bluuebunny/crossref_metadata_2025 huggingface dataset:
ProtoArray® Protein Microarrays: Rapid Identification of PARP Substrates
Authors: Nolan Edward M , Freeman-Cook Lisa
Published: June 2021

Poly(ADP‐ribose) polymerases (PARPs) play a diverse role in many physiological and pathological pathways, particularly DNA damage repair. To date, ~18 different PARP enzymes have been identified, with PARP1 being the best studied. PARP1 is activated by DNA damage and catalyzes the addition of ADP‐ribose units onto acceptor proteins, setting off the base excision repair pathway.

PARP inhibitors have recently garnered attention in cancer treatment as therapeutic adjuvants used in conjunction with DNA damaging chemotherapeutics. Identifying the substrate targets of the various PARP enzymes would better define their function, help identify overlap between different PARP activities, and potentially advance clinical applications for PARP inhibitors.

ProtoArray® Human Protein Microarrays contain more than 9,000 purified full‐length human proteins, providing broad coverage of the proteome for a wide range of discovery applications. We report here the development of a rapid and comprehensive assay for PARP substrate identification using ProtoArray® microarrays. Using PARP1 as a model, we identified ~110 known and novel PARP1 substrates.

Our results highlight protein microarrays as a platform suitable for the global discovery of PARP substrates, enabling a more detailed understanding of their function and facilitating their potential clinical exploitation as druggable targets.
CAM/CAMKII‐dependent survival signaling in bone marrow derived macrophages
Authors: Tano Jean-Yves Kouame , Vazquez Guillermo
Published: June 2021

The balance between macrophage survival and apoptosis is crucial at all stages of atherosclerotic lesion development. We here report that survival mechanisms recently described by us in THP‐1 derived macrophages, are also operational in primary cultures of bone marrow derived macrophages (BMDMs). Similarly to TNF, treatment of BMDMs with oxidized‐LDL results in activation of the canonical survival pathways PI3K/AKT and NFkB as well as the p38α MAPK route. We used pharmacological maneuvers, western blotting and TUNEL to examine the potential mechanisms that may contribute to regulate these survival signaling routes. Treatment with calmodulin (CAM) and CAM‐dependent kinase II (CAMKII) inhibitors significantly reduced activation of AKT and IkBα, and this was positively correlated with an increase in the number of TUNEL positive cells. Surprisingly, inhibition of PI3K using LY294002 markedly reduced not only AKT activation, but also the phosphorylation of IkBα and p38α suggesting a role of PI3K/AKT as a signaling node from which other survival pathways are activated. Altogether, these results suggest that in an inflammatory setting, macrophages trigger compensatory survival mechanisms that involve activation of the PI3K/AKT, NFkB and p38 MAPK routes in a CAM/CAMKII dependent manner. Supported by the NIH R01‐HL111877‐01
Alpha7 nicotinic acetylcholine receptor in macrophage apoptosis and efferocytosis
Authors: Lee Robert , Vazquez Guillermo
Published: June 2021

The development of atherosclerotic lesions depends on an important inflammatory component mediated mostly by macrophages. Notably, apoptosis of macrophages within the lesion and efficient clearance of apoptotic cells are key factors in lesion progression. In this context we are interested in defining whether non‐neuronal nicotinic acetylcholine receptors (nAChR) play a role in modulation of macrophage survival and clearance of apoptotic cells. Specifically, the α7nAChR (α7) was found to mediate, in vitro, survival pathways in endothelial cells and lymphocytes. However, it remains to be determined whether macrophage α7 can modulate cell survival and efferocytosis. The objective of our study is to explore the contribution of pathways downstream of α7 stimulation to macrophage survival as well as effective efferocytosis using a combination of pharmacological and biochemical approaches. In bone marrow‐derived macrophages we observed activation of ERK1/2 upon treatment with nicotine which was abrogated by α‐bungarotoxin, suggesting that the actions of nicotine are mediated by α7. These findings will help outline the potential roles of α7nAChR in regulation of macrophage apoptosis and efferocytosis. Supported by R01HL111877‐01
α1A‐adrenergic receptor is required for normal prostate development and successful conception in mice
Authors: Healy Chastity L , Wright Casey D , OˈConnell Timothy D
Published: June 2021

In the prostate, α1A‐adrenergic receptor (α1A‐AR) mediated smooth muscle contraction is essential for normal function. Conversely, in benign prostate hyperplasia, α1‐AR antagonists are used to block smooth muscle contraction and provide relief from urinary symptoms. This identifies a clear role for α1‐ARs, particularly the α1A‐AR, in the prostate, but less is known about how α1‐ARs regulate prostate development. While breeding α1‐single and double knockout (α1AKO, α1BKO, α1ABKO) mice, we observed that α1AKO‐breeding pairs were significantly (P<0.01) less successful in establishing a pregnancy. Pairing α1AKO mice with WT or α1ABKO mates demonstrated that the lack of successful pregnancies was due to dysfunction in α1AKO males, not females. Gross dissection of the urogenital track in adult α1AKO males revealed distinct discoloration of seminiferous tubules and under‐developed prostate glands. Further, histologic analysis of serial prostate sections stained with hematoxylin and eosin isolated from WT and α1AKO male mice 3–12 weeks of age indicated abnormalities in prostates isolated from α1AKO males as early as 3 weeks of age. A lack of stromal development, thinning of epithelium, and fluid retention was observed in α1AKO prostates at all ages. These data indicate that the α1A‐AR is required for normal prostate development and function. NIH (TDO and CDW) and the PhRMA Foundation (CDW) supported this work.
Regions of PTH(1–34) and PTHrP(1–34) Important for Human PTHR1‐Mediated Signaling
Authors: Thomsen William , Cupp Meghan , Nayak Surendra
Published: June 2021

Parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP), acting through osteoblast PTHR1, play important roles in bone remodeling. Activation of osteoblast PTHR1 leads to regulation of multiple signaling pathways including Gs/cAMP/PKA, Gq/IP/calcium/PKC, and G‐protein‐independent and beta‐arrestin‐mediated Erk1/2 phosphorylation. In vitro and in vivo studies using reported biased PTH peptides have lead to conflicting conclusions regarding which signaling pathway(s) are involved in bone formation and/or resorption. In this study, several PTH and PTHrP peptide analogs were evaluated in parallel assays capturing cAMP accumulation (DiscoveRX HitHunterTM assay), beta‐arrestin recruitment (DiscoveRX PathFinderTM assay), and Erk1/2 phosphorylation (CisBio TRF Cell'erkTM assay) using CHO‐K1 cells stably expressing human PTHR1. The specific aim of this investigation was to determine regions important for activation of these signaling pathways. Collectively our results suggest that both N‐terminal and C‐terminal regions are critical for both stimulation of cAMP accumulation and beta‐arrestin recruitment. In addition, peptide mediated stimulation of Erk1/2 phosphorylation parallels ability to promote beta‐arrestin recruitment.
In silico evaluation, synthesis and characterization of a boron containing arylethanolamine which reverts the mouse motor deficit induced by MPTP administration
Authors: Soriano-Ursúa Marvin Antonio , Picazo Ofir Picazo , Correa-Basurto José , Padilla-Martinez Itzia I , Trujillo-Ferrara José G
Published: June 2021

The development of potent and selective dopamine receptors (D2DR) ligands is an attractive task. In this work, we designed boron containing arylethanolamine derivatives from a molecular docking study on D2 receptor models. We selected and synthesized the boron containing compound 5‐(2‐ammonioethyl)‐2,2‐ dihydroxybenzo[d][1,3,2]dioxaborol‐2‐uide and its chemical characterization was done. The intraperitoneal administration of this compound slightly disrupted the locomotor activity of CD1 or C57‐BL/6 mice. However, locomotor deficit induced by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP, a neurotoxin which damages dopaminergic neurons in the substantia nigra) in an acute regimen on C57‐BL/6 mice, was reverted in the first 24h, after the new compound administration. Altogether, results strongly suggest that this new compound acts as an agonist on the dopaminergic system of mice. This work was partially supported by grants from SIP‐IPN, CONACYT and INNOVAPyME/CONACyT programs.
Cocaine hijacks sigma‐1 receptors from the ER to the nuclear membrane to interact with lamina A/C: Effect on MeCP2 gene expression
Authors: Chuang Chien-Ying , Tsai Shang-Yi , Hayashi Teruo , Hung Jan-Jong , Chang Wen-Chang , Su Tsung-Ping
Published: June 2021

The sigma‐1 receptor (Sig‐1R) is an endoplasmic reticulum (ER)‐resident non‐opioid receptor that is expressed in the nervous system and can regulate cellular differentiation, neuroplasticity, and neuroprotection. Nuclear lamins in the nucleus are essential for the nuclear envelope assembly, DNA synthesis, and gene transcription. We tested here the hypothesis that cocaine, via Sig‐1Rs, may affect the gene expression of methyl CpG binding protein 2 (MeCP2) which relates to cocaine addiction. In glial and NG108‐15 cells, cocaine increases the nuclear envelope invaginations (NEI) and grooving, suggesting that the nuclear‐cytoplasmic exchange is enhanced by cocaine. Confocal microscopic data show that cocaine and another Sig‐1R agonist DHEAS cause the translocation of Sig‐1Rs from the ER into the nuclear membrane. After cocaine treatment, Sig‐1Rs localize mainly in the outer membrane of the nucleus and could increase the chance to interact with lamin A/C. Cocaine is known to cause addictive behavior by upregulating (MeCP2) in the brain. In rat primary cortical neurons, cocaine causes an increase in MeCP2. This effect is antagonized by Sig‐1R antagonist BD1063. Our results suggest that cocaine increases MeCP2 gene expression by translocating Sig‐1Rs into the nuclear outer membrane to increase the NEI and grooving to facilitate the Sig‐1R‐lamin A/C interaction to regulate the MeCP2 gene expression.
Acute toxicity profile of boron containing arylethanolamines and their precursors in mice
Authors: Amezcua-Gutierrez Marcos A , Lopez-Cabrera Yessica , Cipres-Flores Fabiola J , Soriano-Ursua Marvin A , Trujillo-Ferrara José G
Published: June 2021

Arylethanolamines modulate the activity of catecholamine systems. Our working group has found that the addition of functional groups with boron to arylethanolamines confers them greater potency and efficacy on their receptor‐targets in smooth muscle or central nervous system (CNS). However, the use of boronic acids in drug development is limited due to disputes on the toxicological profile of these compounds. This study evaluated the acute toxicity of boronic acids, arylethanolamines and boron containing arylethanolamines by determining its toxic (DT50) and lethal dose (LD50) in CD1 male mice with 22±2 g as well as by general observations from necropsy of treated animals. In our study,3 of the 7 tested boronic acids induced dose‐dependent events presumably mediated by action on the CNS (sedation, hypnosis and/or shaking motion) in live mice, but also eye damage and gastrointestinal effects (as gastric‐gut dilatation and fecal retention)were observed in death mice. On the other hand, the boron containing adducts induced acute toxicity in higher or similar dose than other arylethanolamines, excepting one tested compounds which induced sedation. These data allows a framework for the toxic evaluation of these compounds which can be considered potential therapeutic agents. Supported by grants from SIP‐IPN and CONACYT programs.
Evaluation of novel biomarkers of nephrotoxicity in rats co‐administered melamine and cyanuric acid
Authors: Bandele Omari , Camacho Luisa , Ferguson Martine , Reimschuessel Renate , Stine Cynthia , Black Thomas , Olejnik Nicholas , Keltner Zachary , Scott Michael , Da Costa Goncalo Gamboa , Sprando Robert
Published: June 2021

