bluuebunny/crossref_metadata_2025 huggingface dataset:
Sodium Caprate Enhances Transcellular LPS Transport via Clathrin‐Mediated Endocytosis in Rat Jejunum
Authors: Akiba Yasutada , Kaunitz Jonathan D.
Published: April 2020

Lipopolysaccharides (LPS) entry from the intestinal lumen to the circulation is associated with systemic inflammation. We have found that LPS is transcellularly transported to portal vein during long‐chain fatty acid (LCFA) exposure via CD36‐ and lipid raft‐mediated pathways. We examined the effects of medium‐chain fatty acids (MCFA) on LPS transport in rat jejunum.

FITC‐LPS was applied to the mucosal bath of Ussing chambered muscle‐stripped jejunal and distal colonic mucosa‐submucosa preparations, in which short‐circuit current (Isc) and tissue electrical resistance (TER) were measured. Serosal appearance of FITC‐LPS was measured with or without luminal application of sodium caprate (C10, 10 mM). FITC‐dextran 4kDa (FD4) m‐to‐s movement was also separately measured.

Luminal application of caprate increased FITC‐LPS m‐to‐s transport without any changes in FD4 m‐to‐s movement and TER in the jejunum, whereas caprate remarkably increased FITC‐LPS transport with increased FD4 movement and decreased TER in the distal colon, suggesting that caprate enhances transcellular LPS transport in the jejunum, but increases paracellular permeability in the distal colon. In the jejunum, C10‐mediated FITC‐LPS m‐to‐s transport was inhibited by pretreatment with luminal Pitstop2 (30 μM), a selective inhibitor of clathrin‐mediated endocytosis (CME), whereas the CD36 inhibitor sulfosuccinimidyl oleate (SSO, 0.1 mM), the lipid raft inhibitor methyl‐β‐cyclodextrin (MβCD, 1 mM), or the dynamin inhibitor Dynasore (50 μM) had no effect. In contrast, luminal OA (30 mM) with taurocholate (TCA, 0.1 mM) also increased FITC‐LPS transport with SSO, MβCD and Dynasore‐sensitive, but Pitstop2‐insensitive manner. In vivo, jejunal luminal perfusion of C10 with FITC‐LPS increased portal venous FITC‐LPS appearance 15 min after perfusion, higher than by OA/TCA, which was then inhibited by co‐perfusion of Pitstop2.

These results suggest that luminal MCFA and LCFA differentially induce transcellular LPS transport in the jejunum; MCFA via CME, but LCFA via the CD36/lipid raft/caveolin pathway. Dietary fatty acids may impact LPS‐associated diseases, including metabolic disease and multiple organ failure.

Support or Funding Information

VA Merit Review and Shire Pharmaceuticals
Stimulation of the β2 adrenergic receptor rapidly increases phosphorylation of the Na+‐Clcotransporter (NCC)
Authors: Poulsen Søren Brandt , Cheng Lei , Penton David , Murali Sathish Kumar , Kortenoeven Marleen , Loffing Johannes , Fenton Robert A.
Published: April 2020

Adrenergic signaling is proposed to modulate activity of the thiazide‐sensitive Na+‐Clcotransporter (NCC) by direct effects on the renal distal convoluted tubule (DCT). However, there are conflicting data on whether, α1, β1 or β2‐adrenoceptors mediate these effects by activation of the with‐no‐lysine kinases (WNKs) and STE20/SPS1‐related proline alanine‐rich kinase (SPAK) kinase pathway or by other signaling pathways. Here, we determined using RT‐PCR that the β2‐adrenergic receptor was abundantly expressed in mpkDCT cells (a model of the DCT) and in DCT cells isolated from parvalbumin EGFP mice by fluorescence‐activated cell sorting. In mice administered salbutamol (β2 adrenergic receptor agonist used to treat asthma) for 30 min, phosphorylated NCC levels increased, indicating increased activity. However, mice developed hypokalemia, a condition that also increases NCC phosphorylation. To investigate direct effects of β2 adrenergic signaling on DCT cells independent of K+, the effects of salbutamol on mpkDCT cells and in kidney slices/tubule suspensions were examined. Salbutamol gradually increased NCC phosphorylation in the pharmacologically relevant concentration range of 0.01–10 μM, an effect observed after 15 min and maintained at 60 min. Changes in NCC phosphorylation were associated with increased cAMP levels in tubule suspensions, kidney slices and mpkDCT cells. A role for the NCC modulatory PKA/I‐1/PP1 pathway was suggested by attenuated effects of salbutamol on phosphorylated NCC during PKA/PP1 pathway inhibition or in slices from I‐1 KO mice. Inhibition of the WNK‐SPAK pathway with a WNK inhibitor also attenuated, but did not prevent, the salbutamol‐induced phosphorylation of NCC. In conclusion, β2 adrenergic receptor signaling modulates NCC phosphorylation in vivo and ex vivo via PKA and WNK‐dependent pathways. We speculate that this response may be a compensatory mechanism to limit electrogenic renal K+ secretion during β2 adrenergic receptor stimulation of muscle. Furthermore, frequent use of salbutamol in e.g. asthmatic patients may lead to renal NaCl retention and influence blood pressure.

