bluuebunny/crossref_metadata_2025_split huggingface dataset:
Selective Enrichment of Cell Surface Proteins Using ProMTag to Detect O‐glycosylation‐Dependent Changes
Authors: Minden Jonathan S. , Bachert Collin , Linstedt Adam , Lucas Amber 
Published: May 2022

O‐linked glycosylation, which is regulated by the enzyme GalNAc transferase, plays a vital role in cellular function by regulating membrane protein composition and sorting. Changes in O‐linked glycosylation are very common in diseases such as cancers, diabetes, and Alzheimer’s. It is important to study the effects of changes in O‐link glycosylation on cell membrane composition and function to understand how these changes affect the onset, phenotype, and progression of these diseases. However, membrane proteins are notoriously challenging to analyze at the proteomic level due to their hydrophobicity and the inability to separate the membrane proteins from other proteins in the cell.Here we introduce a new sample preparation workflow that enriches membrane proteins for mass spectrometry analysis to understand changes in protein composition of the plasma membrane as the result of changes in O‐linked glycosylation. To do this, we used the bifunctional tag ProMTag to label and capture plasma membrane proteins via their extracellular domains in cells treated with GalNAc Transferase inhibitors. One end of the ProMTag forms a reversible, covalent bond with protein the other end of the ProMTag is capable of forming an irreversible, covalent bond with a solid bead support via the click chemistry pair of methyltetrazine and trans‐Cyclooctene (TCO). The extracellular domains of proteins from intact wild type HEK cells and HEK cells treated with GalNAc Transferase inhibitors were ProMTagged, captured, cleaned up, and eluted for comparison by mass spectrometry (MS) analysis. MS confirmed enrichment of plasma membrane proteins after sample preparation and protein changes in the plasma membrane as a result of GalNAc Transferase inhibition were characterized. This work establishes a method for enrichment of plasma membrane proteins and reveals how changes in O‐linked glycosylation via GalNAc Transferase inhibition leads to changes in plasma membrane protein composition due to altered membrane protein stability and sorting.
How to Make a Vampire: Part II. Getting Ready to Feed
Authors: Prufrock Kristen A. , DeLeon Valerie B. , Smith Timothy D. 
Published: May 2022

The mammalian feeding system is made up of multiple components that influence facial variation, including the masticatory muscles, bony jaw apparatus, and teeth. However, it is not fully understood how the development of these components are coordinated and are impacted by life history and diet.Bats are the only mammals that continue to nurse until they are almost adult size, and often after they become volant. Common vampire bats (Desmodus rotundus) have a relatively prolonged juvenile period among bats, can nurse up to nine months, and later solely subsist off of blood. Feeding system maturity may therefore be delayed in bats, and particularly delayed in Desmodus, as a result of their lengthy juvenile period and liquid diet. In this study, we examine the developmental maturity of the cranium and feeding apparatus in the late fetus of a common vampire bat (Desmodus rotundus) relative to an adult (its mother).The heads of each specimen were microCT scanned twice. Conventional microCT was used to image mineralized tissues. Diffusible iodine‐based contrast‐enhanced CT (diceCT) imaging was used to image soft tissues. Cranial length and palate length measurements were acquired from unstained scans to evaluate size and jaw apparatus development. Masticatory muscles (e.g., medial pterygoid, zygomaticomandibularis) and permanent tooth crypt volumes were acquired from stained tissue scans to evaluate masticatory muscle and tooth development. Developmental maturity of these structures was assessed by calculating a percentage of the growth completed (i.e., [fetal muscle volume/adult muscle volume]*100).In the fetus, permanent tooth crypt volume is already comparable to the adult condition despite cranial length and palate length being <75% of adult size. Masticatory muscle volume maturity is highest in the lateral pterygoid muscle (~35%), whereas other masticatory muscles are far less developed (e.g., medial pterygoid volume maturity, ~20%; temporalis profundus volume maturity, ~20%; temporalis medius volume maturity, ~6%).These results reveal highly precocious permanent tooth development in Desmodus. Although this may facilitate transition to hematophagy, Desmodus do not fly until eight to ten weeks, so the precocious incisors may primarily aid the infant to grip its mother’s skin. Differential development of the masticatory muscles may also be influenced by the need for young bats to use their mouths to help anchor themselves to their mother early in life. This may explain the advanced maturity of the lateral pterygoid muscle, which would protrude the jaw and help to facilitate a more secure grip.
Sexually dimorphic role of smooth muscle cell estrogen receptor alpha in aging‐associated arterial stiffness
Authors: DuPont Jennifer , Vorn Jennifer , Lu Qing , Duffy Colleen , Martin Gregory , Hill Michael 
Published: May 2022

