bluuebunny/crossref_metadata_2025 huggingface dataset:
Epithelial monolayer development and tight junction assembly on nanopillar arrays
Authors: Yeste Jose , Illa Xavi , Shashikanth Nitesh , Guimerà‐Brunet Anton , Villa Rosa , Turner Jerrold R.
Published: May 2022

Nanostructured materials provide an outstanding opportunity to both stimulate and measure cellular processes. In the context of tight junctions, it was previously reported that transient application of a nanotopographic surface over the apical brush border membrane of epithelial monolayers triggers redistribution of ZO‐1, claudins, and F‐actin that increases paracellular macromolecular flux. In excitable tissues, nanomaterials have been used to apply and measure electrical signals, such action potentials. As a first step towards translating these technologies for use in analysis of epithelial function, we sought to culture monolayers composed of transporting epithelia over nanopillar arrays without perturbing cellular structure or function. Madin‐Darby Canine kidney I (MDCKI) cells were cultured on collagen‐coated silicon chips with ~1.5 µm diameter nanopillar arrays. Fluorescence and scanning electron microscopy were used to assess the impact of height on nanopillar‐epithelial interactions. Monolayers formed over and were largely unaffected by short nanopillars. These nanopillars were located beneath basal epithelial surfaces and were not preferentially located within lateral intercellular spaces or beneath ZO‐1‐containing junctions. In contrast, tall nanopillars that exceeded cell height disrupted MDCK monolayer growth. Cells interacted with, encircled, and extended cytoplasm over the top of tall nanopillars, and dense ZO‐1 and F‐actin accumulations occasionally surrounded apical membranes adjacent to nanopillars. Finally, when grown over arrays composed of nanopillars 1 – 2 µm less than cell height, MDCKI grew more often between nanopillars. As a result, nanopillars were more frequently present within lateral intercellular spaces beneath junctions. Apical complex structure was intact, as assessed by fluorescence microscopy of ZO‐1, occludin, claudin‐2, F‐actin, and E‐cadherin. Apical microvilli were also unaffected. These data establish the conditions required for growth of mature, correctly assembled epithelial monolayers in which nanopillars are present within lateral intercellular spaces. This sets the stage for synthesis of functionalized nanopillar arrays for perturbation and analysis of epithelial biology.
Understanding the Role of Cancer Glycosylation on Antibody Binding to MUC16
Authors: Karnwal Saloni , Brooks Teresa , Patterson Michelle , Brooks Cory L.
Published: May 2022

Protein glycosylation is a common post‐translation modification where carbohydrates are anchored to a protein. Glycans play critical roles in mediating cell‐cell interactions, protein‐receptor signaling, and protein folding during translation. One of the hallmarks of cancer is aberrant glycosylation, where shortened glycans specific to cancer are observed. Tumorigenesis, tumor progression, and migration correlate with abnormal glycosylation. Overexpression of the membrane‐bound glycoproteins, MUC16, has been linked to poor prognosis and metastasis in pancreatic and ovarian cancer patients. A common feature of tumor‐associated MUC16 is altered glycosylation, revealing cryptic epitopes where antibodies can bind. These tumor‐specific antibodies can form the basis of new cancer immunotherapies. This project aims to establish an expression system using genetically modified Chinese Hamster Ovary (CHO) cell lines to produce cancer‐associated MUC16 with truncated O‐glycans. A CRISPR/Cas9 modified CHO cell line (CHO‐S□cosmc/mcherry+) was obtained, which has had the gene mcherry inserted into the cosmc locus, a mutation that is hypothesized to produce truncated O‐linked glycans. Here we report the purification and Enzyme‐Linked Immunosorbent Assay (ELISA) analysis of recombinant MUC16 produced in these cell lines. MUC16 in CHO (ExpiCHOwildtype and CHO‐S□cosmc/mcherry+)cell lines were transiently transfected and purified through the Nickel affinity chromatography. The purified protein was analyzed through SDS‐PAGE. Quantitative analysis of MUC16 binding to three different therapeutic antibodies was performed using ELISA. The results revealed that MUC16 produced in CHO‐Smcherry+displayed a higher binding affinity towards the antibodies than ExpiCHOwildtype. The recombinant proteins produced will be further used for examining the role of cancer‐associated glycans binding to therapeutic antibodies.
The Effect of Protein Supplementation During Time Restricted Feeding in Overweight and Obese Adults
Authors: Bowie Rebecca , Komp Mary , Thomas Ada‐Miette , Boudrey Sydney , Taylor Hannah , Martin Brooke , Martin Grace , Baum Jamie I.
Published: May 2022

Objective

Obesity in the United States continues to increase. Time restricted feeding (TRF) is a dietary intervention that has potential to serve as an effective weight loss strategy, however more data is needed to establish the effectiveness of a TRF eating pattern on health outcomes related to obesity. To our knowledge, manipulation of macronutrient distribution during a TRF intervention and the effect of TRF on sleep and mood have not been studied. Therefore, the objective of this study was to determine the effect of protein supplementation during TRF on mood, sleep, and appetite in overweight and obese adults.

