bluuebunny/crossref_metadata_2025 huggingface dataset:
A Survey of Student Use of a Novel Software Tool for Teaching Neuroanatomy, Draw It to Know It
Authors: Barger Bradley , Fisch Adam
Published: June 2021

Draw It to Know It is a unique software tool designed to aid student learning of neuroanatomy through guided drawings of neurological concepts. The current study is the first analysis of this commercially‐available, software learning tool. In this study, we provided the software to 1497 students from 37 different institutions in schools of rehabilitation therapy (physical, occupational, speech), medicine, osteopathy, and chiropractic medicine. Students were offered the Draw It to Know It software, but were not mandated or incentivized to use it. We then collected voluntary survey responses from the students who had used the software. We also collected survey responses from instructors in whose classes the software was provided to the students. From the survey responses we found students and instructors both had very positive interpretations of the software (Student mean on five‐point Likert scale=4.07, Instructor mean on five‐point Likert scale=4.43). Students strongly agreed that the software was a valuable learning tool, and many reported a desire to see more guided drawings in other basic science courses (Student mean on five‐point Likert scale=4.08). Among students who self‐reported as visual learners, the use of drawings had a positive response, but even among students who did not self‐identify as visual learners, the software was positively received.
Different student perceptions of the effectiveness of Team Based Learning exercises between undergraduate health sciences and medical students
Authors: Ball Alexander , Hawke Thomas , Helli Peter , Wainman Bruce
Published: June 2021

In the fall of 2013 we substituted a didactic lecture for a Team Based Learning exercise (TBL) in an undergraduate Anatomy and Physiology course with an enrollment of 285. We also substituted a didactic lecture for an on‐line video, and ran a TBL exercise during the previously scheduled lecture slot for the neuroanatomy block of an MD class with an enrollment of 160. The exercise consisted of 1) an individual readiness assessment (five MCQs) test (iRAT), 2) review of the iRAT questions, 3) separation of the students into teams to discuss a case study, and 4) a team effort in answering a multiple choice question based on the case study (gRAT). The iRAT and gRAT scores were recorded using an iClicker (undergraduate) or Scantron card (MD). The results were similar for the undergraduate class over two years. 80% of undergraduate students felt that the iRAT helped keep them on task, 54% felt that the iRAT prepared them for the MCQ Midterm exam, and 42% felt that the TBL allowed them to discuss concepts that they would not otherwise have considered. However, more than 53% of students ranked the TBL as the least liked activity in either this course or any other course they have taken. The paradoxical response was attributed by undergraduate students to the stress and the time required to prepare for the exercise (54%). In contrast, the MD students ranked the TBL exercise (67%) higher than the iRAT (63%) and just as valuable as dissection (67.3%) for their learning. We conclude that factors determining how students rank the TBL learning activity include the size and makeup of the TBL groups, whether the activity is part of a summative evaluation, and the experience of the student learner.
Endothelial and Cancer Cells Have Differing Amounts of TGF Beta Receptors Involved in Angiogenesis
Authors: Duffy Iain , Varacallo Patricia , Klerk Harrison , Hawker James
Published: June 2021

Control of angiogenesis is critical in diseases such as cancer, heart disease, or wound repair. Growth factors such as transforming growth factor beta (TGFß) contribute to angiogenesis and cancer. Multiple TGFß family receptors play a role in tumor function and angiogenesis. We show data that the type I receptor Activin‐Like Kinase 1 and the co‐receptor endoglin can signal in endothelial cells via the TGFß ligand. Others have shown that other ligands such as Bone morphogenetic protein 9 (BMP‐9) and Activin A can also signal through ALK‐1. Further, ALK‐1 and endoglin may also be involved in cell‐cell interactions between endothelial and other cells. We screened endothelial cells and also several cancer cell lines for expression of various TGFß family receptors to begin to understand which receptors may be involved in angiogenesis and interaction of tumor cells with endothelial cells. Endothelial cells show similar expression of ALK‐1, ALK‐5, and endoglin and lower expression of BMP receptor II and TGFßRII. Cancer cells show much lower expression of type II receptors. We are exploring expression and function of TGFß family receptors in angiogenesis and interactions with cancer cells
Study of Relationship between Blood Pressure and Visual Acuity among Nigerians.
Authors: Taura Magaji , Adamu Lawan , Modibbo Musa
Published: June 2021

The study was aimed to determine prevalence of different types of visual acuity (VA) and blood pressure (BP) among Hausa ethnic population of northwestern Nigeria. The association of sex with VA and BP were investigated. The relationship between VA and BP was also evaluated. A total of 195 subjects (120 males and 75 females) with mean age of 20.14 ± 1.86 (18‐25 years) participated. BP and VA were determined using standard protocols. Fisher's exact (Fe) test was used to investigate the relationship using SPSS version 20. P < 0.05 was set as level of significance and +/‐ 1.96 as a critical value for the post hoc. The result shows that normal (6, 6) visual acuity had higher prevalence in both right (81%) and left (81.5%) and other forms were the least (19% right and 18.5% left). For BP 42.1% and 43.1%, 45.1% and 39.5%, 11.3% and 0%, 1.5% and 17.4% were optimal, normal, high normal and hypertensive for both systolic and diastolic respectively. Gender was found to be associated with systolic BP (Fe value= 34.47, P< 0.001), diastolic BP (Fe value= 16.54, P< 0.001), right VA (Fe value= 15.21, P< 0.001) and left VA (Fe value= 13.10, P= 0.001). A statistically significant relationship was observed between systolic BP and right VA (Fe value = 33.91, P< 0.001)