Although standard methods are used to detect impaired renal function, novel biomarkers have been identified that detect early changes in renal integrity. Recently, infants and pets experienced renal dysfunction after ingesting food tainted with melamine (MEL). Animal studies demonstrated that co‐ingestion of MEL and cyanuric acid (CYA) caused the formation of melamine‐cyanurate crystals that obstruct renal tubules. Using multiplexed ELISAs and RT‐PCR, we evaluated the ability of clusterin, KIM‐1, NGAL, osteopontin and TIMP‐1 to distinguish the effects of dosing schedule and matrix in rats gavaged with a 1:1 ratio of MEL and CYA either 45 min (staggered) or <1 min (consecutive) apart or dosed in feed. Due to treatment‐related nephrotoxic effects, animals in the staggered group were removed from the study on day 9. Compared to consecutive gavage and feed groups, urinary protein levels and gene expression for each biomarker in the staggered group were substantially elevated. The results were consistent with the treatment‐related effects on body and kidney weights, amount of melamine‐cyanurate crystals in renal tubules, and SCr and BUN levels in each treatment group. Our results demonstrate that the kidney biomarkers can detect the effects of compounds that block renal tubules and differentiate the effects of dosing schedule and matrix. Studies were funded by an interagency agreement between the FDA and the NIEHS.
The Nrf2 activator, tBHQ, inhibits the early production of IL‐2, but not CD69 induction, in primary murine splenocytes and human Jurkat T cells
Authors: Zagorski Joseph W , Klaassen Curtis D , Rockwell Cheryl E
Published: June 2021

Nrf2 is a transcription factor that is activated by cell stress, such as the presence of oxidative compounds. After activation and translocation to the nucleus, Nrf2 binds to and induces the transcription of target genes, including HMOX, NQO1, among others. Previous studies have shown that Nrf2 skews the differentiation of murine CD4+ T cells. The purpose of the present studies was to determine the acute effects of Nrf2 on early transcriptional and translational events following T cell activation in primary mouse splenocytes and human Jurkat T cells. The Nrf2 activator, tBHQ (10μM), significantly reduced IL‐2 secretion by mouse splenocytes 24h after stimulation with anti‐CD3/anti‐CD28. Comparable reduction in IL‐2 secretion was observed in human Jurkat T cells treated with the same concentration of tBHQ. In contrast, Nrf2 had little effect on CD69, an early marker of T cell activation, and only modestly decreased CD25 induction in Jurkat cells. Taken together, the current studies suggest that Nrf2 activation inhibits IL‐2 secretion, but has little effect on CD69 induction in activated murine splenocytes and Jurkat T cells. Collectively, the studies suggest differential effects of Nrf2 on early transcriptional and translational events following CD3/CD28 stimulation in T cells, which further suggests that Nrf2 does not suppress overall T cell activation as a whole. (This work is supported by NIH grant: ES018885).
Excessive erythrocytosis compromises the blood–endothelium interface in erythropoietin‐overexpressing mice
Authors: Richter Vincent , Savery Michele D. , Gassmann Max , Baum Oliver , Damiano Edward R. , Pries Axel R.
Published: June 2021

Elevated systemic hematocrit (Hct) increases cardiovascular risk, such as stroke and myocardial infarction. One possible pathophysiological mechanism could be a disturbance of the blood–endothelium interface. The objective of this study was to examine the effects of an elevated systemic Hct on the thickness of the blood–endothelium interface (‘glycocalyx’, or ‘endothelial surface layer – ESL’) as well as flow resistance. We combined intravital microscopy of a transgenic mouse line (tg6) suffering from excessive erythrocytosis (Hct 0.85), microviscometric analysis of hemodynamics, and an in silico flow simulation model. In tg6 mice the glycocalyx/ESL was nearly abolished (thickness: wild‐type control: 0.52 μm, tg6: 0.13 μm), but was readily restored to normal thickness after systemic hemodilution to normal Hct. However, the corresponding increase in vessel diameter had only minor effects on peripheral flow resistance. This suggests that the pathological effects of elevated Hct may be caused by biological corollaries of a reduced ESL thickness and alterations of the blood–endothelium interface, which could include increased white cell–endothelium interaction and/or increased activation of the coagulation system.
Influence of exercise on adult hippocampal neurogenic subpopulations and its effects on behaviour
Authors: Gradari Simona , Pérez-Domper Paloma , Ortega Sylvia , Trejo Jose L
Published: June 2021

The therapeutic interest of the beneficial effects of exercise prompted us to analyze those effects influenced by the duration of the training and the time elapsed between training and evaluation. In particular, the differential sensitivity of learning/memory vs emotional behavior, and of distinct subpopulations of immature neurons depending on their age. To this goal, we have carried out a 2 weeks forced exercise paradigm, either immediately before or 2 months before evaluation; we have also employed a novel analysis of multiple birthdating, behavioral assessment and stereology.

We found that exercise effects are significant at different times depending on the type of behavior examined: the anxiolytic effects are detectable immediately after training, and also 2 months after exercise, while the pro‐cognitive effects are discrete and appreciable only immediately after training. Exercise affects adult hippocampal neurogenesis inducing an increase in the number of new neurons, but at least one subpopulation of immature neurons correlated negatively with the anxiolytic effect, opposite to the rest of immature neurons. These data show that although the sensitivity of new neurons to physical exercise is similar, its role on the animal's behavior is different. These results provide new evidences for the involvement of new adult‐borne immature neurons in different behavioral tasks, depending on their age.
Stronger vasoconstrictor response to aspirate from patients undergoing stent implantation into native coronary arteries than into saphenous vein grafts
Authors: Kleinbongard Petra , Baars Theodor , Möhlenkamp Stefan , Konorza Thomas , Kahlert Philipp , Erbel Raimund , Heusch Gerd
Published: June 2021

Background

Stent implantation into atherosclerotic coronary arteries releases particulate debris and soluble substances which contribute to impaired microvascular perfusion.

