bluuebunny/crossref_metadata_2025 huggingface dataset:
Understanding Brain/Face Integration from Neuropsychiatric Disorders
Authors: Martínez-Abadías Neus , Hostalet Noemí , Gonzàlez Rubèn , González Alejandro , Sevillano Xavier , Canales Rodríguez Erick J. , Salvador Raymond , Pomarol-Clotet Edith , Fatjó-Vilas Mar
Published: April 2020

Understanding how genetic and environmental variation alter the integrated development of the face and the brain has important implications for medicine and human evolution. Neuropsychiatric disorders offer an ideal model for investigating the association between facial and brain phenotypes with their underlying genotype. Schizophrenia (SZ) and Bipolar Disorder (BD) are severe psychiatric disorders caused by pathological processes occurring early in neurodevelopment. Disruptions in signaling pathways that are common to brain and face development may explain why these disorders are associated with abnormalities of brain structure and a high prevalence of minor facial dysmorphologies. We assessed facial shape with its neuroanatomical and genetic correlates in a sample with magnetic resonances and genetic data available for 188 subjects (75 healthy controls, 67 SZ and 46 BP patients). Geometric Morphometrics revealed that facial shape variation assessed from 3D facial reconstructions significantly differentiated between SZ/BP patients and controls with moderate accuracy (60–65%). ANOVA tests detected a significant association between facial shape and specific measures of the brain superior frontal regions (thickness, volume and area), whereas the explained facial shape variation increased from 1% to 4.8% in SZ and 6.5% in BD when we included in the model the interaction between a SNP in GLI3 gene and these brain measures. We conclude that facial shape may be considered as an indirect marker of deviances in brain development, and GLI3 as a modulator of brain‐face relationships; probably due to the key role of this gene in the Shh signaling pathway regulating brain and face development.

Support or Funding Information

Ministerio de Ciencia, Innovación y Universidades, Proyectos I+D+i «Retos Investigación» RTI2018‐097845‐J‐I00; 2017SGR1630; 2017SGR1271; CD16/00264.
Effects Of J147 On Post‐Operative Cognitive Dysfunction
Authors: Oberman Klaske , Villafranca Ernest , Schoemaker Regien G.
Published: April 2020

Background

Postoperative cognitive dysfunction (POCD) is a major complication in patients undergoing surgery. The inflammatory response necessary for wound healing seems derailed and is reflected in the brain as neuroinflammation and consequently cognitive decline. J147 has anti‐dementia and anti‐aging effects that may be attributed to anti‐inflammatory properties.

Aim

investigate effects of J147 treatment in an animal model for POCD.

Materials and Methods

Male Wistar rats (n=60; ±350g) were randomly divided over 1. non‐surgery; 2. control surgery; 3. surgery + acute J147 (20 mg/kg) and 4. surgery + chronic J147 (30 mg/kg/day). Acutely treated rats received one single oral administration of J147 before surgery, chronically treated rats received oral J147 through their food during the whole experiment. Behavioral testing consisted of an Open Field test (OF), Novel Object and Novel Location Recognition test (NOR/NLR) and the Morris Water Maze (MWM) to assess exploratory behavior, object memory, spatial learning and memory, and cognitive flexibility, resp. Two weeks after surgery, rats were sacrificed, and blood and brain tissue is collected for later analyses of (neuro)inflammation.

Results

All rats survived surgery and were subjected to behavioral testing. The OF showed that chronic treated animals payed significant more visits to the center (12.6±1.5 vs 9.7±1.4 in control surgery), at similar distance moved. No difference in spatial learning capacity, reflected in the learning curve of the MWM test, was seen between the groups. Platform crossings in the MWM indicated that chronic treatment can restore the spatial memory deficits caused by surgery (1.6±0.2 vs 1.0±0.1). NOR test did not discriminate between groups. In NLR, control surgery rats performed at chance level (60±5 vs 50%), whereas performance in chronic treated rats significantly increased above chance level; 70±6%. For the acute treated group no significant differences were seen compared to the control surgery group in all the tests.

Conclusion

Our data indicate that chronic J147, but not acute J147 prevents cognitive impairment following abdominal surgery in our POCD model. Therefore, chronic J147 treatment could provide a promising novel therapeutic approach in POCD in patients. To study the underlying mechanism of (neuro) inflammation, plasma cytokines levels and microglia activation in the brain will be determined.

Support or Funding Information

The study is supported by funding from ABREXA
From Post‐Doc to Faculty Position: The IRACDA FIRST Program A Teaching Post‐Doctoral Fellowship
Authors: King Adrienne Lester
Published: April 2020

