bluuebunny/crossref_metadata_2025 huggingface dataset:
Flat or flexible? Evolutionary cranial biomechanics and the origins of modern archosaur skulls
Authors: Holliday Casey M. , Wilken Alec , Sellers Kaleb , Cost Ian , Middleton Kevin
Published: May 2022

The divergent specializations of living archosaurs, crocodilians and birds, represent two of the great transformations in vertebrate evolution. Despite hailing from common ancestor with a tall skull, braced palate and relatively unspecialized jaw muscles, these two living clades followed disparate paths in cranial biomechanics and feeding functional morphology during the Mesozoic era, resulting in the flat, rigid skulls in crocodylians and the bulbous and beaked, flexible skulls of birds. Here we use new approaches in imaging, 3D modeling, morphometrics, lever mechanics, finite element modeling and phylogenetic comparative methods of living and extinct reptiles to show how morphological and functional changes in the palate, cranial joints and jaw musculature occurred within the fossil record of these lineages that were key features in acquisition of the modern forms. We show how jaw muscles reoriented their resultants and moments about jaw joints during skull flattening, palatocranial suturing and feeding specializations in Jurassic crocodyliforms. These morphological changes resulted in a decrease in mechanical efficiency of the feeding apparatus that was compensated for by an increase in jaw muscle sizes and canalized muscle architecture. Meanwhile, along the line to living birds, the acquisition of enlarged brains in coelurosaur dinosaurs resulted in a shifting of temporal and palatal muscle positions, a canalization of vertically‐oriented of muscle forces and consequently reorientation of their moments about palatocranial joints. These changes in loading environment, along with a concatenate increase in the propulsive and bending properties of the palate, facilitated cranial kinesis in neoavian birds. Together, these examples of evolutionary innovation in reptile cranial biomechanics reveal powerful new approaches to testing biomechanical and evolutionary hypotheses in any cranial or appendicular musculoskeletal system.
Express and Purify Human CRY2 for Functional Analyses
Authors: Pinal Iliana M. , Lopez Obed , Madariaga Alberto , Lazarski Aleksander , Xiao Chuan
Published: May 2022

The circadian rhythm is a biological clock responsible for regulating the daily cycles of essential physiological and metabolic processes, such as sleep wake cycles, by responding to light and dark changes. Every cell has its own circadian rhythm that is synchronized by a master clock in the brain. At the cellular level, circadian rhythms are controlled by a time‐delayed transcriptional‐translational feed‐back loop (TTFL), which consists of several core proteins. These proteins include circadian locomotor output cycles protein kaput (CLOCK), brain and muscle ARNT‐like 1 (BMAL1), cryptochrome (CRY), and period (PER). CLOCK and BMAL1 initiate the TTFL by activating the transcription of several circadian genes including CRYs and PERs that will be translated in the cytosol. With accumulation after a certain time, CRYs/PERs complexes will translocate back to the nucleus to negatively inhibit CLOCK/BMAL1 activities to finish the TTFL. There are two paralogs of CRY within the human genome: CRY1 and CRY2, both of which have essential roles in the negative feedback. The purpose of this project is to study the structure and function of human CRY2. Regardless of many advances in this field including available structure of CRYs, the detailed interaction between CRY2 to other circadian core proteins are still limited. Through this research we would be able to express and purify large quantities of hCRY2 that will be used for the future interaction studies. The results will deepen our understanding of the mechanisms behind circadian rhythm and its associated diseases, facilitating the development of prevention and treatment strategies.
Adenovirus increases arrhythmia susceptibility during acute cardiac infection
Authors: Padget Rachel L. , Blair Grace A. , North Michael D. , King D. Ryan , Zeitz Michael J. , Tanenbaum Mira N. , Hoeker Gregory S. , Swanger Sharon A. , Poelzing Steven , Smyth James W.
Published: May 2022

Myocarditis is responsible for 42% of sudden cardiac death in young adults, yet mechanisms underlying virally induced arrhythmia remain elusive. Adenovirus is a leading etiological agent of myocarditis but species‐specificity has limited development of animal disease models. Normal electrical impulse propagation in the heart is achieved through intercellular coupling via gap junctions, composed primarily of connexin43 (Cx43). Changes in Cx43 expression, localization, and/or function underlie the arrhythmias of sudden cardiac death. Given that gap junctions also propagate innate and adaptive antiviral immune responses, we hypothesized that adenovirus would target Cx43 to facilitate replication. Our prior work has demonstrated that Cx43 expression and function are indeed reduced rapidly during adenoviral infection limiting gap junction communication in human epithelial and cardiac cells. We are now employing a recently described cardiotropic strain of mouse adenovirus, MAdV‐3, to develop our work into whole animal studies and investigate virally‐induced alterations in cardiac electrophysiology and the molecular mechanisms of the resulting arrhythmogenic substrate. Adult mice were inoculated with MAdV‐3 and sampled after 7 days to model acute cardiac infection. We find MAdV‐3 viral genomes are specifically enriched in heart tissue, confirming cardiotropism. No cardiomyopathy was apparent by echocardiography or histopathology, consistent with human acute myocarditis patients. We find reductions in cardiac ion channel and connexin mRNA transcript levels after infection, and at the protein level, Cx43 is phosphorylated at residues known to reduce function. Ex vivo optical mapping experiments illustrate decreased conduction velocity in infected hearts. Turning to primary adult mouse cardiomyocytes, we detect prolonged action potential duration and impaired K+ current in infected cells by patch clamping. Confocal and super‐resolution localization microscopy of infected cardiac tissue and isolated cardiomyocytes reveals remodeling of gap junctions with alterations in complexing with scaffolding proteins and other ion channels at the cell‐cell junction. Finally, employing human iPSC‐derived cardiomyocytes and human adenovirus type‐5, we find increased Cx43 phosphorylation and, using optical mapping, perturbation of intercellular coupling during infection, just as we see with MAdV‐3. Our data demonstrate that reduced cellular coupling and ion channel function during adenoviral infection generates an arrhythmogenic substrate prior to an appreciable immune response or cardiomyopathy development. MAdV‐3 infection therefore provides a novel model of cardiac infection and myocarditis and provides physiologically relevant insight into mechanisms of virally‐induced arrhythmias.
ATP Attenuates α1‐Adrenergic and Peptidergic Vasocontraction in Isolated Porcine Cerebral Arteries
Authors: Luchkanych Adam M. , Morse Cameron , Tomczak Corey R. , Olver T. D.
Published: May 2022

