bluuebunny/crossref_metadata_2025 huggingface dataset:
Epicardial ventricular pacing induces a local inflammatory response in the outer layers of the left ventricle
Authors: Yamazaki Katrina Go , Covell James , Villarreal Francisco
Published: June 2021

Abnormal heart wall motion as seen with dyssynchronous contraction of the left ventricle (LV) yields adverse chamber remodeling characterized by regions of wall thinning and hypertrophy. We hypothesized that heart wall abnormalities secondary to local epicardial ventricular pacing trigger local inflammation, reactive oxygen species (ROS) generation, matrix metalloproteinase (MMP) activation and fibrillar collagen degradation in the epicardial half of the LV wall. To examine this hypothesis we pursued LV pacing studies in anesthetized open‐chest dogs (n=5). Myeloperoxidase activity was significantly increased by 2.3 fold in the epicardial half (3±0.6 SEM, relative units=RU) of the LV compared to the endocardial half (1.3±0.2 RU). Our results also demonstrated a 2 fold increase in MMP‐9 activity in the epicardium (530±151 RU) compared to the endocardium (290±106 RU). In these same samples, ROS generation was increased by 2.2 fold (52±10 RU vs 24±7 RU). These results correlate with 3 fold more collagen damage in the epicardium (237±57 RU) when compared to the endocardium (78±22 RU). These results provide evidence that LV dyssynchrony can induce an inflammatory/injury response in the outer half of the LV wall, which may trigger localized remodeling. Supported by NIH HL‐43617.
Evidence for a novel neuronal projection from the esophagus to the airways in guinea pigs
Authors: Mazzone Stuart Brett , McGovern Alice Elizabeth
Published: June 2021

The aim of the study was to characterize calretinin expressing nerve fibers found to be associated with tracheal parasympathetic ganglia in guinea pigs. Retrograde tracing from the trachea with cholera toxin B identified parasympathetic preganglionic neurons in the nucleus ambiguus and dorsal motor nucleus of the vagus nerve, sensory neurons in the nodose, jugular and dorsal root ganglia, and sympathetic postganglionic neurons in the superior cervical and stellate ganglia. Although some calretinin‐expressing neurons were identified in each of these locations, neurons specifically traced from the airways never expressed calretinin. Calretinin‐immunoreactive nerve fibers disappeared from tracheal ganglia following 48 hours in organotypic culture (confirming an extrinsic origin) but persisted when tracheae were co‐cultured with the esophagus intact. A population of esophageal myenteric plexus neurons was immunoreactive for calretinin and co‐expressed acetylcholine but not nitric oxide synthase. Electrical stimulation of the esophagus in vitro evoked contractions of the trachea that were significantly (P<0.05) inhibited by hexamethonium. Taken together, these data suggest that a subset of esophageal neurons, that express calretinin and contain acetylcholine, provide excitatory input to tracheal cholinergic ganglia in guinea pigs.
Intrinsic neurogenesis in the cerebral cortex continues in adult guinea pig and gives rise to new GABAergic neurons
Authors: Clough Rich W , Xiong Kun , Luo Xue‐Gang , Patrylo Peter R , Struble Robert G , Yan Xiao‐Xin
Published: June 2021

We have identified neuroblast‐like and immature neurons appearing to be born within the adult guinea pig cerebral cortex. Morphologically diverse cells expressing doublecortin (DCX) were confined to layer II and adjacent borders. Cells (often in clusters) varied in size, shape, label‐intensity, nuclear morphology and with somata appearing in contact with one another. DCX cells co‐expressed polysialylated neural cell adhesion molecule (PSA‐NCAM) and neuron‐specific type III beta‐tubulin. Larger DCX+ cells had well‐developed processes and expressed neuron‐specific nuclear protein (NeuN). Some DCX cells displayed reduced DCX staining but elevated NeuN expression, suggesting a transitional state of maturity. “Transitional” cells also showed immunoreactivities to GABA and GAD67. Cells expressing mitotic markers (proliferating cell nuclear antigen, Ki67, or 5‐bromo‐2′‐deoxyuridine following short survival times, were also encountered across the cortex with prevalence in layers I and II. Co‐localization of BrdU and DCX was seen in several cells following longer survival periods. Our findings suggest the superficial layers of the guinea pig cerebral cortex to be neurogenic throughout life and to provide new GABAergic neurons during adulthood. Supported by Southern Illinois University and Illinois Department of Public Health.
The diagnosis of brachial plexus lesion: anatomical knowledge as an essential tool in poorly imaging facilities conditions
Authors: Gashegu Julien Kagabo
Published: June 2021

The diagnosis of the lesion of brachial plexus is a challenge in country where imaging facilities are not well developed. Here we present 3 cases of different lesions of the brachial plexus after a road traffic accident.

The first case presents a lesion of the 3 primary trunks of the right brachial plexus. The clinical examination showed paralysis of all terminal nerves of the brachial plexus and the collateral branches of both anterior and posterior brachial plexus.

The second patient showed paralysis of all muscles of the shoulder and muscles of the anterior compartment of the arm. This clinical feature is in accordance of the upper brachial trunk lesion. The third patient showed a paralysis of muscles of the hand being innerved by median nerve or ulna nerve. He showed also paralysis of muscles of the anterior compartment of the forearm and anaesthesia of the medial region of hand and forearm. This clinical feature is in accordance with a lesion of inferior primary trunk. All diagnosis were made by analysis of the clinical features. The anatomical knowledge is very important in the clinical examination as one can confuse the signs of terminal nerves lesion with the trunk's lesion.
Influence of tumor stroma on normal keratinocyte marker profile
Authors: Lacina Lukas , Cada Zdenek , Dvorankova Barbora , Kolar Michal , Strand Hynek , Smetana Karel
Published: June 2021

Study objective:

This study reports on marker profiling changes of normal keratinocytes under influence of mesenchymal cells derived from tumor stroma in vitro, and especially on induction of stem‐cell‐like features in affected epithelia.

