bluuebunny/crossref_metadata_2025 huggingface dataset:
High fat diet‐induced obesity promotes cerebrovascular autoregulatory dysfunction in aged mice
Authors: Toth Peter , Tucsek Zsuzsanna , Bailey-Downs Lora , Sosnowska Danuta , Gautam Tripti , Sonntag William E. , Ungvari Zoltan , Csiszar Anna
Published: June 2021

Diet‐induced obesity in the elderly promotes the development of vascular cognitive impairment, which develops in the absence of Alzheimer‐type senile plaques as a result of decreased cerebral blood flow due to vascular alterations. To test the hypothesis that obesity in aging impairs local vasoregulatory mechanisms in cerebral arteries, young (3 mo) and aged (24 mo) C57BL/6 mice were fed a high fat diet (HFD) or a standard diet (SD) for 6 months. In middle cerebral arteries (MCA) isolated from obese aged mice stepwise increases in transmural pressure (from 10 to 160 mmHg) elicited impaired steady‐state myogenic constriction as compared to responses obtained in vessels of young SD‐ and HFD‐fed and aged SD‐fed mice. MCAs from aged obese mice also exhibited impaired dynamic myogenic responses and flow‐induced constriction. Furthermore, obesity impaired metabolic vasodilation in response to adenosine in young mice, whereas its effect was exacerbated in aged mice. Collectively, aging exacerbates obesity‐induced cerebrovascular autoregulatory dysfunction, which likely promotes microvascular damage contributing to the development of vascular cognitive impairment in aged obese individuals. (Funding: AHA, AFAR, NIH).
Na+/H+‐exchanger‐1 inhibition delays diabetic cataract formation and prevents retinal apoptosis and oxidative stress
Authors: Lupachyk Sergey , Drel Viktor R , Stavniichuk Roman , El-Remessy Azza B , Obrosova Irina G
Published: June 2021

Na+‐H+‐exchanger‐1 (NHE‐1) controls intracellular pH and glycolytic enzyme activities, and its expression and activity are increased by diabetes. We evaluated the role for NHE‐1 in diabetic cataract formation and retinal oxidative‐nitrosative stress and apoptosis. Control and STZ‐diabetic rats were treated with/without the NHE‐1 inhibitor cariporide (C, Sanofi‐Aventis, 10 mgkg−1d−1) for 1(lens biochemistry) and 3.5 mo. In in vitro studies, bovine retinal pericytes and endothelial cells were cultured in 5 or 30 mM glucose, with or without 10 μM C, for 7 d. C treatment delayed, but did not prevent, diabetic cataractogenesis. This was probably related to inhibition of oxidative‐nitrosative stress in the lens. Diabetes‐induced accumulation of glucose and sorbitol pathway metabolites was not affected. The number of TUNEL positive cells was increased 4.4‐fold in the retina of diabetic rats, and this increase was attenuated, but not completely prevented, by C. Nitrotyrosine fluorescence and TUNEL positivity were increased in pericytes and endothelial cells cultured in 30 mM glucose, and both nitrosative stress and cell death were at least partially prevented by C. In conclusion, NHE‐1 contributes to diabetic cataract formation and retinal oxidative‐nitrosative stress and apoptosis. The findings identify a new therapeutic target for diabetic ocular complications.
Nuclear transcription factor kappa B (NF‐kB) mediates ROS and PKC‐induced decrease in TRPC6 protein expression in human glomerular mesangial cells (HMCs)
Authors: Ma Rong , Wang Yanxia , Ding Min
Published: June 2021

We recently demonstrated that high ambient glucose significantly decreased the expression of TRPC6 at protein level in cultured HMCs through a ROS/PKC pathway. The present study was carried out to examine if NF‐κB is a downstream molecule of ROS/PKC in the pathway of suppressing TRPC6 protein expression in HMCs. Serum‐starved HMCs were treated with 200 μM H2O2 for 6 hours or with 1 μM PMA for 2 hours significantly reduced TRPC6 protein expression. These ROS and PKC effects were significantly prevented by pre‐treating the cells with NF‐κB activation inhibitor at 10 μM for 30 min. Both H2O2 and PMA, but not PMA inactive analog 4α‐PDD phosphorylated the inhibitory κBα (IκBα) and stimulated the degradation of IκBα in a time dependent manner. Furthermore, activation of NF‐κB by knockdown of IκBα using siRNA significantly reduced abundance of TRPC6 protein in HMCs, a reminiscent of H2O2 and PMA effect on TRPC6 protein expression. Importantly, either H2O2 or PMA treatment dramatically increased nuclear expression of NF‐ κB p65/p50 subunits in a time dependent manner in HMCs, suggesting activation of NF‐κB in response to both ROS and PKC activation. These results suggest that NF‐κB is a mediator downstream ROS and PKC for suppression of TRPC6 expression in HMCs.

(Supported by NIH)
Renin Production and Release Associated with Cyclic ADPRibose‐ Mediated Signaling: Evidence from CD38 Gene Knockout Mice
Authors: xiong jing , Xia Min , Boini Krishna , Li Pin-Lan
Published: June 2021