Support or Funding Information

The Novo Nordisk Foundation, The Leducq Foundation and the Danish Medical Research Council.
Systemic Treatment with Cannabinoid Receptor 2 Agonist to Treat Osteoporosis in a Rodent Model of SCI
Authors: Grayson Bernadette E. , Pride Yilianys , Sereduck Susan , Chade Alejandro , Grill Raymond , Tucci Michelle
Published: April 2020

Osteoporosis is a skeletal disease resulting in a reduction of bone mineral density and bone mass with disorganization of trabecular bone architecture, leading to an increased incidence of bone fracture. Nearly all spinal cord injury (SCI) persons will develop osteoporosis following injury, and further up to 50% of all persons with SCI will sustain a fracture during their lives. The mechanism driving osteoporosis following SCI is elusive. The cannabinoid system modulation of bone metabolism through CB1/2 has been increasingly of interest for the preservation of bone mass and density in models of osteoporosis. Here we used early and sustained treatment with selective cannabinoid 2 receptor (CB2) agonist, HU‐308, in a mouse model of SCI to prevent and reverse SCI‐induced osteoporosis. First, we determined the efficacy of acute treatment with CB2 agonist, HU‐308, to stave off osteoporosis with treatment for 40 days post‐injury. In thoracic‐complete transected mice, the bone density was significantly reduced in comparison to injury‐naïve mice. In the distal tibia, HU‐308 was effective to preserve trabecular bone density as measured by Micro‐CT in comparison to SCI mice treated with vehicle (VEH) (P < 0.05) but less so in the proximal tibia. In the femur, 40 days post‐injury, there was no efficacy of HU‐308 to preserve bone density in SCI‐HU‐308 treated mice in comparison to VEH‐treated SCI. Histologic inspection supports substantial loss of trabecular bone. In order to determine if HU‐308 could reverse bone loss, we treated SCI mice with HU‐308, CB2‐agonist significantly after the onset of SCI‐induced osteoporosis (i.e., 3 months post‐injury) for 40 days; this resulted in profoundly improved bone mineral density in SCI mice treated with HU‐308 in the tibia and trends in the femur. Additionally, increased periosteal bone formation was observed in the femurs of SCI‐HU‐308 treated animals following the delayed treatment (P < 0.05) and absent in the SCI‐VEH treated animals. We also sectioned undecalcified bone and stained with Alizarin to demonstrate preservation and rebuilding of bone with HU‐308 in the acute and delayed treatment paradigms. Acutely, there appears to be preservation of the cortical bone and not the trabecular bone; the VEH‐treated mice showed loss of mineralization in both trabecular and cortical bone. Delayed treatment with HU‐308 shows bone formation with a higher degree of trabecular and cortical mineralization in comparison to VEH‐treated SCI mice. Taken together these data suggest that the cannabinoid system contributes to the pathophysiology of SCI‐induced osteoporosis and that CB2 agonism may be a viable therapeutic strategy to prevent or slow osteoporosis in SCI patients.

Support or Funding Information

This work is supported by Department of Defense Grant Award Number: W81XWH‐16‐1‐0349 to BEG.
Adaptation of Puromycin‐Associated Nascent Chain Proteomics to Arabidopsis thaliana and Pisum sativum
Authors: Noras Aleksandra Izabela , von Arnim Albrecht
Published: April 2020

Translation is a biological function present in all forms of life that allows for the production of proteins from strands of messenger RNA (mRNA). In plants, such as Arabidopsis thaliana or Pisum sativum (garden pea), translation is tightly regulated because of the plants’ sessile nature and need to adapt to a variety of stresses. PuNCH‐P is a proteomic technique that offers a snapshot of the translational status of cells in both a noninvasive and time sensitive manner. It was previously developed for mammalian tissues but had not been adapted to plants. PuNCH‐P delivers an estimate of the rate of protein production using a technique complementary to ribosome footprinting or polysome loading. This method circumvents the need for in‐vivo labeling such as in SILAC or BONCAT methods, and also avoids high sequencing costs associated with ribo‐seq methods. Nascent peptides attached to ribosomes are isolated from a frozen tissue sample by centrifugation through a sucrose cushion. These peptides are then C‐terminally labelled with biotin‐puromycin. The incorporation of biotin allows for downstream peptide visualization on an immunoblot (Western Blot) using streptavidin‐HRP as a detection antibody. Peptides may also be captured with streptavidin coated beads. Here, we validate the PuNCH‐P method for Arabidopsis and peas. It is well known that heat shock causes an overall decrease in the translational activity of plants due to mechanisms such as the sequestering of cytoplasmic mRNAs into stress granules and the shift to translating heat shock proteins (HSPs). Here we present methods to efficiently label, capture and detect biotin‐labeled nascent peptides from plants. With heat treatment as a proof of concept, we show that puromycylation recapitulates the shift in the patterns of translation expected from polysome profiling. These data set the stage for the subsequent proteomic analysis of nascent peptides in plants.

Support or Funding Information

NSF MCB 1546402
The Role of the Actin‐Binding Protein Cortactin in the Activation of T Cells: Implications for T‐Cell Acute Lymphoblastic Leukemia
Authors: Martinez Ramon Castellanos , Reyes Salvador Valle , Schoor Michael
Published: April 2020

