bluuebunny/crossref_metadata_2025 huggingface dataset:
Non‐coding FLT3 Mutation is Associated with Lower Galectin Levels in Breast Cancer Patients
Authors: Funkhouser Avery , Blair Bailey , Goodwin Jane , Strigenz Alec , Martin Julie , Funk Christopher R. , Edenfield W Jeffery , Blenda Anna V.
Published: May 2022

Fms Related Receptor Tyrosine Kinase 3 (FLT3) is a class III Receptor Tyrosine Kinase (RTK) which binds to the cytokine Flt3 ligand. It acts to promote cell survival, proliferation, and differentiation of hematopoietic stem cells, and is implicated in the development of acute myeloid leukemia. Galectins are a family of β‐galactoside binding proteins which are involved in many cellular pathways including apoptosis and cell migration as well as the innate immune system. Previous studies have shown that galectins are upregulated and aid in cancer pathologies and current clinical trials are underway to use galectin inhibitors in cancer therapy. RTKs are known to have a variety of direct and indirect interactions with galectins. We have found that breast cancer patients with non‐coding mutations of the FLT3 gene have serum concentrations of galectins ‐1 and ‐9 that align with published healthy values, compared to other breast cancer patients without the mutation which had significantly elevated levels of these galectins. Concentrations of galectin‐1 and ‐9 of 40 breast cancer patients were measured by ELISA. Analysis of 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes from the same patients was conducted using multiplex PCR. Correlations between the galectin serum levels and mutation presence were elucidated by statistical analysis. We found that mean levels of galectin‐1 and ‐9 were at 23.4 ng/mL and 8.4 ng/mL respectively in breast cancer patients. However, patients with a non‐coding FLT3 mutation had levels of galectin‐1 (mean 16.2 ng/mL) and galectin‐9 (mean 5.0 ng/mL) significantly lower than those in cancer patients without the mutation (p‐value, 0.0392 and 0.0266 respectively). Moreover, they were similar to previously published galectin values for healthy controls (18.1 ng/mL and 7.0 ng/mL respectively). While galectins are known to influence spatial organization and trafficking of this class of RTKs, no specific relationship has been elucidated between galectins ‐1 and ‐9 and FLT3. Our results indicate a potential interaction of clinical significance between FLT3 and galectins ‐1 and ‐9, the mechanism of which needs to be investigated further.
The Magnitude of Long‐Term Facilitation is Influenced by Modifications to Mild Intermittent Hypoxia Protocols in Participants with OSA
Authors: Panza Gino , Kissine Dylan , Puri Shipra , Lin Ho‐Sheng , Matieka Jason
Published: May 2022

Introduction

Mild intermittent hypoxia (MIH) with sustained hypercapnia initiates long‐term facilitation (LTF) of minute ventilation in humans. To date the impact of different protocols administered acutely, and repeatedly for a select number of days, on the magnitude of LTF in humans with obstructive sleep apnea (OSA) has not been investigated. Thus, the primary aim of this study was to investigate the impact of two different MIH protocols administered over 10 days on the magnitude of LTF.

Methods

Twenty‐seven males with OSA participated in the investigation. Both protocols were characterized by 12 episodes of hypoxia of similar intensity (PETO2 ≍ 50 mmHg). However, one protocol (P1) was comprised of 2‐minute hypoxic episodes and the other protocol (P2) was comprised of 4‐minute hypoxic episodes. In addition, during P1 PETCO2 was maintained 2 mmHg above baseline throughout the protocol, while PETCO2 was sustained 4 mmHg above baseline during the completion of P2. During the completion of both protocols, minute ventilation was measured initially under conditions of normoxia and hypercapnia (i.e. 2 mmHg or 4 mmHg above baseline) prior to exposure to MIH. Following exposure to MIH, minute ventilation was recorded for an additional 30 minutes (i.e. end recovery period). Data was averaged for the initial 2 days and final 2 days of the protocol. Absolute measures of minute ventilation, and measures standardized to baseline measures on the initial days, were compared between protocols.

Results

Thirteen participants completed P1and 14 participants completed P2. Absolute measures showed that minute ventilation during end‐recovery was greater than baseline on the initial and final days of both protocols (P < 0.01). However, minute ventilation during end‐recovery on the initial and final days was greater after the completion of P2 compared to P1 (P < 0.01). This difference remained after measures of minute ventilation were standardized relative to baseline (P < 0.05). Minute ventilation during end‐recovery on the final days was also greater compared to the initial days after completion of P2 (P < 0.05). This was not the case following completion of P1 (P > 0.3). This difference remained after the data was standardized to baseline measures (P < 0.05).

Conclusion

Our results showed that LTF of minute ventilation can be initiated using two different protocols that varied in regard to episode duration and the degree to which PETCO2is maintained above baseline. We also showed that increases in episode duration and sustained PETCO2 results in a greater magnitude of LTF on a given day. Moreover, these modifications appear to enhance the magnitude of LTF following repeated daily exposure.
Cardiac AMPK is not required for cardioprotection by metformin in pulmonary hypertension induced right heart failure
Authors: McNair Ben D. , Shorthill Samantha , Polson Sydney , Cook Ross , Bruns Danielle
Published: May 2022

