bluuebunny/crossref_metadata_2025 huggingface dataset:
Performing in situ hybridization and quantitative PCR to assess eya2 localization and expression in zebrafish
Authors: Larson Nicole , Baxter Gloria , Tessier Violet , O'Brien Jenean
Published: May 2022

Rhabdomyosarcoma (RMS) is the most common soft‐tissue sarcoma found in children (Skapek, 2019) and is classified as a skeletal muscle cancer (Hsu, 2021). A common transcription factor that increases cell proliferation (how cells grow and divide) in RMS is called SIX1 (Hsu, 2021). The SIX1 protein has coregulators, such as EYA2, that regulate the increase or decrease of SIX1’s function (Sousounis, 2020). Because the coregulator EYA2 can play a critical role in the increase of SIX1's function (Blevins, 2015), we hypothesize that an increase in EYA2 will lead to uncontrolled cellular proliferation. This uncontrolled cellular proliferation could lead to the development of tumors, specifically RMS. In order to understand EYA2’s role in tumor growth and cancer, it is important to study the coregulator under normal homeostatic conditions. Specifically, understanding how muscle tissue normally utilizes eya2 during embryonic development, when cells are proliferating, can provide insight into eya2’s potential roles in muscle tumor development. Zebrafish are ideal research specimens for this research because of their easy to visualize muscular system. A technique used to visualize mRNA expression and localization in zebrafish tissues, is in situ hybridization (ISH) (Chu, 2019). Our first objective was to confirm that we can perform ISH in our laboratory. We used ISH to observe that Eya2 mRNA was generally located in the head and upper torso regions of the zebrafish at both 24 hours post fertilization (hpf) and 48 hpf, which supports previously published results (Thisse, 2004; Thisse, 2004). Additionally, we observed an increase in eya2 mRNA levels by ISH from 24 hpf to 48 hpf. We utilized quantitative PCR (qPCR) (Heid, 1996) to quantify these changes in eya2 mRNA levels. Our qPCR results indicate there was an increase in the coregulator eya2 mRNA expression from 24 hpf to 48 hpf. Overall, both research techniques, ISH and qPCR, demonstrated an increase in eya2 transcript levels from 24 hpf to 48 hpf, suggesting that this time frame would be appropriate for future studies of eya2 function in zebrafish development.
Cardiac and pulmonary expression of renin, prorenin, mast cells and macrophages in a rat model of fat embolism syndrome
Authors: Siddiqui Gulnaz , Hamidpour Soheila , Mateescu Valerica , Monaghan Nichols Paula , Wacker Michael , Poisner Alan , Salzman Gary , Molteni Agostino
Published: May 2022

Fat embolism induced in rats by intravenous injection of triolein(T) causes pulmonary inflammation, fibrosis, and vasculitis after 48 hours ort 10 weeks. Administration of Aliskiren, Captopril and Losartan drugs, which interfere with the renin angiotensin aldosterone system, ameliorates the pulmonary damage. Macrophages and mast cell number decreased with drugs administration. Hearts do not exhibit inflammation or vasculitis although significant emboli are detected at 48 hours. Pulmonary expression of renin, prorenin(R/P) positive cells are also increased after T injection. This study investigates whether the presence of R/P cells correlates with macrophages and mast cell invasion in the hearts and lungs.

Methodology

24 Sprague Dawley rats were divided into four groups and treated with 0.2 ml of T or Saline. Lungs and hearts from 3 animals from each group that were sacrificed 48 hours or 10 weeks, fixed in formalin and stained with Hematoxylin‐Eosin, Trichrome or immunostained with antibodies to CD117, CD68 to identify mast cells and macrophages. Sections of the heart and the lung were evaluated at 400X magnification. Cells were counted, histopathology examined and statistical significance evaluated using GraphPad software.

Results

While the lungs showed the previously reported, histopathological damage caused by the inflammation, vasculitis, fibrosis and cell necrosis, the hearts did not show such damage, thus confirming previous observations.

Contrary to the lungs where the number of CD117, CD68 and R/P positive cells was significantly increased at both interval times, no difference in CD117, CD68, R/P positive cell numbers were observed in the heart. In the lungs, two distinct types of macrophages were identified, one large and the second one of normal size. Such histopathological changes were not observed in the hearts. Hearts exhibited distinct locations of CD117 and CD68 positive cells, with multiple cells located primarily near the myocardium and few in the adventitia of the coronary arteries, in contrast to lungs where the cells were mostly located around the arteries.

Conclusion

The study confirms the previously reported histopathological difference of the T induced damage in the lungs compared to that of the hearts in this model of fat embolization. This finding highlights the variation in cellular response of diverse organs to fat embolism. This difference is not only related to the number of the inflammatory cells but also to their location in different organs as evidenced by the difference in the distribution and location of R/P cells in hearts and lungs. Future studies will investigate the underlying mechanisms that lead to pulmonary susceptibility while preserving cardiac integrity in response to a FE induced inflammatory response in order to better design a therapeutic intervention for the fat embolism syndrome.
Using De Novo Designed Protein Switches in C. elegans
Authors: Berg Adam P. , Kaefer Joseph H. , Wakefield Connor M. , Faber Jakob L. , Novak Walter R. , Sorensen Erika B.
Published: May 2022