Methods

Using a distal protection/aspiration device, arterial blood was retrieved during stent implantation in 19 patients with a stenosis in native coronary arteries (NA) and in 20 patients with a saphenous vein graft (SVG) stenosis. The constrictor response of rat coronary and mesenteric arteries to aspirate was quantified in a myograph as isometric force development (mN).

Results

At 80 mmHg, the diameter was 250±28 μm for coronary and 186±16 μm for mesenteric arteries. The KCl (120mM) ‐ induced maximal vasoconstriction was comparable for coronary and mesenteric arteries (6.1±2.3 mN vs. 7.1±1.4 mN; NS). The vasoconstriction provoked by aspirate was greater in coronary than in mesenteric arteries. Aspirate released during stent implantation into NA induced a stronger vasoconstriction than that into SVG (coronary: NA 6.9±0.8*†, SVG 5.2±0.7†, mesenteric: NA 5.6±0.9*, SVG 4.2±0.5 mN; *p<0.05 NA vs. SVG, †p<0.05 coronary vs. mesenteric arteries).

Conclusion

Human coronary aspirate released during stenting of NA has a stronger vasoconstrictor potential than that from SVG. The vasoconstrictor response to human coronary aspirate is more profound in rat coronary than in mesenteric arteries.
Role of superoxide in cerebrovascular endothelial dysfunction with aging
Authors: Yoshizawa Mutsuko , Seals Douglas R , Zigler Melanie L , Ahsan Sanjana , Sindler Amy L
Published: June 2021

We tested the hypothesis that excessive superoxide contributes to cerebral vascular endothelial dysfunction with aging. Superoxide production (EPR spectroscopy) from the posterior communicating artery was greater in old (~27 mo, n =4) vs. young (~6 mo, n = 4) C57 male mice (2872 ± 458 vs. 1064 ± 476 AU, p < 0.05). Vascular endothelial function, as assessed by endothelium‐dependent dilation (EDD) (posterior communicating artery dilation to acetylcholine [ACh]) was impaired in old vs. young (49 ± 11 vs. 81 ± 3%, p < 0.05), as a result of reduced nitric oxide (NO)‐mediated dilation (EDD to ACh alone minus EDD to ACh + L‐NAME: 21 ± 9% vs. 70 ± 8%, p < 0.05). Incubation with the superoxide dismutase mimetic, TEMPOL, had no effect on EDD in old mice (45±12%), and decreased EDD in young mice (68±5%, p < 0.05 vs. control). Dilation to sodium nitroprusside, a measure of vascular smooth muscle sensitivity to NO, did not differ with age. These results suggest that superoxide bioavailability is increased and NO‐mediated EDD is impaired with aging in the cerebrovascular circulation of C57 mice. However, our data fail to show a direct role for excessive superoxide in age‐related cerebrovascular dysfunction. In young mice superoxide appears to act as a necessary signaling molecule regulating cerebral vasodilation.
JNK Negatively Regulates Adhesion of Monocytes to Human Endothelial Cells through VCAM‐1
Authors: Nizamutdinova Irina Tsoy , Chang Ki Churl , Kim Hye Jung
Published: June 2021

TNF‐α increased VCAM‐1 expression in endothelial cells (EC) and induced adhesion of monocytes and cancer cells to EC. VCAM‐1 siRNA showed significant reduction in the adherent monocytes or cancer cells by TNF‐α. We investigated the mechanisms that regulate VCAM‐1 expression. We found that JNK inhibitor (SP600125) up‐regulated VCAM‐1 increased by TNF‐α. Transfection with dominant negative (DN) JNK1 plasmid resulted in increase of VCAM‐1, whereas JNK1‐overexpressing (OE) vector resulted in inhibition of VCAM‐1 expression, suggesting that JNK negatively regulates VCAM‐1 expression. We tested whether JNK signaling affects IRF‐1 and/or GATA6, which are transcription factors that mediate TNF‐α induction of VCAM‐1 but not ICAM‐1. The DN‐JNK1 enhanced TNF‐α up‐regulation of IRF‐1 whereas JNK1‐OE displayed down‐regulation; but, neither DN‐JNK1 nor JNK1‐OE affected GATA6 level. Chromatin immunoprecipitation assay confirmed that the DN‐JNK1 transfection increases the binding of IRF‐1 to the VCAM‐1 promoter whereas JNK1‐OE inhibits IRF‐1 binding to the VCAM‐1 promoter, but didn't have effect on GATA6. Finally we found that MKP‐7, a negative regulator of JNK was involved in the TNF‐α‐mediated VCAM‐1 expression.
Opposite effect of TNF‐alpha and activin A on erythroid gene expression through different pathways in hematopoietic progenitor cells
Authors: Huang Huei-Mei , Chang Ju-Ling , Chung Ming-Hui
Published: June 2021

Cancer and inflammation related anemia is mediated by proinflammatory cytokines. However, the molecular mechanism of this cytokines‐inhibited erythropoiesis remains unclear. This study shows that proinflammatory cytokines, TNF‐α and IFN‐γ, inhibit activin A‐activated ζ‐globin promoter, but not IL‐1α, IL‐10 and IL‐13 in hematopoieticprogenitor cell line K562. The p38 inhibitor SB203580 decreases activin A‐activated ζ‐globin promoter, but did not alter the effects of TNF‐α and IFN‐γ. The NF‐κB inhibitor Bay117082 reduces TNF‐α‐ and IFN‐γ‐inhibited ζ‐globin promoter activity, but did not alter the effects of activin A. Activin A inhibits c‐Jun expression, c‐Jun binding to AP‐1 binding sites of c‐Jun promoter and c‐Jun promoter activity through p38 pathway. TNF‐α induces c‐Jun expression to reverse activin A inhibitory effects via NF‐κB/c‐Jun complex binding to AP‐1 bonding sites of c‐Jun promoter. Furthermore, wild‐type c‐Jun enhances and c‐Jun dominant negative mutant eliminates TNF‐α‐inhibited ζ‐globin promoter activity. Thus, activin A down‐regulates c‐Jun through p38 to induce globin gene expression. TNF‐α activates NF‐κB/c‐Jun to inhibit the effects of activin A on hematopoieticprogenitor cells.
Lipoplasty technique affects the cellular fate potential of adipose derived stem cells: A comparison of Body Jet and Body Jet with Smart Lipo techniques
Authors: Murphy Daniel P , Moore Caitlyn , Genoese Angela , Debo Matthew , Falcone Victoria , Adams Christopher , D'Angelo Marina
Published: June 2021