Methods

Overweight and obese men and women (36.6±7.3 years; BMI: 32.8±6.5) participated in this randomized, controlled 12‐week dietary intervention. Participants were allocated to one of two groups: 1) control, TRF (n=7) and 2) TRF with whey protein supplementation (25 g/d; n=9). Protein supplements were consumed at the breaking of the fasting period each day. Anthropometrics, sleep (via Pittsburgh Sleep Quality Index Global Sleeping Score; PSQI GSS), total mood disturbances (TMD; via Profile of Mood States (POMS) including six affect states of depression, fatigue, anger, tension, confusion, and vigor subscales), and appetite (using visual analog scales) were assessed at 0, 4, 8, and 12 weeks. Sleep was also measured at 0 and 12 weeks via wrist Actigraphy. Body composition was measured via DXA at 0 and 12 weeks. Data was analyzed using two‐way ANOVA to assess the relationship.

Results

Overall, both interventions improved total body fat percentage (p<0.05) over 12 weeks. There was a significant difference in mood between intervention groups (p<0.001) and the control group total mood disturbance was significantly different (p<0.05) between week 1 and 12. However, there was no change in mood with protein supplementation over the span of 12 weeks. Sleep, self‐reported via PSQI, did not change from week 1 and week 12 within or between each of the interventions. Appetite did not show a significant difference between interventions or time points.

Conclusions

This pilot study suggests that protein supplementation during TRF has the potential to improve body composition in overweight and obese individuals. However, additional research is needed to determine the long‐term effect of protein supplementation in combination with TRF on mood, sleep, and appetite.
Sex differences in cerebrovascular dysfunction and hypertension in offspring of hypertensive pregnant rats
Authors: Smith Jonna B. , Cromartie Whitney E. , Powell Madison , Duncan Jeremy W. , Cunningham Mark W.
Published: May 2022

BACKGROUND

Offspring from preeclamptic women have a greater risk of hypertension and cerebrovascular dysfunction later in life. One of the major contributors of cerebrovascular dysfunction is impaired cerebral blood flow (CBF) autoregulation. In the USA, 1 in 25 pregnancies are preeclamptic. Preeclampsia, as defined as new‐onset hypertension during pregnancy, is an inflammatory condition characterized by elevated interleukin 17 (IL‐17), mitochondrial reactive oxygen species (mtROS), mitochondrial dysfunction (mt‐Dys), and intrauterine growth restriction (IUGR). Preliminary work from our laboratory demonstrates that offspring from preeclamptic rats have sex differences. Males have hypertension and a greater impairment of CBF autoregulation, while females have no change in blood pressure with impaired CBF autoregulation. The exact mechanisms for the sex differences in cerebrovascular dysfunction and hypertension is unknown and is the focus of this study. We hypothesize that changes in cerebral mt‐Dys, cerebral mtROS, and circulating IL‐17 contributes to the sex differences in hypertension and cerebrovascular dysfunction in male and female offspring from preeclamptic rats.

METHODS

In this study, we compared male and female offspring from normal pregnant and preeclamptic Sprague Dawley rats. All offspring were divided into controls (CON) and IUGR by sex. Hypertensive male and non‐hypertensive female offspring’s with impaired CBF autoregulation were examined at 17 weeks of age for changes in cerebral mitochondrial electron transport chain (ETC) protein complexes, cerebral manganese superoxide dismutase (MnSOD), and circulating IL‐17.

RESULTS

Female CON (151±5% IU/Protein/male CON) and IUGR (149±8 %IU/Protein/male CON) offspring have increased MnSOD compared to CON (100±7 %IU/Protein/male CON) and IUGR (122±4 %IU/Protein/male CON) males (p<0.05). No changes in female ETC protein complexes between IUGR and CON. Male IUGR have a decrease in complex II (71±5 vs. 100±8 %IU/Protein/male CON, p<0.05) and V (57±2 vs. 100±10 %IU/Protein/male CON, p<0.05) ETC proteins, and elevated IL‐17 (944±370 vs. 412±115 pg/mL, ns) compared to CON males.

CONCLUSION

Male IUGR offspring have mt‐Dys and elevated IL‐17, which may contribute to hypertension and a greater impairment in CBF autoregulation. Female IUGR offspring may be protected due to an increase in mitochondrial antioxidants. In summary, studying the dimorphic sex differences in the mechanisms of hypertension and cerebrovascular dysfunction in offspring of preeclamptic women, may improve the offspring’s risk of hypertension, cardiovascular disease, stroke, and cognitive dysfunction later in life.
Optimizing 6‐Hydroxydopamine Concentrations for induction of a Parkinson’s Disease Like Behavior in Zebrafish
Authors: Romero Adrian , Hunt Justin C. , Bill Brent R. , Hamouda Ayman K.
Published: May 2022

Parkinson’s disease is an increasing problem that affects the quality of life for over 10 million people worldwide. Affected individuals undergo worsening symptoms that begin with motor function impairment and can eventually result in death. Parkinson’s Disease is associated with the loss of Tyrosine Hydroxylase (TH) expressing dopaminergic neurons. TH is the rate limiting enzyme in catecholamine neurotransmitter synthesis including dopamine. Therefore, chemically induced animal models have focused on mitigation of movement abnormalities and loss of dopaminergic neurons as indicators for drug screening. We chose the 6‐hydroxydopamine (6‐OHDA) chemically induced Parkinson’s Disease model in zebrafish as a tool for drug screening. Previous work on this model had been conducted with varying drug concentrations and methodologies. All demonstrated reductions in TH expressing neurons; however, the behavioral assessments varied in both method, dose of OHDA (1‐750 μM 6OHDA), and whether behavioral abnormalities were observed. The objective of the current study is to determine the minimal 6‐OHDA dosage that is sufficient to induce a significant difference in behavioral effects while minimizing morphological toxicity. Zebrafish larvae were treated daily with 6‐OHDA with ascorbic acid from 2 to 5‐day post fertilization (dpf). We observed significant difference in behavior at ~20 μM 6‐OHDA, and developmental delays in swim bladder formation at 85 μM 6‐OHDA. The swim bladders were developed by 5 dpf when we assessed swim behavior; however, other signs of delay may still be present that would complicate results. Several studies focused on doses of 150 μM 6‐OHDA or higher; however, at these doses we observed severe morphological issues including edema, curved spines, and death. Future work will focus on the TH‐expressing neuron pathology as assessed by immunohistochemical and Western blotting methodologies.
Multivalent Vaccine Strategies against Polydrug Use in Opioid Use Disorders
Authors: Song Daihyun
Published: May 2022