Right ventricle (RV) dysfunction dictates survival in pulmonary hypertension (PH) and other clinical conditions. Metformin, a clinically relevant AMP activated protein kinase (AMPK) activator, has been proposed as a therapy for PH induced right heart failure (PH‐RHF) due to its well‐reported cardioprotective properties. Indeed, our group has recently shown that metformin is protective in PH‐RHF, but the mechanisms of protection in the RV remain unknown. The objective of this study was to use determine whether the protective properties of metformin require cardiac AMPK. We used Cre‐Lox recombination to create a cardiomyocyte specific AMPK deleted with temporal regulation of expression. AMPK knockout (KO) and wild type (WT) control mice were placed in a hypobaric hypoxia (HH) chamber (~17,000ft) for 4 weeks to induce PH‐RHF, with or without metformin (200mg/kg/day) in drinking water. Cardiomyocyte specific AMPK deletion exacerbated RV and LV remodeling in response to HH compared to WT, with significant sex differences evidenced by males undergoing worse remodeling than females. AMPK deletion exacerbated cardiac output and RV stroke volume compared to WT mice. Metformin rescued cardiac remodeling in male KO mice but did not attenuate remodeling in female mice. We suggest that metformin is a potential therapeutic target to attenuate maladaptive cardiac remodeling induced by PH‐RHF, but only in male mice. Metformin does not require cardiac AMPK for cardioprotection. The mechanisms by which metformin affords protection in PH‐RHF are still unclear, but may include AMPK activity in non‐myocyte cell populations. Ongoing work will elucidate the role of AMPK activation in fibroblasts as well as other systemic and cardiac mechanisms which may explain how metformin protects against PH‐RHF.
The Selection of Novel Drugs for the Inhibition of Sterol Carrier Protein‐2
Authors: Perez Victoria , Sabin Amber
Published: May 2022

The endocannabinoid system has been increasingly understood to have important functions in the central nervous system, having a role in the regulation of stress, anxiety, metabolism, food intake, pain, and inflammation. Developing drugs to regulate this system is important not only for the treatment of diseases caused by imbalances of endocannabinoids within the body, such as migraines and even schizophrenia linked to endocannabinoids, but also generalized treatments for pain or stress. The Sterol Carrier Protein‐2 (SCP‐2) has been found to have a role in the regulation and transport of endocannabinoids within the body, and is therefore a target of interest for drug development. Previously a study screened small‐molecule libraries for compounds that could possibly bind and inhibit human SCP‐2 and computationally docked them with a human SCP‐2 PDB structure, resulting in the identification of 4 promising compounds. However, when these compounds were tested against human SCP‐2, they did not bind and behave as expected, concluding that our understanding of the structure of SCP‐2 is not complete. Since this study, a new SCP‐2 homology model has been developed that is theorized to better represent human SCP‐2. This model will be used to reevaluate high‐scoring potential inhibitors from the previous study to determine if there are any significant differences in binding energies, and to identify if a different set of compounds score higher. This will be carried out by conducting docking simulations using AutoDock Vina to determine the binding affinities of the previously screened compounds when docked to the new homology model. Additionally, the results of this study will be cross‐referenced with a dynamics‐based analysis of the homology model to determine if and how the dynamics of the proteins could be modified by ligand binding. This will be conducted using Essential Site Scanning Analysis (ESSA) which calculates how much each residue contributes to the global dynamics of the protein and can be used to predict allosteric sites, as well as ClustENMD which perturbs the structure to generate alternate conformations to identify cryptic pockets and critical dynamics. With these results, we will be able to give further insight into how SCP‐2 can be inhibited as well as give a dynamics based analysis that could be useful in further studies.
Butyrate Analogues Mimicking Hypoxia by the Chemical Stabilization of Hypoxia Inducible Factor (HIF)
Authors: Ornelas Alfredo , Wang Ruth X. , Welch Nichole , Henen Morkos A. , Vögeli Beat , Colgan Sean P.
Published: May 2022

Short chain fatty acids and specifically butyrate (BA) have been well documented to show multiple beneficial influences on health. Typically, the concentration of colonic butyrate can range from 10‐20 mM and 95‐99% is metabolized into energy by the mucosa. BA plays a key role in epithelial barrier regulation, ameliorates inflammation and regulates cell growth and differentiation. There are multiple mechanisms on how it plays such an important role in gut health, many related to its regulatory capacity for gene expression. Our laboratory recently reported a direct influence of BA on the stabilization of the transcription factor hypoxia‐inducible factor (HIF). BA acts directly and non‐competitively inhibits HIF prolyl hydroxylase 2 (PHD2), responsible for the rapid degradation of HIF in normoxia (NX). However, the efficacy of BA on HIF stabilization is limited due to its rapid metabolism as fuel. Based on these observations, we hypothesize that analogues structurally related to butyrate stabilize HIF with a longer biological half‐life. A small library of BA derivatives was screened in search of a non‐endogenous analogue with higher potency and/or half‐life for HIF stabilization. In vitro screenings at a physiologically‐relevant concentration (5 mM) were performed in intestinal epithelial cell lines T84 and CaCo2 in NX and HIF‐1a ELISA was used to determine protein abundance. Several hits were observed, with 4‐mercapto‐butyrate (SHBA) being the most promising candidate. The best BA analogues were validated through the induction of classic HIF gene targets, including BNIP3 and CaIX by q‐PCR. SHBA exhibited higher induction of these targets compared to BA. Dose response and time‐course studies were performed to compare SHBA and BA. Notably, SHBA exhibits a much longer half‐life as observed in the stabilization of HIF and induction of gene targets at least up to 72 h compared to BA (24 h). In vivo studies in C57BL/6 mice explored the induction of classic HIF targets and specifically erythropoietin (EPO) in circulation following administration of BA and SHBA at similar doses. These studies revealed that induction of EPO with a single dose of SHBA exceeded that of native BA. Thus, a non‐endogenous BA derivative (SHBA) stabilizes HIF at a higher potency and longer half‐life than BA and will be useful as a template for the development of a HIF stabilizing agent.
Essential Amino Acid Regulation of Protein Turnover to Support Skeletal Muscle Health with Age
Authors: Komp Mary , Wolfe Robert R. , Borsheim Elisabet , Walker Sam , Baum Jamie I.
Published: May 2022