The potential of stem cells to replace damaged or malfunctioning tissues has been studied for some time, and recent studies using adipose‐derived mesenchymal stem cells (ADSCs) have shown promise for adult stem cells as a rich, clinically‐relevant population. Our lab has previously determined that the quantity and quality of ADSCs varies depending on patient age and sex, anatomical location of harvest and whether it was a previously treated site with lipoplasty. A clinically‐relevant question is whether method of lipoplasty affects pluripotency of the stem cells. Our lab has shown that harvesting ADSCs using BodyJet and BodyJet with SmartLipo lipoplasty techniques produces a high yield of viable stem cells. Using established plating densities, corrected for stem cell viability measured by flow cytometry, we have reared isolated ADSCs, confirmed by flow cytometry and confocal microscopy, harvested with the BodyJet and BodyJet with SmartLipo techniques. This result suggests that both BodyJet and BodyJet with SmartLipo lipoplasty techniques yield clinically useful ADSC's. Funded, in part, under a grant with the Pennsylvania Department of Health and intramural funds.
miR‐1 and miR‐301a Overexpression Impairs Collagen Gel Contraction in Human Mesenchymal Stem Cells
Authors: Klassen Jessica Rae , Neumann Shannon , Guirgis Mina , Ngo Melanie , Li Yun , Dixon Ian , Freed Darren
Published: June 2021

Previous data in our lab suggests that human mesenchymal stem cells (MSCs) display a myofibroblast phenotype in vitro, coincident with a reduction in miR‐301a expression. We hypothesize that miR‐301a and miR‐1 signalling plays a role in modulating the MSC‐myofibroblast phenotype.

The protocol received IRB approval. Human bone marrow derived MSCs were plated on collagen gel substrates and transfected with pre‐miR‐1 and pre‐miR‐301a. The reduction in collagen gel area was analyzed 48 hours post transfection and qRT‐PCR was used to analyze RNA profile and expression of myosin isoforms.

MSCs were found to express both non‐muscle myosin (NMM) IIA and IIB, and readily contracted collagen gels in the basal state, which was increased with TGF‐β1. While overexpression of miR‐301a or miR‐1 both resulted in reduced collagen gel contraction, miR‐1 had a greater effect attenuating contractility. Overexpression with both miR‐1 and miR‐301a decreased NMMIIA expression, while no change was observed with NMMIIB expression.

Overexpression of miR‐301a and miR‐1 reduced MSC contractility through reduced myosin expression. It is likely that miR‐1 and miR‐301a target additional proteins in these cells.
EFFICACY OF ANTI‐TNF‐ALPHA THERAPY IN PREVENTING UTERINE ADHESION FORMATION IN RATS
Authors: DEMIRBAG Suzi , Uysal Bulent , Surer Ilhami , Oter Sukru , Korkmaz Ahmet
Published: June 2021

Objective

Postoperative adhesion formation is a consequence of wound healing and sometimes can result in severe complications. The aim of this study was to evaluate the effect of TNF‐alpha inhibition on the development of postoperative uterine adhesions.

Methods

Twenty one female Wistar‐Albino type rats were used in this study. The rats were divided into three groups as sham‐operated, adhesion and adhesion plus the TNF‐alpha inhibitor etanercept. A lower midline abdominal incision was performed and standard uterine lesion was created by electrocautery. Starting immediately after adhesion induction, the treatment group received daily intraperitoneal injections of 2 mg/kg etanercept for a total of 3 days. At day 7, peritoneal washing fluids of rats were obtained for the determination of TNF‐alpha levels. Then all of the rats were sacrificed and uterine adhesions were staged.

Results

The total adhesion score and tissue malondialdehyde levels were found to be increased whereas superoxide dismutase and glutathione peroxidase activities decreased significantly in the adhesion control group (P<0.05 ). TNF‐alpha inhibition, significantly reversed these findings (P<0.05).

Conclusions

Anti‐TNF‐alpha therapy reduces the prevalence of peritoneal and uterine tube adhesion formation in rats possibly by suppressing TNF‐alpha production as well as limiting oxidative stress.
Rac1‐dependent actin remodeling as a molecular mechanism shaping spine density
Authors: Bongmba Odelia Yuh Ngala , Tejada-Simon Maria Victoria
Published: June 2021

Abnormal dendritic spine morphogenesis has been associated with several psychiatric conditions linked to autism. A consistent abnormality observed in the brain of some of these patients and animal models is related to neurons showing a high density of long, thin, immature spines. Although these abnormalities have been correlated with impaired cognitive abilities in autistic and other syndromes, the causes of these malformations are not yet well understood. The formation and maintenance of dendritic spines require a very dynamic actin cytoskeleton, regulated by small GTPases such as Rac1. Previously, by creating a tissue‐specific Rac1 deficient mouse, our laboratory reported that Rac1 is (1) necessary for normal dendritic and spine development, (2) critical for hippocampus‐dependent learning and memory, and (3) essential for long‐term plasticity. Following up our previous findings, herein we demonstrate that ablation of Rac1 indeed leads to reduced spine density and altered actin remodeling pathway. Thus, regulation of Rac1 may provide a functional link among deficient neuronal morphology, aberrant synaptic plasticity and cognition impairment, phenotypes associated with certain autistic disorders.
Gender differences in the lateral pterygoid muscle: Implications for the etiology of temperomandibular disorder
Authors: Heller Aaron , Hessel Kara , Wright Barth
Published: June 2021