The number of annual fatal drug overdoses reached more than 100,000 between April 2020 to 2021, of which the majority involved individual opioids or polydrug mixtures containing opioids. A series of conjugate vaccines targeting fentanyl, carfentanil, oxycodone, heroin, and their analogs or metabolites have shown preclinical proof of efficacy, selectivity, and safety. Individual vaccines consist of haptens, small molecules that mimic the structure of the targeted drug, conjugated to carrier proteins, such as detoxified diphtheria toxin (CRM) or keyhole limpet hemocyanin (KLH). These studies identified lead haptens based on fentanyl (Fen), carfentanil (Carf), oxycodone (Oxy), and heroin (Her). Preclinical studies showed the efficacy of each vaccine (Fen‐CRM, Carf‐CRM, Oxy‐KLH, and Her‐KLH) in blocking their respective target drug distribution to the brain and their behavioral effects in mice and rats. While multivalent vaccine formulations are commonly used as a safe and effective strategy against infectious diseases, multivalent vaccination strategies against polydrug use have not yet been fully explored at treating substance use disorders. In the current study, the balb/c mice were vaccinated with a combination of Fen‐CRM, Carf‐CRM, Oxy‐KLH, and Her‐KLH as a quadrivalent vaccine. ELISA showed that quadrivalent vaccine increased fentanyl‐, carfentanil‐, oxycodone‐, and heroin‐specific IgG titers in the immunized mice. Correspondingly, this simultaneous immunization prevented the distributions of fentanyl, carfentanil, oxycodone, and heroin to the brain when the mice were challenged by the mixture of these drugs. These data demonstrate that individual efficacy of monovalent vaccine was preserved in a multivalent formulation without interference. Amid opioid epidemics, multivalent vaccine strategies will provide a broad spectrum of protection against multiple opioids with a single vaccination.
Qualitative analysis of the role of teacher rapport on autism spectrum disorder student engagement while scanning for frontal lobe activation using fNIRS
Authors: Lahodny Avery , Critchfield‐Jain Isabella , Gentillon Rachel , Read Chloe , Banner Liz , Wisco Jonathan
Published: May 2022

Introduction

Previous research has indicated that students with Autism Spectrum Disorder (ASD) show lower levels of engagement in the classroom and often struggle to focus in most learning environments compared to neurotypical students. It has been noted that the teacher plays a crucial role in creating a safe learning environment for ASD students. Successful teachers establish rapport with their students in order to create this safe learning environment. When teachers provide encouragement to students or make an effort to engage them through personal conversations, the student is more likely to engage in the material. The purpose of this study was to determine what teacher behaviors result in the highest brain activation in the frontal lobe of ASD students.

Methods

Teachers and students wore functional Near Infrared Spectroscopy (fNIRS) NIRSIT systems (Soterix Medical, Inc.) during a 30‐minute simulated math lesson consisting of three phases ‐ didactic instruction, facilitated practice problem solving, and supervised independent study. Teachers with 3 or fewer years experience teaching ASD students, and those with more than 3 years teaching high‐functioning ASD high school students ranging in grade‐level math from 6th grade to 12th grade were recruited for the study. Blood Oxygenation Level Dependence (BOLD) activation of the participants were monitored to indicate what teaching and learning moments resulted in the most frontal lobe activation of the students, and the results were compared between groups. In addition, we used grounded theory thematic analysis to analyze the video recordings of the learning space.

Results

We observed that teacher rapport was an important factor in engaging students. Friendly remarks and encouragement allowed for students to feel comfortable to engage in learning the material, resulting in a successful lesson flow with increased participation from the students. When students were engaged with material due to teacher rapport, frontal lobe BOLD signal increased. In addition, some students were actively engaged when answering questions or providing explanations for answers, while others showed concomitant brain activation while quietly listening to the teacher or the other student.

Conclusions

The creation of a friendly learning environment is an important component in engaging students and should be emphasized for teachers seeking to engage both ASD students. The results of this study illustrate that students present different behaviors in a learning environment and can be actively engaged even when their behaviors deviate from what is typically defined as engagement.
Repetitive Element Transcripts are Associated with Inflammation in Older Adults
Authors: Smith Meghan E. , Cavalier Alyssa N. , McEntee Cali M. , Wahl Devin , McWilliams Gabriella T. , Seals Douglas R. , LaRocca Thomas J.
Published: May 2022