Variables
Systolic blood pressure
Total

Optimal
Normal
High normal
hypertensive

Right visual acuity
Sub normal I (5,6)
Count
26
3
1
0
30

Expected Count
12.6
13.5
3.4
0.5
30.0

Residual
13.4
‐10.5
‐2.4
‐0.5

Std. Residual
3.8
‐2.9
‐1.3
‐0.7

Normal (6,6)
Count
53
83
20
2
158

Expected Count
66.4
71.3
17.8
2.4
158.0

Residual
‐13.4
11.7
2.2
‐0.4

Std. Residual
‐1.6
1.4
0.5
‐0.3

Sub normal II (others)
Count
3
2
1
1
7

Expected Count
2.9
3.2
0.8
0.1
7.0

Residual
0.1
‐1.2
0.2
0.9

Std. Residual
0.0
‐0.7
0.2
2.7

Fisher's Exact Value = 33.91, P< 0.001

and left VA (Fe value = 32.44, P< 0.001). Similar relationship was noticed with diastolic BP

Variables
Diastolic blood pressure

Optimal
Normal
Hypertensive

Right visual acuity
Sub normal I (5,6)
Count
26
3
1

Expected Count
12.9
11.8
5.2

Residual
13.1
‐8.8
‐4.2

Std. Residual
3.6
‐2.6
‐1.8

Normal (6,6)
Count
57
70
31

Expected Count
68.1
62.4
27.5

Residual
‐11.1
7.6
3.5

Std. Residual
‐1.3
1.0
0.7

Sub normal II (others)
Count
1
4
2

Expected Count
3.0
2.8
1.2

Residual
‐2.0
1.2
0.8

Std. Residual
‐1.2
0.7
0.7

Fisher's Exact Value = 28.71, P=< 0.001

In conclusion, gender had an association with VA and BP. Moreover, VA could be influenced by BP
Intervertebral Canals of Cervical Spine
Authors: Zhandarov Kirill
Published: June 2021

Relevance

In our abstract we give characteristics and make a research of intervertebral canals of cervical spine.

Objective

To give anatomical characteristics of intervertebral canal and to make a research of cervical spine canals.

Material and Methods

37 anatomical preparations were studied, which included 185 intervertebral canals aged 58 ‐ 78 years old (mean age was 52 years old). The complex of the preparations was an intact block of the skull base and cervical spine to the C6‐level. Histological research of connective canal structures was provided.

Lateral and medial apertures particular forms, borders, sizes and diameters were studied, anatomical structures of canal walls were described. Ligaments were found and researched in most of canal areas.

Results

The true cervical intervertebral canals are 5 pairs in segments C2 ‐ C7. Intervertebral canals were measured: medium‐statistical parameters are as follow ‐ length is min 1.46 max 2.0 mm, external aperture is min 0.8 – max 1.4, internal aperture is min 0.4 – max 0.8. Histological research of found structures we discovered gives the right to call their ligaments, the ligaments sizes are as follow: diameter is from 0,1 ‐ to 0,3; length is from 0,4 ‐ to 0,6. Canals studied can be divided into three groups: with clearly identified true ligament (46%); the ligament with matted together false ligaments (fibrotic bands), which compress spinal nerve (43 %); canals without ligament but with the presence of bone and fibrous growths (ossifications), which compress spinal nerve (11 %).

Conclusion

The characteristic of “intervertebral canals of cervical spine” anatomical structures was given. Discovered ligaments introforaminalis can be an additional factor of spinal roots compression.
Renaturation of RibonucleaseA (RNaseA): Walking in the Footsteps of a Nobel Prize Winner
Authors: Carter Timothy
Published: June 2021

The importance of 3‐dimensional structure in the biologic activity of macromolecules can hardly be overstated. Understanding the principles that underlie this phenomenon is accordingly recognized as one of the core objectives of the ASBMB‐developed educational goals for basic competency in the field. In reading the literature on protein folding I was struck by the overall methodological simplicity and intellectual elegance of the groundbreaking work of Christian Anfinsen and his colleagues during the 1950s and 1960s in studying the factors that allowed accurate refolding of denatured RNaseA (e.g., Anfinsen et al., Proc. Nat. Acad. Sci. U. S. 47, 1309 1961 & Epstein et al., Arch. Biochem. Biophys. Suppl. 1 223‐231, 1962). Not the least of these advantages include the remarkable hardiness of this enzyme and the use of catalytic activity as a surrogate for elaborate physical and chemical methods to confirmation correct folding. These considerations suggested that it would be possible to repeat and extend Anfinsen's classic experiments in the teaching laboratory. Over the past six years I developed a series of lab exercises designed to provide beginning biology majors the requisite technical and intellectual background to repeat the essential aspects of the published studies, and then to use this experience to ask their own questions about physical and chemical factors that might influence the rate and extent of protein folding. The series of 7 lab exercises (with alternatives to shorten or extend the total number of labs) are suitable for well‐prepared and highly motivated college freshmen, but could also be used effectively in upper division and even graduate biochemistry courses. The basic units will be described, along with samples of student data, and copies of the lab manual will be made available.
Inquiry‐Based Cell Culture Laboratory Course for Undergraduates
Authors: Carastro L Michael
Published: June 2021