Methods:

We compared phenotype of human tumors with in vitro model where growth and phenotypic pattern of normal human keratinocytes were monitored under influence of various types of fibroblasts including those prepared from stroma of malignant tumors. We visualized panel of keratins, endogenous lectins and binding sites for endogenous lectins, proliferation marker Ki67, membrane protein Ber‐EP4, nucleolar protein nucleostemin and markers of poorly differentiated embryonic stem cells Oct‐4 and Nanog.

Results:

The results demonstrated that normal human keratinocytes were strongly influenced by tumor stromal cells. The observed epithelial cells subsequently expressed tumor markers as keratin 8 and epithelial antigen Ber‐EP4 as well as some putative stem cell markers (e.g. keratin 19, nucleostemin etc.)

In conclusion, the cancer stromal cells significantly influenced the phenotype of normal keratinocytes, suggesting so the role of epithelial – mesenchymal interaction in cancer cells survival and spreading.
Conformational dynamics of mutant forms of formamidopyrimidine‐DNA glycosylase from Escherichia coli
Authors: Fedorova Olga , Kuznetsov Nikita , Koval Vladimir , Douglas Kenneth , Zharkov Dmitry
Published: June 2021

Formamidopyrimidine‐DNA glycosylase (Fpg) repairs oxidative DNA damage by removing oxidized purines such as 8‐oxoguanine (8‐oxoG) from DNA. Fpg possesses three activities resulting in the excision of the damaged deoxynucleoside: hydrolysis of the N‐glycosidic bond, β‐elimination, and δ‐elimination. Phenylalanin‐110 is one of the residues inserted in the DNA helix after evertion of 8‐oxoG into the enzyme's active site. We have studied conformational dynamics and pre‐stationary kinetics of Fpg and its mutant forms F110W and F110A by the stopped‐flow approach with tryptophan (Trp) and 2‐aminopurine (2‐aPu) fluorescence detection. Multiple transient changes in Trp and 2‐aPu fluorescence were observed, indicating sequential conformational changes in both protein and DNA molecules during the catalytic cycle. An induced fit model is proposed that provides a mechanism for substrate recognition and cleavage. Distortion of the DNA helix coupled with the evertion of oxidized base and insertion of amino acids occured in the initial steps of specific site recognition. Fpg‐F110A was not able to proceed beyond base eversion, while Fpg‐F110W revealed that the enzyme likely partially everts normal G in the search for a lesion.

Supported by grants from the Wellcome Trust, RFBR, and INTAS.
A novel tRNA degradation pathway that acts on mature tRNA
Authors: Chernyakov Irina , Whipple Joseph M. , Kotelawala Lakmal , Grayhack Elizabeth J , Phizicky Eric M
Published: June 2021

Under normal conditions, tRNA biosynthesis results in extremely stable species that are exquisitely adapted for their specific roles in translation. Newly transcribed tRNAs undergo multiple processing steps and several quality control checks before use in the cytoplasm. We recently provided evidence for a mechanism to examine the integrity of mature tRNAs in the yeast Saccharomyces cerevisiae, since mature tRNAVal(AAC) in a trm8‐Δ trm4‐Δ strain (lacking 7‐methyl‐guanosine and 5‐methyl‐cytidine) is rapidly degraded and deacylated upon shift to 37°C, resulting in a growth defect (Alexandrov et al., 2006, Mol. Cell 21:87–96).

We have examined the components required for this tRNA degradation pathway as well as the generality of this pathway for different tRNA species and modifications. We find that degradation of tRNAVal(AAC) is mediated by Met22, as well as the 5′–3′ exonucleases Rat1 and Xrn1, which also have established roles in rRNA and snoRNA processing, and mRNA turnover. Furthermore, we find that this MET22‐dependent pathway mediates the degradation of at least three other tRNA species defective in other tRNA modifications. This evidence firmly establishes the existence of a pathway that monitors the integrity of mature tRNAs.

Funded by NIH grants 5T32GM068411 and GM52347.
Structural and biochemical consequences of disease causing mutations in human mitochondrial tRNAMet
Authors: Jones Christie , Jones Christopher I , Agris Paul F , Spremulli Linda L
Published: June 2021

The translational apparatus of human mitochondria requires only 22 tRNAs, which are encoded by the mitochondrial genome. The tRNA genes are hotspots for disease causing mutations. The single tRNAMet (hmtRNAMet) has 3 known disease causing mutations. One of these (U8C) results in the loss of a specific Mg2+ binding site, leading to an incorrectly folded tRNA that is poorly aminoacylated. A second mutation (A37G) in hmtRNAMet is associated with Leber's Hereditary Optical Neuropathy. The A37G mutant, located in the anticodon loop of hmtRNAMet, retains the wild‐type structure, aminoacylation, and binding to EF‐Tu. Because of the proximity of this mutation to the anticodon, it is likely to affect the decoding process. The third mutation (G53A in the T‐stem) results in abnormal mitochondria with defects in energy production. The G53A mutation alters the structure of the tRNA as determined by chemical probing and thermal denaturation. However, these structural alterations are overcome by the human mitochondrial methionyl‐tRNA synthetase and this mutated tRNA can be aminoacylated efficiently. The G53A mutant has a decreased ability to bind EF‐Tu suggesting that it may have a defect in polypeptide chain elongation. The effect of this mutation on the association and dissociation rate constants for its interaction with EF‐Tu and its subsequent delivery to the ribosome is being investigated. (Supported by NIH Grant GM32734)
Exploration of the roles of IF3mt in mammalian mitochondrial translation
Authors: Christian Brooke Elizabeth , Spremulli Linda L.
Published: June 2021