Growing evidence shows that expression of non‐coding repetitive sequences, or repetitive element (RE) transcripts, increases with age and may be related to biological aging itself. In fact, RE transcript levels can be used to accurately predict age, and they are reduced by healthy aging interventions. However, the exact mechanistic links between RE dysregulation and aging are unknown. Some evidence indicates that RE transcripts may stimulate age‐related inflammation (via formation of RE‐derived double‐stranded RNA, an innate immune activator) but there is currently no evidence of this in human subjects. Therefore, we determined if serum interleukin 6 (IL‐6), a key circulating marker of inflammation, was associated with RE transcript expression (assessed via RNA‐seq) in 92 healthy, middle aged to older adults (64 ± 7 years, 47M/ 45F). We found that serum IL‐6 was positively related to expression of every major type of RE (LINEs, SINEs, DNA transposons, LTRs, and Satellites; mean r value = 0.4, p < 0.0005). To examine transcriptome/gene expression differences associated with high vs. low serum IL‐6, we analyzed differential gene expression in subjects with the highest vs. lowest IL‐6 levels (6 per group, age/sex‐matched). We then performed gene ontology analysis to determine biological processes associated with differentially expressed genes. Interestingly, we found that higher IL‐6 was associated with reduced gene expression signatures of immune function and increased gene expression signatures related to cell death and differentiation. To assess a potential upstream link between RE expression and inflammation, we looked for correlations among RE and innate immune sensor transcript levels. We found a positive relationship between RE expression and levels of IFIH1, the gene that encodes MDA5, a proinflammatory sensor of double‐stranded RNA (p < 0.0005, mean r value = 0.5). MDA5 expression correlated weakly with LINE expression (r = 0.36), but more strongly with satellite and LTR expression (r = 0.66), suggesting that these RE types may be involved in age‐associated inflammation. These are the first data to show a relationship between IL‐6 and global RE expression in older adults, and to characterize the possible mechanisms underlying this relationship.
Identification of Peptide Inhibitors of Gαs that Block its Effector Binding Site
Authors: Luebbers Alex , Maziarz Marcin , Garcia‐Marcos Mikel
Published: May 2022

Heterotrimeric G proteins (Gαβγ) are molecular switches that alternate between “on” (GTP‐bound) and “off” (GDP‐bound) states to control the flow of signals from the extracellular milieu to the cytoplasm. Gαs is a prototypical Gα subunit of heterotrimeric G proteins. When bound to GTP, Gαs promotes the synthesis of cAMP in the cytosol through stimulation of the enzymatic activity of adenylyl cyclases. Dysregulation of this mechanism leads to human diseases. For example, naturally occurring mutations like R201C lock Gαs in an active conformation that leads to signaling hyperactivity in cancer. Despite the critical role of Gαs in signaling and its involvement in disease, there are currently no chemical or peptide tools to specifically inhibit its signaling activity. We reasoned that peptides that bind to active Gαs and block the effector binding site would inhibit its ability to signal through adenylyl cyclases. For this, we screened peptides from a phage display library and identified two unrelated peptides that bound to active Gαs. These peptides bound efficiently to GTP‐bound Gαs or to the constitutively active Gαs R201C mutant, but not to inactive GDP‐bound Gαs. Fluorescence polarization experiments revealed an equilibrium dissociation constant (KD) of approximately 1 μM for the binding of these peptides to Gαs‐GTP. Mutations in the adenylyl cyclase binding region of Gαs greatly reduced binding of these two peptides, indicating that they may work as competitive inhibitors of adenylyl cyclase stimulation. Consistently, both peptides blocked Gas‐mediated stimulation of adenylyl cyclase activity in vitrowith μM potency, but had no effect on Gαs‐independent stimulation of adenylyl cyclase activity by forskolin. Protein‐protein binding experiments revealed that one of the two peptides bound to Gαs but not to Gα subunits of other G protein families, whereas the second peptide bound to Gαi‐GTP in addition to Gαs‐GTP. In summary, we have identified a peptide inhibitor specific for Gαs‐GTP that acts by binding to its effector binding site and precluding adenylyl cyclase stimulation. The peptide identified here could be leveraged as a genetically‐encoded probe or derivatized as a cell‐penetrating peptide to inhibit Gαs‐mediated cell signaling.
The laryngeal adductor reflex (LAR) is modulated by the serotonin 5‐HT1A agonist 8‐OH‐DPAT
Authors: Young Kellyanna C. , Musselwhite M. N. , Frazure Michael L. , Greene Clinton L. , Iceman Kimberly E. , Pitts Teresa
Published: May 2022

Airway protection is maintained through a constellation of behaviors. The laryngeal adductor reflex (LAR) responds to mechanical or chemical stimulation of the laryngeal orifice with rapid adduction of the vocal folds. While there are abundant serotonin receptors on laryngeal motoneurons, the effects of serotonin agents on the LAR have not been investigated. To this end, a dose response protocol of 8‐OH‐DPAT (5HT1A receptor agonist) was performed on adult cats (n = 2). Animals were anesthetized using an intravenous dose of sodium pentobarbital and then tracheostomized. Electromyogram (EMG) activity of laryngeal muscles was recorded and LARs were induced by infusing water into the larynx during both normocapnia (0% CO2) and hypercapnia (10% CO2) conditions. A series of six cumulative doses (1 – 300 μg/kg) were delivered intra‐venously. 8‐OH‐DPAT is thought to activate the serotonergic raphe neuron autoreceptors at lower doses and the post‐synaptic receptors on target neurons at higher doses. LAR occurred as a response to water stimulus in 50±23% of control trials. LAR occurrence was modulated by 8‐OH‐DPAT in a dose‐dependent manner: LAR frequency at lower doses may have decreased but this was variable, and LAR frequency appeared to be increased at higher doses (LAR occurred in 13±18%, 17±23%, 50±70%, 57±9%, 84±7%, and 60±4% of stimulus trials at 1, 3, 10, 30, 100, and 300 μg/kg doses, respectively). LAR can be used as a strong clinical predictor of swallow impairment; while other airway protective behaviors require activity across multiple cranial nerves, the LAR is regulated by only laryngeal branches of the vagus nerve. Thus, the LAR is a promising biomarker for testing novel therapeutic agents. These results suggest that pharmacological interventions may be capable of facilitating laryngeal function in patients suffering from dysphagia.
Synthetic Assembly of Bacterial Divisome Machinery in Vitro
Authors: Ferreira Colby N. , Camberg Jodi L.
Published: May 2022

