bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypoglycosylated Follistatin‐Like 1 Attenuates the Loss of Coronary and Cerebral Vascular Functional Capacity in Ossabaw Swine with Cardiometabolic Heart Failure with Reduced Ejection Fraction
Authors: Tharp Darla L. , Kelly Shannon C. , Thorne Pamela K. , Amin Amira , Silva Kleiton A. , Mueller Christina M. , Ivey Jan , Olver T. Dylan , Ruiz‐Lozano Pilar , Emter Craig A.
Published: May 2022

Ischemic heart disease is a leading cause of death, often resulting from heart failure caused by myocardial infarction. Diabetic cardiomyopathy is increasingly problematic clinically, with the combination of metabolic syndrome and chronic inflammation greatly increasing the risk for both coronary and cerebral vascular disease. Follistatin‐like 1 (FSTL1) is a protein that, when non‐glycosylated, displays regenerative properties including pro‐angiogenic effects and prevention of abnormal vascular remodeling. Thus, the objective of the current study was to assess the therapeutic benefit of human recombinant non‐glycosylated FSTL1 protein on both coronary and cerebral vascular function in a pre‐clinical Ossabaw swine model of myocardial infarction (MI). We hypothesized FSTL1 would attenuate the development of coronary and cerebral vascular dysfunction in an experimental setting of cardiometabolic heart failure with reduced ejection fraction (HFrEF). Intact female Ossabaw swine (2 months old) were fed a Western Diet for 4 months to develop metabolic syndrome. At 6 months of age, animals were subjected to 90 minutes ischemia followed by reperfusion (I/R) to induce MI. One month post‐MI, ALZET osmotic pumps were implanted and either vehicle (MI group) or FSTL1 (MI+FSTL1 group) was delivered over two weeks, with terminal vascular experiments performed 2 months post‐MI. In vitroassessment of isolated coronary (n=5‐6 for infarct, border, and remote regions of the left ventricle; 139.5 ± 4.9 μm diameter) and cerebral (n=2‐6 for middle cerebral artery second order pial; 385.3 ± 18.7 μm diameter) arteriole function was examined using pressure myography. Dose response curves for: 1) U46619 (thromboxane A2 agonist); and 2) NS‐1619 (large‐conductance calcium‐activated potassium channel activator; BKCa) were conducted. A 2x2 ANOVA (Group X Dose) was used to determine significance at the p < 0.05 level. Cardiometabolic HFrEF was indicated by a combined ejection fraction of 38 ± 2% and a fasting HOMA‐IR of 3.8 ± 0.4 (vs. 0.6 ± 0.1 historical control). In coronary arterioles, the NS‐1619 induced vasodilatory capacity was increased ~18‐fold in MI+FSTL1 animals relative to MI (100 ± 13% vs. 1832 ± 3% in MI and MI+FSTL1, respectively) in the remote region of the heart (interaction effect), but not the infarct nor border regions. In cerebral arterioles, the vasoconstrictor capacity in response to U46619 was increased ~2‐fold in MI+FSTL1 relative to MI animals (100 ± 19% vs. 213 ± 25% in MI and MI+FSTL1, respectively; interaction effect). In conclusion, FSTL1 partially attenuates the loss of functional capacity in both coronary and cerebral arterioles, demonstrating its potential to improve vascular function in an experimental setting of cardiometabolic HFrEF.
Patient‐specific Autoantigen Sample Preparation and Analysis Using the Reversible Protein Tag ProMTag
Authors: Minden Jonathan S. , Ascherman Dana , Lucas Amber
Published: May 2022

Autoimmune diseases affect >20 million people in the US today. Currently, disease‐specific autoantibodies are thought to be the best biomarkers for diagnosis. Conventional immunoprecipitation methods have been used to identify autoantigens from the most common autoimmune diseases. However, these diseases account for only 6.5 million of the 20 million patients suffering from autoimmune diseases, leaving many without diagnoses until irreversible damage occurs. The remaining 13.5 million patients have >70 autoimmune disorders without well characterized autoantibodies. The state‐of‐the‐art diagnostic test of these remaining diseases relies on gel electrophoresis of immunoprecipitated radiolabeled proteins, which cannot be identified by MS due to safety issues and the overwhelming presence of immunoglobulins.

We have created an immunoprecipitation method that uses serum from patients with any autoimmune disorder to identify patient‐specific autoantigen proteins. This method uses a reversible click chemistry tag, called ProMTag. One end of the ProMTag forms a reversible, covalent bond with protein by coupling to lysines and amino termini. The other end of the ProMTag can form an irreversible, covalent bond with a solid bead support via a click chemistry, methyltetrazine‐TCO, pairing. In this study, the proteins of cell lysates that contain potential autoantigens were labeled with ProMTag. The ProMTagged‐proteins were exposed to patient antibodies bound to Protein A beads, thus capturing the ProMTagged autoantigens. All proteins were released from the Protein A beads, including ProMTagged‐autoantigens and untagged‐antibodies. The ProMTagged‐autoantigens were subsequently coupled to TCO beads, and the untagged‐antibodies were washed away. The linkage between the ProMTag and autoantigens was then reversed, yielding autoantigen proteins with greatly reduced antibody contamination ready for MS analysis. MS analysis successfully identified autoantigens from patient serums with rheumatoid arthri. This autoimmune biomarker discovery method can accelerate sample testing for known autoantigens and facilitating rapid discovery of novel autoantigens for both diagnostic and predictive biomarkers.
Exercise Training Attenuates Radiation‐Induced Impairments in Endothelium‐Dependent Vasodilation of Coronary Arterioles in Rats
Authors: Kunkel Olivia , Baumfalk Dryden , Horn Andrew , Behnke Brad
Published: May 2022