The overall goal of the Intuitional Research and Academic Career Development Awards (IRACDA) program (Fellowships in Research and Science Teaching or FIRST) has been and continues to be the combination of a traditional mentored postdoctoral research experience at Emory University or Morehouse School of Medicine, research‐intensive institutions, with an opportunity for the fellows to develop teaching skills that involve instruction in teaching methods and a mentored teaching experience at one of the Atlanta University Center (AUC) Schools (Spelman College, Clark Atlanta University or Morehouse College, all Minority Serving Institutions(MSIs)). The combined program facilitates the progress of underrepresented minority postdoctoral candidates into research and teaching careers in academia and provides all program fellows with experience at Minority Serving Institutions (MSIs) that promotes the next generation of scientists at MSIs. The program also strongly reinforces interaction among faculty at the AUC schools and their counterparts at the two research intensive institutions participating in this program. The objectives of the Program are four‐fold: (1) to enhance research‐oriented teaching at the AUC schools; (2) to increase or enhance the research backgrounds of developing scientists to conduct high quality research in an academic environment; (3) to further promote interaction between Emory University, the AUC schools, and Morehouse Medical School that will lead to further collaboration in research and teaching; and (4) to increase the number of well‐qualified underrepresented minority students entering competitive careers in biomedical research. The program has benefited the MSIs by providing support for development and recruitment of new faculty some of which are FIRST fellows and providing new courses and training opportunities for MSI students. Evaluation of the Program shows that the fellows in the FIRST Program publish at the same rate as their peers supported on T32 grants at Emory University and other institutions with IRACDA programs. The evaluation also shows that FIRST fellows enter academic positions and receive post‐fellowship grant awards at the same rate as T32 fellows (based on NIH data). As a recipient of the IRACDA FIRST fellowship, I am currently a faculty member at Georgia State University which is the largest university in the state of Georgia. It is the 15th most diverse university in the country with a minority enrollment of 46 percent. It has recently been classified as a Title V Hispanic‐serving institution. I have an active research program, teach, and provide service to the university. I continue to fulfill the purpose of the IRACDA program by developing a diverse group of highly trained scientists to address the nation’s biomedical research needs through my faculty position.
Influence of Incubation Temperature on Chicks Ventilation and Oxygen Consumption
Authors: Rocha Aline Cristina Gonçales , da Silva Welex Cândido , Bícego Kênia Cardoso , Gargaglioni Luciane Helena
Published: April 2020

The first third of the incubation is determinant for the embryonic development and the temperature is considered one of the most important factors that can affect the quality and the survival rate of the embryos. The aim of this study was to evaluate the effect of changes in the temperature of incubation (36 °C‐cold; 37.5 °C‐thermoneutral and 39 °C‐hot) during the first 5 days on ventilation (VE) body temperature (Tb) and oxygen consumption (VO2) of 2 days old broiler males (M) and females (F) in basal conditions and under exposition to hypoxia (10% O2) or hypercapnia (7% CO2). No difference among groups was observed during room air conditions. Under hypercapnia, the VE of females exposed to cold incubation was lower compared to other treatments (P =0.0402 compared to control and P= value=0.0193 compared to hot), as well as the respiratory frequency (fR) when compared with hot treatment (P =0.0387), but not to the control situation (P = 0.1109). VO2 of females during CO2 exposure was higher in 36 °C group compared with other groups (P = 0.0004 compared to control; P =0.0078 compared to hot). VE and of control males was lower when compared to hot (P = 0.0344) and VT of the same group was lower than cold (P‐value=0.0171) and hot (P =0.0324) treatments. There was no difference in fR and VO2 among groups. Hypoxia promoted a higher VE (P = 0.0439) and VT (P =0.0041) in cold exposed females compared to hot. VO2 was higher (P = 0.0290) as well when compared to 37.5 °C group. No difference was observed in respiratory responses of males exposed to hypoxia. Therefore, temperature of incubation can affect the ventilation in young broiler chickens under hypercapnic and hypoxic conditions in a sex‐dependent manner.

Support or Funding Information

FAPESP, CNPq and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ Brasil (CAPES) ‐ Finance Code 00
Saturated Fatty Acid‐Enriched Diet Affect Liver Transcriptional Response of Anti‐oxidant Genes of Perinatal Undernourished Rats
Authors: Costa-Silva João H. , Simões-Alves Aiany C. , Fernandes Mariana P. , Leandro Carol G. , Pirola Luciano , Morio Béatrice
Published: April 2020

In this work, we evaluated the impact of a saturated fatty acid (SFA)‐enriched diet (HL) in the post‐weaning offspring subjected to maternal protein restriction in liver genes of redox balance and inflammatory pathway. Wistar rats were mated and during gestation and lactation, mothers received control (C) or low protein diet (LP, 8% protein). After weaning, rats received either C or HL (+59% SFA) diets up to 90 days of life. It was verified that circulating markers of lipid metabolism and liver function were not significantly altered between groups. The LP group submitted to the post‐weaning HL diet showed an increase in liver protein and lipid oxidation (p<0.01). This group also presented greater gene transcription of NF‐KB1 and TNF‐α (p<0.05) compared to controls. Liver mRNA contents of pro‐oxidant enzymes (XDH, NOX1 and RAC1) were not different between LP and controls animals. Post‐weaning HL diet did not alter the pattern of expression of SOD1, SOD2, CAT, GPX1, GPX2, UCP2 genes in LP groups, except for CAT, which was significantly higher in LP group fed HL diet compared to C (+66%, p<0.05). In protein level, CAT was lower in LP compared to control animals under both post‐weaning C and HL (p<0.05). In contrast, SOD2 protein content was twice higher in LP groups (p<0.05). However, GPX1/2 protein expression was similar between groups. Regarding transcription factors, no differences were noted in NRF2 and FOXO3 mRNA contents. However, SFA enhanced PGC1α (+207%, p<0.05) and PPARγ (+151%, p<0.05) levels in LP groups vs. C. Thus, our data suggest that SFA‐enriched diet induces oxidative stress, activation of inflammatory pathways, however without metabolic disturbance when the animals were submitted to a low protein diet during critical periods of development.

