bluuebunny/crossref_metadata_2025 huggingface dataset:
Expanding Program Specific Anatomy Education During Residency: New Opportunities and Challenges
Authors: Agur Anne
Published: June 2021

The anatomy component of medical school curricula is a dynamic entity that continues to evolve. The challenge for anatomists is to be able to deliver succinct curricula in an ever‐changing environment where hours have already been dramatically reduced and the remaining time redistributed in various ways to provide a longitudinal learning experience for the medical students. This has resulted in increased demands for specialized program specific anatomy education during residency. The focus of this presentation will be on strategies to integrate anatomy education into residency training in a changing curricular landscape and to delve into best practice scenarios that can be used to design and implement the programs. Course design and content, ongoing assessment of knowledge acquisition, anatomy and clinical faculty collaboration, role of the residents and inclusion of case based content will be discussed.
Moving through Barriers: Unlocking the Mysteries of How Enzymes Really Work
Authors: Klinman Judith
Published: June 2021

Delineating the physical principles that underlie enzyme catalysis remains a major challenge at the biology/chemistry interface. Recent investigations in enzymology have moved beyond the historical focus on “transition state stabilization”, toward an understanding of the important interplay between protein motions and the generation of highly precise active site interactions. The almost universal contribution of nuclear tunneling to enzymatic C‐H activation reactions offers an especially powerful tool to quantify the role of reduced barrier width in catalysis and its inherent link to enzyme flexibility. The ability to detect positionally specific networks of protein motions implicates an evolutionary tuning of large regions of the protein that extend beyond the active site. These studies are beginning to uncover design principles for the generation of de novo catalysts.

(Supported by funding from the NIH, GM025765)
Changing Tongue Depressor Position Between Rat Incisors of Both Jaws Varies Gastric Motility
Authors: Lorber Mortimer
Published: June 2021

The periodontal ligament between the wall of every tooth socket & the root of the tooth which it suspends allows slight tooth movement with each bite. This stimulates the ligaments' trigeminal mechanoreceptors (TMs) linked to the vagal motor n. stimulating motility of the antral region, demonstrating a periodontogastric motility reflex (Lorber, Can J Physiol Pharmacol 67:29‐35, 2000). To see whether maxillary or mandibular rat incisors might vary in performing this reflex, eight adult female rats were fasted 24‐48 h. During normal occlusion their molars occlude but not their incisors (Fig. 1A). Under pentobarbital, data were collected during three five minute periods. The first was the baseline(B) in which resting antral motility was observed. For the procedure period(P), a tongue depressor was placed vertically(V) (Fig. 1B) or horizontally(H) (Fig. 1C) between the incisors of each jaw. For (P) the mandible was closed every second for five minutes and antral motility observed. The last period was the postprocedural(PP) to note whether the antral effect extended beyond (P). Comparisons of the mean+1sd of (P) and (PP) with (B) revealed that in (V) the three values were 1.00+0.72, 1.39+0.94, 1.71+ 1.33, ANOVA P<0.04. In (H) the values were 1.00+1.45, 2.43+1.08, 3.98+1.17,ANOVA P<0.04. Comparing (H) and (V) by dependent t‐tests showed that (H) is greater in PP (P<0.05), presumably due to delayed greater stimulation of apically located TMs than those on the sides of the dental root, whereas the V position causes anterior stimulation of only the maxillary TMs and posterior stimulation of only the mandibular TMs. This may be because the primary location of the TMs is at the root apex from which they ramify, so fewer branches are stimulated at any location about the sides of the dental root than at its tip.
A Novel Approach to Identify Genes Related to FGID
Authors: Gombash Sara , Cowley Christopher , Fitzgerald Julie , Iyer Chitra , McGovern Vicki , Fried David , Williams Kent , Burghes Arthur , Christofi Fedias , Gulbransen Brian , Foust Kevin
Published: June 2021

To investigate novel genes involved in functional gastrointestinal disorders, we disrupted a master regulator of mRNA splicing, the survival motor neuron (SMN) protein. SMN disruption leads to widespread changes in gene expression but has not been evaluated for effects in the gastrointestinal (GI) tract. Mice with a Nestin‐cre transgene were crossed to cre reporter mice to analyze expression in the enteric nervous system (ENS). Gene expression was detected in excitatory and sensory neurons, interneurons, and intraganglionic glia throughout the myenteric plexus but not muscle. Next, nestin‐cre mice were crossed to mice with a floxed Smn allele on a SMNΔ7 background (SMN2+/+;SMNΔ7+/+;SmnF7/‐) to produce Nestin‐F7 mice. In organ baths, SMN reduction disrupted colon longitudinal muscle contraction during high frequency electrical field stimulation (EFS). Muscle relaxation was consistently greater in control tissue. The addition of L‐NAME had no effect on differences between control and Nestin‐F7 responses. Tissues showed no morphological changes or cell loss. Gastric emptying and intestinal transit were significantly slower in Nestin‐F7 mice compared to controls. Nestin‐F7 mice had dry, infrequent stool that corresponded to reduced colonic motility in the bead latency test. Collectively, these data suggest that SMN reduction results in impaired GI function. Future studies will identify specific enteric cell types sensitive to SMN reduction and evaluate mRNA expression in those cells.
Neurochemical Phenotype of Afferent Neurons of the Nucleus Tractus Solitarius in Response to Esophageal Distension in the Rat
Authors: Ambler Jason , Babic Tanja , Travagli R.Alberto
Published: June 2021

The nucleus tractus solitarius (NTS) receives visceral information from the gastrointestinal, cardiovascular and respiratory systems via type‐A and type‐C fibers. NTS nucleus centralis (cNTS) receives afferent inputs from the esophagus and previous studies have shown that esophageal afferents display diverse properties. It has also been shown that gastro‐esophageal vagal fibers are under modulatory control of group III metabotropic glutamate receptors (mGluRs). The aim of this study was to determine whether esophageal distention activates cNTS neurons that express group III mGluRs.

