bluuebunny/crossref_metadata_2025 huggingface dataset:
Antioxidant N‐Acetylcysteine attenuates the reduction of cardiac Brg1 protein expression in Type1 Diabetic Rats
Authors: Xu Jinjin , Lei Shaoqing , Liu Yanan , Irwin Michael G , Xia Zhong‐yuan , Hei Ziqing , Gan Xiaoliang
Published: June 2021

Hyperglycemiainduced oxidative stress contributes to the development of diabetic cardiac complications. Brahma‐related gene 1(BRG1) is a key gene in regulating the expression of important endogenous antioxidant enzymes, including heme oxygenase‐1(HO‐1) which is central to cardioprotection, while cardiac HO‐1 is reduced in diabetes. We hypothesized that cardiac Brg1 expression is reduced in diabetes which can be restored by antioxidant treatment with N‐acetylcysteine (NAC). Control and streptozotocin‐induced diabetic rats were treated with NAC in drinking water or placebo for 4 weeks. At termination, cardiac function was assessed by echocardiography, and plasma and heart tissue free 15‐F2t‐isoprostane as well as myocardial Brg1 and HO‐1 protein expression were assessed. Plasma and cardiac free15‐F2t‐isoprostane in diabetes were increased, while cardiac Brg1 and HO‐1 protein expression were significantly decreased. Diabetes decreased the ratio of left ventricular peak velocity of early and late diastolic filling (E/A) and reduced isovolumetric relaxation time, indicative of diastolic dysfunction. NAC significantly increased cardiac Brg1 and HO‐1 protein expression and improved cardiac function. It is concluded that cardiac Brg1 is reduced in diabetes and enhancement of cardiac Brg1 may represent a novel mechanism of NAC cardioprotecion.
Acetaminophen and Isoniazid treatment leads to oxidative stress and cytotoxicity in cerebellar granule neurons possibly by CYP2E1 activation
Authors: Valencia‐Olvera Ana Carolina , Morán Julio , Camacho‐Carranza Rafael , Espinosa‐Aguirre Javier
Published: June 2021

Increasing evidence suggests that brain cytochrome P450 (CYP) can contribute to the in situ metabolism of xenobiotics. Xenobiotics can be metabolized by CYPs into more reactive products that can induce cytotoxicity and cell death. In addition, normal CYP enzymatic activity produces reactive oxygen species (ROS) that contribute to cell damage through oxidative mechanisms. CYP2E1 is a CYP isoform that can generate ROS and induce cytotoxicity in multiple tissue types. The aim of this study was to determine whether neuronal CYP2E1 induction by isoniazid and acetaminophen and/or its metabolism can induce significant brain cell impairment. Exposure of primary cultured cerebellar granule neurons to isoniazid increased CYP2E1 expression, ROS production and cell death. These results were also observed with acetaminophen treatment. The oxidative stress and cytotoxicity observed with both treatments was attenuated by simultaneous exposure to 4‐methyl pyrazole and diallyl sulfide, which are CYP2E1 inhibitors, or to a mimetic of superoxide dismutase/catalase, Eukarion‐134. These results suggest that the induction and activity of brain CYP2E1 could represent a risk of in situ xenobiotic‐induced oxidative neuronal damage. This work was supported by Fundación Miguel Alemán A.C. and DGAPA‐UNAM (IN214908), the first author is supported by a CONACYT fellowship.
Phosphodiesterase‐4‐inhibition stabilizes microvascular endothelial barrier functions and microcirculation in a model of polymicrobial sepsis
Authors: Flemming Sven , Schick Martin , Wunder Christian , Christoph‐Thomas Germer , Schlegel Nicolas
Published: June 2021

In sepsis breakdown of endothelial barrier functions contributes to disturbed microcirculation, organ failure and death. Increased cAMP levels have been demonstrated to protect endothelial barrier functions in inflammation. In the present study effects of systemically intravenously applied phosphodiesterase‐IV‐inhibitor rolipram (PD‐IV‐I) on microvascular endothelial barrier functions and microcirculation were tested in a rodent model of polymicrobial sepsis using the colon‐ascendens stent peritonitis (CASP) model. Animals with CASP showed clinical and laboratory signs of sepsis and peritonitis with increased IL‐6‐levels compared to sham‐operated animals. In postcapillary mesenteric venules PD‐IV‐I led to stabilisation of endothelial barrier properties as revealed by extravasation of intravenously applied FITC‐albumin. Only low‐dose application of 0,3 mg/h of PD‐IV‐I led to barrier protective effects in the CASP model whereas high‐dose therapy of 1,2 mg/h PDI‐IV‐I had adverse effects. Accordingly microcirculatory flow in mesenteric venules was only improved under low‐dose PD‐IV‐I therapy. Severe side effects of PD‐IV‐I application on blood pressure and cardiac output were not observed. The present data provide further evidence that systemic application of PD‐IV‐I can become a clinically relevant and applicable approach to stabilize endothelial barrier functions in sepsis.
Echocardiographic evaluation of Hif‐1α knock‐out mice in the early stage of diabetic cardiomyopathy
Authors: Papousek Frantisek , Bohuslavova Romana , Kolar Frantisek , Sedmera David , Skvorova Lada , Neckar Jan , Pavlinkova Gabriela
Published: June 2021

This study examined the role of hypoxia inducible factor 1α (HIF‐1α) in cardiac responses to diabetes. Functional, geometrical and morphological changes of left heart ventricle were evaluated after 5 weeks of streptozotocine‐induced diabetes using wild type and heterozygous Hif1a knock‐out (Hif1a+/−) male and female mice. Transthoracic echocardiography followed by the measurement of systemic blood pressure was performed under isoflurane (2%) anesthesia. Hearts were dissected and left ventricles were processed for subsequent RT‐qPCR and morphological analyses. Echocardiography did not reveal any difference between both genotypes in non‐diabetic mice, while diabetes resulted in a significant decrease of fractional shortening (by 11%) in Hif1a+/− mice of both sexes. The morphological analysis did not show any significant difference among the groups. However, the combination of diabetes and HIF‐1α insufficiency affected the gene expression profile of the heart including reduced vascular endothelial growth factor A expression. These results suggest that HIF‐1α deregulation during early stage of diabetes contributes to the development of cardiomyopathy.