Skeletal muscle (SM) is vital for both long term health and quality of life. Recent research suggests an increase in catabolic signals with age triggers pathologic conditions, such as sarcopenia. Although results from in vitro studies model how essential amino acids (EAA) can regulate muscle protein synthesis (MPS), the relevance of these models to muscle protein breakdown (MPB), and the presence of physiological plasma amino acid concentrations (PAA) remain to be established. Therefore, the objective of this study was to determine the effects of EAA with (0.2, 1.0, and 3.0 x PAA) in a young (passages 2‐10) and aging (passages 16‐24) C2C12 murine muscle cell model. We hypothesized that increased levels of EAA will increase MPS in aging cells and suppress MPB via the mTORC1 pathway when compared to young cells. Myoblasts were seeded (1x105) into 6‐well plates and differentiated into myotubes when they reached 80% confluency. Myotubes were serum and AA starved for 24 hours before receiving one of the following treatments: control (CON), 0.2 x PAA, 1.0 x PAA, 3.0 x PAA with or without rapamycin (100 nm; RAP) for 1, 6, and 24 hours. All treatments were performed in triplicate and then each experiment was repeated three times, yielding nine wells per treatment. Proteins related to sensing of AA and protein synthesis and degradation were assessed using Western Blot. Rapamycin inhibited phosphorylation of p70 S6 kinase 1 (p70S6K1) and 4E binding protein 1 (4E‐BP1), downstream targets of mTORC1, in both CON and AA‐treated young and aged cells. Phosphorylation of 4EBP1 and p70S6K1 was significantly increased (p<0.05) in both young and aged cells following all PAA treatments. However, there was no significance after 24 hours of AA supplementation in aged cells. Particularly, after 6 hours of treatment all doses significantly increased (p<0.0001) phosphorylation of p70S6K1 in both young and aged cells. 0.2 x PAA and 3.0 x PAA elicited a significantly greater response in 4EBP1 phosphorylation in young cells compared to the CON after 6 hours of treatment, whereas a dose of 3.0 x PAA induced a similar response in aged cells. In conclusion, in a C2C12 myotube model, increasing EAA concentrations downregulates the expression of translation initiation factors in young and aging cells. Specifically, it takes a 3.0 x PAA to elicit a response in aging cells, whereas young cells respond to lower physiological concentrations of 0.2 x PAA and 1.0 x PAA. Therefore, it can be proposed increasing EAA may be effective for adjusting the rate of MPS and MPB via the mTORC1 pathway in both young and aging muscle cells.
A humanized anti‐cocaine mAb blocks the cardiovascular effects of cocaine in rats
Authors: Koch Sheryl E. , Marckel Jordan , Rubinstein Jack , Norman Andrew B.
Published: May 2022

Cocaine use and abuse is associated with significant morbidity and mortality. Further, its use has significant effects on cardiovascular function in humans and has been associated with development of chest pain, myocardial ischemia and in rare cases cardiac death. Currently, the FDA has no approved pharmacotherapy for cocaine abuse. We have developed and generated a humanized anti‐cocaine monoclonal antibody (mAb), h2E2, which dramatically decreases brain cocaine concentrations by sequestering cocaine in the plasma. As h2E2 proceeds into clinical trials, a significant question remains as to whether the increased plasma cocaine concentrations, increased circulation time of cocaine, and the decrease in cocaine excretion into the urine affects cardiac function. Therefore, the objective of this study was to determine the dose, duration and effects of cocaine administration in the presence and absence of mAb h2E2 on echocardiographic variables in an in vivo rat model.

Echocardiography was performed on Sprague‐Dawley rats with indwelling catheters using the Vevo 2100 ultrasound machine with a 20mHz probe. Baseline echocardiographic long axis M‐mode images were collected prior to an i.v. cocaine push and at subsequent time points thereafter. Initial studies determined the dose‐dependent effect of cocaine on cardiac parameters including ejection fraction (EF), heart rate (HR) and cardiac output (CO). The most significant increase in EF was observed with 1.68mg/kg cocaineHCl, which was then used in the subsequent experiments.

As noted, cocaine administration increased EF, with inversely proportional changes in left ventricular systolic volume. In addition, cocaine produced an immediate decrease in HR, which did not fully return to baseline over the 45 minute time course, likely due to the anesthesia, as the saline control group demonstrated comparable effects. Similarly, cocaine caused an immediate decrease in CO, which normalized over time.

Pre‐administration of a single equimolar dose of h2E2 significantly blunted the effect of cocaine on EF, including both the initial drop as well as the subsequent increase. At one week and one month post h2E2, there was no significant blunting of the increased in cocaine‐induced inotropy. However, the initial decrease in EF was still blocked after one week. The initial cocaine‐induced drop in HR was blocked by h2E2, even at 1 week post‐injection. h2E2 similarly blocked the increase in CO, however this effect was not detectable after 1 week or 1 month.

