletal system elements are responsible for many of the palpable bony contours seen on the surface of the body, preliminary knowledge of the skeletal system is essential for learning surface anatomy. Students are taught the basics of the physical examination using standardized patients in conjunction with surface anatomy and osteology laboratory exercises. Since medical imaging has come to occupy a central role in the delivery of patient care, imaging exercises reinforce the osteology and demonstrate important imaging features of common or important pathologies. Weekly assessment of the students’ progress in the acquisition of surface anatomy and imaging skills consists of surface anatomy testing using fellow students as “patients”, osteological practical exams, and identification of normal and abnormal imaging. Evaluation of this integrated approach to the acquisition of clinical skills is highly rated by students and clinical preceptors.
Evaluating an Anatomy‐Specific Tool for Blooming Exam Questions
Authors: Thompson Andrew R. , O'Loughlin Valerie Dean
Published: June 2021

Bloom's taxonomy is commonly used to assess the cognitive level of exam questions and other course assignments. Our previous research found that ‘Blooming’ pathology exam questions was difficult when using guidelines that were not discipline‐specific. Achieving consistency in scoring required that the observers independently rank and then discuss each exam question. Although this strategy is highly reliable, it is inefficient and may impose a limit on the number of questions that can be evaluated. In addition, the discussion based style of this approach makes it problematic for outside researchers to replicate the results.

Building on research by Crowe et al. (2008), we developed a Blooming Anatomy Tool (BAT) that provides tailored guidelines for Blooming anatomy exam questions. To test the efficacy of the BAT, two groups of instructors Bloomed a series of anatomy exam questions, each receiving different scoring criteria. The first group was given a worksheet that broadly outlined Bloom's taxonomy, while the second group received the BAT. After comparing the Bloom levels assigned by individuals in each group to a key generated by the authors, we found that the BAT had a positive impact on accuracy and consistency in determining Bloom categories. We suggest that researchers utilizing Bloom's taxonomy for assessing course materials should consider seeking out or developing a discipline‐specific rubric.
Digital Atlas for Ultrasound‐Guided Regional Anesthesia Nerve Blocks of the Trunk
Authors: Stone Aaron , Johnson Marjorie , Ganapathy Su
Published: June 2021

Ultrasound is a more recent tool anesthesiologists use to provide regional anesthesia. Ultrasound‐guided regional anesthesia has the following benefits: allows for the real time imaging of target nerves and surrounding structures, is used independently of surface landmarks, facilitates purposeful needle movements, improves the quality of sensory block, the onset time, the success rate compared to neurostimulation, and uses fewer needle attempts for nerve localization, which reduces the risk of nerve injury. The purpose of this project is to create a digital atlas of the sensory innervation of the trunk, to help trainees improve their sonoantomy interpretation. The atlas will include cadaver dissections, Visible Human Project images and surface anatomy to demonstrate the relationship between ultrasound images and the corresponding surface and internal anatomy. The blocks and their indications will be described in text and demonstrated visually with a narrated video. The atlas will include the following blocks: thoracic epidural, spinal, paravertebral, intercostal, ilioinguinal, transversus abdominis plane, transversalis fascia block, and the rectus sheath block. The atlas will be available online for trainees prior to practical training sessions in the lab. Future projects would test the efficacy of the atlas and look for ways to improve the teaching methods of ultrasound‐guided regional anesthesia.

Grant Funding Source: N/A
Spatial ability and cognitive load demands during visual learning and testing: A transcranial Doppler ultrasound study.
Authors: Loftus Jay , Wilson Timothy D.
Published: June 2021

Determining the efficacy of learning technologies is often based solely on learning outcomes or performance of students. Technologies that fail to aid students in these outcomes are often abandoned. The present study examines the relationship between the demands of cognitive processing and the underlying physiological response. The aim of this study is to demonstrate the affects of increased cognitive processing on learner cerebral blood flow. Utilizing learners of high (HSA) and low spatial ability (LSA), we hypothesize is that cognitive processing of visual information will be mitigated by spatial ability. Using transcranial Doppler ultrasonography to measure blood flow velocity, it is believed a negative correlation will exist between mean blood flow velocity and spatial ability. Similarly, a negative correlation between mean blood flow velocity and performance will exist. Overall, this suggests cognitive processing will be more efficient and effective in learners with higher spatial abilities.