Rats were anesthetized and an esophageal balloon was placed 5 mm from the lower esophageal sphincter. The esophagus was extended for 1s every 10s for 30min. 90min following esophageal distention, rats were perfused with saline, followed by 4% paraformaldehyde. Brainstem sections were reacted for c‐fos and mGluR8 immunoreactivity (IR). In five control rats, 8±1.8 NTS neurons/section were c‐Fos IR and 9±2.3% were also mGluR8 IR. In cNTS, 0.2±0.05 neurons/section were labelled for c‐fos, 5.7±2% of these neurons were also labelled for mGluR8. Following esophageal distension, 17±3.1 NTS neurons/section were c‐Fos IR and 22±1.5% were also mGluR8 IR (p<0.05 vs. control for both). In cNTS, 2.9±0.5 neurons/section were labelled for c‐fos, i.e. 27±1.9 % of these neurons were also labelled for mGluR8 (p<0.05 vs. control for both).

These data show that esophageal distension activates mGluR8‐ir neurons in cNTS, suggesting that integration of sensory inputs from the esophagus may be modulated by group III mGluRs.

Supported by NIH grant DK‐55530 and a “Frontiers in Physiology Research Teacher Professional Development Fellowship Award” from APS
High‐fat Diet Causes Loss of Nitric Oxide Motor Neurons and Impairs Inhibitory Neuromuscular Communication in the Mouse Distal Colon
Authors: Bhattarai Yogesh , Fried David , Gulbransen Brian , Galligan James
Published: June 2021

High‐fat (HF) diet increases the risks of gastrointestinal (GI) motility complications, however, the mechanisms that underlie these complications are still not entirely understood. We tested the hypothesis that HF diet causes oxidative stress, loss of nitric oxide (NO) motor neurons in the myenteric plexus, disrupted inhibitory neuromuscular communication and impaired GI motility.

Methods

Distal colon segments from mice fed a control (10 Kcal%) or HF diet (60 Kcal%) were used in vitro. Alexa fluor 546 and 680 conjugated maleimides were used to assess the oxidized/reduced glutathione ratio in myenteric NO neurons. NO neuronal density was assessed using nNOS immunohistochemistry. Basal NO availability was measured using NO induced fluorescence of DAF‐FM diacetate. Electrical field stimulation was applied across varying voltages (60‐120V) and train (200‐1000ms) durations to evoke inhibitory junction potentials (IJPs) in colonic circular smooth muscle cells. Amplitude and area of IJP were examined to assess fast and slow IJPs respectively.

Results

We observed an increase (17%, p<0.01) in oxidized/reduced glutathione fluorescence ratio in nNOS neurons of HF mice. HF mice had lower (28%, p<0.01) nNOS neurons per ganglionic area than controls. Fluorescence intensity of DAF‐FM diacetate was also lower (20%, p<0.01) in HF mice. HF mice showed a significant loss (52%, p<0.05) in IJP area, when stimulated at higher train duration (800‐1000ms).

Conclusion

We conclude that HF diet causes oxidative stress, leading to loss of inhibitory NO neurons, lower NO availability, and disrupted neuromuscular transmission in the mouse distal colon. Supported by NIH (DK094932).
Hepatic expression of redox factor‐1 and thioredoxin‐1: effects of aging and environmental heat stress
Authors: Bloomer Steven , Sholomskas Leslee , Roche Kathryn , Kregel Kevin
Published: June 2021

Aging is associated with reduced tolerance to physiological stressors such as hyperthermia. In animal models, heat stress is associated with increased oxidative DNA damage in the livers of old rats. In this study, we evaluated the expression of redox factor‐1 (Ref‐1), a DNA repair enzyme, and thioredoxin‐1 (Trx‐1), an antioxidant protein. We hypothesized that these proteins would be induced by heat stress in young animals, and that aging would attenuate this response. Young (6 mo) and old (24 mo) male Fischer 344 rats were exposed to a two‐heat stress protocol, and livers were harvested at several time points after the second heat stress. Ref‐1 and Trx‐1 were evaluated by immunoblot and immunohistochemistry. In young rats, Ref‐1 was induced immediately (0 h) after heat stress, and returned to control levels at 2h. We observed no heat‐induced elevation of Ref‐1 in old rats; however, Ref‐1 was increased ~2‐fold in old, compared to young rats at all time points. At 2h after heat stress, Trx‐1 was increased in old rats, but there was no change in young rats. In tissue sections, Trx‐1 was localized to Kupffer cells in both age groups. Also, we observed frequent ductular reactions in the old rats that were positive for both Ref‐1 and Trx‐1. The impairment in the induction of Ref‐1 suggests a mechanism for the increased oxidative DNA damage observed in old rats after heat stress. Furthermore, the observation of ductular reactions positive for both Ref‐1 and Trx‐1 demonstrates a proliferative cellular niche that develops with aging.
Effects of PCB126 on the Development of Non Alcoholic Fatty Liver Disease
Authors: Boucher Marie‐Pier , Chapados Natalie
Published: June 2021

Persistent organic pollutants (POPs), are chemicals that were mostly used historically as pesticides, solvents, flame retardants, and other applications. Among POPs, polychlorinated biphenyls (PCB) have been recognized to be of environmental and potential toxicologic concerns. Specifically, PCB126 could act as endocrine disruptors and have recently been associated with hepatic fat accumulation. Fatty liver results from alteration in lipid synthesis and elimination mechanisms such as very low density lipoprotein (VLDL) production and de novo lipogenesis. The purpose of the study was to investigate the effects of PCB126 on the molecular development of non‐alcoholic fatty liver disease (NAFLD) in animals. Male sprague‐Dawley (275‐300g) were injected with a single dose of PCB126 (1 ug/kg) or Corn Oil (control group). Seven days later, liver triglyceride (TAG) content was measured along with protein quantification of microsomal triglyceride transfer protein (MTP) and sterol regulatory element binding protein 1c (SREBP1c).