The initiation of mammalian mitochondrial protein synthesis is poorly understood. Two initiation factors with homology to bacterial IF2 and IF3 have been identified. IF3mt is a 29 kDa protein with an internal region having homology to prokaryotic IF3. This homology region consists of an N‐terminal domain, a linker and a C‐terminal domain. The homology domains are preceded and followed by short extensions. No information is currently available on the regions of IF3mt important for its activity. Based on homology models of IF3mt, mutations were designed in the N‐terminal, C‐terminal, and linker domains to identify the functions of these regions. Mutation of residues 66–70 and 121–124 in the N‐terminal domain and residues 194–197 in the C‐terminal domain had no effect on the ability of IF3 to promote initiation complex formation on mitochondrial 55S ribosomes. Mutation of residues 143–147 in the linker region and residues 184–186 in the C‐terminal domain caused a significant reduction in activity indicating an important role for this region for initiation complex formation. Mutation of histidine 170 and aspartic acid 171 to alanine caused a nearly complete loss of activity. However, sucrose density centrifugation indicates that this variant can bind to 28S subunits. Mutation of the nearby region (residues 175–177) also resulted in a significant loss of activity. The causes for these defects are under investigation.
Caveolin‐1 facilitates cyclooxygenase‐2 degradation through a p97/valosin‐containing protein dependent pathway
Authors: Shyue Song‐Kun , Chen Su‐Fen , Liou Jun‐Yang , Wu Kenneth K
Published: June 2021

Cyclooxygenase (COX)‐2 participates in various physiological functions and involves in tumorigenesis. Although transactivation of COX‐2 has been well studied, the factor that responsible for COX‐2 degradation remains mostly unknown. In this study, COX‐2 degradation induced by caveolin‐1 (Cav‐1) is demonstrated by upregulated endogenous and bacteria induced COX‐2 in lung and colon of caveolin‐1 null mice. Ectopic expression of caveolin‐1 in HT‐29 cells resulted in suppressed endogenous and IL‐1β induced COX‐2 expression. Cav‐1‐induced COX‐2 degradation was implicated to enhanced protein degradation by the ubiquitin‐proteosome pathway. Via immunoprecipitation, we identified valosin‐containing protein (VCP/p97) as the chaperon protein which interacts with both Cav‐1 and COX‐2 and specifically mediates COX‐2, but not COX‐1, degradation. Suppression of VCP via siRNA resulted in increased COX‐2 level with prolonged half‐life. Moreover, deletion of the COX‐2 C‐terminal 32 amino acids upregulated its expression level and lengthened its half‐life. These data reveal a novel degradation mechanism that Cav‐1 and VCP synergistically orchestrate COX‐2 degradation, suggesting a role of Cav‐1 in anti‐tumorigenesis.
Translational control of PPARβ in macrophage differentiation
Authors: Cambiaghi Tavane David , Luchessi Augusto Ducati , Curi Rui
Published: June 2021

Previous studies have shown that the 5′‐UTR of the PPARβ transcript negatively regulates its expression. The mechanisms of translational control of PPARβ expression mediated by its 5′‐UTR in PMA‐differentiated THP‐1 cells were investigated. PMA treatment of THP‐1 cells caused a time‐dependent increase PPARβ production. A polysomal profile assay revealed reduction of the protein synthesis in response to PMA, indicating inhibition of translation initiation. The activity of eIF2α was analyzed by western blotting and showed an intensive dephosphorylation followed by a recover of the phosphorylation state during the period of higher production of PPARβ. This recover was preceded by dephosphorylation of eIF4E‐binding protein (4E‐BP). This process inhibits cap‐dependent translation initiation since dephosphorylated 4E‐BP binds to eIF4E. The mRNAs that contain internal ribosome entry site (IRES) are preferentially translated under conditions where eIF4E activity is decreased. A structural analysis of the PPARβ 5′‐UTR showed the presence of seven uORFs (upstream open reading frames) that can control translation of this transcript. Therefore PPARβ expression may be controlled by both mechanisms. Financial support: FAPESP, CNPq and CAPES.
Dissescting the role of SBP2 in selenocysteine incorporation
Authors: Donovan Jesse , Caban Kelvin , Copeland Paul R
Published: June 2021

The SECIS binding protein, SBP2, is required for the co‐translational incorporation of selenocysteine (Sec) at specific UGA codons. The C‐terminal 447 residues of SBP2 (CTSBP2) are sufficient to promote Sec incorporation and have been divided into a functional domain (FxD) and an RNA binding domain (RBD). SECIS and ribosome interactions through the RBD are well characterized but it is not known how the FxD participates in Sec incorporation. To determine the role of the FxD in Sec incorporation we created chimeras using the domains of CTSBP2 and the analogous region of SBP2‐like protein (SLP) to determine if the FxD in SLP has sufficient identity with SBP2 to promote Sec incorporation. Using an in vitro luciferase reporter specific for Sec incorporation, we showed that the FxD in SLP cannot promote Sec incorporation. A homology driven approach was used to mutate FxD residues in groups of five and mutant proteins were assayed for their ability to promote Sec incorporation, SECIS binding, and ribosome binding. We identified three regions critical for Sec incorporation that implicate the FxD in ribosome binding and suggest that SBP2's ribosome interaction is dynamic. Additionally, we expressed the FxD and RBD as separate recombinant proteins and assayed them for their ability to promote Sec incorporation. Interestingly, we found that both domains together in a Sec incorporation assay are competent but gel filtration and EMSA studies failed to show a stable interaction of the two domains. We currently hypothesize that the FxD acts to allow Sec‐tRNASec access to the ribosomal A‐site and are developing assays to test this.

This work was supported by PHS grants T32AI007403 (JD), FGM081920A (KC), and GM077073 (PRC).
Functional Analysis of the ClC‐2 hyperpolarization‐activated Cl− channel in Mouse Colon.
Authors: Catalan Marcelo A , Sepulveda Francisco V , Melvin James E
Published: June 2021

ClC‐2 is a hyperpolarization‐activated Cl− channel expressed in epithelial cells lining the gastrointestinal tract. ClC‐2 expression has been proposed to compensate for the intestinal Cl− secretion deficit in cystic fibrosis due to mutations in the Cl− channel CFTR.