This laboratory course enables development of meaningful understandings of Biochemistry & Molecular Biology (BMB) principles through a series of cell culture‐based experiments. The latest iteration of this course, Tissue Culture (CHE470), includes exercises and lectures on BMB aspects of cell culture, including: cell growth kinetics, transfection, inducible gene expression and biochemical assays. The unified series of experiments consists of (1) electroporation, (2) clonal selection, (3) induction of gene expression, (4) genomic DNA isolation and PCR screening, and (5) RNA isolation and Real Time‐PCR, and (6) protein isolation and enzyme assays. Course grades are based upon a report on the semester‐long series of experiments in the form of a BMB peer‐review journal style manuscript, three in‐class exams, an oral presentation, and some laboratory technique exercises. Because the integration of the circular tetracyclin‐inducible expression vector (pcDNA4/TO/mycHIS‐LacZ) is random, students' experimental data are quite varied. This provides an opportunity for inquiry‐based learning through the formulation of hypotheses to explain their observations and data. Student outcomes were measured by anonymous feedback through an online survey system using a numeric rating scale (5 = strongly agree; 1 = strongly disagree). The Fall 2013 students cohort (n = 6) reported they “know more about this subject” (mean = 5.00, dev. = 0.00), that their “skills in this area have improved” (mean = 5.00, dev. = 0.00), their “appreciation of this subject increased” (mean = 5.00, dev. = 0.00), and that “learning objectives of the course were met” (mean = 4.86, dev. = 0.38). Survey data are consistent with students finding this course engaging, informative and beneficial.
Protein Structure‐Function Relationships of T4 Lysozyme in an Advanced Biochemistry Laboratory
Authors: Watt Terry , Toro Tasha
Published: June 2021

The relationship between protein structure and function is a fundamental concept in biochemistry, but one that students do not often have a chance to directly test. To illustrate this relationship in an undergraduate laboratory course requires a protein that has been extensively characterized from both a structural and functional standpoint. We have developed a short experimental series around T4 lysozyme that allows students to purify and characterize the activity of T4 lysozyme variants. By combining the class results with available crystal structures and previously reported activity data, students are able to draw conclusions regarding the specific effects particular mutations have on the protein's structure and function. The experimental series can optionally be extended to incorporate mutagenesis of the T4 lysozyme, protein expression, and/or additional structural modeling techniques. We present results showing robust expression, purification, and activity of several T4 lysozyme variants amenable to a variety of biochemistry laboratory courses. The experiment is relatively inexpensive, scalable for larger class sizes or multiple sections, and modular so that the desired aspects can be selected and emphasized.
Eureka! Energizing the Next Generation of Clinical Researchers
Authors: Sawarynski Kara , Baxa Dwayne , Dereski Mary
Published: June 2021

OUWB medical students are required to develop and implement an independent Capstone research project. Students are guided in the project through concurrent research design and methods courses, and by faculty mentors. Quantitative, qualitative and systematic reviews projects fall under areas such as community/public health, patient/medical education, and bench/clinical research. Although medical student research is not novel, the curricular inclusion of required projects with concurrent associated courses is a medical education practice that is quickly gaining momentum. Our M1 year courses are designed to educate students on many aspects of research not typically included in a medical curriculum (from research question and outcomes identification, project design and feasibility, navigating the literature, ethics and regulatory board approval, to planning for data security and appropriate analysis). We use multiple instructional techniques including online modules, labs and workshops, didactic sessions, and self‐directed learning which addresses increasing accreditation requirements. In an effort to provide the best foundation for undergraduates entering into the changing medical education landscape, we would like to encourage research mentors to incorporate more student participation in the entire research process. We believe that students that spend four years of their medical education invested in high impact activities will likely continue on a path of life‐long learning. We anticipate that each of our graduates will have an exceptional impact on the patients and communities that they serve and will act as multipliers to continue the growth of research developments and socially accountable activities within their future peer groups.
Molecular Dynamics Study of the Opening and Closing Mechanisms for DNA Polymerase I
Authors: Miller Bill , Parish Carol , Wu Eugene
Published: June 2021

The quick and accurate replication of genetic information (DNA) is critical to all life. DNA polymerase, a key enzyme for DNA replication in organisms, catalyzes the reaction that extends the primer strand of DNA during replication. Before catalysis, DNA polymerase has the daunting task of matching a template nucleic acid base with its corresponding Watson‐Crick base (dNTP) that must be added to the primer strand. The enzyme must have high specificity in order to distinguish one correct base for incorporation from three incorrect bases. Crystal structures of DNA polymerase have shown the enzyme in an “open” conformation in the absence of a dNTP in the active site and “closed” when a proper Watson‐Crick base pair is matched. Although crystal structures have given insight into these conformations of DNA polymerase in these three states, traditional biochemistry experimental techniques are unable to capture the details of the transition between these states and how the polymerase traverses the potential energy surface from open to closed and back again. In this study, we have utilized recent computational advances to simulate the opening and closing of the fingers domain starting from the of Bacillus stearothermophilus DNA polymerase Klenow fragment using dynamics on the microsecond time scale. We have fully characterized both processes, and determined the key events and movements critical to these large‐scale conformational changes. These simulations aid in the elucidation of the structural changes of DNA polymerase on an atomistic level not currently available with experimental measures.
Characterization of S. cerevisiae Phenotypes Associated with Cancer‐Causing Mutations in Hsh155p
Authors: Zoerner Douglas , Luo George , Carrocci Tucker , Tretbar U Sandy , Hoskins Aaron
Published: June 2021