Results

Exposure to PCB126 resulted in significant increases of hepatic TAG concentrations (64%, P <0.001) in rats compared to respective control groups. Rats with fatty livers depicted lower MTP (40%, P <0.02) and higher SREBP1c (27%, P < 0.05) protein content levels compared to Placebo group in rats.

Conclusion

It seems that exposure to PCB126 has an important emerging role in the pathophysiology of NAFLD by 1) altering elimination mechanisms such as VLDL synthesis and secretion, mainly through MTP; and 2) increasing hepatic TAG synthesis mechanisms through SREBP1c.
Liver specific knockout of biliverdin reductase‐A (BVRA) enhances high fat diet induced hepatic steatosis and type II diabetes in mice
Authors: Alamodi Abdulhadi , Hosick Peter , Drummond Heather , Hinds Terry , Stec David
Published: June 2021

Biliverdin reductase‐A (BVRA) is the enzyme responsible for the reduction of biliverdin to bilirubin. In order to determine the role of intracellular heptatic bilirubin generation in the development of dietary induced hepatic steatosis, we developed mice with hepatocyte specific deletion of BVRA. Floxed BVRA mice were crossed with mice specifically expressing the Cre recombinase in hepatocytes under the control of the albumin promoter (Alb‐Cre). Liver specific BVRA knockout (KO) mice exhibited a significant decrease in hepatic BVRA protein measured by both Western blot and immunofluorescence. Liver BVRA KO mice also exhibited a significant decrease in hepatic BVR activity as compared to WT mice (4 + 1 vs. 14 + 3 µmoles bilirubin/min/mg protein, P<0.05). Mice were placed on a high fat (60%) diet at 6 weeks of age for 12 weeks. At the end of the 12 week period, liver specific BVRA knockout (KO) mice exhibited significantly greater levels of fat in the liver as compared to control mice measured by echoMRI (21 + 2 vs. 10 + 3 %, P<0.05). A similar increase in hepatic steatosis in the liver of BVRA KO mice was also determined by Oil Red O staining of liver sections (50 + 3 vs. 22 + 5 %, P<0.05). Liver BVRA KO mice also exhibited higher fasting blood glucose levels at the end of the 12 week study as compared to control mice (155 + 3 vs. 120 + 2 mg/dL, P<0.05). These results suggest that intracellular BVRA generation of bilirubin is an important factor to protect the liver against hepatic steatosis and the development of type II diabetes in response to a high fat diet.
Protective effects of L‐cysteine against LPS‐induced liver injuries.
Authors: Kitagawa Tomomi , Yokoyama Yukihiro , Kokuryo Toshio , Nagino Masato
Published: June 2021

An activation of Kupffer cells (hepatic resident macrophages) and hepatic stellate cells (HSCs) plays an important role in lipopolysaccharide (LPS)‐induced liver injury. It is known that the upregulated production of ET‐1 by Kupffer cells in response to LPS stress activates HSCs and induces subsequent pathophysiological change in the liver. Recently, it has been reported that some amino acids exhibit anti‐inflammatory effects. In a preliminary experiment, using 20 amino acids, we investigated the effect of amino acid administration on ET‐1 production in the mouse macrophage cell lines (RAW 264.7). Among the amino acids, L‐cysteine, L‐glutamine, L‐leucine, L‐alanine, and Glycine suppressed LPS‐induced ET‐1 production in a dose‐dependent fashion. The effect was strongest by an administration of L‐cysteine. L‐cysteine also suppressed a ET‐1‐induced contraction of collagen gel lattices cultured with HSCs. Moreover, an increased [Ca2+]i in response to ET‐1 administration in HSCs measured with the fluorescent dye fura‐2 was strongly attenuated with the presence of L‐cysteine in a culture medium. In an in vivo experiment, Wister rats were pre‐treated with L‐cysteine or saline and received intravenous injection of LPS. Increased levels of liver function tests following LPS was attenuated by L‐cysteine treatment. These results indicated that L‐cysteine may lead to a beneficial effect in LPS‐induced liver injury.
Functional Analysis of Seasonally‐Changing Gut Microbiotas in Hibernating Ground Squirrels
Authors: Carey Hannah , Butz Daniel , Assadi‐Porter Fariba
Published: June 2021

Host diet influences the gut microbiota in species that regularly consume food. We showed that winter fasting in hibernating ground squirrels increases relative abundance of bacterial taxa that can degrade host glycans (e.g., mucins) and reduces abundance of taxa that prefer plant glycans. Here we determined the functional significance of seasonally changing microbiotas by gavaging summer squirrels and aroused hibernators in early and late winter with 13C‐inulin (25 mg/kg), a plant‐derived fiber resistant to digestion by mammalian enzymes. Subsequent measurement of δ13CO2 in breath over 2‐4 h was used an index of bacterial degradation of 13C‐inulin. Compared with summer, peak changes in δ13CO2 in hibernators were delayed and/or reduced by 60‐90% after 1 month of hibernation (early winter) and nearly abolished after 4 months (late winter). Intraperitoneal injection of 13C‐inulin in summer squirrels produced minimal changes in breath δ13CO2, confirming the bacterial nature of the δ13C signal. The results suggest that over the hibernation season, the gut microbiota loses the capacity to degrade a plant glycan, consistent with the shift away from taxa that prefer dietary substrates. Measurement of δ13CO2 in breath is a useful tool to determine functional capacities of gut microbiotas in species that undergo marked changes in food intake. Further insight will be obtained from {1H‐13C}‐NMR based metabolomics and 13C‐isotope‐assisted labelling studies to track bacterial molecules in the host. Supported by funds from UW‐Madison Graduate School and the School of Veterinary Medicine.
Glycomacropeptide Shows Prebiotic and Immune Modulating Properties in Phenylketonuria and Wild Type Mice
Authors: Sawin Emily , Aktas Busra , DeWolfe Travis , Stroup Bridget , Murali Sangita , Steele James , Ney Denise
Published: June 2021

