bluuebunny/crossref_metadata_2025 huggingface dataset:
Contribution of Reactive Oxygen Species to NLRP3 Inflammasome Activation in Glomeruli of Mice with Hyperhomocysteinemia
Authors: Abais Justine , Boini Krishna M. , Xia Min , Li Pin‐Lan
Published: June 2021

Our previous studies have shown the importance of NADPH oxidase‐derived reactive oxygen species (ROS) in hyperhomocysteinemia (hHcys)‐induced glomerular injury. Using SOD mimetic TEMPOL, catalase, and uric acid to reduce superoxide (O2•−), hydrogen peroxide (H2O2), and peroxynitrite (ONOO−), we have recently shown that these ROS are involved in hHcys‐induced inflammasome activation in podocytes. The present study was designed to further test whether in vivo scavenging of O2•−and H2O2 attenuates NLRP3 inflammasome activation and glomerular injury in mice with hHcys. Uninephrectomized C57BL/6J wild type mice were fed either normal chow (ND) or a folate free (FF) diet for 4 weeks to produce hHcys. Confocal microscopy showed that hHcys increased the colocalization of inflammasome proteins NLRP3 with ASC or caspase‐1 in the glomeruli compared to ND fed mice. Inflammasome formation mainly occurred in podocytes as shown by colocalization of NLRP3 with podocyte marker podocin. In vivo scavenging of O2•− by TEMPOL and removal of H2O2 by catalase substantially inhibited NLRP3 inflammasome formation in glomeruli of hHcys mice. Correspondingly, TEMPOL and catalase treatment inhibited hHcys‐induced caspase‐1 activation, respectively by 25.9% and 33.8%, and decreased IL‐1β production by 38.8% and 35.7%. ESR analysis demonstrated that TEMPOL produced a 44.3% decrease of renal O2•−production, while catalase produced a 42.4% decrease. Importantly, reduction of both O2•−and H2O2 was found to protect glomerular function and structure, preventing hHcys‐induced proteinuria, albuminuria and glomerular morphological pathology. Together, these results indicate that endogenously produced O2•− and H2O2 contribute to NLRP3 inflammasome formation and activation in vivo at early stage hHcys and that inhibition of these ROS may prevent NLRP3 inflammasome activation and subsequent glomerular injury (supported by NIH grants DK54927 and 1F31AG043289).
Acute Inhalation Exposure of Nano‐Titanium Dioxide induces Cardiac and Mitochondrial Dysfunction in Mice
Authors: Nichols Cody , Shepherd Danielle , Croston Tara , Thapa Dharendra , Lewis Sara , Jagannathan Rajaganapathi , Yi Jinhai , Nurkiewicz Timothy , Hollander John
Published: June 2021

While nanotechnology offers innovation in products from cosmetics to drug delivery, exposure to engineered nanomaterial (ENM) is increasing. Unfortunately, health impacts of ENM are not fully realized. Titanium Dioxide (nanoTiO2) is one of the most widely produced ENM due to its variety of uses. The goal of this study was to determine the effects of nanoTiO2 inhalation exposure on cardiac and mitochondrial function. Male FVB mice were exposed to nanoTiO2 at an estimated total lung deposition of 11 μg. Twenty‐four hours after exposure cardiac function was assessed using a Vevo 2100 echocardiography system. Diastolic dysfunction was observed, supported by increased diastolic radial strain, increased E/A ratios (>;2.0), and increased early mitral flow deceleration time (p<0.05, for all three). Inductively coupled plasma atomic emission spectroscopy detected an 80% increase in Ti content in cardiac tissue following exposure suggesting particle translocation as a potential source of cellular stress. To identify subcellular mechanisms mitochondria were isolated and respiration rates assessed. A decrease in active (State 3) respiration rate following exposure was observed (p<0.05). In summary, nanoTiO2 inhalation exposure is associated with cardiac diastolic dysfunction and mitochondrial functional alterations. (Supported by NIH DP2DK08309, NIH R01ES015022, RC1 ES018274, DGE‐1144676, NIH T32HL090610)
Plasma protein protects endothelial glycocalyx mediated by sphingosine‐1‐phosphate
Authors: Zeng Ye , Adamson Roger , Curry Fitz‐Roy , Tarbell John
Published: June 2021

It has long been known that albumin reduction in endothelial cell (EC) media induces collapse or shedding of the surface glycocalyx layer. This leads to impairment of the endothelial transport barrier and other features of vessel homeostasis. The electrostatic interaction between arginine residues on albumin and negatively charged glycosaminoglycans in the glycocalyx has been hypothesized to stabilize the glycocalyx structure. But sphingosine‐1‐phosphate (S1P), which is bound to albumin, also plays important roles in maintaining vascular barrier integrity and homeostasis. Here, the role of S1P in maintaining glycocalyx stability was investigated in this study. Using confocal microscopy on EC in vitro, we observed that heparan sulfate (HS), chondroitin sulfate (CS) and the ectodomain of syndecan‐1 were shed from the surface after removal of plasma protein, but were retained in the presence of S1P or a general MMP inhibitor. S1P receptor 1 (S1P1) antagonism abolished the protection of the glycocalyx by S1P. GAGs released to culture media and the general MMP activity increased significantly after removal of plasma protein, but could be diminished by S1P. Thus, S1P plays a critical role in protecting the glycocalyx via S1P1, and inhibits the protease activity‐dependent shedding of CS, HS and the syndecan‐1 ectodomain. Our results provide new insight into the role for S1P in protecting the glycocalyx and maintaining vascular homeostasis.