T‐cell acute lymphoblastic leukemia (T‐ALL) is an aggressive neoplasm due to high rates of organ infiltration and relapse, which are major obstacles for curing the disease. Infiltration of T‐ALL cells requires extravasation, a migratory process highly dependent on actin cytoskeleton dynamics. Cortactin is an actin‐binding protein that regulates actin remodeling and thus cell migration. Recently, we showed that cortactin overexpression in B‐cell ALL was correlated with infiltration, steroid treatment failure and relapse. However, it is unknown whether cortactin is expressed in leukemic T cells and what role it plays in T‐ALL biology. We analyzed cortactin expression in murine and human normal T cells and T‐ALL cell lines, and cortactin functions using different in vitro and in vivo methods such as transendothelial migration and xenotransplantation assays. We observed that the human leukemic T cell lines Jurkat, CEM and Molt‐3, as well as the leukemic murine T cell line 6645/4 expressed the 80 kDa WT isoform of cortactin. Importantly, all cell lines had significantly higher levels of cortactin than normal resting CD3+ cells. In addition, leukemic T cells transmigrated, infiltrated and colonized bone marrow organoids significantly more than normal T cells. Of note, in vivo xenotransplantation assays revealed that infiltrated leukemic T cells diminished cortactin expression suggesting that cortactin is needed for initial organ invasion, but not for subsequent functions within the organ. As leukemic cells display hyperactive TCR signaling, we examined cortactin and HS1 levels after activation of T cells. To this end, we used α‐CD3 and α‐CD28 to stimulate resting normal CD3+ cells and leukemic T cells, and found that in CD3+, Jurkat and CEM cells, cortactin was up‐regulated during activation (~1000 fold‐over resting) in a time‐dependent manner, reaching a peak after full activation at 72 hours. Cortactin upregulation was also seen after phorbol 12‐myristate 13‐acetate and ionomycin stimulation in all cells. Importantly, levels of the hematopoietic homologue of cortactin, HS1, diminished as cortactin expression increased in CD3+ cells. Taken together, our results show that cortactin is overexpressed in leukemic T cells to endow them with enhanced migratory abilities. Cortactin is upregulated in both leukemic and normal T cells upon activation suggesting that cortactin might be an important protein for effector functions of activated T cells such as cytokine secretion.
The curious case of ligamentum arteriosum: It is more than a ligament
Authors: Quaye Benedicta , Kummer Wolfgang
Published: April 2020

Introduction

The ligamentum arteriosum is generally considered to be a mere a remnant of the embryonic bypass (ductus arteriosus) from the pulmonary circulation to the aortic arch, obliterating soon after childbirth. This study set out to elucidate the morphology, innervation, and neurochemistry of this structure.

Method

The mediastinum of wildtype mice (C57BL/6J, n=5), reporter mouse strains indicating the expression of the nicotinic receptor subunit α3 (chrna3‐eGFP, n=4) and of choline acetyltransferase (chat‐eGFP, n=4), human (n=10) and pig (n=9) ligaments obtained from body donors (4 male, 6 female, aged 69–97 years) and commercial abattoir, respectively, were subjected to single‐ and double‐labeling immunofluorescence using antibodies against structural markers such as α‐smooth muscle actin (αSMA), protein gene product 9.5 (PGP 9.5), neurofilament 68 (NF68), against neuropeptides such as neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), substance P (SP), and calcitonin gene‐related peptide (CGRP), and against transmitter synthesizing enzymes such as tyrosine hydroxylase (TH; nordrenergic), choline acetyltransferase (ChAT; cholinergic), and nitric oxide synthase (NOS). Paraffin embedded human samples were stained with hematoxylin and eosin, Masson‐Goldner trichrom and elastica‐van Gieson dyes.

Results

In mice and human ligaments, remnants of a lumen and of an internal elastic membrane were observed. The lumen of the “ligament” was not completely obliterated, even though it was not patent from one side to the other. Contrary to a canonical ligament, the ligamentum arteriosum is mainly made up of collagen and abundant αSMA‐positive cells in all three species. Some human samples contained a fibrous core, sometimes calcified. Blood vessels and nerve bundles were abundant in the adventitia. In mice, the point of attachment of the ligament to both the aortic arch and pulmonary trunk received a noticeable amount of chrna3‐eGFP+ but no cholinergic innervation. PGP 9.5‐, TH‐, NPY‐, CGRP‐, and SP‐immunoreactive nerve fibers were observed in this rank order of frequency. In pig, VIP+ fibers were additionally observed. In human ligaments, PGP 9.5+, Nf68+, TH+, NPY+, VIP+, CGRP+, and SP+ fibers were observed.

Conclusion

The so‐called ligamentum arteriosum retains a continuous smooth muscular wall until senescence with noticeable, presumably postganglionic sympathetic (TH+, noradrenergic) and to a lesser extent sensory C‐fiber innervation (CGRP+, SP +) long after childbirth. It is clearly not a passive structure and the dense innervation could influence impedance/compliance (reported to be the best prognostic parameter for patient survival in pulmonary hypertension (Hunter et al., Am Heart J. 155:166–74, 2008) of the vascular segments to which it is attached.
Modulation of the tongue muscle activity in response to bronchopulmonary C‐fiber activation following mid‐cervical contused in the rat
Authors: CHANG HSIAO-SEN , LEE KUN-ZE
Published: April 2020

The present study was designed to investigate the tongue muscle activity in response to bronchopulmonary C‐fiber activation following mid‐cervical spinal contusion in the rat. The esophageal pressure and electromyogram of extrinsic tongue muscles (genioglossus and hyoglossus) were monitored before and after inhalation of capsaicin (25 and 100 μg/ml) at the acute, subchronic and chronic injured stages after mid‐cervical contusion. The results demonstrated that pre‐inspiratory and inspiratory burst amplitude of tongue muscle were generally enhanced at the acute and subchronic injured stages in mid‐cervical contused animals vs. uninjured animals. Inhalation of capsaicin usually caused a rapid shallow breathing pattern associated with a reduction in pre‐inspiratory and inspiratory activity of the tongue muscle in uninjured animals at the acute injured stage; however, this altered breathing pattern and decreased tongue muscle activity were blunted in contused animals. During the chronic injured stage, the expiratory duration was prolonged to a similar degree in both uninjured and contused animals after inhalation of capsaicin. Notably, inhibition of genioglossus muscle activity during capsaicin inhalation is more severed in chronically contused animals. These results demonstrated that cervical spinal cord injury is associated with alteration of supraspinal respiratory activity, which may modulate upper airway motor outputs and bronchopulmonary C‐fiber mediated respiratory reflexes. The compensatory increase in the tongue muscle activity at the acute injured stage may be benefit to the maintenance of the upper airway patency; however, the susceptible tongue muscle activity during bronchopulmonary C‐fiber activation may increase the risk of airway obstruction during the chronic injured stage.