Despite extensive studies, the intracellular regulatory mechanism of renin production and release is still poorly understood. The present study was designed to test whether CD38‐ADP‐ribosylcyclase signaling pathway contributes to the regulation of renin production and release. We found that the expression of CD38 and the activity of ADP‐ribosyl cyclase to produce cyclic ADP‐ribose (cADPR) were nearly abolished in the kidney from CD38−/− mice, indicating that CD38 gene is a major enzyme responsible for the generation of cADPR in vivo. Mice lacking CD38 gene showed increased plasma renin activity (PRA) in either conscious or anesthetized state (P<0.05). Low salt intake markedly increased, but high salt intake significantly decreased renin release in both CD38+/+and CD38−/− mice. Compared to low salt intake, the decrease in PRA in response to high salt intake in CD38−/−mice was much larger than CD38+/+ mice. In acute experiments, isoprenaline infusion and renal perfusion pressure (RPP) reduction were used to test renin release in both mouse stains. It was demonstrated that PRA increased upon isoprenaline infusion in CD38−/− mice by 3 folds compared to CD38+/+ mice. Accompanied with such increase in PRA, glomerular filtration rate (GFR), renal blood flow (RBF), urine volume (UV) and sodium excretion (UNa) more significantly decreased in CD38−/− than CD38+/+mice. Similarly, more increases in PRA but more decreases in GFR, RBF, UV and UNa were observed in CD38−/− than CD38+/+mice when they had a low renal perfusion pressure (RPP). Finally, it was found that increase in RBF induced by intravenous injection of losartan (20 mg/kg) in CD38−/− mice with high PRA was significantly attenuated compared to CD38+/+ mice. In conclusion, the CD38‐cADPR‐mediated signaling may importantly contribute to the maintenance of low PRA and thereby participates in the regulation of renal hemodynamics and excretory function (Supported by NIH grants HL‐091464, HL‐75316 and DK54927).
Inhibition of medullary prostaglandin E2 (PGE2) by non‐steroidal anti‐inflammatory drugs (NSAIDs) adversely affects medullary blood flow
Authors: Kennedy-Lydon Teresa M , Crawford Carol , Sawbridge Liam , Unwin Robert J , Wildman Scott SP , Peppiatt-Wildman Claire M
Published: June 2021

NSAIDs are known to be nephrotoxic and act to inhibit cyclooxygenases (COX) and thus production of PGE2. Here we use the kidney slice model to investigate (i) the effect of PGE2 on in situ vasa recta pericytes (ii) the ability of PGE2 to attenuate ET‐1 and Ang‐II‐evoked vasoconstriction of vasa recta, (iii) the effect of NSAIDs on in situ vasa recta pericytes, and (iv) whether NSAIDs attenuate PGE2 and bradykinin‐mediated vasa recta dilation. Kidney slices were collected as previously described1. Real‐time images of in situ vasa recta were recorded and vasa recta diameter at pericyte and non‐pericyte sites measured off‐line. PGE2 evoked dilation of vasa recta specifically at pericyte sites and significantly attenuated pericyte‐mediated constriction of vasa recta evoked by both ET‐1 and Ang‐II. NSAIDs, indomethacin>SC560> celecoxib>meloxicam, evoked significantly greater constriction of vasa recta capillaries at pericyte sites than at non‐pericyte sites and indomethacin significantly attenuated the pericyte‐mediated vasodilation of vasa recta evoked by both PGE2 and bradykinin. These data indicate PGE2 is key in attenuating vasa recta diameter and thus medullary blood flow (MBF). Inhibition of PGE2 that occurs during chronic NSAID treatment causes constriction of vasa recta by pericytes. Thus pericytes may be key in NSAID‐evoked reduction in MBF and any ensuing nephrotoxicity.
Boldine treatment ameliorates kidney damage in rats with 5/6 nephrectomy chronic renal failure
Authors: Velarde Victoria , Gomez Gonzalo , Cespedes Carlos , Vio Carlos P
Published: June 2021

Chronic renal failure (CRF) consists in a progressive decrease in renal function, and morphological alterations. Current therapies only slow the progression of the disease. Oxidative stress is increased in CRF, and we propose that Boldine, a natural product, antioxidant and inhibitor of connexin hemichannels, could ameliorate the adverse effects associated to oxidative stress in CRF.

Rats with CRF (5/6 nephrectomy)were treated with boldine (50mg/kg/day, gavage) for 28 days. Kidney function was evaluated by urinary protein/creatinine (UProt/UCr) and oxidative stress by thiobarbituric acid reactive substances (TBARS). Markers of kidney damage (Col III, ED‐1, FN and α‐SMA) and connexins (Cx) 43 and 37 were evaluated by Western Blot and immunohistochemistry.

In CRF+Boldine rats the ratio Uprot/Ucr (4.5±1.3 vs 13.3±4.1, p< 0.05), TBARS (39.5±3.7 vs 62.4 ±1.1 nmol/g, p< 0.05) and the levels of Col III, ED‐1, FN and α‐SMA were lower than in CRF rats. Cx‐43, increased in CRF rats and slightly decreased in CRF + Boldine; on the other hand, CX‐37, was high in control rats and decreased in CRF and CRF + boldine rats to undetectable levels.

These results suggest that boldine protects the kidney in rats with chronic renal failure induced by 5/6 nephrectomy, through a mechanism under study, and that in the future, boldine could be used as a nutraceutic for CRF.

Funded by Grants Puente VRI 2/2011 and FONDEF D07I1086
Roles of BCL‐2/BCL‐XL in regulation of BCR‐ABL‐induced mitochondrial oxidative stress
Authors: Zhang Hui , Chen Gang , Lu Weiqin , Liu Terrence , Chen Zhao , Trachootham Dunyaporn , Arlinghaus Ralph B. , Huang Peng
Published: June 2021