In conclusion, there were no significant adverse cardiac effects noted with h2E2 administration and co‐administration with cocaine resulted in significant blunting of both cocaine’s inotropic and chronotropic effects. Elevated plasma cocaine concentrations are pharmacologically inert in the presence of h2E2.
Slc2a5 (GLUT5) upregulation in hyperuricemia drives risk for fructose induced NAFLD
Authors: Hoque Kazi M. , Halperin Kuhns Victoria L. , Woodward Owen M.
Published: May 2022

Epidemiological and observational studies have found a strong correlation between hyperuricemia (clinically elevated serum urate levels) and the onset of metabolic and systemic diseases. To investigate a potential causal role for urate in metabolic disease, we created a humanized mouse model utilizing a loss of function mutation in a critical intestinal urate transport protein, ABCG2. Male animals from this model were hyperuricemic and displayed fat deposition in the liver and significantly elevated serum glucose and insulin levels, hallmarks of metabolic dysfunction, and similar to what is often observed in high fructose diet models of NAFLD (Nonalcoholic fatty liver disease). However, the relationship between hyperuricemia, liver pathogenesis, and altered intestinal fructose uptake is largely unexplored.

Using the Q140K+/+ hyperuricemia model we observed higher gene expression of Slc2a5 as well as increased abundance of its protein product, the apical fructose transporter GLUT5, in enterocytes. In addition, ex‐vivo intestinal fructose transport studies revealed a corresponding significant increase of fructose transport across the intestinal tissue of Q140K+/+ mice (16.497 ±5.955 vs. 34.684 ±5.31 µM/cm2). We hypothesized that HNF4a, the critical transcription factor that regulates the expression of many gut transport proteins, and has potential binding sites in the human and mouse Slc2a5 promoter region, might be sensitive to urate levels. Treatment of the human intestinal Caco‐2 cell monolayers with urate increased the protein abundance of both GLUT5 and HNF4a in a dose‐dependent manner, potentially explaining the observed increased mRNA levels of Slc2a5 in our model. We next explored the possibility that the GLUT5 abundance was further altered due to secondary urate effects on the critical transporter scaffold NEDD4 like E3 ubiquitin‐protein ligase (NEDD4L). Nedd4L gene expression was decreased, and its negative regulator, Sgk2, was significantly increased in intestinal tissue from the hyperuricemic model. Follow‐up experiments demonstrated SGK2 protein abundance is increased in Caco‐2 cells by extracellular urate. Further experiments are needed to confirm if hyperuricemia induced up‐regulation of SGK2, and subsequent down‐regulation of NEDD4L would prevent GLUT5 ubiquitination and increase its residence and abundance at the apical membrane.

In conclusion, we find that hyperuricemia induced via common variants in the key intestinal urate transporter, ABCG2, can cause NAFLD and metabolic phenotypes indistinguishable from high fructose diet models of NAFLD. Here we demonstrate that urate may significantly upregulate the capacity for fructose reabsorption through the upregulation of Slc2a5 transcription and GLUT5 abundance, a finding that suggests human carriers of ABCG2 variants are not only at higher risk for hyperuricemia and gout but also the development of metabolic syndrome.
Morphometric Aspects ot the Alba and Lunate Lineas and Correlations with the Physiological Diastasis of the Rectus Abdominis Muscles: Prospective Study
Authors: Barros Mirna D. , Leite Eduarda C. R. , Uehara Elisa Y. T. , Mendes Carlos J. L.
Published: May 2022

Introduction

The anatomical study of the aponeuroses of the anterolateral abdominal wall (AAW) is described, but the AAW aspects remain without clarification. The decussation of the myoaponeurotic of the external oblique (EO), internal oblique (IO) and transversus abdominis (TA) muscles form the abdominal rectus muscle sheath. Theses muscles are not tangential in their respective lateral margins, then they form the semilunar linea (LS), and on the medial margin of the abdominal rectus muscle they form the alba linea. Even having few citations on the width of the LS and the LA, the literature presents clinical aspects, classifications and proposals for the correction of the pathological width of this line: the diastasis. The measurements of the LA and the LS need reliability for the establishment of criteria for normality or for disorder.

Objective

The aim of this study was to measure the width of the LA and LS, the length of the LA, the distance from the umbilicus (CU) to the anterior margin of the lumbar vertebra (VL) in the plane passing through the CU and to establish whether there is a correlation between the measurements of the LS, LA and the distance from the CU to the anterior margin of the VL.

Method

Trough the tridimensional reconstruction of computerized tomographies of the AAW in adults, measurements were made of the distances to the LA and LS in five levels from the xifoid process to the pubic symphysis using Osirix MD software.

Results

A total of fifteen individuals were studied as the pilot sample for the calculation of the sample, with the standard deviation for the average and 95%IC. The calculation led to the evaluation of 29 individuals (15 men and 14 women). The analyzed outcomes were: 1. Dpx_sp_reta; 2. Dpx_sp_pela_LA; 3. Dcu+VL; 4. DLA_NSu1; 5. DLA_NSu2; 6. DLA_NSu3; 7. DLS_dir_NSu3; 8. DLS_esq_NSu3; 9. DLA_NU; 10. DLS_dir_NU; 11. DLS_esq_NU; 12. DLA_Niu1; 13. DLS_dir_Niu1; 14. DLS_esq_Niu1; 15. DLA_Niu2; 16. DLS_dir_Niu2; 17. DLS_esq_Niu2. Considering that the DLA – distance from alba linea, DLS – distance from semilunar linea, sp – pubic symphysis; vL – lumbar vertebra; NSu – supraumbilical level; Niu – infraumbilical level. The maximum and average measurements were established for each outcome for men and women and the measurements (DLA_NSu1) e (DLA_Niu1) presented significant differences between men and women.

