bluuebunny/crossref_metadata_2025 huggingface dataset:
Endothelial‐dependent activation of smooth muscle PDGF Receptors enhances PASMC proliferation in IPAH
Authors: Wu Kang , Tang Haiyang , Wu Xiaomin , Lin Ruizhu , Wang Ziyi , Zhang Qian , Ayon Ramon J. , Wang Jian , Makino Ayako , Yuan Jason X.‐J.
Published: June 2021

Platelet‐derived growth factor (PDGF), is one of the major growth factors found in mammalian serum, which plays an important role in cell growth. It has been previously demonstrated that abnormal activation of the PDGF signaling pathway can contribute to aberrant pulmonary vascular remodeling and pulmonary hypertension via active PDGF receptors. The overall goal of this study was to examine the expression of the PDGFR isoforms in both pulmonary artery smooth muscle cells (PASMC) and pulmonary artery endothelial cells (PAEC) and to identify whether PDGF produced in PASMC or PAEC promotes PASMC cell proliferation. We observed that the protein and mRNA expression level of both PDGFRα and PDGFRβ were upregulated in IPAH patients in comparison to normal PASMC. However, expression of PDGFRα and PDGFRβ was not detected in PAEC. PDGF treatment enhanced the activity of PDGFRα and PDGFRβ, as determined by phosphorylation of PDGFRα and PDGFRβ in IPAH PASMC compared to control. Furthermore, the PDGF‐induced rise in cytosolic free Ca2+ was also increased in IPAH PASMC in comparison to normal PASMC. Treatment with normal PAEC cultured conditioned medium enhanced PDGFRα activation in IPAH PASMC than normal PASMC, while normal PASMC cultured conditioned medium had no effect on p‐PDGFRα and p‐PDGFRβ. We further found that the expression level of PDGFA and PDGFB were increased in IPAH PAEC compare to normal PAEC. Additionally, the expression level of PDGFA and PDGFB in PAEC were increased in normoxic condition compare to hypoxia. Normal PAEC cultured conditioned medium promotes cell proliferation in PASMC; proliferation was markedly higher in IPAH PAEC cultured conditioned medium, but not in PASMC cultured conditioned medium. The data showed that the expression and activity of PDGF receptor were increased in IPAH PASMC. Overall these results suggest that IPAH PAEC secret high‐levels of PDGF to the PASMC, which can affect active PDGF receptor, promoting PASMC proliferation which can lead to pulmonary hypertension.

Support or Funding Information

This study was supported in part by the grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL135807 and HL125208), the National Natural Science Foundation of China (81630004, 81470246, 81220108001 and 81520108001), the Guangzhou Department of Education Yangcheng Scholarship (12A001S), the Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme of China, China Scholarship Council (201608440324) and the Actelion ENTELLIGENCE Young Investigator Award.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interaction of Chitosan Derivatives with Organic Cation Transporter 1 and 2
Authors: Soodvilai Sirima , Sajomsang Warayuth , Chatsudthipong Varanuj , Soodvilai Sunhapas , Opanasopit Praneet
Published: June 2021

The present study was to investigate the interactions of chitosan derivatives including quaternized chitosan (QC), N‐benzyl‐N,O‐succinyl chitosan (BSCS), N‐naphthyl‐N,O‐succinyl chitosan (NSCS) and N‐octyl‐N,O‐succinyl chitosan (OSCS), with organic cation transporter 1 (OCT1) and OCT2. The transport function of OCT1 and OCT2 was monitored by measurement accumulation of radiolabeled methyl‐4‐phenylpyridinium acetate (3H‐MPP+) in OCT1 and OCT2 heterologous expressing cell and endogenous expressing cells (HepG2 cells and human renal proximal tubular; RPTEC/TERT1 cells). The results revealed that of BSCS, NSCS and OSCS at concentration of 1,000 μg/ml significantly reduced 3H‐MPP+ cellular accumulation compared with control whereas QC showed no effect on OCT1 or OCT2‐mediated 3H‐MPP+ accumulation. Among the chitosan derivatives, OSCS showed the highest inhibitory potency (for OCT1; IC50 = 395±1.10 μg/ml, for OCT2; IC50 = 1,241±1.22 μg/ml). BSCS, NSCS and OSCS significantly reduced maximal transport (Jmax) of OCT1 whereas they increased Kt of OCT2 indicating these compounds inhibited OCT1 and OCT2 transport function by non‐competitive and competitive binding mechanism, respectively. In addition, OSCS significantly reduced 3H‐MPP+ accumulation in HepG2 cells and RPTEC/TERT1 cells with the inhibitory potency of 1,208±1.09 μg/ml and 1,529±1.11 μg/ml, respectively. These results suggested that OSCS may alter OCT1‐ and OCT2‐mediated cationic drug clearances.