Introduction

Competing influences regulate blood flow control in the brain. In the skeletal muscle vasculature, purinergic ATP signaling attenuates α1‐adrenergic and peptidergic‐induced vascular smooth muscle contraction (i.e., phenomenon referred to as functional sympatholysis); however, whether this occurs in the cerebrovasculature remains unknown. The purpose of this experiment was to examine the effect of ATP on vascular responses to α1‐adrenergic‐ and peptidergic‐receptor activation in cerebral arteries. We hypothesized that ATP would attenuate the α1‐adrenergic‐ and peptidergic‐mediated vasocontraction in isolated pial arteries.

Methods

Female pigs (n=5) were euthanized and their brains harvested. Thereafter, 1A branches of the middle cerebral artery were dissected for wire‐myography. Dose‐response curves for the α1‐adrenergic agonist phenylephrine (PE;1e‐10‐1e‐4M) and the peptidergic agonist neuropeptide Y (NPY;1e‐12‐1e‐6M) were performed in the absence or presence of ATP (1e‐6M). Paired one‐tailed t‐tests were used to compare the overall magnitude of contraction (area under the curve; AUC) and physiological maximal responses between conditions.

Results

Data are mean±SD. The AUC of PE‐mediated cerebral vasocontraction was attenuated by ATP (untreated=67±25 vs. ATP pre‐treatment=33±23 AU; p=0.03). However, the mean reduction in the maximal response to PE in arteries pre‐treated with ATP was not significant (untreated=43±15 vs. ATP pre‐treatment=27±11%; p=0.10). The AUC (untreated=62±31 vs. ATP pre‐treatment=37±23 AU) as well as maximal NPY‐mediated cerebral vasocontraction (untreated=65±20 vs. ATP pre‐treatment=49±21%) was attenuated by ATP (all p≤0.05).

Conclusion

These data indicate ATP attenuates PE‐ and NPY‐mediated vasocontraction in isolated pial arteries. Thus, similar to the skeletal muscle vasculature, purinergic signaling attenuates vasoreactivity to α1‐adrenergic and peptidergic receptor activation in the cerebrovasculature. Functional sympatholysis may assist in coupling cerebral blood flow to brain metabolism under basal conditions as well as in the setting of heightened sympatho‐excitation.
Visualizing an orphan receptor GPR158 and it complex with neuronal regulator RGS7‐Gβ5 via cryoEM
Authors: Patil Dipak N. , Singh Shikha , Laboute Thibaut , Strutzenberg Timothy , Qiu Xingyu , Wu Di , Novick Scott , Robinson Carol , Griffin Patrick , Hunt John , Izard Tina , Singh Appu , Martemyanov Kirill
Published: May 2022

G protein‐coupled receptors (GPCR) form the largest family of proteins (∼800 GPCRs) encoded in mammalian genomes that detect extracellular signals to program cellular response. They are essential to understanding physiology, disease, and drug development. More than 100 GPCRs are still awaits to identify its endogenous ligand and hence classified as orphan GPCRs. However, these orphan GPCRs have been implicated in several disorders from cancers to neurological diseases. Yet, in many cases, their mechanisms, ligands, and signaling reactions are poorly understood. One such orphan receptor in nervous system is GPR158 that is highly expressed in the brain where it controls synapse formation and function. GPR158 has also been implicated in depression, carcinogenesis, and cognition. However, the structural organization and signaling mechanism of GPR158 are largely unknown. We employed single‐particle cryogenic electron microscopy (cryoEM) to obtain structures of GPR158 in the apo state, and in complex with RGS7‐Gβ5, a regulator of GPCR signaling. The structure reveals highly unique domain organization of GPR158, not documented previously in any GPCRs structures. The homodimeric organization of protomers, phospholipids interaction and the presence of an extracellular Cache domain, an unusual ligand‐binding domain in GPCRs provide insights into the unusual biology of this orphan receptor and the formation of GPCR‐RGS complexes. These structures could help to deorphanize the receptor by serving the template for structure‐based discovery of its ligands that would leads to exploration of mechanistic of GPR158 mediated neuronal signaling.
Specificity of Binding and Antitumor Efficacy of a Covalent Microtubule Stabilizer
Authors: Risinger April L. , Yee Samantha S.
Published: May 2022