Support or Funding Information

This study was supported by FACEPE (Brazil), CNPq (Brazil) and CAPES/COFECUB (Brazil/France).
The Effect of Serum of Cerebral Small Vessel Disease Patients on Brain Endothelial Cells
Authors: Cifu' Adriana , Janes Francesco , Domenis Rossana , Fabris Martina , Curcio Francesco
Published: April 2020

INTRODUCTION

Cerebral Small Vessel Disease (CSVD) is characterized by perforating cerebral arterioles, venules and capillaries, and a consequent damage of cerebral white and deep grey matter. The pathogenesis of CSVD is unknown and could be asymptomatic in early phases. CSVD is a typical disease of elderly population and in stroke patients. However, the increasing use of MRI in last years, led to incidental findings in young people without classical vascular risk factors. To better understand the role of brain endothelium in onset of Cerebral Small Vessel Disease, we evaluated the effect of CSVD patients’ serum on human brain endothelial cells (HBEC‐5i).

METHODS

HBEC‐5i were treated for twenty‐four hours with 20% of serum from six CSVD patients or six age‐ and sex‐matched controls. Trypan blue exclusion method was used to evaluate proliferation rate and viability of treated cells compared to untreated cells. The effect of CSVD patients’ serum on HBEC‐5i, was evaluated by droplet digital PCR in terms of the mRNA expression of interleukins involved in inflammatory response(IL1β, IL1α, IL6, IL8, and IL10).

RESULTS

The treatment with both patients’ and controls’ serum reduced the proliferation rate of cells, however no difference was observed between the two treatment. The viability, instead, was not influenced by serum treatment. The mRNA expression of pro‐inflammatory cytokines (IL1β, IL1α, IL6) was higher in HBEC‐5i treated with serum of CSVD patients compared to both untreated cells and cells treated with serum of controls. On the other hand, the mRNA expression of IL10 , an anti‐inflammatory molecule, increased only in cells treated with controls’ serum. None treatments affected the mRNA level of IL8.

CONCLUSIONS

In brain endothelial cells, the serum of CSVD patients, differently from serum of controls, promotes the expression of pro‐inflammatory cytokines. These preliminary data suggest that inflammation could play a role in pathogenesis of CSVD.

Support or Funding Information

This project was supported by Grant Interreg ARTE (J22F170001005)
The state of the antioxidant system in cows at different densities of radioactive contamination of the soil
Authors: Anipchenko Polina , Vasiliev Roman , Gaponova Viktoriya , Vasiliev Roman , Ponomarenko Nikolai , Troshin Evgenii , Tsuganov Andrey , Ugatova Natalia
Published: April 2020

One of the negative factors of ionizing radiation on the body is the formation of active oxidants, which are protected by the antioxidant system. The effect of high doses of radiation on the antioxidant system of animals has been studied well, but there is not enough information about the long‐term effects of low doses. The study of the effect of pre‐threshold radiation doses on the main indicators of antioxidant protection of cattle was the purpose of our research. The research was carried out in JSC "Osminskoye" of Slantsevsky district and JSC "Plemzavod " RAPTI" of Luga district of Leningrad region on healthy lactating cows aged 3–4 years in the amount of 8 heads from each farm. Radio ecological characteristics of agricultural land JSC "Osminskoe" was as follows‐the density of radioactive contamination of the soil on 137Cs more than 1 Ci/km2, the power of the exposure dose on the ground 35–48 μr/h. Similar indicators of land in JSC "Plemzavod" RAPTI " were less than 0.5 Ci/km2 and 6–19 μr / h. Both farms use their own fodder base (hay, silage, root crops), concentrated feed produced by JSC "LKKZ", the animals are kept in summer walking, which allows us to talk about a similar dose load due to external and internal irradiation. In cows, catalase activity was determined in serum, and malone dialdehyde concentration and superoxide dismutase activity were determined in plasma. The results of the study showed that the activity of superoxide dismutase in cows from JSC "Osminskoye" was 4.15±0.46 U/mg Hb, catalase ‐ 14.54±1.03 mkat/l, and the level of Malon aldehyde ‐ 3.71±0.68 μmol/l. Similar indicators in animals from JSC "Plemzavod "RAPTI" were as follows ‐ 6.3±0.75 U/mg Hb, 9.77±0.62 mkat/l and 2.23±0.12 μmol/l. Intergroup differences of the studied indicators were statistically significant. Analyzing the results of the study, we can say that chronic stimulation of the antioxidant system by active oxidants leads to a decrease in the level of superoxide dismutase in cows by 34.1%. Probably, as compensation, there is an activation of other enzymes, in particular, an increase in catalase activity by 1.5 times. However, the compensatory capabilities of other elements of the antioxidant system are not able to fully neutralize free radicals, as evidenced by an increase in the concentration of malon dialdehyde by 66.4%.The study shows that cows long‐term in conditions of higher pre‐threshold doses of external and internal radiation occurs chronic oxidative stress, manifested by an imbalance of individual links of the antioxidant system.
Modeling of the PTF1‐J and PTF1‐L Heterotrimeric Transcription Factor Complexes
Authors: Coats Ward
Published: April 2020

Students in Biomedical Research Studies at Thomas Jefferson High School in Dallas, Texas participate in an ongoing research program in collaboration with the University of Texas Southwestern Medical Center to study the structure and function of a transcription factor complex. Ptf1a is a transcription factor that is crucial to the development of the embryonic pancreas. Its functional form is in a trimeric complex composed of a heterodimer of E12/E47 and Ptf1a (basic helix‐loop‐helix transcription factors), and either Rbpj or Rbpjl (Rbpj is also present in Notch signaling). The Rbpj form of the complex (PTF1‐J) is required for the early stage of pancreatic development. Subsequently, the Rbpjl‐form (PTF1‐L) is required for the formation of mature acinar cells of the exocrine pancreas. The Thomas Jefferson students using 3D printing technology has generated models for the PTF1‐J and PTF1‐L transcription factor complexes. The models provide the orientation of the Ptf1a/E12 heterodimer relative to the Rbpj and Rbpjl proteins and characterizes interactions formed between Ptf1a (tryptophan 298) and a hydrophobic pocket present in the beta‐trefoil domain of Rbpj or Rbpjl. Determination of the structures for the PTF1‐J and PTF1‐L complexes will help elucidate the structural determinants and the mechanism for the strong cooperative DNA binding of these complexes.