Up‐regulation of BCL‐2/BCL‐XL in chronic myeloid leukemia (CML) has been observed in numerous studies. Besides cell survival function, recent studies have indicated that BCL‐2/BCL‐XL can regulate cellular redox balance. Previous studies have identified that BCR‐ABL elevates mitochondrial reactive oxygen species (ROS), and results in the persistent oxidative stress in CML cells. However, the efficiency of BCL‐2/BCL‐XL in preventing oxidative stress‐induced cell damage and their contribution in BCR‐ABL‐induced mitochondrial ROS generation are still unknown. The goal of this study is to investigate the role of BCL‐2/BCL‐XL in regulating mitochondrial ROS and preventing oxidative stress‐induced mitochondrial damage in CML. To achieve this, BCR‐ABL inducible cells, BCR‐ABL stably transformed cells, Human CML cells and a CML cell model with heterogenic BCL‐2/BCL‐XL expression were used. In this study, BCL‐XL has been identified as the major survival factors responsive to cellular oxidative stress. BCL‐2/BCL‐XL differentially protects mitochondrial integrity under oxidative stress. BCL‐2 but not BCL‐XL plays a role in maintaining mitochondrial ROS generation. These data together demonstrate that BCL‐2 and BCL‐XL play important but differential roles in regulating BCR‐ABL‐induced mitochondrial ROS and protecting mitochondria against oxidative stress.
Novel KCC3 mouse models to study ACCPN, a degenerative neuropathy disorder
Authors: Ding Jinlong , Ponce-Coria Jose , Delpire Eric
Published: June 2021

Among the K‐Cl cotransporters, KCC2 and KCC3 are expressed in the central nervous system where they play an important role in modulating GABA neurotransmission by lowering the intracellular neuronal Cl− concentration. Mice with KCC3 deletion are viable but exhibit a locomotor deficit due to a peripheral neuropathy called ACCPN (or Agenesis of Corpos Callosum with Peripheral Neuropathy). In order to better define the contribution of KCC3 function in the development of ACCPN, we created two novel mouse models. The first model is a knock‐in mouse harboring a single amino acid substitution (E289G), producing a full‐length but non‐functional protein. Surprisingly, this new mutant mouse was non‐viable. Genotyping of embryos and blastocysts demonstrated absence of homozygous E289G mice as early as E3.5. As both males and females were able to produce heterozygous E289G animals, these data indicate that the mutation might affect cell division and/or migration. The second mouse model allowed for tissue‐specific deletion of KCC3 through CRE recombination. To target deletion of KCC3 in specific cell types, we mated KCC3 floxed mice with a variety of transgenic CRE lines. Deletion of KCC3 in small sensory neurons, driven by the Nav1.8 Na+ channel promoter, resulted in mice that exhibited a small but significant motor coordination phenotype.
Caveolin‐3 regulates subcellular targeting of Kir2.1 channels
Authors: Yang Zhaogang N/A , Hu Keli
Published: June 2021

Inwardly rectifying potassium channels, Kir2 in particular, are crucial in shaping the action potential and stabilizing the resting potential. However, little is known about the subcellular localization of the Kir2.1 channel, an important component of the background current, IK1, in cardiac myocytes. The present study was designed to determine whether Kir2.1 channels are localized to the caveolar plasma membrane and regulated by caveolin‐3. We found that about 40% (43.81±3.75%, n=3) of the cellular Kir2.1 was retained in the caveolin‐enriched fractions in the adult rat cardiomyocytes. In contrast, clathrin heavy‐chain was detected across a broad range of the gradient fractions. Cholesterol was shown to be enriched in the cavelin‐rich fractions. Co‐immunoprecipitation from cardiac myocytes indicated that Kir2.1 was associated with caveolin‐3. Expression of caveolin‐3 in HEK293T cells transfected with Kir2.1 significantly increased Kir2.1 level in the caveolin‐enriched fraction by 60% (63.26±8.33%, n=3). Further, immunofluorescent staining showed that a significant portion of Kir2.1 colocalized with caveolin‐3 in both transfected HEK293T cells and cardiomyoctes. In conclusion, we demonstrate that a significant portion of Kir2.1 channels targets to the caveolin‐enrich plasma membrane of rat cardiac myocytes, and the caveolae‐targeting of recombinant Kir2.1 channels is regulated by caveolin‐3.
N‐type Ca2+ channel regulation by the light chain 1 (LC1) of the microtubule associated protein B (MAP1B)
Authors: Gandini María Alejandra , Henríquez Daniel R. , Sandoval Alejandro , González-Billault Christian , Felix Ricardo
Published: June 2021

MAP1B is a protein complex consisting of heavy and light (LC) chains that play an important role in regulating neuronal morphogenesis and function. Recent studies have shown that LC1 interacts with NMDA and HT5 receptors at the postsynapsis regulating their trafficking and surface expression. However, much less is known regarding its role at the presynaptic level where voltage‐gated N‐type Ca2+ channels couple membrane depolarization to neurotransmitter release. Here, using a strategy that combines electrophysiology with biochemical and molecular biology techniques, we investigated whether LC1 interacts with the N‐type channel and whether this interaction has any physiological significance. Hence, using confocal immunofluorescence, co‐localization of these proteins was revealed in hippocampal cells from neonatal mouse, and their interaction was confirmed by pulldown assays using whole brain homogenates. Likewise, heterologous expression of recombinant N‐type channels and LC1 in HEK‐293 cells revealed a significant decrease in whole cell current density without apparent changes in current kinetics, suggesting a diminished number of functional channels in the cell surface. Furthermore, pulldown experiments as well as confocal microscopy showed an interaction between LC1 and the channel in the heterologous system. These results reveal a functional coupling between LC1 and the N‐type channel.