Background

Radiation is an important and effective anti‐cancer therapy, but is associated with many side effects. Radiation exposure may cause damage to the heart and coronary vasculature. Exercise training is currently being investigated in cancer research as a treatment or adjuvant to anti‐cancer therapies, and has been shown to mitigate deleterious changes in coronary vascular function, such as with aging. We hypothesized that impairments in endothelium‐dependent vasodilation of coronary resistance arterioles that occur in response to radiation exposure would be mitigated with exercise training.

Methods

12 male Fischer 344 rats (3‐4 months old) were randomly assigned to one of four groups: control (CON; n=3), exercise (EX; n=3), control radiated (RAD; n=3), or exercise radiated (EX RAD; n=3). After increasing intensity over the first 3 weeks, EX and EX RAD animals ran on a motorized treadmill 5 days/week for 60 minutes/day, at an intensity of 15 m/min and 15° incline for the remainder of 12 weeks of training. After the conclusion of exercise training, all groups of animals were subjected 2 Gy whole body radiation by X‐Rad 350 x‐ray irradiator and euthanized within 8 hours. Hearts were harvested and left anterior descending (LAD) arteriole sections were isolated and fixed on glass pipets. Vessels were pressurized at 60 cm H2O. Once a minimum of 20% tone was developed, vessels were exposed to cumulative doses of bradykinin (10‐13‐10‐7 M), an endothelium‐dependent vasodilator, and vessel diameter was recorded at each dose.

Results

No differences were detected for CON vs. EX or CON vs. EX RAD. Vasodilation for RAD was significantly reduced vs. CON (p = 0.007). EX RAD was significantly increased vs. RAD (p = 0.025).

Conclusions

There were no differences between CON and EX; however, vasodilation was impaired for radiated animals, suggesting even one dose of 2 Gy radiation can cause significant impairment in endothelium‐dependent vasodilation of coronary arterioles. Exercise training attenuated this impairment and restored endothelium‐dependent vasodilation to levels not different from control animals. These findings are significant, given that exercise training may be used as a simple and safe method to preserve coronary arteriole function in patients receiving radiation therapy.
Loss of Autotaxin inhibits Toll‐like receptor4‐mediated immune mechanisms and promote spontaneous colitis in Il10‐ko mice
Authors: Rhee Sang H. , Kim Su Jin , Howe Cody , Jeong Sihyun , Im Eunok
Published: May 2022

Autotaxin (Atx) is a secreted enzyme converting lysophosphatidylcholine (LPC) and sphingosyl‐phosphorylcholine into lysophosphatidic acid and sphingosine 1‐phosphate, respectively. Given the high affinity of LPC to cholesterol and the enrichment of cholesterol and sphingolipids in lipid rafts wherein LPS sensor Toll‐like receptor 4 (TLR4) and its co‐receptor CD14 reside, we hypothesized that Atx deficiency inhibits TLR4‐mediated innate and adaptive immunity; thereby, accelerating the susceptibility to microbe‐induced intestinal inflammation.

To study this hypothesis, we generated myeloid cell lineage‐restricted Atx‐knockout (ko) mice (Atx‐dME/dME) to investigate TLR4‐mediated immune and inflammatory responses and examined LPS‐receptor complex formation through fluorescence resonance energy transfer technique, confocal microscopy, and flow cytometry. Phagocytic activity of macrophages was assessed. Lamina propria CD4+ T cells and bacterial load in the intestinal mucosa were examined. An impact of Atx deficiency in inflammatory bowel diseases (IBD) was investigated using Atx‐dME/dME;Il10‐/‐ mice that have both Atx‐deletion in myeloid cells and a global Il10 deletion. We examined the serum samples from IBD patients.

With peritoneal macrophages from Atx‐dME/dME mice, we identified that Atx‐ko disrupted the integrity of lipid rafts at the plasma membrane, resulting in the inhibition of TLR4 complex formation. Accordingly, the recruitment of adaptor molecules to TLR4 was suppressed, and TLR4‐mediated responses were substantially reduced in Atx‐ko macrophages. TLR4‐induced innate immunity such as phagocytosis was attenuated in Atx‐ko macrophages. The activation of CD4+ effector T cells and regulatory T cells was diminished in the lamina propria lymphocytes of Atx‐dME/dME mice compared to that of Atx‐wt littermates. Consequently, Atx‐dME/dME mice had a higher bacterial prevalence in the intestinal mucosa compared to controls. Just like the notion that commensal microbes translocated from the lumen into the intestinal mucosa can elicit spontaneous colitis in Il10‐/‐ mice, combining Atx‐dME/dME with Il10‐/‐ mice (Atx‐dME/dME;Il10‐/‐) did accelerate spontaneous colitis development. Atx‐dME/dME;Il10‐/‐ mice had gross inflammation occurring throughout the colon, massive neutrophil infiltration and necrosis in the colonic mucosa, and increased mortality (Log‐rank P=0.0046), while Atx‐wt;Il10‐/‐ littermates are normal. Notably, ATX serum protein level was lower in UC (n=26) and CD (n=34) patients compared to the level in normal subjects (n=26) (P<0.001), indicating an association of reduced ATX levels with intestinal inflammation.

