bluuebunny/crossref_metadata_2025 huggingface dataset:
A Comparative Study for Advancing Understanding of Interdisciplinary Learning in STEM
Authors: Eastwood Jennifer L , Schlegel Whitney M
Published: June 2021

Preparing students to make informed and ethical decisions on complex problems is a primary aim of higher education (AAC&U, 2007). Socioscientific issues (SSI) are complex learning contexts incorporating social interaction, scientific and sociomoral reasoning, self‐directed learning, and reflection. SSI provides learning theory and practice frameworks for enhancing learning in STEM. We investigated whether students in an interdisciplinary biology major (IDL), which engaged students in a SSI‐directed core curriculum, would reason differently with complex problems than students in a traditional biology major (BIO). Additionally, we questioned how their perceptions of their college experiences would differ. Assessments included open‐ended questionnaires administered to 95 participants of both majors and interviews with a subset of 16 participants. We found that while IDL and BIO groups made similar decisions on complex problems, the IDL group showed more sophisticated reasoning, suggesting that the IDL learning environment was more effective for enhancing decision‐making skills. IDL students’ citation in questionnaires and interviews of case studies from their coursework suggests that the IDL environment provided a repertoire of examples from which to reason with new problems. We will discuss the broader implications of these and other findings for the teaching of science in higher education.
Pedagogy Affects Final Student Learning Outcomes For Non‐Majors Science Courses
Authors: Parzer Harald F , Schlegel Whitney M
Published: June 2021

It has been demonstrated that context is critical for meaning making and that understanding is enhanced with authentic, interactive problem‐solving opportunities. Learning theory and evidence of student learning assert that student learning outcomes in undergraduate courses should be in part predicted by years of college study. Undergraduates enrolling in introductory non‐majors biology courses come from all class levels as well as diverse academic majors. Do different class levels and academic majors predict learning outcomes in non‐majors biology courses and are these differences associated with pedagogy? This study examined final grade outcomes by class level and other relevant student demographic factors in two introductory non‐major biology courses with similar course content focus, yet different pedagogical approaches. Students randomly enrolled in either a traditional lecture course (content‐centered, CC) or a highly interactive team and case based course (student‐centered, SC). Results showed an effect of class level and academic major on final grade outcome in the CC learning environment but not in the SC learning environment. Student attitudes towards team and case based learning can partially explain student performance in the SC course. The implications of this study for the structuring of courses and curricula and evidence‐based teaching practice will be discussed.
Biological sex and tympanic dehiscence
Authors: Bilal Alina Susana , Jelaca-Tavakoli Maria
Published: June 2021

In this study, the presence of tympanic dehiscence is studied using 72 trephined Peruvian crania (39 male and 33 female) from the Hrdlicka Collection housed at the San Diego Museum of Man. The objective of this study is to determine whether tympanic dehiscence alone could be used to determine sex in human osteological remains. The analysis of the tympanic plates shows that 41 or 57% of the 72 crania have the trait. From the 41 crania with tympanic dehiscence, 19 are male and 22 are female or 46% and 54% respectively, suggesting that tympanic dehiscence is more likely to occur in females. However, because of the significant number of males possessing the trait, tympanic dehiscence cannot be used alone as a reliable sex‐determining feature. Therefore, it is suggested that for accurate results, anatomists and biological anthropologists should only use tympanic dehiscence to determine sex if it is used in collaboration with other skeletal features. San Diego Museum of Man.

Grant Funding Source : N/A
Vascular Variation in the Triangular Interval of the Axilla
Authors: Feldman Eleanor Jane , Shukla Anand , Marquez Samuel
Published: June 2021

The anatomical gateways that allow the neurovascular structures to course between the axilla and posterior body regions are considered clinically important in surgeries. The triangular interval has gained considerable popularity with the increase in use of the motor branches of the triceps muscle for muscle transfers. The radial nerve and profunda brachii artery are the contents traditionally described residing in the triangular interval. To document the inherent variation found in this particular gateway, this study surveyed 40 triangular intervals from 20 donor bodies, representative of an urban population, in order to investigate the path of the radial nerve and the profunda brachii within the axilla. Results show that in 100% of the subjects, the radial nerve coursed deep to the triangular interval. Variation in the path of the profunda brachii, however, was observed. In 12.5% of the sample the profunda brachii was observed deep to the quadrangular space and not in the triangular interval. Bilateral asymmetry was observed within 60% of the variable triangular intervals. These findings demonstrate the highly variable nature of the profunda brachii's path within the axilla between as well as within individuals.

Grant Funding Source : SUNY Downstate
Study of the Vascular Morphology of the lower limb nerves used as grafts
Authors: Omaña Rodrigo Enrique Elizondo , Lopez Santos Guzman , Cruz Juan Jose Bazaldua , GArza Alejandro Quiroga
Published: June 2021

A macroscopic and microscopic analysis of the vascularity of the main lower limb nerves used as vascularized nervous grafts, was carried out. It was followed out a descriptive, transversal and non‐comparative study to analyse the saphenous, the sural, the deep peroneal and the superficial peroneal nerves vascularity. This study was performed in two stages: 1) an in situ vascularity study and 2) a morphometric study. By the in situ study, it was demonstrated the following: The total length of the lower limb nerves used as graft, was in a decrescent order: sural nerve (μ= 29.26 cm, ±4.05 cm), superficial peroneal nerve (μ=28.61 cm, ±3.97 cm), deep peroneal nerve (μ=26.64 cm, ±5.21 cm) and saphenous nerve (μ=25.12 cm, ±4.42 cm). The morphometric study demonstrated that the dominant artery with the major diameter belongs to the saphenous nerve, with a diameter of 1.13 mm; then we have the superficial peroneal nerve with a 1.01 mm diameter, followed by the deep peroneal nerve with 0.91 mm, and finally the sural nerve with the smallest diameter, 0.85 mm. The saphenous nerve has the most appropriated morphological characteristics to be used as a vascularized graft.
A Novel Structure Potentially Associated with Greater Trochanteric Pain Syndrome
Authors: Taylor Victor , Crofford Theodore , Seidel Hudson , Reeves Rustin
Published: June 2021