The eukaryotic spliceosome is composed of five small nuclear ribonucleoproteins (snRNPs) and many other associated protein splicing factors. The human U2 snRNP‐associated protein, SF3B1, allows the U2 snRNP to associate with and define the branchpoint region of precursor‐messenger RNA (pre‐mRNA). Recent evidence has shown that mutations to SF3B1 are linked to a variety of different myelodysplastic syndromes (MDS). These mutations are found in regions of high conservation between SF3B1 and the yeast homolog, Hsh155p. We have constructed a genetic system in S. cerevisiae to interrogate the effects of these mutations on splicing and cell proliferation. We used plasmid shuffling to introduce Hsh155p mutant proteins into a HSH155Δ yeast strain and growth assays to assess the impact of the mutations on cell proliferation. Several of the mutations surveyed thus far (H331D, K335N, P369E, D450G) have no obvious effect on yeast growth despite being correlated with MDS in humans. This may imply that the effects of these mutations are subtle and do not allow splicing to become limiting for yeast growth. To further understand the effects of these mutations, a Reverse Transcription‐Polymerase Chain Reaction (RT‐PCR) analysis will be used to compare pre‐mRNA and mRNA levels of several transcripts from Hsh155p mutant strains relative to the WT. The results from these experiments will be critical for establishing yeast as a model system for studying MDS. This research has been supported by startup funds from the U. Wisconsin‐Madison, WARF, and the Department of Biochemistry.
An epigenetic switch of let‐7 targeted oncofetal genes
Authors: Jnbaptiste Courtney , Gurtan Allan , Lu Victoria , Bhutkar Arjun , Sharp Phillip
Published: June 2021

MicroRNAs (miRNAs) are post‐transcriptional regulators that fine‐tune gene expression. Despite the modest 2‐fold repression that miRNA activity generally confers on a target, miRNAs are critical for many biological processes.

By regulating core transcription factors and their downstream effectors, miRNAs modify transcriptional networks. However, description of the interactions between miRNAs and transcriptional networks has been limited, and the magnitude of these interactions has not been investigated.

We sought to investigate the transcriptional programs regulated by miRNAs in adult murine fibroblasts. We previously reported that Dicer deletion results in up‐regulation of let‐7 targets. For some targets, the magnitude of up‐regulation is 50‐100 fold, much greater than that typically reported for miRNA‐mRNA interactions, suggesting effects additional to the loss of post‐transcriptional repression by miRNAs. The activation of these let‐7 targets upon Dicer loss correlated with H3K4me3 and H3K36me3 chromatin marks, indicating enhanced transcription. Further studies indicate that despite complete restoration of the expression and post‐transcriptional activity of miRNAs, the transcriptionally activated let‐7 targets fail to revert to their original wildtype expression levels. Furthermore, these genes maintain a chromatin signature consistent with enhanced transcription. Thus in fibroblasts, global miRNA loss leads to epigenetic changes of an oncofetal gene network, that continue in the absence of the initiating event. Ongoing studies involve experimental and computational strategies to identify mediators of the observed transcriptional effect.

This work was funded by USA Public Health Service grant RO1‐CA133404 from the NIH to Dr. Phillip Sharp.
Characterizing Polyadenylated uaRNAs Suggests a Potential Role for Pabpn1
Authors: Chiu Anthony , Wu Xuebing , Subtelny Alexander , Sharp Phillip
Published: June 2021

Divergent transcription is observed at most active mammalian promoters, yet productive elongation is primarily limited to the mRNA in the downstream sense direction. The upstream antisense RNAs (uaRNAs) tend to be shorter and have lower steady state levels than the corresponding sense transcripts. We previously proposed that a U1‐PAS axis flanking gene promoters might regulate promoter directionality, in which early termination near the TSS of uaRNAs lowers their steady state whereas sense mRNAs tend to terminate later due to U1‐mediated suppression of promoter‐proximal polyadenylation signal (PAS) motifs.

Given that the polyadenylated uaRNA transcripts are unstable (half‐lives less than 30 minutes), we sought to understand what additional factors may be regulating the degradation of these PAS‐containing transcripts. Treatment with polyA inhibitor cordycepin suggested that the polyA tail may be promoting destabilization of some uaRNAs. Moreover, genome‐wide polyA‐tail length sequencing revealed that while mRNAs tend to peak at approximately 80 adenosine residues, uaRNAs terminating at PAS‐motifs appeared to be more widely dispersed, some of which have longer polyA tails than mRNAs. To identify what proteins may be involved, we knocked down various factors using shRNAs and found Pabpn1 knockdown stabilized several uaRNAs. We speculate that Pabpn1 may be playing an important role on destabilizing certain categories of noncoding RNAs and are now investigating that hypothesis genome‐wide.

This work was funded by USA Public Health Service grant PO1‐CA042063 and R01‐GM34277 from the NIH to Dr. Phillip Sharp.
Identification of mRNA Localization Motifs through Cell Fractionation and Alternative Splicing Analysis
Authors: Taliaferro J , Vidaki Marina , Gertler Frank , Burge Christopher
Published: June 2021

The biogenesis of mammalian mRNAs involves several regulated steps, including alternative splicing and alternative cleavage and polyadenylation. In addition, certain mRNAs can be localized to distinct regions of the cell, contributing to protein localization and complex formation. This is particularly important in polar cell types, such as neurons. Although several hundred mRNAs are present in neuronal projections, only a handful of RNA sequences are known to target an mRNA to a particular location. Using mechanical subcellular fractionation, RNA‐seq, and alternative splicing analysis, we have identified hundreds of mRNA regions associated with localization to neuronal projections in neuroblastoma cells and primary cortical neurons. These regions are overwhelmingly found in 3′ UTRs, and in particular are associated with alternative last exon splicing events and result from a characteristic alternative isoform organization. Localization‐associated 3′ UTRs are shorter, more conserved, more gene‐distal, and contain a higher propensity for secondary structure than other 3′ UTRs. Furthermore, they are sufficient to direct reporter RNA into neuronal projections and are enriched in binding sites for trans‐factors known to be involved in RNA localization. Knockdown of expression of these factors inhibits the localization of many transcripts. These data demonstrate the tight interplay between each step in gene expression as specific alternative splicing decisions in the nucleus determine the cytoplasmic localization fate of the transcript.
Role of Extracellular MicroRNAs in Atherosclerosis
Authors: Nguyen M , Karunakaran D , Geoffrion M , Rayner K
Published: June 2021