Support or Funding Information

Dallas Junior League: Grant for Innovative Teaching

Texas Medical Association Award for Excellence in Science Teaching
Preliminary structural characterization of a methyltransferase involved in rhodoquinone biosynthesis
Authors: Vergunst Kathleen , Bergin Kathleen , Roger Andrew , Shepherd Jennifer , Langelaan David
Published: April 2020

The primary process of energy production for most eukaryotes involves oxidative respiration via the electron transport chain (ETC). In the ETC, electrons are moved by the electron carrier ubiquinone through membrane‐bound protein complexes that pump protons across the mitochondrial membrane, which creates a proton chemical gradient. This gradient is used to produce adenosine triphosphate, which is the primary source of energy in the cell. Oxygen is the final electron acceptor of the ETC for aerobic organisms, but for organisms that live in low‐oxygen environments, other strategies are required for the ETC to function. One strategy used by many species of bacteria and protists is to produce rhodoquinone, which is a derivative of ubiquinone. Rhodoquinone allows the ETC to function in the absence of oxygen by allowing it to use fumarate as the final electron acceptor. RQ is synthesized by the enzyme RquA, which likely uses ubiquinone as a substrate. Our goal is to structurally characterize RquA to better understand its mechanism of catalysis. We used E. coli to overexpress RquA derived from Pygsuia biforma andRhodospirillum rubrum with a variety of fusion tags and found that RquA expresses to a high level but is located in inclusion bodies. We have developed refolding procedures to isolate RquA and are now characterizing its structure as well as the substrates required for RQ production.

Support or Funding Information

This research is funded by the New Frontiers in Research Fund ‐ Exploration Stream
In vitro and in vivo evidence for the role of lipid rafts in Ca2+‐gating of the Transient Receptor Potential channels in sensory neurons
Authors: Szőke Éva , Horváth Ádám , Bíró-Sütő Tünde , Sághy Éva , Payrits Maja , Erostyák János , Makkai Géza , Szolcsányi János , Helyes Zsuzsanna
Published: April 2020

Transient Receptor Potential (TRP) cation channels as TRP Vanilloid 1 and TRP Ankyrin 1 (TRPV1 and TRPA1) are nocisensors playing important role to trigger pain. These cation channels serve as thermosensors and several exogenous and endogenous chemical ligands activate them. Capsaicin and resiniferatoxin (RTX) activate TRPV1, allyl‐isothiocyanate (AITC) and formaldehyde activate TRPA1 receptors. Lipid rafts are defined as liquid ordered plasma membrane microdomains rich in cholesterol, sphingomyelin and gangliosides. Sphingomyelinase (SMase) decreases membrane sphingomyelin by hydrolization, myriocin inhibits the glycosphingolipid synthesis. Our aim was to examine their in vitro actions on TRP channels, and the potential analgesic effect of SMase and myriocin in in vivo mouse models.

The effect of SMase and myriocin was analysed on isolated trigeminal (TG) neurones by measuring agonists‐induced Ca2+ transients with ratiometric technique, and on TRPV1‐ or TRPA1‐expressing CHO cells by measuring 45Ca‐uptake. Mechanonociceptive and thermonociceptive thresholds of the animals in RTX‐induced thermal, and mechanical hyperalgesia, and formaldehyde‐evoked hyperalgesia model were measured. The analgesic effect of SMase and myriocin was measured in capsaicin‐evoked acute nocifensive response test.

It has been revealed that intracellular Ca2+ enhancement evoked by capsaicin (TRPV1), AITC and formaldehyde (TRPA1) was inhibited after SMase and myriocin incubation in TG neurons and receptor‐expressing cells. Treatment with SMase and myriocin significantly diminished the RTX‐induced thermal, and mechanical hyperalgesia, the formaldehyde‐evoked hyperalgesia and the capsaicin‐evoked nocifensive reaction.

On the basis of all these findings it can be concluded that the lipid membrane environment modulates the function of TRPV1 and TRPA1 channels and lipid raft interfaces modulate the channel opening. Targeting this interaction might be a tool for drug developmental purposes.

Support or Funding Information

János Bolyai Fellowship, KTIA_NAP_ 20017‐1.2.1‐NKP‐2017‐00002, GINOP‐2.3.2‐15‐2016‐00050, EFOP‐3.6.2‐16‐2017‐00008, 20765/3/2018 FEKUTSTRAT
Craniofacial growth and development in a mouse model of Down syndrome
Authors: Singh Nandini , Reeves Roger H. , Richtsmeier Joan T.
Published: April 2020