The ‘Latching Orthogonal Cage/Key pRoteins’ (LOCKR) system is a de novo designed protein Switch and Key. The Switch functions to cage a functional protein sequence, such as a degron, in the off or “locked” state. Upon Key addition, the Switch is turned on or “unlocked,” which exposes the degron, simultaneously degrading the Switch and any proteins fused to the Switch via the proteasome. Key expression can be tailored for tissue‐specific expression, which gives LOCKR exquisite spatial protein control. LOCKR provides novel post‐translational control over a protein of interest but has not yet been applied to a multicellular organism. We are developing the LOCKR technology for conditional protein depletion in the model organism C. elegans. Although genetic RNAi and similar gene editing techniques benefit from ease of delivery and can target essentially any gene, they have various limitations, including 1) slow protein turnover can prohibit use of RNAi; 2) genetic perturbation of essential genes via genetic deletion prevents characterization of later functions. The degronLOCKR system requires two parts: 1) a gene fused to the ‘degronSwitch’ and 2) an inducer peptide (the Key). The Switch can be fused to any gene of interest using CRISPR‐Cas9 gene editing. We fused degronSwitch to dhc‐1 (dynein heavy chain), an essential cytoplasmic motor protein along with mScarlet and a 3xFLAG tag to aid in protein quantitation via microscopic and Western analysis. We also cloned a negative control, which lacks degronSwitch, to analyze the effect of inserting the Switch in vivo. To complement the degronSwitch, we designed six tissue‐specific Keys for Mos‐Single Copy Insertion (MosSCI), which allows for spatial control of degronSwitch protein degradation. We predict that ubiquitous expression of Key with the degronSwitch fused to dhc‐1 will result in embryonic lethality, whereas germline‐specific Key will permit viability but result in meiotic defects. These phenotypes are easily identifiable and serve as effective proof of concept. Future work includes expanding the LOCKR system to incorporate temporal control of the degronSwitch fusion protein by toggling the Key on and off using heat‐shock promoters and RNAi against the Key. This work establishes a novel and tissue‐specific method for regulating protein function in an animal model.
TNFα Reduces the Maximum Respiratory Capacity of Mitochondria in Human Airway Smooth Muscle Cells
Authors: Yap Jane Q. , Mahadev Bhat Sanjana , Ramirez Ramirez Oscar A. , Delmotte Philippe , Sieck Gary C.
Published: May 2022

Previously, we reported that airway inflammation mediated by pro‐inflammatory cytokines such as TNFα induces hypercontractility and increased ATP consumption in human airway smooth muscle (hASM). We also found that TNFα induces an increase in maximum O2 consumption rate as well as mitochondrial biogenesis and an increase in mitochondrial volume density. However, when normalized to mitochondrial volume, maximum O2 consumption rate per mitochondrion is reduced in hASM after TNFα exposure. Unfortunately, standard respirometry (e.g., Seahorse) measures only the averaged maximum O2 consumption rate of a large number of cells. In the present study, we used a quantitative histochemical technique to determine the maximum velocity of the succinate dehydrogenase reaction (SDHmax) together with mitochondrial volume density in individual hASM cells. SDH is a key enzyme in the tricarboxylic acid (TCA) cycle as well as complex II in the electron transport chain (ETC), and SDHmax reflects the maximum respiratory capacity of individual mitochondrion. We hypothesized that TNFα decreases SDHmax per mitochondrion in hASM cells. hASM cells were dissociated from bronchial biopsies obtained during thoracic surgeries in patients with no history of asthma or chronic obstructive pulmonary disease. The hASM cells were treated with TNFα (20 ng/ml) or vehicle (DMSO) for 24 h, and SDHmax was measured using a solution containing 80 mM succinate (maximum substrate concentration) and 1.5 mM nitro blue tetrazolium (NBT) as the reaction indicator. As the SDH reaction proceeded, the hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using an oil‐immersion ×60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. The change in optical density (OD), reflecting the accumulation of NBT diformazan reaction product in the hASM cell, was measured every 15 s over a 10 min period. Based on the Beer‐Lambert equation, the slope of the change in OD was used to determine SDHmax expressed as mM fumarate/L tissue/min. To determine mitochondrial volume density, the same hASM cells were also loaded with MitoTracker Green (500 nM) and imaged in 3D. We found that mitochondria were more fragmented after TNFα exposure although mitochondrial volume density increased. When SDHmax was normalized to mitochondrial volume, we found that SDHmax per mitochondrion decreased in hASM cells, consistent with the reduction in maximum O2 consumption per mitochondrion that we previously observed. Thus, the increased energetic demand induced by inflammation (TNFα) are met by mitochondrial fragmentation leading to biogenesis and an increase in mitochondrial volume density rather than an increase in mitochondrial respiratory capacity.
Kisspeptin Reduces Airway Hyperreactivity and Remodeling in Asthmatic Ovariectomized Mice
Authors: Borkar Niyati A. , Ambhore Nilesh S. , Balraj Premanand , Sathish Venkatachalem
Published: May 2022

Rationale

Epidemiological data show changes in asthma occurrence with age and sex. After puberty, increased severity in women shows sex‐skewed occurrence of asthma in females vs. males. This implies the importance of female sex‐steroids, particularly estrogen in asthma pathophysiology. Greater asthma in women may represent detrimental mechanisms upstream to or independent of estrogen. Moreover, data from CNS studies show kisspeptin (Kp) signaling is modulated by estrogen. Our recent in vitro studies show Kp via its receptor, KISS1R regulates airway smooth muscle (ASM) cell proliferation. However, the relevance of endogenous estrogen on Kp/KISS1R signaling and their effect on regulating airway hyperresponsiveness (AHR) and remodeling is not yet explored. Accordingly, in this present study, we utilized intact female vs. ovariectomized (OVX) mice to isolate estrogen influence from the Kp effect in regulating AHR and remodeling in a murine model of allergic asthma.