Atherosclerosis is a maladaptive inflammatory disease driven by the interplay between excess cholesterol accumulation in the arterial wall and the immune system. Macrophages are fundamental to the propagation of atherosclerosis due to their capacity to engulf modified lipoproteins and induce a pro‐inflammatory state. Macrophages can secrete miRNAs in exosomes as a form of intercellular communication during inflammation and infection. miRNAs are small noncoding RNAs that post‐transcriptionally regulate gene expression. Herein, we hypothesize that atherogenic macrophages secrete miRNAs in exosomes to mediate cell‐cell communication, which regulates the atherogenic response. To test this hypothesis, we first examined the miRNA expression profile of exosomes from control and atherogenic macrophages. Among the 88 differentially expressed miRNAs, 68 were down‐regulated whereas 20 were up‐regulated in exosomes from cholesterol‐loaded macrophages as compared with the control. qPCR confirmed the enrichment of several miRNAs such as miR‐146a, miR‐128, miR‐185, and miR‐503 in exosomes derived from atherogenic macrophages, whereas miR‐150 was downregulated in these exosomes. Bioinformatic pathway analysis suggests that atherogenic exosomal miRNAs may regulate cell migration and adhesion pathways via targeting the 3′UTR of migration/adhesion genes (i.e. integrin α3, VE‐cadherin, CXCR4). Interestingly, live cell imaging showed that exosomes secreted from cholesterol‐loaded macrophages were internalized by naïve macrophages and exogenous C. elegans miR‐39 could transfer from atherogenic macrophages to naïve cells. Thus, miRNAs secreted from atherogenic macrophages may promote atherosclerosis by regulating macrophage migration in the vessel wall and the role of these exo‐miRNAs will be tested in vitro and in vivo.
A Novel Isoform of CPEB2 Regulates the Metastasis of Triple Negative Breast Cancer
Authors: Johnson Ryan , Vu Ngoc , Chalfant Charles , Park Margaret
Published: June 2021

While breast cancer has an overall 5‐year survival rate of 90%, triple negative breast cancer (TNBC) represents an anomalous subset of the disease with a greatly reduced (30%) 5‐year survival rate. The enhanced mortality and morbidity associated with TNBC arises from the high metastatic rate, which requires the acquisition of anoikis resistance (AnR). As the dysregulation of mRNA splicing is becoming a recognized phenomenon in cancer and aberrant cell signaling, the alternative mRNA splicing events were interrogated in AnR TNBC cells using next generation sequencing. The mRNA splicing of cytoplasmic polyadenylation element binding 2 (CPEB2), a translational regulator, was identified as dysregulated in AnR TNBC. Specifically, the inclusion of exon 4 into the mature mRNA to produce high levels of the CEPB2B isoform correlated with AnR, which translated to human breast cancer tumors. Furthermore, the molecular manipulation of CPEB2 isoforms had a robust effect on AnR and the metastatic potential of TNBC. Finally, next generation sequencing indicated a subset of mRNA species related to both tumor suppression and oncogenesis were differentially regulated by specific CPEB2 splice variants. Overall, our findings demonstrate that the regulation of CPEB2 mRNA splicing is a key step in the acquisition of AnR for TNBC as well as a major driving force in metastasis.
Amino Acid Positions Important For Substrate Specificity in Plant Sucrose Transporters
Authors: Reinders Anke , Ward John
Published: June 2021

Plant sucrose transporters (SUTs) are members of the GPH family within the Major Facilitator Superfamily (MFS). They are proton‐coupled symporters in the plasma membrane or vacuole (lysosome) membrane and function in sucrose uptake into the cytoplasm. SUTs function throughout the plant but are especially important for sucrose uptake into vascular tissue. Plants contain small gene families of SUTs that can be classified into three basic types (I, II, III) both by their amino acid sequence and by their substrate specificity. Type I SUTs are less selective and transport a range of beta glucosides, including the fluorescent coumarin glucosides esculin and fraxin, in addition to sucrose. Type II SUTs are more selective for sucrose and do not transport esculin or fraxin. To identify amino acid positions that control substrate specificity, we used gene shuffling between a Type II SUT OsSUT1 and a non‐functional version of OsSUT1 in which 64 positions had been mutated to the corresponding positions in the Type I SUT StSUT1. Gene shuffling libraries were expressed in yeast and assayed for their ability to confer uptake of esculin using fluorescence activated cell sorting (FACS). Mutation of five amino acids in OsSUT1 resulted in esculin transport. The amino acid changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was analyzed by expression in Xenopus oocytes and electrophysiology and was almost identical to that of type I SUTs. We are now investigating the role of these positions in more detail.
Fatty acid binding to CD36 affects oxLDL uptake
Authors: Hamilton James , Miguel Paz , Jay Anthony
Published: June 2021