Background

Glycomacropeptide (GMP) is a 64 amino acid (AA) glycophosphopeptide with application to the nutritional management of phenylketonuria (PKU), obesity and intestinal disorders. GMP is a putative prebiotic based on its carbohydrate content including sialic acid.

Objective

To determine the prebiotic properties of GMP by characterizing cecal and fecal microbiota populations and immune responses in C57Bl/6 mice.

Methods

Weanling PKU (Pahenu2) and wild type (WT) mice were fed isoenergetic AA, GMP or casein diets for 8 weeks. The cecum content and feces were collected for microbial DNA extraction to perform 16S microbiota analysis by Ion Torrent PGM sequencing. Plasma cytokines were determined using a Bio‐Plex Pro assay and splenocyte T cell populations were assessed using flow cytometry.

Results

Pairwise comparison suggests that changes in cecal microbiota are primarily diet dependent. The GMP diet resulted in a significant reduction in Proteobacteria, genera Desulfovibrio, in both WT and PKU mice with genotype‐dependent changes in Bacteroides or Firmicutes. The percentage of stimulated spleen cells producing interferon‐gamma (IF‐γ) was significantly reduced in mice fed GMP compared to casein. Plasma concentrations of IF‐γ and tumor necrosis factor‐α were lowest in mice fed GMP.

Conclusions

GMP is a prebiotic based on modulation of the intestinal microbiota compared with casein and AA diets in mice. The shift in microbial populations with GMP appears to be beneficial based on lower indices of inflammation with GMP.
Effects of Probiotic VSL#3 on Cytokine and Tight Junction Protein Expression in Intestinal Epithelial Cells
Authors: Stewart Lauren , Crumley Benjamin , Walton Kristen
Published: June 2021

In the gut, benefits of probiotic bacteria may be mediated through multiple mechanisms, including alteration of the composition or metabolism of the gut microbiota, direct action on host intestinal cells, or modulation of the immune system. 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 VSL#3 on pro‐ and anti‐inflammatory cytokine and tight junction protein expression in Caco‐2 colonic epithelial cells. Cells were treated for 4 hours with VSL#3 (1x108 CFU/ml) and expression of the anti‐inflammatory cytoking transforming growth factor‐beta‐1 (TGFβ‐1), pro‐inflammatory interleukin (IL)‐6 and the housekeeping gene GAPDH was analyzed by semi‐quantitative PCR. Expression of the tight junction protein claudin‐2 was detected by immunofluorescent staining. VSL#3 treated Caco‐2 cells expressed increased levels of TGFβ‐1 mRNA compared to untreated controls. Preliminary experiments suggested a similar increase in TGFβ‐1 in murine CMT‐93 cells. In contrast, IL‐6 mRNA expression was not increased by VSL#3. Immunofluorescent staining showed increased claudin‐2 staining in cells treated with VSL#3 for 24 hours compared to untreated controls. In conclusion, the probiotic mixture VSL#3 stimulates expression of TGFβ‐1 and claudin‐2 in Caco‐2 cells, providing a potential mechanism for protective effects of VSL#3 on the intestinal epithelium. This project was supported by the Missouri Western State University PORTAL program.
Mechanism of Copper Absorption Investigated in Caco2 and HuTu80 Enterocyte Models
Authors: Ricarte Adrian , Valadez Angelica , Arabpour Auriana , Kidane Theodros , Linder Maria
Published: June 2021

How dietary copper is absorbed by enterocytes in the small intestine is still not well understood, and certain aspects continue to be controversial. To shed further light on this process, we used two different human enterocyte models, first determining the expression of mRNA for genes that might be involved. As determined by qPCR, both Caco2 and HuTu cells expressed CTR1, DMT1, and Steap 3, but not Steap 4. Caco2 cells also expressed some Steap 2, but not dCytB; HuTu cells expressed some dCytB. CTR1 expression (relative to 18S RNA) was 2.5x greater in Caco2 cells, whereas that of DMT1 was 2x greater in HuTu80 cells. Rates of Cu uptake were determined with 5 uM 64Cu‐labeled Cu(I)‐histidine and cell monolayers with tight junctions growing in bicameral chambers. Uptake across the apical membrane of both cell lines was inhibited about 50% by 50 uM Ag(I). Interestingly, substitution of sodium sulfate for sodium chloride in the uptake solution had little or no effect, implying no chloride dependence. In iron‐deficient HuTu cells, uptake of 5 uM 59Fe(II) was inhibited ~50% by a combination of drugs targeting DMT1. However, the same drug combination and conditions enhanced rather than inhibited 64Cu uptake. These initial findings continue to support the concept that CTR1 is important for copper absorption. They also imply that neither DMT1 nor a chloride‐dependent channel play a significant role.
Effects of Serotonergic Activation on Respiratory Motor Recovery following Chronic Cervical Spinal Cord Injury
Authors: Hsu Shih‐Hui , Lee Kun‐Ze
Published: June 2021