This work was supported by Conacyt‐Conicyt binational cooperation program.
Acid induces TRPV4‐mediated calcium influx in mouse esophageal keratinocytes
Authors: Berrout Jonathan , Luo Jialie , Hu Hongzhen , O'Neil Roger G
Published: June 2021

Exposure of the inner esophageal epithelial layer to an acidic environment has long been held as a major component of gastroesophageal reflux disease. The study aimed to determine if TRPV4, a Ca2+‐permeable channel, and Ca2+ signaling may play a role in acid sensing as part of this process. Primary culture mouse esophageal keratinocytes were grown to confluency on coverslips and intracellular Ca2+, [Ca2+]i, monitored with the fluorescent indicator, Fura 2‐AM, via microscopic imaging. In control WT cells, addition of GSK1016 790A (30 nM), a selective TRPV4 agonist, or exposure of cells to hypotonic media, a stimulus known to activate TRPV4, induce a rapid elevation in [Ca2+]i. These responses were abolished in cells cultured from TRPV4 knockout mice, thereby demonstrating functional TRPV4 channels in the keratinocytes. In separate studies, cells exposed to low pH media showed little or no [Ca2+]i response to pH 6 media, a modest response to pH 5 media, and a strong response to pH 4 media. However, in cells cultured from TRPV4 knockout mice, the response to all pH states was largely abolished. We conclude that espophageal keratinocytes express TRPV4 and that this channel is activated by acidic pH (pH 4 – 5) and, thus, may be part of the mechanism underlining gastroesophageal reflux disease.
Inhibition of Epithelial Sodium Channel Activity by Inter‐α‐Trypsin Inhibitor
Authors: Ness Emily Christine , Lazrak Ahmed , Jackson Justin M , Garantziotis Stavros , Matalon Sadis
Published: June 2021

Entry of sodium (Na+) ions in lung epithelial cells through amiloride sensitive Na+ channels (ENaC) is the first and limiting step in vectorial Na+ transport, a crucial step in lung fluid homeostasis. Previous studies have shown that serine protease inhibitors decrease ENaC activity by inactivating channel‐activating proteases. Inter‐α‐Trypsin Inhibitor (IαI) is a serum protease inhibitor which is present in the alveolar spaces following lung injury. We therefore tested whether IαI decreased ENaC activity. In the first series of experiments we measured the open probability (Po) of ENaC channel in alveolar epithelial cells of mouse lung slices by patching them in the cell attached mode. Incubation of lung slices with IαI (0.1 mg/ml) for 2 h decreased the Po of both ENaC and cation channels by about 50%. Incubation of lung slices with an antibody against IαI, had no effect. In the second set of experiments, we incubated Xenopus laevis oocytes, injected with human α, β, and γ‐ENaC cRNAs for two days, with IαI and measured amiloride‐sensitive current‐voltage relationships. IαI (0.1 mg/ml) decreased amiloride‐sensitive ENaC currents by about 70% with a half‐time of inhibition of about 12 min. Trypsin (2 μM), added after IαI, reversed the effects of the IαI indicating that IαI decreased the number of active ENaC channels but not total protein levels. These data suggest, that IαI may contribute to ENaC down‐regulation in various forms of inflammatory and oxidative lung injury.
Influenza M2 Inhibits CFTR Activity through its Ion Channel Function
Authors: Londino James David , Atkins Colm , Noah James , Lazrak Ahmed , Matalon Sadis
Published: June 2021

We previously reported that co‐expression of the Influenza M2 hydrogen (H+) channel, and CFTR in Xenopus oocytes downregulates CFTR activity and expression. We now hypothesize that the H+ion channel activity of M2 decreases vesicular pH which in turn alters the maturation or degradation of CFTR. To test this hypothesis we constructed M2 mutant cRNAs containing bulky amino acid side chain substitutions blocking H+ transport. When injected in Xenopus oocytes, these cRNAs expressed similar levels of protein as the wild type (wt) (determined by western blotting) and trafficked to the plasma membrane (confirmed by immunoluminescence). When these mutant cRNAs were co‐expressed with CFTR, little to no decrease in cAMP‐activated, glyH‐101 sensitive, Cl‐ currents were observed. The decrease in total membrane protein was also significantly attenuated. Next we made an M2 cRNA endoplasmic reticulum (ER) targeted construct which does not reach the plasma membrane (Sakaguchi et al. 1996). No decrease in CFTR activity was observed when coexpressed with this construct. Further, incubation of CFTR cRNA injected Xenopus oocytes with ammonium chloride, which increases the pH vesicular organelles, significantly decreased CFTR activity in a dose dependent manner. Overall, these studies suggest that activation of M2 by acidic organelles, and the subsequent alkalization of vesicular pH, is essential to CFTR downregulation.

Funding provided by NIHR01‐HL031197
Modulation of ciliary beat frequency, not bend angle, by phosphodiesterase 1 during β2‐agonist stimulation in mouse bronchiolar ciliary cells
Authors: Nakahari Takashi
Published: June 2021

The ciliary beating of the bronchiole plays a key role in the mucociliary transport. Bronchiolar ciliary cells were isolated from lungs of mice (C57Bl/6J) by an elastase treatment and their beating cilia were observed by a high‐speed camera (500Hz). A β2‐agonist, procaterol, increases ciliary beat frequency (CBF) and ciliary bend angle (CBA) mediated via cAMP. Procaterol consentration‐response studies demonstrated that the CBA curve shifts to a low concentration from the CBF curve; 10 pM procaterol increases CBA, but 10 nM procaterol increases CBA and CBF. However, isobutylmethylxanthine (IBMX) induced no shift of the CBA curve, suggesting that different phosphaodiesterase (PDE) subtypes regulate cAMP concentration in microdomains regulating CBA and CBF. A PDE1 inhibitor (8‐methoxymethyl IBMX) increased CBF, but not CBA. The procaterol actions were examined in ionomycin (IM)‐stimulated cells, because PDE1 is Ca2+‐dependent. Procaterol (10 nM) increased CBA, but not CBF during IM stimulation. Previous studies revealed that the outer dyneins control CBF and the inner dyneins control CBA. Immunoelectronmicroscopy demonstrated that PDE1 is detected around nine doublet tubles in the bronchiolar cilia. In conclusion, in bronchiolar cilia, PDE1 is suggested to control cAMP accumulation in the microdomain regulating outer dyneins in the axoneme.
Activation of ciliary beating by a mucolytic agent, ambroxol, in bronchioles of mice
Authors: Suzuki Chihiro , Matsumura Hitoshi , Shimamoto Chikao , Nakahari Takashi
Published: June 2021