Women are twice as likely as men to develop temporomandibular disorder (TMD) and the lateral pterygoid muscle has been implicated in this pathology. We examined gender differences in lateral pterygoid muscle origin and insertion areas, and vector force magnitudes (VFM), which are the moment contributions from the superior and inferior head of the muscle given their 3D geometry and insertion site. In 12 male and 17 female cadavers, we used a microscribe to collect 3D outlines of the origin of the superior and inferior head and the mandibular insertion of the lateral pterygoid muscle, as well as 13 bony landmarks. Linear measurements of muscle and bone were calculated using Geomagic Studio v.11.0 software, origin and insertion areas were calculated with the program Area Centroid, and VFMs were calculated using the program Boneload. We scaled the VFMs and areas, with skull size using a geometric mean composed of 9 linear craniofacial measurements connecting the 13 bony landmarks. We found a significant increase in the adjusted VFM for the superior head of the lateral pterygoid in females (0.0875) compared to males (0.0808) (p=0.016). This suggests, given identical muscle loads, that the female superior head has a potential for generating stronger muscle forces than males. This may contribute to the propensity of females to develop TMD.
SETD6 lysine methylation of RelA couples GLP activity at chromatin to tonic repression of NF‐kB signaling
Authors: Levy Dan , Kuo Alex , Chang Yanqi , Schaefer Uwe , Kitson Christopher , Cheung Peggie , Espejo Alexsandra , Zee Barry , Liu Chih Long , Prinjha Rab , Garcia Benjamin , Bedford Mark , Tarakhovsky Alexander , Cheng Xiaodong , Gozani Or
Published: June 2021

Covalent modification of histones by protein lysine methyltransferases (PKMTs) influences diverse biological and disease processes. Greater than fifty PKMTs are predicted to be present in the human proteome; however, the catalytic activity and substrate specificity for the majority of these enzymes is unknown. Here, we perform a biochemical screen of over forty human candidate PKMTs and demonstrate that SETD6 (SET domain containing 6) monomethylates chromatin‐associated RelA at lysine 310 (RelAK310me1). RelA, a subunit of NF‐kB, a pivotal transcriptional regulator of inflammation, is repressed when methylated by SETD6. SETD6 attenuates RelA‐driven gene expression and cellular programs including inflammatory responses in primary immune cells. We identify the ankryin repeat of GLP (G9a‐like protein) as a recognition motif for RelAK310me1. SETD6‐dependent binding of GLP to RelAK310me1 promotes a repressed chromatin state at RelAK310me1‐occupied genes through GLP‐mediated H3K9 methylation. This repressive pathway is overridden by NF‐kB‐activation linked PKCZeta‐mediated phosphorylation of RelA at serine 311 (RelAS311ph), which blocks GLP binding to RelAK310me1 and drives target gene expression. Together, our findings identify SETD6 as a novel PKMT and regulator of NF‐kB signaling, and demonstrate a new paradigm for how integrated crosstalk between modifications on transcription factors and histones modulates key physiologic and pathologic programs.
Lipid mobilization and milk production in lactating northern elephant seals
Authors: Fowler Melinda , Debier Cathy , Costa Daniel , Crocker Daniel
Published: June 2021

Northern elephant seals (Mirounga angustirostris) incorporate fasting into several life history stages: post weaning development, molting, breeding and lactation. The mobilization and utilization of particular fatty acids may impact both the mother and pup. This study investigated the mobilization of fatty acids from blubber to understand partitioning of fatty acids between maternal use and milk in northern elephant seals. Blubber and milk fatty acid samples were taken from lactating northern elephant seals in early and late lactation. Overall, patterns of specific fatty acid mobilization conformed closely to previously established patterns in laboratory studies. Proportions of milk and blubber were found to display a high degree of similarity both early and late in lactation. Monounsaturated fatty acids (MUFAs) were the least mobilized from the blubber and increased in the proportion it comprised of the milk across lactation. Polyunsaturated fatty acids (PUFA) and saturated fatty acids (SFA) were more mobilized from the blubber, but proportions decreased in the milk. This suggests that of the MUFA mobilized, the majority is directed to milk synthesis and PUFA and SFA may be partitioned for mother's metabolism. MUFA designation to milk may impact milk viscosity to facilitate secretion from the mammary gland, as well as contribute to the development of diving and thermoregulatory capabilities in the pup.
The Effects and function of the Hypothalamic‐pituitary‐thyroid axis on fasting Northern elephant seals
Authors: Martinez Bridget , Sonanez Jose Guadalupe , Rodriguez Ruben , Viscarra Jose , Vasquez-Medina Jose Pablo , Ortiz Rudy M
Published: June 2021

Northern elephant seals sustain long bouts of food deprivation while remaining physically active with relatively high metabolic rates, and unlike hibernating mammals, they also remain normothermic. In terrestrial mammals, food deprivation is associated with reduced thyroid hormone (TH) levels and metabolism; however, elephant seals pups exhibit sustained, if not, elevated TH. The functional relevance of this apparent paradox is not understood. To address the hypothesis that increased TH levels in prolong‐fasted seals are associated with elevated TH receptor expression, we measured the changes in fasting‐induced TH levels and the mRNA expression of the TH receptor, THrβ‐1, in muscle and blubber. To better ascertain the relevance of the changes in THrβ‐1, we also measured the protein content and mRNA expression of products associated with changes in THrβ‐1. Preliminary data shows almost a three‐fold increase in THrβ‐1 gene expression in both muscle and blubber and we observed an elevation in tT3, tT4, and fT4 over the course of the fast decrease in total T3:T4 ratio over the 49 days. Furthermore, both Deiodinase 1 and 2 increased in muscle tissue suggesting an increase in availability of t3 to meet the higher demands of an increase in the receptor. It would be pertinent to attempt to clarify the mechanism of the hypothalamic‐pituitary axis and its overall function and effect on metabolism.
Neuromuscular electrical stimulation increases muscle protein synthesis rates in type 2 diabetic men
Authors: Burd Nicholas A. , Wall Benjamin T. , Dirks Marlou L. , Verdijk Lex B. , Snijders Tim , Hansen Dominique , Senden Joan M. , Vranckx Pascal , Dendale Paul , van Loon Luc J.C.
Published: June 2021

