bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biochemical analysis of human ERK2 mutants reveals important residues in MEK1‐ERK2 binding and phosphorylation
Authors: Ye Ming , Hotan Quynn , Glatz Gwendolyn , Finkelstein Maxwell T. , Hayashi Scout , Miller Emma P. , Lau Benjamin , Li Kevin , Chen Han , LeBlanc Julia , Zaidi Hussain , Fera Daniela 
Published: May 2022

The Ras‐Raf‐MEK‐ERK (MAPK) pathway is a signal transduction cascade used to regulate cellular processes including cell cycle progression and proliferation. Aberrant activation of this pathway is implicated in cancer development, and treatment with Raf, MEK and ERK inhibitors often leads to adaptive resistance. Combination therapies have been shown to offer benefits; however, the MEK‐ERK interface remains poorly understood, hindering structure‐guided approaches to the design of potent MAPK pathway inhibitors targeting this complex.To identify important residues for the MEK1‐ERK2 interaction, we performed site‐directed mutagenesis on ERK2. We used circular dichroism to assess the secondary structure of the ERK2 mutants. We also used biolayer interferometry binding experiments coupled with phosphorylation assays to evaluate the impact of the mutated residues on the formation of the MEK1‐ERK2 complex and activity.Circular dichroism showed no differences in secondary structure for any of our ERK2 mutants. Of all the mutations generated, the L234D mutation in ERK abrogated binding and phosphorylation by MEK1 the most. Other mutants showed some reductions in binding or activity but require further analysis. Of note, L234 is located on an ERK2 α‐helix adjacent to the phosphorylation lip, consistent with MEK1 binding this face during phosphorylation. Our results suggest that this α‐helix may play critical roles in the MEK1‐ERK2 complex. Studying the impact of additional mutations in this and additional regions will develop our understanding of the MEK‐ERK interface and inform the design of allosteric inhibitors that can modulate MEK‐ERK complex formation.
Immune Cell Regulation by Macrophage Derived Small Extracellular Vesicles in Pain
Authors: Pande Richa , Jean Toussaint Renee , Muir Roshell , Haddad Elias E. , Ajit Seena 
Published: May 2022

BackgroundDevelopment of chronic pain is related to aberrant neuroimmune signaling. Small extracellular vesicles (sEVs) are 30‐150nm size particles of endosomal origin. They play an important role in neuroimmune crosstalk facilitating transfer of biomolecular cargo between cells. Our studies show that sEVs from RAW 264.7 macrophage cells can be therapeutic or prophylactic in mouse model of inflammatory pain. We investigated the mechanistic basis of how sEVs from lipopolysaccharide (LPS) stimulated (Exo+) and unstimulated (Exo‐) cells impact primary glial cells, dendritic cells (DCs) and adaptive immune cells such as CD4+T cells. We hypothesize that macrophage derived sEVs can potentiate T cell activation directly, or indirectly by the upregulation of major histocompatibility complex‐II (MHC‐II) and co‐stimulatory molecules on DCs, attenuating pain hypersensitivity. We will also test if sEVs confer protection to glial cells in the context of inflammation.MethodsPrimary mouse DCs, microglia and astrocytes were incubated with 1µg Exo‐/+sEVs for 24 hours. Splenocyte derived CD4+ T cells were treated with 0.1ug Exo‐/+ sEVs for 72 hours. Flow cytometry and qPCR was used for analysis.ResultsCharacterization of RAW sEVs showed the presence of sEV markers CD81 and Alix. sEV treatment of DCs resulted in an increase in CD80+/CD86+ MHCIIhiCD11c+/hi DCs. Treatment of CD4+T cells with sEVs increased Tgf‐βexpression. Pretreatment with Exo+ sEV increased Tfgf‐β expression in glial cells upon LPS stimulation. An increase in PPAR (Peroxisome proliferator activated receptor) expression was observed upon sEV treatment of glial cells.ConclusionUpregulation of MHC‐II and costimulatory molecules on DCs as well as PPAR upregulation in glial cells upon sEVs treatment indicates that sEVs from RAW 264.7 cells can modulate immune response. These results indicate sEVs modulate innate cells such as microglia and DCs and adaptive immune cells such as CD4+ T cells may contribute to therapeutic effect in attenuating chronic pain.