Support or Funding Information

This research was supported by the Commission of Higher Education (Thailand), the Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. Program (Grant No.PHD/0139/2557), the TRF through the Natural product‐based drug discovery and development (Grant No. IRN58W0004), Mahidol University (Thailand). The authors would like to thank Prof. Stephen H. Wright for providing rbOCTs‐CHO‐K1 cells.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long‐term high‐intensity sound stimulation inhibits Ih in CA1 pyramidal neurons.
Authors: de Deus Júnia Lara , Cunha Alexandra Olímpio Siqueira , Ceballos César Celis , Leão Ricardo Xavier
Published: June 2021

Afferent neurotransmission on hippocampal pyramidal cells can change their intrinsic membrane properties and affect many ion currents. One of the most plastic neuronal currents is the hyperpolarization activated cationic current (Ih), which changes in CA1 pyramidal cells in responseto many types of physiological and pathological processes. Several evidences demonstrated that hippocampal CA1 pyramidal neurons are affected by auditory stimulation. Recently we demonstrated that long‐term potentiation (LTP) is depressed by high‐intensity sound stimulation. Here we investigated if a long‐term high‐intensity sound stimulation could affect intrinsic membrane properties of CA1 pyramidal neurons. Our results showed that Ih is depressed by longterm (2 minutes per day, for 10 days) high intensity sound (110 dB) exposure. This resulted in a decreased resting membrane potential, increased membrane input resistance and time constant, and decreased action potential threshold. In contrast, CA1 pyramidal neurons from sound‐exposed animals fired more action potentials than neurons from control animals, but this effect was not caused by a decreased Ih. Interestingly, a single episode (1 minute) of 110 dB sound stimulation which also inhibits hippocampal LTP did not affect Ih and firing in pyramidal neurons, suggesting that these are long‐term responses to high intensity sound exposure. Our results show that prolonged exposure to high‐intensity sound affects intrinsic membrane properties of hippocampal pyramidal neurons mainly by decreasing the amplitude of Ih.

Support or Funding Information

FAPESP, CNPQ and Capes

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Electroacupuncture Modulates Reflex Elevation in Blood Pressure through Adenosine Receptor A2A, but not A1 in Rostral Ventrolateral Medulla of Rats
Authors: Guo Zhi‐Ling , Malik Shaista
Published: June 2021

Acupuncture potentially offers a non‐pharmacological approach to reduce high blood pressure (BP). Our previous studies have shown that electroacupuncture (EA) attenuates reflex elevation in BP induced by gastric distension (GD) through modulation by several transmitters (e.g., opioids and GABA) in the rostral ventrolateral medulla (rVLM). Although adenosine is released during neuronal activation in the rVLM, its role in acupuncture‐cardiovascular regulation is unknown. Adenosine primarily modulates neuronal function through P1 (specifically, A1, A2, and A3) receptors. Since selective activation of A1 and A2A rVLM receptors respectively induces pressor and depressor responses, we hypothesized that adenosine in the rVLM contributes to EA modulation of sympathoexcitatory reflexes through an A2A but not an A1 adenosine receptor mechanism. Repeated GD every 10 min was performed in Sprague‐Dawley male rats under ketamine and α‐chloralose anesthesia. EA (2 Hz, 0.5 ms, 1–4 mA) was applied at the P5–P6 acupoints, overlying the median nerve, for 30 min after establishing two consistent reflex responses to GDs. We found that repetitive GDs evoked consistent sympathoexcitatory pressor responses. EA (n=5) but not sham‐EA (n=5) at P5–6 (insertion of acupuncture needles into acupoints without electrical stimulation) significantly (P<0.05) attenuated GD‐induced elevations in BPs. EA modulation of sympathoexcitatory cardiovascular reflexes was reversed significantly after rVLM microinjection of SCH 58261 (1 mM in 50 nl; A2A receptor antagonist; n=8; P<0.05), but not by the vehicle (5% dimethylsulfoxide; n=6). Microinjection of DPCPX (3 mM in 50 nl; A1 receptor antagonist; n=4) into the rVLM did not influence EA inhibition of GD‐induced pressor responses. GD‐evoked sympathoexcitatory cardiovascular reflexes, in the absence of EA, were unaltered by administration of SCH 58261 into the rVLM. Neurons labeled with adenosine A2A receptors were co‐localized with neurons stained with tyrosine hydroxylase (i.e., catecholaminergic neurons) in the rVLM. These data suggest that EA modulates sympathoexcitatory cardiovascular responses through an A2A receptor mechanism in the rVLM.

Support or Funding Information

NCCIH grant, AT009347.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of baroreflex during endotoxemic shock
Authors: Amorim Mateus Ramos , Borges Gabriela Silva , Mota Clarissa Maria Dias , de Siqueira Branco Luiz Guilherme
Published: June 2021

Septic shock is a prevalent and life‐threatening condition which is often associated with major hypotension, resulting in multiple organs failure. The infusion of high doses of endotoxin lipopolysaccharide (LPS) is a well‐accepted experimental model of septic shock. Considering that baroreflex is a mandatory mechanism involved in the beat‐to‐beat hemodynamic regulation, we hypothesized herein that baroreflex plays a role buffering endotoxin‐induced hypothension in conscious freely moving rats. Thus, in the present study, we evaluated the cardiovascular outcomes of iv injection of endotoxin (1.5 mg.kg−1 or 2.5 mg.kg−1) or saline in male Wistar rats (280–350g, CEUA #2017.1.585.58.9). The baseline levels of mean arterial pressure and heart rate were not different among all groups. As expected, endotoxin induced hypotension (P<0.001) and tachycardia (P < 0.001). These effects were independent of dose. Interestingly, the drop of mean arterial pressure was significantly higher in rats previously submitted to baroreceptors afferents removal (4 days before recordings) than in (sham) rats with intact baroreceptor afferents (P<0.001). The present data are consistent with the notion that baroreflex is key lessening endotoxin‐induced hypothension. The neural mechanisms involved in this control deserve further investigation aiming new strategic therapeutic tools to revert this critical condition.