Purpose

Greater trochanteric pain syndrome (GTPS) is commonly diagnosed, and is thought to be at least in part caused by repetitive friction between the greater trochanter (GT) and iliotibial tract (ITT). We propose that the ITT has an important insertion into the proximal femur, the ITT‐ Femoral Insertion (ITT‐FI), which is the major contributor to this friction, rather the tibial extension of the ITT.

Method

Hips (n=20) were dissected on embalmed cadavers. The ITT‐FI was identified as the ligamentous structure remaining after excising the gluteus maximus (GM) and tensor fasciae latae (TFL) muscle fibers from the ITT, transection of the ITT at mid femur, leaving the ITT origin at the iliac crest and insertion on the femur (near the GM) intact. Measurements recorded include: length from the iliac crest to the femoral insertion of the ITT, the width of the ITT‐FI fibers, and the relative width of the pelvis as determined by the distance from anterior superior iliac spine (ASIS) to ASIS. Photographs were taken.

Results

The proposed structure originates at the iliac crest and inserts inferior to the GT. The pressure between the ITT and the GT changed little if at all as palpated before and after isolating the ITT‐FI. Average measurements of the structure in length was 21.3cm, and width at the iliac crest was 4.6cm. Average width of the pelvis was 25.3cm.

Conclusion

The femoral insertion of the ITT is identified, measured in dimension, and appears to be the chief cause of ITT static pressure on the GT. Future studies are needed to quantify this assertion.

Grant Funding Source: none
Fenestrations of the Aortic Valve Cusps: An Anatomical Explanation?
Authors: Losenno Katie L , Johnson Marjorie , Chu Michael W A
Published: June 2021

Fenestrations of the aortic cusps are a common finding in older adults. In most cases, they are benign although there are reports of spontaneous rupture and acute onset of aortic insufficiency. The process of fenestration development is poorly understood however, it is possible that variation in cuspal anatomy may be an important factor. The aortic valves from 40 adult cadaveric specimens were harvested and examined for fenestrations. The cuspal height, free margin length, and attached margin length were measured and leaflet perimeter and area were calculated. 37.5% of all specimens were found to have aortic valves with at least one fenestrated cusp. Of those with fenestrations, 73.3% were in the left coronary cusp, 53.3% were in the right coronary cusp and 33.3% were in the non‐coronary cusp. Preliminary data suggests that cusps with a smaller perimeter are more prone to fenestrations. Previous studies have suggested that the, often larger, non‐coronary sinus and cusp are subject to greater stresses during the cardiac cycle which in turn leads to increased collagen deposition within the cusp. Although counterintuitive to the increased stresses, the non‐coronary aortic cusps in this series had less fenestrations, perhaps suggesting that increased stresses may actually prevent fenestration development. The initial findings warrant further investigation with a larger sample size and further statistical analysis.

Grant Funding Source: Queen Elizabeth II Graduate Scholarship in Science and Technology
Fatty Marrow and Increased Loaded Cancellous Bone was Resistant to OVX induced Bone Changes in Female Rats
Authors: Lin Zhiming , Tian Xiaoyan , Chen Daihe , Lin Jin , Chen Min , Liu Jonathan , Shi Hexiu , Men Ping , Liu Gang , Jee Webster
Published: June 2021

Objective

Previous studies determined ovariectomy (OVX) induced cancellous bone loss and raised cage (RC) bipedal stance partially prevented OVX induced bone loss at red marrow bone sites in adult rats; however, these effects in the distal tibial metaphysis (DTM) with fatty marrow, a site similar to adult long bone metaphyses (non‐veterbral bone sites) with low risk of postmenopausal fracture, have yet to be determined.

Methods

Six‐month‐old female S‐D rats were divided into basal, sham, OVX, RC and combination of OVX plus RC exercise for 4 and 8 weeks, cancellous bone histomorphometry on fluorescent labeled undecalcified cross sections of DTM were performed.

Results

In DTM, OVX did not induce cancellous bone loss, but significantly increased bone turnover. RC exercise alone had no effect on cancellous bone mass, but significantly reduced bone resorption. Unlike in high turnover red marrow sites, the combination of RC exercise and OVX only partially prevented bone loss, but combining with raised cage exercise and OVX in DTM totally inhibited OVX induced cancellous bone loss by depressing bone resorption (reduced eroded perimeter).