Cranial Dysmorphology in the Palatine, Vomer, and Pterygoid Plates of Fgfr2mice
Authors: Pennings Amanda N. , Ogg Miette L. , McNulty Margaret A. , Holmes Megan A. , Mussell Jason C. , DeLeon Valerie B. 
Published: May 2022

The fibroblast growth factor and receptor system (FGF/FGFR) regulates cell proliferation and differentiation for various tissue types in the body – including osseous tissue. Mutations in the FGFR2 gene are associated with craniosynostosis, leading to alterations of the shape of bones in the head and face. We have previously demonstrated premature fusion of the spheno‐septal synchondrosis in Fgfr2C342Y/+ mice, a model for Crouzon syndrome. Here, we further describe the specific morphology of individual bones as a result of the Fgfr2C342Y/+ mutation.We used microCT scans of mutant and wild‐type littermates at two developmental stages (adult and postnatal day 14) to create 3D virtual reconstructions of the skull in the program 3DSlicer. After transforming each scan to a uniform, orthogonal orientation, we collected coordinate data for 38 fixed craniometric landmarks to estimate the shape of the cranium. We used the geomorphpackage in R to perform a Procrustes superimposition and principal components analysis (PCA). We used the resulting shape coordinates in a two‐way Procrustes ANOVA to test the effects of Age and Genotype. In addition, we isolated key cranial elements (palatine, vomer, and pterygoid plates of the sphenoid) to compare between genotype groups at the P14 stage using a mesh‐to‐mesh comparison. This allowed us to test our expectation that fusion of the spheno‐septal synchondrosis has a greater impact on the vomer than on the palatines or pterygoid plates.Our morphometric analysis and two‐way Procrustes ANOVA confirmed previous reports of statistically significant cranial shape differences between wild‐type and Fgfr2 mutants at both timepoints. Vomer shape was significantly altered in the mutant P14 cranium when compared to other key cranial elements (palatine, and sphenoidal pterygoid plates), suggesting that fusion of the spheno‐septal synchondrosis has a greater impact on the vomer compared to the other bones in the skull. Our mesh‐to‐mesh comparison showed that the vomer is both dorso‐ventrally and rostro‐caudally compressed in mutants relative to wild‐type mice at this early stage. Additionally, the palato‐sphenoid suture appears prematurely fused in the mutants.This study provides insight into the skeletal phenotypic mechanisms that characterize diseases involving mutations of the FGFR2 gene, such as Crouzon syndrome. The dysmorphology of individual bones affect other osseous structures due to morphological integration within the skull. Elucidating the underlying mechanisms of the phenotypic expression of FGFR2 syndromes supports further translational research.