Down syndrome (DS) is a developmental disorder caused by trisomy of human chromosome 21. Ts65Dn, a widely used mouse model of DS, has facilitated the study of mechanisms underlying the genotypic and phenotypic anomalies associated with DS. Previous studies have shown the adult cranium and brain to be markedly smaller in size in Ts65Dn mice relative to their euploid littermates. To determine how postnatal developmental patterns contribute to cranial variation in trisomic mice, we quantitatively compared growth trajectories from birth (postnatal day 0, P0) to postnatal day 6 (P6) in Ts65Dn and euploid mice. We hypothesized that trisomy affects cranial growth rate in Ts65Dn mice, causing size and shape differences between Ts65Dn and euploid mice. Multivariate regression and principal components analyses of craniofacial shape and size show some subtle differences between the trisomic and euploid mice at P0, but by P6 the trisomic mice are considerably smaller than the euploids. Interestingly, Ts65Dn at P6 are closer in both size and shape to P0 euploid mice than to euploid mice at P6 or newborn Ts65Dn. This could occur through changes in growth rate of Ts65Dn mice, resulting in the trisomic P6 mice retaining features common to younger, typically developing mice. Further research focusing on prenatal ages will provide a better understanding of how differences in timing of developmental events, such as delays in migration and mitosis of cranial neural crest cells in mouse model of DS, contribute to trisomic craniofacial growth and development.

Support or Funding Information

R01‐HD038384 (RHR)
Single cell RNA sequencing (scRNAseq) of severely atherosclerotic coronary artery from Ossabaw miniature swine with metabolic syndrome
Authors: Eggenberger Caleb , Badin Jill K. , Alloosh Mouhamad , Sturek Michael
Published: April 2020

Introduction

Coronary atherogenesis involves multiple signaling pathways and underlying gene expression. The atherogenic plasma milieu (excess lipoproteins, glucose, etc.) injures the endothelium, which is the initiating event of coronary plaque development. Subsequently, smooth muscle cells proliferate and migrate from the wall into the neointima, and blood cells and macrophages infiltrate the vessel wall and contribute to the growing plaque. Our goal was to characterize the gene expression in the heterogeneous cell population in this dynamic coronary artery pathology.

Methods

We performed single cell RNA sequencing (scRNAseq) on cells dispersed from the proximal 45 mm of the left anterior descending coronary artery of healthy pigs compared to separate plaque and wall cell dispersions from arteries of Ossabaw swine that had metabolic syndrome and were atherosclerotic for 23 months. Primary analysis scRNAseq data used t‐distributed stochastic neighbor embedding (T‐SNE) plots for an integrated analysis of the 3 cell preparations.

Results

Metabolic syndrome was confirmed by obesity, dyslipidemia, and hypertension. In vivo intravascular ultrasound (IVUS) of the entire artery revealed every stage of atherosclerosis from fatty streak to complex lesions having up to 70% occlusion with fibrofatty and calcified lesions. Cells (3,510) from healthy coronary artery expressed predominantly markers for endothelial (PECAM1), endocardial (CDH5, POSTN), and smooth muscle cells (ACTA2, TAGLN, CNN1, MYL9, CALD1, RGS5, MHY11). Overall, the healthy coronary artery cells were much more homogeneous than atherosclerotic plaque and wall cells. Wall cells (8,224) in atherosclerotic arteries expressed ~no (0.1%) endothelial, many smooth muscle, and fibroblast genes. Plaque cells (8,049) expressed ~no (0.1%) endothelial genes, robust smooth muscle marker genes, and a high abundance of early marker genes for chondrocytes, osteoblasts, monocytes, neutrophils, and macrophages.

Conclusion

Profound gene expression patterns were shown with scRNAseq in a clinically relevant model of coronary atherosclerosis. The striking heterogeneity of gene expression and marker genes for chondrocytes and osteoblasts in plaque cells indicate significant transdifferentiation and suggest further progression to severe coronary artery calcification.
Impaired Oxygenation of the Right Ventricle during Development of Pulmonary Hypertension in Swine is not due to Loss of Nitric Oxide
Authors: Merkus Daphne , Steenhorst Jarno J. , van Duin Richard W. , Cai Zongye , Duncker Dirk J.
Published: April 2020

Background

Pulmonary hypertension (PH) increases workload of the right ventricle (RV), resulting in RV hypertrophy. Eventually the RV is unable to cope with the high afterload, resulting in RV failure and death. The transition of RV hypertrophy to failure is incompletely understood, but we have recently shown that myocardial oxygen supply did not increase commensurate with the increase in myocardial oxygen demand. This mismatch between oxygen supply and demand is exacerbated during physical activity when workload and hence oxygen demand increases. Here, we aim to investigate whether the contribution of the vasodilator nitric oxide (NO) and/or the vasoconstrictor endothelin (ET) to regulation of right ventricular coronary blood flow (RV‐CBF) is impaired in swine with PH.

Methods

PH was induced by banding of the confluent of the inferior pulmonary vein in juvenile swine (N=5), while 5 healthy swine served as controls. Swine were chronically instrumented to measure pressure in the RV, pulmonary artery, left atrium and aorta, and right coronary blood flow (RV‐CBF) rest and during graded treadmill exercise. Exercise was performed in the presence and absence of eNOS‐inhibition or ET‐receptor blockade 12 weeks after pulmonary vein banding (PVB).

Results

PVB resulted in PH as evidenced by an increase in pulmonary artery pressure (PVB: 39±3 mmHg, CON: 21±1 mmHg, both P<0.05) that was accompanied by RV hypertrophy (Fulton index PVB: 0.58±0.02, CON: 0.44±0.02, P<0.05). The relation between myocardial oxygen supply (RV‐CBF*arterial oxygen content) and myocardial work (arterial elastance, pulmonary artery pressure/SV) rotated downward in PVB. However, despite increased plasma ET‐levels (PVB:8.8±0.5 pg/ml CON: 6.7±0.5 pg/ml, P<0.05), ET‐receptor blockade did not alter the relation between myocardial oxygen delivery and myocardial work. Conversely, eNOS‐inhibition resulted in a downward rotation of this relation that was more pronounced in PVB as compared to CON.