Conclusions

In DTM, the inhibition of OVX induced bone loss can be attributing to a combination of fatty marrow content and increased mechanical loading with decreased resorption, which may partly explain why non‐vertebral bone sites are less at risk to fracture in postmenopausal patients.
ZIPK modulates canonical Wnt/β‐catenin signaling through interaction with NLK/TCF4
Authors: Togi Sumihito , Muromoto Ryuta , Matsuda Tadashi
Published: June 2021

Zipper‐interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase that has been implicated in apoptosis and transcriptional regulation. Here, we identified Nemo‐like kinase (NLK) as a novel ZIPK‐binding partner and found that ZIPK regulates NLK‐mediated repression of canonical Wnt/β‐catenin signaling. Indeed, siRNA‐mediated reduction of endogenous ZIPK expression reduced Wnt/β‐catenin signaling. Furthermore, ZIPK affected the formation of NLK‐Tcell factor 4 (TCF4) complex. Importantly, ZIPK siRNA treatment in human colon carcinoma cells, in which Wnt/β‐catenin signaling was constitutively activated, resulted in a reduction of β‐catenin/TCF‐mediated gene expression and cell growth. These results indicate that ZIPK may serve as a transcriptional regulator of canonical Wnt/β‐catenin signaling through interaction with NLK/TCF4.
WT1 Interacts with p53 and Inhibits p53‐induced p21 gene expression
Authors: Ko Kyoung-Won , Choe Yun-Jeong , Lee Sun-Young , Kim Ho-Shik
Published: June 2021

Wilms’ tumor suppressor gene 1 (WT1) encodes a transcription factor important for normal cellular development and cell survival. Previous studies have reported both physical and functional interaction between the WT1 and p53 proteins. However, the relationship between WT1 and p53 has not yet been fully clarified. Here we show that WT1 retards p53‐induced transcriptional activation of p21.

Using domain deletion mutant constructs of p53, we determined that C‐terminal region containing DNA‐binding domain of p53 was necessary for the interaction between WT1 and p53. In osteosarcoma U2OS cells, WT1 enhanced cell death by nutlin‐3, an antagonist of MDM2, and restrained p53‐mediated p21 expression. Consistently, p53‐induced transactivation of p21 was attenuated by WT1. Moreover, small interfering RNA‐mediated knockdown of p21 expression enhanced nutlin‐3‐induced cell death. Taken together, WT1 interacts with p53 and inhibits p53‐ induced p21, leading to potentiation of p53‐induced cell death.

This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (2011‐ 0027115).
Identification of RNA Polymerase ω subunit in Chlamydia trachomatis
Authors: Allette Kimaada , Fan Huizhou , Bao Xiaofeng
Published: June 2021

The bacteria Chlamydia trachomatis is responsible for sexually transmitted infections. Because of its intracellular developmental process, it poses difficulties for researchers trying to understand it. The goal of this project is to provide evidence that an omega (ω) factor of the RNA polymerase is present in Chlamydia trachomatis. In general, the ω factor aids in the folding of the RNA polymerase's β′ subunit and thus, the assembly of the β′ subunits with α, α, β to form a functional core enzyme in the conformation of α, α, β, β′ and ω. Through BLAST analysis, our lab has found a putative ω subunit in Chlamydia that has a resemblance to E.coli ω subunit. We are in the process of expressing the putative ω with α, β, β′ and σ 66 in E. coli BL21 with or without ω. Whether the protein is true ω or not will be judged by its ability to aid in the yield of the recombinant chlamydial RNA core enzyme.
Acetylation of Replication and Repair Proteins Regulates Genome Fidelity
Authors: Balakrishnan Lata , Bambara Robert A
Published: June 2021

In nuclear DNA transactions, Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki fragments and long patch base excision repair of DNA damage. Acetylation of the two nucleases by the acetylase p300 allows for the displacement and more efficient processing of long flaps. Additionally, DNA polymerase δ (pol δ) and RPA are also acetylated by p300. Acetylation of pol δ stimulates its strand displacement function, thereby promoting the creation of longer flaps. RPA on acetylation shows higher binding affinity to ssDNA. Significantly, acetylation of the main repair enzyme, DNA polymerase β (pol β), pushes repair into the long patch mode. Differential regulation of the activities of replication/repair by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as insuring correct processing. Such intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the re‐synthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. Thus we propose acetylation to be a regulatory mechanism which promotes higher fidelity of DNA transactions, ensuring genome stability.
Dependence of DNA‐protein cross‐linking via guanine oxidation upon local DNA sequence
Authors: Perez Zitadel Anne , Stemp Eric
Published: June 2021

Oxidative damage plays a causative role in many diseases and DNA‐protein cross‐linking is one important consequence of such damage. Here we examined how local DNA sequence influences DNA‐protein cross‐link formation induced by guanine oxidation. Oxidative DNA‐protein cross‐linking was induced via the flash quench technique. Following oxidation of pBR322 DNA by flash quench, several restriction enzymes (PpuMI, BamHI, EcoRI) were used to probe for the expected damage at guanine sites. These three endonucleases were strongly inhibited by DNA‐protein cross‐linking, whereas the activity of the AT‐recognizing enzyme AseI was unaffected. The % inhibition observed for the endonucleases decreased in the order: PpuMI (5’‐GGGTCCT ‐3’ and 5’‐AGGACCC‐3’) > BamHI (5’‐GGATCC‐3’) > EcoRI (5’‐GAATTC‐3’), consistent with the observed and predicted tendencies for guanine to undergo 1‐electron oxidation: 5’‐GGG‐3’ > 5’‐GG‐3’ > 5’‐GA‐3’. Thus, in mixed DNA sequences, the guanine sites most vulnerable to oxidative cross‐linking are easiest to oxidize. Research support was provided by the National Science Foundation, the Denault‐Loring Research Fellowship, and Mount St. Mary's College.
Investigation of the Role of the Proteasomal Subunit Rpn10 in pre‐mRNA Splicing
Authors: Merkhofer Evan C , Johnson Tracy
Published: June 2021