Methods

Wild Type C57BL/6J intact female and OVX mice were procured from Jackson Laboratory, USA, and challenged intranasally with a mixed‐allergen (MA) for 28 days. On alternate days, animals from the treatment group received intranasal Kp‐10 (1.2mg/kg), vehicle group received PBS alone. On day 29, mice were anesthetized and subjected to SCIREQ flexiVent to measure lung mechanics like airway resistance (Rrs), elastance (Ers), compliance (Crs), tissue damping (G), and tissue‐elasticity (H). Following this, bronchoalveolar lavage (BAL) was performed for differential leukocyte count (DLC) and lung tissues were processed for histology using hematoxylin and eosin (H&E) and trichrome staining. Additionally, laser‐capture microdissection (LCM) assisted qRT‐PCR analysis in ASM cells was performed for genes relevant to airway remodeling.

Results

MA‐challenged intact female and OVX mice showed a significant increase in Rrs, Ers, G, H, and reduced Crs compared to PBS, more pronounced effects were observed in intact females. Kp‐10 treatment significantly reversed the MA‐induced changes with a decrease in Rrs, Ers, G, H, and increased Crs. Interestingly, in OVX mice we observed significant effects of Kp‐10 to improve the lung mechanics compared to intact females. Notably, Kp‐10 alone exposed group did not show any changes in lung mechanics compared to PBS. DLC in BAL showed Kp‐10 treatment significantly reduced MA‐induced inflammatory cell infiltration. Futhermore, histology studies showed Kp‐10 treatment significantly reduced MA‐induced smooth muscle mass and collagen deposition in airways.

Conclusion

Overall, this study shows the modulatory role of estrogen in regulating kisspeptin signaling, thereby AHR and remodeling. Thus, providing the relevance to higher asthma incidence and severity in women compared to men.
Low Glucose Induced Alzheimer’s Disease Phenotype in Induced Pluripotent Stem Cell‐Derived Neurons is Due to Decreased O‐GlcNAcylation
Authors: Huang Chia‐Wei , Hart Gerald
Published: May 2022

Sporadic Alzheimer’s disease (sAD) is the most common type of neurodegenerative disease. Recent studies show that detectable impairment of brain glucose metabolism occurs years before onset of AD symptoms, and the dysregulated O‐GlcNAc levels likely arisen from impaired glucose metabolism correlates with AD pathogenesis. So far, the mechanism of sAD and the role of OGN in AD pathology remained largely unknown due to a lack of human sAD model. We have established a human sAD model in which the pathological features are reproduced by glucose deficiency that better represents sAD as a metabolic disease. We generated human cortical neurons from human induced pluripotent stem cell and treated mature neuron with glucose reduction media to study the effect of low glucose on the degenerative status of neuron. Fluoro JADE C staining and cell viability assays reveals low glucose treatment at 2mM leads to dramatic increases of degeneration cell on day 3 and 5 of treatment and decreased cell viability on day 7. Interestingly, western blotting and immunofluorescent staining results demonstrate that long‐term low glucose treatment induces two major AD features in cortical neuron, including accumulation of abnormal hyperphosphorylated tau and increasing amyloid beta production. In addition, glucose deficiency also causes decreased neurite coverage, synapse loss, and neuron network activity disruption detected by immunofluorescent staining and multi‐electrode array electrophysiological analyses. Furthermore, we find that O‐GlcNAc levels are significantly reduced soon after low glucose treatment and last till the end of experiment. Artificially increasing O‐GlcNAc level by thiamet‐G (TMG) in low glucose treated neurons, rescues low glucose‐induced AD phenotypes. Moreover, our data show that O‐GlcNAc dysregulation results in mitochondrial dysfunction, which occurs before any other degenerative phenotype appeared, and may be one of the underlying mechanisms of sAD onset and pathogenesis. Taken together, we established a human sAD model that mimics the main features of AD pathogenesis, which agrees with clinical observations of sAD patients. Therefore, this platform can serve as a tool to better understand molecular processes involved in sAD. Our results also suggest that dysregulated O‐GlcNAc levels by glucose deficiency is involved in the onset and progression of sAD.
Endothelial Function Does Not Show Signs of Impairment in Young Athletes At Two Months Post‐Covid‐19 Infection
Authors: Gao Zhaohui , Fernandez‐Mendoza Julio , Stavres Jonathon , Brandt Kristen , Blaha Cheryl , Cauffman Aimee , Arnold Amy C. , Luck J. Carter , Hamaoka Takuto , Sinoway Lawrence , Cui Jian
Published: May 2022

It has been almost two years since the Covid‐19 pandemic hit, with over 5 million deaths globally. It is known that endothelial activation and dysfunction play a crucial role in Covid‐19 pathogenesis. Evidence of widespread endothelial injury is found in advanced cases of Covid‐19, especially in people with cardiovascular comorbidities. However, the effects of Covid‐19 on endothelial function in unique cohorts such as young athletes have been largely unexplored. This study evaluated endothelium function by noninvasive flow‐mediated dilation (FMD) in a sample of 14 college athletes (10M/4F, 18‐23 yrs, BMI, 23.8 ± 1.6 kg/m2) after testing positive for Covid‐19. All athletes had Covid‐19 with mild or no symptoms and did not require hospitalization. The study was performed 24 ± 10 days (13‐44 days) after having Covid‐19 positive tests. We also involved 10 BMI similar healthy young subjects as controls (7M/3F, 20‐25 yrs, BMI, 24.2±2.0 kg/m2). Baseline brachial artery diameters and the largest diameters after post cuff release were measured to calculate FMD. Brachial blood flow mean velocities and diameters were also recorded within 20 seconds after cuff release for calculating the shear rate (8 * mean velocity/ diameter). The baseline brachial artery diameter was significantly larger in the athlete group versus the control group (4.32±0.63 mm vs. 3.54±0.42 mm, p=0.02). The FMD was significantly lower in the athlete group than in the control group (6.49±2.05% vs. 11.05±4.08%, p=0.02). This finding is consistent with the previous reports that FMD was significantly lower in healthy athletes compared with controls. However, FMD adjusted for the ratio of flow‐mediated dilation and shear rate did not show a statistical difference between groups (0.058±0.028 %/s‐1 vs. 0.076±0.056 %/s‐1, p=0.33), even after evaluating baseline diameter as a covariate. Despite the need for extended acute and chronic observations and a healthy athlete control group, our findings suggest endothelial resilience in response to Covid‐19 infection in young athletes, which may explain their mild or asymptomatic status.
Impaired Sympathetic Neural Recruitment During Skeletal Muscle Metaboreflex Activation in Women with Posttraumatic Stress Disorder
Authors: D'Souza Andrew W. , Yoo Jeung‐Ki , Takeda Ryosuke , Manabe Kazumasa , Badrov Mark B. , Parker Rosemary S. , Anderson Elizabeth H. , Wiblin Jessica I. , North Carol S. , Suris Alina , Shoemaker J. K. , Fu Qi
Published: May 2022