Bacterial cells grow and reproduce through coordination of many proteins collectively known as the cell division machinery. This machinery facilitates the division and physical separation of one cell into two identical daughter cells. The components of this cell division machinery are similar between bacterial taxa. In Escherichia coli, the cytoskeletal protein FtsZ promotes the formation of a division septum at the cell center and assembles into a large protein structure called the Z‐ring. FtsZ protomers in the Z‐ring form large linear polymers that assemble in a head to tail arrangement. Protein interactions between FtsZ in the Z‐ring and additional cell division proteins direct peptidoglycan synthesis and facilitate septation of the cell. FtsN, which may trigger septation, is a bitopic membrane protein with a small cytoplasmic domain and large periplasmic domain containing a peptidoglycan‐binding SPOR domain. FtsN interacts with other cell division proteins on both sides of the cytoplasmic membrane. In the cytoplasm, FtsN interacts with FtsA, an actin homolog, which tethers FtsZ polymers to the membrane. In the periplasm FtsN interacts with the FtsQBL complex and may help to recruit peptidoglycan synthetases such as FtsI. One proposed linear mechanism for activating cell wall synthesis begins with FtsA, which binds to FtsN in the cytoplasm, and causes FtsN to relay information to proteins in the periplasm, such as FtsQBL, to begin remodeling of the cell wall. Thus, it is critical that FtsN localizes to the future site of septation in order to carry out this function. Here we use purified proteins, including FtsZ, FtsA, and FtsN, to reconstitute the membrane‐spanning division complex in vitro and assemble a synthetic divisome scaffold. This reconstituted divisome subassembly allows us to probe direct protein interactions, protein conformations, assembly architectures, and nucleotide requirements in the cell division pathway, thus providing novel insight into the mechanics of division.
Characterizing the Effects of High Fat and High Carbohydrate Diets on Feeding Behavior and Energy Expenditure in Mice
Authors: Linden Benjamin , Herz Hussein , Chu Yi , Mokadem Mohamad
Published: May 2022

Objective

We sought to characterize how different compositions of the diet can alter feeding behavior and energy expenditure in mice, ultimately driven by changes in sympathetic activation and thermogenesis.

Hypothesis

Mice on a high fat diet (HFD) will consume more calories/day compared to high carbohydrate diet (HCD) and standard chow fed mice. Mice on HFD will have decreased expression of UCP1 in both brown and white adipose tissue. There will be decreased expression for several lipolytic enzymes in the white adipose of HFD mice.

Methods

A total of 21 mice were divided into three ad libitum fed diet groups: HFD, HCD, and standard chow. Each group was fed their respective diet for 7 weeks. Changes in weight and percent body fat were recorded by NMR. At week 5, glucose tolerance tests were performed on all mice. Both a 24‐hour heat production and respiratory exchange ratio were collected on each group using a metabolic cage system. Mice were sacrificed at the conclusion of week 7 and organs were collected for molecular analysis.

Results

We show that mice on both ad libitum HFD and HCD consume significantly more calories/day when compared to standard chow. Both HFD and HCD fed mice have a significantly increased body weight and percent body fat when compared to standard chow; however, the HFD mice had a far greater increase in weight compared to HCD fed mice despite no difference in calorie consumption. Mice on a HFD had a lower 24‐hour average heat production per body mass when compared to standard chow and HCD mice and a significantly impaired glucose tolerance test. Furthermore, UCP1 mRNA expression is significantly elevated in brown adipose tissue (BAT) of HCD mice compared to that of HFD and standard chow mice. There were no differences in UCP1 expression in subcutaneous or visceral white adipose tissue (WAT) among all groups. There were also no differences in tyrosine hydroxylase (TH) expression in BAT between all mice. Three markers of lipolysis were decreased in visceral WAT of HFD mice compared to HCD and standard chow counterparts.

Discussion

Mice consumed significantly more calories when fed a HFD or HCD. These mice gain significantly more weight compared to chow with HFD mice gaining weight far more than the HCD group. This major difference in weight gain despite no significant difference in calorie consumption between HFD and HCD suggests differences may be determined by energy expenditure. This evidence is further supported by the fact that HFD fed mice had a significantly lower daily heat production compared to HCD and standard chow mice. We also show that HCD increases thermogenesis in mouse BAT which may be protective against weight gain. Finally, lipolysis signaling seems to be impaired in HFD mice, but no data supports that this is due to differences in sympathetic activation. Further work will include analyzing the same tissues in isocaloric, restricted diet fed mice.
Increased Insulin Resistance in 10‐month compared to 5‐month Old Male Hepatic Androgen Receptor Knockout Mice
Authors: Lofton Taylor , Levey Josephine , Wilson Andre , Carr Kiana , Falzarano Claire , Andrisse Stanley
Published: May 2022

Insulin resistance is an important component of type 2 diabetes and metabolic syndrome. The liver plays an essential component in the metabolism of insulin and androgen signaling. Sex differences exist in type 2 diabetes with men having a higher prevelance than women. In our study we placed male liver androgen receptor knock‐out mice (Liv‐ARKO) on chow diets to determine if Liv‐ARKO had an effect on insulin resistance on younger age (5 month) compared to older age (10 month).