The removal of non‐coding sequences from protein‐coding pre‐messenger RNAs is required for proper gene expression. This highly conserved process, commonly known as RNA splicing, is mediated by a large ribonucleoprotein complex known as the spliceosome. Dysregulation of RNA splicing is common in disease, as up to 15% of mutations leading to disease have been attributed to an effect on pre‐mRNA splicing. The DExD/H‐box family of ATPases, including Prp5, play a critical role in mediating ATP‐dependent RNA remodeling in splicing. One pressing question that remains is what factors are required for rapid ATP‐dependent rearrangements of the spliceosome that underlie high fidelity splice site recognition. Using Saccharomyces cerevisiae, our studies of the mechanisms of spliceosomal rearrangements have identified striking genetic interactions between the Rpn10 subunit of the proteasome and the ATP‐dependent spliceosomal RNPase Prp5. Deletion of rpn10 rescues the slow growth phenotype exhibited by strains containing prp5 ATPase mutations, and leads to an increase in splicing in vitro. Furthermore, we show that Rpn10 can regulate protein levels of multiple spliceosome components, including Cus2, and rpn10 shows strong genetic interactions with this gene. These intriguing results suggest a novel role of the regulatory subunit of the proteasome in mediating rearrangements between components of the spliceosome.
Yeast Two‐hybrid Reveals a Potato Polypyrimidine Tract Binding (PTB) Protein Interacts with a Nova‐like Protein
Authors: Shah Shweta , Butler Nathan , Hannapel David J , Rao A Gururaj
Published: June 2021

PTB proteins are RNA‐binding proteins that contain four RNA recognition motifs. In potato, four PTB‐like genes, designated as PTB1, PTB6, TC201749 and PTB7, have been identified. In plants, these proteins play an important role in alternative splicing. In order to understand the multifunctional role of PTB proteins of plants we performed yeast two‐hybrid screening using PTB1 protein as bait. PTB1 protein was found to interact with a Nova‐like protein, termed StNova‐like protein. Nova (neuron‐oncological ventral antigen) protein is a RNA‐binding protein that contains three K‐homology domains. In humans, these proteins play an important role in regulation of neuronal RNA metabolism but the role of Nova‐like proteins in plants has not been explored. In this study, the StNova‐like gene has been cloned into the pMAL‐c2E vector, expressed in T7 express cells and purified over Ni‐NTA resin. Further, putative interactions between StPTB proteins and StNova were confirmed by pull‐down experiments and by blue‐native PAGE. Using a random 21‐mer phage‐peptide library, we also identified a consensus peptide sequence motif [S/G][I/L][L/I][S] that binds to PTB1, suggesting that the PTB1 protein might be interacting with the [S][I][I][S] sequence present in StNova‐like protein. Our studies suggest that in plants, Nova‐like proteins may be involved in processes of RNA metabolism mediated by PTB proteins.
The novel role of Sav1 in aggresome formation
Authors: Sakai Nobuya , Fujiwara Yoko , Shiraki Takashi , Koshimizu Taka-aki , Shibata Katsushi
Published: June 2021

Intracellular deposition of misfolded protein aggregates into ubiquitin‐rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Recent genetic screens of fly mutants and molecular analysis have revealed that the Hippo/Salvador/Warts pathway controls both cell proliferation and cell death. Specifically, adaptor protein Salvador is the key component of this pathway. Using proteomics screening, we identified the molecular chaperone Hsp70 as a novel protein associated with Sav1 (mammalian homologue of Salvador). Sav1 targets to the aggresome and its sequestration to the aggresome is enhanced by the proteasome inhibitor MG132. Furthermore, Sav1 co‐localizes with Hsp70 and CHIP (carboxy terminus of Hsp70‐interacting protein) that has been shown to mediate substrate ubiquitination and degradation by the proteasome. This study suggests that Sav1 has a previously unexpected critical role in the aggresome pathway.
Par‐6 activates atypical Protein Kinase C by pseudosubstrate displacement
Authors: Graybill Chiharu , Prehoda Kenneth E.
Published: June 2021

The phosphorylation activity of atypical Protein Kinase C (aPKC) is central to the establishment of cell polarity by Par complex. Like other PKC family members, aPKC contains a pseudosubstrate domain that binds to the active site in the kinase domain and acts as an internal inhibitor. Despite the presence of the cis‐acting inhibitor, another Par complex member, Par‐6, is thought to repress aPKC activity. To examine the precise mechanism by which the pseudosubstrate domain and Par‐6 regulate aPKC activity, we reconstitute the system in vitro and performed a detailed kinetic analysis. We confirm that the pseudosubstrate domain is responsible for the autoinhibition. Surprisingly, rather than acting as an inhibitor, Par‐6 activates aPKC by displacing the pseudosubstrate from the kinase domain. Par‐6 activation of aPKC is consistent with our observation that Par‐6/aPKC complex, but not aPKC alone, is competent to release its substrate from the cell membrane in Drosophila S2 cells. Our data support a model in which Par‐6 displacement of aPKC pseudosubstrate domain is required for the diverse cell polarities mediated by the Par complex. NIH Grant 068032
EXAMINATION OF EFFECT OF DIET, PH, AND AGE ON CHITINASE ACTIVITY IN THE GUT OF THE YELLOW‐BELLIES SLIDER TURTLE TRACHEMYS SCRIPTA
Authors: Garrett Adrianne Marie , Tansey John , Bouchard Sarah
Published: June 2021

