bluuebunny/crossref_metadata_2025 huggingface dataset:
Sustained Mentorship Promotes The Development Of Active Learning Strategies In Undergraduate Biology Classrooms: Evidence Gained From The Promoting Active Learning (PALM) Network
Authors: Wolyniak Michael J. , Wick Sue
Published: April 2020

Here we report on the work of the Promoting Active Learning and Mentoring Network (PALM), a National Science Foundation‐supported project started in 2016 and dedicated to leveraging the expertise found in life science professional societies to provide long‐term mentorship in developing active learning techniques in the classroom. PALM mentees work one‐on‐one with mentors that are seasoned in active learning strategies and travel to their mentor’s institution to observe classes, plan modifications for their own classes, and strategize the best active learning techniques that are suited to their class’ particular needs. A monthly online journal club and regular opportunities to meet in conjunction with the Society for the Advancement of Biology Education Research (SABER) and ASCB meetings provide a dynamic teaching and learning community for mentees and mentors alike. After their mentorship, mentees are encouraged to work with their colleagues on active learning strategies and to become a PALM mentor to further the spread of effective active learning strategies to an ever‐wider audience. We have assessed the efficacy of PALM mentorship through the use of the Classroom Observation Protocol for Undergraduate STEM (COPUS) protocol (Smith et al., 2013) on pre‐mentorship and post‐mentorship videos of mentee teaching. The COPUS results have revealed a consistent shift in mentee teaching practices from a strict focus on lecture to a more diffuse combination of classroom activities in which the student is more directly active and engaged in the class and the instructor is more directly engaging students in a more personalized manner. While the specific changes in classroom practice vary between individual mentees due to the distinct needs and environments of each class, there is a consistent picture of more student‐centered classrooms that are more engaging and dynamic for both student and instructor. Overall, the COPUS data suggests that the long‐term mentoring strategies brought forth by PALM are an effective and sustainable means to promote the development of active learning techniques among undergraduate biology instructors.

Support or Funding Information

‐National Science Foundation Award #1624200
RNA Sequencing of Gas6 induced Preeclamptic Rat Placenta
Authors: Hirschi-Budge K. M. , Tsai K. Y.F. , Llavina S. , Hill J. , Arroyo J. , Reynolds Paul R.
Published: April 2020

Preeclampsia (PE) is an obstetric complication that affects 2–8% of pregnancies and remains a leading cause of morbidity and mortality for both the mother and fetus. Recent research has focused on biomarkers that may indicate disease onset and models for the study of PE. One such potential biomarker is the growth arrest specific protein 6 (Gas6). Gas6 binds to the TAM family of tyrosine kinase receptors, with primary affinity for the Axl receptor. Axl‐mediated signaling is implicated in cellular proliferation and migration, and we’ve demonstrated that Axl activation by Gas6 during pregnancy induces PE in the rat, including symptoms such as increased blood pressure, proteinuria, altered trophoblast invasion, and increased inflammatory cytokines.

Our objective was to elucidate changes in gene expression induced by Gas6/Axl in the developing placenta. Pregnant Holtzman Sprague Dawley rats were treated with Gas6, or Gas6 and the Axl inhibitor R428, from gestational day (GD) 7.5–17.5. Control rats received saline injections during the same period. Tissues were collected at GD 18 and snap frozen in liquid nitrogen. Placental RNA was isolated and submitted to the sequencing center at Brigham Young University. Quality filtering was performed prior to alignment, and alignment for each sample was between 98.0–98.4%. Significance was noted as an adjusted p value of < 0.05.

Analysis of the RNA sequencing data identified 123 genes that were affected by Gas6 administration. Of these, 44 genes were not responsive to Axl inhibition, 29 were returned to baseline levels, and 50 were mildly to moderately responsive to Axl inhibition. Examination of enriched gene ontologies identified several impacted biological processes including cellular proliferation and apoptosis, RNA stability, lipoxin processes and metabolism, and transcription in response to stress. These initial findings highlight several options for clarifying the molecular mechanisms of PE mediated by Gas6. Further research should examine translational effects in greater detail.