Ambroxol has been used as a mucolytic agent in pulmonary diseases. In this study, we studied the effects of ambroxol on ciliary beat frequency (CBF) and ciliary bend amplitude (CBA) of the bronchioles. Mice were anestetized by pentobarbital sodium (70mg/kg, ip) and bronchiolar ciliary cells were isolated from lungs by an elastase treatment. In a HCO3−‐containing solution, ambroxol increased CBA and CBF in a concentration dependent manner: ambroxol (10μM) increased CBA and CBF by 30 % within 15 min. However, in a HCO3−‐free solution, it did not induce any increase in CBA and CBF, although the switch from the HCO3−‐containing solution to the HCO3−‐free solution increased them. Measurement of intracellular pH (pHi) demonstrated that ambroxol (10 μM) increased pHi gradually in bronchiolar ciliary cells. These suggest that an increase in pHi induced by ambroxol increases CBA and CBF. To confirm the effects of pHi on CBA and CBF, an NH4+ pulse was applied. An increase in pHi increased CBA and CBF, whereas a decrease in pHi decreased them. Diisothiocyanostilbene‐disulfonic acid (DIDS) abolished the increases in CBA and CBF induced by ambroxol. In bronchiolar ciliary cells, ambroxol increases pHi via activation of HCO3−/Cl− exchanges, which increases CBA and CBF.
Inflammatory gene expression in BMDM macrophages in response to fungal metabolites.
Authors: Stallworth Akida , Osei-Bonsu Daniel , AZIM ANSER C
Published: June 2021

PU.1, a member of the Ets family of transcription factors, is a requirement for the proper differentiation of hematopoietic cells during developmental stages. Toll like receptors, a member of the family of pattern recognition receptors (PRRs) bind and recognize LPS triggering a signaling cascade with known activation of the NF‐kappa B canonical pathway to regulate genes involved in microbial clearance. Of the thirteen human TLRs, TLR 2 and TLR 4 have also been shown to recognize both bacteria and fungi and generate a inflammatory response producing COX‐2 and nitric oxide and a variety of cytokines and chemokines including IL‐1 and TNF‐α. Recently we have shown its activation in response to lipopolysaccharide bacterial endotoxin (LPS) in the regulation of pro‐inflammatory gene expression like cyclooxygenase 2. Aspergillus niger and Penicillium notatum both opportunistic fungi, have been shown to have clinical pathologies in immunocomprimised patients which enter the host through spore inhalation. We hypothesize that the fungal metabolites from Aspergillus niger and Penicillium notatum bind and signal through TLR 2 and TLR 4 in both the Raw 264.7 and bone marrow derived macrophage (BMDM) cell which leads to a two fold up‐regulation of COX‐2 protein, nitric oxide synthase (NOS), and tumor necrosis factor alpha (TNF‐α). In addition we believe that there is a link between the expression of these pro‐inflammatory mediators involving transcription factor PU.1, whose regulation is controlled by NIK, in response to the fungal metabolites as has been shown previously in response to LPS from our earlier work.
Annexin A7 interactions with SNARE protein SNAP23 are regulated by calcium and protein phosphorylation during surfactant secretion in alveolar type II cells
Authors: Chander Avinash , Gerelsaikhan Tudevdagva , Vasa Pavan Kumar
Published: June 2021

We have previously postulated that annexin A7 (A7) facilitates the obligatory membrane fusion between lamellar bodies and plasma membrane during surfactant secretion in alveolar type II (T2) cells. Recently, we reported that several secretagogues of lung surfactant promote protein kinase‐dependent co‐localization of A7 with the lamellar body marker protein ABCA3 in T2 cells. We now report that secretagogues also promote association of A7 with SNAP23, one of the SNARE proteins. Confocal immuno‐fluorescence microscopy of T2 cells showed a time‐dependent increase in co‐localization of A7 and SNAP23 in T2 cells stimulated with PMA or calcium ionophore A23187. The increased co‐localization was prevented by pre‐treatment of T2 cells with PKC inhibitor, BisI. Supporting evidence for interactions of A7 and SNAP23 was derived from in vitro binding studies. The binding of bacterially expressed A7 with GST‐SNAP23 was Ca2+‐dependent. The binding was observed at 0.2μM Ca2+, which increased further in Ca2+ concentration‐dependent manner. Thus, physiologic conditions causing calcium elevation would increase interaction of A7 with SNAP23. In vitro phosphorylation of A7 with PKC also increased the Ca2+‐dependent binding to GST‐SNAP23. We conclude that calcium elevation and increased protein phosphorylation that occur in secretagogue‐stimulated T2 cells would facilitate A7 interactions with SNAP23 during surfactant secretion. We speculate that A7 phosphorylation and association with lamellar bodies and SNARE proteins is to facilitate the membrane fusion process.
Quantitative live cell imaging of nanoparticle translocation into/across lung alveolar epithelial cells
Authors: Sipos Arnold , Fazlollahi Farnoosh , Kim Yong Ho , Chow Robert H. , Borok Zea , Kim Kwang-Jin , Crandall Edward D.
Published: June 2021

Trafficking of inhaled engineered nanomaterials (ENM) and ambient ultrafine particles from airspaces of the lung into the systemic circulation and internal organs is poorly understood. In order to better define the kinetic parameters of such ENM trafficking, we developed a highly sensitive, confocal, live cell imaging approach utilizing fluorescently labeled polystyrene nanoparticles (PNP; 20nm, carboxylated and NIR dye‐labeled) and primary cultured rat alveolar epithelial cells (RAEC). Nanoparticle uptake was analyzed in single cells: in xyz series, cytoplasmic nanoparticle concentration was measured stack‐by‐stack and integrated over the entire volume of cytoplasm. [PNP] in bathing fluid was assessed by microfluorimetry and correlated with live cell cytoplasmic concentration. Glass coverslip‐seeded RAEC took up 3.9%, and Anopore filter‐seeded RAEC took up 2.6%, of apical PNP over 24 hours. In both cases, cytoplasmic [PNP] leveled off (at ~4 and ~3 hours, respectively), suggesting that PNP accumulation inside RAEC reached a steady state. For filter‐grown cells, trafficking across RAEC monolayers from apical to basolateral fluids over 24 hours was ~1.4% of apical PNP. We conclude that live cell imaging can be a useful tool for visualization and quantification of nanoparticle uptake into/egress from RAEC in real time with high spatial resolution.