The Slider Turtle, Trachemys scripta, undergoes an ontogenetic dietary shift during its life. During this shift, juvenile turtles prefer to eat aquatic invertebrates, which contain chitin in their exoskeletons. Digestive enzymes, like chitinase, are needed to help break down the exoskeleton allowing access to the underlying nutrients. A fluorescent chitinase assay for exochitinase was adapted for these studies. Enzymatic activity was detected in extracts of stomach, small intestine, large intestine, and pancreas obtained from five year old T. scripta fed a mixed diet. This work aimed to establish the existence and location of an endogenous chitinase and identify its optimal pH ranges in T. scripta hatchlings. Hatchlings typically have a more carnivorous diet which may result in an increase of chitinase within their gut. The procedure used on the five year old turtles is being repeated on hatchling turtles to determine if activity levels vary with age. This work was sponsored by the Merck AAAS Undergraduate Science Research Program.
Solution Structures of CaM‐iNOS and CaM‐eNOS peptide complexes: Effects of a phosphomimetic CaM mutation
Authors: Piazza Michael , Dieckmann Thorsten , Guillemette J Guy
Published: June 2021

NMR spectroscopy is an efficient method for studying the dynamics and structures of protein‐protein and protein‐peptide complexes. Nitric oxide synthase (NOS) functions in key physiological processes. Calmodulin (CaM), is required to fully activate the enzyme. Studies have shown CaM to act like a switch that causes a conformational change in NOS through a process that is thought to be highly dynamic. To investigate the dynamic properties of CaM‐NOS interactions, we determined the solution structure of CaM bound to the inducible NOS (iNOS) and endothelial NOS (eNOS) CaM‐binding region peptides. In addition, we investigated the structural and functional effects of CaM phopsphorylation on NOS using a phosphomimetic Y99E CaM. Our results show that Y99E CaM significantly reduces maximal synthase activity of eNOS by 40% while having little effect on nNOS and iNOS activity. A comparative NMR study between Y99E CaM and wt‐CaM bound to the eNOS peptide was performed. This investigation provides important insights into the effects that CaM phosphorylation has upon the binding and activation of the NOS enzymes. This work was funded by the NSERC of Canada.
The role of oxygen in bone regeneration
Authors: Sammarco Mimi , Simkin Jennifer , Castillo Jaime , Muneoka Ken
Published: June 2021

One in every 200 people in the U.S. will suffer an amputation. Amputation has protracted effects on vocational outcome, individual productivity, and personal independence far beyond the initial trauma. Amputation of the digit tip within the terminal phalangeal bone of rodents, monkeys, and humans results in near‐perfect regeneration from a cell population called the blastema, which has been well characterized in both adult and neonatal mice. However, it remains unclear why amputations within the more proximal phalangeal elements, or within limbs, fail to produce the same regenerative result. The objective of this study is to gain insight into the role of oxygen in bone regeneration by manipulating oxygen to alter the regenerative process. Using the murine amputation model, we have developed a novel multi‐tissue in vitro slice culture model that is used with mixed gas to control oxygen tension in vitro. To our knowledge, this is the first time such a model has been used in digit regeneration and shows promise as a model for studies involving bone remodeling. Using this model we have determined that 3% oxygen delays bone redifferentiation while 20% oxygen promotes redifferentiation into bone. Incubation of the blastema at 3% oxygen, prior to incubation at 20% oxygen, further accelerated the rate of bone formation. Our results show that the use of sequential incubation in 3% and 20% oxygen can be used to accelerate the regenerative process of bone when used at the correct temporal interval during the regenerative process. Research enabled by this work will lead to a defined mechanistic understanding of oxygen's role in bone regeneration and identify therapeutic targets to enhance regenerative capacity.
Requirements of Gαi1 and Gαi3 for BDNF and NGF induced PI3K/AKT and MAPK signaling, neuronal survival and migration
Authors: Cao Cong , Marshall John
Published: June 2021

Our previous study has indicated an unexpected and critical role of Gαi proteins in mediating receptor tyrosine kinase signaling (Cao et al., Science Signaling, 2009). Here we discovered a novel function of Gαi proteins in BDNF/NGF signaling. Gαi proteins are required for Brain‐Derived Neurotrophic Factor (BDNF) and Nerve Growth Factor (NGF) to transduce signals from Trk receptor to stimulate MAPK (p38, JNK and ERK) and PI3K/AKT/mTORC1 signaling. Knockout of Gαi1 and Gαi3 completely blocked BDNF and NGF induced PI3K/AKT/mTORC1 and MAPK activation in MEFs (mouse embryonic fibroblasts). Both Gαi1 and Gαi3 associated with the Grb2‐associated binding protein 1 (Gab1), and were required for the interaction of Gab1 with SHP2 as well as the interactions of Grb2 with SHP2, Shc, MEKK1 and MEKK2, in response to BDNF and NGF. Loss of Gαi1 and Gαi3 impaired the phosphorylation of Gab1, SHP2 and Shc in response to BDNF and NGF. Importantly, siRNA knockdown of both Gαi1 and Gαi3 inhibited BDNF and NGF induced AKT/mTORC1 and MAPK activation in cerebellar granule neuron, and BDNF induced neuronal survival, neuronal migration and neurite outgrowth were also largely impaired in cerebellar neurons with Gαi1 and Gαi3 deficiency. Overall, these results showed that Gαi1 and Gαi3 lie downstream of Trk receptor, but upstream of Gab1‐Shc‐Grb2‐Shp2, mediating MAPK and PI3K/AKT/mTORC1 signaling as well neuronal survival and migration.
The role of PHLPP in regulating cell migration in pancreatic cancer
Authors: Smith Alena , Gao Tianyan
Published: June 2021