Support or Funding Information

Financial support: FAPESP, CAPES and CNPq

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Orcokinin Neuropeptide NLP‐14 Regulates Sleep in C. elegans
Authors: Barrett Natalie , Riazati Niknaz , Buscemi Kristen , Brown Amelia , Nelson Matthew
Published: June 2021

Caenorhabditis elegans displays two distinct sleep states: Developmentally timed sleep (DTS), which occurs during a life stage called lethargus (Raizen et al., 2008) and stress induced sleep (SIS), which occurs following exposure to environmental stress that damages cells. Both sleep states have been shown to be regulated by distinct neuropeptide signaling pathways. We have identified a new neuropeptide pathway that regulates SIS, neuropeptide‐like protein‐14 (NLP‐14). NLP‐14 shares sequence similarities (~69%) with a class of neuropeptides in arthropods called orcokinins (Nathoo et al., 2001), which are highly conserved within Ecdysozoans. In cockroaches, injection of exogenous orcokinin into the accessory medulla, a circadian pacemaker in the brain, results in a circadian phase shift (Hofer and Homberg, 2006). Interestingly, we have found that ectopic over‐expression of nlp‐14 during adulthood induces behavioral quiescence which resembles sleep. Specifically, when nlp‐14 over‐expression is induced under the control of a heat shock promoter, animals become quiescent for locomotion and show prolonged defecation cycles. Additionally, we find that nlp‐14(tm1880) reduction in function mutants display decreased SIS phenotype. Introduction of NLP‐14:GFP transgenes into this background rescues the SIS defects. The tm1880 allele eliminates 7 of the 12 encoded peptides in the NLP‐14 protein, leaving 5 peptides (NLP‐14‐2(2), NLP‐14‐4(2) and NLP‐14‐5(2)). We are currently attempting to remove the remaining peptides one at a time using the CRISPR‐cas9 system.

Support or Funding Information

John P. McNulty Scholars Program

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rare Case of an Accessory Left Vertebral Artery on the Aortic Arch
Authors: Granite Guinevere , Lashley Kerrie
Published: June 2021

Vascular variations of the head and neck are common, but often go unnoticed because they generally do not present clear clinical symptoms. Unawareness of the presence and location of such variations may result in iatrogenic complications or even fatalities. Medical physicians and surgeons should be cognizant of such deviations due to their clinical significance.

Vertebral artery variations are prevalent in both number and origin, but not frequently documented in the literature. In this case study, a White 65‐year‐old male presented with an accessory left vertebral artery originating on the aortic arch, between the left common carotid artery and the left subclavian artery. This is the most common place for variations of this type, however, this individual also presented a normal left vertebral artery branching off of the left subclavian artery. This accessory left vertebral artery courses cranially, entering the foramina transversarium at the C4‐C5 level. The left vertebral artery also has an atypical course. It enters into the foramina transversarium at a higher level than its aberrant analog. With a prevalence rate of 2.04%, this rare anatomical variant illustrates the clinical importance of extensive anatomical understanding of head and neck vascular variations.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using a Virtual Reality Laboratory to Identify Methods for Improving Neuroanatomy Learning
Authors: Shermon Suzanna , Stout Randy
Published: June 2021

Virtual reality (VR) is a 3‐dimensionsal (3D) digitally‐created environment that is widely used as a training and instructional tool (e.g. surgical preparation, space mission rehearsal, flight training, and even museum exhibits). With the use of a headset and handheld controllers, the user can explore, move around in, and interact with the virtual environment. Of importance to our research, the newest generation of consumer VR systems allows the participant to use their hands to manipulate virtual objects in a very intuitive manner.

The goal of our research is to eventually use VR to improve neuroanatomy instruction for medical students. In particular, we hypothesize that with proper application of VR technology, students could learn structures more efficiently, gain deeper understanding of how structure is related to function, and enhance the interactions between students and course leaders (instructors). To test how the design of the virtual learning process and environment affect retention, motivation, and depth of learning for complex concepts in neuroanatomy, we are constructing a virtual laboratory with the use of Blender and Unity softwares. We are adapting a 3D model of the human brain to use as the learning materials for the virtual learning laboratory (VLL) that we are developing with Unity™. Our current build of the Virtual Learning Laboratory is accessed via Oculus Rift+Touch™, in which the students will participate in gamified and active learning by manipulating virtual brain anatomical models using the Touch Controllers ™. We have assembled a 3D brain in Blender, built a prototype VLL, and are currently refining the interface in preparation for testing human participants.