Supported by grants PIRG02‐GA‐2007–224760, GACR 301/09/0117 and AV0Z50520701.
NRF‐1 and its target genes are increased by exercise: Potential role of NRF‐1 in type 2 diabetes
Authors: Mukwevho Emmanuel
Published: June 2021

Nuclear respiratory factor (NRF)‐1 is a mitochondrial transcriptional factor whose function of late shown to be involved in glucose transport (Ramachandran et al., 2008, Baar et al., 2003) and set it as a potential therapeutic modality in the treatment and management of type 2 diabetes. In this study we sought to access NRF‐1 and its target genes expression during exercise which are crucial in glucose transport and lipid oxidation.

Five to six weeks old male Wistar rats were exercised to identify the time points for optimum increase in the levels of NRF‐1 and its target genes. Gastrocnemius muscle was harvested after 0, 2, 4, 6, 8, 10, 12 and 15 hours (h) post exercise and non‐exercise rats (control). Primers were used to amplify the region of following genes: Nrf‐1, Glut 4, Carnitine palmitoyltransferase (Cpt‐1& Cpt‐2), Peroxisome proliferator‐activated receptor gamma co‐activator 1 (Pgc‐1), Mef2a, Acetyl‐CoA Carboxylase‐1 (Acc‐1). Relative mRNA expression was normalized to actin reference gene.

Cpt‐1, Nrf‐1, Mef2a, Glut4, Cpt2 and Pgc‐1 showed 2.5, 8, 1.2, 4.1, 4.6, 3.5 fold increases respectively after 8h post exercise compared to control whereas Acc‐1 showed 3.1 fold decrease in gene expression ratio after 6 h post exercise compared to control. NRF‐1 binding to cpt‐1 and mef‐2 increased 3 and 3.5 fold respectively 6h post exercise compared to controls.

These results show that NRF‐1 was increased by exercise and also its binding to target genes which has huge implication in emeliorating type 2 diabetes and insulin resistance.
Hydrogen sulfide attenuates homocysteine induced neurovascular dysfunction
Authors: Kamat Pradip , Kalani Anuradha , Givvimani Srikanth , Tyagi Suresh C , Tyagi Neetu
Published: June 2021

High levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), are associated with neurovascular diseases such as vascular dementia. Endogenously, hydrogen sulfide (H2S) is produced as a metabolite of Hcy by cystathionine β‐synthase, cystathionine γ‐lyase, and 3‐mercaptopyruvate sulfurtransferase. H2S is a potent anti‐oxidant, anti‐inflammatory, anti‐hypertensive and vasorelaxing agent. However, the effect of H2S has not been explored in Hcy induced neurovascular dysfunction. Therefore, the present study was designed to explore the protective role of H2S in neurovascular dysfunction induced by Hcy. To test this hypothesis we employed 8 weeks old male wild type (WT), WT+aCSF WT+ Hcy (5μmol in aCSF) intra‐cerebral injection (one time only prior to NaHS treatment), WT+Hcy +NaHS (sodium hydrogen sulfide, precursor of H2S, 30 μmol/L in 0.9% normal saline). NaHS was injected intra‐peritoneally once daily for a period of 7 days after Hcy treatment. Memory function was assessed by novel object recognition test. The brain tissues were analyzed for neuronal loss, oxidative‐nitrosative stress, matrix metalloproteinases, tight junction proteins, tissue inhibitor of matrix metalloproteinases, cell death, neuroinflammatory and synaptic markers by Western Blot, qRT‐PCR and IHC. Hcy administration induced oxidative‐nitrosative stress, apoptosis, neuronal loss, neuro‐inflammation along with vascular dysfunction. Interestingly, administration of NaHS significantly improved neurovascular dysfunction in Hcy treated groups thereby suggesting its potential therapeutic role against HHcy‐related neurovascular diseases.
Effects of Exercise Ancestry on Multiple Generations of Mature C57BL/6 Mouse Offspring
Authors: Guth Lisa M , Venezia Andrew C , Marini Michael P , Beltran Estefan P , Spangenburg Espen E , Roth Stephen M
Published: June 2021

The objective was to examine if exercise (EX) ancestry vs. sedentary (SED) ancestry alters body/tissue mass, glucose metabolism, or muscle mRNA in two generations of mature mouse offspring. C57BL/6 mice (F0) were exposed to EX (wheel running, N=20) or a SED condition (no wheel, N=20) for 12 weeks before breeding. EX males bred with EX females and SED males bred with SED females to obtain F1 pups. F0 EX animals had wheel access through weaning. F1 pups bred with like‐ancestry F1 offspring to obtain the F2 pups. F1 and F2 offspring were sacrificed at 28 wk without EX exposure. Body and tissue masses were recorded, intraperitoneal glucose tolerance was measured, and mRNA expression of several metabolic genes was examined in muscle. In F1 males, liver and soleus mass were lower in EX (p < 0.05). F2 EX males also had lower liver and soleus masses (p < 0.05). In females, no differences in tissue mass were observed. However, F1 EX females had higher baseline glucose (p < 0.05) while F2 SED females had better glucose tolerance (p < 0.05). No differences in glucose or glucose tolerance were observed in male offspring. Pgc1a mRNA expression was higher in F1 EX males and Cox1 mRNA expression was higher in F2 SED males. There were no other differences in mRNA expression. These findings suggest limited, generation‐ and sex‐specific effects of EX ancestry on tissue mass, glucose metabolism, and muscle mRNA in two generations of mature C57BL/6 offspring.

