bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment Of Different Trophoblast Cell Lines As In Vitro Models For Placental Development
Authors: Daoud Georges , Barrak Joanna , Abou‐Kheir Wassim
Published: November 2023

Background

The placenta is a temporary organ of pregnancy that is involved in insuring optimal growth and development of the fetus. Since the 1980s, there has been a growing interest in the isolation of villous trophoblasts from human placenta for primary culture. Although very interesting, isolated primary trophoblasts have the disadvantage of being extremely difficult to maintain in culture which motivated investigators to generate villous and extravillous trophoblastic cell lines. Nevertheless, when aspiring to extrapolate the results they obtain in the trophoblastic cell lines to the trophoblasts in the placenta, the scientific community must be cautious as to whether these cell lines are truly representative of the physiologic setting.

Aim

Our study raises questions regarding the validity of using the choriocarcinoma cell lines (BeWo, JEG‐3, JAR) vs. the extravillous cell line (HTR‐8/SVneo) as in vitro model systems for human trophoblast studies.

Methods

Immunofluorescence staining was used to investigate the expression of CK7, e‐cadherin and vimentin in BeWo, JEG‐3, JAR and HTR‐8/SVneo cell lines. RT‐PCR allowed the measurement of mRNA levels of CK7, epCAM and vimentin in BeWo, JEG‐3, JAR and HTR‐8/SVneo while western blots assessed the protein expression of CK7, e‐cadherin and vimentin in BeWo, JEG‐3, JAR and HTR‐8/SVneo.

Results

BeWo, JEG‐3 and JAR cell lines all expressed CK7, e‐cadherin and epCAM and did not express vimentin while HTR‐8/SVneo cell line showed lower expression of CK7 and e‐cadherin and expressed vimentin. Additionally, two populations were observed in HTR‐8/SVneo cell line: a CK7+/Vimentin‐ population vs. CK7‐/Vimentin+ population.

Conclusion

Our study indicated that even though BeWo, JEG‐3 and JAR cell lines have proved to be epithelial trophoblastic cell lines, HTR‐8/SVneo has been disqualified in that regard and results based on this cell line should be controlled in primary trophoblast models.

Support or Funding Information

Medical Practice plan (MPP), American University of Beirut Medical Center, Beirut, Lebanon
Ablation of the Carotenoid Cleavage Enzyme β‐Carotene‐15,15′‐Dioxygenase (BCO1) Reduces Serum Testosterone and Prostatic Androgen Receptor Signaling in Mice
Authors: Smith Joshua W. , Ford Nikki A. , Thomas‐Ahner Jennifer M. , Moran Nancy E. , Bolton Eric C. , Clinton Steven K. , Erdman John W.
Published: November 2023

β‐carotene‐15,15′‐dioxygenase (BCO1) cleaves dietary carotenoids at the central 15,15′ double bond, most notably acting on β‐carotene to yield retinal. Ablation of this gene in mice fed carotenoid‐rich diets has been shown to affect tissue carotenoid accumulation. However, Bco1 ablation also impacts diverse physiologic endpoints independent of dietary carotenoid feeding, including expression of genes controlling androgen metabolism. Using the Bco1−/−mouse model, we sought to probe the effects of Bco1 ablation on testicular steroidogenesis, serum testosterone levels, and prostatic androgen signaling. Male wild‐type (WT) and Bco1−/− mice were weaned and raised on carotenoid‐free, semi‐purified AIN‐93G diets before sacrifice at 10–14 weeks of age. In three independent trials, Bco1 ablation significantly and consistently decreased serum testosterone and altered prostatic homeostasis. Organ weights of the prostate (all lobes) and seminal vesicles were significantly lower in Bco1−/−than WT mice (−18% and −29%, respectively; p<0.001). Bco1−/− mice had significantly reduced levels of serum testosterone (−79%; p<0.05). Additionally, Bco1 ablation decreased testicular mRNA expression of Hsd17b3 (−32% p<0.01), while increasing Hsd17b2 expression (+34%, p<0.05), suggesting a possible inhibition of testosterone synthesis. Furthermore, prostatic NanoString gene expression analysis revealed that Bco1 loss significantly (p<0.005) disrupted androgen receptor signaling, cell cycle progression, and proliferation. Reduced mRNA expression (by qPCR) of the androgen‐induced gene Msmb (−30%, p<0.05) and reduced protein expression of the proliferation marker Ki67 (by immunofluorescence microscopy; −47%, p<0.001) support these findings. While BCO1 has been suggested to function in physiologic processes more diverse than carotenoid cleavage and production of vitamin A, this is the first demonstration that Bco1 ablation impacts murine prostatic proliferation and androgen receptor signaling.

Support or Funding Information

Funded by NIH grant PHS‐1‐R01 CA125384
Acute excess caloric intake negatively affects the rhythmic expression of the human growth hormone gene and its association with the circadian‐linked transcription factor Bmal1
Authors: Vakili Hana , Jin Yan , Cattini Peter A.
Published: November 2023