Conclusion

The results may lead to a relevant discussion on the concept and the classifications of diastasis, producing a deeper knowledge of the morphological variations of the abdominal wall and the normal and unnormal functional aspects of these muscles and fáscias. This anthopometric anatomical parameter will fill in a blank with relevant data for the determinations of the criteria of normality, anatomical variation or disorder.
Assessment of Skeletal Muscle Oxygenation during Exercise in Patients with Peripheral Artery Disease Using Near Infrared Spectroscopy: Impact of Skin Color and Adipose Tissue Thickness
Authors: Pae Byung Joon , Hirai Daniel , Monroe Jacob , Motaganahalli Raghu , Song Qifan , Roseguini Bruno
Published: May 2022

Introduction

Near infrared spectroscopy (NIRS) is a potentially valuable tool to examine disease severity and the effects of therapeutic interventions on microvascular leg oxygenation in patients with peripheral artery disease (PAD). However, multiple factors can confound the interpretation of NIRS signals and therefore limit its clinical applicability, including adipose tissue thickness (ATT) and skin melanin content. We tested the hypothesis that the changes in the concentration of oxygenated [oxy‐Hb+Mb] and deoxygenated [deoxy‐Hb+Mb] hemoglobin + myoglobin would be reduced in the calf of black PAD patients during exercise when compared to white patients. Similarly, we hypothesized that patients with a thicker calf ATT would exhibit a lower change in oxy[Hb+Mb] and deoxy[Hb+Mb] during exercise relative to patients with low ATT.

Methods

Twenty‐four participants with a history of exertional calf pain (18 males, 6 females, 67±7 years; ankle‐brachial index: 0.6±0.1) were asked to complete two graded tests on the treadmill. The protocol consisted of a constant walking speed of 2 mph and progressive increases in grade of 2% every 2 min. The NIRS device was secured to the skin over the medial gastrocnemius muscle of the most symptomatic leg. Oxy[Hb+Mb] and deoxy[Hb+Mb] were measured continuously at rest and during exercise. The final 20‐s averages of stages 1, 2 and peak exercise were utilized for analysis. Magnetic resonance imaging of the calf was used to measure ATT. The average ATT among all individuals (7±3 mm) was used to divide participants into ‘low’ (n=12) or ‘high’ (n=12) ATT groups. A three‐way repeated measures analysis of variance was used to evaluate the effects of skin color, ATT, and stage of the treadmill test (i.e. stage 1, stage 2, peak walking time) on the changes from baseline in calf oxy[Hb+Mb] and deoxy[Hb+Mb].

Results

Seven participants were black and the remaining 17 were white. The average ATT at the medial gastrocnemius site was 4±1 mm in the low ATT group and 9±2 mm in the high ATT group. A significant interaction was observed between skin color, ATT, and exercise stage for the change from baseline in oxy[Hb+Mb] (p=0.049). Post‐hoc testing revealed that among white participants, the change in oxy[Hb+Mb] during exercise was significantly lower in the high ATT group when compared to the low ATT group (p=0.039). Among black participants, the change in calf oxy[Hb+Mb] was similar between high and low ATT (p=0.57). There was a trend for a main effect of ATT on the changes in deoxy[Hb+Mb] during exercise (p=0.1), but the main effects of skin color (p=0.2) and the interaction between skin color, ATT, and exercise stage (p=0.5) were not significant.

Conclusions

In conclusion, the changes in NIRS‐derived calf oxy[Hb+Mb] during exercise are impacted by both skin color and ATT in patients with symptomatic PAD.
Increased fatty acids in Type 1 Diabetes disrupts C‐peptide’s transport and downstream effect
Authors: Jacobs Monica , Summers Suzanne , Spence Dana
Published: May 2022

C‐peptide, a hormone that is co‐secreted from the pancreatic beta‐cell, has been shown to alleviate diabetic complications attributed to poor microvascular blood flow. In this mechanism, C‐peptide binds to the red blood cell (RBC), increasing adenosine triphosphate (ATP) release and subsequent nitric oxide (NO) release, a potent vasodilator. Prior research has indicated that C‐peptide must be complexed with zinc (Zn2+) and albumin to elicit this response. However, in type 1 diabetes (T1D), increased basal levels of fatty acids result in high concentrations of fatty acids bound to albumin. These increased concentrations of fatty acids seen in T1D may have implications in C‐peptide’s transport and therapeutic effect. Here, the binding of C‐peptide, Zn2+ and albumin are measured with increased levels of a myristic acid, a common fatty acid seen in vivo.3D‐printed technologies were employed to fabricate an ultrafiltration device to measure the association between C‐peptide, Zn2+ and albumin. C‐peptide derived ATP release from RBCs was quantified via chemiluminescence on a spectrophotometer. Results indicate that C‐peptide binding to albumin is disrupted when myristic acid is in solution (3.68 nM free, n=12). In addition, C‐peptide derived ATP release from RBCs is decreased as concentrations of myristic acid increased. These discoveries indicate disruptions in C‐peptide’s transport and effect with increased fatty acids. In addition, these findings open an unprecedented area of study regarding other modifications in the bloodstream of people with T1D that may disrupt C‐peptide transport and delivery.
Effect of Knock‐out of the a4 Isoform of Vacuolar H+‐ATPase following CRISPR/Cas9 on Activation of Cathepsins in MDA‐MB231 Breast Cancer Cell
Authors: Hinton Ayana M.
Published: May 2022