This work was supported by NIH grants HD062868 and AG000268.
Caffeine improves anaerobic performance in a 90‐s Wingate test in habitual caffeine users
Authors: Ogden Kevin , Haak Jodie L , Kieffer H. Scott , Marlow N
Published: June 2021

The purpose of this study was to evaluate the effects of caffeine on a maximal anaerobic exercise protocol using the 90‐s Wingate Test (WAnT90). 29 (11 females, 17 males) anaerobically‐conditioned athletes volunteered for a randomized counterbalanced double‐blind study. Subjects were classified as either habitual consumers (n=15) or caffeine naïve (n=13) based on daily caffeine consumption habits. One h after ingesting caffeine (5mg/kg) or placebo participants completed a WAnT90 using a resistance of 0.05g/kg. In addition to peak power (PP), total power (TP), and total power decline (PDT), power and power decline were calculated for each 30‐s interval of the test (TP30, TP60, TP90, PD30, PD60, PD90). Paired t‐tests compared caffeine vs placebo in each group, and significance was set at p<0.05. In the habitual caffeine consumption group TP, TP30, and TP60 were significantly greater in the caffeine trial. In the caffeine naïve group PD30 was greater in the caffeine trial; there were no other significant effects of caffeine on this group. This study suggests an ergogenic effect of caffeine for those who regularly consume caffeine but not for those who do not. These results imply a possible learning effect for caffeine; further research should focus on a mechanism.
Beneficial effect of exercise combined with licorice flavonoid oil supplementation on visceral adipose tissue
Authors: Tanaka Masayuki , Kanazashi Miho , Tanaka Minoru , Kondo Hiroyo , Murakami Shinichiro , Fujita Naoto , Fujino Hidemi
Published: June 2021

Objective

We investigated the beneficial effect of exercise combined with licorice flavonoid oil (LFO) on visceral adipose tissue, and the lipid metabolism in skeletal muscle and liver.

Methods

Male Sprague‐Dawley rats divided into four groups: control, exercise, control with LFO, and exercise with LFO groups. The rats subjected to exercise ran on a treadmill 5 times a week. LFO was administered 1500mg/kg body weight daily. After 7 weeks, overnight fasted rats were killed, and samples were collected. Adipocyte cell size in the epididymal white adipose tissue and the fat‐oxidation related enzyme, CS, HAD and CPT‐2, activities in the plantaris muscle and liver were measured.

Results

Both exercise and LFO reduced the adipose tissue mass and adipocyte cell size. In addition, exercise combined with LFO more effectively reduced the adipose tissue than only exercise or LFO. Furthermore, exercise increased the muscular CS and HAD activities, and LFO increased the hepatic CPT‐2 activity.

Conclusion

Exercise combined with LFO effectively reduced visceral adipose tissue, because the combination enhanced the muscular and hepatic lipid metabolism, respectively. These results suggested that the combination might be effective to decrease the risk from obesity. Supported by Grants‐in‐Aid for Science Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
Periaxonal Application of Capsaicin as a Novel Method for Selective Afferent Renal Denervation
Authors: Foss Jason , Engeland William C , Osborn John W
Published: June 2021

Because the differential role of afferent and efferent renal nerves in hypertension is unclear, we developed a method of afferent renal denervation (ARDN) by periaxonal application of capsaicin (renal‐CAP). Sprague‐Dawley (SD) rats were subjected to SHAM surgery or renal‐CAP and sacrificed after 10 days. Immunohistochemistry (IHC) staining for calcitonin gene related peptide (CGRP; a marker of afferent fibers) and tyrosine hydroxylase (TH; a marker of efferent fibers) showed near complete depletion of CGRP from the kidneys with no effect on TH. We next tested the hypothesis that afferent renal nerves regulate arterial pressure (AP) and fluid balance during chronic salt loading. SD rats were subjected to SHAM surgery or renal‐CAP, instrumented with telemeters for measurement of AP and heart rate (HR), and placed in metabolic cages. Rats were fed a 0.1% NaCl diet for 2 weeks, 4% NaCl for 3 weeks and 8% NaCl for 2 weeks. There were no differences in AP or sodium/water balance between SHAM and renal‐CAP rats throughout the protocol; however, SHAM rats exhibited progressive bradycardia that was attenuated in renal‐CAP rats (p<0.05). IHC showed partial depletion of CGRP after 7 weeks. We conclude that renal‐CAP is an effective method of ARDN, afferent renal nerves play a minimal role in the regulation of AP and fluid balance in normotensive rats, and afferent renal nerves mediate sodium dependent decreases in HR.
A questionnaire study to evaluate brain fog in orthostatic intolerance
Authors: Ross Amanda , Schwartz Christopher , Shanks Lindzi , Jason Leonard , Medow Marvin , Rowe Peter , Stewart Julian
Published: June 2021