Physical Therapy Student Knowledge Retention from a Remotely Taught Anatomy Course
Authors: Nesbitt Allison , Briedwell Teresa 
Published: May 2022

IntroductionA whole‐body donor anatomy course is a foundational learning experience for additional musculoskeletal courses for Doctor of Physical Therapy (DPT) students. The summer 2020 human anatomy course at the University of Missouri was taught remotely with daily asynchronous and synchronous components. The DPT students were evaluated with online multiple‐choice lecture and picture‐based lab practical exams. Due to the novel nature of the course, student knowledge retention and interpretation of three‐dimensional anatomy is unknown.AimThe purpose of this study was to evaluate student retention of anatomy knowledge from the remotely taught course, test if students can identify structures on anatomical donors in a laboratory setting that they previously identified on picture online exams and survey the students’ prior anatomy experiences.MethodsA 90‐minute in‐person laboratory session was held seven months after the completion of the remote anatomy course for first year DPT students. Students’ previous anatomical course work and opinions of remote and lab learning were assessed with a survey. Prior to entering the anatomy lab, the students completed a multiple‐choice retention quiz of eight questions from the four‐unit exams that all students previously answered correctly. During the laboratory session, students worked in pairs to answer fifteen questions and identify some of the same structures from the retention quiz on the donors.ResultsEvery student had taken at least one undergraduate course as a requirement for DPT school admission or as part of their major. All the students described benefits to a lab‐based anatomy course compared to a remote course and a majority emphasized the hands‐on and spatial learning in their comments. Most students (73.7%) also believed there were some advantages from the remote course, including having additional study time, cost savings, and more individualized learning. Fifty‐nine of the 60 students in the remote anatomy course took the retention quiz and the average score was 86.7% (range 62.5% ‐ 100%). Students performed well on the musculoskeletal questions on the lab identification quiz.ConclusionThe physical therapy students from this remote anatomy course had slightly higher retention scores than most studies of health professions students’, particularly in topics that were repeated throughout their first‐year courses.SignificanceThe study supports the importance of whole‐body donor anatomy lab learning for students and the importance of flexibility, and ample inclusive access to learning materials for students.
Development of Cancer‐on‐a‐chip Technology for the Study of Tumor Microenvironment and Metabolism
Authors: Ericson Daniel L. , Azukas Joshua , Strey Helmut H. , Rubenstein David 
Published: May 2022

Cancer metabolism has become an area of intense interest. In the 1920s, Otto Warburg showed that cancer cells metabolize glucose to produce lactate and ATP in the presence of oxygen, i.e. aerobic glycolysis, also known as the Warburg Effect. It is now appreciated that cancer cells display a complex metabolic phenotype. Metabolic reprogramming by oncogenes and tumor suppressor genes has been linked to the altered metabolic function of cancer cells. However, the tumor microenvironment (TME), which has different nutrient gradients for glucose, oxygen, and amino acids, has a dramatic effect on cancer cell growth and proliferation. In vivo studies have demonstrated a heterogeneous metabolic phenotype that is difficult to characterize. In addition, the exchange of nutrients and metabolites between the TME, vasculature, and tumor cells has been difficult to fully recapitulate. As a tumor grows, nutrients diffuse from the blood vessels to the cancer cells, in a process partially described by A. Krogh in 1919 through a reaction‐diffusion differential equation. Krogh’s work showed that for typical tissues the oxygen decay radius is around 150μm and we find approximately the same ‘Krogh radius’ for high glucose consumption cancer cells (e.g. MDA‐MB‐231). Thus, around 150μm from a blood vessel exists an oxygen deprived environment that imposes tremendous competitive stresses on the cancer cells for survival. There is rising evidence that this selection pressure favors the transformation to metastasis and causes invasive tumor cell lines to exhibit high glycolytic rates and overall metabolic reprogramming. However, most studies focused on defining the metabolic function of tumor cells have been conducted in vitro, under conditions that do not represent the physiological conditions accurately.Here, we present a cancer‐on‐a‐chip technology that produces a well‐defined 3D TME that can be exposed to precise nutrient stresses such as varying oxygen or other nutrient gradients. In addition, we can determine the metabolic phenotype of cancer cells with varying oncogenic changes and degrees of aggressiveness. Our technology should be able to address the question of the role of the TME on metabolic reprogramming and in particular glycolysis. Our design is based on growing tumor microenvironments inside 360µm ID capillary tubing that can be accessed by 360µm PEEK tubing. To allow/control perfusion through the microenvironment we embed a 70µm nylon fiber into a photocross‐linkable gelatin gel in which the cells are dispersed (MDA‐MB‐231/GFP). After exposing the gel to UV‐light for a few seconds, the nylon fiber is pulled, leaving a perfusable microchannel. Using this technique, we have successfully perfused cell‐laden hydrogels over weeks while observing cell‐growth in a fluorescence microscope.