Rhythmicity of biological functions is fundamental for optimal adaptations to environmental cues. Growth hormone (GH) is a major metabolic homeostatic factor that is secreted with a circadian pattern, but whether it is produced rhythmically is unknown. We used transgenic (TG) mice containing the intact human GH gene locus (human GH/CS‐TG) to investigate the rhythmicity of human GH gene (hGH‐N) expression. Human GH RNA expression oscillates over a 24‐hour cycle. Moreover, we have previously reported that excess caloric intake is associated with a decrease in human GH synthesis with reduced hGH‐N promoter function. Sequence analysis of the hGH‐N promoter revealed the presence of an enhancer motif (E‐box) and as such a putative binding site for circadian transcriptional machinery (Bmal1 and Clock). Results from electrophoretic mobility shift assay support participation of Bmal1/Clock and the hGH‐N E‐box DNA element in a common complex. Transfection studies combined with site‐directed mutagenesis revealed that this E‐box DNA element contributes to basal hGH‐N promoter activity. Furthermore, Bmal1/Clock were able to transactivate the hGH‐N promoter. The ability of Bmal1 to associate with the hGH‐N promoter region containing the E‐box element was confirmed in the human GH/CS‐TG mouse pituitary in situ by chromatin immunoprecipitation assay. Occupancy was reduced in mice fed a high fat diet during the light cycle. This decrease was not seen, however, during the dark cycle. Initial assessment of the mechanism suggests an impact of excess caloric intake on the chromatin and/or DNA structure. In summary, the presence of an E‐box DNA element as well as association of Bmal1 with the hGH‐N promoter region in situ supports the notion that hGH‐N is a cyclic gene. Acute excess caloric intake for three days was sufficient to reprogram the circadian rhythm mediated by chromatin remodeling of the hGH‐N promoter region and interfer with recruitment of Bmal1 to the E‐box element. Further investigation of the rhythmic expression of human GH may provide insight into the changes in GH availability under pathophysiological conditions such as metabolic changes associated with overeating and obesity.

Support or Funding Information

Funded by the Canadian Institutes of Health Research
G Protein‐Coupled Receptor Kinase 2's Effect on TNFα Signaling Modulates Wound Healing in Intestinal Epithelial Cells
Authors: Steury Michael D , McCabe Laura , Parameswaran Narayanan
Published: November 2023

G protein‐coupled receptor kinases (GRKs) are serine/threonine protein kinases critical in the regulation of GPCR lifecycle. Recently GRKs have been shown to have a large interactome and can play a critical role in cell signaling pathways as well as in consequent cell physiological processes. In this study, we examined the role that GRK2 in TNFα signaling in colon epithelial cell biological processes including wound healing, proliferation, apoptosis, and gene expression. Using an siRNA based knockdown approach in SW480 cell line, we find that GRK2 mediates rate of wound closure under basal conditions. GRK2 did not affect either apoptosis or proliferation of these cells. Interestingly, upon examining the levels of various gene transcripts, we observed that matrix metalloproteinases (MMP) 7 and 9 as well as urokinase plasminogen activator (uPa) (proteins involved in cell migration) was markedly upregulated in GRK2 knockdown cells. To assess the mechanism by which GRK2 affects these physiological processes, we examined the role of GRK2 in TNFα‐induced MAPK and NFkB pathways. Our results demonstrate opposing roles of GRK2 in MAPK and NFkB pathways‐while GRK2 mediates NFkB pathway, it inhibits ERK pathway. Our results further demonstrate that GRK2‐mediated regulation of MMP9 is via the ERK pathway. Together, these data suggest that GRK2 plays a critical role in cellular migration and/or wound healing by modulating MMP7, 9 and uPA levels potentially via differential regulation of MAPK and NFkB signaling pathways. Our results provide a novel role for GRK2 in TNFa signaling in colon epithelial cells.
Soy protein in the prevention of hepatic steatosis in obese OLETF rats
Authors: Rector R Scott , Schuster Colin M , Phillips Kathryn E , Eck Kevin J , Meers Grace M , Butteiger Dustie N , Krul Elaine S , Thyfault John P
Published: November 2023

Soy protein has shown beneficial effects on hepatic lipid metabolism and prevention of hepatic steatosis. Here we sought to test our hypothesis that a soy protein versus dairy protein‐based diet would significantly improve hepatic mitochondrial function and attenuate hepatic steatosis development in hyperphagic Otsuka Long‐Evans Tokushima Fatty (OLETF) rats. Five week old male OLETF rats were randomized to isocaloric diets containing 21% kcal as protein from milk protein isolate (MPI), soy protein isolate (SPI), or 50:50 MPI/SPI (M/S) (N=9–10/group) for 16 weeks. SPI significantly (p<0.05) attenuated fat mass and percent body fat by ~10% compared with M/S, but did not differ from MPI. Cumulative food intake was not different between SPI and other groups, but was significantly less in MPI rats compared with M/S (p<0.05), which corresponded with lowered serum insulin (p<0.05 vs SPI and M/S); serum glucose did not differ among groups. SPI reduced serum cholesterol and serum thiobarbituric acid reactive substances (TBARS) content (p<0.05) and tended to lower serum free fatty acids and increase serum antioxidant capacity compared with the other groups (p=0.1). Histological examination of liver revealed significant attenuation (p<0.05) in hepatic steatosis in the SPI group compared with MPI and M/S and biochemical assessment of liver triglyceride content showed a similar trend. Lipidomics analyses also indicated significant reductions (p<0.05) in hepatic diacylglycerols and phosphatidylcholine in SPI vs MPI. Intriguingly, reduced hepatic steatosis with SPI was associated with dramatic reductions in markers of hepatic de novo lipogenesis (acetyl‐coA carboxylase and fatty acid synthase protein content) but not with improvements in hepatic mitochondrial content or function. In fact, MPI livers displayed higher hepatic mitochondrial respiration, β‐hydroxy‐acyl‐CoA dehydrogenase activity and citrate synthase activity (p<0.05) compared with SPI and M/S groups. Furthermore, milk protein and soy protein appear to impact different pathways in hepatic lipid metabolism, with milk protein upregulating hepatic mitochondrial content/function and soy protein downregulating hepatic de novo lipogenesis. In summary, soy protein may be more beneficial than milk protein in the attenuation of hepatic steatosis development in hyperphagic obese OLETF rats.