We investigated the hypothesis that interactions of unesterified fatty acid (FA) with the plasma membrane protein CD36 affect uptake of oxLDL. Because the binding of natural FA to CD36 have not been systematically studied, we first characterized FA binding to CD36 by the biophysical method surface plasmon resonance (SPR). After covalently linking the extracellular domain of CD36 to an SPR chip surface, aqueous FA (solubilized in cyclodextrin) was pulsed across the surface, generating association and dissociation binding curves. We studied structurally different long chain FA: a saturated (palmitic acid); unsaturated (oleic acid); trans‐oleic acid; (elaidic); a polyunsaturated [docosahexaenoic acid (DHA)], and an oxidized FA [9S‐hydroxy‐10E,12Z‐octadecadienoic acid (HODE)]. With the exception of HODE, SPR detected FA binding to CD36, and showed that multiple sites are present with rapid association and dissociation kinetics that were similar to HSA. To study the role in oxLDL uptake of these FA, we used a fluorescent oxLDL (Dii‐oxLDL) live‐cell assay with confocal microscopy imaging. CD36‐mediated uptake in serum‐free media was very low but greatly increased with serum. Addition of FA in serum‐free media increased oxLDL binding and uptake to levels found with serum, and affected CD36 plasma membrane (PM) distribution. Binding/uptake of oxLDL exhibited dose dependency of added FA, with the exception of DHA, which binds avidly to CD36 but did not activate uptake. HODE, which binds poorly to CD36, did not affect oxLDL uptake. The high affinity FA binding to CD36 has important implications that binding at some or all sites could affect its conformation, binding of other ligands, and functional properties.
Admixture in Horse Breeds Illustrated from Single Nucleotide Polymorphism Data
Authors: Torres Gutierrez Cesar , Brandvain Yaniv
Published: June 2021

More than 300 horse breeds exist in the modern world. Many of these horse breeds are thought to have closely related evolutionary paths, but with the horse genome being sequenced recently (2007), genome‐wide single nucleotide polymorphism (SNP) studies confirming evolutionary relationships are lacking. This project determines whether there is admixture between horse breeds that are thought to be evolutionary related by using the program Treemix to create a phylogenic tree, as well F3 statistics to identify admixture in the multiple horse breeds. Allele frequencies across 34,648 SNP sites formatted from the genotype of 36 breeds (795 individuals in total) obtained with an Illumina Beadchip, were used for our analyses. We found evidence for admixture events between several breeds of horses, which was confirmed with F3 statistics tests (three population test). These findings will facilitate future studies of the origin of the domestic horse and its evolutionary path by providing new insights on each breed and how they are related to each other. In the future we will use denser chips to obtain more SNP sites allowing us to find regions of introgression and determine whether or not they are related to health or performance traits.
Phosphoproteins with Stability against All Urinary Phosphatases as Potential Biomarkers in Urine
Authors: Zhao Mindi , Gao Youhe
Published: June 2021

Urine, by accumulating all kinds of changes, was proposed to be a better source for biomarker discovery. As one of the most common post‐translational modifications, phosphorylation plays a vital role in many biological activities. However, the urine phosphoproteome has been largely neglected due to the low abundance of phosphoproteins and the presence of various phosphatases in urine. The low level of background phosphorylation in urine is actually advantageous, as urinary phosphopeptides/proteins that are stable to the phosphatases present in urine have the potential to serve as valuable disease biomarkers. Using a TiO2 enrichment strategy, this study aimed to create a comprehensive proteomic profile of human urinary phosphoproteins and to characterize the changes in the urine phosphoproteome after incubation of urine with cell lysates. In total, 106 phosphorylation sites corresponding to 64 proteins, including 80 previously unidentified human urine protein phosphorylation sites, were identified by mass spectrometry. Fifteen phosphopeptides, together averaging 47% of the total phosphopeptides, were found in samples from all individuals. Addition of cellular proteins to urine did not significantly change the phosphorylation level of urine proteins. But there were still a few phosphopeptides from cell lysates survived urinary phosphatases; such phosphopeptides represent potential biomarkers in urine.
Proteomic analysis of the interacting partners of different S6Ks isoforms.
Authors: Pavan Isadora , Freitas Lidia , Springer Alice , Yokoo Sami , Carnielli Carolina , Paes‐Leme Adriana , Simabuco Fernando
Published: June 2021

The mTOR signaling pathway has been associated with several diseases and metabolic disorders in humans, including obesity, diabetes and several cancers. The S6Ks proteins have shown an important role in mTOR signaling, acting as effectors of this pathway. Nevertheless, the functional differences between the S6Ks isoforms is still an open question. The S6Ks family is composed of two genes, S6K1 and S6K2, encoding the isoforms p70‐S6K1, p85‐S6K1 and p54‐S6K2. The objective of this study was to investigate the interactomes related to the different S6Ks. Isoforms p70‐S6K1 and p54‐S6K2 were overexpressed in HEK293 cells, immunoprecipitated and samples submitted to mass spectrometry. The identified proteins were grouped into several classes, such as, protein synthesis, ribosomal proteins, metabolism, transcription, methylation, RNA splicing and processing, stress response, inflammation, phosphatases, cell cycle and others. Moreover, we found the known phosphorylation motif of S6Ks (RXRXXT/S) in several of these interacting partners, suggesting new possible targets of this kinase. Furthermore, the interacting partners of S6Ks were classified according to their subcellular localization. Finally, validation by western blotting was performed for some of the interacting partners. In conclusion, here we show that p70‐S6K1 and p54‐S6K2 present different interactomes, which may be related to their function in the cell.
Phosphatidylinositol 4‐phosphate dynamics during phagocytosis
Authors: Levin Roni , Hammond Gerald , Balla Tamas , Fairn Gregory , Grinstein Sergio
Published: June 2021