Substrate‐Dependent Differential Regulation of Mitochondrial Bioenergetics and ROS emission in the Heart and Kidney Cortex and Outer Medulla
Authors: Tomar Namrata , Sadri Shima , Zhang Xiao , Kandel Sunil , Yang Chun , Audi Said , Cowley Allen W. , Dash Ranjan K. 
Published: May 2022

RationaleThe kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) is determined by the availability and utilization of particular respiratory substrates. Although the heart and kidney represent the two major energy consuming organs in the body, the substrate dependency of their mitochondrial OxPhos has not been characterized for these two organs within the same species and same individual animal.MethodThe effects of different substrate combinations on the kinetics and efficiency of OxPhos were characterized in mitochondria isolated from the heart, renal cortex, and renal outer medulla (OM) of adult Sprague‐Dawley rats. The substrate combinations included pyruvate+malate (PM), glutamate+malate (GM), palmitoyl‐carnitine+malate (PCM), alpha‐ketoglutarate+malate (AM), and succinate±rotenone (Suc±Rot). Mitochondrial bioenergetics (O2 consumption and membrane potential) and H2O2 emission were studied using two protocols: addition of a fixed [ADP] and sequential additions of incremental [ADP] in the presence of different substrates to determine how these substrates influence mitochondrial functional markers in each of these tissues.ResultsResults show that the kinetics and efficiency of mitochondrial OxPhos are highly dependent on the substrates used and this dependency is tissue‐specific. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates used in comparison to kidney mitochondria. Renal cortex mitochondrial respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. It was also found that heart mitochondria do not synthesize ATP efficiently from ADP with Suc in the absence of Rot (blocker of complex I). This may be due to (i) accumulation of oxaloacetate (OAA) and/or (ii) occurrence of reverse electron transfer (RET). In contrast, cortex and OM mitochondria achieved the same respiratory rates with Suc±Rot suggesting that efficient conversion of ADP to ATP by kidney mitochondria can occur in the presence of Suc. Similar differences were observed in mitochondrial membrane potential. However, differences in H2O2 emission in the presence of Suc±Rot were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. The bioenergetics and H2O2 emission data observed with Suc±Rot indicate that OAA accumulation and RET play a more prominent regulatory role in heart mitochondria than in kidney mitochondria.ConclusionThis study provided novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial bioenergetics and H2O2 emission in a tissue‐specific manner.
Computational Modeling of Substrate‐Dependent Differential Regulation of Mitochondrial Bioenergetics in the Heart and Kidney Cortex and Outer Medulla
Authors: Zhang Xiao , Tomar Namrata , Sadri Shima , Audi Said , Cowley Allen W. , Dash Ranjan K. 