Support or Funding Information

Support: MU School of Medicine Summer Research Fellowship (CMS), DuPont Nutrition & Health, NIH R01DK088940 (JPT) and VHA‐CDA2 IK2BX001299 (RSR).
Deep‐sea water administration efficiently inhibits the growth of Helicobacter pylori through the inhibition of caspase‐1/inflammasome/pyroptosis signaling
Authors: KOU WENG SI , YANG JYH CHIN , CHIEN CHIANG TING
Published: November 2023

The autophagy, apoptosis and inflammasome/caspase‐1 signaling pathways play a critical role in pathogenesis of H. pylori. Deep‐sea water (DSW) rich in micronutrients and minerals and with antioxidant and anti‐inflammatory qualities may be developed as marine drugs to provide gastric protection and therapeutic potential against H. pylori infection. Here, we investigated the effect of DSW on H. pylori‐induced autophagy, apoptosis and caspase‐1‐mediated epithelial damage, as well as H. pylori colonization in vitro (AGS cells) and in vivo (BALB/c mice). Our results found that H. pylori‐infection activated caspase 3‐induced apoptosis and caspase‐1/IL‐1β mediated pyroptosis and downregulated LC3 II‐regulated autophagy in AGS cells and gastric tissue of mice. In addition, we observed increased oxidative stress, NADPH oxidase gp91phox, CD68, caspase‐1/IL‐1β, and apoptosis, but decreased autophagy, in the gastric mucosa of H. pylori‐infected mice. We have further demonstrated that treatment with DSW led to anti‐H. pylori activity. Treatment with DSW for 14 days eradicated H. pylori infection in H. pylori‐infected mice. Taken together, we suggest that DSW is capable to inhibit H. pylori–activated inflammasome/caspase‐1 signaling, caspase 3‐mediated apoptosis and downregulated LC3 II‐mediated autophagy effectively eradicating H. pylori infection.
The mechanosensitive ion channel Piezo2 is selectively expressed in enterochromaffin (EC) cells of the human and mouse gastrointestinal mucosa
Authors: Knutson Kaitlyn , Wang Fan , Alcaino Constanza , Bernard Cheryl , Farrugia Gianrico , Beyder Arthur
Published: November 2023

BACKGROUND

Enterochromaffin (EC) cells are enteroendocrine cells in the gastrointestinal (GI) epithelium that release serotonin (5‐HT) in response to a variety of stimuli including mechanical force. In turn, EC cell 5‐HT affects GI secretion, motility and sensation. The molecular mechanism of EC cell mechanotransduction is unknown. The recently discovered mechanosensitive ion channel Piezo2 is important in several mechanosensory cell types.

AIM

to determine if the mechanosensitive ion channel Piezo2 is expressed and functional in EC cells.

METHODS

Murine small bowel was collected, fixed in 4% paraformaldehyde, and frozen. Human and mouse RNA from human jejunum and mouse duodenum, jejunum and ileum was analyzed by quantitative RT‐PCR for Piezo2 mRNA. Piezo2 and 5‐HT were also identified by fluorescence immunolabeling. QGP‐1, a human enteroendocrine cell model, was analyzed by RT‐PCR for the presence of Piezo2 and immunolabeling was done to detect Piezo2 and 5‐HT. Piezo2 mechanically activated currents were recorded from QGP‐1 cells in whole‐cell configuration. Voltage‐clamped QGP‐1 cells were subjected to a series of mechanical perturbations using a piezo‐electrically driven glass probe at steps of 0.3 μm for stimulation.

RESULTS

Human jejunum and all segments of mouse small bowel mucosa (duodenum, jejunum, ileum) expressed Piezo2 mRNA. In human jejunum and all segments of mouse small bowel Piezo2 co‐localized with 5‐HT in >90% of 5‐HT containing cells. No Piezo2 immunoreactivity was seen outside of 5‐HT positive enterochromaffin cells. The enteroendocrine cell model QGP‐1 expressed both Piezo2 and 5‐HT by immunohistochemistry and RT‐PCR showed message for Piezo2. Application of force to the surface of voltage‐clamped QGP‐1 cells elicited whole cell fast inward non‐selective cation currents of ~200 pA (at −60 mV) that were blocked by Gd3+ with kinetic properties compatible with Piezo2 channels.

CONCLUSIONS

Piezo2 is specifically expressed in human and murine small bowel EC cells and in the enteroendocrine cell model QGP‐1. QGP‐1 cells have a mechanosensitive inward cation current with kinetic and pharmacological properties similar to Piezo2. Our data suggest that Piezo2 is involved in EC cell mechanotransduction.

Support or Funding Information

Supported by NIH DK106456, DK84567, DK052766
Effect of Pediococcus pentosaceus at different doses on in vivo model of acute ulcerative colitis.Effect of Pediococcus pentosaceus at different doses on in vivo model of acute ulcerative colitis
Authors: AGUILAR LORENA TORRES , FRAGOSO LOURDES RODRIGUEZ , REYES‐ESPARZA JORGE
Published: November 2023