Pancreatic cancer is the fourth leading cause of cancer in the United States. However, it has the worse prognosis of any malignancy with a 4% 5 year survival rate. Contributing factors include but not limited to late stage diagnosis due to vague symptoms and the degree of aggression associated with this type of cancer. Surgical resection is impossible in 50% of cases due to the advanced stage at the time of diagnosis. Pancreatic Ductal Adenocarcinoma (PDA) has a high rate of chemotherapy and radiation resistance increasing the need for innovative treatment modalities. Hyperactivation of PI3K/Akt pathways is commonly associated with many types of human cancers including pancreatic cancer. Recently, we have identified a novel protein phosphatase, PHLPP (PH domain Leucine repeat Protein Phosphatase) that dephosphorylates Akt and terminates Akt signaling directly. Our previous studies show that loss of PHLPP expression results in an increase in cell proliferation and a decrease in apoptosis. Here, we investigate the role of both PHLPP isoforms namely PHLPP1 and PHLPP2 in regulating pancreatic cancer cell migration and metastasis. Stable pancreatic cancer cell lines were generated using lentivirus‐based shRNAs to knockdown endogenous PHLPP expression. Alteration of Akt‐mediated signaling is analyzed in PHLPP knockdown cells. In addition, cell migration and invasion assays are performed to determine if loss of PHLPP expression promotes both tumorigenesis and metastasis in pancreatic cancer. The knowledge gained in these studies will provide a novel therapeutic avenue using PHLPP as a potential target.

Funded by NIH R01CA133429, and American Cancer Society RSG0822001TBE.
The phosphoramidase competency of prototypical phosphatase motifs
Authors: Haney Mark P. , Hengge Alvan C.
Published: June 2021

The discovery that phosphorylation of proteins occurs on nitrogen by particular kinases raises the question of whether a separate class of phosphoramidases also exists, or, if known phosphatases carry out the hydrolysis of phosphoramidates. The phosphoramidase activity of a number of phosphatases with different catalytic motifs was studied using the substrates N‐phenylphosphoramidate (N‐PhPAM) and phosphoryl imidazole (PIm). The phosphatases assayed were: the protein tyrosine phosphatase YopH; alkaline phosphatase; the dual‐specificity phosphatase VHR; PHPT1, the only known phosphohistidine phosphatase; and, the serine/threonine phosphatase PP1. The catalytic efficiencies, kcat/Km (s−1M−1), were compared for the respective phosphoramidase and phosphatase activities for each enzyme. Ratios of catalytic efficiencies (kcat/Km)/(kcat/Km) of phosphoester (pNPP or phenyl phosphate) over PIm are: YopH – 43; AP – 4.1; VHR – 0.23; and PHPT1 – 0.0065. PP1 exhibited no phosphoramidase activity. The results show that most phosphatase catalytic motifs display catalytic promiscuity by cleaving both phosphoesters and phosphoramidates, but with a pronounced preference for one substrate type versus the other. (Funding from NIH GM47297)
Oxidative stress responses in Caenorhabditis elegans with reduced mrck‐1 expression
Authors: Rigoulot Stephen , Blondeaux Sarah , Russo Matthew , Erickson Patti T.
Published: June 2021

MRCK‐1 (myotonic dystrophy kinase‐related Cdc42 binding kinase) acts downstream of the Rho GTPase CDC‐42 to determine cell polarity and is critical for C. elegans embryonic development. Given the conserved GXXXXCGKTC redox‐sensitive motif in the phosphoryl‐binding loop of CDC‐42, a role for MRCK‐1 in cellular responses to reactive oxygen species (ROS) is being explored. An RNA interference (RNAi) construct of 190 bp targeting exon 5 of the mrck‐1 gene on chromosome V was cloned into the RNAi expression vector pL4440 to enable production of double‐stranded RNA. Mrck‐1 RNAi induction in worm strains with oxidative stress responsive green fluorescent protein (GFP)‐ promoter fusions, including hsp‐16.2, sod‐3 and gst‐4, enables the measurement of oxidative stress induced GFP expression. Ongoing experiments include exposing worm strains to temperature, ultra‐violet (UV) light and chemical stressors to elucidate a possible phenotype related to diminished MRCK‐1 function under oxidative stress conditions. This work was supported by the Henson Undergraduate Research Grant program.
Development of biosensor for protein tyrosine kinases
Authors: Wei Lan-Yi , Yuan Chiun-Jye
Published: June 2021

Protein tyrosine kinase plays an important role in many cellular events, and many diseases are found to be associated with the dysregulation of various protein tyrosine kinases. However, the conventional methods for the detection of the activity of tyrosine kinase were expensive, specific instrument required, and high risk of radioactive contamination. In this study, A simple, quick and label‐free detection method was developed and developed. The general concept of this detection method is based on the fact that tyrosinase‐based amperometric biosensor can catalyze an electrochemical response of tyrosine but not its phosphorylation form, phosphor‐tyrosine. Further studies showed that the tyrosinase‐based biosensor could recognize tyrosyl residue within either dipeptides or heptapeptide p60c‐src substrate I, a specific peptide substrate for Src kinase. The linear dynamic range of detection to p60c‐src substrate I was studied with a sensitivity of 0.71μA μM‐1cm‐2. The effect of various kinase inhibitors in the Src activity was also studied.