The overall goal of this project was to evaluate the ability of the ClC‐2 Cl− channel to stimulate intestinal fluid secretion. Distal colon sheets from ClC‐2 +/+ and ClC‐2 −/− mice were mounted in Ussing chambers and Cl− secretion was induced by two different paradigms: a) cAMP‐ and b) Ca2+‐increasing secretagogues. Neither cAMP nor Ca2+ ‐ mediated Cl− secretion was affected in ClC‐2 −/− colon. In contrast, the amiloride‐inhibited short circuit current (ISC) increased in the early distal colon of ClC‐2 −/− mice.

Based on our findings, the ClC‐2 channel appeared not to be necessary for Cl− secretion in colonic epithelium. On the other hand, increased amiloride‐sensitive ISC in the early distal colon of ClC‐2 −/− mice suggested that there was an increased expression of the epithelial Na+ channel ENaC, the key component of the electrogenic NaCl absorption process. We conclude that a compensatory NaCl absorptive mechanism was activated in the early distal colon of ClC‐2 −/− mice, implying that the ClC‐2 channel might be important for colonic NaCl absorption.

This work was supported in part by grants from NIH (DE09626) and Takeda Pharmaceutical Company, Inc.
Tyrosine nitration of L‐type Ca channels prevents activation of the cyclic AMP Response Element (CRE)
Authors: Kang Minho , Akbarali Hamid I
Published: June 2021

Calcium influx through Cav1.2 has been implicated in signal transduction and gene expression. We have recently shown that colonic inflammation results in decreased Ca currents due to nitration of c‐terminal tyrosine residues that prevents src‐kinase mediated phosphorylation. In hCav1.2b transfected cells, depolarization by KCl and/or treatement with the Ca channel agonist BayK8644 induced phosphorylation of the cAMP response binding protein (CREB). Phospho‐CREB binds to CRE sites on promoter regions of several genes. A dual luciferase assay was used to determine activation of CRE in hCav1.2b transfected cells. CRE activation was increased by 2.4‐fold by KCl‐induced depolarization that was blocked by nifedipine. Nitration of hCav1.2b by peroxynitrite (150 uM) prevented KCl induced CRE activation. Both Forskolin and Ionomycin enhanced CRE activation by 2.8‐fold. Mutation of c‐terminal tyrosine residues (Y1837‐1861‐2134F) prevented CRE activation. These data suggest that tyrosine phosphorylation of the COOH terminus of Cav1.2 plays an important role in the downstream signaling for the activation of transcription factors. Supported by NIDDK046367.
An inhibitory action of ASIC1a peptide on BK channels
Authors: Petroff Elena
Published: June 2021

Acid Sensing Ion Channels contain a sequence that is identical to scorpion toxins blocking potassium channels, and this sequence is critical for ASICs' inhibition of large conductance calcium‐ and voltage‐activated potassium (BK) channels (Petroff et al., PNAS 2008, 118(4), 1571). In this study, a peptide containing the ASIC1a toxin‐like sequence (without the Cys to avoid peptide aggregation) also reduced BK current amplitude. Mutating the peptide sequence replacing Arg and Lys with Ala prevented BK inhibition. Peptide inhibition was reversible and concentration‐dependent, and although pH 6 solution only marginally reversed inhibition the rate at which current increased following peptide washout was faster at pH 6 than 7.4. Moreover, the peptide competed with the known BK channel blocker, tetraethyl ammonium. Extracellular K+is known to reduceIbTx and CTx block. Raising the K+ and Rb+ concentrations, but not Na+, Cs+, Li+ or Ca2+, reduced BK inhibition by the peptide. A parallel exists inCTx block ofShaker K+ channels; external K+, but not Na+ reduced block, indicating the presence of an extracellular K+ coordination site located near the toxin binding site. In addition, the peptide inhibition of BK channels was voltage‐dependent only in the presence of intracellular K+. These data suggest that ASIC1a peptide interacts with the K+ conduction pathway in the BK channel and may act as a BK channel blocker.
Coordinate activation of cardiac and pulmonary fibroblasts by hypoxia‐induced pulmonary hypertension in a transgenic mouse model
Authors: Brown R. Dale , Ambler S. Kelly , Stenmark Kurt R.
Published: June 2021

Activation of adventitial fibroblasts and myofibroblasts is an important determinant of pulmonary vascular remodeling in hypoxia‐induced pulmonary hypertension (PH). PH leads to right ventricular hypertrophy and failure. We hypothesized that hypoxia activates cardiac fibroblasts in concert with pulmonary fibroblasts. Transgenic mice expressing fibroblast phenotype‐sensitive promoter:reporter constructs (Collagen I promoter:Enhanced Green Fluorescent Protein, Coll I:EGFP; fibroblast phenotype; Smooth Muscle alpha‐Actin promoter:Red Fluorescent Protein, SMA:RFP, myofibroblast phenotype) were exposed to hypobaric hypoxia (6 weeks, 10% pO2). Fibroblast activation was monitored by reporter fluorescence in frozen tissue sections and by FACS analysis of enzymatically dispersed cells. Hypoxic exposure significantly elevated cardiac expression of SMA:RFP with a modest enhancement of CollI:EGFP. Increased RFP expression was greatest in right ventricle. Increased expression of RFP and EGFP were also observed in perivascular and parenchymal regions of lung. Distinct lung cell populations expressing fluorescent reporter proteins were observed by FACS analysis (EGFP+ > RFP+EGFP+ > RFP+). Coordinate activation of cardiac and pulmonary fibroblasts in response to hypoxia offers opportunities for novel fibroblast‐targeted therapies in PH. Supported by NIH HL014985.
Toll Like Receptor 3 expression and function on Peripheral Blood Mononuclear Cell in pregnant women with HBV Infection
Authors: Song Xiuxia , Li Shuzhen , Wang Suping
Published: June 2021