Funded by: NIH; Hastings and Whittier Foundations
Mitochondrial Biogenesis in Hypoxic Rat Pulmonary Artery Endothelial Cells (PAECs): Possible Involvement of DNA Oxidation
Authors: Ruchko Mykhaylo V. , Gorodnya Olena M. , Pastukh Viktor M.
Published: June 2021

Like many stimuli promoting cell growth and proliferation, hypoxia uses reactive oxygen species (ROS) as signaling molecules. There is emerging evidence that for nuclear genes, controlled ROS‐mediated DNA damage and repair in specific promoter sequences is necessary for normal transcriptional regulation. Mitochondrial (mt) biogenesis in hypoxia requires mtDNA transcription and replication, but the specific mechanisms are unknown. Here we tested the idea that, in PAECs, these key events in hypoxia‐induced mitochondrial biogenesis, similar to hypoxia‐regulated nuclear gene expression, are associated with oxidative base modifications in the mtDNA D‐loop region involved in mtDNA transcriptional control. Using Fpg‐sensitive qPCR and quantitative Southern blot analyses, we found that although hypoxia did not cause alkali‐sensitive lesions in either the D‐loop or coding regions of mtDNA, Fpg‐sensitive modifications were present exclusively in the D‐loop sequence, with the coding region devoid of oxidative base modifications. Slot blot and real time RT‐PCR analyses revealed increased mtDNA copy number and mitochondrial mRNA transcripts after hypoxic exposure. Hypoxia also increased TFAM binding to D‐loop region as shown by ChIP assay. These observations support the concept that controlled oxidative DNA damage in the D‐loop region may be involved in mitochondrial biogenesis. Supported by NIH.
Respiration drives phase synchronization between blood pressure and R‐R interval following loss of cardiovagal baroreflex during vasovagal syncope
Authors: Stewart Julian Mark , Ocon Anthony James , Medow Marvin Scott
Published: June 2021

Thoracic hypovolemia and hyperpnea contribute to the nonlinear time‐dependent hemodynamic instability of vasovagal syncope. We used a phase synchronization index (PhSI) to describe coupling between systolic BP (SBP), RR‐interval (RR), and ventilation, and a directional index (DI) of coupling. We hypothesized normal AP‐RR PhSI during early 70° head‐up tilt, indicating intact cardiovagal baroreflex (CVB), but decrease as faint approached. Data were recorded supine and upright in 15 control (C) and 15 fainters (F). Data were evaluated during baseline, early tilt, late tilt, faint, and recovery. During late tilt to faint, F but not C showed SBP‐RR PhSI decrease (from 0.65±0.04 to 0.24±0.03) which increased at faint (0.80±0.03) coinciding with a change in phase difference from positive to negative. Starting in late tilt and through faint, F but not C exhibited increasing phase coupling between respiration and AP and between respiration and RR. DI indicated respiratory driven AP and RR in F. The initial drop in the SBP‐RR PhSI and directional change of phase difference at late tilt indicates loss of cardiovagal baroreflex. The subsequent increase in SBP‐RR PhSI is due to a respiratory synchronization and drive on both AP and RR. Cardiovagal baroreflex is lost prior to syncope and supplanted by respiratory reflexes, producing hypotension and bradycardia.
The effects of melatonin on endoplasmic reticulum stress during brain development in rat
Authors: Jung Daeun , Hong Yunkyung , Hong Yonggeun
Published: June 2021

Endoplasmic reticulum (ER) stress is induced in various physiological or pathological conditions. During embryonic development, ER stress occurs in order to organize neuronal architecture. Melatonin, pineal hormone, has roles for neuronal protection as well as cell death inhibition. The purpose of this study was to confirm the effect of melatonin treatment (10 mg/kg/day) into the Sprague Dawley rats under conditioned photoperiod (L/D, LL, and DD) on ER stress during brain development. As a result, maternal body weight was lower in the melatonin‐treated rats (MT) than control rats (Con) in LL as well as DD group at embryonic day 20 (P<0.05). Body weight of pups was higher in MT than Con group under LD and DD condition, whereas lowest in MT group under LL condition (P<.05). mRNA expression of eIF2a, PERK and XBP‐1 were significantly decreased in MT compared to Con group at embryonic day 20 (P<.05) Interestingly, expression of LC3 mRNA was significantly decreased in MT group of LL and DD condition at the same time point (P<0.05). Taken together, endogenous and/or exogenous melatonin reduced autophagy activation as well as ER stress during embryonic development suggesting that melatonin is beneficial to formation of nervous system.
Perinatal Nicotine Exposure Increases Hypoxic‐Ischemic Brain Injury in Neonatal Rats
Authors: Li Yong , Tong Wenni , Xiao Daliao , Zhang Lubo
Published: June 2021