Posttraumatic stress disorder (PTSD) is a debilitating psychiatric disorder that is more prevalent in women than men and has been linked to an elevated risk of developing cardiovascular disease. Although the physiological mechanisms are unclear, it is thought that exaggerated neuro‐cardiovascular reactivity is partially responsible. Indeed, women with PTSD demonstrate exaggerated muscle sympathetic nerve activity (MSNA) responses at the onset of exercise. However, it is unknown whether PTSD modifies the underlying sympathetic action potentials (AP) coding patterns that comprise the integrated MSNA signal during fatiguing exercise in women. We therefore hypothesized that women with PTSD will exhibit exaggerated MSNA and AP recruitment compared to women without PTSD during fatiguing handgrip exercise. MSNA and AP discharge patterns (via microneurography and a continuous wavelet transform) were assessed in eleven women with PTSD (43±11 [SD] years) and thirteen women without PTSD (Controls; 40±8 years) during isometric handgrip exercise (IHG) to fatigue at 40% of maximal voluntary contraction (MVC) force, followed by 2 minutes of post‐exercise circulatory occlusion (PECO), and three minutes of recovery. The IHG time was divided equally into five stages. IHG duration (P=0.867) and MVC (P=0.226) were similar between both groups. PTSD did not modify the increase in MSNA and AP frequency or incidence during IHG and PECO (all group‐by‐stage interactions: P=0.294‐0.380). Conversely, throughout IHG and PECO, AP content per burst was greater in the Controls compared to women with PTSD (main effect of group: P=0.026). Furthermore, relative to Controls, women with PTSD were unable to increase the number of AP clusters per burst during the first (Controls: ∆1.3±1.2 vs. PTSD: ∆‐0.2±0.8 clusters/burst; P=0.016) and last minute (Controls: ∆1.2±1.1 vs. PTSD: ∆‐0.1±0.8 clusters/burst; P=0.022) of PECO, and recruited fewer subpopulations of previously silent axons during the first (Controls: ∆5±4 vs. PTSD: ∆1±2 clusters; P=0.020) and last minute (Controls: ∆4±2 vs. PTSD: ∆1±2 clusters; P=0.021) of PECO. Lastly, although an interaction effect was observed for the AP cluster size‐latency relationship during IHG and PECO (P=0.042), post‐hoc analyses revealed that PTSD did not modify this relationship (all P=0.658‐0.999). These data indicate that the central features of sympathetic outflow are impaired in women with PTSD during skeletal muscle metaboreflex activation.
Efficacy Assessment and Mechanism Exploration of Haptoglobin Phenotyping and Peptide Supplementation Strategy for Restenosis
Authors: Wu Chieh‐Hsi , Pan Chun‐ Hsu , Chu Irene
Published: May 2022

Restenosis is a common complication after balloon angioplasty, and the major mechanism is associated with abnormal proliferation, migration and cellular inflammation of vascular smooth muscle cells. However, its therapeutic diagnosis and treatment as well as the fundamental molecular mechanisms have not been fully established. Different haptoglobin (Hp) phenotypes have been clinically confirmed to be related to the incidence of restenosis, and various Hp subunits have also been found to have a variety of cellular activities, which have the potential to inhibit restenosis. Previous studies have shown that patients with Hp 1‐1 phenotype have the least incidence of restenosis. Therefore, the main axis of this study is to evaluate the efficacies and safeties of the Hp subunits as protein drugs for preventing restenosis, as well as exploring their possible regulatory mechanisms. We have constructed the expression vectors of different Hp subunits to understand the regulatory activities of these subunits on critical signaling pathways. Rat carotid artery balloon‐injury model was used to evaluate the efficacies, additive effects, and possible toxicities of potential Hp subunit on preventing restenosis. The outcomes of this project will help us to understand the feasibility, efficacy and safety of the Hp subunit as protein drugs for preventing vascular restenosis.
Role of the Phosphate Transport Facilitator XPR1 in Bone Homeostasis and Phosphate‐Dependent FGF23 Secretion
Authors: Cabuzu Daniela , Ramakrishnan Suresh , Bonny Olivier
Published: May 2022

Background

Evidence point at Xenotropic and Polytropic retrovirus Receptor 1 (XPR1) as a protein involved in the cellular phosphate export pathway and phosphate homeostasis: systemic constitutive deletion of Xpr1 in mice is lethal and kidney‐specific deletion of Xpr1 induces hypophosphatemic rickets with hypercalciuria. XPR1 is expressed in the bone, but its function remains elusive. We hypothesized that XPR1 is crucial for bone homeostasis and that is involved in the sensing mechanism of phosphate in the bone.