The aim of this study was to analyze the expression and function of TLR3 on PBMC in pregnant women with HBV infection. The expression of TLR3 mRNA on PBMC in 28 HBsAg positive pregnant women and 26 normal pregnant women was examined by semi‐quantitative RT‐PCR. The concentration of IL‐2, 6 in both serums of HBsAg positive and normal pregnant women were analyzed by ELISA. TLR3 mRNA was expressed at a higher level in HBsAg positive pregnant women(group A) than in normal pregnant women(group B). Group A were divided by their newborns having HBV intrauterine infection or not, the rate of TLR3Ct/β ‐actin Ct was not significant difference between the two groups. IL‐2 in group A serum was higher than group B,while IL‐6 was opposite to IL‐2. IL‐6 in serum of group A with their newborns having HBV intrauterine infection highly increased compared with not having HBV intrauterine infection women's. Correlation analysis showed that IL‐2 increased with the TLR3 mRNA increasing. The results demonstrated that TLR3 might predominantly promote Th1 immunological reaction against HBV infection.
Deciphering the indolence of osteomyelitis: Evaluation of the uptake of Staphyloccocus aureus and silver nanoparticles into osteoblasts
Authors: Wagner William , Benjamin Joseph C. , Webb Lawrence X. , Coldren Faith , Kute Timothy , Martin Eileen , Smith Thomas L. , Smith Beth P.
Published: June 2021

Chronic osteomyelitis caused by Staphylococcus aureus is difficult to treat because the bacteria persists inside osteoblasts. The first aim of this study was to assess the uptake potential of S. aureus into osteoblasts. Because novel therapies are needed to combat the increasing antibiotic resistance of S. aureus, the uptake of silver nanoparticles (a possible therapy) into osteoblasts also was examined. Osteoblasts were incubated with carboxyfluorescein‐succinimidyl ester (CFSE) labeled S. aureus or silver nanoparticles and stained for confocal microscopy. Incubated osteoblasts were evaluated using flow cytometry for their FITC fluorescence profile (S. aureus) or SSC (side scatter) changes (nanoparticles). Osteoblasts were incubated with nanoparticles at different concentrations to determine their cytotoxicity. Flow cytometry showed that the FITC fluoresence was higher in infected osteoblasts due to incorporation of CFSE‐labelled S. aureus while SSC was greater due to the incorporation of silver nanoparticles. Confocal microscopy images confirmed the uptake of both S. aureus and nanoparticles. Nanoparticles demonstrated minimal cytotoxicity at low concentrations (MTS assay).

Aided by a grant form the Orthopaedic Research and Education Foundation and the Translational Science Institute at Wake Forest University Health Sciences
Peptide analogs based on the innate immune system molecule, CAP37, are potent antimicrobials against Pseudomonas aeruginosa and attenuate Pseudomonas LPS‐mediated effects in vitro
Authors: Pereira Heloise Anne , Brevetti Joseph , Hinsley Heather , Xhaja Anisa , Harris Christina , Clark Richard , Svoboda Pavel , Pohl Jan
Published: June 2021

CAP37 is a neutrophil‐derived protein with strong antimicrobial activity against Gram negative bacteria. The principal antimicrobial domain of CAP37 resides between amino acids 20 and 44. A synthetic peptide based on amino acids 20‐44 (CAP37/20‐44) mimics the antibacterial activity of the native protein. Recently, two further analogs of CAP37/20‐44 were synthesized in which the cysteine residues at positions 26 and 42 were replaced with serine residues. The objective of this study was 1) to evaluate all three CAP37 peptides for their antimicrobial activity against Pseudomonas aeruginosa including clinical isolates with known antimicrobial resistance profiles to conventional antibiotics and 2) to determine their ability to bind and neutralize Pseudomonas lipopolysaccharide (LPS) or endotoxin. The Limulus Amebocyte Lysate assay was used to determine LPS binding. LPS neutralization was determined by measuring the ability of the peptides to attenuate the release of cytokines such as tumor necrosis factor‐alpha from the murine macrophage cell line, RAW264.7. Our findings demonstrate that all three synthetic peptides showed bactericidal activity against Pseudomonas including the clinical isolates and that they bound and neutralized the toxic effects of LPS in vitro. We conclude that synthetic CAP37 peptides have strong potential as therapeutics against Pseudomonas infections. Support: NIH UO1AI075391.
RENAL PATHOLOGY AND MORPHOMETRIC STUDIES ON NATURAL HISTORY OF BXSB LUPUS MICE
Authors: Ahmed Atif A , Jones Olcay Y
Published: June 2021

Introduction

BXSB is a prototypic murine model for Systemic Lupus Erythematosus manifested. Although few scoring systems have been reported in the literature, there has been no consensus on how to evaluate murine lupus nephritis.

Methods

Post mortem renal tissue sections from male BXSB mice at 10, 20 , 30, 40 and 50 weeks of age (collectively n=45 ) were evaluated for histopathology on H&E, and PAS staining. Quantitative measurements of glomerular cell count, glomerular tuft area, and glomerular sclerosis areas were obtained on an average of 10 glomeruli per mouse by using computerized image analysis provided by Nikon. Controls were age matched male C57Bl/6 (n=3 per group) or female BXSB (n=3 at 30 weeks) mice.

Results

There is progressive increase in glomerular cellularity and tuft size with age in mice at 10 and 20 weeks of age, compared to controls. Sclerosis was evident starting at 30 weeks. Four major step of progression were appreciated: 1) increased glomerular cellularity with increased glomerular size at 10th and 20th week of age; 2) expansion of matrix at 20 to 30 weeks of age; 3) progressive sclerosis with corresponding decreased cell counts, in mice at 30 to 40 weeks of age; and 4) end‐stage disease with pan‐sclerosis and glomerular obliteration in older mice.