Support or Funding Information

This work was supported by a grant from the Flight Attendant’s Medical Research Institute (FAMRI, PRR and JAA) and a BYU Mentoring Environment Grant (JAA and PRR).
Regulation of Expression of β‐lactamase (ampC) and Lytic Transglycosylase by a Cascade of RpoE7‐>RpoH3 Sigma Factors Regulates Ampicillin Resistance in Azospirillum brasilense Sp245
Authors: Pandey Parul , Dubey Ashutosh Prakash , Mishra Shivangi , Mishra Muktinath , Singh Vijay Shankar , Tripathi Anil Kumar
Published: April 2020

Bacteria colonizing rhizosphere of plants face an intense competition for the nutrients present in the root exudates. This competition also involves production of antibiotics as well as of the enzymes that prevent inhibitory effects of antibiotics. Extra‐cytoplasmic function (ECF) sigma factors are known to initiate the transcriptions of genes involved in the adaptation against several stresses including cell envelope stress caused by antibiotics. Azospirillum brasilense is a plant growth promoting rhizobacterium, which colonizes the rhizosphere of a large number of non‐legume crops. The genome of A.brasilense Sp245 encodes 23 different sigma factors; one σ54 (RpoN), one σ70 (RpoD) and 21 alternative sigma factors of σ70 family. Out of 21 alternative sigma factors, 5 belong to RpoH, 10 to RpoE (ECF), and 6 to FecI type of σ factors. When the gene encoding RpoE7 was inactivated, the rpoE7::km mutant showed resistance to higher concentration of β‐lactam antibiotics compared to its parent. Inactivation of rpoE7 led to a decrease in the activity of its own promoter as well as of its divergently organized gene encoding a lytic‐transglycosylase. Transcription start site of rpoE7 was identified by 5’RACE, and studies with lacZ fusions with the promoters of rpoE7 and lytic transglycosylase genes indicated that both the promoters were regulated by RpoE7. Since β‐lactamases are known to be involved in conferring resistance to β‐lactams, we examined the expression of β‐lactamase paralogs in the parent and rpoE7::km mutant, and observed upregulation of one β‐lactamase (AmpC) in rpoE7::km compared to the wild type. The promoter activity of ampC and its regulator ampR were also increased in the rpoE7::km mutant. We also noted that RpoE7 regulated the expression of AmpC indirectly via RpoH3 sigma factor. Altogether, these data suggest that ampicillin resistance in A. brasilense is regulated by a regulatory cascade of RpoE7 and RpoH3.

Support or Funding Information

Parul Pandey, Ashutosh Prakash Dubey, Shivangi Mishra and Vijay Shankar Singh are Thankful to ICAR, CSIR, and UGC respectively for the research grant.
Altered Coagulation Parameters and D‐Dimer Measurements in Sepsis are useful in Scoring the Risk Stratification
Authors: Joshi Divya , Bontekoe Emily , Rondina Matthew , Middleton Elizabeth , Blair Antoinette , Siddiqui Fakiha , Hoppensteadt Debra , Fareed Jawed
Published: April 2020

Background

Sepsis is characterized by a simultaneous activation of inflammation and hemostasis in response to microbial infection. This systemic inflammatory response is due to the release of pro‐inflammatory cytokines, pro‐coagulants and adhesion molecules from immune cells and/or damaged endothelial tissue. Simultaneous activation of coagulation and fibrinolysis eventually leads to consumption coagulopathy and severe vascular dysfunction. This study was designed to profile coagulation parameters in a defined clinically confirmed sepsis population in conjunction with an IRB approved clinical trial.