Financially supporting by NSC‐97‐2311‐B‐009‐001 MY3.
Purification and identification of Polyisoprenylated Methylated Protein Methyl Esterase from Porcine Brain
Authors: Duverna Randolph , Aguilar Byron J , Amissah Felix , Lamango Nazarius S
Published: June 2021

Polyisoprenylated methylated protein methyl esterase (PMPMEase) is a key enzyme in the polyisoprenylation pathway (PP). This PP is important for the post‐translational modification of proteins which regulate cell growth, differentiation and apoptosis. Porcine Liver Esterase (PLE), an isoform of PMPMEase has been isolated and characterized. A Recent study strongly suggests the existence of a brain isoform of PMPMEase. The principal objective was to isolate, identify and characterize the brain isoform of porcine liver esterase. Using various chromatographic techniques, brain PMPMEase was partially purified to few bands. Active fractions were analyzed by SDS‐PAGE and the coomassie bands digested with trypsin, and analyzed using MALDI‐TOF/TOF Mass Spectrometry (MS) followed by Mascot database searches. The enzyme was identified from SwissProt, NCBI and MSDB databases as an endoplasmic reticulum, single‐pass calcium‐independent phospholipase A2. This is interesting given the similar characteristics between the known lipid substrates of the enzyme and the polyisoprenyl moieties of the putative protein substrates. This information will now be used to produce recombinant enzyme for substrate and inhibitors kinetics studies.
Anatomy for Physician Assistants: A program director's perspective
Authors: Luke Sharon
Published: June 2021

The Review Commission on the Education of the Physician Assistant, Inc. (ARC‐PA), the body that accredits physician assistant (PA) programs in the United States, requires that all entry‐level PA programs instruct their students in Anatomy. The commission does not dictate, however, how programs provide that instruction.

Students generally complete a didactic phase in physician assistant education that spans 12–15 months. Subsequent to the didactic phase of the program, students complete a clinical phase, in which they apply the principles of medicine they learned during the didactic phase to patient management encounters.

This session will review the most popular ways that programs provide Anatomy instruction to their students, and what components of Anatomy instruction are most important to program directors in relation to curriculum mapping and learning outcomes for their programs.
Anatomy for physician assistants: how we tackle this beast
Authors: Mabee John
Published: June 2021

Although Physician Assistants (PA) are dependent medical practitioners, they practice medicine with a degree of autonomy requiring a knowledge base of sufficient breadth and depth to function as primary care providers. Factual knowledge in the basic sciences must at a minimum allow for effective examination of patients, performance of common technical skills, interpretation of common imaging studies, and of course, safe medical practice. The USC Primary Care PA Program of the Keck School of Medicine delivers a 6 unit integrated Basic Medical Sciences (BMS) course to first year PA students during the first semester of a six‐semester program. Anatomy (33% of BMS) covers regional and radiographic anatomy, and introductory histology. Learners also rotate through four, 2‐hour prosection‐driven cadaver laboratory sessions. The remainder of the BMS course is comprised of systems physiology (52%), and adjunct topics (15%) (eg. introductory genetics and pathology). Course objectives for anatomy were derived from a mix of published standard‐setting documents for medical students and the course director's clinical and academic experience. Learning strategies include lecture, assigned readings, audiovisual resources, and cadaver laboratory. The course is taught by a PA‐PhD, and laboratory sessions are taught by Biokinesiology and Physical Therapy anatomy faculty.
Ligand‐induced conformational changes in cytochrome P450 3A4 detected by luminescence resonance energy transfer (LRET)
Authors: Sineva Elena V , Rumfeldt Jessica , Bakulina Anastasia Yu , Halpert James R , Davydov Dmitri R
Published: June 2021

Recent advances in solving crystal structures of cytochromes P450 have revealed a remarkable plasticity that allows the enzymes to accommodate substrates of different shapes and sizes. However, the data on conformational mobility of cytochromes P450 in solution are limited, and the role of conformational plasticity in their allosteric behavior remains obscure. The major human drug‐metabolizing cytochrome P450 3A4 (CYP3A4), exhibits both homo‐ and heterotropic cooperativity. We probed ligand‐induced conformational changes in the enzyme with LRET. For this purpose we produced a number of mutants containing only two cysteine residues accessible to modification. Unchanged ligand binding properties of the Cys64/Cys468 mutant prompted its use in the further studies. Successive labeling of this mutant with the phosphorescent dye erythrosine and a far‐red fluorophore DY‐731 allowed us to create an intramolecular pair of LRET donor and acceptor. This double‐labeled protein was used to probe the conformational response of the enzyme upon interactions with a series of ligands. The changes in the efficiency of LRET induced by the substrates 1‐pyrenebutanol and bromocriptine were negligible. In contrast, a pronounced effect of such ligands as α‐naphtophlavone or quinidine reveals a conformational transition induced by these heterotropic activators. (Supported by GM054995).
VEGF Signaling in Health and Disease
Authors: Iruela-Arispe Luisa
Published: June 2021

Vascular Endothelial Growth Factor (VEGF), also known as vascular permeability factor, is a homodimeric 46kDa protein that signals through two main tyrosine kinase receptors, VEGFR1 (flt) and VEGFR2 (flk, KDR). In addition to these receptors, the non‐tyrosine kinase receptors: Neuropilin1 and Neuropilin2 can bind to VEGF and contribute to signaling. Activation downstream VEGF receptors have been recognized to initiate a series of events resulting in the morphogenesis of blood vessels. However, amongst all these receptors, VEGFR2 is known to be the major mediator of VEGF‐responses associated with angiogenesis. In fact, pharmacological blockade of either the VEGF or VEGFR2 results in the suppression of angiogenesis, as shown in several pathological models. These findings have resulted in the generation of antibodies and small molecule inhibitors. Many of these molecules are currently in the clinic to suppress the angiogenic cascade in cancer, vascular retinopathies and other afflictions. Interestingly, the interventions have worked well in some settings, but are not as effective in other situations. The basis for this disconnect is unclear at this point. Our laboratory has recently uncovered a novel mean for VEGFR2 activation that does require ligand and is also independent of the kinase activity of the receptor. This ligand‐independent, kinase‐independent mode of VEGR2 activation results in downstream signaling but is not affected by current modes of pharmacological blockade of the pathway. In this symposia, we will present our findings, disclose the pathway and discuss its potential impact to several pathological conditions.
The role of phosphatidate phosphatase in the yeast chronological life span
Authors: Park Yeonhee , Han Gil-Soo , Carman George M.
Published: June 2021