Collectively, we found that Atx deficiency suppresses TLR4‐mediated innate and adaptive immune mechanisms; thereby accelerating the susceptibility to microbe‐induced gut inflammation.
Retention of Physical Therapy students in relation to Human Anatomy failure in a private university in Santiago, Chile
Authors: Orquera Paulo , Cariseo Carolina , Santana Andres , Gold Marjorie , Orellana Mathias , Valenzuela Juan J.
Published: May 2022

The subject of human anatomy is a critical subject in terms of the failure rate in many universities in the world, this attributed to the complexity and the large amount of new content for the student, so knowing the failure and how it affects the continuity of the student in the career, it is important to have new and better guidelines in the subject human anatomy. Our research was carried out during 2020 with a retrospective correlational design, in which the data of Physical Therapy students in the admission cohorts of the years 2017, 2018 and 2019 of the University of the Americas, Campus Santiago Centro, were analyzed. Chile. Of the 3 cohorts analyzed in this study, the cohort with the most gender differences between admission was that of 2019, with respect to failing, the cohort with the highest failing was that of 2019, while the highest percentage of desertion in relation to reprobate was the year 2018. Regarding what was seen in this study, failing this subject individually or associated with another subject of the same semester, for students is frustrating which could be a condition for desertion, we know that desertion can have many aspects, but we do believe that by taking some remedial measures such as; creating support programs, optimizing the delivery of content, active learning methodologies, among others, could improve the results of the human anatomy.
Investigating Potential Therapeutic Activity of Designed Histone Derived Antimicrobial Peptides through Hybridization and Antibiotic Cocktails
Authors: Alvarez Valentina E. , Perry Brianna E. , Darling Louise E. , Elmore Donald E.
Published: May 2022

Modern medicine is contingent on innovation in order to best face the myriad of challenges to human health and the evolution of organisms such as bacteria. Due to increasing antibiotic resistance to small molecule antibiotics in bacteria, there is a need for alternative therapeutic routes and options. One potential alternative is antimicrobial peptides (AMPs), which have a broad range of antimicrobial activity and are found as part of the innate immune response of many organisms. Through research and exploration of these AMPs we look to improve their antimicrobial activity. Our current research investigates these two approaches utilizing novel histone derived antimicrobial peptides (HDAPs). These peptides are based on buforin II (BF2), DesHDAP1, and DesHDAP2. In one approach we hybridize two peptides into a single, longer AMP. In the other approach we combine AMPs with conventional antibiotics. Studies have shown that some hybrid peptides are more effective against bacteria and less toxic to mammalian cells than the lone parent peptides. Similarly, using two therapeutics can yield synergistic effects, proving more beneficial combined than alone. Thus far, the hybrid peptides demonstrate increased antibacterial activity compared to the individual parent peptides, and some show much greater activity depending on the order of the parent peptides and the amino acid linker utilized. DesHDAP2 has antimicrobial activity when used alone comparable to that of BF2. Trials running DesHDAP2 in combination with several conventional antibiotics with differing mechanisms of action have noted potentially synergistic combinations. Ongoing work will determine if there are any harmful side effects to eukaryotic cells from these two therapeutic approaches.
Conformations of p90 Ribosomal S6 Kinase (RSK) Activation
Authors: Kobori Evan , Chen Katherine , Taylor Susan
Published: May 2022

Protein kinase have evolved to be dynamic macromolecular switches that alternate between inactive and active conformations. Although most kinase are typically phosphorylated and activated by other kinases, relatively little is known about the detailed mechanistic steps required for one kinase to activate another. The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine kinases that lie downstream of the Ras‐MAPK pathway and regulate cell proliferation, cell survival, cell growth, and cell motility. RSK is an interesting model system to study kinase activation because it contains two distinct kinase domains, an N‐terminal kinase (NTK), and a C‐terminal kinase (CTK) in the same polypeptide chain. The NTK is a member of the AGC kinase superfamily, and the CTK is a member of the CAMK family. A flexible linker bridges the NTK and CTK, and embedded within the linker is the highly conserved tail of AGC kinases (AGC tail) which contains the conserved turn and hydrophobic motifs (HF). RSK has a complex activation mechanism that includes sequential phosphorylation events and requires two additional kinases: extracellular signal‐regulated kinase (ERK2) and 3‐phosphoinositide‐dependent protein kinase 1 (PDK1). ERK2 phosphorylates the CTK and the turn motif. The phosphorylated CTK is then able to phosphorylate the linker at the HF, this enables PDK1 to bind the linker and then phosphorylate and activate the NTK2. The precise structural details of RSK kinase activation are not fully understood and a more rigorous biochemical characterization could provide valuable insight into how kinase complexes assemble and how the linker facilitates multiple binding and phosphorylation events.