Materials & Methods

Plasma samples from septic shock patients (n=78) were collected in citrated tubes within 72 hours of ICU admission under an IRB approved protocol in conjunction with ongoing trial at the University of Utah and Veteran’s Affair FFC Health Care System VAMC. Normal controls were comprised of commercially available 25 male and 25 female samples of citrated plasma (George King Biomedical, Overland Park, Kansas City). D‐Dimer was measured using a commercially available sandwich ELISA methods. PT/INR, aPTT and fibrinogen measurements were based on clot‐based assays. All results were compiled as mean ± SD and SEM.

Results

D‐Dimer levels were markedly increase in sepsis patients (23‐fold) in comparison to control. Significant elevation of clotting parameters such as PT/INR (2.0‐fold), aPTT (2.5‐fold) and fibrinogen (2.0‐fold) was noted in the sepsis patients in comparison to controls. These differences were significant (p value ≤0.0009) for all of the parameters except fibrinogen (p value 0.4694). There was no correlation between D‐dimer and fibrinogen in the sepsis patients.

Summary & Conclusion

In comparison to the control group, the sepsis patients showed wide variations in all of the parameters investigated in this study. The marked prolongation of PT and aPTT are suggestive of both the extrinsic and intrinsic pathway defects and consumption of clotting factors. The aPTT data showed wider scatter in comparison to PT data. The fibrinogen levels were also elevated and nearly 1/3 of the patients showed >1000 mg/dL levels. Marked elevation of D‐Dimer is indicative of endogenous fibrin formation and its consumption consistent with activation of secondary fibrinolysis. These results further underscore the importance of including the clotting parameters and D‐dimer in scoring the coagulopathies associated with sepsis such as disseminated intravascular coagulation.
Nitrated Fatty Acids Reduce Inflammatory Cell Recruitment in Bleomycin‐Induced Lung Injury
Authors: Krishnan Anirudh , Wilkinson Melissa L. , Gow Andrew J.
Published: April 2020

Acute lung injury (ALI) leads to infiltration of inflammatory cells and impaired lung function. Matrix Metalloproteinase 9 (MMP9) is a zinc‐dependent endopeptidase that degrades the extra cellular matrix and increases the porosity of the lung. CD11b is a marker for macrophage activation and migration. Nitro‐oleic fatty acid (OANO2), an electrophile that reduces cardiovascular inflammation has not been examined in lung injury. OANO2 modifies cysteine residues via Michael addition altering protein function. The goal of this study were to determine if OANO2 administration would reduce MMP9 expression and cellular infiltration following ALI. An intratracheal bleomycin (ITB) ALI model was used. Two groups of 6 C57BL/6‐J mice were treated intratracheally with bleomycin (3U/kg) or PBS (50uL). Half of the animals received OANO2 (50μg) in the same instillation and 72 hours later. The lungs were collected 7 days post ITB treatment. As previously seen, mice treated with ITB lost a significant amount of body weight; however, the addition of OANO2 mitigated this loss (−2.3 +/− 0.94 vs −0.4 +/− 0.83 g). Histology revealed cellular infiltration, deposition of proteinaceous debris, and airway epithelial damage in ITB mice that was reduced with OANO2 administration. The number of CD11b+ cells (marker for migratory macrophages) and MMP9+ cells was determined by immunohistochemistry. ITB increased number of CD11b+ (69 +/− 17.1 vs 195 +/− 17.1 #cells/HPF). OANO2 treatment reduced CD11b+ cells most in ITB animals (121 +/− 17.1#cells/HPF). ITB increased MMP9+ cell numbers compared to controls (71 +/− 15.7 vs 124 +/− 15.7 #cells/HPF). However, MMP9 expression was increased with OANO2 administration in PBS animals (304 +/− 15.7; 166 +/− 15.7 #cells/HPF), though this increase was not significant in ITB mice. Increased MMP9 expression was confirmed by western blot of whole lung tissue, where both ITB OANO2 and control OANO2 animals displayed high expression. Increased MMP9 expression may result from reduced self‐degradation as a consequence of OANO2 mediated enzyme inhibition. Overall, OANO2 increased MMP9+ cells and decreased CD11b+ cell populations. These findings suggest that treatment with OANO2 may oppose ITB‐mediated pro‐inflammatory cellular activation possibly by inhibition of extracellular peptidases, making it a potential therapeutic agent for acute lung injury.