Conclusions

The renal pathology in BXSB follows a predictable pattern that resembles human renal pathology. Computerized quantitative measurement of glomerular cell counts, tuft size and extent of sclerosis is a reliable objective tool to study lupus nephritis in mouse models and effect of experimental treatment options.
High Resolution SSO Tissue Typing by Luminex Technology in Bone Marrow (HSC) Allotransplant Recipients
Authors: Lewis Robert E , Cruse Julius M , Holliday Jay A , Singleterry Will L , Suggs Jeanann L
Published: June 2021

Bone marrow or HSC transplantation requires stringent exploration of HLA tissue type to detect histoincompatibility between potential donor and recipient. This study investigates the value of using Luminex® technologies with LABType® SSO HD from One Lambda to efficiently determine high resolution tissue type. SSO by Luminex® is being compared with SSP to determine the relative level of resolution. Current results suggest that a combination of the two is requisite to achieve HLA‐A, ‐B, ‐C, and ‐D allele specific typing. SSO is preferable for multiple typings, whereas SSP is applicable for individual specimens. Virtual typing based on archival high resolution data for our patient population suggests that the SSO‐Luminex method would yield concordant results with SSP. Therefore, the ability to HLA type multiple (i.e., up to 16) samples simultaneously renders SSO‐Luminex superior to SSP, especially for family studies, in bone marrow or HSC allotransplantation.
Temporal genomic transcriptional profiling of DSS induced colitis by microarray
Authors: Fang Kai , Bruce Megan , Kevil Christopher G.
Published: June 2021

We have previously reported that the dextran sodium sulfate (DSS) induced colitis model involves a complex relationship between inflammation and angiogenic activity in that inflammation and angiogenesis serve to perpetuate chronic inflammation. Here we performed genome transcription profile analysis of 3% DSS induced colitis using the Affymetrix mouse genome 430 2.0 Array from colon tissues taken at day 0 (D0), 2 (D2), 4 (D4), and 6 (D6). Several inflammatory genes were greatly up‐regulated, such as Interleukin‐6 (0.9, 8.6 and 80.7 fold increase at D2, D4, and D6), Mmp 10 (1, 3, 22 fold increase at D2, D4, and D6), and Mmp 13 (2 and 21 fold increase at D4 and D6). Angiogenesis related genes were also significantly up‐regulated, such as HGF (0.4 and 2.8 fold increase at D4 and D6), Map3k8 (3 fold increase at D6), and Epiregulin (3.7 fold increase at D6). Increased inflammatory and angiogenic gene expression was observed in a proportional manner with earlier changes in inflammatory gene expression. Several genes were also significantly down regulated such as Trpm6 (38%, 43% and 71% at D2, D4 and D6), Repin1 (16%, 54%, and 76% at D2, D4 and D6), and Pnpla6 (41%, 59% and 56% at D2, D4 and D6). Our study demonstrates that inflammatory and angiogenic activities are linked during experimental colitis and delineate distinct time points of genome transcriptional change which may suggest new targets for the treatment of colitis.
Age‐Dependent Impairment in Tissue Perfusion in the Foxn1nu/nu Nude Mouse: Potential for Better Predicting the Efficacy of Cell‐Based, Proangiogenic Therapies
Authors: Brooks Benjamin David , McKee Jeff
Published: June 2021

The mouse model of hindlimb ischemia is used routinely to evaluate proangiogenic therapeutics. Most studies employ young mice (<12 wk) even though peripheral arterial disease is not a clinical issue in young patients. Young (12 wk), wild type mice are more efficient at angiogenesis versus old (2 yr) mice as shown by significantly higher hindlimb perfusion (measured by laser Doppler imaging, LDI) and capillary density, which were attributed to an age‐dependent impairment in endothelial function and VEGF expression (Rivard et al 1999). This suggests that old mice are more relevant for use in nonclinical development and may be more sensitive in detecting therapeutic effects as compared with young mice. The purpose of this work was to determine if a similar effect of age on angiogenesis was detectable in immunocompromised mice, specifically the Foxn1nu/nu nude mouse, which is used routinely to evaluate cell‐based, proangiogenic therapeutics. Foxn1nu/nu nude mice, 10 or 34 wk, underwent surgically induced, unilateral hindlimb ischemia. Hindlimb perfusion, measured by LDI was performed on days 1, 4, 7, 14 and 21 post‐surgery. Laser Doppler ratios (ischemic to nonischemic limb) were significantly lower in the old mice on days 4, 7, 14 and 21 compared to the young mice. These data indicate that older Foxn1nu/nu nude mice recover hindlimb perfusion more slowly, presumably due to an age‐dependent impairment in angiogenesis. Baxter provided all research support.
Molecular basis of IL‐10 attenuation of TNF‐α induced NFκB pathway activation and cardiomyocyte apoptosis
Authors: Dhingra Sanjiv , Sharma Anita K , Al‐Shudiefat Abd Al‐Rahman , Arora Rakesh C , Singal Pawan K
Published: June 2021

We have reported that tumor necrosis factor‐α (TNF‐α) increases oxidative stress and apoptosis in cardiomyocytes by upregulating p38 mitogen activated protein (MAP) kinase phosphorylation. Interleukin‐10 (IL‐10) blocked these effects of TNF‐α by upregulating ERK 1/2 MAP kinase phosphorylation. However, the precise site of this IL‐10 action is still unknown and this was investigated in the present study. Cardiomyocytes isolated from adult Sprague Dawley rats were exposed to TNF‐α (10ng/ml), IL‐10 (10ng/ml) and IL‐10α (ratio 1) for 4hrs. Hydrogen peroxide (H2O2) and antioxidant trolox were used as positive controls. Exposure to TNF‐α resulted in an increase in the production of reactive oxygen species, number of apoptotic cells, caspase‐3 activation and PARP cleavage. Increased oxidative stress by using H2O2 also caused apoptosis. These changes due to TNF‐α were associated with an increase in IKK and NFκB phosphorylation. IL‐10 by itself had no effect, but it prevented TNF‐α induced changes. Trolox also mitigated TNF‐α induced changes. Pre‐exposure of cells to IKK inhibitor, prevented TNF‐α induced changes and inhibition of ERK 1/2 MAP kinase attenuated protective role of IL‐10. The study shows that IL‐10 prevents TNF‐α‐induced NFκB activation and proapoptotic changes in cardiomyocytes by inhibiting IKK phosphorylation through the activation of ERK 1/2 MAPkinase. (Supported by CIHR).
CGRP Selectively Activates a Subpopulation of Nodose Sensory Neurons with Tonic Action Potential Firing Properties
Authors: Fazan Rubens , Lu Yongjun , Chapleau Mark W
Published: June 2021