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon, whose characteristic symptoms include rectal bleeding or bloody diarrhea, abdominal pain and weight loss. Factors associated with the development of CU is the change of intestinal microbiota, for example declining species of Pediococcus. Therefore a new alternatives for the treatment of UC is the use of probiotics. Probiotics are live microorganisms which when administered in adequate amounts exert a beneficial effect on human health. There are several strains of Pediococcus pentosaceus with probiotic properties. The aim of this study is to evaluate the efficacy of different doses of Pediococcus pentosaceus on in vivo model of acute ulcerative colitis induced with dextran sodium sulfate (DSS). Thirty‐six male BALB/c mice weighing 20–22 g were housed in groups of 6 mice/cage. Acute colitis was induced by administration of DSS (molecular weight 40 kDa) 3.5% wt/vol in drinking water ad libitum for 7 days. At day 2 during induction of colitis, four groups were administered via gastric tube once daily at different doses of Pediococcus pentosaceus (1×107, 1×108, 1×109, and 1×1010 Colony Forming Units(CFU)). The clinical course of colitis was monitored by a daily disease activity index consisting of the three parameters weight loss, stool consistency, and perianal bleeding. The animals were sacrificed on the seventh day, the colon length was measured and histology were performed using H & E staining. P. pentosaceus at dose of 1×107, 1×108, 1×109, 1×1010 CFU didn't change the weight in animals with ulcerative colitis. However, the animals treated with P. pentosaceus at dose of 1×1010 delay the onset of diarrhea and bleeding during the induction of damage and showed a disease activity index by 41% less than the UC group. Furthermore, this group has a 21.3% less colon shortening than the CU group (p <0.05). The groups treated with P. pentosaceus at different doses (1×107, 1×108, 1×109 and 1×1010 UFC) didn't show damage to the submucosa of the colon, because the damage is only present in the area of the mucosa and muscularis mucosa. In conclusion Pediococcus pentosaceus treatment at the dose of 1×1010 CFU delayed the onset of some of the symptoms of acute ulcerative colitis in animals.

Support or Funding Information

Lorena Torres Aguilar es fellowship from CONACYT
Effects of probiotic bacteria on cytokine expression in cultured intestinal epithelial cells
Authors: Lyle Sarah , Erickson Tony , Walton Kristen LW
Published: November 2023

The benefits of probiotic bacteria in intestinal physiology and disease may be mediated through multiple mechanisms, such as affecting the composition of the gut microbiome, having direct action on host intestinal cells, or modulating mucosal immune responses. The commercially available probiotic VSL#3 includes a mixture of eight species, mostly lactobacilli and bifidobacteria. VSL#3 has been shown to have clinical benefits in ulcerative colitis patients, and has also been demonstrated to alter expression of mucins and other genes in cultured intestinal epithelial cells. This study aimed to assess effects of Lactobacillus acidophilus or VSL#3 on pro‐ and anti‐inflammatory cytokine and tight junction protein expression in CMT‐93 and Caco‐2 colonic epithelial cells. Cells were treated for 4 hours with L. acidophilus or VSL#3 (1×108 CFU/ml) and expression of the anti‐inflammatory cytokine transforming growth factor‐beta‐1 (TGFβ‐1), pro‐inflammatory interleukin (IL)‐6 and the housekeeping gene GAPDH was analyzed by semi‐quantitative PCR. The number of viable cells was analyzed after 24 hours of exposure to probiotic bacteria using a cell survival assay. Expression of claudin‐2 and localization of nuclear factor kappa‐B (NFkB) were detected by immunofluorescent staining. Probiotic‐treated CMT‐93 cells showed less induction of IL‐6 than LPS‐treated cells. TGFβ expression was not altered by probiotic treatment compared to untreated controls. Immunofluorescent staining showed increased claudin‐2 staining in cells treated with VSL#3 for 24 hours compared to untreated controls. In conclusion, probiotic bacteria stimulate reduced IL‐6 production compared to a strong inflammatory stimulus in murine intestinal epithelial cells. In addition, VSL#3 stimulated claudin‐2 expression in CMT‐93 cells, providing a potential mechanism for protective effects of VSL#3 on the intestinal epithelium.

Support or Funding Information

This project was supported by the Missouri Western State University PORTAL program.
Effect of Salba‐Chia (Salvia Hispanica L), an Ancient Seed, in the Treatment of Overweight and Obese Patients with Type 2 Diabetes: A Double‐blind, Parallel, Randomized Controlled Trial
Authors: Jenkins Alexandra L , Brissette Christy , Jovanovski Elena , Au‐Yeung Fei , Ho Hoang Vi Thanh , Zurbau Andreea , Sievenpiper John , Vuksan Vladimir
Published: November 2023

Preliminary studies indicate that consumption of Salba‐chia (Salvia hispanica L) improves management of type 2 diabetes and acutely suppresses appetite. In this study, we assessed the effect of Salba‐chia on body weight and related cardiovascular disease risk factors in overweight or obese adults with type 2 diabetes over 6 months.

Using a double blind, parallel, controlled trial design, 77 overweight or obese patients with type 2 diabetes (HbA1c 6.8–8.5%; BMI 25–40kg/m2) were recruited. Both groups followed a 6‐month calorie‐restricted diet; one group consumed 30g/1000kcal of Salba‐chia, the other 36g/1000kcal oat bran‐based control, per day. Primary endpoint was change in body weight over 6‐months. Secondary endpoints included changes in waist circumference, body fat %, HbA1c, blood pressure, lipids, C‐reactive protein, and obesity‐related gut hormones.

Fifty four participants completed the full 6‐month protocol and 58 (31 Control, 27 Salba‐Chia) were included in the final analysis. At 6‐months, participants on Salba‐chia lost significantly more weight than those on oat bran‐based control (1.9±0.5kg and 0.3±0.4kg, respectively; p=0.020), accompanied by a greater reduction in waist circumference (3.5±0.7cm and 1.1±0.7cm, respectively; p=0.027). C‐reactive protein was reduced by 1.1±0.5mg/L (39±17%) on Salba‐chia, compared to 0.2±0.4mg/L (7±20%) on control (P=0.045) and plasma adiponectin was increased on Salba‐chia by 6.5±0.7% compared to 0.0±0.6% on control (P=0.022). There were no significant differences between treatments on blood pressure, lipids and HbA1c, and fat distribution, however, a within intervention reduction of android (3.7±2.8%, P=0.031) and gynoid fat (6.9±3.9%, P=0.047) was observed in the Salba‐chia group, but not in control.