BackgroundAging is associated with rising arterial stiffness (AS), an independent risk factor for cardiovascular disease. The development of aging‐associated AS follows a sexually dimorphic pattern with women developing AS later in life. Estrogen and estrogen receptor alpha (ERα) are protective in the premenopausal state, potentially due to the regulation of the vasoprotective angiotensin II type 2 receptor (AT2R), however, the role of estrogen/ERα in aging‐associated AS has not been defined and the mechanisms of the sexually‐dimorphic development of aging‐associated AS are unknown.Methods/ResultsAll studies were conducted in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. We generated novel smooth muscle cell‐specific ERα‐KO mice and measured aortic pulse wave velocity (PWV), an index of AS, in 3, 12, and 18 month old male and female intact and KO mice. Male mice develop increases in AS from 3 to 12 months compared to 3 to 18 months in females (p<0.05). SMC‐ERα‐KO protected females from the aging‐associated increase in AS (p<0.05). Circulating estrogens also declined in 18 month old females vs. 3 mo. (p<0.05), suggesting a role for unliganded SMC‐ERα in aging‐associated AS. SMC‐ERα‐KO females were also protected from the aging‐associated decline in aortic AT2R expression observed in intact females (p<0.05). To further investigate the potential role of unliganded SMC‐ERα in AS, we performed ovariectomy (OVX) or sham surgeries in young female SMC‐ERα‐Intact and KO mice and measured PWV. Results demonstrated an OVX‐driven increase in AS that was prevented by SMC‐ERα‐KO (p<0.05). This was also associated with an OVX‐mediated decrease in aortic expression of the AT2R, which was prevented by SMC‐ERα‐KO. To determine the in vivo role of AT2R in the vascular protection observed in 12 month old female mice, we treated 12 month old male and female mice with AT2R antagonist PD‐123319 (3mg/kg/day) or vehicle via osmotic minipumps for 4 weeks. Chronic inhibition of the AT2R increased AS in 12 month old females (p<0.05), but not males, suggesting that AT2R‐mediated protection contributes to the delay in aging‐associated AS in female mice. In vitro studies also demonstrated that estrogen upregulates AT2R mRNA and protein in female mouse aortic SMC, supporting an estrogen‐mediated mechanism of AT2R driven vascular protection in females.ConclusionThese data indicate sex differences in the time course of the development of aging‐associated arterial stiffness and suggest the presence of unliganded SMC‐ERα contributes specifically to aging‐associated arterial stiffness in females via reductions in vascular AT2R. Further, these results implicate that AT2R agonist therapy may improve cardiovascular outcomes in aging women to retain the beneficial downstream effects of estrogen signaling, without the potential detrimental effects of hormone replacement therapy.
Design of Experiment (DOE) for Optimization of PLGA Nanoparticles
Authors: Gupta Ritu , Xie Huan , Sarkar Mahua , Chen Yuan 
Published: May 2022