Microtubule stabilizers, including the taxanes paclitaxel and docetaxel, are a mainstay in the treatment of solid tumors, including breast and ovarian cancers. Although these agents are often effective as first‐line therapy, they are susceptible to common drug resistance mechanisms, including the upregulation of drug efflux pumps. Therefore, there is a critical need to develop novel classes of microtubule targeted agents that retain activity in taxane‐resistant settings. The C22,23‐epoxy taccalonolides are a class of plant‐derived microtubule stabilizers that covalently and irreversibly bind microtubules, allowing them to retain efficacy in a variety of taxane‐resistant cancer models. We employed cellular‐based binding studies to fully map the taccalonolide pharmacophore and confirm the target specificity of this covalent compound. The identification of β‐tubulin residues that are critical for taccalonolide binding in a cellular context has provided the opportunity to generate functional taccalonolide mimetics through a synthetically tractable approach.

Herein, we demonstrate that taccalonolide AF is less susceptible to p‐glycoprotein‐mediated drug efflux than paclitaxel in two separate ovarian cancer models and that it has superior cellular persistence after drug washout, likely due to irreversible target engagement. While taccalonolide AF demonstrated potent and persistent in vivo antitumor efficacy against the taxane‐resistant NCI/ADR‐RES ovarian cancer model as a flank xenograft when administered systemically, it was associated with a narrow therapeutic window. Additional studies showing that intratumoral injection of the taccalonolides into flank tumors provided long‐term antitumor efficacy months after the final dose prompted a study of their utility in the targeted delivery to a disseminated orthotopic ovarian cancer model representing localized metastasis. Indeed, we found that taccalonolide‐treated animals showed a significant decrease in micrometastasis of taxane‐resistant ovarian cancer cells to the spleen, as detected by quantitative RT‐PCR, without any evidence of systemic toxicity. We propose that the irreversible mechanism of microtubule stabilization elicited by the taccalonolides has the unique potential for intraperitoneal treatment of locally disseminated taxane‐resistant disease, which represents a significant unmet clinical need in the treatment of ovarian cancer patients.
A Novel Method for Measuring Lung Metabolism of Precision‐cut Lung Slices
Authors: Guo Chanjian , Herbert Julia , Kelty skye , Laskin Debra , Gow Andrew J.
Published: May 2022

Pulmonary diseases are associated with changes in metabolism. Cellular metabolism is dynamically regulated by signaling cascades at molecular levels and associated with the pathology at organ level. Improved understanding of cellular metabolism is necessary to investigate its role in disease and identify the potential targets for therapy. We have developed a real‐time method to adapt to Agilent XFe96 metabolic analyzer for precision‐cut lung slices (PCLS) so that compound effects on metabolism can be screened across multiple technical replicates. PCLS were cut in 3 mm maximum diameter to allow for fitting and adhering to XFe96 plate wells, which were pre‐coated with matrix gel. Once the PCLS were attached to the well bottom, the XFe96 cartridge probe can hold the tissue to measure, since the measurement volume is about 2‐6 µl, 200 micrometers (µm) above the lung tissue. This method generated reproducible oxygen consumption and extracellular acidification data in precision‐cut lung slices. We have optimized the conditions of oligomycin (OM) and Trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), rotenone and antimycin A to produce precision measurements. We have been able to utilize this technique to establish baseline metabolic rates for extracellular acidification and oxygen consumption and the dependency of those rates on mitochondrial function. In comparison with current cellular metabolic measurements, this method measures the whole tissue instead of an individual cell population. We have utilized this method to measure the effects of LPS treatment on PCLS metabolic function. Specifically, we have been able to demonstrate that LPS treatment not only leads to increased nitric oxide production in PCLS, but also reduces both glycolysis and spare respiratory capacity (SRC). This new method allows for the investigation of lung metabolism ex vivo and the screening of therapeutic targets for their interaction with lung metabolism.
Structural basis for interactions between RPTPζ/PTPRZ and the perineuronal net component tenascin‐R
Authors: Bouyain Samuel , Matthews Russel T.
Published: May 2022

Perineuronal nets (PNNs) are complex extracellular matrix structures found exclusively surrounding a subset of neurons in the central nervous system and are established key regulators of developmental neural plasticity and cognitive functions. Unfortunately, our current understanding of the roles that PNNs play in the central nervous system far exceeds our grasp of their molecular architecture. The identities of several PNN components are known and nets include the glycosaminoglycan hyaluronan along with hyaluronan‐binding link proteins, lectican family members, and the extracellular matrix protein tenascin‐R (TNR). Recently, it was shown that disruption of the gene encoding the receptor protein tyrosine phosphatase Ptprz1/RPTPζ altered the appearance of nets from a reticulated structure to puncta on the surface of cortical neuron bodies in adult mice. The structural alterations found in adult ptprz1‐/‐ mice mirror those found in tnr‐/‐ mice, and TNR is absent from the net structures that form in dissociated cultures of ptprz1‐/‐cortical neurons. These findings raise the possibility that TNR and RPTPζ cooperate to promote the assembly of PNNs. Here, we show that TNR associates directly with a fibronectin type III domain in the extracellular region of RPTPζ and provide a structural basis for the interactions between TNR and RPTPζ. Furthermore, we show that mutating residues at the TNR/RPTPζ interface impairs the formation of PNNs in neuronal cultures. Overall, this work sets the stage for analyzing the roles of protein‐protein interactions that underpin the formation of nets.
An optogenetic GDI drives GPCR and Gα independent macrophage migration
Authors: Olupothage Koshala , Kankanamge Dinesh , Karunarathne Ajith
Published: May 2022