Maternal cigarette smoking increases the risk of neonatal morbidity. This study determined whether perinatal nicotine exposure causes heightened brain vulnerability to hypoxic‐ischemic (HI) injury in neonatal rats via altered expression patterns of angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors. Nicotine was administered via subcutaneous osmotic minipumps from gestational day 4 to postnatal day 10. HI brain injury was determined in 10‐day‐old pups. Nicotine treatment significantly enhanced HI brain infarct size in male pups. In fetal brains, nicotine caused downregulation of AT2R, but not AT1R, expression. The downregulation of AT2R persisted in male pup brains. In female brains, there was an increase in AT2R, but a decrease in AT1R. Intracerebroventricular (ICV) administration of AT1R antagonist losartan or AT2R antagonist PD123319 exaggerated HI brain infarction in both male and female pups. AT2R agonist CGP42112 (ICV) abrogated nicotine‐induced increase in HI brain infarction in male pups. PD123319 (ICV) uncovered nicotine's effect on HI brain infarction in females. The results suggest that perinatal nicotine exposure causes aberrant AT2R gene expression in the developing brain, resulting in heightened brain vulnerability to HI injury in neonatal male rats in a sex‐dependent manner. (Supported in part by NIH grants HL82779 and HL83966).
Low sodium intake is associated with low birth weight and size only when given in the second half of gestation
Authors: de Siqueira Flavia Ramos , de Souza Regiane Machado , de Oliveira Ivone Braga , Furukawa Luzia Naoko S. , Heimann Joel Claudio
Published: June 2021

This study examined the effects of maternal low sodium intake during different periods of pregnancy on dams and male (M) and female (F) offspring.

Methods

Female Wistar rats (n= 6 ‐ 8/group) were fed low (LS ‐ 0.15%) or normal (NS ‐ 1.3% NaCl) sodium diet since the 1st day of gestation until delivery or LS during the 1st (LS10) or 2nd half of gestation (LS20).

Results

(mean±SEM, p< 0.05): Maternal body weight (g) was lower in LS and LS20 than in NS and LS10 at the 3rd week of gestation (LS = 380±8.1; LS20 = 406±13.2; NS = 435±6.1, LS10 = 461±12.9). No difference was found in water and food intake among the groups. Also, no differences were observed in the litter size among experimental groups and no correlation between the number of pups and maternal body weight at the 3rd week of gestation. At birth, body weight (g, n=14 to 27/group) (M: NS = 6.5±0.1, LS = 5.5±0.1, LS10 = 6.1±0.1, LS20 = 4.8±0.1, F: NS = 6.1±0.2, LS = 5.2±0.1, LS10 = 5.6±0.1, LS20 = 4.1±0.1) and naso‐anal length (cm, n=14 to 27) (M: NS = 5.1±0.02, LS = 4.9±0.03, LS10 = 5.0±0.03, LS20 = 4.6±0.03, F: NS = 4.9±0.05, LS = 4.8±0.04, LS10 = 4.9±0.03, LS20 = 4.4±0.03) were lower in LS and LS20 than in NS offspring.

Conclusion

Although maternal dietary intake was not influenced by dietary sodium content, maternal low sodium diet during the second half of gestation induced lower birth weight and size. Supported by CAPES and FAPESP
Specific targeting of pancreatic β‐cells by simultaneous binding of GLP‐1 and α2‐adrenergic receptors with a multivalent ligand
Authors: Ananthakrishnan Kameswari , Weber Craig , Hart Nathaniel , Vagner Josef , Limesand Sean , Lynch Ronald
Published: June 2021

Diabetes Mellitus is characterized by a decline in β‐cell mass and function. The lack of specificity of β‐cell targeting agents poses a challenge to observe changes in β‐cell mass and thus monitor disease progression. We propose β‐cell specificity can be enhanced by using multivalent ligands that simultaneously bind to multiple unique surface receptors that, as a combination, identify a cell type of interest. We synthesized a ligand composed of Yohimbine (Yhb) (α2 adrenergic receptor (α2AR) antagonist) linked to Glucagon‐like Peptide 1(GLP‐1). Receptors to both ligands are expressed on βTC3 cells at a ratio of ~ 1.5:1 (GLP‐1R/α2AR). Microscopy of a fluorophore tagged ligand indicated that binding was rapid with removal of ligand from the cell surface seen within 2–3 min. From population based competition assays, the binding affinity for monovalent GLP‐1 was ~10 nM, whereas GLP‐1/Yhb exhibited 2 distinct binding sites. The high affinity binding (9 pM) is correlated to MVL crosslinking the complementary receptors causing a decrease in ligand off rate, resulting in a 1000X increase in apparent affinity. The low affinity binding (225 nM) is consistent with weak binding of the GLP‐1 moiety in the MVL to the spare GLP‐1 receptors in βTC3 cells. Our findings suggest that GLP‐1/Yhb acts as a specific targeting agent to β‐cells as it exhibits enhanced binding to and retention in β‐cells compared to other cells in the imaging field.

Supported by: Juvenile Diabetes Research Foundation, and the Arizona Biomedical Research Commission
Backward design helps faculty develop case studies
Authors: Cliff William
Published: June 2021

During instructional design, teachers often plan learning activities that fail to align with desired student learning outcomes and assessment. This investigation examined the approach taken by science faculty when developing a case study about embryonic sex determination. Faculty participants in a case study workshop used the backward design method of Wiggins and McTighe to establish learning outcomes, acceptable evidence for learning, and a plan for learning activities. Participants worked in groups of three or four to complete a case design template that was collected and analyzed. When asked to select the three most suitable student learning outcomes for the case from Wiggins and McTighe's six facets of understanding, participants chose perspective (18/22 groups), engagement (13/22), explanation (11/22), interpretation (11/22), application (8/22) and self‐knowledge (4/22). The preference for perspective suggests that faculty recognized that critical analysis is an important facet of understanding for a case requiring nuanced judgments about science, technology and ethics. When given a choice of approaches for assessing student learning, participants most often selected performance tasks to assess student perspective; quiz or test questions to assess student explanation or interpretation; and observation or dialogue to assess student engagement. These selections indicate that faculty were generally able to match appropriate forms of assessment to desired student learning outcomes. This investigation shows that, contrary to ordinary practice, backward design can help faculty choose learning outcomes and assessment methods that are suitably aligned with the understanding called for by a case study.
Are they really just big boned?: How increased body mass in human juveniles affects bone joint morphology in the lower limb
Authors: Kelso Rebecca K Scopa
Published: June 2021