Methods

We established an inducible whole body Xpr1 KO mouse model. The mice were placed in metabolic cages for urine and blood analysis, followed by femur uCT and histomorphometry. The Fgf23 mRNA and protein (c‐terminal and intact FGF23) levels were assessed from tibia and femur incubated ex vivo for 24h with increasing phosphate concentrations. Primary osteoblast cells were isolated from control and Xpr1 KO mouse calvaria.

Results

Conditional total body Xpr1KO mice are constantly decreasing their body weight upon Xpr1 deletion, and develop profound hypophosphatemia, compared to their control littermates. Histomorphometry analysis and uCT on femurs of Xpr1 KO mice show high trabecular bone density, despite low bone formation rate and low number of osteoclasts. Increased intact FGF23 secretion by tibia and femur was observed in control mice in response to increased phosphate concentration in the media. However, the bone samples from Xpr1KO mice are resistant to phosphate induced FGF23 secretion. No changes were observed for the c‐terminal FGF23 in response to increased phosphate concentration, for both the control and Xpr1 KO mice.

Osteoblasts were successfully prepared from control and Xpr1 KO mouse calvarie and Xpr1 expression was inactivated by tamoxifen treatment.

Conclusion

XPR1 is essential for phosphate homeostasis and FGF23 secretion upon phosphate exposure. Osteoblasts will now be used to further study phosphate dependent FGF23 secretion.
Human Fetal Brain Magnetic Resonance Imaging (MRI) Tells Future Emergence of Autism Spectrum Disorders
Authors: Ortug Alpen , Guo Yurui , Feldman Henry Arthur , Ou Yangming , Dieuveuil Harrison , Baumer Nicole Tara , Faja Susan Kaminski , Takahashi Emi
Published: May 2022

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder which can result from a range of observed symptoms pertaining to social, emotional, and cognitive processing difficulties especially regarding communication of their own feelings, opinions, and thoughts with others. Although a diagnosis received by 1 in 68 children in the United States, a cause for autism is still unknown. Both genetic and environmental factors during prenatal and postnatal development are believed to account for the emergence of ASD. It is known that early treatment improves language and cognitive abilities later. However, currently, the earliest diagnosis of ASD can be made only around 18 months of age. It is critical to be aware of an altered initial brain anatomy in ASD for better predicting what aspects of brain function will likely preferentially and precociously deteriorate, which would allow for potentially earlier/better prevention and treatment. We hypothesized that prospective ASD patients will show region‐specific abnormalities in developmental stages. We have used retrospective clinical brain MRI of the fetuses who were diagnosed as ASD in later age to obtain earliest MRI‐based regional volumetric biomarkers. Thirty‐nine fetuses (mean age 24.87 ± 5.05 gestational week [GW]) were included in this study (9 prospective ASD patients, 20 neurotypical controls, 6 non‐ASD controls with neurological comorbidities observed in the prospective ASD patients, and 4 non‐ASD controls with non‐neurological comorbidities observed in the prospective ASD patients. After preprocessing, the images were segmented automatically with an atlas‐based automated anatomical labeling method. A set of three‐factor ANOVA was performed for lobar and regional comparisons of the segmented brain regions. The insula/insular lobe showed statistically significantly larger volume in ASD than that in all three control groups in the lobar comparison. In the regional comparison, the ASD group had statistically significantly larger amygdala, hippocampal commissure, and insula compared to the non‐ASD controls with neurological and non‐neurological comorbidities. Our results suggest that an increased volume of the insula/insular lobe may be a strong prenatal MRI biomarker that could predict the emergence of ASD later in life. The larger amygdala and hippocampal commissure could be additional biomarkers for identifying such prospective ASD patients.
Effect of cellular stress on retinoid homeostasis in the small intestine
Authors: Zalesak‐Kravec Stephanie , Yu Jianshi , Pilli Nageswara , Kane Maureen A.
Published: May 2022

Vitamin A is an essential diet‐derived nutrient. Retinoids are compounds with Vitamin A activity and are essential for many biological processes, including proper cell function and tissue organization. The main active metabolite of Vitamin A is all‐trans retinoic acid (RA), which is critical for nuclear receptor mediated signaling for cell proliferation, differentiation, and apoptosis. Retinoid signaling and RA are essential for intestinal immune homeostasis. Intestinal epithelial cells (IEC) produce high levels of RA, which is crucial for RA‐dependent signaling, lineage commitment, and barrier function. Vitamin A deficiency can alter intestinal immune and IEC homeostasis.

There are many mechanisms in place to maintain retinoic acid homeostasis, including the expression of the main intracellular chaperone protein cellular retinol binding protein, type 1 (CRBP1). CRBP1 binds to retinol and retinal, protects them from non‐specific oxidation, and facilitates their delivery to the appropriate enzymes for RA biosynthesis. CRBP1 has been shown to be decreased in diseases that display dysfunctional proliferation and differentiation, including cancer, inflammatory disorders, fibrosis, and infection. Reduction of CRBP1 directly correlates with reduction in the active metabolite, RA, and restoration of CRBP1 expression has been shown to increase RA levels and positively impact RA‐dependent outcomes. Therefore, monitoring CRBP1 and retinoid metabolite levels in response to cellular stress will provide useful information about the effect of CRBP1 loss on retinoid signaling.

While previous studies have explored the effect of cellular stress on RA levels, the effect of stress on the entirety of the retinoid signaling pathway is not well understood. The objective of these studies is to systematically determine the effect of cellular stress on retinoid homeostasis.