Activators of G protein signaling (AGS) proteins play diverse functional roles in eukaryotic cells, including asymmetric cell division and membrane protein trafficking. The group II AGS (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) consisting of four G‐protein regulatory (GPR) GoLoco motifs that bind to Gαi/o in the G protein heterotrimer. AGS3 binding should therefore release Gβγ from the heterotrimer. However, the ability of AGS3 to directly signal through Gβγ in a living cell has yet to be demonstrated. To investigate the role of AGS3 in Gβγ signaling independent of G protein‐coupled receptors, we engineered optogenetic AGS3 using one or more consensus GPR sequences. This consensus sequence has shown higher binding efficacy towards Gαi GDP comparative to the wild type. Upon expression, our Opto‐AGS3 showed localized PIP3 generation and directional migration in RAW 264.7 cells due to Gβγ generation, indicating non‐canonical Gβγ signaling of AGS3. Our results also provide GPCR and Gα independent optogenetic control of Gβγ signaling with subcellular spatial and millisecond temporal control.
Adult‐Onset Exercise Improves Mitochondria Efficiency of the Heart in Female Offspring Born from Dams Fed a High‐Fat and a High‐Sucrose Diet
Authors: Mockler Sarah , Gonzalez Kassandra , Chiñas Merlin Andrea , Perez Yessenia , Jia U‐Ter Aondo , Chicco Adam J. , Chung Eunhee
Published: May 2022

A wealth of studies have shown that an adverse intrauterine and perinatal environment is a strong predictor for cardiometabolic diseases in offspring. Conversely, maternal exercise before and during pregnancy has been shown to diminish the risk of offspring developing metabolic disorders later in life. A few promising human and animal studies demonstrated that adult‐onset exercise in offspring can overcome the detrimental effects on offspring health associated with maternal obesity. However, it is unknown whether exercise initiated by adult offspring can mitigate the negative cardiometabolic health induced by maternal obesity. Female CD‐1 mice were fed either a high‐fat, high‐sucrose diet (HFHS) or a refined low‐fat, low‐sucrose diet (LFLS) for eight weeks before pregnancy and gestation, and during lactation. All dams were sedentary throughout the study. At 21 days, female offspring were weaned onto their maternal diet. At 15 weeks, offspring were randomly divided into exercise and sedentary groups which yielded 4 groups: LLS, LLE, HHS, and HHE (first letter denotes maternal diet, second letter offspring diet, and third letter indicates sedentary (S) and exercised group in offspring(E)). For exercise groups, mice had unrestricted exercise wheel access for 10 weeks. Glucose tolerance test (GTT) was performed at 25 weeks and tissues were collected to evaluate cardiac mitochondrial respiratory function at 26 weeks. Maternal diet did not affect body weight (BW) at weaning. Diet did not affect the daily average distance or hours run. BW was significantly higher in the HHS compared to other groups, but exercise did not alter final body weight. This was also true for fat mass. Exercise induced cardiac hypertrophy in mice fed LL, but not in HH mice. Surprisingly, mice in HHE had impaired glucose tolerance compared to other groups. However, no difference was found between LLE and LLS for GTT. In freshly isolated cardiac mitochondria, HHS significantly lowered the coupling efficiency of carbohydrate (pyruvate) and fatty acid (palmitoylcarnitine) oxidation compared to LLS. Exercise training prevented these effects in the HH mice, but had no exercise effect on OXPHOS coupling with either substrate in LL mice. These results demonstrate that adult‐onset exercise can prevent the deleterious effects of maternal obesity on cardiac mitochondrial function in offspring, but may alter glucose handling by mechanisms that merit further investigation.
Acetyl‐CoA Electron Density: Acyl‐CoA Reactivity and Crystallographic Data
Authors: Hemme Colin , Lohman Jeremy
Published: May 2022

Acetyl‐CoA and other acyl‐CoAs are the building blocks used in carbon‐carbon bond forming reactions underlying fatty acid and polyketide biosynthesis and many other biological activities. Since many enzymes use acetyl‐CoA, understanding how the substrate binds in the active site is key to determining interactions leading to enzymatic catalysis and substrate selectivity. We examined all of the PDB deposited crystal structures that report acetyl‐CoA bound and report stats for the reactive acetyl‐thioester moiety. We quantified this data by generating the real‐space R‐factor (RSR) and real‐space correlation coefficient (RSCC) by running a few rounds of refinement with Refmac to normalize the data. We examined the modeled thioester ends in comparison to the rest of CoA by creating a Linux script that splits acetyl‐CoA into the nucleic acid, pantetheine, and acetyl‐thioester moieties. We also analyzed the 3’phosphate of acetyl‐CoA to examine how splitting the molecule affects the RSR/RSCC values of small portions of the molecules along with the ribonucleotide containing substrates NAD+, NADH, NADP, and NADPH. Preliminary analysis of the output data reveals the nucleic acid and pantetheine RSR/RSCC values indicate good fits of models to electron density, but there is a portion of structures where RSR/RSCC values indicate the acetyl‐thioester moiety is poorly fit. We also find that the RSR method of calculation shows a significant difference between the acetyl‐CoA thioester and the 3’phosphate where the 3’phosphate is better than the average of the entire structure while the thioester is poorly fit. This is also consistent with the RSR values of NADP and NADPH 3’ phosphates. Conversely, the RSCC values of the acetyl‐CoA thioester end and the 3’phosphate were both equally lower than the entire structure. It appears there is an overfitting of the acetyl‐thioester fitting in crystal structures, likely due to a pressure to claim mechanistic inference from the structures.
The role of cytoskeleton regulators in embryonic facial morphogenesis
Authors: Ibrahim Isra , Richman Joy M.
Published: May 2022