Grant Funding Source: None
‘Hidden’ states are pervasive in RNA folding: detection and dissection through mutate‐and‐map experiments
Authors: Das Rhiju , Doniach Sebastian , Ali Mona , Cordero Pablo , VanLang Chris
Published: June 2021

Experiments that probe RNA biophysical behavior are typically interpreted in terms of the smallest number of states that account for observed data. In contrast, extensive computational work has suggested that most RNAs sample many dramatically different secondary structures at equilibrium frequencies of at least a few percent. Data bearing on this prediction are scarce. We will present detailed evidence from a novel experimental approach indicating that such alternative states are pervasive but have been missed in prior experiments. We have applied the mutate‐and‐map method, which couples comprehensive single‐nucleotide mutagenesis and single‐nucleotide‐resolution chemical mapping, to a dozen systems including riboswitches, the putative noncoding RNA HAR1, and RNAs of random sequence. The data are strikingly information rich, in some cases yielding not just models of alternative structures but also their energy differences: a view of the low‐lying energy landscape. Understanding whether these newly revealed states are tolerated, destabilized, or leveraged by functional RNAs in the cellular milieu is a critical next challenge.
Selected vs. Constructed response in assessing anatomy knowledge‐outcomes from a brief study
Authors: Vasan Cheryl , Vasan Nagaswami
Published: June 2021

Anatomy teaching in medical school has changed in the last few decades. However, assessing students’ knowledge has not changed despite the awareness of Bloom's taxonomy. For economical reason we rely on multiple choice questions‐MCQ (selected response) that only tests knowledge. However, the psychometric properties such as critical thinking and skill development which are part of academic achievement and progress are not assessed. Contrarily, constructed response testing demands knowledge construction and higher level of thinking; however, for faculty more time consuming to score. We conducted a pilot study. We made a MCQ exam which was also formatted as short answer that requires content specific knowledge and critical thinking. We administered this approach to a small group of incoming first year medical students taking preparatory anatomy course. In the midterm exam, constructed exam score was much lower compared to selected response score. However, in the final exam their score in the constructed exam improved considerably compared to the midterm score. Student reported that their improved performance was due to changes in their study and learning anatomy than relying on being familiar with the material. While this outcome is encouraging feasibility of this approach to a large class needs to be explored. Supported by the Office of Education and the Department of Cell Biology and Molecular Medicine.
Texas Tech System (TTS) for student peer teaching in anatomy: didactic small group sessions
Authors: Schneider Brandt L , Lee Vaughan H
Published: June 2021

In a continuing effort to promote independent learning in first year medical students, the TTS for laboratory anatomy has been modified and expanded to include didactic sessions. Here we share the initial experience of modifying the TTS for peer teaching of didactic concepts in small groups. Two didactic sessions (heart development and head & neck clinical cases) were designed for groups of ten students to discuss the conceptual (comprehension and application) processes of assigned objectives. A pair of students was assigned an objective or case for each session and provided resources including: notes with integrated online videos, narrated PowerPoint presentations, and/or textbook tutorials. Five point quizzes were administered and the results for each didactic session were collected. The class average for the heart development quiz was 75.4 and for the H&N quiz was 88.0. Student perceptions were assessed on course surveys.

Conclusions

1. Conceptual details for the heart development session were overambitious and difficult for students at this level. 2. Timing relative to the course exam is critical for student appreciation. 3. Students prefer the case studies, recognizing a more direct correlation to exams. 4. Students perceive these activities detract from their preparation for exams. This project was funded by the Department of Cell Biology and Biochemistry, TTUHSC.
Understanding Human Cranial Nerves through a Patterned Based Comparative Approach
Authors: Bohn Jacqueline A , Morton David A , Nielsen Mark T
Published: June 2021

Learning the cranial nerves is a rigorous challenge encountered by first year medical students. Not only must they learn and remember the names, numbers, modalities, pathways, and nuclei of the twelve cranial nerves, in addition they must understand the clinical significance and clinical implications of these complex cranial structures. Educational studies have demonstrated that incorporating patterns as a basis to learning enhances the learning process and improves retention of the learned material. Understanding simple patterns, especially if the patterns are anatomically based, can greatly reduce the learning curve associated with learning complex anatomical topics — like the cranial nerves. Using comparative and developmental anatomy, clear, simple patterns are established to illustrate that cranial nerves are similar to their simpler spinal nerve counterparts. Understanding these patterns takes the random memorization out of the learning process and introduces a logic that brings a deeper meaning to the organization and structure of the cranial nerves. With this logic the learning process becomes easier and more memorable. With improved learning comes improved application in a clinical setting, which should be the ultimate goal.

Grant Funding Source: Department of Neurobiology and Anatomy
Using the antiphosphatase Paladin to understand the phosphoregulation of neural crest development
Authors: Roffers‐Agarwal Julaine , Hutt Karla J. , Gammill Laura S.
Published: June 2021

The neural crest is a migratory embryonic cell population that arises in the dorsal neural tube and gives rise to diverse cell types including peripheral nerves, glia and melanocytes. While the network of transcription factors (TFs) responsible for neural crest development has been established, expression of these TFs does not guarantee that a dorsal neural tube cell will become a migratory neural crest cell. We propose that subsequent modulation of protein activity through phosphorylation also regulates the establishment of neural crest cell fate. Paladin, a novel protein containing two consensus phosphatase active site motifs, is expressed throughout neural crest development and influences the expression of a subset of neural crest TFs. Both gain and loss of Paladin function disrupt early neural crest migration. Mutational analysis reveals that critical catalytic cysteine residues are dispensable for Paladin function, revealing Paladin as an antiphosphatase. Together these data identify Paladin as a unique regulator of neural crest specification and migration. We are currently utilizing Paladin to identify new neural crest regulatory factors that are modulated by phosphorylation using both in vitro and in vivo assays, thus expanding our understanding of an underappreciated level of control within the neural crest developmental program. Funding: NIH F32DE019973; K22DE015309.