The current trial demonstrates the potential of Salba‐chia to promote weight loss, and improve obesity‐related risk factors, while maintaining good glycemic control. The results suggest Salba‐chia may represent a promising supplementation to conventional therapy in the treatment of overweight and obesity in diabetes.

Support or Funding Information

Canadian Diabetes Association Operating Grant, Salba‐chia was provided by Salba Smart Natural Products, LLC
Thinking Positive: The Effect of Emotional Context on Respiratory Sensory Gating
Authors: Chan Pei‐Ying Sarah , Ju Ya‐Jhih
Published: November 2023

Respiratory sensory gating function has been found decreased by induced negative emotional stimulation in healthy adults. The increased ratio of the respiratory‐related evoked potentials (RREP) N1 peak amplitude for the second occlusion (S2) versus the first occlusion (S1), S2/S1, is indicative of decreased central neural gating mechanism. In this study, we tested the hypothesis that positive emotional context would enhance respiratory sensory gating function in healthy individuals.

We tested respiratory sensory gating in a group of healthy individuals by the paired inspiratory occlusion paradigm of two transient obstructions of 150 milliseconds (msec) with 500 msec interval under both pleasant and neutral emotional contexts. The S2/S1 amplitude in the pleasant context and the neutral context were 0.57 and 0.45, respectively. There was a significant decrease in the S2/S1 ratio under the pleasant context (p < .05). After post‐hoc analysis, there was a significant decrease in S2 N1 peak amplitude in the pleasant compared to neutral context (p < .05).

Our results demonstrated that positive emotional context enhances brain inhibitory mechanisms by filtering out repetitive respiratory stimuli. Further investigation on how positive emotional context modulates respiratory sensation in clinical patients is necessary.

Support or Funding Information

MOST 103‐2420‐H‐182 ‐003 ‐MY2 CMRPD1B0332
Ventral Medullary Inspiratory Neurons Do Not Contribute to Increased Inspiratory Drive during Swallow
Authors: Pitts Teresa , King Suzanne , Poliacek Ivan
Published: November 2023

Schluckatmung (swallow‐breath) is believed to be an evolutionarily preserved strategy from deglutition to eupnea. The schluckatmung is composed of both alterations in respiratory phase timing as well as a transient increase in inspiratory motor drive. This increased inspiratory motor drive creates a negative trans‐diaphragmatic pressure that moves the bolus from the pharynx into the esophagus. Our previous work showed that inspiratory neurons in the region of the dorsal respiratory column (DRC) increase instantaneous firing rate during the schluckatmung, consistent with an important role for this population of neurons in swallowing. We hypothesized that this role of DRC neurons in the generation of the schluckatmung is unique, and not shared by other inspiratory neurons in the ventrolateral medulla. A multi‐channel electrode array was used to record ventral respiratory column (VRC) neurons in anesthetized, spontaneously breathing cats. Swallowing was induced by injection of water into the oropharynx. Electromyograms (EMG) were recorded from pharyngeal, laryngeal, and chest wall muscles, and intra‐esophageal pressure was recorded from the mid‐thoracic esophagus. Unlike previously reported dorsal respiration column neurons, VRC inspiratory neurons significantly decreased their firing rate during swallow compared to eupnea. Suppression of VRC inspiratory neurons could play an important role in phase timing for swallow and phase‐lag of esubsequent inspirations for breathing and cough. Supported by HL 111215, P30‐GM103507.

Support or Funding Information

Supported by HL 111215, P30‐GM103507.
Expiratory Diaphragm Activity Reduces Atelectasis Formation
Authors: Hedenstierna Goran , Pellegrini Mariangela , Roneus Agneta , Segelsjo Monica , Larsson Anders , Perchiazzi Gaetano
Published: November 2023

Objective

We hypothesized that the major inspiratory muscle, the diaphragm, also acts during expiration as a brake to maintain lung aeration and prevent collapse. We tested this hypothesis in a porcine model of surfactant‐depleted lung.

Methods

Acute lung injury was induced in 8 anesthetized, tracheostomized pigs by repeated lung lavages, targeting a PaO2/FiO2 of 250 mmHg. After stabilization, the animals were switched to spontaneous breathing (SB) and underwent a decremental continuous positive end‐expiratory pressure (PEEP) trial of 15, 12, 9, 6, 3 and 0 cmH2O. Diaphragmatic electric activity during ongoing expiration (EAdiexp) and at no‐flow end‐expiration (EAdimin) (six electrodes esophageal‐gastric catheter) and the trans‐diaphragmatic pressure (gastric minus esophageal pressure, Pdi) were measured. Para‐diaphragmatic dynamic‐CT scans (dCTs) were obtained. The dCT scans were collected at end‐expiration and the amount of collapse (atelectasis) in that cut was calculated. The atelectatic tissue was defined as the sum of voxels with a density between −100 and +100 Hounsfield Units. In 4 pigs, the same protocol was repeated during mechanical pressure control ventilation (PCV) after complete muscle paralysis.

Results

In spontaneously breathing pigs, a linear correlation was found between expiratory EAdi and expiratory Pdi (R2 > 0.82, p < 0.01). During mechanical ventilation (PCV) with muscle paralysis, no electrical activity of the diaphragm was seen.