Support or Funding Information

MOST (105‐2628‐B‐110‐002‐MY3 & 108‐2636‐B‐110‐001), Higher Education Sprout Project (07C030111), NSYSU‐KMU Joint Research Project (107‐I001)
The Effects of C‐Phycocyanin and Lycium barbarum Polysaccharides on Aspirin‐Induced Apoptosis in Rat Gastric Mucosal Cells
Authors: Chao Jane C-J , Liu Yu-Chen , Hsu Chien-Yeh , Chen Yu-Shan
Published: April 2020

Non‐steroidal anti‐inflammatory drugs (NSAIDs) cause gastrointestinal injury through both topical and systemic effects, which may generate reactive oxygen species and lead to apoptosis. Aspirin, a NSAID, is widely used for cardio‐protection and causes gastrotoxicity in long‐term administration. C‐phycocyanin (CPC) is a biliprotein pigment of the blue‐green alga Spirulina platensis, and possesses antioxidant, anti‐inflammatory, and wound‐healing properties. Lycium barbarum polysaccharides (LBP) extracted from wolfberry act as an antioxidant and immunomodulator. This study investigated the effects of CPC and LBP on aspirin‐induced apoptosis in rat gastric mucosal RGM‐1 cells. Rat RGM‐1 cells were pretreated with different concentrations of CPC and/or LBP (100, 250, and 500 μg/mL) for 24 hours, and gastric damage was induced by 21 mM aspirin for 3 hours. Apoptotic protein expression was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting. The results showed that aspirin significantly increased c‐Jun N‐terminal kinase (JNK), p38 phosphorylation, and Bax expression, while LBP decreased JNK activation and Bax expression compared with aspirin treatment alone. However, CPC had no effects on these apoptotic markers. In conclusion, LBP may decrease aspirin‐induced apoptosis in rat gastric mucosal RGM‐1 cells by suppressing the activation of JNK signaling pathway.

Support or Funding Information

The study was supported by the Ministry of Science and Technology, Taiwan (grant no. MOST105‐2320‐B‐038‐036‐MY3).
Cardiovagal Baroreflex Sensitivity is Reduced in Middle‐Age and Older Men with Low Testosterone
Authors: Babcock Matthew C. , Cornwell William K. , Witten Teresa L. , Brubaker Ashley , Hildreth Kerry L. , Moreau Kerrie L.
Published: April 2020

BACKGROUND

Male aging is associated with a general decline in serum testosterone (T) concentrations. Endogenous low T is associated with increased cardiovascular disease risk. Following orchiectomy or androgen suppression, rats have reduced cardiovagal baroreflex sensitivity (BRS), a key antecedent to the development of hypertension and other cardiovascular diseases. BRS is determined by mechanical (i.e. arterial stiffness) and neural factors, and heart rate variability (HRV) may provide insight into the contribution of these mechanical and neural factors. However, the effects of low T on BRS in middle‐age and older men is unknown.

PURPOSE

Determine whether BRS and HRV are reduced in middle‐age and older men with low T.

METHODS

Eight healthy older men between the ages of 50–75 years (BMI: 28.7±6.3 kg/m2, systolic blood pressure: 130±9 mmHg, diastolic blood pressure: 78±7 mmHg; mean±SD) with either low (<300 ng/dL; n=4) or normal (≥400 ng/dL; n=4) T underwent simultaneous recordings of blood pressure (finger photoplethysmography) and R‐R interval (electrocardiogram) during supine rest. Spontaneous cardiovagal BRS was assessed in sequences of ≥4 cardiac cycles in which R‐R interval and systolic blood pressure changed in the same direction. Time (standard deviation of R‐R intervals of normal sinus beats; SDNN, and root‐mean‐square of successive differences in R‐R interval differences; RMSSD) and
frequency (low frequency and high frequency power) domain indices of HRV were determined during six minutes of paced breathing at 0.2 Hz. Serum T was measured in the morning under fasting conditions.

RESULTS

T concentrations were significantly lower in the low T group compared to the normal T (269.3±40.5 vs. 561.0±109.8 ng/dL; p=0.002). Age (56±3 vs. 57±5 years; p=0.836) and mean arterial pressure (96±9 vs. 98±8 mmHg; p=0.794) were similar between groups. Cardiovagal BRS was significantly lower in the low T compared to normal T group (
8.2±3.1 vs. 16.2±4.2 ms/mmHg; p=0.021). Cardiovagal BRS was strongly correlated with T concentrations when considered as a continuous variable (R=0.71, p=0.049), but cardiovagal BRS was not correlated to age (R=0.06, p=0.891). No indices of HRV were significantly different between groups (p≥0.20 for all).

CONCLUSIONS

These preliminary data indicate that spontaneous cardiovagal BRS is reduced in men with low T. The absence of differences in HRV suggest that differences in BRS may be influenced by
mechanical (i.e. arterial stiffness), rather than neural mechanisms.