In Saccharomyces cerevisiae, the PAH1‐encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol, plays a crucial role in the synthesis of the storage lipid, triacylglycerol, and in the regulation of phospholipid synthesis. The viability of yeast in stationary phase is closely related with functions of mitochondria. In this study, we showed that the pah1Δ mutant had a significantly shorter chronological life span than wild type when grown in synthetic growth medium and incubated in water. In the absence of fermentable carbon sources, the pah1Δ mutant utilized ethanol, but had a great defect in the utilization of glycerol and lactate as a non‐fermentable carbon source. Moreover, the pah1Δ mutant exhibited higher sensitivity to hydrogen peroxide which is known to generate reactive oxygen species. These results suggest that the role of PAH1‐encoded PA phosphatase is involved in cell viability that is mediated by the mitochondrial function. Supported by NIH grant GM 28140.
Acyl chain dependent product inhibition by phosphatidylserine synthases
Authors: Kimura Atsuko Kakio , Kim Hee-Yong
Published: June 2021

Phosphatidylserine (PS), the major anionic phospholipid in eukaryotic cell membranes, is synthesized by PS synthase 1 (PSS1) and PSS2 through the serine‐base exchange reaction from phosphatidylcholine (PC) and phosphatidylethanolamine (PE), respectively. Membrane PS is critically involved in key cell signaling processes such as Akt, PKC and Raf‐1 activation. The membrane PS profile is well maintained although mechanisms for the regulation of the PS profile are not clearly understood. In this study, we investigated the effect of PS acyl chain on the feedback mechanism in which activities of PSSs are inhibited by product PS. We found that PSS1 and PSS2 have significantly different inhibitory response to two PS molecular species, oleic acid (OA, 18:1n‐9) containing (OA‐PS) and docosahexaenoic acid (DHA, 22:6n‐3) containing PS species (DHA‐PS). PSS1 was inhibited more strongly by OA‐PS, while PSS2 was more by DHA‐PS. Together with the differential expression between PSS1 and PSS2 in variety of cells and tissues, these results indicate that the level of individual PS molecular species is regulated through feedback mechanism by which synthetic activity of each enzyme is inhibited differently.

Intramural Program of National Institutes of Health
Endoplasmic reticulum stress induces the degradation of stearoyl‐CoA desaturase protein in human retinal pigment epithelial cells via ubiquitin‐proteasome pathway
Authors: Samuel William , Kutty R. Krishnan , Duncan Todd , Redmond T. Michael
Published: June 2021

Perturbations to endoplasmic reticulum (ER), the central organelle for protein biosynthesis and maturation, can cause ER stress, and if unchecked could lead to apoptosis. Stearoyl‐CoA desaturase (SCD), an ER resident protein and a key enzyme in the biosynthesis of monounsaturated fatty acids, is known to regulate cellular functions by controlling the ratio of saturated to monounsaturated fatty acids. SCD has been reported to be degraded through both ubiquitin‐proteasome‐dependent and‐independent pathways when overexpressed in cells. The purpose of this study was to determine whether ER stress affects the expression of SCD protein. Human retinal pigment epithelial (ARPE‐19) cells treated with N‐(4‐hydroxyphenyl)retinamide (4HPR), tunicamycin and thapsigargin, compounds known to induce ER stress, showed a time‐dependent decrease in the expression of SCD protein, while, BiP/GRP78, a protein marker for ER stress, was markedly increased. The decrease in SCD protein expression was completely blocked by the proteasome inhibitor, MG132. Immunoprecipitation analysis of 4HPR‐treated cell lysate using ubiquitin antibody showed that ER stress increased the ubiquitination of proteins including SCD. Thus, these data show that ER stress mediates the degradation of SCD in human RPE cells via the ubiquitin‐proteasome dependent pathway

Support: Intramural Research Program, NEI, NIH
Molecular dissection of syndecan‐1 mediated triglyceride‐rich lipoprotein clearance
Authors: Gonzales Jon C , Foley Erin M , Gordts Philip L , Esko Jeffrey D
Published: June 2021

Recently, genetic experiments in mice have confirmed that the heparan sulfate proteoglycan syndecan‐1 acts as a receptor for triglyceride‐rich lipoprotein (TRL) clearance in the liver. Binding depends on the heparan sulfate chains based on the accumulation of plasma TRLs in mice bearing mutations in heparan sulfate biosynthesis. To gain further insight into interaction of TRLs with syndecan‐1, we used an adenovirus reconstitution strategy. Reconstitution of mice lacking syndecan‐1 with virus containing wild‐type syndecan‐1 restored clearance in vivo, whereas viruses containing syndecan‐4 or mutant syndecan‐1 lacking the heparan sulfate chains did not. The intracellular C‐terminal domain also proved necessary for uptake. To identify the major apolipoprotein on the surface of TRLs that mediates binding to the heparan sulfate chains, we measured binding of TRLs isolated from mice lacking the major apolipoproteins, ApoB100, ApoB48, or ApoE. Particles derived from ApoE−/− mice did not bind, whereas no significant difference was noted in binding of ApoB100‐ and ApoB48‐ deficient TRLs compared to TRLs from wild‐type mice. These studies demonstrate that apoE is the dominant ligand for binding of TRLs to the heparan sulfate chains of syndecan‐1 and explains why dual deficiency of syndecan‐1 and LDL receptor results in a synergistic accumulation of plasma triglycerides.