To gain a better understanding of the FL RSK2‐ERK2 signaling complex we have used cryoEM and hydrogen deuterium exchange mass spectrometry (HDX) to gain insight into the overall shape and solvent accessibility of the overall complex. We have determined a low‐resolution density map of the RSK2‐ERK2 complex, revealing a compact shape. Additionally, all three kinase domains are relatively well protected from solvent, with the exception of the activation loops of the CTK, and the aC helix of the NTK. While the tails of RSK2 and linker (AGC tail) are highly solvent exposed. We hypothesize that the linker region needs such solvent accessibility in order to enable the various interactions and phosphorylation events necessary for RSK activation. We have also utilized peptide array binding experiments to determine the residues within the linker that are necessary to enable ERK2 phosphorylation of the turn motif, CTK phosphorylation of the HF, and PDK1 binding and subsequent phosphorylation of the NTK activation loop. ERK2 appears to bind to the linker and NTK in a multivalent fashion via the C‐lobe (300‐320) and the N‐lobe. The CTK seems to primarily interact with HF in the linker. And PDK1 binds to both the HF and a conserved FDX1‐2Y/F motif in the linker.
Associations Between Heart Rate and Skeletal Muscle Oxygen Saturation (SmO₂) in Upper and Lower Extremities During Self‐Paced Walking
Authors: McCarthy Ryan , Locke Emily , Kadry Ahmed , Torad Ahmed , Elwan Mostafa , Appiah‐Kubi Kwadwo , Towler Christopher , Boolani Ali
Published: May 2022

This study aims to determine the association between heart rate and skeletal muscle oxygen saturation (SmO₂) during 30 minutes of self‐paced walking on 4 different days. It was hypothesized that there would be a significant association between heart rate and SmO₂, as increased exercise intensity would result in increased peripheral muscle O₂ consumption and increased heart rate. Participants (age= 18‐40) were recruited from Clarkson University as part of an ongoing larger study and informed consent was signed. Heart rate was measured using a pulse oximeter after every five minute interval of the 30 minute walking period and was recorded in an online survey on surveymonkey.com. The muscle oxygen monitor (MOXY) near infrared sensors were placed on the left and right medial gastrocnemius heads as well as on the left biceps brachii muscle to record SmO₂ levels. Data was analyzed using a bivariate Pearson correlation to determine if there was a significant correlation between SmO₂ and heart rate while walking. Heart rate was negatively associated with SmO₂ in the left medial gastrocnemius head (R= ‐0.139, p= 0.044, n= 211) while there was no significant relationship seen in the right medial gastrocnemius head (p> 0.05). In the left biceps brachii muscle, there was a positive association between heart rate and SmO₂ (R= 0.148, p= 0.035, n= 204). The findings of our study suggest that as walking intensity increases, O₂ consumption in the upper and lower extremities has a weak, but significant association with heart rate. These findings suggest that as blood is shunted from the upper extremity to lower extremity to meet lower extremity demands during walking, O₂ utilization by muscles in the upper extremity is decreased. Further research is needed to elucidate how these relationships change when performing activities that require both upper and lower extremity muscles as well as with varying intensities of exercise.
Ephrin Ligands are Upregulated in the Saliva of SARS‐CoV‐2 Infected Patients
Authors: Egal Erika , Helms My N. , Liou Theodore G. , Mimche Patrice
Published: May 2022

Background

Patients infected with SARS‐CoV‐2 may develop severe lung disease characterized by respiratory distress, systemic thrombosis, or death. Molecular mechanisms responsible for severe cases of COVID‐19 remain unclear. We investigated the role of the Eph family receptor‐interacting ligands (ephrin‐A1 and ephrin‐B2) in COVID‐19 patients. Ephrin binding to its tyrosine kinase Eph receptor plays important roles in injury and inflammation. Serum levels of ephrin‐A1 ligand correlates positively with COVID‐19 severity (Mendoza et al, CellPress 2021). Based on this recent report, and the emerging role of Ephrin‐Eph signaling in lung injury, we hypothesize that ephrin‐mediated cytokine signaling is upregulated in epithelial and immune cells assayed from saliva samples obtained from seriously ill COVID‐19 patients seeking emergency evaluation and treatment.

Methods

Saliva samples were collected from patients (± SARS‐CoV‐2) in the University of Utah Emergency Department (ED) with IRB‐approved written consent. Saliva samples were incubated 1:1 in Streck Preservative (prior to EpCAM and CD45 labeling) or with 0.008% glutaraldehyde (following ELISA and multiplex immunoassays) in order to inactivate virus.

Results

Flow cytometric evaluation of saliva for EpCAM+ and CD45+ cells indicated that SARS‐CoV‐2 positive samples contain both epithelial and immune cell types (see Fig 1). Uninfected saliva samples (control) primarily contained EpCAM+ cells. COVID‐19 samples (n=67 patients) expressed higher levels of type 1 interferon [IFNγ1 and IFNα2 (p< 0.001)] vs control (n=64 patients). Interferon induced protein [IP‐10] was also upregulated in COVID‐19 patients (p<0.001), as well as several cytokines in the interleukin family [IL‐1β, IL‐6, IL‐8 (p<0.001) and IL‐12p70 (p<0.05)]. TNF‐α did not differ significantly between the ± SARS‐CoV‐2 samples evaluated. Using a subset of the +SARS‐CoV‐2 samples (n=20) and uninfected controls obtained from the ED (n=20), we show that both ephrin‐A1 and ephrin‐B2 ligands are significantly elevated in +SARS‐CoV‐2 samples vs. ED control samples (p<0.001). Ephrin‐B2 levels correlated negatively with IL‐12p70 (Pearson Correlation =‐0.53, p=.01).