Grant Funding Source: NIH
C‐Jun N‐terminal Kinase (JNK) maintains tissue integrity during cell rearrangement in the gut
Authors: Nascone‐Yoder Nanette , Dush Michael
Published: June 2021

Tissue elongation is a fundamental morphogenetic process that generates the proper anatomical topology of the body plan and vital organs. In vertebrates, c‐Jun N‐terminal Kinase (JNK) is a downstream effector of Wnt/PCP signaling that is required for tissue elongation, but the precise cellular role of JNK in this context has remained elusive. Here, we show that JNK activity is indispensable for the rearrangement of endoderm cells that underlies the elongation of the Xenopus gut tube. Whereas other Wnt/PCP mediators, such as Rho, are necessary to induce cell intercalation and remodel adhesive contacts, we found that JNK is required to maintain cell‐cell adhesion and establish parallel microtubule arrays; without JNK activity, the reorganizing endoderm dissociates. Depleting microtubules phenocopies JNK inhibition, consistent with a model in which JNK regulates microtubule architecture to preserve adhesive contacts between rearranging cells. Thus, in contrast to Rho, which generates actomyosin‐based tension and cell movement, JNK signaling is required to establish cytoskeletal stability and maintain tissue cohesion; both factors are required to achieve proper cell rearrangement and gut extension. Our results shed new light on the means by which intra‐ and intercellular forces are balanced to promote topological change, while preserving structural integrity, in numerous morphogenetic contexts.
Sphingosine‐1‐phosphate signaling in inflammation and cancer
Authors: Spiegel Sarah
Published: June 2021

The sphingolipid metabolite sphingosine‐1‐phosphate (S1P) and the kinases that produce it have emerged as critical regulators of numerous fundamental biological processes important for inflammation and cancer. Activation of sphingosine kinases (SphKs) by a variety of agonists increases intracellular S1P. S1P can be secreted out of cells and bind to and signal through five specific G protein‐coupled receptors, designated S1PR1–5. It is now well established that this “inside‐out” signaling by S1P is important for many human diseases. The roles of the S1PRs in cell and organismal physiology will be discussed in detail and particularly their roles in chronic intestinal inflammation and colitis‐associated cancer (CAC). Focus will be on activation of SphK1 and how the SphK1‐S1P‐S1PR1 axis is at the nexus between NF‐κB and Stat3 and connects chronic inflammation and CAC. The development of inhibitors of SphK1 and small molecules that target specific subtypes of S1P receptors will also be discussed. The effectiveness of the pro‐drug FTY720 (known as Fingolimod or Gilenya), approved for the treatment of multiple sclerosis, has become the gold standard for S1P‐centric drugs, and will be used to illustrate the therapeutic value of modulating SphK1 and S1P receptor functions which holds great therapeutic promise.

Supported by NIH grants R37GM043880, R01CA61774, and U19 AI077435.
IMPROVEMENT OF P. CHRYSOGENUM BY UV MUTATION FOR OVERPRODUCTION OF CELLULASE AND LIGNINASE
Authors: Oladayo Musa Babalola , Magbagbeola O. Abiola , Albert Ebuehi O , Imaga O Ngozi , Miriam Ezikpe N
Published: June 2021

The improvement of microbial strains for the overproduction of lignocellulolytic enzymes production by mutagenesis has attracted attention in the commercial fermentation industry. The present study was designed to modify a Penicillium chrysogenum strain, overproduce cellulase and ligninase, and see the relationship between exposure time and distance of delivering UV rays to fungal strains for the improvement of enzyme production.

UV radiation source was placed above the surface of P. chrysogenum suspension spores. Radiation was delivered at 30W at various distances between 10cm ~ 50cm for 10 ~ 60mins intervals of 10min. Mutants obtained from the irradiation were selected and cultured in mineral salt media for enzyme assays. Reducing sugars released were determined as well as enzyme activity of the cellulase (FPase and CMCase) and ligninase (Laccase, MnP, LiP). Growths on plates were also monitored and studied.

The FPase and CMCase activity of the mutant strains were two folds higher than that of the parent strains. Similar trend was observed with the LiP, MnP and Laccase where there was 5 fold, 15 fold, and 9.6 fold respectively.