We have established three models of cellular stress, including hypoxia, oxidative stress, and inflammation, in two human intestinal epithelial cells lines, HIEC‐6 and Caco‐2. To mimic hypoxia, cells are treated with 0‐100 μM CoCl2 for 24 hours. To mimic oxidative stress, cells are treated with 0‐100 μM H2O2 for 24 hours. To mimic inflammation, cells are treated with 0‐1 μg/mL lipopolysaccharide for 48 hours. These conditions were chosen because there was no effect on cell viability. CRBP1 and RA levels will be measured through targeted mass spectrometry methods developed by our lab. Additionally, expression levels of genes associated with retinoid metabolism will be measured. Our current data shows that RA levels decreased in response to hypoxia, oxidative stress, and inflammation in both HIEC‐6 and Caco‐2 cells.
The sialyltransferase ST6Gal1 is upregulated in a cytokine‐mediated manner during pancreatitis
Authors: Silva Austin D. , Marciel Michael , Bellis Susan L.
Published: May 2022

Pancreatitis is a life‐threatening medical disorder that occurs when abnormally activated pancreatic enzymes damage surrounding pancreatic acinar cells, leading to the release of several pro‐inflammatory cytokines. Subsequently, inflammation of the pancreas promotes the dedifferentiation of acinar cells towards a ductal‐like, progenitor phenotype, to heal from pancreatic injury, in a process known as acinar to ductal metaplasia (ADM). Recent studies by our group suggest that the sialyltransferase, ST6Gal1 promotes ADM. ST6Gal1 adds sialic acid to N‐glycosylated surface receptors and thereby regulates cell signaling and phenotype. In the current study, we report the novel finding that ST6Gal1 is upregulated in the pancreatic tissues of patients with chronic pancreatitis and mice with experimental pancreatitis, whereas ST6Gal1 expression is undetectable in normal acinar cells. These results are the first to show stark modulation of ST6Gal1 within pancreatic inflammation. While the upregulation of ST6Gal1 has been noted across multiple cancers, the mechanisms by which ST6Gal1 expression is modulated within an inflammatory milieu are not well understood. To evaluate inflammation‐induced ST6Gal1 upregulation, we injected mice with cerulein to evoke pancreatitis. We first examined levels of pro‐inflammatory cytokines through immunohistochemistry in cerulein‐treated mice and observed striking increases in the release of both IL‐6 and IL‐1β. Accordingly, we proceeded to treat multiple pancreatic acinar cell line models with IL‐6 and IL‐1β to assess their respective capability to upregulate ST6Gal1. These two cytokines stimulated increases in ST6Gal1 mRNA and protein in all cell models. It's known that there are three major isoforms of ST6Gal1, each driven by a different promoter; thus, we investigated which of the three isoforms was increased following cytokine treatment. We found that the isoform driven by the P3 promoter, a ubiquitously expressed isoform known as “YZ”, was the only isoform seen to be upregulated following cytokine treatment. To further understand the mechanisms by which IL‐6 and IL‐1β mediate their upregulation of ST6Gal1, we used an online transcription factor binding predictor to assess which transcription factors bind to the P3 promoter of the YZ isoform of ST6Gal1. On this P3 promoter, we found binding sites for STAT3, which is activated by IL‐6, and NF‐κB, which is activated by IL‐1β. Furthermore, we observed STAT3 and NF‐κB pathway activation following cytokine treatment. Importantly, STAT3 and NF‐κB inhibitors blocked cytokine‐induced ST6Gal1 upregulation in multiple cell models, thus confirming that IL‐6 and IL‐1β mediated their upregulation of the YZ isoform through the STAT3 and NF‐κB pathways, respectively. Taken together, these results not only show ST6Gal1 modulation within pancreatitis but detail the mechanisms by which this occurs, thus providing significant insight into an area of research that has not been well characterized.
Nigella Sativa Can Reverse Statin‐Induced Renal Injury in Rats: An Ultrastructural Study
Authors: Oules Jon , Thomann Charity , Youakim Magdy , Amin Shaimaa , Hassan Sherif
Published: May 2022

Introduction and Hypothesis

Atorvastatin (ATO) and its siblings in the statin drug class are widely prescribed as a lipid lowering agent. However, ATO in high doses can induce renal toxicity via oxidative stress. Thymoquinone (TQ), an extractive of Nigella sativa,is a natural antioxidant that has been shown to protect the kidney via its anti‐inflammatory effects. The aim was to study the anti‐inflammatory effect of posttreatment TQ and whether it could reverse the ATO‐induced oxidative injury to the rat kidney.

Methods

We used 4 groups of adult male Sprague Dawley rats (control; TQ‐treated; ATO‐post treated; ATO‐ post treated plus TQ‐treated). Each group was formed of 10 rats. Ultrastructural, and morphometric analyses of the rat kidneys were performed. Statistical analysis was done using SPSS. One‐way analysis of variance (ANOVA) and post hoc tests (Bonferroni test) were used to compare the means of different groups, expressing data as mean ± standard deviation (SD) and P values of less than 0.01 were expected to be significant.

Results

Morphometric analyses of the ultrastructural examination revealed that the histopathological changes induced by post treatment ATO has improved in the ATO/TQ‐treated group compared to the ATO‐ post treated group.