Small GTPases are downstream of the non‐canonical WNT JNK‐PCP pathway. The most well studied members of this group are RhoA, Rac1 and CDC42. These small GTPases regulate cell shape, orientation, and migration through regulating the actomyosin cytoskeleton. In addition, components of this pathway have been linked to non‐syndromic cleft lip with or without cleft palate. The objective of this study is to test whether small GTPase signaling is required and sufficient for facial morphogenesis at stages where lip fusion is taking place.

Methods

The faces of E4.5 chicken embryos were dissected to include the two facial processes involved in lip fusion, the frontonasal and maxillary processes (Fig. 1). After a dose‐response experiment to find the optimal concentrations, the organ cultures were treated with either an antagonist of RhoA signaling (ROCKi 20µM) or an antagonist of Rac1 (Rac1i 100µM) or an agonist (RhoA/ Rac1/CDC42 activator 0.5µg/ml). Organ cultures were photographed and measured at 0 and 24hrs. Percentage growth differences were analyzed using Prism Graphpad software (v. 9.2).

Results

In the control cultures, frontonasal mass width decreased by 23% and increased in cranio‐caudal height by 17% over 24‐hour period. The nasal slits did not change. The maxillary prominences increased in height and width by 12% and 37% respectively over 24‐hour period similar to growth in vivo. The ROCKi treated cultures prevented the narrowing of the frontonasal mass and increased the length of the nasal slits. ROCKi treatment had no effect on the height of the frontonasal mass nor maxillary prominences. In contrast, the RAC1i had no effect on facial growth. The agonist significantly reduced frontonasal mass width compared to ROCKi. In addition, the agonist significantly decreased growth of the maxillary prominences.

Conclusions

The changes in facial morphogenesis may be explained by active cytoskeletal remodeling, particularly in the RhoA pathway. We excluded a requirement for RAC1 in facial morphogenesis in our system. Our data also suggest that different regions of the face have different requirements for specific small GTPase signaling. The frontonasal mass is dependent on RhoA signaling but not the maxillary prominences. Over activation of all GTPases shows that correct levels are necessary for normal morphogenesis. Overall, we have revealed a complex interplay between small GTPAses in midfacial morphogenesis.
Evaluation of a Combination of an HDAC Inhibitor and a Cell Cycle Inhibitor in Pancreatic Cancer Cells
Authors: Bhutkar Shraddha P. , Yadav Anjali , Dukhande Vikas V.
Published: May 2022

Pancreatic cancer is one of the deadliest cancers with 5‐year survival rate of ~ 10 percent. Late diagnosis, early metastasis, and resistance to therapeutics necessitate a better treatment approach for pancreatic cancer. Alterations in epigenetic profile through DNA and histone modifications enable tumor growth and metastasis. Evading the control of growth suppressors that regulate cell cycle is also a major hallmark of cancer pathophysiology. We aimed to study a combination therapeutic targeting histone deacetylase (HDAC) and key cell cycle regulator cyclin‐dependent kinase (CDK) in pancreatic cancer. We used panobinostat, a pan‐HDAC inhibitor approved for multiple myeloma and abemaciclib, an FDA‐approved CDK4/6 inhibitor for breast cancer. Our preliminary in vitro data in pancreatic cancer cells depicts synergistic cytotoxicity when treated in combination. In addition, combination treatment resulted in a significant decrease in proliferation and clonogenic survival of pancreatic cancer cells. Our mechanistic studies on the effects of this potent combination on histone modifications, gene expression, and cell cycle regulation will provide insights on drug synergy in pancreatic cancer cells. The results of our in vitro studies show that combining drugs that pharmacologically target two critical aspects of pancreatic cancer proliferation has a high potential for treating this lethal disease and as such merits further investigation.
Morphological Studies of the Left Ventricle in the Creatine Deficiency Mice Model
Authors: Shults Nataliia V. , de Souza Aline M. , Baydyuk Maryna , Sandberg Kathryn , Huang Jeffrey K.
Published: May 2022

Introduction

Heart failure is the major cause of death characterized by changes in myocardial energetics, including a reduced level of creatine. In organs with high energy demand such as the heart, creatine is taken up by cardiomyocytes via creatine transporter with subsequent transformation into phosphocreatine, which controls by creatine kinase. This system functions as shuttles high‐energy phosphates from mitochondria to contractile elements such as the myofibril, which utilize 70 % of total produced energy by mitochondria. For the past decades, many studies tried to understand whether a reduced level of creatine affects the myocardial function leading to the failing heart. However, most of the results from these studies have been contradictory. The present study describes the detailed morphologic changes of cardiomyocytes of the left ventricle (LV) of mice with whole‐body creatine deficiency due to knockout of the biosynthetic enzyme (guanidinoacetate N‐methyltransferase, GAMT).