Conclusion

Saliva is low risk to obtain and is a biologically relevant sample that can be used to reliably measure changes in epithelial and immune cell responses of COVID‐19 patients. Specifically, our study showed that ephrin‐A1 and ephrin‐B2 ligands are upregulated in the saliva of COVID‐19 patients in a TNF‐α independent manner and that ephrin‐B2 correlates negatively with IL‐12p70 (a potent pro‐inflammatory cytokine). Future studies are aimed at establishing ephrin ligands as a biomarker for severity of lung injury and improving understanding the patho‐physiology of SARS‐CoV‐2 infection.
In Vitro Transcription Factor Binding Site Predictions Using Support Vector Machine Classification
Authors: Pomales‐Matos Diego A. , Rosado‐Tristani Diego A. , Carrasquillo‐Dones Emmanuel A. , Rodríguez‐Martínez José A.
Published: May 2022

Transcription factors (TFs) are sequence‐specific DNA‐binding proteins essential in regulating gene expression. Determining TF DNA‐binding specificity can help to study gene regulatory networks within cells and how genetic variation can disrupt normal gene expression. One method for characterizing TF specificity is through Support Vector Machines (SVMs) by analyzing chromatin immunoprecipitation followed by DNA‐sequencing (ChIP‐seq) data. However, this can also be achieved using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) data, a method that also aids in determining TF‐DNA preferences. During this project, I implemented a gapped kmer SVM to study TF‐DNA binding preferences by using data from SELEX‐seq. I used a large scale‐gapped kmer, a sequence‐based SVM for analyzing TF specificity. It works by creating a predictive model that is trained with bound and unbound sequences from SELEX data. For purposes of this project, we used the T‐box transcription factor 5 (TBX5). After training the model for TBX5 and testing its performance, it had an AUROC value of 0.8248, indicating a significant degree of reliability. Likewise, the sequences with highest scores contained motifs for the TBX5. Given these results, we concluded that SVM was successfully implemented. In addition, SELEX data had not been previously used to train SVM based predictive models, meaning SELEX data is compatible and useful for developing predictive models.
Membrane Protein Comparison Between Cell Membranes and ExtracellularVvesicle Membranes of S. pneumoniaeProvide Insights into Extracellular Vesicle Formation and Shedding
Authors: Biedka Stephanie , Eutsey Rory , Hiller Luisa , Minden Jonathan S.
Published: May 2022

Extracellular vesicles (EVs) are complex, cell‐derived nanoparticles generated by all cell types. EVs are composed of lipid bilayer membranes and their associated membrane proteins, nucleic acids, and luminal proteins. The mechanism by which Gram‐positive bacteria shed EVs is still unknown. EVs from the Gram‐positive human pathogen S. pneumoniae, which is a major cause of otitis medi and pneumonia, are of particular interest because of how they EVs modulate the host immune response. To uncover possible mechanisms for EV production and shedding in S. pneumoniae, we have performed a comparative proteomics analysis of EV membrane proteins versus whole‐cell membrane proteins.

Membrane proteins were enriched from intact S. pneumoniae cells or their EVs using a ProMTag labeling and capture workflow. ProMTag is a bifunctional protein tag where one moiety of the tag is able to form a reversible, covalent link to primary amines on proteins. The other moiety is methyltetrazine, which can form an irreversible, covalent bond with trans‐Cyclooctene (TCO) on the surface of beads to capture ProMTagged proteins for cleanup and elution. Using this workflow plasma membrane proteins can be tagged, captured, washed to remove non‐plasma membrane proteins, and then eluted in their original, unmodified state.

In this study, intact cells and EVs from S. pneumoniae cultures were separated and the extracellular domains of membrane proteins in these two fractions were labeled with ProMTag. The membrane proteins were then enriched, washed, and eluted using the ProMTag workflow. These membrane protein populations were then TMT labeled and analyzed using mass spectrometry. Comparative analysis revealed membrane proteins that are concentrated or absent in EV membranes relative to bulk plasma membrane from whole cells, indicating a selective process for EV formation in S. pneumoniae. With this information, we present a new model for EV formation and shedding in S. pneumoniae.
Understanding the endocrine response to macronutrients in the context of a ketogenic diet
Authors: Deru Landon , Warren Cali , Wright Braxton , Coleman Jacob , Duersch Austin , Chan Hi’ilei , Hales Katelynn , Bailey Bruce , Bikman Benjamin
Published: May 2022