There was no significant difference in FPase activities (P>;0.05) for the different exposure time and distance from source of radiation, however, there was in the case of CMCase. There was no significant difference (P>;0.05) for Laccase and Manganese Peroxidase, however there was significant difference for Lignin peroxidase. The distance at which Ultraviolet Irradiation is delivered and the exposure time could significantly affect the mutation of fungi for production of lignocellulolytic enzymes.
DNA‐nuclear protein interactions around‐224 A/G Single Nucleotide Polymorphism in the Neuropeptide Receptor Y2 (NPY2R) Gene in Predisposition to Hypertension
Authors: Albino‐Rodriguez Elinette M , Katsuya T , Deng A , Dutil J
Published: June 2021

Hypertension (HTN), is an important risk factor for cardiovascular and kidney diseases. Multiple genes as well as environmental and lifestyle factors influence the risk of developing high blood pressure. Previous work in animal models and human populations identified Neuropeptide Y receptor 2 (NPY2R) as a candidate gene for HTN. In two independent Japanese populations, the GG genotype single nucleotide polymorphism (SNP) located 224 bp upstream of the transcription start site was associated with an increased risk of HTN. The aim of this study was to assess the DNA‐nuclear protein interactions surrounding the NPY2R‐224 A/G SNP. Two versions of double stranded oligonucleotide probes (50bp) corresponding to the sequence flanking NPY2R‐224A/G were synthesized and incubated with different amounts nuclear extracts for an electrophoretic shift assay (EMSA). In addition, we used CTCF antibody for a gel supershift assay. The DNA/protein reactions were loaded on a 4% non‐denaturing polyacrylamide gel. Interestingly, the EMSA showed an allele‐specific binding with the oligonucleotide containing an A nucleotide in ‐224 position. However, the oligonucleotide containing the G nucleotide in ‐224 position shows no DNA/nuclear protein interaction. Also, we were able to detect a supershift using the CTCF antibody. The data provides strong evidence for a functional role of NPY2R in genetic predisposition to HTN.
Functional studies of point mutation G295R on the catalytic core of human DNA polymerase η
Authors: Valdez Adali Joanna , Kumar Mukesh , Jiang Xiaohua
Published: June 2021

The human DNA polymerase η (hPolη) specializes in bypassing UV‐induced cyclobutane pyrimidine dimers (CPD) formed from adjacent thymine bases by pairing the corresponding adenines during a process known as translesion synthesis (TLS). Patients with the variant form of Xeroderma Pigmentosum (XPV) have a deficiency in hPolη and are very sensitive to sunlight and develop skin cancer in their early ages. Research shows that mutations in hPolη of XPV patients are directly linked to skin cancer development. To understand the mechanism of how those mutations cause defects in protein function, I have introduced a point mutation found in XPV patients in the catalytic core (1–432 amino acids) of hPolη by site‐directed mutagenesis. Point mutation G295R was successfully introduced in to the polymerase and is being expressed in E. coli cells for protein purification. Support for this research comes from the Research Student Grant of the Department of Chemistry, Tenessee Tech University, Cookeville, TN.
Cooperative role of MMP‐3 and HP1 in heat shock protein transcription
Authors: Eguchi Takanori , Calderwood Stuart
Published: June 2021

Transcriptional induction of HSP70 is controlled by stress responsed phosphorylation of heat shock transcription factor (HSF1) (1). Here we report novel transcriptional induction system of HSP70 mediated by cooperative action of heterochromation protein (HP1) and intracellular matrix metalloprotease‐3 (MMP‐3). MMP family have been studied as extracellular proteases; however, a recent study revealed a novel role of MMP‐3 in transcription in cellular nuclei and direct interaction between MMP‐3 and HP1 (2). HP1 family was firstly discovered as a heterochromatin forming factors; however, HP1 is a versatile protein involving also in transcription activation upon heat stress in drosophila. In this study, transcriptome analysis in MMP3‐transfected HEK293 cells, HSP70B was revealed as the most inducible factor by intracellular MMP3 among whole mRNA. By deletion mutant analysis, full length and PEX domain of MMP3 induced HSP70B mRNA. Co‐transfection studies revealed cooperation between the PEX and HP1 for Hsp70B mRNA induction and promoter activation. HSF1 enhanced the action of MMP3 activating the Hsp70B promoter in reporter assay but dominant negative HSF1 reversed it, suggesting crucial interaction between these proteins. Overexpressed PEX domain co‐localized with HP1 and remarkably accumulated into nucler stress bodies upon heat stress observed in immunocytochemistry. Furthermore, HP1 remarkably reduced clonogenicity of HeLa cells, while knock down of MMP‐3 repressed proliferation in HCS‐2/8 cells. Together with these results, it was clarified that MMP3 and HP1 transcriptinally induce HSP70 and are involed in stress response and tumorigenesis.
Sirtuins mediate cohesion of silenced domains in Saccharomyces cerevisiae
Authors: Chen Yu‐Fan , Gartenberg Marc
Published: June 2021