Grants: Supported by NIH Grant HL094889 and HL 28607.
Loss of endothelial caveolae leads to eNOS uncoupling and impaired flow‐mediated dilation in human coronary arterioles in diabetes
Authors: Cassuto James , Feher Attila , Szabo Andras , Bagi Zsolt
Published: June 2021

Diabetes mellitus (DM) leads to impaired endothelial synthesis of nitric oxide (NO). We aimed to investigate the role of caveolae ‐ cholesterol‐rich membrane lipid rafts ‐ in leading to eNOS uncoupling in human coronary arterioles in DM. Flow‐mediated dilation (FMD) of isolated, pressurized coronary arterioles was compared between DM (n=18) and non‐DM (n=16) patients undergoing heart surgery. Coronary arterioles of non‐DM patients exhibited NO‐dependent FMD (35±8%), which was abolished in DM patients (−7±4%). Incubation of DM arterioles with sepiapterin (a tetrahydrobiopterin (BH4) precursor) or peroxynitrite scavengers, uric acid or FeTMPyP restored FMD in an NO‐dependent manner. In DM patients, increased protein 3‐nitrotyrosine (3‐NT) formation was detected, with a greater degree of colocalization with caveolin‐1. Electron microscopy analysis revealed a prominent 3‐NT staining in endothelial cell membrane and demonstrated a reduced number of caveolae in coronary arteries of DM patients. Also, aorta from mice lacking caveolae showed a reduced NO‐dependent dilation to acetylcholine, which was restored by sepiapterin. Thus, we suggest that impaired NO‐mediated FMD of human coronary arterioles in diabetes is due to the peroxynitrite‐dependent destruction of caveolae, which leads to decreased bioavailability of BH4 and eNOS uncoupling.
Role of Thrombospondin‐1 In the Remodeling Induced by a Chronic Increase in Flow in Resistance Arteries
Authors: Grenier Céline , Caillon antoine , Grimaud Linda , Ayer Audrey , Toutain Bertrand , Blanc‐Brude Olivier , Henrion Daniel , Loufrani Laurent
Published: June 2021

Background

Chronic increases in blood flow in resistance arteries induce outward vascular remodeling due to increased NO‐dependent dilation and MMP activation. Although thrombospondin‐1 (TSP‐1) interacts with a variety of matrix proteins and cell‐surface receptors, controversy remains about its vascular functions. We investigated the role of TSP‐1 in high flow (HF)‐mediated remodeling of resistance artery.

Material and methods

Mesenteric arteries were ligated in vivo in order to generate high‐flow arteries compared with normal flow (control) vessels located at distance on wild type (WT) and TSP‐1 deleted mice (TSP‐1 KO). Arteries were isolated 4 days to 1 week after ligature. Diameter and arteries reactivity were studied in vitro in an arteriograph. Genes implicated in inflammation and immune cells recruitment were determined in HF and NF arteries.

Results

Arteries remodeling, due to high flow (found in WT), were not significant in TSP‐1 KO arteries. Dilatory (sodium nitroprusside) and contractile (Phenylephrine) tone, were unaffected by the absence of TSP‐1.

We also demonstrated, after 4 days of ligature, an increase in CD68, Cox1, Cox2, Gp91phox, p47phox, p22phox, expression level between NF and HF WT arteries that was not observed in KO arteries.

Furthermore, we also observed a increase in neutrophils and lymphocytes number in response to chronic high flow in WT arteries, not observed in HF KO arteries.

Conclusion

The absence of TSP‐1 induced a decrease in flow‐induced remodeling with a reduction of the inflammatory response and of the endothelium‐mediated dilation involved in HF remodeling.
Comparison of cardiovascular responses to yoga and slow paced breathing
Authors: Bertisch Suzie , Hamner Jason , Tan Can , Taylor J. Andrew
Published: June 2021

One in twelve U.S. adults practice yoga annually. Some data suggest improvement in cardiovascular health with yoga training. Though a shift in cardiovascular control has been purported with slow yogic breathing, there are sparse data on its specific effects compared with other slow breathing patterns. We investigated whether slow yogic breathing differentially impacts the interrelations among respiration, heart rate, and blood pressure in seven long‐term yoga practitioners (>;10 yrs). To determine whether control pathways are distinctly different across breathing patterns, a Granger causality test was used to define the pathway(s) linking respiration, heart rate and blood pressure during 0.25 Hz, 0.1 Hz, and yogic breathing. Yogic breathing ranged from 0.03–0.10Hz and encompassed respiratory maneuvers including breath‐holds, segmented breathing, and ujjayi. The strongest cardiovascular control path was from respiration to heart rate to blood pressure and this was significantly stronger during 0.25 Hz breathing. However, yogic and slow paced breathing did not differentially impact this cardiovascular control path. Thus, it appears in experienced yogic breathers, breathing frequency is a key factor in respiratory‐mediated cardiovascular fluctuations.
Improvement of salt‐sensitive hypertension in SSMstnem1Mcwi Rats
Authors: Stepp David W. , Dunlap Robert Hunter , Mintz James D.
Published: June 2021

Exercise is known to lower blood pressure (BP) by mechanisms which are not well‐understood. Exercise increases muscle mass, which may improve metabolism or perfusion to improve BP. To determine the effects of increased muscle mass on BP regulation, BP was monitored via telemetry in salt‐sensitive rats in which myostatin (SS‐Mstn) had been genetically deleted (PhysGen). At 24–26 weeks of age, SS‐Mstn were had larger muscles (gastrocnemius, 60%, gluteus maximus, 30%) compared to SS‐controls. On 0.4% salt diet, BP was higher in SS‐control (161+4 vs. 123+8 mm Hg, p<0.05) compared to SS‐Mstn. When salt was elevated to 4%, the BP increase was smaller in SS‐Mstn (5+1 vs 14+3 mm Hg, p<0.05). Lowering dietary salt to 0.1% caused a larger drop in SS‐control than SS‐Mstn (19+6 vs. 3+2, p<0.05). Heart rate was similar in both groups. Kidney mass was lower in SS‐Mstn (1.4+0.1 vs 1.8+0.3, p<0.05) and peak dilator responses to acetylcholine in aortic rings were larger in SS‐Mstn (57+3 vs. 35+7 %, p<0.05) with no difference in dilation to nitroprusside. Constrictor responses to phenylephrine (in vivo and in vitro) were similar between groups as was the drop in BP to ganglionic blockade. Plasma 1‐methylhistidine, a marker of muscle turnover, was increased in SS‐Mstn and reduced in SS‐Mstn. Taken together, these data suggest that an increase in muscle mass is of significant benefit in the regulation of BP.
Renal denervation abolishes age‐dependent hypertension in female intrauterine growth restricted rats.
Authors: Intapad Suttira , Brown Andrew D. , Fahling Joel M. , Dasinger John Henry , Ojeda Norma B. , Alexander Barbara T.
Published: June 2021