VR and Augmented Reality (AR) are already being used as tools in neuroanatomy education. We expect that the virtual learning laboratory that we have developed will allow optimization of the benefits that VR can provide to the learning process. This tool for pedagogical research will be important as improved VR technology and user interfaces are being rapidly developed.

Support or Funding Information

The Academic Medicine Scholars Program at New York Institute of Technology, College of Osteopathic Medicine.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ACE Inhibitor Reaction‐Induced Acute Angioedema Leading to Macroglossia: Two Case Studie
Authors: Kesterke Matthew John , Stigall Garret , Carmichael Ryan , Bhattacharjee Ritesh , Reddy Likith V
Published: June 2021

Macroglossia, either congenital or acquired enlargement of the tongue, is a rare condition that leads to functional deficiencies in speech, mastication/deglutition, and airway patency. Acute‐acquired cases of macroglossia are potentially life threatening and have myriad etiologies. Presented here are two cases of macroglossia in adults caused by acute angioedema resulting from reaction to angiotensin‐converting enzyme (ACE) inhibitors. The mechanism of ACE inhibitor‐induced angioedema is attributed to dysfunction in the kallikrien‐kinin system, a protease pathway converting high molecular weight kininogens to kinins such as bradykinin, which can lead to inflammation of the cardiovascular system. This condition, while rare in adults, is more easily diagnosed, monitored, and treated if detected early. However, many clinicians fail to educate patients on the warning signs of acute angioedema: swelling of the tongue, submandibular and pharyngeal spaces, and/or difficulty breathing. Furthermore, patients often do not recognize the symptoms of ACE inhibitor reactions, leading to delays in diagnosis and treatment. Management of acute angioedema and subsequent macroglossia includes hospitalization and intubation for airway maintenance, partial glossectomy, and subsequent glossal rehabilitation and speech therapy. Both cases presented here led to anoxic brain injury and necessitated intensive surgical intervention, demonstrating the need for better patient‐clinician communication for positive outcomes of ACE inhibitor use. Current research on the kinin system and its role in the cardiovascular system is limited, so baseline clinician‐patient education represents the best first‐line defense against life‐threatening cases of acute angioedema.

Support or Funding Information

This research is supported in part by funding from the Texas A&M University College of Dentistry and the Baylor University Medical Center at Dallas.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Generation and partial characterization of cancer cell lines from Mexican breast cancer patients
Authors: Valencia María Lourdes , De Leon Roberto , Lüthy Isabel Alicia , Angulo Aracely , Ríos Omar , Velázquez Carlos Arturo
Published: June 2021

Numerous cell models have been approached to study cancer biology. Some of those models are primary cell cultures, as well as, cancer cell lines derived from tumors. Several breast cancer cell lines are available for scientific research. Nevertheless, those cell lines do not represent all the cancer types and subtypes, neither their origin corresponds to most of the ethnic groups of the world. The main goal of this study is to generate and partially characterize cancer cell lines from tumor tissue obtained from Mexican patients with breast cancer, which can be used as models to study cancer biology and to characterize the phenotype and genotype of breast cancer affecting Mexican population. At present, four primary cell cultures (ID010, ID012, ID013 and ID014) have been established from primary tumor tissues representative of infiltrating ductal carcinomas, such as luminal B and HER‐enriched molecular subtypes. In addition, all the primary cell cultures showed the expression of cytokeratins, which suggests the presence of epithelial cells. The development and partial characterization of new breast cancer cell lines with different molecular subtypes, will allow us a better comprehension about the variations in morbidity, mortality and susceptibility to anti‐cancer drugs among different ethnicities.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanism of Fgf‐Mediated Lhx2 Upregulation
Authors: Shah Neera , Watson Billy , Pira Charmaine , Oberg Kerby
Published: June 2021

Introduction

During development, fibroblast growth factors (FGFs) secreted from the apical ectodermal ridge (AER) direct limb outgrowth. FGFs also coordinate limb patterning through cross‐talk with other signaling molecules. The LIM homeobox transcription factor LHX2 is a suspected regulator of patterning cross‐talk, is expressed at the distal tip of the limb bud subjacent to the AER, and is a primary response target of FGF (Figure 1). However, the mechanism by which FGFs upregulate LHX2 is unknown. There are four major FGF signaling pathways through which regulation may occur: Jak/Stat, PLCγ, AKT, and Ras‐related (Figure 2). Based on the suspected roles of each signaling pathway, we hypothesize that FGF mediates LHX2 upregulation through the Ras‐related pathway.

Methods

To determine whether FGFs regulate LHX2 through the Ras‐related pathway, we injected the chemical inhibitor Salirasib (2.5 mM) into the anterior, posterior, and distal tips of the right limb of chick embryos at Hamburger‐Hamilton stage 21–22 (Figure 3). We also examined the potential function of the other Fgf pathways using pathway‐specific inhibitors. The embryos were harvested 24 hours after treatment and assessed for morphology. Whole mount in situ hybridization for LHX2 was then performed (Figure 4).