When PEEP was lowered, with concomitant decrease in lung volume, the EAdiexp increased significantly throughout the expiration. The EAdimin increased when PEEP was reduced from 12 to 0 cmH2O. Gas flow was lower at any given time point of expiration during SB without muscle paralysis than during PCV with muscle paralysis. Atelectasis increased in size with the lowering of PEEP but significantly more so during PCV than during SB so that the difference between SB and PCV increased with decreasing PEEP (Figure 1).

Conclusions

Our findings show that the diaphragm electrical activity is well related to the diaphragmatic muscle force and that the diaphragm acts as an expiratory brake to limit the fall in end‐expiratory lung volume and to prevent lung collapse. Thus, the diaphragm is an active muscle also during expiration and loss of its function, as during mechanical ventilation and muscle paralysis, may be a contributing factor to unsuccessful respiratory support. We also speculate that expiratory work of breathing may be an additional component of work, added to the inspiratory work that contributes to respiratory muscle fatigue.

Support or Funding Information

Supported by grants from the Swedish Research Council, the Swedish Heart and Lung Fund, and institutional funds at Uppsala and Bari Universities
Ex Vivo Elastase Treatment Disrupts Parenchymal Structure And Enhances Airway Narrowing in Precision Cut Lung Slices
Authors: van Dijk Eline Margaretha , Culha Sule , Bidan Cecile , Menzen Mark , Gosens Reinoud
Published: November 2023

Background

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease caused primarily by smoking. COPD patients suffer from a loss of lung function due to airway inflammation, airway remodelling and in some patients emphysema development. Alterations in the parenchymal structure, due to the presence of elastolytic enzymes and oxidative stress may affect airway mechanics in COPD. Here, we hypothesize that ex vivo treatment of lung slices with elastase or H2O2 enhances bronchoconstriction caused by a disrupted parenchymal structure.

Aim

To investigate the relationship between the parenchymal structure and airway narrowing in an ex vivo precision cut lung slice (PCLS) model after treatment with elastase or H2O2.

Methods

Lungs of C57Bl/6J mice (n=8) were sliced (250 μm) and treated ex vivo with elastase (2.5 μg/ml) or H2O2 (200 μM) for 16 hours. Following treatment, slices were washed twice with medium and incubated for another 24 hours after which the slices were collected. To determine airway narrowing, methacholine‐induced contraction studies were performed. Parenchymal structure was assessed by confocal microscopy after which mean linear intercept (Lmi) was determined. Gene expression levels of markers of alveolar type I and II cell repair, known to be decreased in COPD, were assessed using RT‐PCR.

Results

Following elastase, but not H2O2 treatment, slices showed a significant increase in Lmi (basal 75.91 μm vs elastase 90.18 μm (p=0.04) and H2O2 84.95 μm (p=0.76)), reflective of an altered parenchymal structure. In addition, elastase enhanced methacholine‐induced airway narrowing as shown by a significant decrease in pEC50 (−5.78 vs −6.68 log M, p=0.01). H2O2 did not change pEC50 (−5.78 vs −5.99 log M, p=0.64), but showed a trend for increasing Emax (44.12 vs 67.35 % contraction, p=0.06). Elastase did not alter Emax significantly (44.12 vs 61.11 % contraction, p=0.12) Expression levels of alveolar type I and type II markers Aqp5 and RAGE were decreased (p<0.05) following elastase treatment, whereas H2O2 –treatment decreased the expression of RAGE, Con43, T1α, PAI1 and SFTPC mRNA (p<0.05).

Conclusion

Treatment of PCLS with elastase, but not with H2O2 enhances airway narrowing in response to methacholine. This is associated with decreased gene expression levels of several markers of alveolar repair and a larger Lmi. These findings support our hypothesis that the link between increased bronchoconstriction and a disrupted parenchymal structure can be mimicked using PCLS ex vivo. PCLS may be a promising model to study therapies aimed at lung repair.

Support or Funding Information

This study was sponsored by a Vidi grant by NWO (016.116.309)
Regulation of Kv7.5 Potassium Channels by Protein Kinase C in Human Airway Smooth Muscle Cells
Authors: Haick Jennifer M , Brueggemann Lioubov I , Byron Kenneth L
Published: November 2023

Kv7.5 potassium (K+) channels were recently found to be expressed in airway smooth muscle cells (ASMCs). Bronchoconstrictor agonists inhibit the outward flux of K+ through these channels, an effect which has been implicated as a mediator of their bronchoconstrictor actions in human airways. To test the hypothesis that Kv7.5 channels in ASMCs are regulated by bronchoconstrictor agonists via protein kinase C‐ (PKC‐) mediated phosphorylation, FLAG‐tagged human Kv7.5 channels were overexpressed in human trachealis smooth muscle cells (hASMCs). Function of the channels was assessed by measuring outward Kv7.5 currents using whole cell patch clamp electrophysiology. Indirect activation of PKC by the bronchoconstrictor agonist histamine (1 μM) reduced Kv7.5 currents by 34.6 ± 7% at −20 mV (n=6, p<0.05). This effect was abolished upon blockade of the histamine H1 receptor with chlorpheniramine (30 μM). The PKC activator phorbol 12‐myristate 13‐acetate (PMA, 1 nM), but not its inactive analog 4α‐PMA (1 nM), also reduced Kv7.5 currents (75.5 ± 7.6% at −20 mV, n=4, p<0.05). Phosphorylation of the channel protein was assessed by immunoprecipitating the channels (using anti‐FLAG antibodies) and Western blotting with anti‐phosphoserine PKC substrate antibodies. Both histamine and PMA significantly increased Kv7.5 channel phosphorylation and this effect was reduced by the PKC inhibitor Ro‐31‐8220 (1–10 μM). Two putative PKC‐phosphorylation sites on Kv7.5, serine 441 (S441) and threonine 505 (T505), were mutated so that they can no longer be phosphorylated. Neither Kv7.5‐T505A or Kv7.5‐S441A currents were inhibited by histamine (<8% inhibition at −20 mV, n=5, p>0.05, and <14% inhibition at −20 mV, n=7, p>0.05, respectively), however both mutant channels remained sensitive to PMA (58.3 ± 16.8% at −20mV, n=5, p<0.05, and 63 ± 7.5% at −20 mV, n=6, p<0.05, respectively). Despite the insensitivity of Kv7.5‐S441A currents to histamine treatment, Western blot analyses indicated that histamine still induced serine phosphorylation of Kv7.5‐S441A channels. Determining the mechanism by which Kv7.5 channels are regulated in hASMCs will aid in the understanding of how these channels contribute to the regulation of airway diameter and how Kv7 channels in ASMCs may be effectively targeted for treatment of diseases associated with airway obstruction, such as asthma.