AbstractDesign of Experiments (DOE) is useful tool for formulation optimization. In present study, Design Expert® software (version 13) was used for optimization of Poly(dl‐lactide‐co‐glycolide) (50:50) (PLGA) nanoparticles (NPs). Capecitabine, an approved anticancer drug with short half‐life, was used as model drug. Capecitabine NPs can minimize dosing frequency and improve patient compliance. Effect of two numeric (drug and polymer amount) and one categoric factor (pH of W2 phase) was studied on NP Size and Entrapment Efficiency (EE). Drug amount and pH significantly influenced EE. Polymer amount significantly influenced NP size.MethodsPLGA NPs were prepared by double emulsion technique. Aqueous drug solution (W1) was emulsified with polymer solution in ethyl acetate using probe sonicator (Misonix) for 2 minutes. Formed primary emulsion was sonicated with second aqueous phase (W2) containing stabilizer Kolliphor®P188 for 1 minute to produce double emulsion. Organic solvent and excess water were evaporated at 600 rpm, 60°C for 1.5 hr and NPs were washed thrice (14,000 rpm, 4°C for 45 min) Lyophilized NPs were sonicated with acetonitrile for 5 min and EE was analyzed using UV/Vis spectrometer at ʎmax304nm. Particle size was determined using Malvern Nano‐ZS.ResultsExperimental data were fitted into models of increasing polynomial complexity. EE data is best described by two‐factor interaction model with R² of 0.9145. ANOVA results show model is significant. Predicted R²of 0.8076 is in reasonable agreement with adjusted R² of 0.8824. Drug amount, pH of W2 phase and their interaction are significant (p<0.05). Higher drug amount decreases EE which maybe because polymer amount was not sufficient to encapsulate increased drug amount and more drug migrated to W2 phase. Lower EE at pH 4.5 might be described by drug leaching into W2 phase due to better drug solubility. Diagnostic normal plot, perturbation plots, interaction plots and 3D‐surface plot confirm model robustness. Size data was best fitted to linear model showing polymer amount being significant. Higher polymer amount might have decreased dispersion efficiency of first emulsion into W2 phase and led to fusion of semi‐formed particles to larger size.Numerical OptimizationThe desired goals were set to minimize polymer amount (to increase drug amount per unit weight of NPs), to minimize drug amount and the pH was set at 7.4 to maximize EE. Predicted optimum responses were checked experimentally. Batch prepared with 120mg polymer and 25mg capecitabine has average size of 111.3 nm (predicted value 116.8nm) and EE of 38% (predicted value 40%), both with 5% coefficient of variance.ConclusionDOE gives an opportunity to look at individual and combined impact of various factors. It can provide valid conclusions with minimal experimental runs, time, and cost. Similar DOE approaches can be used for other PLGA‐drug formulations optimization.Conflict of InterestAuthors declare no conflicts of interest.
Novel Therapeutics Modulate Cardiac Leukocyte Populations Following Myocardial Infarction
Authors: Staffenson Melanie , Kumar Sushil , Sepe Joseph , Coussens Lisa , Habecker Beth 
Published: May 2022

Myocardial infarction (MI) triggers an inflammatory response that transitions from pro‐inflammatory to reparative over time, a progression that is essential for cardiac healing. Sympathetic nerve loss in the heart after MI predicts the probability of serious ventricular arrhythmias, and restoring nerves prevents arrhythmias. Although noradrenergic transmission is pro‐inflammatory in several contexts, acetylcholine (ACh) can be anti‐inflammatory; and cardiac sympathetic nerves release ACh along with norepinephrine after MI. Recent studies have discovered sympathetic regulation of global inflammatory responses, and our lab has shown previously that treatment with either Intracellular Sigma Peptide (ISP) or the small molecule HJ‐02 following ischemia‐reperfusion (IR) injury restores sympathetic innervation to the cardiac scar and prevents arrhythmias. However, whether sympathetic reinnervation modulates leukocyte homing and function in the heart post‐IR was unknown.C57BL/6J mice were treated on days 3‐10 after IR with ISP or HJ‐02, and we used quantitative multiplex immunohistochemistry (mIHC) to identify the leukocyte phenotypes present in the heart two weeks following IR. Reinnervated hearts had decreased inflammatory (M1‐like) macrophage levels [Percent of total macrophages ± SEM: VEH, 47.29%±1.446; ISP, 37.97%±1.590; HJ‐02, 29.72%±1.226] and elevated reparative (M2‐like) macrophages [VEH, 52.71%±1.446; ISP, 62.03%±1.590; HJ‐02, 70.28%±1.226]. HJ‐02 stimulated a significantly greater shift from inflammatory to reparative cell types compared to ISP.However, it is unknown whether it is a direct effect of the treatments on immune cells or the restoration of sympathetic nerves in the infarct causing the changes in leukocyte populations. To answer whether the treatments are the instigator, we isolated and cultured primary peritoneal macrophages from unoperated C57BL/6J mice, treated with ISP or HJ‐02, and examined macrophage marker expression using flow cytometry. Preliminary results suggest that both ISP and HJ‐02 expand isolated peritoneal macrophages in situ, with a predominantly M2‐like phenotype. M2‐like macrophages promote wound‐healing, suggesting these novel therapeutics may be beneficial for the heart after MI.
Myristic Acid‐Trans‐Activator of Transcription Dual Conjugation Improves Intracellular Delivery of Protein Kinase C Beta II Peptide Inhibitor Cargo in Isolated Rat Polymorphonuclear Leukocytes
Authors: Singh Sunit G. , Verwoert Alexis , Nair Arjun , Johnson Devani , Humayun Annam , Clair Logan , DiLisi Taylor , Harrell Kayla , Dean Tameka , Chen Qian , Barsotti Robert , Young Lindon 
Published: May 2022