Assembly of yeast heterochromatin requires Sir2, the founding member of an evolutionarily conserved family of NAD+‐dependent protein deacetylases known as the sirtuins. These enzymes play key roles as metabolic sensors involved in cellular lifespan, and they have been linked to cancer and numerous human diseases. Recently we uncovered an unanticipated function of yeast Sir2 within heterochromatin: the protein is both necessary and sufficient for cohesion of heterochromatic domains. Tethered Sir2 fragments mediate cohesion in the absence of other heterochromatic Sir proteins whereas synthetic heterochromatin domains that lack Sir2 do not mediate cohesion at all. We have mapped the domain of Sir2 that is responsible for cohesin enrichment at heterochromatic regions and we have identified point mutations in this domain that abolish heterochromatic cohesion. Here we show that these mutations bear little impact on heterochromatic repression in most standard phenotypic assays. However, we find that de novo establishment of silencing is delayed. These data suggest that cohesin regulates the assembly or functionalization of yeast heterochromatin. This research was supported by a grant from the NIH (GM51402).
A Biologist's Resource for Protein Expression Plasmids: The Protein Structure Initiative:Biology‐Materials Repository
Authors: Cormier Catherine , Park Jin , Fiacco Michael , Steel Jason , Hunter Preston , LaBaer Joshua
Published: June 2021

The Protein Structure Initiative: Biology‐Materials Repository (MR; http://psimr.asu.edu) sequence‐verifies, annotates, stores, and distributes plasmids created by the Protein Structure Initiative (PSI). As of November 2012, over 60,000 protein expression plasmids and 97 empty vectors from the PSI are available through the distribution website DNASU (http://dnasu.asu.edu). In addition to the PSI collection, DNASU archives over 162,000 plasmids created by our laboratory, by various consortia, and by individual laboratories for distribution worldwide. Each plasmid is fully sequence‐verified and the insert sequence, along with additional annotations such vector information and associated publications, are available through our new and improved clone and vector detail pages. Each plasmid also links to external resources, including the PSI Structural Biology Knowledgebase (http://sbkb.org), facilitating cross‐referencing of plasmids to additional protein annotations and experimental data. Recent developments to DNASU include automation of our Expedited Material Transfer Agreement (MTA) process, which eliminates delays caused by MTA processing. With the added biological focus in PSI:Biology, the distribution of these materials will help researchers expand the knowledge of the role of proteins both in normal biological processes and in disease.
Synthetic Biology and the Production from Brazilian Spider Silk Protein Masp2 in E.coli System
Authors: Michalczechen‐Lacerda Valquiria A. , Vianna Giovanni R. , Murad Andre M. , Rech Elibio , Kaplan David L.
Published: June 2021

Spider silk fibers have high strength, toughness and extensibility. This biomaterial has numerous potential applications in medicine and broader materials‐related needs. With synthetic biology it is possible to construct and express genes encoding recombinant spider silk proteins. These proteins are characterized by their highly repetitive specific structural motifs. However, production of spider silk in Escherichia coli can be difficult because the DNA can be unstable resulting in deletions, and the repetitive sequences can lead to transcription and translation errors. Here we show spidroin protein silk (≥50 KDa) production of the major ampullate silk, Masp2, from the Brazilian spider (Parawixia bistriata) in E. coli. A silk monomer gene was designed and a 16mer plasmid constructed using a “head to tail” cloning strategy. BL21(DE03) with or without a plasmid with copies of glycyl‐tRNA and glycyl‐tRNA synthetase were induced with IPTG. Samples was analysed by SDS‐PAGE. Both bacteria produce the protein. In other studies similar protein sizes have been produced from N. clavipes, with the size directly associated with improved fiber properties. The amino acid sequence of Masp2 from P. bistriata shows differences in composition. Different spider silks may result in different types of materials, which will provide new types of protein‐based biomaterials.

Support: University of Brasilia, CNPq, CAPES, Embrapa Genetics Resources and Biotechnology, the NIH P41 Resource Center (EB002520), Tufts University.
Widespread regulation of translation by elongation pausing in heat shock
Authors: Shalgi Reut , Lindquist Susan , Burge Christopher B
Published: June 2021

Global repression of protein synthesis is a hallmark of the cellular stress response and has been attributed primarily to inhibition of translation initiation. Heat shocked cells globally inhibit protein synthesis, however the extent of translation initiation inhibition cannot explain the degree of translational repression observed.

Exploring translational control during heat stress in mammalian cells using ribosome footprinting, we discovered that severe heat shock triggers elongation pausing of ribosomes after translating ~70 codons on the vast majority of mRNAs. The genome‐wide nature of the phenomenon, its location and the features of protein N‐termini suggested the involvement of ribosome‐associated chaperones.