Conclusion

Posttreatment TQ appears to reduce and reverse renal injury due to ATO‐induced oxidative stress. TQ can potentially play a role in alleviating side effects in patients with kidney disease who are being treated with high dose of atorvastatin.
HH pathway orchestrates the epithelial‐mesenchymal crosstalk during human lung branching morphogenesis
Authors: Belgacemi Randa , Danopoulos Soula , Deutsch Gail , Dormoy Valerian , Al Alam Denise
Published: May 2022

Rationale

Hedgehog (HH) pathway plays an essential role in lung branching morphogenesis in rodents. However, little is known about the role of this pathway in human lung development. We hypothesize that HH pathway is necessary for branching during human lung development and hence in pulmonary hypoplasia.

Methods

Expression of HH pathway components was analyzed in single cell and bulk RNA‐sequencing data from publicly available lung datasets. Fresh human fetal lung tissues between gestational ages 10‐18 weeks were collected. RT‐qPCR, fluorescent in situ hybridization (FISH) and immunofluorescence staining (IF) were performed to determine spatiotemporal localization of HH pathway actors. In addition, distal lung segments were cultured in air‐liquid interface for up to 48 hours in the presence or absence of HH inhibitor (5E1). RT‐qPCR, western blot and IF were performed to investigate the effect of HH pathway inhibition on epithelial‐mesenchymal markers and associated signaling pathways.

Results

Bulk and scRNA‐seq analysis showed an early expression of HH pathway components during human lung development. We noted an inverse expression between activators and inhibitors receptors (PTCH1/HHIP) over time. PTCH1 had a higher expression at 20 weeks compared to 14 weeks while the opposite was observed for HHIP. HHIP FISH staining was restricted to ACTA2+ airway smooth muscle cells while PTCH1 was primarily localized in mesenchymal populations adjacent to the tip of airway epithelial buds where SHH ligand is produced. Inhibiting HH signaling ex vivo reduced branching during development (2.5‐fold vs control, p<0.01, n=8). Consequently, SOX9expression, an epithelial progenitor marker, was increased whereas ACTA2 was decreased (respectively 1.5‐ and 2‐fold vs control, p<0.05, n=8). These results were confirmed by western blot and IF staining. No change was observed for SOX2 gene expression while a significant increase in protein expression was observed. FGF10 increased following HH inhibition (1.5‐fold vs control, p<0.01, n=8), but the expression of ETV4and 5 decreased (1.5‐fold vs control, p<0.05, n=8). Finally, we demonstrated that HH signaling elements (including SHH, PTCH1, GLI2) were downregulated in lung tissues from congenital diaphragmatic hernia patients.

Conclusion

We have determined for the first‐time gene expression and localization of HH pathway during human lung morphogenesis. Moreover, we showed that HH signaling inhibition alters important genes and proteins expression, such as epithelial‐mesenchymal progenitors and associated pathways, required for proper branching processes. Understanding the role of HH pathway on human lung development could lead to the identification of novel therapeutic targets for childhood pulmonary diseases.
Probing Pal‐TolB Interactions Using Site‐directed Mutagenesis
Authors: O'Neill‐Knasick Katherine , Pilo Isabelle , Perdue Janai , Torelli Andrew , Michel Lea
Published: May 2022

Extensive research has suggested that Peptidoglycan‐associated Lipoprotein (Pal) from Escherichia coli (E. coli) plays a role in E. coli sepsis. When exposed to blood sera, for example, Pal is released from the E.coli outer membrane (OM). Released Pal has been found to be toxic and inflammatory, capable of inducing the release of pro‐inflammatory cytokines. Pal is tethered to the OM via an N‐terminal lipid moiety and non‐covalently binds to peptidoglycan (PG) through a site that overlaps with its binding site for TolB, a periplasmic protein and a key member of the Tol‐Pal complex. Due to the many interactions with these cellular components, the mechanism of Pal’s release from E. coli is not well understood. Here, we discuss our production and analysis of a site‐directed mutant of Pal (K150E), which we propose to disrupt the Pal‐TolB interaction. The effects of the mutation are assessed using immunoblotting and enzyme‐linked immunosorbent assays, the results of which we hope will provide insight into the Pal‐TolB interaction and the mechanism of Pal release from E. coli.
Chronic Vagal Nerve Stimulation Rescues Sympathetic Control of the Heart following Myocardial Infarction
Authors: Hadaya Joseph , Chan Christopher A. , Mori Shumpei , Hoover Donald B. , Shivkumar Kalyanam , Ardell Jeffrey L.
Published: May 2022

Cardiac injury, such as myocardial infarction (MI), leads to neurohormonal activation and autonomic remodeling, resulting in reflexive sympathoexcitation and parasympathetic dysfunction, which further contribute to progression of disease. The purpose of this study was to determine whether chronic cervical vagal nerve stimulation (VNS) influences MI‐associated changes in cardiac sympathetic function. Yucatan minipigs were divided into control (n=6), chronic MI (n=7), and chronic MI + chronic VNS (n=8) groups. VNS therapy was delivered to the right cervical vagus nerve and systematically titrated to an optimal intensity based on heart rate dynamics (5Hz, 2.1±0.3mA, 250μs, 17.5% duty cycle) using telemetry in the conscious state. Chronic MI was created using percutaneous embolization of the left anterior descending coronary artery with polystyrene beads under fluoroscopy (Figure 1A). VNS therapy was started 2 days following MI, and the animals maintained for 6‐8 weeks. At terminal study, we evaluated hemodynamic responses to graded bilateral sympathetic chain stimulation (BSS). Left ventricular (LV) strips encompassing regions of scar, border zone, and normal myocardium were fixed in 10% formalin, embedded in paraffin, sectioned, and studied using Masson's trichrome stain (Figure 1B‐C). Structural changes at the peri‐infarct area were scored (0‐3) for myocyte morphology and presence of hypertrophy or myocytolysis. Animals with MI alone exhibited spontaneous ventricular arrhythmias, including two that experienced sudden cardiac death (Figure 1D). At terminal study, there were no significant differences in baseline systolic function among groups. Moderate (4Hz) and high (10Hz) intensity BSS increased LV contractility (dp/dtmax) in all groups (Figure 2A‐B); however, evoked changes were significantly reduced in the chronic MI compared to control or MI + VNS groups. While control animals displayed evidence of contractile reserve at 10Hz vs 4Hz (1297±266 mmHg/s, p=0.03), chronic MI animals did not (631.9±264 mmHg/s, p=0.11), which was improved with chronic VNS therapy (1027±160.6 mmHg/s, p<0.01, Figure 2C). Chronic VNS therapy also significantly reduced MI‐associated structural remodeling in the peri‐infarct area, including the degree of myocytolysis and dysmorphic myocytes (structural abnormality index 0.6±0.2 vs 1.8±0.2, p<0.01). Our findings suggest that chronic VNS ameliorates sympathetic functional remodeling following myocardial infarction and improves cardiac mechanical performance in response to simulated stress. Furthermore, chronic VNS reduces pathologic myocardial remodeling at the peri‐infarct zone, potentially stabilizing the resultant ventricular scar.
Elp1‐Mediated Mechanisms Underlying Trigeminal Ganglion Development
Authors: Hines Margaret , Taneyhill Lisa
Published: May 2022