Phosphoinositide levels undergo acute changes during the course of phagocytosis; phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 3,4,5‐trisphosphate and phosphatidylinositol 3‐phosphate (PtdIns3P) undergo marked changes. However, the fate of phosphatidylinositol 4‐phosphate (PtdIns4P) has not been investigated. Recently, a genetically encoded biosensor was developed to visualize PtdIns4P in live cells. The probe, called P4M2x, consists of two copies of the PtdIns4P‐binding domain of SidM, a Legionella pneumophila effector, tagged with a fluorescent protein. Expression of P4M2x in macrophages revealed tri‐phasic changes of PtdIns4P during phagocytosis. PtdIns4P, normally present in the plasma membrane, underwent a transient, moderate increase as the phagosome sealed, coinciding with the disappearance of PtdIns(4,5)P2. This initial increase was followed by the virtual disappearance of PtdIns4P, which coincided with accumulation of PtdIns3P. At the time when PtdIns3P disappeared, a gradual reacquisition of PtdIns4P was observed, exceeding the levels normally found at the plasma membrane. This reappearance of PtdIns4P was preceded by the sequential acquisition of Rab5 followed by Rab7 –early and late maturation indicators, respectively. The reacquisition of PtdIns4P was accompanied by the appearance of P4M2x‐positive tubules that extended dynamically between phagosomes and the pericentriolar Golgi area.
Characterization of Proteins Associated with the Coenzyme Q Biosynthetic Complex and Analyses of Phosphorylated Coq Proteins in Yeast Mitochondria
Authors: Awad Agape , Allan Christopher , Johnson Jarrett , Shirasaki Dyna , Loo Joseph , Clarke Catherine
Published: June 2021

Coenzyme Q (also termed ubiquinone or CoQ) is an electron carrier in the mitochondrial respiratory chain that functions as an essential component in energy metabolism and serves as a vital lipid soluble antioxidant. Patients with CoQ defects suffer from various health disorders, such as kidney disease, neurodegenerative diseases, ataxia, and cardiovascular complications. Additionally, aging has also been linked to decreased CoQ levels. Saccharomyces cerevisiae serves as an excellent model for studies on CoQ biosynthesis and function because it is both a simple model and is highly conserved with respect to human CoQ biosynthesis. In yeast, several of the eleven proteins (Coq1p‐Coq9p, Yah1p, and Arh1p) necessary for the biosynthesis of CoQ6 have been shown to be associated in a high molecular mass complex that localizes in the inner mitochondrial membrane, and has been termed the 'CoQ‐synthome'. Here we identify other potential protein partners that co‐purify with several of the Coq polypeptides and appear to be required for efficient Q biosynthesis. We use co‐immunoprecipitation techniques, Western blotting, blue native PAGE, lipid analysis, yeast cell growth assays, phosphorylation enrichment, and liquid chromatography‐tandem mass spectrometry to identify novel associated proteins and phosphorylation modifications sites of the Coq polypeptides. Elucidating their biosynthetic role in regulatory processes, such as phosphorylation, will aid our understanding and treatment of multiple diseases caused by CoQ‐deficiencies and will provide novel insight regarding longevity and issues related to aging in humans.
Status of Lipid Peroxidation and the Immune System in the Remote Period after a Sublethal Dose Of Gamma Radiation
Authors: Ilderbayeva Gulzhan , Abduldayeva Aigul , Utegenova Aigul , Ilderbayev Oralbek
Published: June 2021

We studied the intensity of free radical processes in organs (liver, spleen, thymus, lymph nodes of the small intestine, and adrenal glands) and cells (lymphocytes) and immunity system in the remote period to a sublethal dose of γ‐radiation (6 Gy). To investigate the immunity system and the lipid peroxidation of laboratory animals (white rats) in two series of experiments. All rats were divided into 2 groups: I – controls, II group – animals exposed to radiation.

As opposed to the said tissues in hepar, there were material changes. MD sustentation was at the level of intact animals exponent (there is the tendency to rise). Given fact show that there are big compensation abilities of hepatic tissues which enables abnormalities arresting of free‐radical processes induced by the effect of ionized irradiation

In such a manner, at the irradiated rats in the remote period were revealed decrease in the main indicators of immunity, the quantity of T‐lymphocytes, a supressiya of nonspecific phagocytic protection of an organism defense. There was CYC increase in blood serum. Perhaps, it was the result of uprising of organism autoimmune response on conjugated antigen. After the radiation effect, in remote period, there were abnormalities in the peroxidation process of lipids in mainly under investigation immunocompetent organs and tissues.
Hyperglycemia Hinders First Trimester Cytotrophoblast Migration and Induces Apoptosis
Authors: Cawyer C , Yuen S , Pantho A , Drever N , Beeram M , Zawieja D , Kuehl T , Uddin M
Published: June 2021

Objective

Approximately 20% of the diabetic pregnant women develop preeclampsia (preE). In a recent study, we have shown that hyperglycemia impairs CTB function via stress signaling. In the present study, we assess the CTBs apoptotic signaling.

Study Design:

Human extravillous CTBs were treated with 0, 100, 200, 300 or 400 mg/dL glucose for 48h. Some cells were pretreated with a p38 inhibitor or a peroxisome proliferator‐activated receptor gamma (PPARγ) ligand. Other cells were treated with D‐Mannitol. Cell migration was performed by Matrigel migration kit. Cell lysates were utilized to measure PPARγ, p38 MAPK phosphorylation, and apoptotic signaling proteins; Bcl‐2‐associated X protein (Bax), anti‐apoptotic Bcl‐2 and caspase‐9 and cyclooxygenase‐2 (Cox‐2) by western blot.