Methods & Results

The present study used GAMT‐/‐ mice model with the unique metabolic fingerprint of zero creatine similar to those in human GAMT deficiency. The histological and transmission electron microscopy (TEM) studies of LV myocardium in GAMT‐/‐ mice showed cardiac hypertrophy and increased collagen accumulation in the myocardial interstitium compared to the wild‐type (WT) mice. The TEM examination showed irreversible destructive ultrastructure changes of cardiomyocytes in conjunction with insufficient adaptive protein synthesis accompanied by focal and diffuse lysis of myofilaments in cardiac myocytes. The mitochondrial changes were characterized by cristae homogenization and fragmentation in association with cluster formation. The volume of density of mitochondria (VvMt), myofibrils (VvMf), and their ratios (VvMt/VvMf), along with the coefficient of energy efficiency of mitochondria (CEEM) have been examined in GAMT‐/‐ mice in comparison to WT. We established that VvMt, VvMf, VvMt/VvMf, and CEEM were all significantly reduced. Further, destructive changes in the nucleus and nuclei, focal degradation of sarcoplasm, increases autophagy, are all evidence of inadequate adaptive protein synthesis in cardiac myocytes.

Conclusions

These studies of GAMT‐/‐ mice demonstrated that creatine deficiency promotes decreased VvMt/VvMf and CEEM in LV myocytes, which may contribute to the development progression of heart failure and in conjunction with insufficient adaptive protein synthesis contribute to irreversible ultrastructure restructuring of cardiomyocytes with loss contractile myocardial function.
NCAD and ITGB1 Coordinately Regulate Growth Plate Cartilage Architecture
Authors: Greer Sydney E. , Erickson Alek , Dudley Andrew
Published: May 2022

Spatial and temporal regulation of chondrocyte maturation in the growth plate drives long bone growth. A notable feature of growth plate cartilage is the progression of progenitor resting chondrocytes into clonal columns of transit‐amplifying proliferative chondrocytes. Following division of proliferative chondrocytes, daughter cell pairs undergo a unique 90‐degree rearrangement to form columns. Our previous studies using a murine fluorescent membrane reporter system and time‐lapse imaging along with chemical inhibition of cell adhesion suggested a primary requirement for cadherin adhesion at the daughter cell interface in column formation. Studies designed to test the force generating mechanism at the cadherin dependent daughter cell interface produced a paradoxical result with activated myosin motor proteins enriched at the cell‐matrix interface, an observation that suggests integrin adhesion molecules might drive cell rearrangement. The purpose of the current work was to determine the relative contributions of cadherin and integrin mediated cell adhesion in chondrocyte column formation. As a first step, we used live cell imaging to compare the daughter cell rearrangement phenotype in growth plate explants from embryos containing a cartilage‐specific deletion of N‐cadherin (NCAD) or integrin β1 (ITGB1). Deletion of either gene produces similar disruption of column formation, even though the mutant phenotypes show differences in cell shape, rearrangement dynamics, and kinetics of daughter cell separation following rearrangement. The apparent requirement for both NCAD and ITGB1 suggested potential reciprocal regulatory interaction between the two adhesion surfaces. To test this hypothesis, we analyzed phenotypes of an allelic series. We found that chondrocytes mutant for ITGB1 show defects in cell rearrangement and column formation (n=37 embryos) regardless of the NCAD genotype as expected from single mutant studies. Surprisingly, chondrocytes mutant for NCAD and heterozygous for ITGB1 are phenotypically wildtype with organized columns and normal rearrangement (n=13 embryos). These results support a model for which ITGB1, and not NCAD, is the driver of chondrocyte rearrangement and suggest that failure to rearrange in NCAD mutants results from increased ITGB1 function. Thus, rearrangement requires a narrow range of cell‐matrix adhesion strength and NCAD potentially acts as a brake by restricting ITGB1 activity in non‐rearranging resting chondrocytes. Current studies aim to confirm these genetic findings using gene expression analysis and an innovative quantitative adhesion assay as well as identify upstream regulators that control strength of antagonism between the cell adhesion interfaces.
Novel Role of Dual Oxidase 2 as a Mediator of Podocyte Injury in the Diabetic Environment
Authors: Castro Aracely , Ford Bridget M.
Published: May 2022

Glomerular injury is a prominent pathological feature of diabetic nephropathy (DN). In glomerular epithelial cells, or podocytes, hyperglycemia alters slit diaphragm proteins and causes foot process effacement, apoptosis and cell detachment. Oxidative stress has emerged as an important pathogenic mechanism in the development of glomerular injury in DN. However, the mechanisms by which these factors exert their action remain poorly understood.

We provide evidence that the NADPH oxidase of the Nox family, Dual oxidase 2 (Duox2), is present in cultured glomerular cells, including podocytes. Exposure of cultured podocytes to high concentrations of glucose (HG) elicited a rapid upregulation of Duox2 protein expression. Inhibition of Duox2 with specific siRNA prevented the HG‐induced increase in intracellular reactive oxygen species (ROS) generation and hydrogen peroxide (H2O2) production in cultured podocytes.