Female intrauterine growth‐restricted (IUGR) offspring are normotensive after puberty; however, age increases susceptibility to hypertension and is associated with a marked increase in total fat and plasma leptin. Whether age alters renal function or whether leptin mediates actions via the renal nerves is unknown. Thus, we tested the hypothesis that altered renal function and renal innervation contribute to age‐dependent hypertension in female IUGR offspring. Renal function was determined in conscious, chronically instrumented animals with measure of mean arterial pressure (MAP) via radiotelemetry performed 2 weeks post sham or bilateral renal denervation (RDV). Glomerular filtration rate adjusted per kidney weight and filtration fraction did not differ; yet, renal blood flow and effective renal plasma flow were significantly reduced (P<0.05) and renal vascular resistance was significantly increased (P<0.05) in IUGR versus control. Hypertension in female IUGR, (134±2 vs. 118±2 mmHg, P<0.01 versus control) was abolished by RDV (119±2 mmHg, P<0.05 vs. IUGR RDV) normalizing MAP relative to RDV control (120±3 mmHg). Renal norepinephrine content in RDV groups was markedly decreased (P<0.05) confirming effectiveness of RDV. Thus, these data suggest that the renal nerves may serve as an underlying mechanism in age‐dependent hypertension in the female IUGR. HL074927; HL51971; AHA12POST11980021.
Neutrophil depletion attenuates placental ischemia‐induced hypertension in rat
Authors: Lillegard Kathryn E , Bauer Ashley J , Johnson Alex C , Lojovich Sarah J , Gilbert Jeffrey S , Regal Jean F
Published: June 2021

Preeclampsia is characterized by compromised placental perfusion and hypertension during pregnancy. Preeclamptic women exhibit a greater number of neutrophils (PMN) in the vasculature and a decrease in plasma free vascular endothelial growth factor (VEGF) compared to normal pregnancy.

Hypothesis

PMN are critical to placental ischemia‐induced hypertension and decreased VEGF. Using the reduced uterine perfusion pressure (RUPP) model of placental ischemia‐induced hypertension, we determined the effect of PMN depletion on blood pressure and plasma VEGF. PMN were depleted with repeated injections of polyclonal rabbit anti‐rat neutrophil antibody (αPMN). On gestation day 14, clips were placed on the abdominal aorta and uterine arteries to decrease placental perfusion. RUPP and Sham rats received either αPMN or normal rabbit serum (Ctl) on gestation days 13, 14, 16, and 18, and mean arterial pressure (MAP) was measured via arterial catheter on day 19. PMN‐depleted rats had reduced circulating PMN (p<0.05) on day 14 compared to Ctl. MAP increased (p<0.05) in RUPP Ctl vs Sham Ctl rats, and PMN depletion attenuated this increase in MAP in RUPP rats (p=0.04) without any effect on Sham rats. The RUPP‐induced decrease in VEGF was not affected by PMN depletion. Thus, neutrophils are important in placental ischemia‐induced hypertension independent of plasma free VEGF concentrations. Support: NIH R15 HL109843.
Acute Effects of Aldosterone on NCC Are Mediated by SPAK
Authors: Ko Benjamin , Hanson Lauren , Mistry Abinash , Thai Tiffany L , Mallick Rickta , Klein Janet D. , Bailey James L. , Hoover Robert
Published: June 2021

The sodium chloride cotransporter (NCC) is a major site of renal sodium reabsorption and plays a key role in blood pressure regulation. Using mDCT15 cells, we previously demonstrated that aldosterone (aldo) increases NCC activity and phosphorylation at 24 hrs. without affecting total or surface NCC abundance. Here we confirm those findings in vivo, examine the mechanisms underlying them and investigate additional time points. Adrenalectomized rats were given aldo or vehicle via osmotic minipumps. Kidney cortex from rats receiving aldo demonstrated no change in total NCC compared to control but showed a three‐fold change in phospho‐NCC (T53). Similarly, mDCT15 cells showed a three‐fold increase in phospho‐NCC at 12, 24, and 36 hours, corresponding to observed increases in NCC activity as measured by radiotracer uptake with no evidence of changes in total or surface NCC (n=6, p<0.05). mDCT15 cells were treated with shRNA specific for SPAK to generate a cell line with a 68±7% decrease in SPAK protein expression compared to controls. Stimulation of these cells with aldo had virtually no effect on NCC activity (6±3% increase after 24 hour aldo treatment in shRNA SPAK groups versus 52±5% increase after aldo treatment in control groups, n=4, p<0.01). This provides clear evidence that aldo acutely increases NCC activity without changing NCC abundance or distribution via a pathway involving SPAK and NCC phosphorylation.
Separate control of TRPV4 activity and trafficking in the distal nephron
Authors: Mamenko Mykola , Zaika Oleg L. , Boukelmoune Nabila , Berrout Jonathan , O'Neil Roger G , Pochynyuk Oleh
Published: June 2021