Individuals with orthostatic intolerance (OI) often experience ill‐defined neurocognitive impairment, typically referred to by patients as brain fog. The objective of this study was to evaluate brain fog to further insight on its mechanism and potential treatments. Eligible subjects, 14–29 yrs diagnosed with OI, were recruited through ads on online support groups. Participants electronically completed a 38‐item questionnaire designed for this study. Of 116 participants (91% F), 111 reported brain fog. The symptoms most concurrent with brain fog were fatigue (88%), lightheadedness (78%), headaches (75%), and palpitations (68%). The most frequent triggers were fatigue (95%), lack of sleep (94%), prolonged standing (93%), and heat (85%). The best preventive strategies were staying cool (69%), high fluid intake (65%), high salt intake (62%), and compression stockings (44%). The interventions most frequently reported to improve brain fog were intravenous saline (74%), stimulants (64%), salt tablets (52%), IM vitamin B12 (52%), and midodrine (44%). Brain fog is associated with the onset of typical OI symptoms and triggers that exacerbate OI. Yet, some endorsed treatments are not commonly used to treat OI and deserve further study in this population.
A Gradual Rise in Cancer Stem Cells in the Crypt‐Villous Axis of the Colon During Aging
Authors: Majumdar Adhip , Nangia‐Makker Pratima , Yu Yingjie
Published: June 2021

One of the most consistent pathological conditions in the gastrointestinal (GI) tract with advancing age is malignancy, specifically colorectal cancer. Although the underlying cellular and molecular events are not fully understood, we have suggested a role for self‐renewing pluripotent cancer stem/stem‐like cells (CSCs) in this process. Earlier, we demonstrated that the age‐related increase in colonic polyps is associated with a concomitant rise in CSCs. An age‐related rise in CSCs in the colon is also observed in experimental animals. Since CSCs are thought to be the mutated counterpart of normal stem cells, we hypothesize that a gradual increase in CSCs in the crypt‐villous axis of the colon may lead to tumor. To test this hypothesis, we isolated mucosal cells from the upper, middle and lower regions of the colon of young and aged Fischer‐344 rats and subjected them to functional assays for CSCs and for mutational status. We found that mucosal cells from the middle and lower, but not the upper part of the colon formed spheres/spheroids, but was much higher in aged than young animals. This increased spheroid forming ability of mucosal cells from aged rats was also associated with increased expression of CD44 and b‐catenin. In contrast, CK‐20, a marker of differentiation, was augmented in the middle/upper part of aged colon, compared to lower region. In addition, spheres formed by mucosal cells from aged, but not young rats could be expanded to 2nd generation, indicating higher self‐renewal ability of CSCs of aged rats. Frequency of gene mutation was also higher in the lower region of the colon of aged than young rats. We conclude that with aging there is a gradual increase in CSCs in the colonic crypt which may partly be responsible for the age‐related rise in colorectal cancer.
R‐type Ca2+ Channels Contribute to Neural Control of Murine Colonic Motility
Authors: Skylis Laura , Galligan James , Rodriguez‐Tapia Eileen
Published: June 2021

Background

The enteric nervous system controls gastrointestinal motility. Multiple subtypes of voltage‐gated Ca2+channels contribute to neurotransmitter release from enteric neurons.

Objective

We tested the hypothesis that R‐type Ca2+channels contribute to enteric neurotransmitter release in the mouse colon.

Methods

Studies were done in vivo and in vitro using wild type (WT) and a1E Ca2+ channel (R‐type) knockout (KO) mice. Fecal output was measured in vivo. The colonic migrating motor complex (CMMC) was measured in vitro. We used NiCl2 (50 mM) to block R‐type Ca2+ channels , Ω‐conotoxin (CTX, 0.1 mM) to block N‐type Ca2+ channels and tetrodotoxin (TTX, Na+ channel blocker, 0.3 mM) on CMMCs.

Results

There was no significant difference in fecal output between a1E KO and WT mice. There was no difference in the frequency, propagation speed or amplitude of CMMC contractions between colons from WT and a1E KO mice. NiCl2 slightly decreased contraction amplitude in colons from a1E KO and WT mice. CTX and TTX completely blocked neurogenic contractions and disrupted the CMMC pattern, leaving only spontaneous myogenic contractions behind.

Conclusion

These results suggest that either R‐type Ca2+ channels have a minor role in controlling colonic motility, or that there is compensatory upregulation of other Ca2+ types in the constitutive a1E KO mice.

*Supported by: R01DK094932
Role of Endothelin 3 (ET3) in Estradiol 17B‐glucuronide (E217G)‐induced Cholestasis in the Rat
Authors: Rodriguez Myrian , Martinefski Manuela , Tripodi Valeria , Vatta Marcelo , Bianciotti Liliana
Published: June 2021

We previously reported that ET3 induces choleresis through ETB receptors coupled to nitric oxide (NO). It enhances, independently of hemodynamic changes, bile acid dependent and independent bile flows and promotes plasma membrane insertion of the main hepatic transporters involved in bile genesis and increases their mRNA expression (Clin Sci., 125:531, 2013). In this work we aimed to determine whether ET3 played a beneficial role in E217G‐induced cholestasis. Sprague‐Dawley rats were infused with ET3 (5 ng/kg/min) or vehicle for 30 min followed by E217G administration to induce cholestasis. Bile samples were collected every 5 min for 120 min. Bile acids were assessed in bile by capillary electrophoresis. Other set of animals were also pretreated with L‐NAME (NO synthases inhibitor) or BQ788 (ETB receptor antagonist) before ET3 and E217G administration. ET3 prevented E217G‐induced cholestasis and the response was abolished by BQ788 or L‐NAME. Furthermore ET3 also modified bile acid excretion, particularly ursodeoxycholic acid and lithocholic acid as compared with control and E217G. ET3 induced no changes in plasma transaminases, bilirubin, alkaline phosphatase or 5´nucleotidase or in the hepatic histology assessed by light microscopy in stained hematoxylin and eosin liver samples. Present findings show that ET3 through ETB receptors coupled to NO prevents E217G‐induced cholestasis and modifies bile acid profile excretion in the rat. Results further suggest that the ETB receptor may represent a promising therapeutic target in pathophysiological situations where bile flow is impaired.
Regeneration associated pathophysiology following major hepatectomy with subsequent choledoco‐jejunostomy: A comparison with simple hepatectomy
Authors: Takagi Takehiro , Yokoyama Yukihiro , Kokuryo Toshio , Yamaguchi Jumpei , Nagino Masato
Published: June 2021

Background

The hepatectomy with extrahepatic bile duct resection and subsequent choledoco‐jejunostomy (CJ) are often required in bile duct carcinoma. However, the impact of CJ is unknown in the liver regeneration after major hepatectomy.