The trigeminal nerve is the largest of the cranial nerves and relays sensations of pain, touch, and temperature from the face and head back to the brain. Cell bodies for this nerve are positioned in the trigeminal ganglion, which arises from the coalescence of neurons and supporting glia derived from two important embryonic cell populations, neural crest cells and placode cells. While the dual cellular origin of the trigeminal ganglion has been known for over 60 years, the molecular mechanisms controlling trigeminal ganglion development remain obscure. Our prior work has shown that the chick embryonic trigeminal ganglion initially forms from cadherin‐based interactions between neural crest cells and placode cells. In an effort to elucidate pathways regulating cadherin‐mediated trigeminal ganglion assembly, we identified Elongator complex protein 1 (Elp1) as a new regulator of trigeminal ganglion neuron development. Mutations in ELP1cause Familial Dysautonomia, an ultimately fatal disease characterized, in part, by the presence of small trigeminal nerves and a reduced capacity to sense facial pain and temperature. In a mouse model of Familial Dysautonomia, embryos possess trigeminal ganglion innervation deficits and decreased expression of cadherins in trunk neural crest‐derived dorsal root ganglia. While Elp1 is vital for neurogenesis and has been implicated in protein trafficking, cell adhesion, and migration, the role of Elp1 in the trigeminal ganglion is unexplored. We hypothesize that Elp1 regulates trigeminal ganglion development by controlling cadherin levels important for continued interactions between neural crest cell and placode cell derivatives. To address this, we have first generated a spatio‐temporal expression profile of Elp1 in the chick trigeminal ganglion using section immunohistochemistry. Our preliminary data indicate that Elp1 is expressed in migratory neural crest cells, trigeminal placode cells, and trigeminal placode cell‐derived neurons. Additionally, immunocytochemistry of chick trigeminal ganglion explant cultures revealed colocalization of Elp1 and Rab proteins associated with vesicle trafficking. Future experiments will include loss of function assays to investigate the relationship between Elp1 and cadherin trafficking in the trigeminal ganglion. Our results have potential high significance for providing new insights into the function of Elp1 in trigeminal ganglion development and the etiology of diseases like Familial Dysautonomia and others arising from defects in neural crest cells and/or placode cells.
The DeBakey Executive Research Leadership Program Bridges the Depth of Experienced Leaders with the Breadth of Emerging Leaders
Authors: Bennett Trudy K. , Quick Christopher M. , McNeely Andrew C. , Laine Glen A.
Published: May 2022

Although new investigators leading research operations may be well trained to conduct research, they often have limited practical experience leading research teams. To address this unmet need, the Aggie Research Program (ARP) was developed in 2016 to advance the research careers of graduate students and postdoctoral scholars by connecting them with teams of undergraduates seeking research experiences. The requirements for graduate students and postdoctoral scholars to become “Team Leaders” are simple: they must get permission from their faculty mentors to participate, have an active research project, select and train a team of 3‐8 undergraduates, meet with their team once a week, and submit short weekly progress reports. To earn certification of completion, they must also attend monthly Team Leader meetings where they discuss best practices in leadership and complete a “Best Practices Report” to document their innovations to address challenges. This scalable structure allowed the ARP program to grow exponentially until a critical mass was achieved for Team Leaders to congregate in different affinity groups, which could then be formalized in distinct research leadership programs with different disciplinary or vocational focuses. In 2020, the ARP partnered with the Michael E. DeBakey Institute at Texas A&M University to create the DeBakey Executive Research Leadership program for graduate students, postdoctoral scholars, and junior faculty. This program leverages the combined 50 years of leadership experience of the Director and the Executive Director of the DeBakey Institute to distill a set of “research leadership principles” consistent with successful leadership careers at Texas A&M. These principles are introduced at the beginning of each Team Leader meeting to stimulate discussion of current challenges faced by Team Leaders. By bridging the deep experience of established leaders with the broad experience of a diverse cohort of emerging leaders, the DeBakey Executive Research Leadership Program structures a resilient, mutually supportive community that has grown during a pandemic. In the fall of 2021, it not only provided research leadership training for 47 Team Leaders, but also created research opportunities for 229 undergraduates. This multilevel, experience‐based training program provides a strategy to fill a critical gap in research training that cannot be filled by standard leadership or mentoring workshops which are typically too general, too short in duration, and too removed from the context in which they were originally developed.