Reduced association of Hsp70 chaperones with ribosomes was seen following severe heat shock. Pre‐treatment with mild heat stress or overexpression of Hsp70 protected cells from the elongation pause induced by severe heat shock, while inhibition of Hsp70 activity triggered elongation pausing without heat stress. Finally, rewiring of the Hsp70‐translational machinery interactome during heat stress hinted at a mechanism that involves interactions with nascent peptides and the exit tunnel. Our findings suggest that regulation of translation elongation in general, and by chaperones in particular, represents a major component of the cellular stress response.
Comprehensive Mechanistic Analysis of the RNA‐Lariat Debranching Enzyme
Authors: Ransey Elizabeth Marie , Dey Sourav , Das Subha , Macbeth Mark
Published: June 2021

RNA‐lariat Debranching Enzyme (Dbr1p) hydrolyzes the 2′‐5′ phosphodiester bond between the intronic, adenosine branch point and the 5′ end of the intron. Intron lariat debranching is essential for processing of snoRNAs and miRNAs and for efficient intron turnover. Interestingly, Dbr1p null mutants have been shown to be defective in Ty1 retrotransposition in Saccharomyces cerevisiae. Additionally, siRNA knockdown of DBR1 in a eukaryotic cell line has inhibited HIV‐1 replication, making characterization and structure determination imperative to the rational design of a wide range of inhibitors.

We present structural, mechanistic and comparative analyses of two Dbr1 proteins. While S. cerevisiae Dbr1p has been characterized biochemically, complete mechanistic understanding has been limited by the unavailability of physiologically relevant substrates, as well as the lack of a high‐resolution Dbr1p structure. Our comprehensive analysis has utilized in vitro cleavage assays with a variety of branched substrates, site‐directed mutagenesis, inductively coupled plasma atomic emission spectroscopy (ICP‐AES) and fluorescence anisotropy to characterize Dbr1p kinetics, and to identify a unique versatility in substrate recognition and a hetero‐dinuclear active site.

Funding is provided by the CMU Center for Nucleic Acids Science and Technology, NSF GRFP #0750271 and the UNCF/Merck Graduate Fellowship.
AMPK and mTOR in nutrient signaling and autophagy regulation
Authors: Kim Joungmok , Russell Ryan , Yuan Haixin , Guan Kun‐Liang
Published: June 2021

The AMP activated kinase (AMPK) is a key cellular energy sensor. The mammalian target of rapamycin (mTOR) is a central cell growth controller. mTORC1 is regulated by a wide range of cellular signals, including energy, and nutrients. High mTORC1 promotes cell growth while inhibition of mTORC1, such as under nutrient starvation, induces autophagy, which is a self‐degradative process of cellular components in order to maintain essential cellular activity and viability. Extensive genetic and biochemical evidence has shown that the yeast Atg1 kinase plays an essential role in autophagy induction. Mammals contain two Atg1 homologs known as ULK1 and ULK2, which have been implicated in autophagy regulation. We have investigated the role of AMPK (AMP activated protein kinase, a cellular energy sensor) and mTORC1 in ULK1 regulation. We observed that ULK1 activity and function is directly regulated by AMPK and mTORC1. VPS34 is a lipid kinase critically important for intracellular vesicle trafficking and autophagosome formation. VPS34 regulation by nutrient starvation has also been investigated. Our study not only established a molecular basis of AMPK and mTORC1 in autophagy regulation but also revealed a biochemical mechanism for ULK1 activation in response to nutrient starvation.
Turnabout is Fair Play: Mutual Regulation of Palmitoylation and Raft Partitioning
Authors: Levental Ilya , Simons Kai
Published: June 2021

The physical basis of protein partitioning into lipid rafts remains an outstanding question in membrane biology that has previously been addressed only through indirect techniques involving differential solubilization by non‐ionic detergents. We have used Giant Plasma Membrane Vesicles (GPMVs), a plasma membrane model system that phase separates to include an ordered phase enriching for raft constituents, to analyze the structural determinants for raft partitioning of the transmembrane protein Linker for Activation of T‐cells (LAT). LAT enrichment in the raft phase was dependent on palmitoylation at two juxtamembrane cysteines. This palmitoylation requirement for raft association was also shown for the majority of integral raft proteins. Independently, systematic mutations that disrupted raft partitioning showed a causal relationship between raft association and plasma membrane (PM) localization, with non‐raft mutants mis‐sorted to late endosomes and depalmitoylated by the lysosomal protein thioesterase PPT1. These findings implicate lipid‐driven phase separation as one mechanism for protein sorting at the late stage of the secretory pathway and suggest mutual regulation of palmitoylation and raft partitioning of LAT.
Role of HSF1 and FoxO3 in proteotoxic stress induced Macroautophagy
Authors: Murshid Ayesha , Chou shiuh dih , Eguchi Takanori , Calderwood Stuart
Published: June 2021