Results

CTBs migration was inhibited by 蠅 150 mg/dL of glucose compared to basal (100 mg/dL) (p<0.05). The p38 MAPK phosphorylation and PPARγ were upregulated (p<0.05) in CTBs treated with >150 mg/dL glucose. The expression of Bax/Bcl‐2, Cox‐2, caspase‐9 were up‐regulated (p<0.05) in CTBs treated with 蠅100 mg/dL glucose. The SB203580 or rosiglitazone pretreatment showed an attenuation of hyperglycemia‐induced apoptotic signaling proteins. D‐Mannitol had no effect on CTBs.

Conclusions

Hyperglycemia inhibited the migration of CTBs and induced the apoptotic signaling in CTBs. The attenuation of hyperglycemia‐induced apoptotic signaling suggests the involvement of apoptotic signaling mechanisms in CTBs dysfunction.
Characterization of Yeast Bax Inhibitor, BXI1, Function in the Unfolded Protein Response and Calcium Signaling in Saccharomyces cerevisiae
Authors: Cavedon William , O'Brien James , Gonzalez Alfredo , McCarthy Morgan , Austriaco Nicanor
Published: June 2021

Bax inhibitor‐1 (BXI1) is an anti apoptotic gene whose human homolog's expression is upregulated in a wide range of human cancers. Studies have shown that Bxi1p is localized in the endoplasmic reticulum and is involved in the unfolded protein response (UPR) that is triggered by endoplasmic reticulum (ER) stress. BXI1 is thought to act via a mechanism involving altered calcium dynamics. In this poster, we provide evidence that suggests that the Saccharomyces cerevisiae protein facilitates Ire1p clustering in response to ER stress and alters calcium signaling. With DTT induction experiments using Δbxi1 mutants and different UPR GFP reporters, we have confirmed our published data that suggests that BXI1 is involved in the UPR, Intriguingly, our preliminary data suggests that Bxi1p facilitates clustering of Ire1p during ER stress. [Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.]
Structural and Functional Properties of N‐Methyl‐D‐Aspartate Receptor‐Specific ConRlB
Authors: Kunda Shailaja , Yuan Yue , Zajicek Jaroslov , Balsara Rashna , Castellino Francis
Published: June 2021

Conantokins are neuroactive peptides found in the venom of marine snails, containing post‐translationally modified γ‐carboxyglutamate residues (γ). The γ residues are integral for their structure, function, and subunit selective antagonism towards N‐Methyl‐D‐Aspartate Receptors (NMDAR). Conantokin RlB (ConRlB) from Conus rolani additionally contains the post‐translationally modified 4‐transhydroxyproline (Hyp; O) residue. The 10 Hyp residue not only changes the γ positioning in the C‐terminal part of ConRlB, but also changes its helicity (62%) in the presence of Mg2+, compared to Mg2+ bound ConG, which is an end‐to‐end α‐helix. The 3D structure of ConRlB, revealed by high‐field 1H NMR, shows disruption in the helix due to 10 O interfering with Mg2+ binding, thus resulting in solvent accessible backbone amide protons. To assess the role of O and the molecular requisites necessary of their unique structural and functional properties, mutant peptides, ConG[10▼O] and ConRlB[ΔKAO▼NQ] were synthesized. Weaker NOEs between residue 9 and 12 of ConG[10▼O], distinguished the nature of the break in the helix due to O compared to ConRlB. Inhibition of intracellular Ca2+ in WT, GluN2A‐/‐, and GluN2B‐/‐ mice cortical neurons by ConG[10▼O] showed increased GluN2A selectivity compared to ConG. ConRlB[ΔKAO▼NQ] showed diminished GluN2B selectivity, in spite of a similar 3D structure to ConG. ConRlB[ΔKAO▼NQ] lost, and ConG[10▼O] retained its ability, to inhibit NMDA‐mediated P‐CREB compared to their respective parent peptides. In conclusion, the O residue changes the structural and functional properties of conantokins.
The Interplay of Phosphorylation and Ubiquitylation in the Regulation of PTEN
Authors: Chen Zan , Bolduc David , Yan Ming , Jiang Xuejun , Wolberger Cynthia , Cole Philip
Published: June 2021

PTEN is a lipid phosphatase that hydrolyzes PIP3 to PIP2 antagonizing the PI3K‐Akt signaling pathway. As a critical tumor suppressor, PTEN is often down‐regulated in cancer by mutation, epigenetic silencing or post‐translational modification (PTM). One important PTM involves phosphorylation of a cluster of residues: S380, T382, T383, and S385 in the C‐tail of PTEN. Such PTEN phosphorylation is proposed to enhance PTEN's stability, potentially by blocking ubiquitin‐mediated degradation. To clarify the mechanism, we employed semisynthesis to generate non‐phosphorylated (n‐PTEN) and tetra‐phosphorylated PTEN (4p‐PTEN) protein. We assessed the effects of phosphorylation of PTEN on its ubiquitylation by the E3 ligases WWP2 and NEDD4‐1. The ubiquitylation of 4p‐PTEN by WWP2 was significantly slower than that of n‐PTEN, while there was no phospho‐mediated differences in the NEDD4‐1 catalyzed reactions. Single turnover experiments revealed that the inhibition of WWP2‐mediated PTEN ubiquitylation by PTEN phosphorylation involves the specific transfer step of Ub‐WWP2 to PTEN. Pulldown experiments demonstrated that WWP2 binds more weakly to 4p‐PTEN than n‐PTEN. We also used semisynthesis to investigate the roles of specific phosphorylation combinations on PTEN activity which has thus far revealed a gradation of impact with larger numbers of phosphorylations. Overall, this study has provided new insights into the complex relationship of Ser and Lys PTMs in the regulation of a key tumor suppressor protein PTEN by E3 ligases.