Support or Funding Information

Supported by the Summer Undergraduate Research Fellowship and NIH Grants: 5R25ES020721‐09 (MPI)
The role of diet‐induced obesity on Peyer's Patch gene expression
Authors: Phillips Charles L. , Welch Bradley A. , Grayson Bernadette E.
Published: April 2020

Peyer’s Patches (PP) are part of the complex gut‐associated lymphoid tissue located throughout the intestinal wall. PP consists of highly organized ovoid‐shaped follicles, classified as non‐encapsulated lymphatic tissues. PP are populated with B cells, T cells, macrophages and dendritic cells allowing them to function as an organism’s internal surveillance of the intestine. There is limited research on PP in relation to diet‐induced obesity. In the current study, we attempt to close this gap in knowledge. We placed male, Long Evans rats on Chow or high‐fat diet (HFD) for 8 weeks and harvested PP from the small intestine. HFD‐fed rats had significantly increased number of total PP, p<0.05. All rats had significantly reduced number of PP in the duodenum in comparison to either the jejunum or ileum, p<0.0001. We performed gene expression analysis for immune cell markers in mRNA obtained from individual PP in the 3 distinct regions of the intestine. Expression of T cell markers CD4/CD3 was differentially reduced in PP from the distal intestine in comparison to the proximal. Macrophage marker, CD68, was more highly expressed in the duodenum in comparison to the more distal PP. Master regulator of T cell expression, TGFβ, is more highly expressed in duodenal PP in comparison to ileal PP, p<0.05. In summary, gene expression of immune cell markers was most highly associated with the location of the intestinal PP. Surprisingly, HFD did not influence gene expression of immune markers in the PP as was predicted. Further work is necessary to understand whether other inflammatory markers are affected by obesity.
No effect of anti‐hypertensive treatment during the metaboreflex in patients with essential hypertension
Authors: crisafulli antonio , Roberto Silvana , Milia Raffaele , Doneddu Azzurra , Ghiani Giovanna , Magnani Sara , Pinna Virginia , Mulliri Gabriele , Olla Sergio
Published: April 2020

In healthy subjects (HS), when the muscle metaboreflex (MM) is activated, the target mean blood pressure (MBP) is reached thanks to cardiac output (CO) increments, whilst systemic vascular resistance (SVR) plays a minor role
1. Differently, in essential hypertension (EHT) hemodynamics during the MM has been often reported to be dys‐regulated, with exaggerated SVR response (i.e. arteriolar vasoconstriction) and reduced CO
2. We tested the hypothesis that standard anti‐hypertensive treatment could improve this abnormal hemodynamics. 20 patients in pharmacological treatment for EHT (10 females; age 54.5±10.4 yrs, time since diagnosis 8.5±6.9 yrs) participated in this study together with a control group of 20 age‐matched HS. At baseline (BAS), they underwent randomly assigned the following protocol to study the MM activity: 1) post‐exercise muscle ischemia (PEMI), and 2) control exercise recovery CER) test. Exercise was dynamic handgrip performed at 30% of the maximum previously assessed. The EHT group performed again the test at least 7 days later (interval 7–15 days) after pharmacological wash‐out (WHT). Response to the MM for each cardiovascular parameter was assessed as PEMI minus CER level. Hemodynamic variables were evaluated by impedance cardiography. Results demonstrated that in EHT, SVR response did not change at the BAS and at the WHT (338.0±452.7 vs. 371.8±465.3 dynes•s‐1•cm‐5 at BAS and at WHT respectively). Moreover, EHT showed higher SVR response than HS (20.9±399.6 dynes•s‐1•cm‐5, p<0.05 vs. both the BAS and the WHT test). However, the MBP response was similar between groups and conditions. Likewise CO was not different between groups. It was concluded that the anti‐hypertensive treatment was not able to counteract the arteriolar vasoconstriction and the increased SVR response experienced by ETH patients during the MM. Further study is warranted to better understand what causes the hemodynamic abnormalities of EHT patients during the MM activation.