In additional experiments, we established a functional link between Duox2‐derived ROS generation and podocyte injury in response to HG. Impairment of Duox2 function nearly abolished HG‐mediated DNA fragmentation, apoptosis and decrease in/reorganization of slit diaphragm protein expression in podocytes, indicating that Duox2 is required for the deleterious effects of glucose in podocytes.

Our study demonstrates, for the first time, that Duox2 is responsible for increased ROS generation and subsequent alteration of podocyte function in response to HG. Our work serves as proof of concept to demonstrate the utility of targeting Duox2 as a future therapeutic intervention to reduce diabetes‐mediated glomerular lesions.
Direct reprogramming of dermal fibroblasts derived from Northern elephant seals into muscle cells
Authors: Lam Emily , Torres‐Velarde Julia M. , Allen Kaitlin , Arango B. Gabriela , Crocker Daniel , Vázquez‐Medina José P.
Published: May 2022

Ex vivo tissue culture allows the study of complex cellular mechanisms that are relevant to physiological responses while overcoming the challenges presented by studying animals that are not tractable. In a primary cell culture system, certain proliferating cells can be functionally reprogrammed into other cell types via overexpression of key genes. Dermal fibroblasts can be reprogrammed into muscle cells, which are often challenging to obtain but offer a unique system to study metabolic responses, by overexpression of the myogenic transcription factor myod. We isolated cells from Northern elephant seal (NES) skin samples and propagated them in primary culture. NES cells respire, stain positive for fibroblast markers (vimentin and PDGFR), and are amenable to electroporation. We overexpressed GFP‐myod in NES fibroblasts and conducted antibiotic selection and purification by FACS. As expected, expression of myod was higher in transfected cells than in controls according to qPCR analysis (t‐test p< 0.05). Treatment with small molecules (CHIR99021, Forskolin and Repsox) enhanced myod expression. Furthermore, fibroblasts overexpressing myod expressed downstream markers of myogenesis (myogenin, myosin heavy chain 1 and myosin heavy chain 8) and the effect was enhanced when myod‐overexpressing cells were supplemented with small molecules. We are currently evaluating the capacity of myod‐overexpressing cells to differentiate into myotubes and comparing metabolic profiles with primary NES myoblasts. Establishing differentiated muscle fibers from other mature cell types could provide a unique platform to conduct mechanistic studies in species where muscle tissue samples cannot be obtained from live animals.
Cartdridged Food Materials for 3D Printing of Alternative Meat: Regarding Modified Texture of 3D Printed Food according to the Ionic State of Particle Surface exposed on Protein Powder
Authors: Oh Yourim , Lee Nam Keun , Rhee Jin‐Kyu
Published: May 2022

In the field of food processing, 3D food printing can be one of the foremost methods which allows to laminate various type of complex food structures. The technology makes it possible to manufacture food products with desired internal structures, tastes, flavors and nutrition and is attracting attention as an innovative way to customize food to meet each customer's needs. 3D printing can produce a wide range of foods with different textures and viscosities using the cartridge loaded with materials ranging from soft sauces to hard doughs. Additionally, for controlling the material while processing in the 3D printer, the food ink should be easily ejected through the nozzle, and pretreatment is required to homogenize and reduce the particle size. Since the ionic layer on the surface of the powder particles affects the bonding between the powders, zeta potential measured on the surface of these can be used as a standard for monitoring the organization of powdered materials in 3D printed substitute meat. In this study, various prototype of meat substitute was prepared using protein powder‐based materials with various processing parameters on the particle surface, and the texture was evaluated by comparing and analyzing the physical properties. Specifically, the particle surface state of the powder material was monitored by measuring various properties including the zeta potential, and the physico‐chemical and rheological properties of 3D printed meat substitute were analyzed to deduce the relationship for reaching the food processing aptitude.
NKX2‐1 directly regulates Efnb2 to generate a boundary that allocates tracheal cells and defines the position of tracheoesophageal separation
Authors: Lewis Ace E.
Published: May 2022

Tracheoesophageal (TE) fistula is a human congenital condition characterized by an improper connection between the esophagus and trachea. These two tubular organs originate from a single foregut tube and separate from one another beginning at the site of lung bud evagination and progressing rostrally. Genetic studies in mice have identified signaling pathways that initiate dorsoventral patterning of the foregut; NKX2‐1 marks ventral, presumptive tracheal cells whereas high levels of SOX2 expression predominantly mark the dorsal, esophageal‐fated cells. Perturbation of either factor results in loss of that identity, increased expression of the opposing transcription factor, and failure of TE separation. Currently, the downstream mechanisms required to effect morphogenetic separation at the cellular and tissue level remain largely unknown. Here, we show that EPHRIN‐B2, a member of the EPH/EPHRIN signaling family, is restricted to the dorsal, esophageal‐fated cells, and that this domain of expression is required for TE separation and for maintaining the correct allocation of tracheal cells to the nascent trachea. Further, we demonstrate that NKX2‐1 directly binds at the Efnb2 locus and negatively regulates its expression, establishing an NKX2‐1/EPHRIN‐B2 TE boundary. Nkx2‐1 mosaic mutants exhibit ectopic NKX2‐1/EPHRIN‐B2 boundaries which drive ectopic tracheal separation morphogenesis in an EPHRIN‐B2 dependent manner. These results establish NKX2‐1 as a regulator of TE cell sorting and tissue separation, through modulation of EPH/EPHRIN signaling, which then serves to translate tissue identity information into morphogenetic outcomes.
Individualized Out-of-Pocket Price Estimators for “Shoppable” Surgical Procedures: A Nationwide Cross-Sectional Study of US Hospitals
Authors: Berlin Nicholas L. , Chopra Zoey , Bryant Arrice , Agius Josh , Singh Simone R. , Chhabra Karan R. , Schulz Paul , West Brady T. , Ryan Andrew M. , Kullgren Jeffrey T.
Published: May 2022

Objective:

To estimate the nationwide prevalence of individualized out-of-pocket (OOP) price estimators at US hospitals, characterize patterns of inclusion of 14 specified “shoppable” surgical procedures, and determine hospital-level characteristics associated with estimators that include surgical procedures.