A ketogenic diet (KD) consists of long‐term carbohydrate avoidance and the resulting elevation in ketones due to low insulin. This altered energetic state may result in unique hormonal responses to macronutrients. Accordingly, the purpose of this study was to quantify the endocrine response to various macronutrients in the context of a KD. Nine subjects (6 male, 3 female) who adhere to a carbohydrate‐restricted diet (50g or less carbohydrates per day) completed three interventions, each at least one week apart. Each intervention consisted of 5 blood draws. One immediately before and in serial increments every 30 minutes (30, 60, 90, and 120 minutes) after the consumption of a 300 kcal drink which consisted of either all protein (whey), carbohydrate (dextrose), or fat (olive oil). Thirteen analytes were measured including amylin, C‐peptide, ghrelin, gastric inhibitory peptide (GIP), glucagon, glucagon‐like peptide 1 (GLP‐1), interleukin 6 (IL‐6), insulin, leptin, pancreatic polypeptide (PP), polypeptide YY (PYY), and tumor necrosis factor alpha (TNF). Amylin, C‐peptide, GIP, glucagon, GLP‐1, insulin, and MCP‐1 each demonstrated a statistically significant difference between conditions (p = 0.0298 for MCP‐1, p<0.0001 for all others). The glucose load significantly raised amylin, C‐peptide, GIP, and insulin while decreasing glucagon, relative to the fat and protein loads. The protein load, in contrast, uniquely raised GLP‐1 and MCP‐1 relative to the other drinks. In conclusion, adherence to a KD results in varied endocrine outcomes when challenged with protein, glucose, and fat, including incretins and pancreas‐derived hormones.
Sarcospan‐deficient mice exhibit a heightened inflammatory phenotype under obesiogenic conditions
Authors: Crawford Rhiannon Q. , Valera Isela C. , Pindado Jose , Reis Gisienne , Kahmini Aida R. , Mumbi Florence , Parvatiyar Kislay , Parvatiyar Michelle S.
Published: May 2022

To study the metabolic consequences of sarcospan (SSPN‐/‐) deletion we subjected WT and SSPN‐/‐ male mice to high fat diet (60% fat) for 4 months and compared them to mice fed standard chow diet (CD). SSPN‐/‐ mice were partially protected from weight gain and additional studies were performed to understand the role SSPN plays in obesity‐associated inflammation. Initial experiments examined innate immune activation of cultured bone marrow‐derived macrophages (BMDM) obtained from CD SSPN‐/‐ and WT mice. In response to B‐DNA, Poly I:C and lipopolysaccharide (LPS), SSPN‐/‐ BMDM generally exhibited a faster, more robust pro‐inflammatory response (e.g. IL‐6, IFN‐β, TNF‐α) compared to WT. We hypothesized that SSPN acts as negative regulator of the Type‐I interferon response although the pathways involved are still being investigated. To determine whether cultured SSPN‐/‐ BMDM obtained from HFD mice have a dysregulated Type‐I interferon response ‐ isolated, cultured BMDM were treated at time 0‐ with B‐DNA, Poly I:C and LPS and cytokine production was monitored at 0‐, 12‐, 24‐ and 36‐hours post‐treatment. BMDM obtained from HFD SSPN‐/‐mice exhibited a more pronounced response compared to BMDM obtained from non‐HFD mice. Blood serum collected from WT and SSPN‐/‐ HFD and CD mice was analyzed by cytokine array for the presence of pro‐inflammatory markers. Consistent with data obtained from BMDM suggesting heightened responsiveness, SSPN‐/‐ HFD mice had significantly higher circulating levels of C‐X‐C motif chemokine ligand (CXCL10 and CXCL11), chemokine (C‐C motif) ligand 2 (CCL2) and IFN‐γ compared to WT HFD and SSPN‐/‐ CD mice. CXCL10, secreted by several cell types in response to IFN‐γ, was significantly higher in serum of SSPN‐/‐ mice. CCL2 is primarily secreted by monocytes, macrophages and dendritic cells to recruit specific immune cells to sites of inflammation while CXCL10 and CXCL11 are validated biomarkers associated with heart failure and ventricular dysfunction. In summary, the significance of a heightened pro‐inflammatory response in SSPN‐/‐ mice is intriguing and remains to be elucidated.
Elucidating Pharmacological Activities of Volatile Chemotypes in Essential Oil of Morina longifolia
Authors: Yousuf Sajad , Morenikeji Olanrewaju , Kutchy Naseer A. , Singh Akhand P.
Published: May 2022

Morina longifolia(Himalayan Whorlflower) is a glabrous herb, found at an altitude of 3000 – 4000 m in the regions of Himalaya, Kashmir, and Bhutan. Previous reports have shown that the parts of this plant like leaves and flowers have been used for healing wound, improving digestion, and enhancing appetite. However, what compound or phytochemicals promote the medicinal use of this plant has not been elucidated and what other uses in medicine are also largely unknown. Therefore, the objective of this study was to characterize phytochemicals in the essential oil of Morina longifolia using GC‐MS (gas chromatography‐mass spectrometry) and to evaluate their bioactivity against bacteria, fungi, and oxidants. The results obtained from the GC‐MS analysis identified 23 compounds, including α‐copaene (49.19%), caryophyllene (20.03%), δ‐cadinene (10.49%), and α‐humulen (4.10%), with higher concentrations in the plant essential oil. We found that essential oil at concentrations of 0.125 mg/mL to 1 mg/mL was able to control the growth of bacterial and fungal strains. Among the bacteria strains, Kocuria rhizophila(MTCC 1541), Micrococcus luteus(MTCC 1541) and Salmonella typhi(NCTC 786) were sensitive at high concentrations, as their growth was inhibited by oil at concentrations of 0.5 to 1%. The oil also inhibited the growth of fungal strains including Aspergillus niger(MTCC 1344) at a high concentration of 0.5 to 1%, while Candida albicans(MTCC 227) growth was inhibited at a low concentration of 0.125 to 0.250%. In addition, we found free radical scavenging activity (FRSA) of M. longifolia essential oil which was in the range of 10.19 – 89.15%. Using ferric chloride (FeCl3) reducing method to access the reducing power activities of M. longifolia essential oil, our results revealed M. longifolia has more than 50% reduction potential at the concentration of 0.75 mg/ml and confirmed its antioxidant properties. Finally, a 0.50 mg/mL IC50 value was obtained indicating a scavenging of nitric acid activities, at 54.21%. The present study identified the significance of M. longifoliaand its potential use as a source of antimicrobial and antioxidant compounds which could act as an alternative against the menace of growing antibiotic resistance.
Sex Differences in Antioxidant Gene Expression in response to Chronic Arsenic Exposure
Authors: Berry Savannah R. , Dresler Sara R. , Propper Catherine R. , Kellar Robert S. , Traustadóttir Tinna
Published: May 2022