Support or Funding Information

This study was supported by the University of Cagliari and the Italian Ministry of Scientific Research.
Modulation of Aquaporin 2 by Renin‐Angiotensin‐Aldosterone System Peptides in Medullary and Cortical Collecting Duct Cells
Authors: Da Silva Braga Tamires , de Oliveira Lilian Caroline Gonçalves , Casarini Dulce Elena
Published: April 2020

The kidney is responsible for the body water balance and homeostasis. The collecting duct system is important for this role due its responsibility, as water reabsorption and urine production. In the collecting ducts cells, it’s known that the reabsorption of water is made by the water channels named aquaporins, specially aquaporin 2 and aquaporin 3. In those channels the reabsorption is controlled by the peptide hormone vasopressin which induces the water transport due the water channels. Into the homeostatic mechanism, there is the renin‐angiotensinaldosterone system (RAAS) which regulates the water and sodium absorption as well. Previous studies of our group described that Ang 1‐7 and Ang II are inductors to the migration of AQP2 to the membrane in LLC‐PK1 cells. Therefore, the aim of this study is to evaluate if the RAAS peptides, Angiotensin II (Ang II) and Angiotensin 1‐7 (Ang 1‐7), are capable to induce water reabsorption due aquaporin 2 in Inner medullary collecting duct cells (IMCD) and cortical collecting duct cells (M1).

Methods

In this present study we evaluate the expression of aquaporin 2 after the treatment with Ang II and Ang 1‐7in different times (5,15 and 30 minutes) in both cells of the collecting duct system (medullary and cortical). The analysis of the expression was made by western blotting assay.

Results

The western blot technique showed that in both cellular types, there is a difference between the expressions in the cells. M1 cells have showed that after different treatments times, the RAAS peptides decrease the expression of aquaporin 2 differently of the IMCD cells, that showed expression of aquaporin 2 increased when treated with Ang II. In conclusion, the results demonstrated that Ang II and Ang 1‐7can modulate the water channel aquaporin 2 expression.

Support or Funding Information

CAPES, FAPESP and CNPq
The Effects of Diet on Hard Palate Morphology in Humans
Authors: Bushman Jeness , Maddux Scott D. , Menegaz Rachel A.
Published: April 2020

With the Neolithic adoption of agricultural practices around 10,000 BCE came a dietary transition from foraged foods to domesticated crops. A significant reduction in the overall dimensions of the human craniofacial skeleton is attributed to the decreased biomechanical demands associated with agricultural diets. However, the impact of this dietary transition on specific skeletal structures such as the hard palate is poorly understood. Previous experimental work has shown that laboratory rabbits fed a soft, processed diet have a vertically shallow palate with decreased cortical bone thickness and trabecular area. Here we test the hypothesis that compared to hunter‐gatherer populations, human agriculturalists have a similarly reduced palate due to the consumption of softer, more intensely processed foods.

CT scans from a mixed‐sex adult sample of Homo sapiens skulls (n=36) representing two pre‐industrial agricultural groups (ancient Egyptians and pre‐Columbian Peruvians) and two hunter‐gatherer groups (indigenous Australians and native Alaskans) were analyzed using 3D Slicer software. The following dimensions were measured at the anterior, middle, and posterior palate: palate width; vertical palate depth; cortical bone thickness along the oral lamina; and trabecular bone area. Palate variables were analyzed using pairwise Mann‐Whitney U‐tests (α = 0.05). Results show that compared to hunter‐gatherers, individuals from agricultural populations have significantly vertically shallow palates, thinner cortical bone at the middle and posterior palate, and decreased trabecular bone area at the middle palate.