In this study, male Liv‐ARKO mice were kept on chow diets for either 5 months or 10 months and then sacrificed. Some mice were given a dose of 0.5 U/kg insulin before sacrificing to investigate the effects of age on insulin signaling. Western blots were used to determine protein expression in tissue from the liver, skeletal muscle, white adipose tissue. BCA assays were used to standardize the protein concentration in each sample.

Upon activation of the insulin signaling pathway, the insulin receptor substrate (IRS) proteins, initiates activation of the phosphatidylinositol 3‐kinase (PI3k) pathways, resulting in stimulation of protein kinases such as Akt. As seen in a previous study1 approximately 5 month old (22 week old) hepatic‐AR‐knockout chow mice showed that when stimulated with insulin there was an increase in PI3k activity. This study did not take it a step further to show what was going on with p‐AKT.

It was hypothesized that p‐AKT is lowered in both 5 and 10 month old mice compared to mice not given insulin. Additionally, after 10 months, total AKT was increased. Insulin stimulated p‐Foxo was lowered at 5 months but at 10 months p‐Foxo was increased.

Our results suggest that Liver Androgen Receptor is causing decreased insulin action at the level of p‐AKT in both young (5 month) and old (10 month) mice. If insulin is not stimulating p‐AKT, the body may try to make more total AKT to try to compensate; hence the increase in total AKT seen in the 10 month mice. Further investigation should also be performed on the gender differences in these results.
Steroid‐Derived Isonitriles are Potent Cytochrome P450 Ligands that Should Enable Solution NMR as a Structural Technique to Probe P450 Dynamics
Authors: Richard Alaina M. , Pochapsky Thomas C. , Scott Emily E.
Published: May 2022

Human cytochrome P450 (CYP) enzymes are versatile, dynamic, and essential to many aspects of human health. The catalytic versatility of this superfamily of enzymes is driven by structural dynamics, which are often elusive to static structural techniques. For this reason, solution nuclear magnetic resonance (NMR) is likely to provide useful structural information orthogonal to X‐ray crystallography. The challenges of human P450 NMR studies are two‐pronged: 1) Human P450 enzymes are membrane‐bound, and often aggregate or precipitate in aqueous environments, and 2) P450 enzymes contain a paramagnetic heme iron buried within the active site, leading to decreased intensity of NMR correlations corresponding to nearby amino acid residues. We have overcome both obstacles with the human steroidogenic P450 enzyme CYP17A1 in complex with a novel substrate derivative, 3‐formyl pregnane‐20‐isonitrile1. The steroid skeleton of this ligand is similar to that of CYP17A1 endogenous substrates and therapeutics, but in this case the C20 isonitrile functional group directly coordinates both the oxidized (Fe3+) and the reduced (Fe2+) diamagnetic states of the heme iron. As a result, this compound both stabilizes CYP17A1 and suppresses paramagnetic relaxation and will facilitate application of multidimensional solution NMR to CYP17A1. The dissociation constant (Kd) and inhibition (IC50) of this isonitrile have been determined and compared to the prostate cancer drug abiraterone, which is the tightest‐binding CYP17A1 inhibitor to date. Structural characterization of this complex by solution NMR is ongoing, but this strategy should open the door to new methods of structural analysis of dynamic human P450 enzymes.
Evolution of substrate specificity in a maize anthranilate methyltransferase
Authors: Tadfie Hisham , Holland Cynthia
Published: May 2022

Secondary metabolites play a key role in mediating pollination and plant defense against herbivory. One such metabolite, methyl anthranilate, is a volatile compound responsible for the recognizable scent of Concord grapes. Methyl anthranilate is commercially used as a bird repellent for golf courses and as a flavoring agent for grape‐flavored foods, beverages, and pharmaceuticals. A variety of plant species such as Zea mays (maize) and Vitis labrusca (fox grape) synthesize methyl anthranilate from anthranilate, an intermediate in tryptophan biosynthesis. Methyl anthranilate biosynthesis is a classic example of convergent evolution; in maize, this reaction is catalyzed by an anthranilate methyltransferase (AAMT1), while in grape, there is a two‐step pathway. Previous studies have found that the ZmAAMT1 SABATH methyltransferase likely evolved from methyltransferases that recognize the plant pathogen response hormone salicylic acid as a substrate. Using site‐directed mutagenesis, we introduced 12 independent missense mutations at 7 key active site residues with the intention of restoring substrate specificity for salicylic acid. This work will allow us to better trace and understand the evolution of anthranilate‐derived specialized metabolism in plants.
Human Milk Oligosaccharide L‐N‐Tetraose Activates GPR35 on Vagal Afferent Neurons In Vitroand Reduces High Fat Diet‐Induced Obesity in Male Mice
Authors: Zumpano Brucia Danielle L.
Published: May 2022

Obesity is associated with altered gut microbiota; dietary supplementation with prebiotics improves metabolic outcomes in obesity, an effect ascribed to improved microbial dysbiosis. However, alternative mechanisms have not been fully explored. We have shown that prebiotic milk oligosaccharides (MOs) decrease weight gain and food intake in mice fed a high‐fat diet (HFD), and improve cholecystokinin (CCK)‐induced satiety signaling. It has recently been shown that the human MO lacto‐N‐tetraose (LNT) is a ligand for the G‐protein coupled receptor, GPR35. GPR35 is highly expressed by vagal afferent neurons (VAN) where it is co‐expressed with the CCKAR. HFD reduces endogenous GPR35 ligands and sensitivity to CCK, but the role of GPR35 and its ligands eg LNT, in satiety signaling via the vagal afferent pathway have not been investigated. Here we test the hypothesis that LNT reduces HFD‐induced weight gain and hyperphagia in mice via a direct effect on GPR35 expressed by VAN.