Support or Funding Information

Loyola University Chicago
Effects of PACAP administration in the extended amygdala on acoustic startle reflex
Authors: Seiglie Mariel , Cottone Pietro , Sabino Valentina
Published: November 2023

Anxiety‐related disorders are the most common forms of mental disorders; characterized by feelings of excessive worry in the absence of specific external stimuli, they are accompanied by physical, affective and behavioral symptoms.

Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a 38‐amino acid peptide expressed not only in the hypothalamus but also in various extra‐hypothalamic regions including the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST). PACAP and its receptor PAC1 (PAC1R) have been proposed to play a key role in mediating the behavioral and endocrine responses to stress; however, few studies have examined the involvement of the extrahypothalamic PACAP system in the modulation of stress. Our aim was to elucidate the role of the PACAP/PAC1R system of the CeA and BNST in the context of anxiety‐like behaviors.

Acoustic startle response (ASR) is a behavioral test sensitive to states of anxiety and fear. The effects of PACAP bilateral microinfusion into either the CeA or the BNST of male rats on ASR were assessed in this study. In addition, to test the role of the endogenous system, the effects of intra‐CeA and intra‐BNST PACAP antagonist PACAP(6–38) on ASR were assessed, both on basal and on footshock‐induced sensitization of ASR.

Infusion of PACAP into both the CeA and BNST increased ASR. Importantly, doses of the antagonist PACAP(6–38) which per se did not affect ASR, were able to prevent the sensitization of ASR induced by footshock, when infused into either the CeA or BNST.

These data prove an anxiogenic role for the PACAP/PAC1 system of the extended amygdala and suggest that hyperactivity of this system may underlie the anxiogenic effects of stress.

Support or Funding Information

NIH grants # MH093650, MH091945, DA030425; T32 grant GM008541
TDP‐43 interacts with cannabinoid type 1 receptor modulating its subcellular localization and function
Authors: Filipeanu Catalin , Deshotels Matthew , Weed Peter , Ogunlade Blessing , Robinson Anastasia G , Winsauer Peter , Guidry Jessie J.
Published: November 2023

To identify new molecular chaperones of cannabinoid type 1 receptor (CB1R) we used a proteomic approach in HEK293T cells, transfected with two spicing variants of this receptor, CB1R and CB1AR. These two isoforms differ only in the composition of the N‐terminus domain. Surprisingly, the only protein found to interact differently with CB1R and CB1AR was transactive response DNA‐Binding Protein 43 (TDP‐43), which is known to be involved in the pathology of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The specific interactions between CB1R and TDP‐43 were also confirmed by co‐immunoprecipitation not only in transfected HEK293 cells, but also at the endogenous level in rat crude spinal cord. TDP‐43 overexpression decreased CB1R plasma membrane levels by stimulating receptor internalization, but it did not change the total receptor levels. Further, TDP‐43 enhanced CB1R ubiquitination to similar levels, as induced by the full CB1R agonist, CP55940. Also, overexpression of TDP‐43 resulted in reduced effects of CB1R effects on cAMP, P‐ERK1/2, and P‐CREB responses. Present results revealed TDP‐43 as an unexpected molecular chaperone of CB1R and these findings may have significance not only in the regulation of CB1R intracellular trafficking, but also in the treatment of ALS and FTLD.

Support or Funding Information

This work was supported by Startup Funds from Howard University (CF) and USPHS grant DA019625 (PW) from the National Institute on Drug Abuse
MAPK Signaling Plays an Important Role in Differentiating Paraquat‐ and Diquat‐Induced Hepatocellular Injury
Authors: Tong Chris , Fry Maddie , Stamper Brendan D
Published: November 2023

Objective

The objective of this study was to utilize the structure‐toxicity relationship between two quaternary ammonium‐containing herbicides, paraquat and diquat, in order to pinpoint key mechanisms propagating diquat‐induced liver injury.

Methods

Both dose‐ and time‐dependent toxicity screens were performed in an in vitro liver model (TGF‐alpha transgenic mouse hepatocyte cell line; TAMH) to determine toxicologically relevant concentrations of paraquat and diquat. TAMH cells were then exposed to the respective median lethal dose (LD50) for each herbicide, harvested and processed for RNA isolation. Microarrays were then performed using Affymetrix Mouse Gene 2.1 ST array strips and analyzed using DAVID (https://david.ncifcrf.gov). Results from the whole genome expression studies were compared with the MicroArray Quality Control (MAQC)‐II dataset (GSE24363) to validate robust markers for diquat‐induced liver injury.