Autophagy or Macroautophagy and the ubiquitin‐proteasome pathways are the two major catabolic pathways in all cells. Autophagy is a cellular process in which a portion of the cytoplasm becomes enclosed in a membrane‐bound vesicle, the autophagosome, which fuses with lysosomes leading to digestion of the contents of this autolysosomes. The exact mechanism is still not clear. Autophagy appears to switch on when translation is switched off and the processes are reciprocally regulated by TORC1 kinase. In addition to induction of autophagy by nutrient starvation, this process is also triggered by various stresses. Thus, this process of autophagy is regulated by two different mechanisms: nontranscriptional inhibition by mTOR and transcription‐dependent upregulation through FoxO3. Nevertheless, transcriptional mechanisms that physiologically regulate expression of autophagy genes in tissues other than myotubes have not been characterized. We are concentrated here on autophagy stimulated by proteotoxic stresses such as heat shock. We found that FoxO3 transcription factor which is necessary and sufficient for induction of autophagy in skeletal muscle is regulated by the heat shock protein transcription factor, HSF1. Our data show that HSF1 negatively regulates FoxO3 activity in HeLa cells. FoxO3 is shown to be positively regulating autophagosome formation determined by LC3I/II expression. In this study we hypothesize that stress enhances autophagy by activating FoxO3 and MAP‐LC3I expression whereas stress induced HSF1 activation causes increase in protein folding pathway.
Effect of a six week exercise training program on proteolytic enzyme activity, ROS generation and autophagy in the white gastrocnemius and left ventricle of normotensive and hypertensive rats
Authors: McMillan Elliott M , Paré Marie‐France , Quadrilatero Joe
Published: June 2021

Hypertension is a cardiovascular disease associated with increased cell death and proteolytic activity in both skeletal and cardiac muscle. Autophagy is a degradative system that plays an essential role in muscle health and can influence cell death processes. Little is known about the effects of exercise training on autophagy in healthy or diseased states. The purpose of this study was to investigate the effects of exercise training on markers of autophagy, proteolytic activity, and ROS levels in white gastrocnemius (WG) and left ventricle (LV) of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Male WKY and SHR were assigned to a sedentary condition or exercise consisting of treadmill running for 6 wks. In the WG muscle of SHR, greater LC3I protein (p<0.01) and a lower LC3II/I protein ratio (p<0.05) was observed compared to WKY rats. Exercise training did not alter the WG LC3II/I protein ratio; however, exercise training upregulated Beclin (p<0.02), LC3 (p<0.05), and p62 (p<0.04) mRNA and reduced ATG7 (p<0.04) and Beclin (p<0.01) protein levels. Following exercise training, lower caspase‐3 (p<0.001) and cathepsin (p<0.001) activity as well as a trend towards lower ROS production (p<0.08) was observed in the WG. In the LV, SHR had a lower LC3II/I protein ratio (p<0.05) and a greater accumulation of p62 protein (p<0.01) compared to WKY rats. Training increased both Beclin and p62 mRNA levels (p<0.05) in the LV; however, no change in the LC3II/I protein ratio was observed. Exercise training lowered LV caspase‐3 (p<0.001) and calpain (p<0.001) activity, as well as tended to reduce cathepsin activity and ROS production (p<0.07). Together, these results suggest that autophagy is altered in the SHR muscle and heart, and exercise training can alter upstream regulators of autophagy, attenuate ROS production and proteolytic enzyme activity in WG and LV of normotensive and hypertensive rats.
A protocol for screening of autophagy regulatory genes applying cell sorting and next generation sequencing technologies
Authors: Huang Wei‐Pang , Liu Ang‐Yu
Published: June 2021

Autophagy, as a conserved pathway in eukaryotic cells, plays a critical role in maintaining cellular homeostasis by delivering cytoplasmic materials to lysosomes/vacuoles for degradation. Abnormal autophagy activities are associated with several human diseases, such as diabetes, neurodegenerative diseases, and cancers. These observation leads to the possibilities of manipulating autophagy for therapeutic applications. To achieve this purpose, detailed information on the molecular mechanisms of autophagy regulation is essential. In this study, we have developed a protocol to screen for more selective autophagy regulatory genes based on cell image analysis, which is compliable to cell sorter application. We constructed a plasmid for expressing peroxisome‐targeted pH‐sensitive green fluorescent proteins in Saccharomyces cerevisiae. The plasmid was introduced into yeast deletion mutant pools and pexophagy‐deficient cells with high fluorescent signals after induction of pexophagy was sorted out. The identities of the mutated genes of those selected cells were determined by next generation sequencing of the gene barcodes. Our results proved it is able to distinguish atg mutants from wild types cells. We are currently characterizing the isolated autophagy mutants from the nonessential yeast gene deletion mutant pool. Grant support: NSC 101–2311‐B‐002–005
Perillyl alcohol and rapamycin‐mediated inhibition of migration and invasion
Authors: Sundin Tabetha , Mollica Peter , Peffley Dennis , Hentosh Patricia
Published: June 2021