We have recently documented that Ca2+‐permeable TRPV4 channel, which is abundantly expressed in the distal nephron cells, mediates cellular Ca2+ responses to elevated luminal flow. The current research combined Fura‐2 Ca2+ imaging with immunofluorescent microscopy in split‐opened distal nephrons of C57BL/6 mice to probe molecular determinants regulating subcellular distribution and activity of TRPV4. We found that activation of PKA pathway with forskolin does not affect TRPV4‐ mediated Ca2+ responses to flow, while markedly shifting subcellular distribution of the channel towards the apical membrane. These actions are blocked with a specific PKA inhibitor H‐89. Activation of PKC pathway with PMA significantly increases Ca2+ responses to flow without affecting the subcellular distribution of TRPV4. In contrast, inhibition of PKC with BIM‐1 diminishes cellular responses to elevated flow. Concomitant activation of PKA and PKC cascades greatly increases basal Ca2+ levels indicating constitutive TRPV4 activation and this effect is precluded by a selective TRPV4 antagonist, HC‐067047. Therefore, functional status of TRPV4 channel in the distal nephron is regulated by two distinct signaling pathways. While PKA cascade appears to be responsible for TRPV4 trafficking and translocation to the apical membrane, PKC‐dependent pathway increases the activity of the channel on the plasma membrane.
The Degenerin/ENaC channel, encoded by ACCN5, serves a critical function in the vestibulocerebellum
Authors: Kucher Volodymyr , Boiko Nina , Stockand James D.
Published: June 2021

ACCN5 encodes the BLINaC/hINaC member of the Deg/ENaC ion channel family. As we demonstrate using a novel mouse line that we engineered to contain a LacZ cassette knockin that interrupts the third exon of this gene, BLINaC has a unique expression profile for a Deg/ENaC channel. Similar to acid‐sensing channels, it is expressed in neurons. But also similar to the epithelial Na+ channel, it is expressed in epithelial cells, particularly those lining the hepatic bile duct. Thus, BLINaC is the only known mammalian Deg/ENaC channel to be expressed in both neurons and epithelial cells. In the brain, BLINaC is restrictively expressed in a subset of interneurons in the cerebellum. Specifically, BLINaC is expressed in unipolar brush cells (UBC) of lobule IX and X. These cells serve a key function in the vestibulocerebellum. Deletion of ACCN5 in our novel ACCN5 null mouse line results in abnormal coordination and balance as measured with standard tests of behavior. It is likely that this phenotype results UBC dysfunction. These results are consistent with BLINaC serving a critical function in UBC that is key to normal vestibulocerebellar function and motor control of balance.
Interaction between purinergic receptors P2X4 and P2X7
Authors: Pérez‐Flores Gabriela , Levesque Sebastién , Vaca Luis , Lacroix Steve , Rassendren François , Pérez‐Cornejo Gloria Patricia , Arreola Jorge
Published: June 2021

ATP‐gated P2X7 Receptor (X7R) channel is linked to cell death. Function of X7R is regulated by P2X4 Receptor (X4R) independent of X4R channel function and independent of formation of heterotrimeric channel. Mechanism by which this interaction takes place remains unsettled and the physiological consequences of such interaction remain partially characterized. We used Fluorescence Resonance Energy Transfer technique (FRET) and fluorescence labeled X4R and X7R expressed in HEK293 cells to analyze the interaction between these receptors. We found that the FRET signal increased after activation of X7R with 100 uM BzATP. Activation of X4R with 20 μM ATP (does not activates X7R) did not increase the FRET signal. Because the fluorescence label was fused to the carboxyl tail of X7, our result suggests X7R interacts with X4R via a conformational change of the carboxyl tail. This hypothesis was further supported by coimmunoprecipitation of X4R and the X7R carboxyl tail. To test if the X4R/X7R interaction was physiologically relevant we evaluated cell death after activation of X7R in murine macrophages. Resident peritoneal macrophages were isolated from both wild type and X4R knockout (X4RKO) mice. Macrophages were stimulated with ATP and the solution was supplemented with YOPRO. Sudden increase in the YOPRO fluorescence indicated collapse of the nucleus, a irreversible phenomenon that leads to cell death. Death rate was slower in macrophages isolated from WT mice than in macrophages isolated from X4RKO mice. The percentage of cell death was significant greater in WT mice than in macrophages from X4RKO mice. Our results suggest that coexpression of X4R with X7R potentiate X7R‐dependent cell death. In macrophages co‐expression of X4R with X7R may be important for immune regulation. ATP‐induced cell death of immune cells could play an important role in termination of the immune response.
Shank2E mediates dexamethasone‐induced increase in membrane CFTR in human airway cells
Authors: Koeppen Katja , Coutermarsh Bonita , Bomberger Jennifer M , Stanton Bruce A
Published: June 2021

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), which often leads to severely reduced CFTR membrane abundance. Thus, many therapeutic approaches aim to increase membrane CFTR. Dexamethasone (Dex) up‐regulates serum‐ and glucocorticoid‐induced protein kinase (SGK1) and has been previously reported to increase CFTR in the plasma membrane of Xenopus oocytes (Sato et al., 2007) and CFPAC‐1 cells (Caohuy et al., 2009). The goal of this study was to test if Dex also increases membrane wt‐CFTR in human airway cells and to elucidate the underlying mechanism. Dex (50nM, 4h) increased membrane wt‐CFTR by 139%, and increased SGK1 mRNA and protein by 171% and 708%, respectively. We found that SGK1 did not directly phosphorylate CFTR and identified Shank2E, a scaffold protein with two SGK1 phosphorylation sites, as a potential intermediate regulatory protein. siRNA‐mediated knockdown of endogenous Shank2E reduced the Dex‐mediated increase in membrane CFTR by 59%. While over‐expression of wt‐Shank2E slightly enhanced this Dexeffect, over‐expression of a Shank2E mutant without SGK1 consensus sites had a dominant‐negative effect and reduced the Dex‐induced increase in membrane CFTR by 43%. Our results suggest that Shank2E mediates the Dex‐induced increase in membrane CFTR and that this effect is dependent on SGK1 phosphorylation of Shank2E.
S‐adenosylmethionine(SAMe) improves the oxidative stressinduced lung epithelial barrier dysfunction in HIV‐1
Authors: Fan Xian , Raynor Robert , Joshi Pratibha C , Koval Michael , Guidot David M
Published: June 2021