Support or Funding Information

Supported by NIH Grants R01AG049762 and T32AG000279‐16A1
Increasing Academic Rigor in a General Physiology Course and its Effect on Student Mastery of Course Objectives
Authors: Bunker Aaron
Published: April 2020

Thoughtfully designed and well‐written exam questions can serve as a place to collect useful data on student mastery of course objectives. Low academic rigor/challenge has been a recurring issue at a small private (~1200 students), Midwestern, liberal arts college as indicated by National Survey of Student Engagement institutional data and course teaching evaluation data for several years. Data presented here was collected over 4 years from exam questions designed to assess student mastery of course objectives following an increase in course academic rigor starting at year 3 of the 4 years of data collection. A scoring rubric was used assess student mastery of course objectives with 4‐basic levels: Advanced, Proficient, Partially Proficient, and Not Proficient. Following an increase in course academic rigor from year 2 to year 3 the percent of students performing at the Advanced level decreased significantly (p<0.05) for Exam2 (−11%) and Exam3 (−19%) course objective assessment questions. Mean exam scores also decreased significantly (p<0.05) from year 2 to year 3 for Exam2 (−11%) and Exam3 (−14%). Whole class discussions with students during year 3 revealed that the course objective assessment questions for Exam2 and Exam3 were not clearly written to mirror the course changes in academic rigor. Moreover, additional academic support for the increase in academic rigor was not provided for the students. Prior to year 4, exam questions were revised to better reflect changes in course academic rigor, more forms of academic support were added, and two new formative assessment strategies were created. From year 3 to 4 the percent of students performing at the Advanced level increased significantly (p<0.05) for Exam2 (+23%) and Exam3 (+42%) course objective assessment questions. Mean scores also increased significantly for Exam2 (+10%) and Exam3 (+11%) (p<0.05). This data highlights the important role that exam questions play as indicators in changes of student mastery of course objectives following increases in course academic rigor. However, instructors must provide additional academic support for the students that parallels any alterations made in course academic rigor.
Kangxian‐Yixin Granule alleviates myocardial fibrosis in rats with dilated cardiomyopathy through RhoA/ROCK1 pathway
Authors: Wang Zhentao , Wang Hailiang , WU HONG , Han Lihua
Published: April 2020

Objective

Kangxian‐Yixin granule (KXYX) is an effective traditional Chinese medicine prescription for the clinical treatment of dilated cardiomyopathy (DCM). The purpose of this study was to study the role of RhoA/ROCK1 signaling pathway in DCM myocardial fibrosis and the intervention mechanism of KXYX.

Methods

DCM rats were randomly divided into model group (n = 8), KXYX group (n = 8), captopril group (n = 8) and control group (n = 8). After 4 weeks of drug intervention, the cardiac function of rats was detected by echocardiography, and the degree of myocardial fibrosis was observed by Masson staining. The expressions of RhoA, ROCK1, p‐MLC and fibrosis related factors CTGF and Col‐1 in RhoA/ROCK1 signaling pathway were detected by PCR and Western blot. At the same time, the myocardial fibroblast model was induced by angiotensin II, and treated with KXYX and inhibitor fasudil. RhoA/ROCK1 signaling pathway and the expression of fibrosis factors CTGF and Col‐1 were detected by Western blot and PCR.

Results

Compared with the control group, the expression of RhoA/ROCK1 signaling pathway in the model group was up‐regulated, the expression of p‐MLC was overactivated, and the expression of fibrosis‐related factors CTGF and Col‐1 was increased in the model group (P < 0. 05). KXYX could inhibit myocardial fibrosis, inhibit the transformation of fibroblasts into myofibroblasts, and down‐regulate the expression of RhoA/ROCK1 signaling pathway and fibrosis factors CTGF and Col‐1 in DCM rats.

Conclusion

RhoA/ROCK1 signaling pathway plays an important role in DCM myocardial fibrosis, and KXYX can inhibit the up‐regulation of RhoA/ROCK1 signaling pathway, thus inhibiting the development of myocardial fibrosis in DCM rats. The correspondence to Lihua Han;
hlhaa@sina.com.

Support or Funding Information

This work was financially supported by grants from the National Natural Science Foundation of China (Grant No. 81573920)
G Protein Coupled Receptor 75 and Its Role in Pulmonary Hypertension
Authors: D’Addario Catherine , Kitagawa Atsushi , Zhang Frank , Zeldin Darryl , Gruzdev Artiom , Garcia Victor , Schwartzman Michal , Gupte Sachin
Published: April 2020

Pulmonary Hypertension (PH) is a multifactorial disease with poor prognosis and inadequate treatment. Recent studies have shown that the orphan G Protein Coupled Receptor 75 (GPR75) is upregulated in the pulmonary vessels of PH patients, especially in females. Therefore, we hypothesized that GPR75 knock out (GPR75KO) mice will be protected from developing PH. We placed 3‐month‐old female GPR75KO (n=6) and control C57BL/C mice (n=10) in a hypoxic chamber with 10% Oxygen or at atmospheric oxygen for 5 weeks. At the end of 5 weeks, we performed echocardiograms and right heart catheterizations. GPR75KO mice were protected from developing PH (Right Ventricle Systolic Pressure (RVSP) of 26mmHg and Right Ventricle Diastolic Pressure (RVDP) of 2 mmHg), while C57BL/C mice developed PH under hypoxia (RSVP and RDVP of 56mmHg and 12mmHg, respectively). Wire myography was performed on isolated intra‐lobar pulmonary arteries from GPR75KO mice (n=3) and WT mice (n=31). Hypoxic pulmonary vasoconstriction (HPV) was abolished in GPR75KO as compared to WT. HPV response in WT was blocked (P<0.05) by antibody against RANTES (CCL5), a putative GPR75 ligand. These results suggest GPR75 plays a major role in the development of HPV and PH.