Methods

We established a novel rat model, 70% hepatectomy with CJ (Hx+CJ rat). The inflammatory response and regeneration rates were evaluated in the liver using 70% hepatectomy (Hx rat) or hepatectomy with CJ (Hx+CJ rat). The gene expression was analyzed concerning inflammation and regeneration in the liver of these two groups.

Results

Histological examination revealed that progressive periportal inflammatory response in the liver of Hx+CJ rat. The hepatic regeneration rate was significantly lower in the Hx+CJ rat compared to the Hx rat on days 2 and 4 after surgery. The expression of inflammation‐associated genes was higher in the liver of the Hx+CJ rat than the Hx rat. The regeneration‐associated genes, such as hepatocyte growth factor and epidermal growth factor were significantly lower in the Hx+CJ rat than the Hx rat.

Conclusion

These results showed that CJ impairs the liver regeneration after major hepatectomy. CJ induce the progressive periportal inflammation in the liver. The inflammation partly causes the suppression of regeneration‐associated genes in the liver.
Breathing Frequency (fR) Is No Longer Under Chemoreflex Control During REM Sleep
Authors: Burke Peter , Kanbar Roy , Basting Tyler , Viar Kenneth , Stornetta Ruth , Guyenet Patrice
Published: June 2021

Objective

To define the contribution of central chemoreception in general and the retrotrapezoid nucleus (RTN) in particular to the regulation of breathing in REM sleep.

Methods

EEG, neck EMG, blood pressure, fR and tidal volume (VT) were recorded in 23 conscious adult male Sprague‐Dawley rats. RTN was optogenetically stimulated with channelrhodopsin‐2 (ChR2, 20s) or inhibited with archaerhodopsin (Arch; 10 s) during natural sleep and quiet wake periods. ChR2 and Arch were delivered using previously described PRSX8‐promoter containing lentiviral vectors.

Main Results

In quiet wake or non‐REM sleep, hypercapnia (3 or 6% FiCO2) increased both fR and VT; in REM sleep, hypercapnia increased VT but fR was unaffected. Optogenetic inhibition of RTN neurons reduced VT in all three states but reduced fR only during quiet wake and non‐REM sleep. Optogenetic stimulation of RTN neurons always increased VT but raised fR only in quiet wake and non‐REM sleep. Phasic stimulation of RTN at frequencies above resting fR entrained and shortened the breathing cycle except in REM sleep. Phasic RTN stimulation produced active expiration only during wake and increased fR exclusively by reducing the duration of the post‐inspiratory phase.

Conclusions

During REM sleep, VT regulation by both hypercarbia and RTN is retained but the overall hypercapnic ventilatory reflex is reduced because fR is no longer under chemoreceptor control. Furthermore, respiratory drive contributed by RTN is remarkably state‐dependent, active expiration being elicited only during wake and tachypnea only during non‐REM sleep and quiet wake. Support: HL074011.
Effects of Tracheal Distension on Upper Airway Reflex Responsiveness in Cat
Authors: Shen Tabitha , Musselwhite M , Rose Melanie , Bolser Donald , Pitts Teresa
Published: June 2021

A tracheostomy cuff is used in patients to ensure no material enters the lower airway. To clinically determine whether a patient is ready for cuff‐deflation airway protective reflexes are tested, often while the cuff is actively distending the trachea. We hypothesized that tracheal distension alters normal sensory feedback and decreases airway protective reflex efficacy. In adult cats, a balloon was inflated at the 4th cartilaginous ring of the trachea, and electromyography (EMG) activities from the diaphragm, internal oblique, crycoarytenoid and mylohyoid muscles were recorded. Cough was stimulated by mechanical stimulation of the intrathoracic trachea. Swallow was elicited by a water bolus to the oropharynx. The presence of the deflated balloon in the trachea greatly promoted the number of swallows (105±60%). Moderate tracheal distention decreased diaphragm (~20%) and internal oblique (~30%) EMG activity compared to baseline during cough, and partially recovered 10 minutes after distension was removed. Cough number decreased by 52±13% during tracheal distension. No significant effect on crycoarytenoid or mylohyoid (~4%) EMG activity during swallow was observed. However, large tracheal distensions induced depression in swallow EMG magnitude and number. These results indicate that tracheal distension in the physiological range alters sensory feedback that is important in the production of cough but not swallow, although the production of swallow can be negatively affected by distensions that increase the risk of mechanical or ischemic damage to tracheal tissue. Therefore, clinical assessments of cough when a tracheal cuff is or recently has been present may not represent a metric of normal function. Supported by HL 103425.
NMDA receptors and Nitric Oxide Synthase Regulate Encoding of Cough in the nTS
Authors: Farmer David , Mori Nanako , Canning Brendan
Published: June 2021

We sought to locate and characterize the central terminations of cough receptor airway afferents in the nucleus tractus solitarii (nTS) of guinea pigs. Neuronal tracing indicated that cough receptors terminate in locations bordering the medial and intermediate nTS. Microinjection of NMDA receptor antagonists into these locations nearly abolished cough evoked by citric acid (0.001‐2M; 100μl aliquots) applied to the trachea of anaesthetised animals (vehicle: 10±3; AP‐5: 3±2; SDZ 220‐581: 0±0 coughs; n=3‐6).