Published: May 2022

RationaleIt is recognized that the kinetics and efficiency of mitochondrial O2 consumption for ATP production depend on the choice of respiratory substrates. Various substrates differentially generate the reducing equivalents NADH and FADH2 via the tricarboxylic acid (TCA) cycle that feed electrons to the electron transport chain (ETC) driving oxidative phosphorylation (OxPhos). However, the contributions of these substrates and their combinations have not been systematically characterized in either the heart or the kidney, the two major energy consuming organs in the body.MethodBioenergetic responses (respiration and membrane potential) of mitochondria isolated from the heart and kidney cortex and outer medulla (OM) of adult Sprague‐Drawly rats were characterized with different substrate combinations and different ADP perturbations. To better understand these distinct substrate and tissue‐specific mitochondrial bioenergetics, thermodynamically‐constrained mechanistic computational models were developed by integrating the kinetics and regulation of mitochondrial substrate transport and oxidation, TCA cycle, and ETC/OxPhos, incorporating published data and the novel mitochondrial bioenergetics data from our laboratory. Intrinsic model parameters such as Michaelis‐Menten constants were assumed identical and fixed for the heart and kidney, while extrinsic model parameters such as maximal enzymatic rates were separately estimated based on experimental data for the heart and kidney mitochondrial bioenergetics.ResultsConsistent with the data, model simulations of the ADP‐induced state 3 responses of the heart and kidney cortex and OM mitochondria yielded dramatically different values, which for a given organ were also very distinct for different substrates. In addition, heart mitochondria energized with succinate without rotenone (blocker of complex I) and saturated [ADP] failed to produce a robust state 3 response. This was in contrast to kidney cortex and OM mitochondria for which responses to succinate±rotenone were similar, indicating that reverse electron transfer via complex I played less prominent regulatory role in the kidney than the heart. In addition, oxaloacetate, a TCA cycle intermediate, was shown to accumulate faster in the heart compared to the kidney, thereby potently inhibiting succinate oxidation more in the heart than the kidney. As such, the model was able to quantitatively characterize how various substrate combinations account for the differential contributions of different substrate oxidation pathways within the mitochondria towards OxPhos and ATP synthesis in the heart and kidney cortex and OM.ConclusionModeling of these data provided a deeper quantitative understanding of key determinants of kinetic and molecular mechanisms involved in the differential regulation of substrate‐dependent mitochondrial bioenergetics in the heart and kidney cortex and OM.
FABP1: A Potential Biomarker for Nephropathy in Type II Diabetes
Authors: Prabhakar Sharma , Deshmukh Hemalata , Cooper Darbie 
Published: May 2022

Aims of the studyNephropathy is an important and frequent complication of diabetes mellitus. Diabetic nephropathy (DN) is also the leading cause of end stage renal disease in most parts of the world. Albuminuria is the hallmark of DN although recent observations suggest that non‐albuminuric nephropathy accounts for as much as 40% of all cases of DN. There is a need for additional and more accurate biomarkers for diagnosis and prognosis of DN. Fatty acid binding protein 1 (FABP1) is normally expressed in the liver but also in the kidney in human and animal DN models. However, it has not been detected in normal rat kidneys. We hypothesized that FABP1 is consistently elevated in the kidneys of experimental nephropathy of type II diabetes and could be a diagnostic biomarker and may have a potential pathogenic role.MethodsMale and female ZSF1 rats were used as an experimental model for nephropathy in type II diabetes and CD rats (non‐diabetic rats) were used as controls. Animals were studied from 8th week and sacrificed at 40 weeks. Kidneys were harvested and tissue homogenates were used for total RNA extraction for Next Gen sequencing and protein extraction for western blots. Serum and urine samples were also collected at the start and end of the study to quantitate the renal function and excretion of markers.ResultsThe ZSF1 rats spontaneously developed obesity, diabetes and hyperlipidemia as expected. Male rats developed more severe hyperglycemia, renal failure and proteinuria (data previously communicated) than females. Next Gen Sequencing of kidney tissue revealed amongst other findings a 1396‐fold increased expression of FABP1. Examination of kidney tissue protein homogenates for the expression of FABP1 showed increased expression especially in the male ZSF1 rats. Examination of urine samples for the presence of FABP1 by ELISA showed remarkably increased levels (12 to15‐fold in males and 6 to 7‐fold in females) increase compared to CD rats. (See figure 1)ConclusionsBoth male and female ZSF1 rats developed metabolic syndrome and nephropathy although the disease was much more severe in male rats. FABP1 was expressed more in male rat kidneys compared to female and control rat kidneys. Additionally, the urine excretion of FABP1 was increased in both male and female ZSF1 rats compared to control rats. These data suggest FABP1 may be a potential biomarker in DN. Additional studies are warranted to examine the mechanisms driving the robust renal FABP1 production/excretion and the possible pathogenic role and potential of FABP1 as a therapeutic target in DN.