Conclusion

PH leads to a mismatch between myocardial oxygen supply and demand. This mismatch is not influenced by ET‐receptor blockade and is aggravated by eNOS‐inhibition, suggesting that NO plays an important role in maintaining RV‐perfusion particularly during exercise.
Autophagy Impairment in Aging Motor Neurons
Authors: Pareja-Cajiao Miguel , Gransee Heather M. , Stowe Jessica M. , Rana Sabhya , Sieck Gary C. , Mantilla Carlos B.
Published: April 2020

Neuromuscular dysfunction is common in old age. A hallmark of aging is the aggregation of damaged cytoplasmic structures, especially in post‐mitotic cells like motor neurons. Autophagy flux may be impaired as a result of deficits in the formation of double membrane autophagosomes, the fusion of autophagosomes with lysosomes, or lysosome‐based degradation. Our overarching hypothesis is that autophagy contributes to neuromuscular dysfunction in old age, and that this effect occurs in motor neurons. We evaluated changes in expression levels of core autophagy proteins necessary for the initiation (Beclin1) and elongation (LC3‐I, LC3‐II, ATG7 and ATG5‐ATG12 complex) phases of autophagsome formation, as well as lysosome degradation (assessed by p62 clearance), in adult male and female C57BL/6 mice at 6, 18, and 24 months of age (reflecting 100%, 90% and 75% survival, respectively). Protein expression was analyzed by Western blotting in the cervical spinal cord segments C2–C6. Expression of p62 was reduced in the cervical spinal cord of adult mice at 18 months compared to 24 months, suggesting increased clearance and autophagy flux at 18 months of age. There was no evidence of an effect of age on the expression of the other autophagy markers. In separate animals, immunohistochemical (IHC) analyses of the C2‐C6 segments was used to determine the relative expression of LC3 and p62 in gray and white matter regions. Across all age groups, LC3 and p62 immunoreactivity was evident in the gray matter with minimal expression in the white matter. The ratio of both LC3 and p62 in motor neurons vs. gray matter was compared using a mixed linear model with animal as a random effect. No effect of sex was evident. Increased expression of both LC3 and p62 was evident in choline acetyl transferase (ChAT)‐positive motor neurons (~2–3 fold) compared to the remainder of the gray matter. There was evidence of a difference in relative LC3 and p62 expression in motor neurons across age (F349,2 =12.5, p<0.001), with a significant increase by 24 months of age (p<0.005). Taken together, these findings suggest that 1) there is an aging effect on autophagy markers in the cervical spinal cord of mice; 2) an increase in autophagy by 18 months of age reflects an increase in autophagosome clearance (reduced p62 expression) not restricted to motor neurons; and, 3) increased motor neuron expression of both LC3 and p62 by 24 months of age reflect autophagsome accumulation and autophagy impairment in motor neurons. These findings are also is in accordance with previous reports suggesting a period of vulnerability between 18 and 24 months of age in mice, where autophagy may play a role in the development of age‐related neuromuscular dysfunction.

Support or Funding Information

Supported by NIH R01 AG057052.
K2P Channel Inhibition Enhances Allergen‐Induced ATP Release by a Mechanism Involving PKC Activation in Human Bronchial Epithelial Cells
Authors: Srisomboon Yotesawee , Maniak Peter J. , Kita Hirohito , O'Grady Scott M.
Published: April 2020

Allergens derived from the common fungus Alternaria alternata induce oxidative stress in human airway epithelial (HBE) cells that leads to the regulated release of ATP and subsequent activation of P2X7 receptors that facilitate Ca2+ uptake into the cell. Sustained increases in [Ca2+]i are required for nuclear processing and secretion of IL‐33, which initiates allergic inflammation by activating ST2 receptors on immune cells to induce Th2 cytokine secretion. Pretreatment of HBE cells with the K2P channel blockers bupivacaine or quinidine produced a significant increase in ATP release following Alternaria stimulation. This response was blocked by inhibiting ATP loading into membrane vesicles by treatment with 200 nM bafilomycin A1 before allergen exposure. Similarly, pretreatment with the PKC inhibitor Go6976 also blocked approximately 50% of Alternaria‐induced ATP release and completely abolished the increase in ATP release produced by pretreatment with K2P channel blockers. The effects of bupivacaine and quinidine were comparable to the effect of phorbol 12‐myristate 13‐acetate (PMA, 2 mM), a well‐characterized PKC activating compound prior to Alternaria stimulation. These results support the conclusion that K2P channel inhibition is associated with PKC activation, which functions to potentiate the effects of Alternaria allergens on vesicular ATP release. We propose that enhanced PKC activity is dependent on membrane depolarization associated with inhibition of constitutive, K2P channel mediated K+ efflux from these cells.