Microinjection of NMDA produced respiratory perturbations including tachypnoea (0.01nmol NMDA) and bradypnoea/apnoea (0.1nmol NMDA) that were eliminated by NMDA receptor antagonists or inhibitors of nitric oxide synthase (NOS): L‐NNA (non‐specific) or N‐propyl‐L‐arginine (NPLA) (neuronal NOS‐specific). NOS inhibitors also reduced citric acid‐evoked coughing (Vehicle: 11±2; L‐NNA: 4±1; NPLA 4±2 coughs; n=6‐8). Citric acid‐induced cough was not attenuated after inhibition of soluble guanylate cyclase (sGC) with ODQ (vehicle: 11±3 coughs; ODQ: 14±2 coughs; n=8) or potentiated after inhibition of phosphodiesterase‐5 (PDE5) with zaprinast (vehicle: 9±3 coughs; zaprinast 10±1 coughs; n=3). Our data indicates that reflex cough is dependent on NMDA receptor activation and/or NO in the nTS. NO signalling occurs independent of either sGC or PDE5, suggesting that alternative signalling mechanisms are at play.

Work supported by NIH.
Lung Lining Interaction Determines the Fate of Multi‐Walled Carbon Nanotubes (MWCNTs) in vivo
Authors: Botelho D , Shaffer M , Porter A , Chung KF , Tetley T , Zhang J , Gow A
Published: June 2021

MWCNTs are coated by lipid when incubated with dipalmitoylphosphatidylcholine (DPPC)—the primary component of lung lining fluid (LLF). This lipid coating allows MWCNTs to integrate into the LLF, and to be phagocytized by lung macrophages. We propose that in vivo, the presence of surfactant protein‐D (SP‐D) is needed for clearance of MWCNTs. MWCNTs, functionalized by carboxylation (C) or acid‐etched (A), (30‐50nm OD, 0.5‐2μm length) were instilled at 1.5μg/g body weight into C57Bl/6 or SP‐D‐/‐ mice. In C57Bl/6, C‐MWCNT induced neutrophilia (120.9 ± 55.9 v 0.2 ± 0.24 x103) and increased cell count (234 ± 57.6 v 100 ± 35.3 x103), 1 day post instillation (not seen with A‐MWCNTs). Lung mechanics were unchanged; but there was a decrease in phospholipid (PL) content and an increase in surfactant protein‐B (SP‐B) in the LLF (A‐ & C‐MWCNTs); consistent with lipid‐coating of MWCNTs and consequent phagocytosis. 3 days post instillation, neutrophilia, PL content and SP‐B levels resolved, but there was an increase in SP‐D; without significant change in lung mechanics (A‐ & C‐MWCNTs). In SP‐D‐/‐ mice, C‐MWCNTs elicited an acute neutrophilic response (63.6 ± 23.4 v 1.4 ± 1.44 x103). SP‐D‐/‐ mice displayed increased PL at baseline, but no reduction in response to MWCNT exposure, and no change to lung mechanics. Neutrophilia had resolved at 3 days in SP‐D‐/‐, but there was impairment of lung mechanics; especially with A‐MWCNTs. Though lipid coating allows for MWCNTs to be phagocytized, in the absence of SP‐D, MWCNTs are not cleared efficiently (as shown by maintenance of PL levels & the presence of MWCNTs in the LLF at 3 days). Failure to clear MWCNTs appears to increase lung collapse by surfactant dysfunction.
Female Rats Are More Sensitive to Enhanced Reinstatement of Alcohol Seeking Following Exposure to Both Alcohol‐related Cues and Yohimbine
Authors: Bertholomey Megan , Torregrossa Mary
Published: June 2021

Exposure to cues previously associated with drug use and to stress can each promote craving and increase the risk of relapse. Preclinical studies that have combined these stimuli in tests of the reinstatement of drug seeking have shown additive effects on craving‐like behavior, which was enhanced in female rats trained to self‐administer cocaine. Therefore, we predicted that exposure to the pharmacological stressor, yohimbine (YOH), and cues previously associated with alcohol reinforcement would produce additive effects on reinstatement and that this effect would be augmented in females. To test this hypothesis, male and female rats were trained to self‐administer a 10% ethanol/0.1% saccharin solution on a fixed ratio 1 schedule of reinforcement in 1‐hour sessions. Delivery of alcohol was paired with a light+tone cue. Responding was then extinguished, and rats were subsequently injected with either 1.25 mg/kg YOH or vehicle in the presence of absence of the light+tone cue during a non‐reinforced reinstatement test. As predicted, female rats showed reinstatement following both cue and YOH exposure alone and twice as great reinstatement when combined. In contrast, males only showed cue‐induced reinstatement which was modestly enhanced by YOH. These finding suggest that female rats are more sensitive to complex environmental factors in provoking a craving‐like response that might increase the risk of relapse to alcohol drinking. Ongoing studies are exploring the mechanisms that underlie sex differences in the ability of stress to enhance cue‐related alcohol seeking with the goal of developing behavioral and pharmacological approaches to preventing relapse. *Supported by K01DA071345, AHA 13BGIA16850030, and The PA Department of Health.
Micelle Sequestrant Polymers as Novel Therapeutics to Treat Obesity
Authors: Sullivan Bradley , Qian Jian , Berkland Cory
Published: June 2021