A loss of skeletal muscle proteins ultimately lowers the capacity for blood glucose disposal. Thus, the development of paradigms that can increase muscle protein accretion will have implications for the treatment of type 2 diabetes at a more advanced age. We aimed to determine the effect of neuromuscular electrical stimulation (NMES) on mixed muscle protein synthesis rates in type 2 diabetic men. Six sedentary, type 2 diabetic men (Age: 70±1 y, BMI: 26.5±1.0 kg·m−2) received a primed, constant infusion of L‐[ring‐13C6]phenylalanine and 60 min of one legged NMES. Biopsies of the vastus lateralis were obtained at 0, 2, and 4 h after NMES to measure mixed muscle protein synthesis rates in the unstimulated (CON) and stimulated (STIM) leg. The electrical current of the NMES protocol consisted of a pulse duration of 500 μs, pulse frequency of 60 Hz, and a 3 sec contraction – 3 sec relaxation time. Muscle protein synthesis rates were increased by 21% and 64% above CON at 0–2 h and 2–4 h in the STIM leg, respectively (P<0.05). Similarly, the cumulative MPS response (0–4 h) was greater in the STIM (0.049±0.008 %·h−1) compared with the CON leg (0.039±0.008 %·h−1). Our data demonstrate that NMES stimulates mixed muscle protein synthesis rates in vivo in humans. This work lends credence to the use of NMES to attenuate muscle protein loss during periods of muscle disuse or to support an increase in skeletal muscle hypertrophy.
Epigallocatechin Gallate Inhibits Endothelin‐1 Stimulation of 3T3‐L1 Adipocyte Glucose Uptake
Authors: Liao Kuo-Yu , Hsu Yung-Chia , Huang Yao-Ming , Ho Low-Tone , Wu Liang-Yi , Kuo Yow-Chii , Kao Yung-Hsi
Published: June 2021

Objective

This study was designed to investigate whether epigallocatechin gallate (EGCG) modulated the acute (30 min) and chronic (6 h) effects of endothelin (ET)‐1 on glucose uptake and glucose transporters in 3T3‐L1 adipocytes.

Methods

Glucose uptake was assayed to measure the uptake of 3H‐2‐deoxyglucose. Western blot analysis was performed to measure levels of Gluts and ET‐1 signaling molecules.

Results

EGCG at 10 μM for 2 h was found to inhibit the acute effects of ET‐1 on glucose uptake and the translocation of glucose transporter (Glut)‐4 from the cytosol to the plasma membrane. EGCG had no effects on the translocation of Glut1 and total levels of Glut‐1 and Glut‐4 proteins. But, we observed that EGCG prevented the chronically ET‐1‐increased levels of glucose uptake and Glut‐1 protein expression, and it had no effects on the ET‐1‐altered levels of Glut‐1 and Glut‐4 translocation. The actions of ET‐1 were shown to be mediated through the extracellular signal‐regulated kinases (ERKs) and protein kinase B (PKB) pathways, but signaling was demonstrated to be prevented by EGCG pretreatment.

Conclusions

These data suggest that EGCG mediates the acute and chronic effects of ET‐1 on 3T3‐L1 adipocyte glucose uptake via the respective alteration of Glut‐4 translocation and Glut‐1 protein levels and via the ERK and PKB pathways.
Dual Regulation of Glucose Homeostasis by the Adaptor Proteins Nck
Authors: Dusseault Julie , Latreille Mathieu , Larose Louise
Published: June 2021

Nck1 and Nck2 adaptor proteins mediate intracellular signaling from activated plasma membrane receptor tyrosine kinases. Previous studies reported that the Nck expression levels vary with insulin sensitivity, suggesting a role for Nck in regulating insulin signaling. We previously showed that Nck1−/− mice fed a high fat diet (HFD) display better glucose disposal ability and hepatic insulin signaling compared with HFD insulin resistant Nck1+/+ littermates. In fact, we found that Nck1 interacts with and mediates signaling from IRE1α, an important component of the Unfolded Protein Response that inhibits insulin signaling by inducing JNK‐dependent phosphorylation of the insulin receptor substrate 1 (IRS‐1) on an inhibitory site. In contrast, we observed that Nck2−/− mice fed a normal chow diet (NCD) or a high fat diet (HFD) display greater glucose intolerance and insulin resistance compared to Nck2+/+ littermates mice. In agreement, we found decreased insulin‐induced hepatic Akt phosphorylation/activation in NCD Nck2−/− mice compared with their littermate controls. All together, our data provide strong evidence that the two Nck adaptors have distinct role in modulating insulin signaling in vivo. Further studies are in progress to define the mechanisms by which Nck differentially regulate glucose homeostasis. Work supported by CIHR and CDA. JD holds a FRSQ postdoctoral fellowship.
Quantum dots(QD) ‐ based nanobiosensor for intracellular glucose ([glu]i) measurements
Authors: Wang Yuchi , Mao Hua , Wong Lid
Published: June 2021