Results

LD50 values for paraquat and diquat were calculated to be 126 μM and 18 μM, respectively. Time‐based toxicity screening indicated that significant differences in toxicity between paraquat and diquat manifested after 9 hours of exposure at LD50 concentrations. Whole genome expression analysis at this time point identified mitogen activated protein kinase (MAPK) signaling to be significantly upregulated following diquat exposure when compared to either paraquat exposure (p < 1E‐03) or control (p < 1E‐06). Mining of the MAQC‐II dataset identified 42 upregulated transcripts specifically associated with diquat toxicity, of which 19 were shared in our TAMH experiments. All 19 of these shared transcripts were associated with MAPK signaling pathways.

Conclusion

Paraquat and diquat are structurally‐related herbicides currently in use that have been shown to be toxic in multiple human organ systems. Studies reporting direct comparisons and potential genotoxicity between these herbicides are lacking. The work presented herein found diquat to be seven times more hepatotoxic than paraquat. Based on the gene expression array studies, the differential toxicity between the two herbicides appears to be at least in part MAPK‐mediated. The identification of MAPK signaling as an important contributor to diquat‐induced hepatocellular injury was validated through MAQC‐II dataset mining. Future work is currently underway to investigate how phosphoprotein targets within MAPK signaling cascades are altered between paraquat and diquat, and to subsequently integrate results from the protein‐based experiments with gene expression data.

Support or Funding Information

This work was supported by the Pharmacy Research Incentive Grant Program through the Pacific University School of Pharmacy.
Transdermal Detection of Low Concentrations of Hydrogen Sulfide
Authors: Rios Lynnette , Friedrichsen Debra , Mowry Curtis , Silaski Grayson , Shekarriz Reza , Kanagy Nancy Lapp
Published: November 2023

We report here a novel approach to measure circulating hydrogen sulfide (H2S) non‐invasively as a potential way to diagnose and monitor endothelial dysfunction and peripheral artery disease (PAD). PAD is a life‐threatening condition caused by arterial constriction and obstruction of blood flow leading to limb ischemia. Current methods to diagnose and monitor PAD lack sensitivity, result in frequent false‐negatives, and require specialized technicians. Recent studies indicate that decreased H2S production is an underlying cause of PAD. In addition, reduced plasma H2S correlates with endothelial dysfunction in untreated hypertension, diabetes, sleep apnea and other cardiovascular diseases. Conversely, excess H2S is cytotoxic causing irreversible damage to mitochondrial function. Therefore, careful monitoring of plasma H2S levels is essential to diagnose and treat vascular disease and to prevent H2S toxicity. This device was designed to measure H2S at the surface of the skin to test the hypothesis that the diffusion rate (and therefore gas phase concentration) of H2S is directly proportional to the concentration of H2S in the blood. Our results demonstrate that the device is sensitive enough to detect H2S at 10 part per billion (ppb) or lower in gas standards and in the headspace of a 3 nM solution of Na2S (H2S donor). In vitro studies used excised rat skin superfused on the subcutaneous surface with a H2S solution. H2S was detected in a stream of N2 gas flowing through a sealed chamber on the epidermal side both by GC/MS measurement and with the sensor only when the superfusing solution contained an H2S donor demonstrating that H2S diffuses through the skin. The diffusion rate of H2S through excised abdominal skin from a male Sprague‐Dawley rat is approximately 1.42×10−9 μmol/s/cm2/μM. The device also detected increases in H2S on the skin surface in anesthetized rats within seconds of intra‐venous injection of Na2S at doses that caused a fall in arterial pressure, with an estimated average diffusion rate of 4×10−8 μmol/s/cm2/μM. Therefore, this noninvasive, highly‐sensitive and portable device has the potential to transform diagnosis and monitoring of early stage PAD and to monitor H2S biosynthesis or exposure in vivo.
Changes in Hydrogen Sulfide Concentrations in Porcine Ocular Anterior Segment Explants Under Elevated Perfusion Pressures: Role Of Prostaglandins
Authors: Robinson Jenaye , Mitchell Leah , Nguyen Olivia , Opere Catherine , Ohia Sunny , Njie‐Mbye Ya Fatou
Published: November 2023

We have evidence that H2S donors can reduce intraocular pressure in rabbits presumably by increasing aqueous humor (AH) outflow. The aim of the present study was to assess the contribution of H2S present in tissues of the trabecular meshwork in the observed increase in AH outflow facility in an ex vivo porcine anterior segment model.

Methods

Porcine ocular anterior segment explants were perfused with Dulbecco's Modified Eagle's Medium maintained at 37° C and gassed with 5% CO2 and 95% air, under an elevated pressure of 15 mm Hg for four hours. Perfusates from the anterior segment explants were collected and immediately assayed for their H2S content using the well‐established Methylene Blue assay. In some experiments, explants were perfused with cyclooxygenase (COX) inhibitors [flurbiprofen (30 μM) and indomethacin (10 μM)]. The concentration of H2S was also measured in the AH (as positive control), and at normal perfusion pressure of 7.35 mmHg in the absence of COX inhibitors.

Results

Increasing perfusion pressure from 7.35 to 15 mm Hg significantly (p < 0.001) increased H2S concentrations from 0.4 ± 0.1 to 67.6 ± 3.6 nM/μg protein. As a reference value, the H2S concentration in the AH was 1.5 ± 0.2 nM/μg protein. In the presence of the COX inhibitors, flurbiprofen (30 μM) and indomethacin (10 μM), the effects of elevated pressure on H2S levels were significantly (p < 0.001) reduced from 67.6 ± 3.6 to 0.8 ± 0.1 and 4.2 ± 0.68 nM/μg protein, respectively.

Conclusion

We conclude that the elevated perfusion pressure‐induced increase in H2S concentrations in the perfusate is dependent upon the endogenous production of prostaglandins. However, the exact mechanism whereby elevated perfusion pressures leads to changes in basal H2S is unknown and merits further investigation.