Methods

Mice were maintained on low‐fat diet (10% kcal fat), HFD (45% fat kcal), or HFD supplemented with 10% LNT for 8 weeks (male, n=5‐9/group). To determine if VAN are directly activated by GPR35 ligands, VAN from adult male Wistar rats were cultured and treated with the GPR35 agonist zaprinast (100 µM), LNT (10 mM), or D‐lactose (constituent of LNT; 10 mM) for 15 minutes after 1 hour pre‐incubation with the GPR35 competitive antagonist CID‐2745687 (100 µM) or vehicle (n=4/treatment). Phosphorylation of extracellular regulated kinase (ERK) relative to total ERK (pERK/ERK) was determined by western blot and used as an indicator of VAN activation.

Results

LNT administration significantly reduced body weight, fat mass, and improved HFD‐induced hyperphagia (p<0.05). Treatment of VAN with the GPR35 agonist zaprinast increased pERK/ERK (p<0.05) relative to vehicle; zaprinast‐induced activation was prevented by the CID‐2745687. LNT, but not D‐lactose, also activated VAN, an effect inhibited by the GPR35 antagonist.

Conclusions

LNT may decrease food intake via a direct effect on VAN, possibly by increasing sensitivity to peripheral satiety signals. This work reveals a potential peripheral therapeutic target for ameliorating diet‐induced weight gain.
Proteomics, phylogenetics, and co‐expression analyses indicate novel interactions in the plastid CLP chaperone‐protease system
Authors: van Wijk Klaas J.
Published: May 2022

The chloroplast CLPC1 chaperone delivers and unfolds substrates to the stromal CLPPRT protease complex for degradation. We previously used an in vivo trapping approach to identify a dozen interactors with CLPC1 in Arabidopsis thaliana.This was achieved by expressing a STREPII‐tagged copy of CLPC1 mutated in its Walker B domains (CLPC1‐TRAP) followed by affinity purification and mass spectrometry. To screen for additional interactors, we carried out a far more sensitive and comprehensive in vivo protein trapping analysis. This identified 59 highly enriched CLPC1 protein interactors, in particular proteins in families of unknown functions (DUF760, DUF179, DUF3143, UVR‐DUF151, HugZ/DUF2470), as well as the UVR domain proteins EXE1 and EXE2 implicated in singlet oxygen damage and signaling. Phylogenetic and functional domain analyses identified other members of these families. These protein families appear to localize (nearly) exclusively to plastids. Several of these DUF proteins are of very low abundance as determined through the Arabidopsis PeptideAtlas http://www.peptideatlas.org/builds/arabidopsis/ showing that enrichment in the CLPC1‐TRAP was extremely selective. Evolutionary rate covariation indicated that the HugZ/DUF2470 family co‐evolved with the plastid CLP machinery suggesting functional and/or physical interactions. mRNA‐based co‐expression networks showed that all 12 CLP protease subunits tightly co‐expressed as a single cluster with deep connections to DUF760‐3. Co‐expression modules for other trapped proteins suggested specific functions in biological processes, e.g. UVR2 and UVR3 were associated with extra‐plastidic degradation, whereas DUF760‐6 is likely involved in senescence. This study provides a strong foundation for discovery of substrate selection by the chloroplast CLP protease system.
Role of cardiomyocytes in right ventricle viscoelasticity with pulmonary hypertension development
Authors: LeBar Kristen , Liu Wenqiang , Roth Kellan , Ahern Matt , Evans Erith , Pang Jassia , Ayers Jessica , Chicco Adam , Wang Zhijie
Published: May 2022

Background

The myocardium is viscoelastic, which means there exists elastic and viscous resistant forces during cardiac motion. It has been shown that cardiomyocyte (CM) or muscle fiber has significant viscoelasticity, and microtubule depolymerization via colchicine treatment reduces the CM’s viscoelasticity. However, most prior study is focused on the left ventricle, and the role of CM in right ventricle (RV) viscoelasticity in healthy and pulmonary hypertensive (PH) states is poorly understood. Our goal is to evaluate the contribution of CM to RV viscoelasticity in healthy and PH rats. We hypothesize that CM contributes to both healthy and PH RV viscoelasticity.

Methods

All procedures were approved by Colorado State University IACUC. 6‐week‐old male rats were treated with 3‐week monocrotaline (MCT) (60 mg/kg) to induce PH, and intact healthy rats served as control. RV function was quantified by echocardiography and in vivo pressure‐volume (PV) measurements. After euthanasia, RV free wall underwent equibiaxial stress relaxation in a relaxant bath to obtain passive, biaxial viscoelasticity pre and post colchicine treatment (0.3mM). The tissue was stretched at 20% of strain and with physiological ramp speed (5Hz). Relaxation modulus and normalized stress at 0.01 s were used to quantify elasticity and viscosity. Student t test was performed.