Thirty‐five percent of the United States population is classified as overweight or obese. Several options exist to treat obese patients; however, currently available therapies are associated with side effects or poor long term compliance necessitating development of more efficacious and accepted therapeutics for treating obesity. We synthesized and evaluated a library of Micelle Sequestrant Polymers (MSP) as novel orally dosed non‐absorbable molecules that bind intestinal micelles (i.e., dietary triglycerides, bile acids, and cholesterol) and prevent their digestive absorption. Individual MSPs were incorporated into a high fat diet (HFD, 45% kcal from fat) at 0.5% (wt/wt) and fed to mice. Feeding mice a MSP containing HFD increased fecal elimination of triglycerides. Interestingly, fecal elimination of bile acids was increased by some, but not all MSP containing HFDs. Of importance, fecal bile acid, fecal triglyceride, and weight loss was increased in mice treated with 300 or 1,500 mg/kg/day of a top performing MSP via daily oral gavage for 5‐days compared to mice fed HFD given control gavage (H2O), although weight loss in the 300 mg/kg group did not achieve the level of statistical significance. The data suggest that MSPs can bind whole intestinal micelles and enhance fecal elimination of bile acids and triglycerides. Taken together, MSPs may serve as a novel weight loss platform for inhibiting excess caloric intake by preventing absorption of dietary triglycerides. Furthermore, increasing fecal elimination of bile acids may also simultaneously serve as a cholesterol lowering agent by increasing de novo bile acid synthesis, a metabolite of cholesterol.
Effect of Cocaine and p21 on the Blood Lipidome
Authors: Scholpa Natalie , Sahin Serap , Wagner John , Cummings Brian
Published: June 2021

Cocaine is powerfully addictive and induces a breadth of physiological effects, even at low, recreational doses. To begin to discern the full extent of these effects, we used electrospray ionization‐mass spectrometry (ESI‐MS) by direct infusion to investigate drug‐induced alterations on the blood lipidome. Lipids were extracted from the whole blood of mice exposed to cocaine or saline using the Bligh‐Dyer method. ESI‐MS data was analyzed with multivariate principal component analysis to determine changes in lipid profile. Significant m/z features indicated alterations in four categories: fatty acyls, glycerolipids, glycerophospholipids and sphingolipids. Features were further identified into classes within each category, with prominent classes being acyl coenzyme A, triacylglycerol, cardiolipin and ceramide, respectively. While the directionalities varied, the greatest number of altered features were sphingolipids. Remarkably, some features correlated to drug‐seeking behaviors in cocaine‐treated mice, specifically locomotor response. How cocaine‐induced effects occur, including alterations in lipid profile, is not fully understood. Our data using a p21 knockout mouse model suggests a role for the cell cycle regulator in cocaine‐induced response in vivo and the effects of genetic ablation of p21 also extended to the blood lipidome. Knockout mice had distinct lipidomic profiles compared to wild‐type mice, indicating that p21 affects the blood lipidome. Again, the majority of significantly altered features were sphingolipids. Moreover, cocaine exposure also altered the blood lipidome in the knockout mice. Collectively, these results suggest not only a role for cocaine, but also for p21 in regulating the blood lipidome.
Mechanism of impaired lipid homeostasis in MCPIP1‐deficienct mice
Authors: Sedinkin Jessica , Chang Yingzi
Published: June 2021

Background

Monocyte chemotactic protein‐induced protein 1 (MCPIP1) is found in monocytes/macrophages in response to MCP‐1 stimulation. Our preliminary data showed that MCPIP1 knockout mice have increased plasma LDL cholesterol and decreased HDL and total triglycerides. We have also found that mice lacking MCPIP1 display a marked loss of adipose and muscle tissues. Our current project was designed to determine whether MCPIP1‐deficiency impairs lipid homeostasis by affecting the apoprotein expression hepatic remodeling.

Methods

C57/BL6 mice at 6‐8 weeks of age were used. Liver histological changes and plasma apoprotein expression were examined in samples collected from wild‐type, heterozygous, and MCPIP1 knockout mice.

Results

hepatic histological staining revealed an increase in collagen and fibrous tissue and a decrease in hepatocytes in MCPIP1 knockout mice. We also found that the expression of LPL, and apoB48 is increased but the apoB100 and apoA‐1 is decreased in the plasma from MCPIP1 knockout mice.

Conclusions

The data collected so far suggest that the impaired lipid homeostasis observed in MCPIP1 deficient mice may occur through hepatic remodeling and alteration of apoprotein expression and/or function.
NEW SELECTIVE PEPTIDYL DI(CHLOROPHENYL)‐PHOSPHONATE ESTERS TO VISUALIZE AND BLOCK NEUTROPHIL PROTEINASE 3 IN HUMAN DISEASES
Authors: Guarino Carla , Legowska Monika , Jenne Dieter , Lesner Adam , Gauthier Francis , Korkmaz Brice
Published: June 2021

The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent PR3. We developed selective phosphonate inhibitors with the structure: Ac‐peptidyl‐P(O‐C6H4‐4‐Cl)2. The peptidyl moiety was deduced from molecular modeling and kinetic studies comparing the substrate specificities of PR3 with a single residue PR3 mutant and HNE. The P4 and P2 positions were essential to discriminate between PR3 and HNE. We then synthesized N‐terminally biotinylated peptidyl phosphonates to identify PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils, despite their low molecular mass, suggesting that the conformation and reactivity of membrane‐bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease).
Biochemical and Pharmacological Characterizations of ESI‐09 based EPAC inhibitors
Authors: Zhu Yingmin , Chen Haijun , Mei Fang , Zhou Jia , Cheng Xiaodong
Published: June 2021