Support or Funding Information

This study was supported by grants from the NIH (R01‐AI128729), awarded to HK and SMO and from the Minnesota Agriculture Experiment Station (AES‐0016097) awarded to SMO.
Dynamic formation of the malate dehydrogenase/citrate synthase multienzyme complex
Authors: Obata Toshihiro , Krassovskaya Inga , Horová Magdaléna
Published: April 2020

The enzymes of Krebs tricarboxylic acid (TCA) cycle form multi‐enzyme complexes mediating metabolite (substrate) channeling. As metabolite channeling can enhance the pathway reactions, the multi‐enzyme complexes are postulated to regulate metabolic flux by dynamic association and dissociation. Especially, the interaction between malate dehydrogenase (MDH) and citrate synthase (CS) is expected to have significant impact on the TCA cycle flux since the forward reaction of MDH to produce oxaloacetate from malate is thermodynamically infeasible with physiological metabolite concentrations. However, the dynamics of this multi‐enzyme complex and its relationship with metabolic pathway fluxes have not been demonstrated in vivo. To this end, plate reader‐based split‐luciferase assay was developed to monitor the dynamics of the protein‐protein interaction in living yeast cells and was applied to observe the dynamics of MDH/CS multi‐enzyme complex during the transition of metabolic status. The sequences coding N‐ and C‐ terminus of highly sensitive NanoLUC luciferase were introduced into the yeast genome by homologous recombination to fuse these peptides to the C‐terminus of CS (CIT1) and MDH (MDH1) enzymes, respectively. The resulted yeast strain showed decent fluorescent signals while wild‐type cells showed no background signal. The substrate concentration and cell concentration in the culture were optimized to obtain constant signals for 120 min of measurement. Using this system, the interaction of MDH1/CIT1 complex was monitored following the changes of growth condition which are expected to induce the alteration in the TCA cycle metabolic flux. In the preliminary results, we observed the alteration of luminescence signal in some conditions which are known to modulate the TCA cycle flux. We are currently testing various conditions to analyze the relationship between the dynamics of the multi‐enzyme complex and metabolic fluxes in the TCA cycle and adjacent pathways. We are also trying to identify the factors affecting the interaction of the enzymes.

Support or Funding Information

This study is supported by National Science Foundation, Faculty Early Career Development Program 1845451 to T.O.
Therapeutic anti‐P2X4 receptor scFv antibody for chronic neuropathic pain
Authors: Kunamneni Adinarayana , Montera Marena , Suri Nikita , Westlund Karin N , Alles Sascha R , Pappu Suguna , Durvasula Ravi
Published: April 2020

The P2X purinoceptor 4 (P2X4) plays a crucial role in signaling leading to chronic inflammatory and neuropathic pains, thus, the P2X4 receptor provides a new potential therapeutic target for their treatment. We have generated a panel of mouse single‐chain Fv‐antibodies (scFvs) recognizing extracellular peptide of P2X4 using cell‐free ribosome display, for use to pain‐signalling neurons. A mouse scFv library was generated, and ribosome display was applied for anti‐P2X4 scFv recombinant antibody selection. After three rounds of bio‐panning, a panel of recombinant antibodies was isolated followed by ELISA, cross reactivity analysis, western blotting and immunofluorescence staining. Anti‐P2X4 scFv clones with high specificity and affinity were successfully selected. Indirect ELISA assay revealed differential P2X4 peptide binding capability and specificity for three scFvs, with no reaction to the negative control anti‐Zika scFv 7‐2. The panning process was efficient in selecting clones of high affinity, as was evident from affinity differences of the scFvs: scFv95 and scFv12 had the highest and second highest affinities, respectively, while scFv103 had lower affinity. The highest affinity P2X4 receptor scFvs (12 and 95) were selected for in vivo behavior testing in the Foramen Rotundum Inflammatory Constriction Trigeminal Infraorbital Nerve injury (FRICT‐ION) Model. Single dose intraperitoneal injection (4 mg/kg, 100 μl) reversed mechanical allodynia and anxiety in mice with long standing neuropathic pain (8–10 weeks). Reversal to naïve baseline within one week was maintained for more than 10 weeks post‐treatment at experiment end. Continued reversal was maintained at baseline for >20 weeks in a second cohort. The Western blot of the medullary brainstem provides evidence of brain penetration of both these scFv antibodies that reduce hypersensitivity and anxiety behaviors. We also performed whole‐cell patch clamp recordings from dissociated trigeminal ganglia neurons of nerve‐injured vs naïve mice and determined the effect of P2X4 receptor scFvs on neuronal excitability. These data provide support for pursuit of P2X4 receptor scFvs as translational therapy for pain relief.

Support or Funding Information

1. GRANT FUNDING NIH NIDCR R21 DE028096 2. Start‐up Funds to Dr. Durvasula 3. Startup Funds to Dr. Westlund
Yoga versus Aerobic Exercise: An Alternative Intervention for Neuropathic Pain Among African Americans with Diabetes
Authors: Swift Janae , Bradley William Christopher , Hubbert Douglas , Edmondson Deborah Ann
Published: April 2020