Lung disease is a major cause of death in people living with HIV. HIV viral proteins can directly induce epithelial dysfunction in the lung via mechanisms involving oxidative stresses such as glutathione (GSH) depletion. Because SAMe is a GSH precursor, we hypothesized that supplementation of SAMe would antagonize oxidative stress‐induced impairment of alveolar epithelial function in HIV‐1 transgenic (Tg) rats. Monolayers derived from alveolar epithelial cells (AEC) isolated from HIV‐1 Tg rats had markedly decreased intracellular GSH, abnormal tight junction (TJ) protein expression in the cell membranes, and increased permeability compared monolayers derived wild type rats. In contrast, treatment with SAMe in the culture medium restored intracellular GSH and improved the localization of TJ proteins in the cell membrane of HIV‐1 Tg monolayers. More importantly, SAMe attenuated the HIV‐1 viral protein‐induced AEC monolayer dysfunction as evidenced by both an increase in the transepithelial electrical resistance and a decrease in FITC‐dextran paracellular flux. Further, monolayers derived from HIV‐1 Tg rats whose diets had been supplemented with SAMe for 8 wks had significantly improved function compared to monolayers from untreated HIV‐1 Tg rats. Our findings raise the possibility that augmenting antioxidant defenses within the alveolar space could improve the lung health of HIV‐infected individuals.
Hydrogen sulfide donors – Differential effects of different donors in paraventricular nucleus of the hypothalamus
Authors: Khademullah C. Sahara , Ferguson Alastair
Published: June 2021

Several hydrogen sulfide (H2S) donors have been produced for commercial use, all of which have been assumed to have similar if not identical effects in their ability to deliver H2S. The identification of H2S‐catalyzing enzymes in the paraventricular nucleus of the hypothalamus (PVN) led us to examine the effects of sodium hydrogen sulfide (NaHS) on the excitability of PVN neurons. Bath application of 50 and 10mM caused 75% of responsive neurons to hyperpolarize (n=28, −13.58±1.51mV), while at 1 and 0.1mM 75% neurons depolarized (n=8, 9.627±2.82mV ). The lack of commercial availability of this donor led us to continue our experiments using sodium hydrosulfide hydrate (NaHS•XH20). Administration of NaHS•XH20 across all concentrations (50, 10, 1, and .1mM) caused 95% of responsive neurons to depolarize (n=37, 10.21±1.33mV). These data show that the administration of NaHS and NaHS•XH20, especially at high concentrations produces different effects in the PVN. While we do not yet know the reason for this difference, these observations may provide some explanation for the different physiological effects of various H2S donors reported in the literature.
Eggshell Calcium Influences Embryonic Growth in Pantherophis guttatus
Authors: Ecay Tom W , Stewart James R , Pyles Rebecca A
Published: June 2021

The eggshells of oviparous reptiles provide structural stability during development and are a significant source of embryonic calcium. Hatchling corn snakes (Pantherophis guttatus) obtain approximately 25% of their calcium from the eggshell, primarily in late development. Removing eggshell calcium by peeling away the outer shell layers at early stages results in significantly shorter and lighter hatchlings. To test whether embryos can access environmental calcium to compensate for shell calcium lost to peeling, we incubated peeled and intact eggs in media with increasing calcium and monitored egg uptake of calcium with a calcium electrode. Intact eggs lose calcium in low calcium media (≤ 5 mM calcium) until about 10 days before hatching when they accumulate calcium. The timing of this calcium accumulation is consistent with expression of calcium transport pathways in tissues lining the inner shell. In contrast, peeled eggs in low calcium slowly accumulate calcium early and accelerate uptake at late stages in parallel with intact eggs. In high calcium media (20 mM) peeled and intact eggs exhibit a parallel pattern of increased calcium uptake at all stages. We conclude that corn snake eggs can access environmental calcium and that eggshell peeling and calcium supplementation is an experimental model to study the role of shell calcium in embryonic development and metabolism. (NSF Grant IOB‐06515695)
Decoding Ubiquitin Networks in regulation of inflammation and autophagy
Authors: Dikic Ivan
Published: June 2021

Ubiquitin acts as a cellular signal that is decoded by its receptors to control normal functioning of the cell via different linkage types. Using the different structural and biochemical properties of different ubiquitin chain linkages, we have developed fluorescence‐based sensors to monitor real‐time localization and dynamics of ubiquitination events in vivo. For example, linear chain‐specific sensors labeled the ubiquitin coat surrounding cytosolic Salmonella and were also able to block TNF‐induced NF‐κB activation; whereas K63‐sensors were enriched at DNA double strand breaks and depolarized mitochondria following Parkin recruitment.