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are essential for bone degradation and synaptic signaling. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231 and may contribute to its metastatic phenotype. We hypothesize that V‐ATPases creates a low pH in a localized environment which promotes tumor cell invasion by activating secreted cathepsins. Studies indicate that cathepsins secreted by cancer cells cleave extracellular matrix proteins and activate other proteases, facilitating tumor cell invasion. Our lab is investigating a connection between the isoforms of the a subunit for V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. Previous data from our lab indicates that inhibition of V‐ATPases by a general inhibitor reduced the activity of secreted cathepsin B, but not cathepsin L. However, although there was a trend, differences in activity for secreted cathepsin B after V‐ATPase inhibition was not statistically significant. Our current data supports the conclusion that there is a significant difference in the effects of V‐ATPase inhibition on secreted cathepsins. Inhibiting V‐ATPases does not appear to affect the activity of secreted cathepsin L, however it reduces the activity of secreted cathepsins B and D. To further investigate the role of V‐ATPases in the activation of cathepsins, we are using CRISPR/Cas9 to knockout the genes of the four V‐ATPase “a” subunit isoforms, a1, a2, a3 and a4. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. The a1 and a2 isoforms are found in intracellular compartments. Thus far we have made the most progress with knocking out the a4 isoform. We hypothesize that the a4 isoform knockout cells will have reduced activation of secreted cathepsins B and D while not effecting activation of cathepsin L.
Topographical Organization and Morphology of Calcitonin Gene‐Related Peptide (CGRP) Axons in the Flat‐Mounts of Whole Mouse Stomach
Authors: Nguyen Duyen , Ma Jichao , Madas Jazune , Mistareehi Anas , Chen Jin , Heal Maci , Tappan Susan , Lin Mabelle , Hunter Peter J. , Powley Terry , Cheng Zixi J.
Published: May 2022

Nociceptive afferents innervate the stomach and send nociceptive signals centrally to the brain and locally to the enteric nervous system. Nociceptive afferents can be detected with a variety of different markers (e.g., CGRP, SP, TRPV1). However, the distribution and morphological structure of nociceptive axons and terminals have not yet been well determined in the flat‐mounts of the whole stomach. In this study, we used calcitonin gene‐related peptide (CGRP) as a marker to label nociceptive afferent axons and terminals in the in the flat mounts of the whole ventral and dorsal stomachs of mouse (n = 6/each side, 3‐5 months). We applied a combination of state‐of‐the‐art techniques, including confocal microscopy, Zeiss Imager microscopy, flat‐mount tissue processing of the whole organ and immunohistochemistry, Neurolucida 360 tracing and integration of the tracing data into a 3D stomach scaffold to determine the distribution and morphology of CGRP‐IR axons and terminals in the whole stomach. We found that 1) CGRP‐IR axons formed extensive terminal networks in both ventral and dorsal stomachs. 2) CGRP‐IR axons dramatically innervated the blood vessels. 3) In longitudinal and circular muscles, CGRP‐IR varicose axons ran in parallel with the direction of the muscles. 4) CGRP‐IR axons formed a complex network between the longitudinal and circular muscle layers. 5) In the myenteric ganglia, CGRP‐IR axons formed varicose terminal contacts with individual myenteric neurons. 6) We did not observe any significant CGRP‐IR myenteric neurons. 7) CGRP‐IR axon innervation of the blood vessels were traced and digitized and integrated into a 3D scaffold. This is the first time that we provided a topographical map of CGRP‐IR innervation of the whole stomach at single cell/axons resolution. The work provides an anatomical foundation for functional studies of CGRP‐IR axons in various regions of the stomach and their remodeling in diseases. The first two authors contributed equally to this work.
Alisol B23‐Acetate Targets SHH Signaling in MED12 Knockdown Breast Cancer Cells to Inhibit Oncogenesis
Authors: Gonzalez Cristian G. , Burleson Marieke
Published: May 2022

Breast cancer currently ranks second in leading causes of death in women in the United States. It is estimated that one in eight women will develop breast cancer over the course of their life, so finding treatment options for this disease is critical. Current treatment strategies include chemotherapy, radiation, and surgery, all of which can cause harmful side effects that can be damaging to the patient’s overall health. Furthermore, these strategies are not always effective, so personalized treatment has become an area of interest to those who are searching for alternative treatment options. With the current genome sequencing technology that is available, researchers are able to identify specific locations in the DNA that could be a potential target area for treatment. One of those locations is the Mediator subunit MED12 that has been shown to be mutated in up to 67% of breast cancer tumors. Our results from previous experiments show mutations in MED12 promote GLI3‐dependent Sonic Hedgehog (SHH) signaling, thus indicating that tumor cells could potentially use this pathway to proliferate when MED12 is mutated. With these findings we hypothesize that MED12 mutations promote hyperactivated SHH signaling in breast cancer cells and that the compound Alisol B 23‐acetate (AB23A), a natural triterpenoid, can target SHH signaling in these cells to suppress oncogenesis. To study the effects of MED12 mutation, the MCF‐7 breast cancer cell line was infected with a lentivirus carrying a shRNA targeted against MED12. Most MED12 mutations in breast cancer cells lead to a loss of function in the protein, so we presume that a knockdown of MED12 would lead to similar presentation that is seen in tumors. To test for the effects of this knockdown on cell growth, proliferation assays were conducted which confirmed that MED12 knockdown promotes oncogenesis. Quantitative PCR was then conducted to determine the effects of MED12 on GLI3 target genes. Our results show that when MED12 is knocked down, there is an increase in the expression of GLI3 target genes. Next, MTT assays were performed to determine the effects of AB23A on both MCF‐7 control and MCF‐7 MED12 knockdown cells. These results indicate that AB23A does not affect the viability of MCF‐7 control cells but does promote cell death in MCF‐7 MED12 knockdown cells. Furthermore, through quantitative PCR analysis, we found that AB23A specifically promotes downregulation of GLI3 target genes, thus indicating that this compound likely targets the GLI3‐dependent SHH signaling pathway. Overall, these results provide insight into AB23A as a potential therapeutic treatment for breast cancer with MED12 mutations.
Characterization and Ischemic Response of Telecytes in the Mouse Renal Medulla
Authors: Zou Yao , Li Guangbi , Hu Gaizun , Yuan Xinxu , Ritter Joseph K. , Li Pinlan
Published: May 2022