Unilateral cervical spinal cord hemisection (i.e., C2Hx) usually interrupts the bulbospinal respiratory pathways and results in attenuation of the phrenic motor output. It has been shown that activation of serotonergic system can induce partial recovery of phrenic activity after acute C2Hx. The purpose of present study was to examine whether manipulation of the serotonin system can still improve respiratory function during the chronic injury phase. Bilateral diaphragm electromyogram (EMG) and tidal volume were measured in anesthetized and spontaneously breathing adult rats at 8 weeks post‐C2Hx or C2 laminectomy. A bolus intravenous injection of a serotonin precursor (5‐Hydroxytryptophan; 5‐HTP, 10 mg/kg) or a potent agonist for serotonin 2A receptor agonist (TCB‐2, 0.05 mg/kg) was used to activate serotonergic system. Current results demonstrated that both 5‐HTP and TCB‐2 significantly induced a persistent increase in activity of the diaphragm EMG ipsilateral to the lesion but not contralateral diaphragm in C2Hx animals or bilateral diaphragm in uninjured animals. However, the tidal volume was not changed after administration of 5‐HTP or TCB‐2, indicating the enhancement of ipsilateral diaphragm activity is not associated with improvement of the tidal volume. These results suggest that activation of the serotonergic system can specifically increase the ipsilateral diaphragmatic motor output, but it is not sufficient to improve respiratory functional recovery following chronic cervical spinal injury.
Swallowing‐Related Activities of Respiratory and Non‐Respiratory Neurons in the Nucleus Tractus Solitarius (NTS) in Cats
Authors: Tsai Hsiu‐Wen , Guannan Zhou , Horton Kofi‐Kermit , Morris Kendall , Gestreau Christian , Nuding Sarah , Segers Lauren , Lindsey Bruce , Bolser Donald , Davenport Paul
Published: June 2021

A coordinated swallow‐breathing (S‐B) pattern is generated by the interactions between swallow and respiration central pattern generators in the brainstem. Dorsal swallowing group neurons in the NTS receive pharyngeal and laryngeal inputs to generate a swallow. The NTS is a principal region for integration of peripheral respiratory afferents for the modulation of respiration. We hypothesized that neurons in the NTS contribute to the generation of a coordinated S‐B pattern. To elucidate the coordination between swallowing and respiration, we examined the swallowing‐related activities of respiratory and non‐respiratory modulated neurons (NRM) in the NTS. We electrically stimulated the superior laryngeal nerve (SLN) or applied water to the pharynx of paralyzed, decerebrate, and ventilated cats to elicit swallows. A multiple electrode array was placed into the NTS to record neuronal spike trains during swallowing and breathing. Two types of responses of NRM neurons were observed during swallowing, their firing rate increased either before or during swallowing (50‐50%). Inspiratory (I) neurons either stopped firing or increased their activity during swallowing. The results suggest NRM and I neurons in the NTS are modulated in different phases of swallow to generate a coordinated S‐B pattern.
Oscillatory Pattern of Breathing in Healthy Humans at Exercise: Effects of Hypoxia, Hyperoxia and Hypercapnia
Authors: Hermand Eric , Pichon Aurélien , Lhuissier François , Richalet Jean‐Paul
Published: June 2021

Periodic breathing has been rarely studied in wakefulness during exercise in healthy humans.

Spectral analyses of 82 healthy subjects breath‐by‐breath ventilation (VE) signals, from hypoxia exercise tests, evidenced a periodic pattern with a period from 11.1 to 12.0 seconds. VE Peak power was higher in the group 'high ventilatory response to hypoxia at exercise' (HVRe) group (vs low HVRe, P<0.001). A prospective study (25 subjects) was performed to evaluate the influence of cardiorespiratory factors on the amplitude and period of ventilation oscillations in various conditions of exercise (20 to 40% maximal aerobic power) and hypoxia (0 to 4,000m altitude). VE period was shorter during exercise (vs rest) and during hypoxia (vs normoxia) (P<0.001). VE peak power was higher at exercise (P<0.001) and hypoxia (P<0.001). It was also positively related in hypoxia with the ventilatory response to CO2 (HCVR). We assessed respective contributions of central and peripheral chemoreceptors in hypoxia under acetazolamide (ACZ), in hyperoxia and in hypercapnic hyperoxia. ACZ reduced VE peak power (P<0.001), for an unchanged period. VE Peak power remained unchanged in hyperoxia (vs normoxia), but increased in hypercapnic hyperoxia (P<0.001). This novel observation suggests that healthy subjects demonstrate a spontaneous periodic breathing, triggered by hypoxic exercise, enhanced in subjects with high HVRe, high HCVR and hypercapnic conditions, suggesting that hypoxia and CO2 play a synergistic role in the modulation of these oscillations.
Inactivity‐induced Phrenic Motor Facilitation Following Intermittent, but not Sustained, Neural Apnea Requires Retinoic Acid and Protein Synthesis
Authors: Baertsch Nathan , Baker‐Herman Tracy
Published: June 2021