Inorganic arsenic is highly toxic metalloid considered a water contaminant that is naturally occurring through geological formations where it leaches into ground water sources and is anthropogenically sourced through pollution and found in surface water as a contaminant. Acute and chronic arsenic exposure can stimulate the production of reactive oxygen species and increase systemic oxidative stress. The aim of the present study was to investigate sex differences in the redox response induced by chronic arsenic exposure in a murine model.

Twenty‐four (12 female & 12 male) 10‐wk old mice were randomized to arsenic treatment or control. Arsenic groups were exposed to controlled arsenic levels (10µM = ppb = ug/L) via drinking water for 8‐wks. Following the treatment, mice were euthanized, and skeletal muscle was collected. The tissue was homogenized and processed for measures of gene expression for seven antioxidant genes (Sod2, Cas1, Gpx, Gst, Gsr, Gclc, Nrf2). One‐way ANOVA was performed with post‐hoc comparisons depending on normality of data. Interactions between sex and arsenic were determined using univariate testing.

There was a significant sex‐by‐treatment interaction for Cas1, Gpx1, Gsr, Gclc, and Nrf2 where the female mice treated with arsenic responded by upregulating these genes in comparison to controls (p<0.05), whereas the males did not. Only Sod2 was lower in the arsenic treated female mice versus controls and there were no effects on Gst across the 4 groups.

These data demonstrate that sex differences only emerged under stimulated conditions (arsenic exposure) and support the protective role of estrogen in antioxidant defenses.
Interplay between intestinal microbiota and PRDM16‐mediated metabolic regulation of intestinal stem and progenitor cells
Authors: Stine Rachel R. , Seale Patrick
Published: May 2022

Distinct metabolic programs regulate intestinal stem cell renewal and differentiation. However, the mechanisms that rewire metabolism during differentiation are poorly defined. Previously, we have shown that the transcription factor PRDM16 is a critical regulator of intestinal metabolism and differentiation. Acute deletion of Prdm16 in adult mice causes intestinal wasting, apoptosis, and an accumulation of poorly differentiated cells in the small intestinal crypt, primarily in the duodenum and proximal jejunum. PRDM16 has emerged as a regulator of metabolic signaling in multiple tissues including thermogenic adipose tissue and the small intestine. RNA‐seq and tracer metabolomic studies show that PRDM16 regulates fatty acid oxidation (FAO) in the intestinal crypt, leading to impaired differentiation of stem and transit amplifying cells into mature cells.

Our current studies have focused on the effects of the intestinal microbiota, which produces significant quantities of metabolites that could affect cells in the digestive tract. We postulated that altering the microbiome of mice in the context of Prdm16 deletion could modify the phenotype. Strikingly, pre‐treatment of animals with an antibiotic cocktail including ampicillin, neomycin, vancomycin and metronidazole almost completely rescues the Prdm16 deletion phenotype. While deletion of Prdm16 typically leads to death within 10 days, antibiotic treated mice survive at least 12 weeks after deletion. An early hallmark of the Prdm16‐deletion phenotype is the induction of the p53 pathway in the crypt. Apoptotic cells are present in high numbers, particularly in transit amplifying progenitors. Gene expression profiling of isolated small intestinal crypts indicates that the early p53 signature that is typical of Prdm16 deletion does not develop in antibiotic treated mice. Furthermore, pretreatment of mice with antibiotics prevents the severe defects normally observed when Prdm16 is deleted ex vivo in organoid cultures. However, treatment of organoids with the same antibiotics only in culture does not lead to a phenotypic rescue. We are currently characterizing how deletion of Prdm16 alters the intestinal microbiota and the effects that those changes could have on metabolite levels in the tissue. Overall, these studies aim to understand how changes in metabolic signaling can affect stem and progenitor cell behaviors and how the microbiome and, potentially, antibiotics themselves can affect the function of the intestine.
Functional Morphology of Sensory Organs of the House Cricket, Acheta domesticus (L.): A Morphological and Physiological Investigation
Authors: Shields Vonnie D. , Weaver Cameron E. , Farmer Shannon
Published: May 2022

Insects have paired antennae that bear many sensory organs or sensilla. These sensilla allow them to gather information about olfactory, gustatory, and mechanosensory environmental cues. Both antennae of male and female house crickets, Acheta domesticus, bear approximately 200 segments. The first aim of this study was to morphologically characterize the sensillum types on the antennae. Scanning and transmission electron microscopy studies revealed that each antennal segment bears seven sensillum types. Four types have external morphological features suggestive of olfactory sensilla, namely an abundance of cuticular pores. Two of these types resemble short to medium‐sized pegs, a third resembles a short peg with longitudinal ridges or “fingers,” while a fourth resembles a short peg recessed in a deep pit. Each antenna also bears two sensillum types devoid of external cuticular pores, suggestive of a mechanosensory function. One of these types is a long hair‐like sensillum with conspicuous ridges, while the other is a small circular depression on the antennal surface. Another medium‐sized sensillum bears diagonal circular ridges and a single apical pore, indicative of a gustatory function.