Results

Preliminary results indicate that inhibition of the Ras‐related pathway caused growth inhibition of the treated limbs in all embryos. In addition, LHX2 expression was markedly decreased in the these limbs. Treatment with a PLCγ‐specific inhibitor did not alter limb growth, while an AKT‐specific inhibitor impaired limb growth and LHX2 expression.

Conclusion

These results suggest that the Ras‐related and AKT pathways control Fgf‐mediated LHX2 upregulation, whereas PLCγ does not. Further studies are needed to confirm these findings and to evaluate the Jak/Stat pathway.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Muscle Fiber‐type Composition of Primates in Relation to Brain Size
Authors: Omstead Kailey M , Muchlinski Magdalena N , Hemingway Holden W , Burrows Anne M
Published: June 2021

There are many metabolic hypotheses for how humans can afford such large brains. The Expensive Tissue Hypothesis (ETH) addresses the metabolic cost of the brain in accordance to the costs of other metabolically “expensive” organs in the body. Skeletal muscle is composed of two main fiber types; type I (slow‐twitch) and type II (fast‐twitch). Type I fibers convert glucose to ATP using oxygen, while type II fibers rely primarily on anaerobic metabolic processes. When active, skeletal muscle is in direct competition with the brain for glucose and is an expensive tissue to maintain. Skeletal muscle acting as expensive tissue however, has not been addressed in context to the ETH. Based on these observations, we hypothesize that primates will have a decrease in type I fibers in accordance to their brain mass to account for the expensive tissue. For this preliminary study, our goal was to measure type I and type II fiber frequencies in various primates using immunohistochemistry to provide us with a muscle fiber composition profile. Two forelimb muscles (deltoid, pectoralis major) and two hindlimb muscles (gastrocnemius, soleus) were biopsied from 10 species of primates (N=11) for histological analysis. Formalin‐fixed paraffin embedded muscle samples were sectioned (12um) and immunostained using type I and type II Myosin heavy‐chain antibodies. Histological analysis of muscle morphology was visualized and quantified using light microscopy. Through muscle fiber‐typing, we found that primates appear to have fewer type I fibers than type II fibers. Results from this study have been implemented as part of a larger study (Muchlinski et al., 2018) to clarify the relationship between muscle mass and brain mass to better illustrate how muscle fiber composition could be used to address evolutionary questions in relation to energetics.

Support or Funding Information

National Society for Histotechnology (NSH)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Capillary density and succinate dehydrogenase in skeletal muscle on chronic kidney disease rats
Authors: Murakami Shinichiro , Sakita Masahiro , Huo Ming , Kondo Hiroyo , Hayashi Satoshi , Kameyama Kazuyoshi , Fujino Hidemi
Published: June 2021

Background and Purpose

Kidney disease increases risk of blood vessel and capillary disease. In case of chronic kidney disease (CKD), these problems may happen slowly over a long period of time. The thing about heart in particular is well known. Chronic kidney disease may be caused by diabetes, high blood pressure and other disorders. Hypertension causes CKD and CKD causes hypertension. Anyone can get CKD at any age and any sex. However, some people are more easily to get kidney disease than others. That two main causes of CKD are diabetes and high blood pressure. Those diseases fail with blood vessel and capillary in various factors. In any case, the blood vessel system is connected with kidney disease deeply. We confirmed muscle capillary architecture on rat last time. We clarified muscle capillary density and mitochondrial activity this time.

Materials/Methods

12 Wistar rats (age; 16wks) were randomly divided into CKD group (n=6) and Sham operation (SHAM) group (n=6). Rats in CKD group suffered from a chronic kidney failure artificially. The details are as follows; 1. When 16wks old, under anesthesia with pentobarbital, we removed the two‐third left kidney surgically. 2. When 17wks old, under anesthesia with pentobarbital, we removed the right kidney surgically. 3. When 23wks old, we removed soleus muscle out. 4. Those muscles were frozen, were stained by SDH, AP and ATPase (pH4.2) method, and observed with light microscope (LM). In SHAM group, they were operated for at the same time, but the kidney was not removed surgically. The kidney was extracted to outside from rat and it was returned to rat without removing it surgically. The microscope image of SDH stained was analyzed in NIH Image software and it was compared. All data were presented as the means ± SEM. All statistical tests were done using an unpaired Student's – test. P<0.05 indicates a significant difference.

Results

On 23 wks old CKD, body weight and soleus weight were decrease. Feeding volumes were same. Urinary albumin/urinary creatine value was increase. Data from measurement of the LM images by the NIH Image software of SDH, the mean SDH activity on CKD rats were significantly higher than that in the SHAM rats. Capillary density on CKD rat was lower than that in SHAM. However, muscle myosin heavy chain types of ratio on CKD were same to SHAM.

Conclusions

These results indicate that muscle requires oxygen, but shows that blood supply to muscle decreases on CKD.

Support or Funding Information

This study was supported by Grants‐in‐Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (Grant no. 17K01879)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Serum Brain‐derived neurotrophic factor promotes repair and regeneration of aged myelinated fibers via aerobic exercise
Authors: Sakita Masahiro , Murakami Shinichiro , Isobe Wataru , Kameyama Kazuyoshi , Hayashi Satoshi , Fujino Hidemi
Published: June 2021

Purpose

Degeneration of the distal peripheral nerve (DPN) with aging is associated with balance and gait disorders. Brain‐derived neurotrophic factor (BDNF) contributes to the repair and remodeling of myelinated fiber. However it is not obvious whether BDNF in the DPN increases with aerobic exercise (AE) in old age and promotes remodeling of MFs. We investigated morphological changes of MFs and BDNF expression levels of both serum and DPN via AE.