Phrenic motor output is enhanced following respiratory neural inactivity (neural apnea), a form of spinal plasticity called inactivity‐induced phrenic motor facilitation (iPMF). Mechanisms giving rise to iPMF differ depending on the pattern of neural apnea since TNFα is required to initiate iPMF following a sustained neural apnea, but not when neural apnea occurs intermittently. Some forms of inactivity‐induced plasticity in other neural networks require retinoic acid (RA) to promote translation of proteins that increase neuronal synaptic strength. Accordingly, we hypothesized that iPMF elicited by intermittent neural apnea requires spinal RA and protein synthesis. Anesthetized and ventilated rats were prepared for electrophysiological recording of phrenic burst amplitude. Rats received intrathecal injection (C4) of the RA synthesis inhibitor DEAB (100μM), the protein synthesis inhibitor emetine (70μM) or vehicle (aCSF) prior to intermittent neural apnea (5 ~1.25 min episodes), sustained neural apnea (30 min) or no neural apnea (time control). Following intermittent neural apnea, iPMF was impaired in rats receiving either DEAB (22±10 %baseline) or emetine (10±9 %baseline) since phrenic amplitude was decreased compared to aCSF (56±6 %baseline; p<0.05) and not different from time controls (7±7 %baseline; p>0.05). In contrast, DEAB and emetine did not impair iPMF following sustained neural apnea (87±18 and 56±12 %baseline, respectively) since phrenic amplitude was not different than aCSF (p>0.05) and increased from time controls (p<0.05). Together, these data indicate that mechanisms giving rise to iPMF differ in their requirement for RA and protein synthesis depending on the pattern of reduced respiratory neural activity. NIH HL 105511
Tight Junction Protein Expression and Junction Development During Differentiation of Normal Human Bronchial Epithelial Cells
Authors: Zaidman Nathan , Panoskaltsis‐Mortari Angela , O'Grady Scott
Published: June 2021

Normal human bronchial epithelial cells (NHBE) possessing a basal cell phenotype (p63+, KRT5/6a+) were differentiated on Transwell polyester membranes under air‐liquid interface (ALI) conditions. After 24 days of differentiation, NHBE cells had developed sufficient transepithelial resistance (TER) to perform voltage clamp experiments and expressed mRNAs associated with airway surface cells (Muc5b, FOXJ1). Additionally, mRNA transcripts for ENaC, CFTR and ATP1A1 all increased during differentiation and the corresponding proteins were appropriately locatized as confirmed by immunohistochemistry. Transcripts associated with epithelial adhesions (adherens and tight junctions) were also measured. Upregulation of Cldn3, Cldn8, and Cldn10 correlated with high TER, while ZO1/2, Jam1 did not exhibit differentiation‐dependent increases in mRNA expression. Examination of paracellular ionic permeability demonstrated sodium selectivity over chloride (PCl/PNa ∼ 83%). Furthermore, comparison of NHBE mRNA to an immortalized human airway cell line (HBEC) unable to develop TER revealed loss of claudin, ZO1, and E‐Cad transcripts as measured by qRT‐PCR. These results suggest that TER development in NHBE cells requires constitutive expression of ZO1, E‐Cad and differentiation induced increases in expression of Cldn3, Cldn8, and Cldn10.
Alveolar Type II Cells from Mice Expressing a Lung Disease‐Associated ABCA3 E292V Mutation Exhibit Alterations in Organellar Homeostasis and Autophagy
Authors: Mulugeta Surafel , Tomer Yaniv , Zhao Ming , Gonzales Linda , Russo Scott , Beers Michael
Published: June 2021

The lipid transporter, ATP binding cassette class A3, ABCA3, plays a critical role in the biogenesis of alveolar type II (AT2) cell lamellar bodies (LBs). A relatively large number of mutations in the ABCA3 gene have been identified in association with interstitial lung disease (ILD). The most common mutation is a valine for glutamate missense mutation at residue 292 (E292V) which results in a functional impairment of lipid transport in vitro. To characterize the cellular consequences induced by this mutation in vivo, a novel knockin (KI) mouse model carrying allelic ABCA3 E292V mutations was produced. Lung ultrastructure from E292V homozygous mice revealed smaller size but increased numbers of LBs accompanied by increased AT2 cell mitochondrial counts. Examination of 16 wk and 32 wk homozygous E292V mice showed age‐dependent increase in bronchoalveolar lavage cellularity as well as reduction in alveolar wall thickness coupled with enlarged alveolar air spaces. While isolated E292V AT2 cells demonstrated no alterations in the unfolded protein response, an age‐dependent increase in autophagy components LC3‐I/LC3‐II and P62 also developed in these mice. Furthermore, AT2 cell lysates from 32 wk mice showed accompanying increases in the levels of marker antigens for both mitochondria (Tom20) and lysosomes (Lamp1). We conclude that expression of the ABCA3 E292V mutation produces changes in AT2 organellar homeostasis which result in time‐dependent disruption of macroautophagy‐dependent cellular quality control pathways. We speculate that these changes contribute to the phenotype of a vulnerable epithelium susceptible to second hit injuries that promote the pathogenesis of ILD.
Mitochondria Homeostasis Plays a Key Role in the Age‐related Susceptibility to Lung Epithelial Injury and Fibrosis
Authors: Bueno Marta , Lai Yen‐Chu , Romero Yair , Brands Judith , St. Croix Claudette , Rojas Mauricio , Shiva Sruti , Chu Charleen , Mora Ana
Published: June 2021

Rationale

Aging is associated with increased susceptibility to lung pathologies. The prevalence of idiopathic pulmonary fibrosis (IPF) has been found to increase with age. While familial disease has been related to disordered telomerase activity and mutations of SPC, the effects of advancing age and lung epithelium dysfunction and fibrosis remains unclear.

Methods

Lung fibrosis and mitochondria function, morphology and dynamics were analyzed in alveolar epithelial cell type II (AECII) of patients with IPF, aging mice and mice deficient in PINK1 (PTEN‐induced putative kinase 1).

Results

We found that AECII in IPF lung have marked accumulation of dysmorphic and dysfunctional mitochondria. These mitochondrial abnormalities were recapitulated in normal mice with advancing age and ER stress stimulation. We found that impaired mitochondria in IPF and aging lungs were associated with low expression of the PINK1. Knockdown of PINK1 expression in lung epithelial cells resulted in mitochondria depolarization, and expression of pro‐fibrotic factors. Moreover, young mice deficient in PINK1 develop similar dysmorphic dysfunctional mitochondria in the AECII and are highly vulnerable to apoptosis and development of lung fibrosis.