In addition to sensors that detect linkage‐specific ubiquitin chains, we have been exploring the fate of intracellular pathogens and how they are targeted by the host autophagy system. Removal of harmful protein aggregates, damaged organelles and microbes is mediated by autophagy, a process by which the cell sequesters cytosolic cargo and delivers it for degradation by the lysosome. Optineurin, a new autophagy receptor, together with p62 and NDP52, mediates selective autophagy of ubiquitin‐coated cytosolic Salmonella enterica. The protein kinase TBK1 (TANK binding kinase 1) was shown to mediate phosphorylation of both LC3‐ and Ub‐binding domains to ensure a rapid engulfment of cytosolic bacteria and efficient autophagic clearance.
Antagonistic roles of CD14 and TLR4 in LPS‐induced colonic epithelial cell apoptosis
Authors: Kuo Wei‐Ting , Yang Hsun‐Yuan , Chen Chi‐Yun , Lu Yen‐Zhen , Ni Yen‐Hsuan , Yu Linda Chia‐Hui
Published: June 2021

Luminal exposure to bacterial LPS induces apoptosis in intestinal epithelial cells via unknown mechanism. Recent studies in monocytes showed that after LPS binding to CD14, phosphatidylcholine‐specific phospholipase C (PC‐PLC), SMase and ceramide activate PKCζ, and recruits TLR4 for complex formation and downstream MAPK and NFκB signals.

Aim

To investigate the receptors and signaling pathways involved in LPS‐induced epithelial apoptosis.

Results

Apical expression of CD14, low levels of MD2, and absence of TLR4 were demonstrated in primary human colonocytes and Caco‐2 cells. Luminal LPS challenge increased cell apoptosis, which may be inhibited by anti‐CD14 or gene silencing of CD14 and PKCζ, but not anti‐TLR4. Inhibitors to PC‐PLC, SMase and PKCζ decreased apoptotic levels. CD14‐mediated ceramide and phospho‐PKCζ levels were increased after LPS challenge, whereas TLR4 signals were not. TLR4 overexpression by plasmid transfection inhibited LPS/CD14‐mediated apoptosis. Moreover, both CD14 and TLR4 were found on mouse colonocytes. Colonic tissues of TLR4‐mutant mice exposed to luminal LPS or bacterial overgrowth display increased mucosal apoptosis and phospho‐PKCζ compared to wild types.

Conclusions

LPS‐induced epithelial apoptosis was dependent on CD14‐mediated lipid secondary messengers, whereas TLR4 plays an antagonistic role. Funding support by National Science Council and NTU.
IgCAM domain 3 is necessary for basal and TNF‐induced MLCK1 trafficking in intestinal epithelial cells
Authors: He Weiqi , Zha Juanmin , Wang Yitang , Turner Jerrold R.
Published: June 2021

Tumor necrosis factor (TNF), a cytokine central to inflammatory bowel disease (IBD), causes tight junction barrier loss via myosin light chain kinase (MLCK) activation. Two splice variants, MLCK1 and MLCK2, which differ by a single immunoglobulin‐cell adhesion molecule (IgCAM) domain, IgCAM3, are expressed in human intestinal epithelia. Based on our previous data showing that MLCK1, which includes IgCAM3, is concentrated at the perijunctional actomyosin ring (PAMR) in vivo, we hypothesized that IgCAM3 is essential for MLCK1 trafficking. Analysis of EGFP‐tagged MLCK1 and MLCK2 stably expressed in Caco‐2 intestinal epithelial monolayers showed that MLCK1 concentrates at the PAMR while MLCK2 distributes more broadly and associates with basal stress fibers. TNF increased MLCK1 trafficking to the PAMR, but had little effect on MLCK2 localization. To assess the potential contributions roles of other MLCK domains to MLCK1 trafficking, EGFP‐tagged MLCK constructs composed of IgCAM domains 1–2‐3, 1–2‐3–4, and 1–2‐4, which lack DXR motifs and enzymatic domains. Both MLCK1‐IgCAM1–3 and MLCK1‐IgCAM1–4 concentrated at the PAMR, while MLCK1‐IgCAM3 and MLCK2‐IgCAM1–4 did not. These data show that IgCAM3 is necessary, but only MLCK1‐IgCAM1–3 is sufficient for MLCK1 trafficking to the PAMR. We speculate that IgCAM3‐mediated trafficking may be required for TNF‐induced barrier loss.
Ablation of intestinal divalent metal‐ion transporter‐1 produces iron‐deficiency anemia
Authors: Shawki Ali , Anthony Sarah R , Niespodzany Eric J , Amratia Avni A , Mackenzie Bryan
Published: June 2021

Divalent metal‐ion transporter‐1 (DMT1)—the product of the SLC11A2 gene—is a widely expressed iron transporter serving intestinal iron absorption and erythroid iron utilization. We tested the hypothesis that DMT1 is essential for intestinal absorption of iron by examining hematological variables, iron stores, and expression of iron‐related genes (by using real‐time qPCR) in a mouse model lacking intestinal DMT1 (i.e. DMT1int/int), males at age ≈ 120 days. Generation of the DMT1int/int model, by crossing floxed DMT1 and villin‐Cre transgenic lines, was described previously [Gunshin et al (2005) J. Clin. Investig.115, 1258–1266]. The DMT1int/int mouse exhibited a severe microcytic– hypochromic anemia—characterized by profound decreases in hematocrit, hemoglobin concentration (DMT1int/int, 2.3 ± SD 1.5 g/dL cf. wildtype, 14.6 ± 0.7 g/dL; n = 7–9), mean corpuscular volume, and serum iron—accompanied by cardiac hypertrophy, splenomegaly, and severely depleted nonheme iron stores (liver, spleen). mRNA expression of the brush‐border ferrireductase Cybrd1 was increased (30 ± 3)‐fold in enterocytes of DMT1int/int mice cf. wildtype mice (mean ± propagated SE, n = 3) and mRNA expression of the basolateral iron‐exporter ferroportin was increased (2.7 ± 1.0)‐fold. Liver Hamp1 (hepcidin) mRNA expression was depressed by 98% ± 42% in the DMT1int/int mouse cf. wildtype. Intraperitoneal iron injection corrected the hematological variables, iron stores, and mRNA expression levels in the DMT1int/int mouse. Our data reveal that tissue‐specific ablation of intestinal DMT1 produces an iron‐deficiency anemia, and confirm that DMT1 is critical for iron homeostasis in the mouse. PHS Grant DK080047
Assessing the value of clay modeling as an effective adjunct to human donor dissection
Authors: Rosentsveyg Juliana Ann , Patel Jasmine , Adar Tony , Marquez Samuel
Published: June 2021