Methods

Eight male wistar rats (aged 95 weeks) were randomly divided into AE and control (Cont) groups. Rats in AE group performed running on the treadmill for 1 hour per day, 6 days per a week during 2 weeks. At 97 weeks of age, the bilateral tibial nerves of 2 groups were removed. To measure myelin thickness and axon diameter, semi‐thin sections of the unilateral specimens were stained with 0.5% Sudan Black B solution following double‐fixation. In addition, each expression level of serum and tissue BDNF in the contralateral specimens was analyzed with enzyme‐linked immunosorbent assay.

Results

Myelin thickness and axon diameter of AE group were significantly larger than those of Cont group (p<0.01). The serum BDNF level of AE group significantly decreased in comparison to that of Cont group (p<0.01). In contrast, BDNF level of AE group was significantly higher than that of Cont group (p<0.01).

Conclusion

It was thought that serum BDNFs that were promoted by AE release into nerve fibers and contribute to the remodeling of myelinated fibers in aged DPNs.

Support or Funding Information

The present study was supported by a Grant‐in‐Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (https://kaken.nii.ac.jp; 26350853).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of 5‐Fluorouracil/Leucovorin Chemotherapy on Cognitive Function in Mice
Authors: Groves Thomas , Alexander Tyler , Anderson Julie , Kiffer Fred , Allen Antino
Published: June 2021

5‐fluorouracil (5‐Fu) and leucovorin (LV) are often given in combination to treat colorectal cancer. 5‐Fu/LV prevents cell proliferation by inhibiting thymidylate synthase (TS), which is an enzyme that catalyzes the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate. While 5‐Fu has been shown to cause cognitive impairments, the synergistic effect of 5‐Fu with LV has not been fully explored. The present investigation was designed to assess how the combination of 5‐Fu and LV affect cognition in a murine model. A total of 30 nine‐month‐old male mice were used in this study; 8 mice received 0.9 % saline injections, 6 mice received LV (90 mg/kg) injections, 8 mice received 5‐Fu (50 mg/kg) injections, and 8 mice received 5‐Fu and LV (50 mg/kg and 90 mg/kg) injections. One‐month post‐treatment, Y‐maze and Morris water maze (MWM) behavioral tasks were performed. Brains were then extracted, cryosectioned, and stained with CD68 and tomato lectin. All animals in the MWM task were able to locate the visible and hidden platform locations in the water maze; however, treatment effects were seen when spatial memory retention was assessed in the probe trials (no platform). A significant impairment in spatial memory retention was observed in the probe trial after the first day of hidden platform training (first probe trial) in animals that received LV, 5‐Fu, and 5‐Fu/LV. However, by day 5, animals that received 5‐Fu only and 5‐Fu/LV showed spatial memory retention in the probe trials, whereas the LV only mice were impaired. We are currently performing microscopy to quantify changes in the activated microglia and brain vasculature. In studies conducted by Mustafa et. al and El Betagy et. al, Lister‐hooded rats were given 5‐Fu and LV and performed the novel location recognition task. For both studies, rats that received 5‐Fu/LV failed to discriminate between the two object locations, suggesting a spatial memory deficit. While these studies are promising, there is still a need to determine how this drug combination affects other aspects of memory and brain function. The present data suggests that LV may exacerbate the cognitive deficits seen with 5‐Fu treatment, as mice that received LV treatment only remained impaired by day 5 of the MWM. Mice that received 5‐Fu and 5‐Fu/LV were also impaired but showed spatial memory retention by day 5.

Support or Funding Information

This work was supported by Pilot Grant under NIH P20 GM109005 (ARA).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of microRNAs associated with apoptosis in neurospheres of glioblastoma cell line subjected to treatment with ionizing radiation and temozolomide.
Authors: Schiavoni Vagner Sarraipo , Rodrigues Andressa Romualdo , de Assis Cirino Mucio Luiz , Neto Fermino Sanzes Lizarte , da Cunha Lourenço Luana Pretti , Peria Fernanda Maris , Bisson Gabriela Silva , Tiezzi Daniel Guimaraes , Tirapelli Luis Fernando , Junior Carlos Gilberto Carlotti , da Cunha Tirapelli Daniela Pretti
Published: June 2021

Introduction

Glioblastomas (GBM) are the most common malignant primary tumors in the brain, and show high mortality rate. Despite the current advances in the therapy, the GBMs are extremely resistant to ionizing radiation and chemotherapy, and the number of relapse is high. Studies bring this eminent tumorigenic potential due to the presence of a subpopulation of neoplastic cells with characteristics of stem cells, called cancer stem cells (CSCs). In gliomas, the isolation of tumor stem cells have been done by antigenic markers and observing the culture conditions of normal neural stem cells in vitro. That is, the floating proliferation of tumor cells when cultured, are analogous to neurospheres derived from normal neural stem cells in defined culture conditions. It is believed that CSCs are responsible for the restoration of the tumor and the low efficiency of the treatment, as these cells demonstrate malignant properties as tumorigenesis, chemoresistance and radioresistance. The practical implication of this finding is that no current therapy is able to suppress or stop the proliferation of these cells. Several microRNAs have been linked to the development and proliferation of glioblastomas, associated with different molecular mechanisms, among them programmed cell death. Studies have shown that these microRNAs in brain tumors exhibit altered expression levels, one of the essential mechanisms for the regulation of tumor stem cells (CSCs).