Conclusion

Our data indicates that PINK1 deficiency and the subsequent swollen mitochondria dysfunction in the presence of ER stress causes defective mitophagy and promotion of fibrosis in the aging lung.
Akt‐Mediated Mitochondrial Oxidative Stress Induces Mitophagy and Development of Pulmonary Fibrosis
Authors: Casey Jennifer , Ryan Alan , Murthy Shubha , Carter A Brent
Published: June 2021

Macrophage mitochondrial oxidative stress is directly linked to pulmonary fibrosis. Mitophagy is the selective engulfment of dysfunctional mitochondria by autophagasomes and can be induced by mitochondrial oxidative stress. Oxidative stress can trigger the PINK1/Parkin pathway, which target mitochondria for mitophagy. Mitophagy is important for quality control for cell homeostasis. In this study we show Akt induces macrophage mitochondrial ROS and is associated with an increase in PTEN‐induced kinase 1 (PINK1) and Parkin. Mitochondrial recruitment of PINK1 was determined to be Akt‐dependent, as knockdown of Akt reduced PINK1 mitochondrial localization. Akt did not modulate mitochondrial dysfunction by altering membrane depolarization; however, macrophages exposed to chrysotile asbestos had a decrease in mitochondrial membrane potential in a time‐dependent manner. Akt enhanced macrophage survival and induced autophagosome formation by an induction of LC3 expression. Biological evidence to support this shows that Akt deficient mice have reduced mitochondrial localized PINK1 and Parkin expression, decreased macrophage survival, and are protected from pulmonary fibrosis compared to WT mice. Moreover, alveolar macrophages obtained from asbestosis and idiopathic pulmonary fibrosis patients had increased cell survival and increased mitochondrial localization of PINK1 and Parkin. These observations suggest a direct link between Akt activation, mitophagy, and pulmonary fibrosis by regulating mitochondrial dynamics.

2R01ES015981‐07 & BX001135‐01
The Hydrophobic Surfactant Proteins Strongly Induce Lipid Curvature
Authors: Hall Stephen , Chavarha Mariya , Loney Ryan , Rananavare Shankar
Published: June 2021

The hydrophobic surfactant proteins, SP‐B and SP‐C, greatly accelerate adsorption by vesicles of the surfactant lipids to form a film that lowers the surface tension in the lungs. Pulmonary surfactant enters the interface by a process analogous to the fusion of two vesicles. Like fusion, several factors affect adsorption according to how they alter the curvature of lipid leaflets, and suggest that adsorption proceeds via a rate‐limiting structure with negative curvature, in which the hydrophilic face of the phospholipid leaflets is concave. The studies reported here tested whether the surfactant proteins might promote adsorption by inducing the lipids to adopt more negative curvature, closer to the configuration of the hypothetical intermediate. We used X‐ray diffraction to determine how the proteins in their physiological ratio affect the radius of cylindrical monolayers in the negatively curved, inverse hexagonal phase. With binary mixtures of dioleoyl phosphatidylethanolamine (DOPE) and dioleoyl phosphatidylcholine (DOPC), the proteins produced a dose‐related effect on curvature that depended on the phospholipid composition. With DOPE alone, the proteins produced no change. With increasing amounts of DOPC, the response to the proteins increased, reaching a maximum 50% reduction in cylindrical radius at 5% (w:w) protein. This change represented a doubling of curvature at the outer cylindrical surface. The change in spontaneous curvature, defined at approximately the level of the glycerol group, would be greater. Our findings show that the surfactant proteins can promote negative curvature, and support the possibility that they facilitate adsorption by that mechanism.
Mesenchymal Stem Cell Exosomes ‐ Potential Regenerative Medicine Application for Tendinopathy
Authors: Ludlow John , Buehrer Benjamin
Published: June 2021

Adipose tissue‐derived mesenchymal stem cells (MSCs) are characterized by their self‐renewing capacity and their ability to differentiate into chondrocytes, adipocytes, and osteocytes. This makes them attractive starting materials for tendon tissue engineering and regenerative medicine applications. While the recognized regenerative properties of stem cells show promise in bioengineering tendon constructs for repairs, there is limited evidence that direct injection of stem cells for tendon healing has beneficial effects. It has been suggested that much of the observed benefit of these stem cell injections arises from stem cell‐secreted factors carried in discreet microvesicles called exosomes. These small vesicles contain bioactive components related to wound healing and present a potential new allogeneic therapy for tendinopathies. Here, we present our findings on the physical and functional characteristics of adipose tissue‐derived MSCs relevant to wound healing and tissue regeneration. Data is presented on the miRNA profile of these exosomes, their ability to mediate cell migration and incorporate into the recipient cell membrane, and their ability to downregulate STAT3 phosphorylation. Having successfully developed exosome isolation procedures and in vitro assays to functionally characterize these particles which are secreted by the cultured MSCs, our goal is to leverage the regenerative and healing properties of adult stem cells by developing exosomes as a non‐surgical and non‐cellular treatment for tendon repair.
Nuclear Reprogramming with Hindbrain Patterning Genes by Nanochannel Electroporation
Authors: Czeisler Catherine , Gallego‐Perez Daniel , Ortiz Cristina , Gygli Patrick , Askwith Candice , Lee L James , Otero Jose
Published: June 2021