The second aim of this study was to determine which odorants elicited positive or negative anemotaxis (orientation toward or away from a food source, respectively), suggestive of possible food lures or repellents. Odorants permeate through the environment and bind to receptor sites in olfactory neurons. Once these molecules bind, chemical signals are converted to electrical ones. Olfactory signals are then transmitted to higher‐order olfactory processing centers in the insect’s brain. Depending on the odorant, this may trigger a behavioral response(s). Odorants may be used to detect and identify food, mating partners, and avoidance of predators. Previous studies in the lab have focused on odorants associated with some fruits, vegetables, and proteins. In this study, we tested some odorants associated with amines derived from ammonia. We hypothesized that such odorants would elicit negative anemotaxis. We carried out each experiment over a 24‐hour period in plastic bins. We found that the crickets elicited positive anemotaxis to the odorants tested, leading us to reject our hypothesis. These findings are important as house crickets are pests and known to contaminate food sources. This finding raises concerns about foodborne illnesses associated with microbial pathogens and can create health concerns especially in developing countries. The results of this study may prove to be helpful in the suggestion of lures as effective biocontrol measures against pests.
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.
Hepatic Stearoyl‐CoA desaturase deficiency‐mediated induction of the insulin‐like growth factor‐binding protein 1 requires FGF21
Authors: McGahee Ayren A. , O’Neill Lucas M. , Lewis Sarah , Liu Zhaojin , Lefers Luke A. , Huff Hailey , Dinerman Samuel J. , Simcox Judith , Ntambi James M.
Published: May 2022

Stearoyl‐CoA desaturase‐1 (SCD1) catalyzes the rate‐limiting step in the biosynthesis of monounsaturated acids and is a key regulator of systemic glucose metabolism. Hepatic deletion of Scd1protects mice from high‐carbohydrate diet (HCD)‐induced hepatic steatosis and adiposity in part by suppressing hepatic lipogenesis, increasing adipose tissue lipogenesis, and increasing systemic glucose uptake. However, the mechanism leading to these phenotypes is unclear. Given that high carbohydrate diets can lead to chronic metabolic diseases such as obesity, diabetes, and hepatic steatosis, it is critical to understand how SCD1 deficiency alters metabolic pathways to produce metabolically beneficial phenotypes. Here we show that hepatic SCD1 deficiency enhances tissue glucose uptake and is correlated with dramatic increases in the expression and plasma levels of the fibroblast growth factor 21 (FGF21), and Insulin growth factor binding protein‐1 (IGFBP1), both liver derived insulin‐sensitizing hormones that are known to regulate whole body lipid and glucose homeostasis. Feeding both male and female LKO mice with triolein, but not tristearin, supplemented HCD reduced Fgf21 and Igfbp1 expression and restored plasma glucose levels. Inhibition of SCD activity in primary hepatocytes induced the expression of Fgf21 and Igfbp1 which was repressed by treatment with oleate but not stearate. Moreover, we show that the hepatic stearoyl‐CoA desaturase deficiency‐mediated induction of the Igfbp1 is through the oleate‐FGF21 axis.
Difficulties Performing Morphometric Assessment on the Costal Cartilage‐Rib Interface in Cadaveric Research
Authors: Gumble Zachary , Bolden Alexys M. , Alsharaf Fatemah , Burkett Jesse T. , Lambert H. Wayne , Zdilla Matthew J.
Published: May 2022

Costal cartilage is commonly used in a variety of reconstructive surgeries such as reconstruction of head and neck anatomy. However, there is disagreement among surgeons regarding the most appropriate costal cartilage from which to harvest (e.g., 5th cartilage versus 6th or 7th; left versus right side). Thus, assessment of costal cartilage morphometry is warranted. In an attempt to assess costal cartilage morphometry in a cadaveric sample, researchers encountered noteworthy inter‐examiner disagreement. This report details the difficulties encountered when attempting to perform morphometric assessment costal cartilage in a cadaveric sample. It is well‐established that the hyaline cartilage of the rib cage commonly undergoes calcific change with age; however, such changes were not appreciable in the cadaveric sample that was assessed. Predominantly, difficulties were encountered in the identification of the cartilage‐bone interface. The disagreement among examiners primarily stemmed from the nature of the costal cartilage and rib color. Some cadaveric specimens had a clear cartilage‐bone interface with evident gross color change between cartilage and bone tissues whereas other cadaveric specimens had relatively little color change between cartilage and bone. This research highlights the importance of cadaveric tissue color in research and underscores an important caveat: incongruent data may result if tissue color is not accounted for in cadaveric research.