Calcitonin gene‐related peptide (CGRP) and its receptors are highly expressed in subpopulations of sensory C‐fibers innervating heart, viscera, and blood vessels including carotid sinuses. While CGRP release and its vasodilator activity have been studied extensively, little is known regarding its effects on the activity of vagal afferent and baroreceptor neurons in nodose ganglia. We hypothesized that CGRP may selectively activate a subpopulation of nodose sensory neurons. Resting membrane potential (RMP) and action potential (AP) responses to depolarizing current injection (0.1‐1.0nA for 1s) were recorded by intracellular microelectrode from neurons isolated from nodose ganglia of C57BL/6 mice. Two types of neurons were studied: 'tonic' neurons that repetitively fire APs during sustained depolarization, and 'phasic' neurons that fire only a single AP at the beginning of the period of depolarization. CGRP (500 nM) depolarized 8 of 10 tonic neurons from ‐52±2 to ‐47±2 mV and increased AP discharge from 17±2 to 29±2 spikes during the period of current injection (n=8, P<0.01). These excitatory effects were fully reversed upon drug washout. In contrast, CGRP did not change RMP or AP discharge in phasic neurons (n=6). Consistent with these findings, CGRP increased cytosolic Ca2+ concentration (Fluo‐4) in a subset of rat nodose neurons. We conclude that CGRP selectively activates the majority of tonic nodose sensory neurons without affecting RMP or AP discharge in phasic neurons. The selectivity of the response likely reflects co‐expression of CGRP receptors with ion channels enabling repetitive spike firing in tonic neurons. (HL14388, VA)
Enhancement of the hypoxic ventilatory response following neonatal maternal separation: the role of testosterone in adult male rats
Authors: Fournier Sebastien , Joseph Vincent , Kinkead Richard
Published: June 2021

Neonatal maternal separation (NMS) disrupts normal neurological development in mammals. The hypoxic ventilatory response (HVR) of adult male rats previously subjected to NMS is ~25% greater than controls while the response of NMS females is reduced by ~30% (Genest et al., 2004). To address the role of sexual hormones on the respiratory phenotype observed in NMS males, we compared the HVR of normal adults (10‐14 weeks old; controls n = 6, NMS n = 6) with that of gonadectomised (GDX) adult males (controls n = 7, NMS n = 6) in which circulating testosterone levels are considerably reduced. Pups subjected to NMS were placed in an incubator 3h/day for 10 consecutive days (P3 to P12). Controls were undisturbed. Rats were then reared until adulthood when HVR was measured with plethysmography (12% O2, 20 min). In controls, GDX had no effect on the HVR. In NMS rats, GDX reduced the HVR 50% below that of controls (GDX x NMS; p = 0.03) with a pattern now similar to the respiratory phenotype of female rats. These results indicate that testosterone is necessary for full expression of the HVR in NMS but not in control males, and likely contributes to the enhancement of the HVR following NMS. Supported by the CIHR.
Elimination of nucleus of the solitary tract (NTS) neurokinin‐1 receptor (NK1R) expressing neurons enhances heart rate adaption and attenuates body weight loss during chronic restraint stress in spontaneously hypertensive rats
Authors: Bechtold Andrea G , Bonham Ann C
Published: June 2021

Chronic stress is associated with cardiovascular hyperreactivity, a risk factor for later cardiovascular disease. Increases in heart rate (HR) and mean arterial pressure (MAP) adapt slowly during daily restraint in the stress sensitive spontaneously hypertensive rat (SHR). Experiments were performed to test the hypothesis that elimination of neurokinin‐1 receptor (NK1R) expressing neurons in the nucleus of the solitary tract (NTS) would enhance adaption of the cardiovascular responses to chronic restraint stress in SHR. The NK1R neurons were ablated by microinjection of the targeted neurotoxin stable substance‐P saporin (SSP) or saporin vehicle (SAP) into the NTS of SHR implanted with telemetry transmitters to measure MAP and HR. Three weeks after NK1R ablation, rats were restrained in a Plexiglas tube for 1 hr/day for 14 d. SSP significantly reduced the increase in HR, but not MAP during daily restraint (p<0.05, 2‐way RM ANOVA). For example, on day 7 the HR area under the curve was 8144±361 (SAP; n=11) vs 6250±260 bpm/60 min (SSP; n=10). SSP treatment also reduced body weight loss during chronic stress. On day 1 after restraint body weight was reduced ‐4.6±1.2 g in SSP vs ‐6.2±1.6 g in SAP rats. By day 14 weight loss was ‐6.0± 4.1 g (SSP) and ‐14.5±2.8 g (SAP). We conclude that in SHR, NTS NK1R neuron ablation enhances adaption of the HR response and attenuates weight loss during chronic restraint stress. (Support: HL‐67183)
Atorvastatin reduces sympathetic nerve activity through the inhibition of Rac1/NAD (P) H oxidase and upregulation of Mn‐SOD in SHRSP
Authors: Kishi Takuya , Hirooka Yoshitaka , Ito Koji , Araki Shuichiro , Konno Satomi , Sunagawa Kenji
Published: June 2021

Background

Statin is consider to protect for neuroplasticity. We demonstrated that oral administration of atorvastatin decreases sympathetic nerve activity (SNA) through the anti‐oxidant effect in rortral ventrolateral medulla (RVLM) of stroke‐prone spontaneously hypertensive rats (SHRSP). We hypothesized that atorvastatin might protect for neuroplasticity by reduction of oxidative stress in RVLM of SHRSP.