Donor dissection remains a fundamental and inimitable component of first‐year medical education. Learning anatomy is an integral part of physician training that warrants the use of a multi‐modal program. Clay modeling (CM) is a unique adjunctive pedagogical tool that offers aspiring physicians the opportunity to improve their understanding of 3D anatomy by physically recreating organs within their proper topographical setting in an interactive and constructive kinetic learning environment. Our study examines the probative value of adding CM instruction to a medical school anatomy dissection course. Ten Downstate Anatomy students attended eight weekly 120‐minute interactive CM classes. Qualitative results suggest that, compared to students in the control group, students in the CM class reported a higher overall satisfaction rate with the anatomy course, stating they were better able to identify anatomical structures and their topographical relationships and felt more confident in answering anatomy questions extrapolated into hypothetical clinical scenarios. Quantitative comparison of pre and post‐test performance showed statistically significant differences within the CM group (p < 0.05). Our preliminary findings suggest CM to be an enhancing adjunct to traditional gross anatomy courses that facilitates deeper learning in a non‐traditional method of delivery. SUNY Downstate Alumni Association
Use of a Wiki as a Collaborative Learning Tool to Promote Active Learning in a Neuroscience Course for First‐Year Medical Students
Authors: Gould Douglas , Mi Misa
Published: June 2021

The use of Wikis in higher education is not new. It has long been recognized – even if difficult to put into action, that the whole is greater than the sum of its parts; in other words, the greater the user base and the greater the participation of that base, the greater the potential for knowledge construction, transfer and retention. Educational agendas, medical education included, are undergoing a shift towards more individualized learning and collaborative knowledge construction, which encourage interactive and reflective learning strategies. This type of active, contextual, integrative learning has many benefits for students beyond content transmission, including: improved understanding of principles and concepts; increased motivation and satisfaction; and enhanced self‐appraisal and self‐efficacy. To capitalize on the significant benefits of collaborative learning, we have developed a learning activity using a neuroscience Wiki to help our first‐year medical students at OUWBSOM review course content and develop a team learning project. Our class of 75 students is divided into 15 teams; each team is responsible for the creation of a 1–3 page topical neuroscience review ‐ Wiki submission. The submission is graded for accuracy, quality of citations and clinical relevance using a thoroughly vetted grading rubric. The submission is 5% of their overall course grade. Evaluation of the effectiveness of the activity in promoting student active learning is ongoing and involves score comparisons with previous groups, student surveys and focus groups.
Teaching anatomy online using retrieval practice, 3D stereoscopic images and virtual human fly‐through videos
Authors: Kolitsky Michael A.
Published: June 2021

An online Human Anatomy and Physiology course used Quizlets (practice quizzes) as a form of retrieval practice pedagogy along with a lab based on reusable learning objects such as 3D anaglyph images made from stereo image pairs in the Bassett Collection at Stanford University School of Medicine Medical History Center and Wright State University School of Medicine Quicktime VR Anatomical Resource. Fly‐through videos were made from the NIH National Library of Medicine Virtual Human series. Red‐blue glasses will be provided to view 3D images.

Higher course grades were observed in lecture and lab than in past courses when no Quizlets were used. Total Quizlet scores in lecture showed a moderate to high correlation using R‐squared analysis with the total exam score. Total Quizlet time (study time) was also linked to the grade received in the lecture course. The average lab exam scores for students using 3D images was 97.1% compared to 92.5% for students choosing to study non‐3D lab images available for students who cannot see in 3D. Although the 3D average lab exam score was slightly higher, it was not statistically significant. Student response, however, to using 3D images was strongly positive.
Historical development of the surgical technique for cleft lip and palate surgery: Bourgery's Treatise on Human Anatomy, 1854
Authors: Barros Mirna Duarte , Andreotti Joao Duvilio B , Levy Stephanie F , Curcio Daniella Franco , Liquidato Bianca Maria
Published: June 2021

The Bourgery's Treatise on Human Anatomy is a compendium on anatomy, surgery, and clinics, comprised of 16 volumes with 726 colored lithographic plates. Based on Bourgery's medical and anatomical experience the treatise is a detailed anatomical encyclopedia, with illustrated description of the surgical procedures.

Objective

to provide an overview of the historical development of the surgical technique for cleft lip and palate corrective surgery, based on Bourgery's treatise. We compared the Bourgery's Vol VII, plate 18, atlas VII, entitled “Becs de Lièvre”, with the descriptions of the current techniques for cleft lip and palate correction. The surgical treatments include bilateral incisions on the edge surface of the cleft, followed by retropulsion fracture of the incisive tubercle with triple swing flap technique. During the XVI century, the use of caustic substances, antimony and nitrate on the edge surface of the cleft was common; in the XVIII century, scalpels and scissors were the instruments most frequently employed in surgical reconstructions, whilst only scissors continued to be used throughout the XIX century. The treatise shows the anatomy of the cleft lip and palate, surgical timing, complications, treatments, and final results. Current surgical techniques are very similar, except for modifications that improve functional and aesthetic results: 1. Millard I and II techniques are used for lip repair; 2. Veau‐Wardill‐Kilner‐Braithwaite, for palate repair in “V” or “Y” shape; 3. McComb technique, extended incision up to the alar wing and sulcus. The Bourgery's Treatise of Human Anatomy is a historical source, from where it is possible to review and learn on surgical techniques for cleft lip and palate usually performed until the XIX century.
Chromatin Dynamics and the Fanconi Anemia DNA Damage Response Pathway
Authors: Neira Karissa L , Mauro Maurizio , Howlett Niall G
Published: June 2021