Objectives

To evaluate the effect of ionizing radiation and temozolomide, alone or associated, on the expression of miRNAs that act as tumor suppressors (miR‐15 and miR‐16) and as oncogene (miR‐21), and adhered cells in neurospheres in culture of glioblastoma cell line (U343‐MG).

Materials and Methods

The trypan blue was used to examine cell viability before and after treatment, and method of real time PCR to check the expression of microRNAs in time 0h, considered to be immediately after the treatments, and 48h after exposure to treatments.

Results and Conclusions

The presented microRNAs are differentially expressed when comparing the different treatment modalities and time. We also observed differences between adhered cells and neurospheres. The oncogene miR‐21 remained with higher expression on treatment with ionizing radiation in neurospheres compared with the group of adherent cells.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endurance exercise training protects against the upregulation of nitric oxide in the striatum of MPTP/probenecid mouse model of Parkinson's disease.
Authors: Bataineh Ziad Mohedeen
Published: June 2021

Parkinson's disease (PD) is a neurodegenerative disorder, caused by the gradual loss of cells in substantia nigra. Nitric oxide (NO) plays an important role in a variety of signal transduction pathways that are crucial for maintaining the physiologic functions of nervous system. The aims of this study are: 1) To investigate the expression of the inducible form of NO (iNOS), and compare it to neuronal nitric oxide (nNOS) in the brain of a chronic mouse model of PD and 2) To study the effect of endurance exercise training on the expression of these markers.

METHOD

Mouse models of PD were obtained using 10 doses of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) (25 mg/kg) and probenecid (250 mg/kg) over 5 weeks. Forty C57BL/6 albino mice were randomly divided into four groups: sedentary control (SC, N = 10), exercise control (EC, N = 10), sedentary PD (SPD, N = 10), exercise PD (EPD, N = 10). At the end of training program, nNOS and iNOS were evaluated in the striatum in all animal groups using immunohistochemistry.

RESULTS

nNOS showed significant increases in striatum (ST) of SPD mice compared to SC mice (P > 0.03). There was also decreased expression of nNOS in EC group compared to SC mice, but this decrease was not significant (P > 0.8). Exercise training significantly decreased the level of nNOS in the EPD compared to SPD, (P > 0.04). Although, iNOS expression followed almost the same trend as nNOS, but exercise training did not significantly decrease the expression of iNOS in both EC and EPD groups, P > 0.2 and 0.3 respectively.

DISCUSSION

The data from this study suggests that 4 weeks of treadmill exercise has a positive impact on the expression of nNOS and iNOS in the striatum of a PD model. This might clear in part the pathogenicity of the diseases and the positive impact of training on PD.

Support or Funding Information

This work was funded from the Deanship of Scientific Research at Jordan University of Science and Technology

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanistic Insight into Species‐specific Redox Partner Interactions in the Vitamin D Carbon‐24 Hydroxylase CYP24A1
Authors: Kumar Amit , Estrada D. Fernando
Published: June 2021

The mitochondrial cytochrome P450 24A1 (CYP24A1) is responsible for side chain hydroxylation of vitamin D, the initial step in deactivation of the bioactive hormone. Mutations in CYP24A1 lead to incomplete clearance of active vitamin D and correlate with idiopathic infantile hypercalcemia, or elevated calcium in blood serum, and can lead to loss of appetite, nausea, vomiting, and failure to thrive in newborns. The regiospecific hydroxylation of the vitamin D side chain at either carbon‐23 (C23) or carbon‐24 (C24) relies in part on differences between species; while human isoforms are both C23 or C24 hydroxylases, other species are either C23 or C24 pure hydroxylases, despite an overall sequence identity of 80% between isoforms. Recent work from our group using solution NMR to evaluate the interaction between CYP24A1 and the redox accessory protein adrenodoxin (from bovine) suggests that species displaying distinct regioselectivity for vitamin D also form distinct P450‐redox partner complexes. Evidence of species‐specific complexes combined with the fact that mix‐species P450‐redox complexes are widely used to examine P450 function, highlights the need to examine this redox complex using proteins from the same species. Therefore, in this work we have used a combined approach of site‐directed mutagenesis, protein NMR, chemical cross‐linking, and vitamin D hydroxylation assays to evaluate the CYP24A1‐adrenodoxin complex from rat (a C24 hydroxylase). Differences in the 2D NMR spectra between bovine and rat adrenodoxin led to the complete re‐assignment of the protein backbone for rat adrenodoxin. Nonetheless, differential broadening of the NMR signal when titrated with rat CYP24A1 bound to 1α‐hydroxyvitamin D suggests the complex is mediated by surface charges. Dependence on surface charges was also verified by site‐directed mutagenesis of both proteins as well as zero‐length chemical cross‐linking using 1‐Ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide. While preliminary, this works demonstrates the combined use of biophysical and biochemical approaches to evaluate nuanced differences in a P450‐redox complex that is critical to the maintenance of proper levels of bioactive vitamin D.