Methods and Results

In SHRSP, intracerebroventricular infusion of atorvastatin (20 μg/kg/day) (ATORVA) for 14 days significantly reduced systolic blood pressure, heart rate and SNA (‐28±3 %, n=5, P<0.01) in comparison with vehicle infusion (CNT). In the RVLM, ATORVA decreased oxidative stress, Rac1 and NAD (P) H oxidase activity. ATORVA also significantly lowered the expression of Rac1, gp91phox, p22phox in membrane fraction, and p47phox in cytosolic fraction. In contrast, the expression level of Rac1 in cytosolic fraction and Mn‐superoxide dismutase (Mn‐SOD) activity were significantly increased in ATORVA.

Conclusion

In the RVLM of SHRSP, atorvastatin inhibits membrane translocation of Rac1 and NAD (P) H oxidase subunits and upregulates Mn‐SOD activity, which contribute to the inhibition of oxidative stress in the RVLM, protection for neural plasticity and the sympatho‐inhibitory effect.
Hypotensive action of adrenomedullin (ADM) receptor blockade in the rostral ventrolateral medulla of spontaneously hypertensive rats
Authors: Colombari Debora Simoes Almeida , Milosavljevic Snezana , Colombari Eduardo , Japundzic‐Zigon Nina , Paton Julian F.R. , Murphy David
Published: June 2021

Microarray data from our laboratory indicate that mRNA levels for the ADM receptor (GRP182) are upregulated by 2.9 fold in SHR compared to Wistar‐Kyoto (WKY) rats. Further, ADM increases mean arterial pressure (MAP) when microinjected into the rostral ventrolateral medulla (RVLM) of anesthetized normotensive rats (Xu and Krukoff, 2004; AJP, 287, p R729). We tested the hypothesis that blockade of ADM receptors in the RVLM of spontaneously hypertensive rats (SHR) would reduced arterial pressure. Animals were anesthetized with urethane (1.4 g/kg, iv) and an ADM antagonist (ADM 22‐52, 100 fmol/100 nl) was microinjected bilaterally into the RVLM. In the SHR MAP decreased by ‐42 ± 3 mmHg after ADM receptor blockade (n=6; P<0.01); this response peaked 20 min after drug delivery. Conversely, ADM 22‐52 injected either into RVLM of WKY rats (n=6) or outside the RVLM in SHR (n=5) failed to change MAP. In arterially perfused rats (n=3), ADM (100 fmol/100 nl) microinjected bilaterally into the RVLM of SHR produced a time‐dependent increase in thoracic sympathetic chain activity (34 ± 4 and 81 ±13%, at 20 and 60 min) but in stark contrast, no change in WKY rats. These results demonstrate that in SHR there is tonic endogenous activation of ADM receptors that directly excite vasomotor sympathetic activity. This may, in part, reflect the high mRNA levels in the RVLM of the SHR compared to WKY rat. Support: CNPq, CAPES (Brazil), The Royal Society.
Mechanisms of the ventrolateral medulla on the control of sympathetic activity by central chemoreflex in rats
Authors: Moreira Thiago S , Takakura Ana C , Menani Jose V , Sato Monica A , Colombari Eduardo
Published: June 2021

In the absence of inputs from carotid bodies and cardiopulmonary receptors hypercapnia markedly increases sympathetic nerve discharge (SND) to the heart and blood vessels. In the present study, splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were recorded in urethane anaesthetized sino‐aortic denervated and vagotomized rats. Hypercapnia (end‐expiratory CO2 from 5% to 10%) increased sSND (δ= 103 ± 7%). Bilateral injection of muscimol (GABA‐A agonist ‐ 2 mM in 30 nl) into the retrotrapezoid nucleus (RTN) eliminated PND and reduced the effect of CO2 on sSND (δ= 56 ± 8%). Injection of AP‐5 (NMDA glutamate receptor antagonist ‐ 100 mM in 50 nl) or DNQX (non NMDA glutamate receptor antagonist ‐ 100 mM in 50 nl) bilaterally into rostroventrolateral medulla (RVLM) reduced PND, but did not change the effect of CO2 on sSND. Bilateral injection of MCPG (metabotropic glutamate receptor antagonist ‐ 100 mM in 50 nL) into the RVLM reduced PND and the effect of CO2 on sSND (δ= 41 ± 7%). In conclusion, RVLM metabotropic receptors and RTN are involved in the increase in sSND produced by CO2.
Tracheal epithelial innervation and sensory neuron number in the nodose and jugular ganglia of rats
Authors: Zellner Leor , Hunter Dawn , Brundage Kathy , Dey Richard
Published: June 2021

Postnatal Fisher 344 rat pups were examined on days 6, 10, 15, and 28. Animals were intra‐tracheally instilled with FITC labeled beads in early postnatal life to retrogradely label nodose and jugular ganglia neurons specifically innervating the tracheal epithelium (airway neurons). Ganglia and tracheae were removed, prepared for their respective assays and labeled using immunocytochemistry for pan neuronal marker, protein gene product‐9.5 (PGP‐9.5). Using Flow Cytometry neurons labeled with PGP‐9.5 (total) and FITC beads (airway) were counted. Using imaging software tracheal epithelium nerve fiber density (NFD) was calculated as percent area. The total number of neurons in the ganglia at PD 28 (165,678 +/‐ 53570; n=13) was significantly increased above neuron numbers at PD 6 (12,495 +/‐2194; n=8), but not at PD 10 (39,009 +/‐ 8,083; n=7) or PD 15 (69,829 +/‐ 16,858; n=10). Airway neuron numbers at PD 28 (2,334 +/‐ 468; n=13) were significantly increased over both PD 10 (913 +/‐ 177; n=7) and PD 15 (722 +/‐ 197; n=10), but not PD 6 (1050 +/‐ 208; n=6). Total tracheal epithelium NFD on PD 10 (0.98728 +/‐ 0.16847; n=6) was significantly higher than on PD 28 (0.29610 +/‐ 0.046046; n=5), but no difference was found from PD 6 (0.78600 +/‐ 0.17932; n=5) or PD 15 (0.71973 +/‐ 0.17210; n=3). The data suggests that there is a divergent relationship between epithelium NFD and airway neuron number at PD 28.