Support or Funding Information

This study was supported by NIH grant 1RO1HL139793 (MLS).
Effects of Arginine Vasopressin and Oxytocin on Neurite Outgrowth and Cellular Signaling in SH‐SY5Y Human Neuroblastoma Cell Line
Authors: Pierce Marsha
Published: April 2020

Introduction

In the central nervous system, arginine vasopressin (AVP) and oxytocin (OT) stimulate neural networks that regulate social behaviors, including social attachment, aggression, and complex social cognition. AVP and OT are neuropeptides that are expressed in the social behavioral network and bind to G‐protein coupled receptors, stimulating cellular signaling patterns that ultimately affect social behaviors at the organismal level. Consensus mammalian AVP and OT sequences are highly conserved. In addition to sequence homology between AVP and OT, there is ~85% structural homology between the OT receptor (OTR) and vasopressin 1a receptor (V1aR) resulting in significant cross‐reactivity between the ligands and receptors. Perturbations in OT and/or OT receptor expression results in social behavioral deficits, and is associated with a number of psychopathologies including autism spectrum disorder, schizophrenia, anxiety, and depression.

Methods

The human neuroblastoma derived SH‐SY5Y cell line demonstrates morphological characteristics of neurons including neurites and formation of functional synapses, endogenously expresses OTR, and is often used as an in vitro model for human neurons. Functional assays were performed using Fluo8‐AM to measure ligand‐induced Ca2+ mobilization and FLIPR Membrane Potential (FMP) assays were performed to assess ligand‐induced hyperpolarization. To assess effects on neurite outgrowth, cells were treated with logarithmic doses of AVP and OT for 24 hours, fixed and stained with phalloidin and DAPI.

Results

In SH‐SY5Y cells, OT was more potent than AVP at both ligand‐induced Ca2+ mobilization and membrane hyperpolarization, which is consistent with OT being more potent and efficacious at the human OTR. Specificity of the signaling at the OTR were confirmed using the vasopressin 1a receptor antagonist SR49059 and the oxytocin receptor antagonist L‐371,257. These data are consistent with previous reports that vasopressin 1a receptors are absent in SH‐SY5Y cells. Additionally, OT demonstrated a dose‐dependent increase in neurite outgrowth, which is inhibited by L‐371,257.

Conclusions

Together, these data demonstrate that OTR mediated signaling pathways lead to neurite outgrowth in SH‐SY5Y cells. Additional studies are needed to assess the specific molecular mechanisms that contribute to neurite outgrowth, and whether functional synapses are formed. Integrative studies of behavior, genetics and ligand‐receptor interaction are crucial for translating signaling activation at the cellular level to effects of AVP and OT ligands on social behavior. Knowledge of how OT alters neuronal structure and function has the potential to both identify mechanisms that produce social dysfunction and to inform the development of therapeutic agents.
PPARα‐targeted mitochondrial bioenergetics mediate repair of intestinal barriers at the host‐microbe intersection during SIV infection
Authors: Crakes Katti R. , Rocha Clarissa Santos , Grishina Irina , Hirao Lauren A. , Napoli Eleonora , Gaulke Christopher A. , Fenton Anne , Datta Sandipan , Arredondo Juan , Marco Maria L. , Sankaran-Walters Sumathi , Cortopassi Gino , Giulivi Cecilia , Dandekar Satya
Published: April 2020

Chronic gut inflammatory diseases are associated with disruption of intestinal epithelial barriers and impaired mucosal immunity. Human immunodeficiency virus‐1 (HIV) causes depletion of mucosal CD4+ T cells early in infection and disruption of gut epithelium resulting in chronic inflammation and immunodeficiency. Although anti‐retroviral therapy (ART) is effective in suppressing viral replication, it is incapable of restoring the ‘leaky gut’ which poses an impediment for HIV cure efforts. Strategies are needed for rapid repair of the epithelium to protect intestinal microenvironments and immunity in inflamed gut. Using an in vivo non‐human primate intestinal loop model of HIV/AIDS, we identified the pathogenic mechanism underlying sustained disruption of gut epithelium and explored rapid repair of gut epithelium at the intersection of microbial metabolism. Molecular, immunological, and metabolomic analyses revealed marked loss of peroxisomal proliferator‐activated receptor alpha (PPARα) signaling, predominant impairment of mitochondrial function and epithelial disruption both in vivo and in vitro. To elucidate pathways regulating intestinal epithelial integrity, we introduced probiotic Lactobacillus plantarum into Simian immunodeficiency virus (SIV)‐inflamed intestinal lumen. Rapid recovery of the epithelium occurred within 5 hours of L. plantarum administration and independent of mucosal CD4+ T cell recovery and in the absence of ART. This intestinal barrier repair was driven by L. plantarum‐induced PPARα activation and restoration of mitochondrial structure and fatty acid beta‐oxidation. Our findings elucidate the critical role of PPARα 1) at the intersection between microbial metabolism and epithelial repair in virally‐inflamed gut and 2) as a potential mitochondrial target for restoring gut barriers in other infectious or gut inflammatory diseases. These data highlight an opportunity to investigate the evolutionary synergism between host mitochondria and gut microbes to restore mucosal health in lieu of full immune recovery.