Background:

Price transparency for shoppable surgical services is a key requirement of several recent federal policies, yet the extent to which hospitals provide online OOP price estimators remains unknown.

Methods:

We reviewed a stratified random sample of 485 U.S. hospitals for the presence of a tool to allow patients to estimate individualized OOP expenses for healthcare services. We compared characteristics of hospitals that did and did not offer online price estimators and performed multivariable modeling to identify facility-level predictors of hospitals offering price estimator with and without surgical procedures.

Results:

Nearly two-thirds (66.0%) of hospitals in the final sample (95% confidence interval 61.6%–70.1%) offered an online tool for estimating OOP healthcare expenses. Approximately 58.5% of hospitals included at least one shoppable surgical procedure while around 6.6% of hospitals included all 14 surgical procedures. The most common price reported was laparoscopic cholecystectomy (55.1%), and the least common was recurrent cataract removal (20.0%). Inclusion of surgical procedures varied by total annual surgical volume and health system membership. Only 26.9% of estimators explicitly included professional fees.

Conclusions:

Our findings highlight an ongoing progress in price transparency, as well as key areas for improvement in future policies to help patients make more financially informed decisions about their surgical care.
Residual dysplasia of the hip after successful ultrasound-monitored treatment: how does an infant’s hip evolve?
Authors: Dornacher Daniel , Lutz Bernd , Freitag Tobias , Sgroi Mirco , Taurman Rita , Reichel Heiko
Published: May 2022

Despite that normal values for the hip joint are reached at the end of ultrasound-monitored-treatment, the development of the acetabulum can be compromised during the growth phase. The acetabular index (AI) measured on a pelvic radiograph has been proven to be a reliable parameter. The aim of this study is to gain a better understanding of the dynamics of once-treated, residually dysplastic hips. This should be achieved by radiographically following these hips up to a milestone-examination at the end of preschool age. A total of 120 hips of consecutive 60 infants were included in this examination, each presenting with a residual developmental dysplasia of the hips (DDH) after successful ultrasound-monitored harness treatment. Radiographic follow-up was assessed retrospectively around 18 months, 3 years and 6 years of age, and the AI was measured. The age-dependent Tönnis classification was applied. The hips were assigned normal, mildly or severely dysplastic. Dependent t-test for paired samples indicated a highly significant improvement of the AI-values, including from the first to the second and from the second to the third follow-up. The percentage distribution into the Tönnis classification changed remarkably: in the first follow-up, 36 of the 120 hips were evaluated ‘severely dysplastic’, in the third follow-up only 1. On the other hand, three hips underwent acetabuloplasty. Even after normal values have been achieved at the end of ultrasound-monitored treatment, there remains a risk of residual dysplasia of the hips. Particularly, when the first radiographic examination shows nonphysiological findings, further close-meshed follow-up is recommended. Level of evidence: retrospective study of therapeutic outcome, consecutive patients, level II.
Incidence and risk factors for acute compartment syndrome in pediatric tibia fractures
Authors: Obey Mitchel R. , Shlykov Maksim A. , Nickel Katelin B. , Keller Matthew , Hosseinzadeh Pooya
Published: May 2022

Acute compartment syndrome (ACS) is a rare complication following traumatic injuries in pediatric patients, and tibia fractures represent the most common cause of ACS. To determine the incidence and risk factors of developing ACS, State Inpatient Databases, State Emergency Department Databases, and State Ambulatory Surgery and Services Databases from the Healthcare Cost and Utilization Project (HCUP), Agency for Healthcare Research and Quality, were used to retrospectively identify patients 1–18 years of age with tibia fractures from 2006 to 2015 (quarter 3). The HCUP Nationwide Emergency Department Sample for nationwide data was also queried. Multivariable generalized estimating equations models were used to determine risk factors associated with development of ACS. A total of 50 640 patients with tibia fractures were studied, and 309 cases of ACS were identified. The incidence of ACS was 0.6 and 5.5% in the all tibia and open tibia fracture groups, respectively. Twenty-three cases of ACS (7.4% of all ACS) were diagnosed after discharge from the index admission, which was more common in teens treated nonoperatively. Predictors of increased ACS risk in the all tibia fracture group included age 13–18 [relative risk (RR): 4.04)], open fractures (RR: 3.83), and motor vehicle crash (MVC) mechanism (RR: 5.69). Nationwide, open and operatively treated fractures had an increased ACS rate (3.98 and 5.51%, respectively). Teenagers, open fractures, and MVC mechanisms were most strongly associated with ACS. ACS can present in a delayed fashion, as evidenced by postindex cases.