Protein kinase C beta II (PKCβII) activation promotes polymorphonuclear (PMN) superoxide (SO) production by phosphorylating serine and threonine amino acid residues on NADPH oxidase (NOX‐2). In previous studies, cell‐permeable myristic acid conjugated PKCβII inhibitor (myr‐PKCβII‐) significantly attenuated PMN SO release induced by phorbol 12‐myristate 13‐acetate (PMA), a diacylglycerol mimetic. Myr‐PKCβII‐ was determined to be superior to unconjugated peptides and nontreated controls, suggesting enhanced intracellular delivery of cargo. We hypothesize that the simple diffusion of myr‐conjugation combined with the endocytotic mechanism of trans‐activator of transcription (Tat) would optimize the intracellular delivery of PKCβII‐ cargo compared to myr‐conjugation alone.In this study, we tested the concentration‐dependent effects of a dual myr‐Tat conjugated PKCβII‐ (myr‐Tat‐PKCβII‐; N‐myr‐Tat‐CC‐SLNPEWNET) on intracellular delivery compared to myr‐PKCβII‐, scrambled myr‐Tat‐PKCβII‐ (myr‐Tat‐PKCβII‐ scram), unconjugated PKCβII‐, and 0.5% dimethyl sulfoxide (DMSO) vehicle control group. Rat PMNs were incubated for 15 min at 37°C with either unconjugated PKCβII‐ (20μM), myr‐Tat‐PKCβII‐ (2μM, 5μM, 7.5μM, 10μM, and 20μM), or myr‐Tat‐PKCβII‐scram (2μM, 5μM, 7.5μM, 10μM, and 20μM). PMN SO release was calculated by the change in absorbance at 550 nm over 390 sec via ferricytochrome c reduction after PMA stimulation (100nM). The efficacy of intracellular drug delivery was evaluated by the magnitude of PMA‐induced PMN SO release attenuation with the PKCβII‐ cargo. Data were analyzed with ANOVA Fisher’s PLSD post‐hoc analysis.Myr‐Tat‐PKCβII‐ 5μM (n=12, 0.392±0.04), 7.5μM (n=11, 0.397±0.05), 10μM (n=5, 0.211±0.05) and 20μM (n=5, 0.121±0.02) demonstrated a concentration‐dependent increase in intracellular delivery compared to DMSO vehicle control (n=84, 0.496±0.02, all p<0.05). Myr‐PKCβII‐ only significantly increased intracellular delivery at the 20μM concentration (n=27, 0.303±0.02, p<0.05) compared to DMSO vehicle control. Intracellular delivery of myr‐Tat‐PKCβII‐ 2μM (n=10, 0.436±0.06) and all concentrations of myr‐Tat‐PKCβII‐scram were not significantly different from DMSO vehicle controls.Results suggest that myr‐Tat dual conjugation is superior to myr‐conjugation alone at intracellular delivery of cell impermeant cargo. Future studies will investigate the concentration‐dependent effects of PKCβII‐ peptide conjugates on PMA‐induced PKCβII activity and translocation to membrane targets, such as NOX‐2, using immunocytochemistry and western blot analysis.
Association of Pre‐Living Kidney Donation Body Mass Index with Post‐Donation Estimated Glomerular Filtration Rate Decline
Authors: Tantisattamo Ekamol , Tanariyakul Manasawee , Polpichai Natchaya , Mutirangura Pornthira 
Published: May 2022