The plant‐derived isoprenoid perillyl alcohol (POH) exerts anti‐cancer properties via effects on mTOR (mechanistic target of rapamycin) signaling in a similar but distinct process as does rapamycin, a well‐studied mTOR inhibitor. Previously, rapamycin was shown to inhibit both matrix metalloproteinases (MMPs)‐2 and ‐9 in a human umbilical vein endothelial cell line. MMPs are key indicators of both angiogenesis induction and tumor cell metastatic activity. We evaluated clinically relevant concentrations of POH and rapamycin for their respective abilities to limit cellular migration and invasion of a highly metastatic human prostate cancer line–DU145. In a wound healing assay, POH or rapamycin significantly prevented DU145 cells from migrating into the wound area during a 24 hr period. Further, both agents inhibited DU145 movement through an ECM layer in a cell invasion assay. Gelatin zymography revealed that POH and rapamycin reduced the activity of secreted MMP‐2 and ‐9 in a time‐dependent manner. Additionally, decreased intracellular MMP‐2 activity was observed after treatment with either agent. Pre‐treatment with the proteasome inhibitor MG‐132 rescued MMP‐2 activity indicating that POH and rapamycin provoke MMP‐2 degradation. Our results establish comparable anti‐invasive abilities of POH–which displays fewer side effects–and rapamycin that are associated with MMP‐2 and ‐9 down‐regulation.

Funding provided by a Golden Key Research Grant.
RpoS is directly controlled by ATP levels
Authors: Peterson Celeste
Published: June 2021

Many cells enter a state of dormancy when deprived of nutrients. For bacteria, this makes them more robust to environmental stresses. In Escherichia coli, reshaping the repertoire of proteins for stress resistance is mostly done by changing gene expression. The master regulator of this response, the sigma factor RpoS, regulates transcription of >;300 genes upon starvation. However, RpoS itself is controlled at the level of protein degradation: under exponential growth it is the most short‐lived protein in E. coli but upon carbon starvation degradation quickly ceases. The molecular steps leading from carbon starvation to a change in RpoS protein stability are not well understood. We systematically block key steps of glycolysis and the citric acid cycle and monitor the effect on RpoS degradation in vivo. Nutrient upshifts trigger RpoS degradation independently of protein synthesis by activating metabolic pathways that generate small energy molecules. Using metabolic mutants and inhibitors, we show that ATP but not GTP or NADH is necessary for RpoS degradation. In vitro reconstitution assays directly demonstrate that ClpXP fails to degrade RpoS but not other proteins at low ATP hydrolysis rates. These data suggest that cellular ATP levels directly control RpoS stability. Connecting ATP levels directly to degradation could provide a reliable and ultrafast signal for starvation.
Probing molecular mechanism of destabilization and misfolding of human apolipoprotein A‐I in familial amyloidosis
Authors: Das Madhurima , Mei Xiaohu , Atkinson David , Gursky Olga
Published: June 2021

Plasma high‐density lipoproteins (HDL) are nanoassemblies of lipids and specific proteins, mainly apolipoprotein A‐I (apoA‐I, 243 a. a.). HDL and apoA‐I remove excess cell cholesterol and protect against atherosclerosis. In familial amyloidosis, mutant apoA‐I is proteolysed and the N‐terminal ~10 kDa fragments deposit as fibrils in vital organs, causing organ damage. There is no treatment for this serious disease and its mechanism is unknown. Recently, we mapped all known human amyloidogenic mutations on the high‐resolution crystal structure of the C‐terminally truncated apoA‐I and proposed the first molecular mechanism of apoA‐I misfolding in amyloidosis. To probe this mechanism, we use recombinant amyloidogenic mutants (e.g. G26R apoA‐I) together with the non‐amyloidogenic variants (apoA‐I Fin fragment 1–158). Protein secondary and tertiary structure and stability in solution and in recombinant HDL are determined by spectroscopy. The results are correlated with the protein propensity to form fibrils. These studies will test our central hypothesis: destabilization of HDL and apoA‐I by mutations that lead to increased solvent exposure of the N‐terminal segment L44‐S55 cause apoA‐I misfolding and amyloid fibril formation, whereas partial protection of this segment (e.g. in apoA‐I Fin) favors catabolism of the destabilized protein. This work was supported in part by NIH grants HL026355 and GM067260.
Rational enzyme redesign for enhancing activity and selectivity of heterologous taxane oxidation in engineered E. coli
Authors: Yadav Vikramaditya Ganapati , Edgar Steven McBride , Stephanopoulos Gregory
Published: June 2021

Plant secondary metabolic enzymes characteristically exhibit substrate promiscuity and poor product selectivity. This compounds the flux dissipation from heterologous pathways comprising them. Their active sites must be redesigned in order to enhance pathway flux. However, as plant secondary metabolites are assembled through permutations and combinations of only a handful of enzymatic reactions, the use of structure‐driven approaches is desirable. The structure‐activity insights gained from redesigning one active site could potentially be extended to reengineering homologous enzymes that act downstream. Ergo, we hypothesized and computationally evaluated the mechanism via which taxadiene‐5α‐hydroxylase catalyzes proto‐oxidation of taxadiene – a gateway precursor to several hundred taxanes, one of which is taxol, a blockbuster anti‐cancer drug – at multiple positions. We then used these conclusions to guide the construction of enzyme mutants with improved activities and selectivities. Some mutants exhibited activity improvements as high as 132%. The highest selectivity improvement achieved was 64%. Synchronous activity and selectivity improvements of 117% and 32%, respectively, were also achieved.