Directly inducing a specific cell fate requires not only the knowledge of which transcriptional regulatory networks orchestrate one's desired cellular phenotype, but also the ability to exogenously introduce these proteins into cells. As has been elegantly demonstrated, nuclear reprogramming methodologies typically require small collections of transcription factors to change a cell's epigenetic framework. To generate desired cell types, expression larger collections of patterning genes are needed to generate specific neuronal fates. However, the ability to directly control the dosage of reprogramming factors that each individual cell would need has not been achieved. We have overcome this problem by implementing nanochannel electroporation for neuronal reprogramming. With NEP precise amounts of reprogramming agents are delivered electrophoretically through the nanochennel, with agent dosing being modulated by electrophertic pulse duration and total number of pulses. We utilized the technology to induce specific neuronal cell types of the hindbrain lineage.
Regulation of the sodium‐activated potassium channel Slack by MAGI‐1
Authors: Pryce Kerri , Agwa Dalia , Tomasello Danielle , Bhattacharjee Arin
Published: June 2021

The sodium‐activated potassium subunit Slack (Kcnt1) is highly expressed in sensory neurons and contributes to firing accommodation. Previous studies have shown that protein kinase A‐induced internalization of Slack channels contributes to hyperexcitability in DRG neurons. These results suggest that the molecular modulators of Slack channel trafficking could serve as novel therapeutic targets in the treatment of pain. In this study, we probed the molecular determinants of Slack channel trafficking by focusing on the evolutionary conserved last 10 amino acids sequence of the C‐terminus. In this region there is a post‐synaptic density‐95/discs large/zonulaoccludens‐1 (PDZ) binding domain. Using a synthetic peptide comprised of these 10 amino acids, we performed patch‐clamp recordings on isolated DRG neurons. We found that incubating neurons with the peptide resulted in hyperexcitability whereas, a scrambled peptide did not alter firing. Biotinylation assays confirmed a significant reduction in channel expression at the membrane upon peptide incubation; suggesting that the anchoring of Slack channels to the membrane is likely mediated through the PDZ‐binding motif. Studies have found MAGI‐1, a scaffolding protein, and Slack to both be enriched in the dorsal horn of the spinal cord. Using co‐immunolocalization assays, we demonstrated that MAGI‐1 localized with Slack at the cell membrane. We next confirmed that MAGI‐1 interacts with Slack via the PDZ binding motif by immunoprecipitation assays. Our results suggest that Slack is stabilized at the cell membrane by its ability to bind PDZ containing proteins. Dissociation from the PDZ scaffold leads to hyperexcitability. MAGI‐1 is a potential Slack anchoring protein and strengthening this interaction may be a novel analgesic strategy for pain.
Effect of Small/Intermediate Conductance Ca2+ Activated K+ Channel Opener on Myogenic Differentiation in C2C12 myoblasts
Authors: Sakamoto Kazuho , Tanaka Shoko , Kimura Junko
Published: June 2021

Skeletal muscle tissue contains unique stem cells called myoblasts or satellite cells, and they play a pivotal role in the repair and maintenance of skeletal muscle by differentiation into muscle cells. However, the regenerative ability declines with age, and invite reduction of skeletal muscle mass (sarcopenia). We are searching for a compound which enhance muscle regenerative activity. Since membrane hyperpolarization facilitates the differentiation in myoblasts, we hypothesized that a compound which hyperpolarizesthe myoblast membrane potential may enhance the myogenic differentiation. It has been reported that a major current component in myoblast is the small/intermediate conductance Ca2+ activated K+ (SKCa/IKCa) channel. In this study, we treated mouse C2C12 myoblasts with DCEBIO, a SKCa/IKCa channel opener. C2C12 myoblasts fuse each other to form myotube in 2% horse serum medium. Our image analysis revealed DCEBIO significantly increased the percentage of fusion in a concentration dependent manner, and this effect was partially attenuated by co‐application of SKCa/IKCa channel blockers, apamin (100 nM) and TRAM‐34(1 μM). Furthermore, DCEBIO (10 μM) treatment increased the protein expression level of myosin heavy chain type II, a muscle specific protein. Our realtime RT‐PCR analysis showed that DCEBIO (10 μM) significantly increased the expression level of myogenin but not of Myo D, two of the essential transcription factors for myogenic differentiation. These suggest that DCEBIO enhanced myogenic differentiation by opening of SKCa/IKCa channel, and activated myogenic regulatory factor. These findings show a novel possibility of potassium channel openers as a muscle differentiation enhancer.
Systems Biology Tools and Data for a Better Understanding of Mechanisms of Toxicity.
Authors: Boue Stephanie
Published: June 2021

Risk assessment in the context of 21st century toxicology relies on the elucidation and understanding of mechanisms of toxicity. While no single experiment allows deciphering toxic mechanisms at the interplay of complex pathways, network models built from the knowledge accumulated in the scientific literature enable a more comprehensive view of the changes to the system caused by toxic or pharmaceutical agents. Moreover well‐designed and rich data collection in the context of toxicology or pharmacology will further advance risk assessment. Bringing the scientific community together and harnessing the wisdom of crowds is the method used by sbv IMPROVER to verify methods and biological networks used in systems toxicology, pharmacology and diagnostic through a series of collaborative competition challenges. We will present past challenges and their outcome regarding methods to classify biological samples based on their transcriptomics profiles and to assess the translatability of key pathways between human and rat cell cultures. Moreover, we will show how we apply crowd‐sourcing methods to verify and fine tune network models of lung biology.

Our goal at Philip Morris International R&D is to develop and assess potentially reduced risk alternatives to combustible cigarettes in order to reduce the harm caused by smoking, which is one of the leading causes of preventable diseases. The next sbv IMPROVER challenge will focus on evaluating large pre‐clinical Systems Toxicology datasets generated from experimental systems exposed to conventional cigarette smoke and the aerosol generated from a prototype noncombustible product's aerosol.