To enable the first [glu]i measurements in cells, the high quantum yield, super‐luminous and photobleaching resistant QD were used to develop a glucose nanobiosensor based on Forster Resonance Energy Transfer (FRET). The [QD‐conA]‐[B‐cyclo‐dextrins‐Au] nanosensor comprises of CdSe as a FRET donor at 640 nm, a 5 um AuNP as a FRET acceptor and concanavalin A (con A)‐B‐cyclo‐dextrins as a glucose binding receptor. When glucose are bounded to con A, it displaces the B‐cyc‐Au as a FRET acceptor by increasing the FRET distance. This restores the quenched QD fluorescence to produce a [glu] dose‐dependent emission. The spectral fluorescence of glu‐QD was calibrated using a 460 nm excitation and 90 degree forward scattering configuration. The respective fluorescence (a.u.) measurements at 0 and 20 mM were 22 and 320000 with a remarkable log‐log linearity in between [glu]. We have also applied the glu‐QD to measure [glu]i in HEK‐293F cells. The glu‐QD nanosensor was loaded into the HEK‐293F cells using a cationic liposome loading protocol. Fluorescence measurements were made using a photon‐counting, inverted fluorescence microscope. As predicted, 10 uM forskolin increased [glu]i of HEK‐293F cells by 30% compared to baseline. This induced increases in [glu]i was inhibited by subsequent application of 1 mM cytochalasin B, a GLUT inhibitor. Dynamic cellular measurements of [glu]i can now be made with this glu‐QD.
The Biochemistry of hemolymph and the concentration of some heavy metals in the exoskeleton of the Helix aspersa snail in two localitites in Kosovo
Authors: Grapci Linda , Bislimi Kemajl , Bojniku Hazbije , Gashi Agim , Halili Jeton , Mazreku Ilir , Bajraktari Ismet , Halili Fetah
Published: June 2021

The concentration of Pb, Cd, Zn and Fe in the soil, their bioaccumulation in the exoskeleton of the Helix aspersa snail, as well as the concentration of total proteins and hemocyanin in its hemolymph was researched.

Snails of the (N=24) sample group were collected in the close vicinity of the “Sharrcem“ Hani i Elezit (N=24) cement factory whereas, those of the (N=24) control group were taken in the village of Zhur near Prizren. The weight of the snails was approximately the same.

The analysis of heavy metals was done with the Perkin Elmer – ICP OES Optima 2100 DV equipment whereas, the concentration of hemocyanin has been indirectly measured through copper with the sodium diethyldithiocarbamate.

The research results show that compared to the location of Zhur, the concentration of lead, cadmium, iron and zinc (ppm) in the soil samples and the exoskeleton of snails from the contaminated location (Sharrcem) snails, are 2–6.5 times higher whereas, the quantity of total proteins (P<0.01) and the hemocyanin, (P<0.05) ) in their hemolymph, is significantly lower.

The preliminary analysis of the results show that the biochemistry of the hemolymph depends on the accumulation level of heavy metals in the orgranism, while exoskeleton of the snail Helix aspersa has a higher capacity for the accumulation of Zn (about 5–20 times more than its concentration on the soil), but not for the Pb, Cd and Fe.
Postural change during hyperthermia modifies thermal perception in men
Authors: Takeda Ryosuke , Okazaki Kazunobu , Imai Daiki , Suzuki Akina , Naghavi Nooshin , Matsumura Shinya , Miyagawa Toshiaki
Published: June 2021

Increase in skin and esophageal temperature (Tsk and Tes) enhances heat dissipative responses and induces behavioral thermoregulatory responses with the perception of both temperatures. We assessed whether the perception of both temperatures was modified with postural change from supine (SUP) to sitting (SIT) during hyperthermia (HT) which attenuates heat dissipative responses.

Methods

Eleven healthy young men underwent measurement of noticeable increase/decrease (± 0.1°C/sec) of skin temperature (warm/cold threshold) at forearm and chest by using a thermode(6.25 cm2), and of subjective thermal sensation (visual analogue scale) in SUP and SIT in normothermia (NT, Tes; 36.6 ± 0.2°C) and HT (Tes; 37.3 ± 0.1°C, lower legs immersion in 42°C water). Tes, Tsk, cutaneous vascular conductance (CVC) and sweat rate (SR) at forearm and chest were measured continuously.

Results

Cold threshold at both sites were decreased and subjective thermal sensation, CVC and SR were increased in HT compared with NT (P < 0.05). In HT, cold threshold at forearm was decreased and subjective thermal sensation was increased while CVC at forearm was decreased in SIT compared with SUP (P < 0.05). Warm threshold at both sites remained unchanged.

Conclusions

Postural change from SUP to SIT during hyperthermia attenuates cold threshold and enhances subjective thermal sensation which is accompanied by attenuated heat dissipative responses.
Quantifying heat balance components in neonates nursed under radiant warmers in neonatal intensive care
Authors: Molgat-Seon Yannick , Daboval Thierry , Chou Shirley , Jay Ollie
Published: June 2021

Neonatal intensive care units use radiant warmers (RW) to maintain stable body temperatures in many newborns. This study examined whether the rate of radiant heat required for heat balance (Rreq) is equal to the rate of radiant heat provided by the RW (Rprov). A systematic evaluation of time‐dependent changes in heat balance components was conducted in 10 newborns (mass: 2829±636 g; age: 9.2±11.0 days; BSA: 0.19±0.03 m2) nursed under RW. Metabolic rate, evaporative heat loss, convective and conductive heat flow, rectal temperature (Trec) and mean skin temperature (Tsk) were measured continuously for 105 min. The rate of body heat storage (S) was calculated using a two‐compartment model of ‘core’ (Trec) and ‘shell’ (Tsk) temperatures. Mean Rprov (1.28±2.59 W) and Rreq (1.25±2.51W) were not significantly different (P=0.63). However, while the resultant mean change in body heat content after 105 min was low (+0.46±2.79 kJ) and not significantly different from zero (P>0.05), an acute time‐dependent change in body heat storage was evidenced by a mean positive heat storage component of +6.64±2.98 kJ and a mean negative heat storage component of −6.17±2.64 kJ. Accordingly, the mean difference between maximum and minimum values for Trec and Tskwere 0.71±0.37°C and 2.18±0.81°C respectively. In conclusion, while RW maintain astable core temperature over a prolonged period, they induce acute bouts of heat imbalance.

Supported by a NSERC Discovery Grant (O. Jay) & a CHEO Dept. of Surgery Research Grant (S. Chou).