Support or Funding Information

NIGMSR00GM112862 (DFE)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cultivation of Hepatic Stellate Cells in 3D ECM to Simulate Physiological and Fibrotic Conditions
Authors: HAN YUAN‐PING
Published: June 2021

Background

Hepatic stellate cells, the major providers of hepatic retinoids and extracellular matrix (ECM), reside in the space of Disse filled with loose basement like ECM, and flanked by hepatocytes and sinusoidal endothelial cells. Upon hepatic injury, stellate cells are activated to express multiple matrix metalloproteinases (MMPs) and to mobilize retinoids for wound healing. In chronic injury, the quiescent stellate cells undergo trans‐differentiation into myofibroblast like cells, marked by loss of retinoids, expression interstitial ECM as well as smooth muscle actin (alpha‐SMA), and gain of contractibility. Although knowledge has been evolved substantially, two fundamental questions remain largely unknown as to how, in normal liver, HSCs maintain quiescence, and how, by injury, HSCs are activated within the ECM milieu.

Results

To recapitulate the sub‐sinusoid microenvironment we embedded rat primary stellate cells in three‐dimensional ECM (3D ECM). Cultured on plastic, the stellate cells activated spontaneously, showing loss of vitamin A droplets and expression of alpha‐SMA. However, in 3D Matrigel, stellate cells retained retinoid droplets, in association with elevated level of lecithin retinoid acyltransferase (LRAT), a key enzyme for retinol esterification. In 3D ECM, stellate cells were arrested at G0/G1 phase, while on plastic the cells underwent cell cycling and stopped at G2/M phase. We then reconstituted the microenvironment of sinusoids by introducing Kupffer cells, the major hepatic inflammatory cells, into 3D ECM with HSCs. Activated Kupffer cells triggered subsequent activation of stellate cells, evidenced by expression of multiple MMPs, buildup of stress fibers, and loss of retinoids. The Kupffer cell‐mediated activation of stellate cells was blocked by interleukin‐1 receptor antagonist, demonstrating interleukin‐1 as a major injury signal from Kupffer cells conducting stellate cell activation and trans‐differentiation. Furthermore, within 3D ECM, but not on the plastic, interleukin‐1 signal was greatly amplified by stellate cells in an autocrine manner, which may exacerbate liver injury by expressing MMPs in the space of Disse. The myofibroblastic cells derived from the 3D ECM culture were contractile in response to endothelin.

Conclusion

The 3D ECM culture provides a model to simulate key physiological functions of stellate cells in normal liver, and also to recapitulate patho‐physiological responses in liver injury. The method may also be used in cell‐based drug screening to identify components targeting liver failure and fibrosis.

Support or Funding Information

National Institutes of Health (NIH) Grants R01s, DK069418‐05 and AR051558, and the Natural Science Foundation of China

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Typing of Hepatitis B Using Fusion Primers and IonTorrent
Authors: Ringlander Johan , Andersson Maria , Lindh Magnus
Published: June 2021

Background

Hepatitis B virus (HBV) chronically infects 250 million people, and is the main contributor to hepatoceullular carcinoma (HCC) in the world. When contracted at adult age, HBV can cause acute hepatitis which sometimes is severe and may result in acute liver failure. Molecular epidemiology is important for the analysis of circulating HBV strains and for clarifying transmission chains. Next‐generation sequencing (NGS) has several advantages over traditional Sanger sequencing, but high costs for NGS, in particular the library preparation step, has restricted its use.

Objective

To develop a cost‐efficient strategy for simplified amplicon sequencing of HBV whole genomes on IonTorrent S5 (Thermofischer, Watlham, MA, USA), and an automated bioinformatics pipeline.

Method

Primers with tails carrying IonTorrent A or P1 adaptor, were designed to target conserved regions using an alignment consisting of fifty HBV strains, reflecting all known genotypes. The primers were located to cover the genome by 10 amplicons of approximately 380 bp size, and will be presented. HBV genomes in serum samples was amplified by duplex PCR in five parallel reactions, followed by sequencing in IonTorrent S5, and bioinformatic processing by CLC Genomics Workbench (Qiagen, Aarhus, Denmark).

Results

All ten overlapping segments of HBV DNA from four patient sera, representing genotypes A, B, C and D were successfully amplified. Sequencing data are yet to be analyzed and will be presented.

Discussion and conclusion

To our knowledge, whole genome sequencing of HBV using fusion primer derived amplicons for IonTorrent have not previously been published. By this technique, whole genome sequencing of HBV can be performed in large scale and at low cost.

Support or Funding Information

Governmental strategic research funds, Sahlgrenska University Hospital.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.