In today’s world, the prevalence of African Americans (AA) with diabetes continues to grow as it is becoming an epidemic. As of 2012, AA adults are two times more likely to be diagnosed with diabetes. Diabetes is most closely associated with being overweight/obese and around 50%–75% of AA that are older than 20 years of age fall into this category. With this disease comes many challenges not only mentally but physically that can decrease the quality of life in individuals that live with this condition. Often times pharmacological management of diabetes is introduced to patients as a means of controlling symptoms, such as neuropathic pain, without discussion of alternative methods. Commonly, side effects of these medicines introduce new problems that are then managed with more medications; this contributes to the rising opioid crisis in the United States. Therefore, alternative methods such as yoga should be employed. Current evidence suggests yoga has many beneficial effects to individuals with diabetes including decreased neuropathic pain. Although this evidence exists for the majority of the population in the United States, few studies have been conducted on AAs using yoga as a means to decrease neuropathic pain. The purpose of this study was to conduct a systematic review regarding neuropathic pain in the AA population and how yoga may be a better alternative to treating this pain versus aerobic exercise only. Using a variety of databases such as PubMed, Google Scholar, and EBSCO Host, studies were reviewed and ranked according to the Oxford Center for Evidence‐Based Medicine. Distal symmetric polyneuropathy (DSPN) is a common neuropathy among individuals with diabetes and this neuropathy can cause pain in the upper and lower distal extremities due to damage of small and large myelinated nerve fibers. While aerobic exercise is commonly used and has shown positive results in helping decrease neuropathic pain as well as promote regrowth of nerve fibers, research shows that yoga can be used as an alternative method for reducing blood glucose levels as well as improvements in nerve conduction velocities leading to a reduction in neuropathic pain. In conclusion, our research provided evidence as to why AA populations should be encouraged to participate in yoga as an effective means of providing relief of neuropathic pain.
Carvedilol protection involves the mitochondrial deacetylase Sirt3 in mitigating Doxorubicin‐induced Cardiomyocyte Injury
Authors: Uche Nnamdi , Dai Qiang , Lai Shuping , Benjamin Ivor
Published: April 2020

Although anthracyclines such as doxorubicin are highly effective chemotherapeutic agents and widely used for various malignancies, their use in the clinic is accompanied by major cardiovascular events. Cardiotoxicity is a well‐recognized and unpredictable consequence of anthracycline therapy that may be permanent and progressive, leading to heart failure in some patients while severely impacting quality of life. The pathophysiology of anthracycline‐induced cardiotoxicity has not been fully elucidated but the most widely accepted hypothesis is the increased reactive oxygen species (ROS) generated by anthracyclines. Data from our lab as well as other recent studies support an alternative model involving mitochondrial dysfunction perpetuated by anthracycline‐induced dysregulation of mitochondrial protein modifications. The mitochondrial deacetylase Sirt3 has been suggested to play a role in cardio‐protection. Carvedilol, a beta‐blocker, had previously been shown to mitigate anthracycline‐induced mitochondrial dysfunction and although the mechanism is unknown, data from our lab suggest involvement with Sirt3. To explore this further, rat embryonic cardiomyocytes (H9c2 cells) devoid of Sirt3 expression were either treated with doxorubicin or pretreated with carvedilol followed by doxorubicin treatment before downstream assays were performed. Protein expression and/or activity assays were performed to assess proteins involved in apoptosis and mitochondrial function and stability including caspase3 and the Sirt3 target Mn/SOD. MitoSOX assay was done to assess mitochondrial ROS generation. Our results suggest that carvedilol prevents or reverses the anthracycline‐induced inhibition of Sirt3, a protein responsible for activating mitochondrial proteins involved with metabolic processes and maintenance of ROS. Further and ongoing studies in the lab will access the role of Sirt3 in cardio‐protection in vivo.

Support or Funding Information

NIH CTSA UL1TR001436
Relative Shedding of Glycosaminoglycans from the Endothelial Glycocalyx During Inflammation
Authors: Lipowsky Herbert H.
Published: April 2020

The endothelial (EC) surface layer (glycocalyx) has been shown to act as a barrier to transvascular exchange of solutes, and adhesion of leukocytes (WBCs) during the inflammatory process. It is a labile structure whose components are readily shed by the action of proteases and endoglycosidases. Evidence to date suggests that during the inflammatory process, activation of matrix metalloproteases on the EC surface may cleave the principal proteoglycans (syndecans and glypicans), and EC derived lyases may result in shedding of the associated glycosaminoglycans (GAGs) heparan sulfate (HS), chondroitin sulfate (CS) and hyaluronan (HA). Shedding of these components of the glycocalyx serves to increase leukocyte (WBC) access to adhesion receptors on the EC membrane. The objective of the present study was to elucidate the relative role of HS (the dominant GAG) and HA as the major barrier to WBC‐EC adhesion.

Shedding of glycans from the EC in post‐capillary venules of the mesentery (rat) in response to topical application of the chemoattractant f‐met‐leu‐phe (fMLP, 10−6 M) was observed by intravital microscopy. The presence and loss of HS, CS and HA was assessed by labeling all components with fluorescently labelled lectin (BS‐1) or HS antibodies, and HA with fluorescently labelled hyaluronan binding protein (HBP). During exposure to fMLP, GAG concentration exhibited an exponential decay. Following a 30 min period about 50% of HBP was lost in contrast to a previously shown loss of 20% of lectin labelled GAGs, and 25% loss of Mab labelled HS. The exponential time‐constant for HBP shedding was 5.8 min, which was one‐third the 14.3 min for BS‐1 labelled GAGs.

The relatively rapid shedding of HA compared to HS was consistent with the hypothesis that HA may form the dominant barrier to WBC‐EC adhesion. In situ observations (in rat mesentery) of fMLP induced transient adhesion of circulating beads coated with antibody to ICAM‐1 (1A29) revealed a time constant for adhesion of 10.9 min, which was less than that for BS‐1 labelled GAGs. Prior observations by Fan et al., (Biorheology, 2019, DOI: 10.3233/BIR‐180204) using stochastic optical reconstruction microscopy suggest that HA lies closer to and parallel to the endothelial membrane, compared to HS which lies further away and normal to the EC surface. Thus, a compact layer of HA near the EC membrane may surround WBC adhesion receptors that are much shorter in length than the total thickness of the glycocalyx. Its more rapid shedding, compared to other GAGs, may be the major factor affecting exposure of WBC adhesion receptors on the EC.

Support or Funding Information

Supported in part by NIH R01 HL39286