Telocytes are currently defined as a novel type of interstitial (stromal) cells with very long and thin prolongations, and these cells can produce exosomes to transmit signals to different cells such as vascular smooth muscle cells, tubular cells and telocytes themselves. So far it remains unclear whether telocytes are present in the renal medulla and whether they are able to regulate renal medullary function. In the present study, we first characterized telocytes in the kidney, in particular, in the renal medulla, given that the renal medullary interstial cells (RMICs) are able to sense pressure and release bioactive lipids lowering arterial blood pressure. Using two selective markers of telocytes, CD34 and c‐kit for immunohistochemical analysis, we found that telocytes are present in the renal medullary interstitium between tubules, vessels or tubular‐blood vessels. Compared to the renal cortex, telocytes are more enriched in the renal medulla. Their long prolongations could be seen in the interstitium along the tubules or blood vessels. Using confocal microscopy, colocalization of CD34 and c‐kit are mainly detected around different tubules or vessels, but not in tubular or vascular cells. Electron microscopy found that many renal medullary telocytes had at least 3 prolongations, where a number of telopods were shown. In some interstitial space with enriched telopods, moreover, a large number of exosomes could be found. We also isolated and cultured telocytes, which were purified from renal medullary interstitial cells used in our laboratories by MiniMACS Cell Separator with anti‐c‐kit antibody beads‐loaded column. In culture, these cells showed typical telocyte features such as more than 3 prolongations, telopods and project connections between cells. Confocal microscopy showed that most cells through MiniMacs column could be double stained by antibodies against c‐kit and CD34, but the original renal medullary institial cells had much fewer cells stained, which was also confirmed by flow cytometry. In a group of mice with 2 kidney‐1 clip (2K1C) hypertensive model, we found that compared to the unclipped kidney, the clipped kidney had largely increased CD34 and c‐kit staining in the renal medullary interstitium, but not much in the renal cortex. These results suggest that telocytes are present in the renal medullary interstitium and in response to chronic ischemia produced by partially clipping renal artery, telocytes are increased. Given that exosomes are substantially enriched in the renal medulla of 2K1C mice, telocytes may be one of the important source for these exosomes that have antihypertensive action (Supported by NIH grants DK054927 and DK120491).
Optimizing Newborn Primate Endocast Reconstruction in 3DSlicer
Authors: Bryson E. R. , King Scot E. E. , McGovern‐Lind Brenna R. , Proffitt Kathryn A. , Smith Timothy D. , DeLeon Valerie B.
Published: May 2022

Natural and artificial endocasts have been used for decades to enable researchers to visualize the endocranial space and approximate the morphology of internal soft tissue structures. Producing an endocast of the newborn primate skull is made difficult by the large fontanelle spaces between growing bones. As there is no definitive endocranial boundary, these spaces have the potential to introduce error into both manual and semi‐automatic methods of endocast reconstruction.

This study examines three methods of capturing endocranial volume (EV) from the endocasts of multiple species of newborn primates. For each specimen an EV was produced using three methods: a) manual, b) semi‐automatic and c) “patched” semi‐automatic segmentation. Manual segmentation is considered the “gold standard” of endocast reconstruction, however it lacks the efficiency of semi‐automatic methods. The “patched” method proposed here is a novel approach to fontanelle closure that is accomplished using the “Baffle Planner” module in 3DSlicer, an open‐access post‐processing software that is freely accessible to all researchers. The interosseous patches constructed from user‐defined landmarks are placed around the ossifying edges of the fontanelle and reflect the curvature of adjacent bones.

In this study we provide guidance for optimizing the parameters of newborn primate endocast reconstruction in open‐access software. Our study found both quantitative and qualitative differences between the models produced by these methods suggesting the chosen method of reconstruction affects the resulting EV measurement. We conclude that a patched semi‐automatic approach with minimal baffles appears to reflect the anatomy with sufficient accuracy, while increasing efficiency and repeatability.
Anti‐Tumor Effect of Embryonic Stem Cell Derived Exosomes in Triple Negative Breast Cancer: Potential Role of TCF7‐E‐Cadherin and VEGF
Authors: Samidurai Arun , Das Anindita , Kraskauskas Donatas , Zhang Bei , Olex Amy , Liu Jinze , Hu Bin , Koblinski Jennifer , Singla Dinender K. , Kukreja Rakesh C.
Published: May 2022

Background

Triple Negative Breast Cancer (TNBC) is an aggressive form of cancer and accounts for 10‐20 % of all breast cancer types. TNBC patients are widely treated with chemotherapeutic drugs such as doxorubicin (Dox), which is a primary standard of care. Dox treatment results in unwanted side effects including cardiac dysfunction and skeletal muscle weakness. We previously demonstrated the anti‐tumor effects and cardioprotective effects of embryonic stem cell derived exosomes (Es‐Exos) in the xenograft model of TNBC. The purpose of this study was to identify the biomolecules present in Es‐Exos, which may be linked to the anti‐tumor effects in the xenograft model.