Support or Funding Information

This study was supported by the National Institutes of Health grant R01 AI123105. KRC received a pre‐doctoral training grant F30 AI150462.
Identification of the amino acids associated with the novel ribonuclease activity of cusativin via protein engineering and LC‐MS
Authors: Thakur Priti , Atway Jowad , Limbach Patrick A. , Addepalli Balasubrahmanyam
Published: April 2020

Ribonucleases (RNases) are widely used in detecting the location of chemical modification in RNA, an approach commonly referred to as, “RNA modification mapping”
[1]. The commonly employed RNases include RNase T1 (guanosine‐specific), U2 (purine‐specific) and A (pyrimidine specific). Recently, uridine‐specific (MC1)
[2], cytidine‐specific (cusativin)
[3] and a non‐specific RNase U2
[4] were identified as potential tools for RNA modification mapping. Of these, RNase cusativin exhibits novel cleavage behavior where it does not cleave the bond between two consecutive cytidine nucleotides (CpC)
[3]. This unique feature of cusativin has encouraged us to probe the nucleobase recognition characteristics of the enzyme binding site, which is composed of B1 and B2 sites. We employed amino acid substitutions (through protein engineering), and liquid chromatography coupled with mass spectrometry (LC‐MS) to analyze the contributions of specific amino acids in the enzyme binding site. The mutant proteins generated by specific amino acid substitutions were investigated for alteration of secondary structure, kinetic changes in enzyme activity, and substrate specificity. While single amino acid substitutions revealed minimal effects on the secondary structure, multiple substitutions resulted in significant changes as revealed by circular dichroism studies. However, the mutant proteins, irrespective of the number of substitutions, exhibited remarkable changes in their kinetic behavior with the tested RNA substrate. The LC‐MS analysis of enzymatic digestion products of RNA indicated altered nucleobase specificity depending on the location of amino acid substitutions. While mutation at one location (V99L) behaved more or less like wild type protein, mutation at different position (N97S) but within same B2 binding site pocket revealed generation of digestion products indicative of CpC bond cleavage. However, the N97S mutant also exhibited digestion products that suggest cleavage of RNA at 3′‐end of guanosine. Another mutant protein with amino acid substitutions in both B1 (R69T) and B2 (V99L and T100R) sites also exhibited significant cleavage of CpC bond. But this behavior is also accompanied with more or less equal affinity towards C and G for RNA cleavage. The implications of this preliminary experimental data on the role of specific amino acids in nucleobase recognition of RNA substrate will be discussed.

Support or Funding Information

Financial support of this work was provided by the National Institutes of Health (GM58843), and the University of Cincinnati.
Longitudinal In Vivo Imaging of Atherosclerotic Disease Development in The apoE Deficient Zucker Rat
Authors: Alvidrez Roberto Ivan Mota , Buglak Nicholas , Anderson Tamara , Norenberg Jeffrey , Bahnson Edward
Published: April 2020

One obstacle hindering the development of therapies to treat diabetic vasculopathies, is access to an affordable and appropriate small animal model that recapitulates human pathology. There are currently no well‐established small animal models to study diabetes and atherosclerosis combined. Longitudinal in vivo imaging of inflammation in atherosclerosis is a promising tool to evaluate progression of cardiovascular complications. However, vascular size of current mouse models of atherosclerosis does not allow for detailed molecular imaging. Radiolabeled DANBIRT has been used as an in vivo molecular imaging tool on environmental exposure, aging, and atherosclerosis. Our group has modified the lipid metabolism in Lean Zucker (LZ: Lepr+/?) rats to promote dyslipidemia and development of atherosclerosis by knocking out the apolipoprotein E (apoE) gene using CRISPR/Cas9. We hypothesize that apoE knockout, LZ rats (apoE−/−, Lepr+/?) will develop atherosclerosis with dietary exposure to high fat, high cholesterol (HFHC) diet. [111In]‐DANBIRT will be able to localize inflammation in vivo and allow longitudinal monitoring of vascular atherosclerotic disease using SPECT/CT imaging.

Methods

apoE−/−;LZ adult rats were imaged for 1hr post injection of ~900 μCi of [111In]‐DANBIRT for SPECT/CT imaging using a NanoSPECT/CT®. Rats were placed on either HFHC or standard rodent chow for 10 weeks starting at 12 weeks of age (baseline). apoE+/+;LZ under normal and HFHC diet were used as controls. Regions of Interests (ROI) are identified and determined for muscle (background tissue), carotid arteries, aortic arch, and descending aorta. Activity is decay corrected and compared between and among experimental groups. Serum chemistry and histologic analysis of en face aortas and aortic root were performed. Data is represented as mean ± SEM. Two‐way ANOVA with Tukey’s correction was performed.

Results

All apoE−/− rats developed severe hypercholesterolemia particularly under a HFHC diet. After 10 weeks of HFHC diet apoE−/−, LZ rats have higher [111In]‐DANBIRT uptake in the descending aorta uniquely compared to baseline (p<0.05) and to normal diet fed rats (ns). Histologic analysis showed apoE−/−, LZ have increased significant atherosclerosis on the aortas and the aortic sinus, which correlates to our imaging results. apoE+/+, LZ rats did not develop atherosclerosis disease as expected.

Conclusion

To our knowledge, we have the first polygenic rat model able to develop atherosclerosis translationally under HFHC. [111In]‐DANBIRT longitudinally evidenced atherosclerotic disease in the abdominal aorta in vivo correlated to ex vivo histologic analysis.