Fanconi anemia (FA) is a rare genetic disease characterized by congenital defects, bone marrow failure and cancer susceptibility. FA is caused by biallelic mutations in any one of fifteen genes. The FA proteins function in the FA‐BRCA pathway to repair DNA damage and to prevent cellular transformation. A hallmark of FA patient cells is increased sensitivity to DNA interstrand crosslinking agents such as mitomycin C (MMC). A key step in the activation of this pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which targets these proteins to chromatin. Following exposure to DNA damaging agents, histones are subject to posttranslational modification, e.g. acetylation, resulting in chromatin remodeling, facilitating the recruitment of DNA repair proteins to sites of DNA damage. In this study, we report that inhibition of class I and II, but not class III, histone deacetylases leads to a failure to activate the monoubiquitination of FANCD2 and FANCI following exposure to MMC. This leads to increased sensitivity to the cytotoxic and clastogenic effects of MMC. In addition, we describe the identification and characterization of a putative FANCD2 H4 binding domain. Interestingly, mutation of this domain leads to polyubiquitination of FANCD2. Together our preliminary results suggest that activation of the FA‐BRCA pathway is dependent upon the deacetylation of histones and on interaction with histone H4.
Innovative immunological assays for diagnosis of Schistosoma mansoni for clinical acute and/or chronic forms
Authors: Queiroz Rafaella Fortini Grenfell , Harn Donald , Coelho Paulo Marcos
Published: June 2021

Control constraints of schistosomiasis include the lack of diagnostic methods with high sensitivity. We initiated a prospective study in southeast Brazil in order to develop sensitive diagnostic methods for Schistosoma mansoni infection, with 3 endemic areas together with 84 travelers infected in a freshwater pool. Sera samples from all those patients are used for the standardization of innovative methods for schistosomiasis mansoni. Comparisons are performed with eggs in feces, IgG titers, encephalomyelitis by NMR and clinical symptoms. We could diagnose acute patients 10 days post‐infection and also more than 95% of positive cases from chronic and low endemicity patients. New methods for IgG detection using purified glycoprotein or recombinant protein or peptides (10 aminoacids) were superior to conventional ELISA. Best results were seen for recombinant protein with 100% of sensitivity. Lastly, monoclonal antibodies were produced for improvement of the new methods by promoting direct diagnosis. Data showed 100% of sensitivity of chronic patients and 98% of acute patients. Financial support: Fapemig, CNPq, Capes, Fiocruz, Fiotec, PDTIS (Brazil). Fulbright, NIH, University of Georgia (USA).
CASPASE‐1 ACTIVATION IS CRITICAL FOR ENDOTHELIAL ACTIVATION AND EARLY ATHEROGENESIS
Authors: Yin Ying , Wang Hong , Yang Xiao‐feng
Published: June 2021

We hypothesized that caspase‐1(casp1)/inflammasome in endothelial cells (ECs) as the innermost cells exposed to circulation can sense hyperlipidemia as a danger signal in early atherosclerosis and initiate EC activation. Our data demonstrated that: 1) early hyperlipidemia induced casp‐1 expression and activation in mouse aorta in apolipoprotein E (ApoE)−/− mice with high fat (HF) diet; 2) proatherogenic oxidized low density lipoprotein(ox‐LDL), two oxLDL active components, such as lysophosphatidylcholine and lysophosphatidic acid, induced casp‐1 activation in human aortic ECs (HAECs); 3) deficiency of casp‐1 attenuated oxLDL‐induced EC activation by inhibiting the up‐regulation of EC adhesion molecules including ICAM‐1, VCAM‐1 and E‐selectin in both mRNA and protein levels in primary mouse aortic ECs; 4) casp‐1 inhibitors attenuated oxLDL‐induced HAEC activation and monocyte adhesion to HAECs. We then generated the ApoE−/−/Casp‐1−/− double knockout mice, and found that the double knockout mice contained significantly less atherosclerotic lesion in mouse aortic arch and less CD11b+/F4/80‐monocyte and CD11b+/F4/80+ monocyte recruitments into mouse aorta. Collectively, our data have demonstrated for the first time that caspase‐1/inflammasome in ECs can sense early hyperlipidemia and become activated, drive EC activation, and promote monocyte recruitment and early atherosclerosis.
A Systematic Study of the Chemical Stress Response Induced by Bleach And Vinegar in E. Coli on Viability and Protein Expression Profiles Using Plating Assays And Matrix‐Assisted Laser Desorption Ionization (MALDI) Biotyper™ System
Authors: Liu Serena , Vardar‐Ulu Didem
Published: June 2021

Bacterial water contamination is a serious global public health problem. Even though most mammals carry Escherichia coli (E. coli) in their guts, certain strains can lead to severe ailments of the gastro‐intestinal tract including diarrhea, the second leading cause of death in children under five. While the contamination frequently originates at the water source, water storage conditions can heavily exacerbate the health risk. Here, we present our systematic study of the effects of two ordinary household chemicals, bleach and vinegar, on E. coli viability and protein expression profiles, as a function of chemical concentration, contact time, E. coli density, and incubation temperature, A combination of standard culture plating assays and spectroscopic measurements were used to assess viability and to determine the minimal chemical concentration required to keep bacterial levels below the EPA's safe drinking water limits. Matrix‐assisted Laser Desorption Ionization (MALDI) Biotyper was used to compare the unique molecular fingerprint of E. coli subjected to multiple chemical stress conditions and harvested at different time points. This work represents the initial step in identifying E. coli proteins sensitive to chemical treatment and could provide insights into the mechanism of stress response in E. coli that would be important in developing strategies to overcome bacterial resistance.