Methods and Results

Orthotopic xenograft model of TNBC was generated by injecting human MDA‐MB‐231‐Tom‐Luc cell line in mammary intraductal T4 region of nude mice. Non‐tumor bearing female mice were used as control (NT‐Con;n=6) as applicable. The animals (n=10/group) were randomized to receive Saline (Con); Dox (5 mg/kg/week); Es‐Exos (50 ug/animal/twice a week; Dox+Es‐Exos; Es‐Exos alone and MEF‐Exos‐(mouse embryonic fibroblast cell derived exosomes 50 ug/animal/twice a week) for up to 4 weeks. Skeletal muscle grip strength declined both in Con and upon treatment with Dox in tumor bearing mice as compared to NT‐Con, which was restored upon treatment with Es‐Exos. Cardiac function measured by echocardiography showed decline in ejection fraction (EF) in saline treated tumor bearing Con and in Dox group, when compared to NT‐Con. Treatment with Dox+Es‐Exos and Es‐Exos improved ejection fraction compared to Con and Dox cohort. Treatment with Es‐Exos significantly reduced the tumor size compared to Dox and saline treated Con groups (Con Vs Dox and Dox+Es‐Exos; p‐value <0.05). Picro Sirius red staining showed increased deposition of collagen in saline treated Con, which was reduced in Dox+Es‐Exos and Es‐Exos groups. RNA seq analysis of Es‐Exos identified several anti‐proliferative genes including a 6‐fold expression of Transcription Factor 7 (TCF7) and 6.5‐fold higher expression of E‐Cadherin (CDH1). Immunohistochemistry analysis of tumor tissue showed abundant expression of VEGF in untreated Con, which was reduced by combined treatment with Es‐Exos and Dox.

Conclusion

Embryonic stem cells derived exosomes contain anti‐proliferative genes which may possibly lead to their anti‐tumor property in the TNBC tumor model.
Specificity Determinants of Lysine Deacetylases
Authors: Toro Tasha B. , Bezue Jada A. , Bornes Kiara E. , Broussard Christian G. , Swanier Jordan S. , Watt Terry J.
Published: May 2022

Lysine acetylation is a reversible post‐translational modification that has been found on thousands of nuclear and cytoplasmic proteins. A family of enzymes, known as lysine deacetylases (KDACs), catalyzes the removal of acetyl groups. This reversible modification is a regulatory mechanism for proteins involved in numerous cellular processes; however, few KDAC‐substrate pairs have been identified, and there is a lack of information regarding how individual KDACs interact with substrates. We hypothesized that differences between KDACs near the active site would influence substrate specificity, allowing members of the family to preferentially react with a unique subset of acetylated proteins. Using a peptide library derived from a previously‐identified substrate of KDAC8, we demonstrated that KDAC1, KDAC6, and KDAC8 have distinct preferences regarding the residues surrounding the acetylated lysine. A combination of activity data using an in vitro assay and molecular dynamics simulations revealed discrete features of the KDACs that contribute to substrate specificity. In KDAC8, but not in other KDACs, an aspartic acid residue (D101) forms an ionic interaction with positively charged residues in the ‐1 position of the substrate relative to the acetyllysine, promoting deacetylation. Additionally, nearby hydrophobic residues further influence substrate preferences in a KDAC‐specific manner. Using interaction models based on these data, we were able to predict biologically relevant peptide substrates for individual KDACs. While the identified residues are undoubtedly not the only KDAC specificity determinants, understanding the role of these close contacts on enzyme preferences has greatly increased our understanding of KDAC specificity and has given us a handle to identify potential protein substrates from cells.
The selective inhibitors for heat shock protein 90 beta reduce the lipopolysaccharide (LPS)‐induced inflammatory response via inhibiting the activation of NF‐κB and ERK MAPK in BV2 microglial cells
Authors: Kliebe Valentin M. , Smith Amanda G. , Hanna Christen , Blagg Brian S. , Lei Wei
Published: May 2022

Neuroinflammation contributes to several health conditions related to the central nervous system, such as Alzheimer’s Disease and pain. Understanding the regulation of neuroinflammation is essential for discovering approaches to treat inflammation‐related diseases. Previous studies demonstrated that the inhibition of Heat shock protein 90 (Hsp90) using 17‐N‐allylamino‐17‐demethoxygeldanamycin (17‐AAG) could reduce neuroinflammation. However, the contribution of Hsp90 isoforms, including Hsp90α, Hsp90β, glucose‐regulated protein 94 (Grp94), and tumor necrosis factor type 1 (TRAP1), to inflammatory signaling remain poorly understood. This study aimed to determine the impact of selective inhibitors for Hsp90β on lipopolysaccharide (LPS)‐induced inflammatory responses in microglial cells. A murine microglial cell line, BV‐2 cells, were pretreated with selective inhibitors for Hsp90β for 1 hour, followed by the stimulation of LPS. The production of Nitric Oxide (NO) was measured using Griess Reagents, and qPCR was performed to detect the expression of tumor necrosis factor‐alpha (TNF‐α) and Interleukin‐1 beta (IL‐1β). In addition, the activation of the nuclear factor‐κB (NF‐κB) and Nrf2 were investigated. The impact of selected inhibitors on the activation of extracellular‐signal‐regulated kinases (ERK), p38 mitogen‐activated protein kinases (p38 MAPK), and c‐Jun‐N‐terminal kinases (JNK) was measured using Western Blots. We found that treatment with Hsp90β inhibitorsstrongly inhibited the production of NO, TNF‐α, and IL‐1β, and the activation of NF‐κB, but showed a modest effect on Nrf2 activation. Additionally, the Hsp90β inhibitors also showed to inhibit the activation of ERK, but no effect on p38 and JNK activation. These findings demonstrate that Hsp90β plays a vital role in the LPS‐induced neuroinflammation in microglial cells.