Support or Funding Information

Supported by the NORC PPF (P30 DK056350) to EMB and the CRTG (Burroughs Wellcome Fund) to RIM.
Establishing Normative Embryonic and Early Postnatal Cranial Bone Volume and Density Values for C57BL/6J Laboratory Mice
Authors: Lesciotto Kate M. , Tomlinson Lauren , Richtsmeier Joan
Published: April 2020

We provide normative data for cranial bone volume and tissue mineral density (TMD) for embryonic and early postnatal C57BL/6J mice that can be used as control data for researchers using experimental mouse models. C57BL/6J mice were selected based on their common usage in experimental research. Our sample includes 3 males and 3 females at four ages: embryonic day (E) 15.5, E17.5, postnatal day (P) 0, and P7. MicroCT images were acquired by the Center for Quantitative Imaging at the Pennsylvania State University using the General Electric v|tom|x L300 nano/microCT system. Prior to data collection, all postcranial bones were electronically removed from the scan using Avizo 9.4, and then background noise was removed from each scan using a 3D median filter. Isosurfaces were reconstructed to represent cranial bone based on a hydroxyapatite phantom that was scanned with each specimen. To maintain consistency between specimens and across age groups, we used 85 mg/cm3 as the minimum threshold to create isosurfaces and segment bone for this study. Twenty cranial bones were individually segmented for each specimen, and volume and TMD were estimated in Avizo. After correcting for multiple testing, no significant differences were found in bone volume or TMD between males and females for any of the examined cranial bones at any age, and data were pooled for analyses. As expected, volume of ossified bone increased with age from E15.5 to P7. The number of ossification centers present in a single individual is highly variable at E15.5 (ranging from 2 – 10), with all bones present by P7. Most of the measured bones consistently increased in TMD with age, with several exceptions. The palatine, vomer, and zygomatic bones each decreased in TMD from P0 to P7. The TMD of the maxilla also decreased slightly from E17.5 to P0, but then increased from P0 to P7. No significant patterns of development related to originating cell population (mesoderm v. neural crest) or mode of ossification (endochondral v. intramembranous) were apparent. These data can serve as a reference standard for future studies that examine craniofacial growth and disease models using mice bred on a C57BL/6J background.

Support or Funding Information

National Science Foundation Grant BCS‐1731909, NIDCR R01DE027677 and R01 DE022988, and the Pennsylvania State University Energy and Environmental Sustainability Laboratories Green Seed Grant
Advancing Anatomy Education Competencies—A Critical Interpretive Synthesis from Five Key Papers
Authors: Pacey Lindsay , Kulesza Randy , Lomiguen Christine , Terrell Mark
Published: April 2020

Introduction

Since the early 1900s, time devoted to studying anatomy in medical schools has substantially decreased. Although the number of students enrolled in health professions education programs is increasing, a shortage of formally trained gross anatomists persists. Consequently, anatomy education and learning must be efficient and effective. Programs training the next generation of anatomy educators need to be sure that graduates obtain particular knowledge and skill sets. Competency‐based education (CBE) may provide one such approach. Extensive research on competency‐based education (CBE) exists in medical education that provides developmental milestones and entrustable professional activities (EPAs). A similar process involving CBE and EPAs would benefit anatomy education program development graduates of such programs, students of teachers from such programs, and ultimately patients.

Aim

The purpose of this research is to identify five key papers in CBE that are most influential for providing a foundation for developing competencies in training the next generation of anatomists as educational specialists.

Methods

Critical interpretive synthesis (CIS) will be utilized to identify, summarize, and integrate five key papers into a primer for emerging anatomy educators on competencies highlighted in literature and through professional societies. A comprehensive search of PubMed/MEDLINE, CINAHL, and ERIC databases published in English from 1990–2020 utilizing terms including competency‐based education, faculty development, and anatomy education. Additional searches through relevant journal article’s bibliographies will also be conducted. Papers’ relevance will be given a numerical value based on relevance and usability. A student learning community will be established to generate a cumulative list of the 20 most highyielding papers regarding anatomy education and CBE. These numerical results will go through several iterative processes, inspired by the Delphi methodology, to generate a top five ranked articles that will then be analyzed and integrated into a single CIS paper for publication.

Results

Main concepts from the five key CBE papers will be amalgamated into one primer through CIS to lay the groundwork for developing competencies for future anatomists as education specialists.

Discussion and Conclusion

Results of this study will aid in helping anatomy educators attain competency and aid in developing and designing curricula to train the next generation of anatomy educators.
Lysophosphatidic acid reduces microregional oxygen supply/consumption balance after cerebral ischemia‐reperfusion
Authors: Weiss Harvey R. , Mellender Scott J. , Kiss Geza , Liu Xia , Chi Oak
Published: April 2020

Lysophosphatidic acid (LPA) is a small phospholipid signaling molecule, whose levels are increased after cerebral ischemia. We hypothesized that exogenous LPA would increase the size of infarct and reduce microregional O2 supply/consumption balance after cerebral ischemia‐reperfusion. This was tested in isoflurane‐anesthetized rats with middle cerebral artery blockade for 1 hr and reperfusion for 2 hr with or without LPA (1 mg/kg, at 30, 60 and 90 min after reperfusion). Regional cerebral blood flow was determined using a C14‐iodoantipyrine autoradiographic technique. Regional small vessel (20–60 μm diameter) arterial and venous oxygen saturations were determined microspectrophotometrically. There were no significant hemodynamic or arterial blood gas differences between groups. The control ischemic‐reperfused cortex had a similar O2 consumption to the contralateral cortex. However, microregional O2 supply/consumption balance was significantly reduced in the ischemic‐reperfused cortex with many areas of low O2 saturation (43 of 80 veins with O2 saturation below 50%). LPA did not significantly alter cerebral blood flow, but it did significantly increase O2 consumption of the ischemic‐reperfused region. It also significantly increased the number of small veins with low O2 saturations in the reperfused region (76 of 80 veins with O2 saturation below 50%). This was associated with a significantly increased cortical infarct size after LPA administration (11.4 ± 0.5% control vs 16.4 ± 0.6% LPA). This suggests that LPA reduces cell survival and that it is associated with an increase in the number of small microregions with reduced local oxygen balance after cerebral ischemia‐reperfusion.