IntroductionObesity is one of the risk factors for chronic kidney disease (CKD). Whether body mass index (BMI) at pre‐living kidney donation is associated with worsening kidney function of the remaining kidney after donation is unclear. We aim to examine the association between pre‐donation BMI and change in post‐donation estimated glomerular filtration rate (eGFR) in living kidney donors.MethodsA single‐center retrospective cohort study included 14 living kidney donors. Associations between pre‐donation BMI and percentage of eGFR decline at 2 and 7 days post‐donation were examined by multiple linear regression.ResultsOf all 14 living kidney donors, mean age±SD was 52.64±13.46 years old and 50% was female. Mean BMI was 26.90±2.66 kg/m2. The majority were overweight (72%) followed by normal weight (21%) and obese (7%). eGFR at 2‐ and 7‐day post‐donation were significantly lower than pre‐donation eGFR (55.59±12.48 vs. 55.29±11.33 vs. 93.39±14.62, p <0.001; Figure 1). Every 1 kg/m2 increase in pre‐donation BMI was associated with 0.1% greater decline in eGFR at 2‐day post‐donation, while eGFR decline was up to 1.4% at 7‐day post‐donation (β2day±SE 0.149±0.716, p 0.861, 95%CI ‐1.668, 1.966; Figure 2A and β7day ±SE 1.368±0.834, p 0.080, 95%CI ‐0.191, 2.928; Figure 2B). After adjusted for age, gender, ethnicity, pre‐donation eGFR, and interaction term between age and pre‐donation eGFR, the magnitude of eGFR decline post‐donation were greater for both 2‐ and 7‐day post‐donation; however, only the 7‐day post‐donation eGFR decline was statistically significant without the interaction between age and pre‐donation eGFR (β2day±SE 0.653±0.977, p 0.525, 95%CI ‐1.657, 2.963, pinteraction 0.045 and β7day±SE 2.068±0.800, p 0.036, 95%CI 0.176, 3.959, pinteraction 0.541).ConclusionsHigher pre‐donation BMI is associated with greater eGFR decline at 7‐day, but not 2‐day, post‐donation. Pathological glomerular hyperfiltration from overweight or obesity may counteract with physiological glomerular hyperfiltration from acquired solitary kidney post‐donation leading to worsening post‐donation eGFR decline.
Role of Digital Media in Enhancing Anatomical Education: A Survey Study Focused on the Cranial Cavity and Its Contents
Authors: Mashayekhi Mohammadsadegh , Laghaei Farimani Pedram , Kassam Farris , Doroudi Majid 
Published: May 2022

IntroductionRecently, there has been an increasing effort to modify traditional learning tools to adopt them with the new demands of students by producing visual learning tools. These efforts have aimed for an equal access to high quality learning opportunities to fulfill the increasing interest in self‐paced flexible studying using digital medial technology. The emergence of COVID pandemics has highlighted these demands for these educational resources. Human gross anatomy education is one of the fields that has been impacted by recent critical situation more dramatically. Thus, it is crucial to create more educational videos in gross human anatomy and study the effects of such videos on anatomy education.ObjectivesThis project aims for understanding the role of self‐paced educational videos in education of human body anatomy with a focus on cranial cavity and its contents.MethodA video exploring the cranial cavity and its contents for learners in professional programs was filmed and edited by Camtasia 2020 software. Different features such as labels, transitions, schematic pictures, and quiz questions were added in the editing process to enhance the students’ experience. The edited video was uploaded on YouTube and shared with students taking human gross anatomy courses. The viewers were asked to complete an online survey that was designed to assess their learning experience using the video.Link to video: https://www.youtube.com/watch?v=ROGgR7mxsr8&t=107sResultsThe video received 2429 views, 92 likes and 29 responses to the survey from Jan 2021 to Dec 2021. Participants watched the video from different countries such as Canada, US, India, and Brazil. Analysis of the data shows that the majority of the participants who completed the online survey found the video and its features enhancing their learning of the anatomy of the human body. More specifically, 82.8% of students selected “to prepare for my anatomy lab” as their main purpose for viewing the video and 93.1% choose “Strongly Agree” when asked if the videos helped them fulfill their purpose.Moreover, on the 1‐5 scale, the average score of 4.59, 4.66, 4.41, and 4.86 were given by students for the quality of labelling, transitions, quiz questions, and overall quality of the video, respectively.ConclusionIn conclusion, the feedback received from this survey strongly suggests that using digital media is beneficial for enhancing the anatomical learning experience of students.
Actionable Mutation Profile of Sun-Protected Melanomas in South America
Authors: Hsieh Ricardo , Nico Marcello M. S. , Camillo Cláudia M. C. , Oliveira Kátia K. , Carraro Dirce M. , Sangueza Martin , Lourenço Silvia V. 
Published: May 2022

Abstract:
Melanomas that arise in sun-protected sites, including acral and oral mucosal melanomas, are likely under the control of unique, specific mechanisms that lead to mutagenesis through various pathways. In this study, we examined somatic mutations in tumors by targeted sequencing using a custom Ion Ampliseq Panel, comprising hotspots of 14 genes that are frequently mutated in solid tumors. Tumor DNA was extracted from 9 formalin fixation, paraffin-embedded sun-protected melanomas (4 primary oral mucosal melanomas and 5 acral lentiginous melanomas), and we identified mutations in the NRAS, PIK3CA, EGFR, HRAS, ERBB2, and ROS1 genes. This study reveals new actionable mutations that are potential targets in the treatment of photo-protected melanomas. Additional studies on more of these melanoma subtypes could confirm our findings and identify new mutations.