Exchange protein directly activated by cAMP (EPAC) is one of two intracellular receptors that mediate the effects of the prototypic second messenger cAMP. Identifying pharmacological probes for selectively modulating EPAC activity represents a significant unmet need within the research field. In this study, we present a thorough biochemical and pharmacological characterization of ESI‐09 based EPAC specific inhibitors, provide convincing evidence that ESI‐09 acts as an EPAC selective antagonist by directly competing with cAMP binding, and argue against the notion that ESI‐09′s effect on EPAC proteins is due to its non‐specific protein denaturing property. Our data show that ESI‐09 dose‐dependently inhibits cAMP‐mediated guanine nucleotide exchange activity in both EPAC1 and EPAC2 with apparent IC50 values well below the concentrations shown to induce “thermal unfolding shifts” reported by Rehmann. Furthermore, structure‐activity relationship analysis reveals that the exact position and number of the chloro‐substituents on the chlorophenyl moiety are important for the potency of ESI‐09 analogs in competing with 8‐NBD‐cAMP for EPAC2 binding and HJC0726 with 3,5‐dichloro‐substituent is five‐fold more potent than ESI‐09 in inhibiting both EPACs. These results suggest that ESI‐09′s action towards EPAC proteins is specific as it is highly sensitive to minor modifications of the 3‐chlorophenyl moiety. In summary, our current study validates our previous publications by demonstrating that ESI‐09 is a specific EPAC antagonist with providing convincing evidence that it acts as a competitive inhibitor.
Shielding of a Non‐Glycosylation‐Dependent Strong Interaction between MMP‐9 and Calreticulin by N‐Glycosylation Allows Efficient Secretion of MMP‐9
Authors: Duellman Tyler , Burnett John , Yang Jay
Published: June 2021

The secreted glycoprotein matrix metalloproteinase 9 (MMP‐9) is a dynamic modifier of extracellular signaling targeting numerous substrates such as cell surface proteins, cryptic signaling molecules, latent extracellular protease inhibitors, and extracellular matrix proteins. N‐glycosylation plays a major role in the early secretory events in the endoplasmic reticulum (ER) regulating protein folding and subcellular trafficking but the role of N‐glycosylation in MMP‐9 secretion has not been explored. In this study we utilized mutagenesis to disrupt N‐glycosylation and analyzed the resulting consequences of N‐glycosylation‐deficiency on MMP‐9 secretion. Residues N38 and N120 are N‐glycosylated. The removal of N‐glycosylation at residue 38 resulted in a reduction in secretion and ER retention presumable due to inefficient protein folding and an increased interaction with the ER‐chaperone calreticulin (CALR). Amino acid replacement studies suggested N‐glycosylation cloaks a non‐carbohydrate‐dependent strong interaction between MMP‐9 and CALR preventing retention of MMP‐9 in the ER. Amino acids at residue 38 with low molecular volume resulted in normal secretion despite the elimination of N‐glycosylation, hydrophobic residues resulted in intracellular retention, and polar residues resulted in reduced intracellular protein stability. Limited mutagenesis of N120 indicated that this N‐glycosylation was essential for secretion. This study identified a novel residue 38‐dependent regulation of MMP‐9 secretion, folding, and stability that are effectively masked by N‐glycosylation allowing normal secretion of the protein.
How Physically Active are Statin Users? The Polish Norwegian Study (PONS)
Authors: Vansal Sandeep , Vaidean Georgeta , Manczuk Marta
Published: June 2021

Introduction

Pharmacological treatment of hypercholesterolemia needs to be accompanied by increased physical activity. Whether this is implemented is not fully known.

Methods

We used cross‐sectional data of 12754 participants, ages 45 to 64 years, in an ongoing cohort study in Poland. Statin use during the last 30 days was recorded. Physical activity assessment was based on the validated International Physical Activity Questionnaire.

Results

There were 1728 (13.55%) statin users. Fewer statin users (37.56%) than non‐users (39.37%) reached the highest physically active category (p < 0.05). Statin users were 1.24 (95% CI: 1.13 to 1.37) more likely to watch TV for more than 20 hours/week (29.72%) than non‐statin users (23.10%) , even when age, gender, BMI, muscle/joint pain and cardiovascular disease status were adjusted for. The proportion of people meeting the recommended activity levels of 500‐1000 MET‐minutes/week, minutes spent sitting, engaging in walking activities, moderate and vigorous activities were not statistically significant between statin users and non‐users, adjusting for age and BMI. Statin‐users were more likely to report poor overall health (8.35%), muscle/joint pain (73.96%) and poor health as a barrier to exercise (14.72%) than non‐statin users (4.69%, 66.12% and 8.29% respectively, all p < 0.001).

Conclusion

Statin‐users do not have more favorable physical activity patterns than non‐users. Further studies are needed to explore the contribution of the “false reassurance” phenomenon and that of medication side effects.
Cadmium Levels in Maternal and Cord Blood and Anthropometric Parameters of Newborns in Nnewi,South‐Eastern Nigeria
Authors: Obi Ejeatuluchukwu , Agbasi Patrick , Orisakwe Orish , Nriagu Jerome
Published: June 2021

The study investigated the cadmium levels in maternal and cord blood and also tried to find out if there was any correlation between cadmium level and anthropometric parameters in the newborns
A total of 119 pregnant women from the third trimester to delivery were recruited from three different hospitals in Nnewi , South Eastern Nigeria. Maternal and umbilical cord blood samples (4ml) were collected at delivery and put into EDTA sample containers and were stored at ‐80C before transporting them to the University of Michigan ,Michigan ,USA for analysis of heavy metals. Cadmium were analyzed in a Class 100 facility using an Agilent 7500c series inductively coupled plasma/mass spectrometer (ICP‐MS)
Cadmium was detected in all the maternal and cord blood samples. The mean cadmium level in cord blood was 1.72 x 10‐3 ppm and that of the maternal blood was1.73 x 10‐3 ppm.The prevalence of abnormal cadmium level in mothers' blood is 73.1% and that of the cord blood was 68.9%.The anthropometric measurements showed that mothers whose blood had abnormal cadmium level delivered newborns with significantly lower (P<0.05) body weight and length compared to those with normal cadmium level. This study reveals the presence of cadmium in both maternal and cord blood more than the permissible cadmium level and this calls for a great concern because of its inherent health implications.