With higher incidence of childhood obesity in the United States, it is clinically important to understand the long‐term mechanical effects of increased loadbearing on joints of the lower limb throughout ontogeny. As part of this documentation, this study looks at the relationship between increased mass and femoral and proximal tibia dimensions. A clinical sample of juveniles considered to have normal mass was used as a control in comparison with juveniles considered clinically obese. Radiographic measurements of the femoral head, distal femur, and proximal tibia were taken, and measurements obtained from the images after correcting for parallax. Preliminary results indicate a high correlation between dimensions from these three regions and observed body mass, but that the femoral head breadth— commonly thought to be a good indicator of body mass—was not the most highly correlated in either the normal‐weight or obese groups. Rather, femoral epicondylar breadth has higher correlations with mass. Furthermore, there is some indication that joint sizes may be adaptive and outpacing weight gain in older children (10–17 yrs). This may indicate overcompensating in joint size growth among obese juveniles, most likely in response to high mechanical loads. The limitations of the data leave the timing of the onset of obesity unknown, however, and therefore the ontogeny of joint hypertrophy cannot yet be established.

Grant Funding Source : n/a
Bifid median nerve variations: A cadaveric study
Authors: Warren Kelly , Norbury John
Published: June 2021

Bifid median nerve (BMN) is an anatomical variation that occurs when the main median nerve trunk splits into medial and lateral trunks prior to entering the hand within the carpal tunnel (CT). The classification of BMN can be further divided into subcategories, which are based on associated abnormalities and location of branching. In this study, the upper extremities of twenty‐nine cadaveric specimens were examined for the presence of BMN in the flexor forearm and hand regions (14 females, 15 males). Of the 58 upper extremities examined, 6 exhibited BMN patterns (10.3%; 4 female, 2 male), with both trunks traveling within the CT into the hand. Two distinct bifid branching patterns were also observed: (B1) mid‐forearm, proximal to carpal tunnel (n=2 females) and (B2) wrist level, within CT (n=2 females, 2 males). In both B1 cases, the smaller medial trunk pierced the flexor digitorum superficialis muscle, traveled within the CT with the larger lateral trunk, and gave off communicating branches to both the lateral trunk and superficial branch of the ulnar nerve after entering the left hand. Incidence of BMN was found to be higher in female cadavers (67%), and in left upper extremities (67%). Knowledge of variable median nerve branching patterns, especially as it is associated with the CT, is important for diagnostic and surgical interventions.

Grant Funding Source: n/a
The fabellofibular and arcuate ligaments of the knee joint: attachments, functional and clinical implicationsfor the posterolateral aspect of the knee joint
Authors: Ghosh Tamojit
Published: June 2021

Summary Of Results

The fabellofibular ligament is attached proximally to the fabella (bony or cartilaginous, found in 8–16 % of human knees) and distally to the lateral basal aspect of the fibular head anterolateral to the insertion of the popliteofibular ligament. It's an independent ligament proven that the lateral inferior genicular blood vessels separate it from the femoral condyle. The Y‐SHAPED arcuate ligament's base is attached to base of the apex of the fibular head ( deep to the fabellopopliteal ligament when it is present), the medial limb courses up and merges with posterior part of the knee joint capsule and the oblique popliteal ligament. It has fibres attached to the lateral knee joint mensicus too adding to its posterior mobility. Its lateral limb courses upwards to be attached to the lateral femoral condyle. The arcuate ligament's relationships with the fabellopopliteal ligament, the deeper part of the lateral collateral ligament and with the absence or presence of fabellae are discussed in detail. Attachments of the popliteofibular ligament and the superior & inferior popliteomeniscal fibres are also discussed.

Summary of conclusions

All the ligaments of the posterolateral aspect of the human knee develop relative to the larger phylogenetic evolution event in which the fibular head regresses downwards and loses it primitive articulation with the lateral condyle of the femur. In posterolateral knee joint injuries the above ligaments should be visually sought out and attempts made to repair them individually. This is especially true for repairs of injuries of the lateral meniscus and lateral collateral ligament where associated injuries of the above smaller ligaments exist.

Grant Funding Source : Indian Institute of Morphology
Lateral and longitudinal ultrastructural study of the recurrent laryngeal nerve in adult rats
Authors: da Silva Ferreira Renata , de Barros Oliveti Maria Carolina Dal Bem , Fazan Valéria Paula Sassoli
Published: June 2021

Despite the importance of the recurrent laryngeal nerve (RLN) in terms of respiration and phonation functions, studies on the normal histology of this nerve in rats are still scanty. The RLN is asymmetric in length, because the different origin on both sides but neither a longitudinal ultrastrucutral study nor information about ultrastructural lateral asymmetry of this nerve is available in rats. Male adult Wistar rats (N=5), had their RLN prepared for transmission electron microscopy and morphometry. Comparisons were made between segments on the same side and same levels between sides. The RLN fascicles are significantly larger proximally, on both sides as is the myelinated fiber number. No other longitudinal differences were observed. Unmyelinated fiber number and density is larger on right, compared to proximal left segment. This lateral difference was not present on the distal segments. No other lateral differences were observed. Our results indicate that the RLN are asymmetric not only macroscopically (origin and length, between sides) but also on its microscopic anatomy, not only between sides but also between proximal and distal segments. This information is crucial for those studies investigating RLN neuropathies since results can vary according to the nerve site studied.

Grant Funding Source: FAPESP, CNPq, CAPES and FAEPA