Results

In vivo RV function measurements showed the development of PH and RV failure in MCT rats (Table 1). Marked RV dilation and decreased fractional shortening confirmed the RV failure establishment. PV loop data with fewer samples (n=3) also implies declines in cardiac output, contractility (Ees) and ventricular‐vascular coupling (Ees/Ea). At the baseline, PH development led to increased viscoelasticity in the RV (Fig. 1). Mechanical data before and after colchicine treatment showed reduced elasticity in both healthy and MCT RVs (Fig. 1A&B). The percentage reduction in elasticity was similar between groups, indicating a similar contribution of CM to RV elasticity. Unexpectedly, we did not observe significant change in viscosity after colchicine treatment in both groups (Fig. 1C&D). Moreover, the control RVs were isotropic, whereas the MCT RVs showed marked anisotropy in elasticity (Fig. 1B).

Conclusion

Our results demonstrated similar impact of CM on RV viscoelasticity before and after PH development, despite the CM hypertrophy. Colchicine reduced elasticity in healthy and MCT RVs, but minimally changed RV viscosity. These findings will deepen the understanding of biomechanical mechanism of RV failure associated with viscoelastic changes.
Increased Integration in Mutant Mice: An Analysis of the Patterns of Covariance through Ontogeny in Fgfr2 Mice
Authors: Ogg Miette L. , Pennings Amanda N. , McNulty Margaret A. , Holmes Megan A. , Mussell Jason C. , DeLeon Valerie B.
Published: May 2022

Mutations in the FGF/FGFR2 gene result in the premature fusion of fibrous and cartilaginous joints in the skull. This results in profound morphological changes in the skulls of both humans and mice with these mutations. Previous studies have indicated that morphological integration of the skull is expected to become more pronounced in dysmorphic syndromes, including FGFR2 syndromes which result in craniosynostosis. In this study, our objective was to test two hypotheses: 1) that the magnitude of morphological integration was greater in Fgfr2C342Y/+ mutant mice relative to wild‐type littermates; and 2) that the magnitude of morphological integration would increase through ontogeny.

Our sample included Fgfr2C342Y/+ mutants and wild‐type littermates at postnatal day 14 (P14) and adult stages. We used micro‐computed tomography (microCT) scans to create image volumes of the skull. Virtual reconstructions of the cranium were produced in 3DSlicer software, using volume renders based on tissue densities to define the surface of the bone. We collected coordinate data for 38 biologically homologous landmarks with approximately equal distribution across face and neurocranium. All morphometric analyses were conducted using the geomorph package in R. The sample was subdivided by Age and Genotype. Landmark coordinates were divided into face and neurocranium modules. Coordinate data for each subset were superimposed with object symmetry using the bilat.symmetry() function. Covariance across modules was tested using the two.b.pls() function for each sample subset.

Results confirmed our expectation that the magnitude of covariation between face and neurocranium increased in the mutant sample relative to the wild‐type sample at both timepoints. Surprisingly, within the mutant sample, integration between face and neurocranium was greater in the younger P14 mice than the older adults. Results of this study can be used to inform researchers about patterns of covariance that can be applied to better understand craniosynostosis syndromes that affect humans.
Pro‐inflammatory Cytokines Increase Glycolysis through Upregulation of Uncoupling Protein and Akt Activation in Colorectal Cancer Cells
Authors: Kim Ji Yeon , Park Bohye , Bettaieb Ahmed , Donohoe Dallas R.
Published: May 2022

Non‐cancerous colonocytes use butyrate, which is a short‐chain fatty acid derived from the fermentation of dietary fiber, as a primary energy source. In contrast, cancerous colonocytes use glucose as their preferred energetic substrate and undergo enhanced glycolysis or the Warburg effect. It has been reported that increased production of pro‐inflammatory cytokines such as interleukin1 beta and tumor necrosis factor alpha (TNFalpha) promote glycolysis. Previously, we have shown that interleukin1 beta increased glycolysis and decreased butyrate oxidation. However, the mechanism underlying how this pro‐inflammatory cytokine increased glycolysis was unclear. Here, we have discovered that Uncoupling Protein 2 (UCP2) appears to mediate the elevation in glycolysis induced by interleukin1 beta. UCP2 is a mitochondrial protein that transports the protons back into the mitochondrial matrix, thus dissipating the proton gradient and reducing ATP production. UCP2 is upregulated in various cancers such as breast, prostate, skin, and colorectal cancers. We hypothesize that interleukin1 beta promotes glycolysis by upregulating UCP2 and activating Akt in colorectal cancer cells. We have found colorectal cancer cells treated with interleukin1 beta showed elevated UCP2 expression and increased Akt activation. Next, we utilized the Seahorse XFe24 analyzer to check whether interleukin1 beta affects proton leak and ATP production in colorectal cancer cells. Preliminary data suggests that interleukin1 beta increases proton leak and decreases ATP production in mitochondria, which is consistent with the upregulation of UCP2 and demonstrates a functional consequence. To study whether UCP2 mediates the interleukin1 beta effect toward increasing glycolysis, we created a stable UCP2 knockdown in colorectal cancer cells and tested whether colorectal cancer cells that had diminished UCP2 from the RNAi knockdown did not show an increase in glycolysis when treated with interleukin1 beta as compared to RNAi mock colorectal cancer cells. Our preliminary data allude to a role for UCP2 in mediating the interleukin1 beta induced increase in glycolysis observed in colorectal cancer cells.