These results support the hypothesis that cross‐sectional palatal morphology is associated with the biomechanical profile of the human diet, and that the hard palate of modern humans has been significantly affected by the adoption of agriculture. The adoption of agriculture decreased the mechanical strain experienced by the hard palate, resulting in a decrease in overall bone volume. We propose that further changes might be expected in palate morphology in more recent human populations, related to increased food processing and reduced dietary breadth seen during the Industrial and Green Revolutions of the mid‐17th through 20th centuries. Additional research is needed to investigate the effect of industrialized agriculture on palatal morphology, as well as the influence of biomechanical loading on the hard palate across life history and growth.

Support or Funding Information

Funding was provided by the American Association for Anatomy Innovations Program.
Characterization of EfhX, a calcium sensor protein in Xanthomonas required for elicitation of the hypersensitive response in Solanum species
Authors: Quay Juliana , Hurlbert Jason
Published: April 2020

We have identified a gene in several strains of Xanthomonas that, when expressed, can elicit a hypersensitive response (hr) in tomato plants. Hr elicitation is normally limited to specific bacterial‐host pairings, but introduction and expression of this gene by bacterial species that do not normally infect tomato serves to elicit hr, indicating that the encoded 170 amino acid protein is crucial to the infectious process. Bioinformatic analysis of the protein, which we have named EfhX (
EF‐
hand containing protein from
Xanthomonas) reveals that the protein is predicted to contain a domain of unknown function spanning the amino terminal region, a single transmembrane alpha‐helix spanning amino acids 60 to 81 and two putative EF‐hand calcium binding domains in the carboxy‐terminal domain of the protein. In order to better understand the function of this novel protein, we have cloned the EfhX gene from Xanthomonas aurantifolia and expressed it in Escherichia coli. We have successfully purified the protein to homogeneity as determined by SDS‐PAGE, anti‐hexahistidine Western Blot and MALDI‐TOF mass spectrometry. Using the purified protein, we have used isothermal titration calorimetry to confirm that the protein has two calcium binding sites, with one site having an affinity (Kd) for calcium of 6.75 mM and the second site having an affinity of 1 uM. We are currently performing circular dichroism spectropolarimetry and gel filtration studies to determine if the quaternary state of the protein changes upon calcium binding. Simultaneously, we have initiated crystallization trials of the purified protein to identify solution conditions suitable for growth of crystals for x‐ray diffraction experiments so that the structure of the protein can be determined.

Support or Funding Information

This work is supported by grants from the National Center for Research Resources (5 P20 RR16461) and the National Institute of General Medical Sciences (8 P20 GM103499) from the National Institutes of Health.
Mice but not rats modulate liver mitochondrial machinery in chronic hypoxia
Authors: Reyes Christian Arias , Joseph Vincent , Soliz Jorge
Published: April 2020

Mice (FVB) permanently living at high‐altitude have increased respiratory frequencies and sustained or increased metabolic rates. On the other hand, rats permanently living at high‐altitude (SD) have impaired respiratory control, high hematocrit and hemoglobin, signs of pulmonary hypertension, and reduced metabolism; all these considered to be detrimental. This is coincident with the fact that common rats are not found in natural high‐altitude environments. At sea level, we reported that mice, but nor rats, exposed to 21 days of hypoxia (12% O2) increase their metabolic rate (O2 consumption and CO2 production), similarly to other rodent species that are considered well adapted to high‐altitude. As mitochondria is the final user of O2 in metabolism, we were interested in the role of mitochondria behind this divergence in physiological adjustments. We used saponin‐permeabilized samples of liver (most energy‐consuming organ in rodents), from male adult FVB mice and SD rats after exposure to hypoxia (12% O2, for 0, 1, 7 or 21 days), to measure the mitochondrial O2 consumption rate (OCR) following the SUIT‐01 protocol in the high‐resolution respirometer O2K (OROBOROS instruments). OCR was measured with substrates activating the NADH, succinate , and fatty acid pathways of the electron transport chain (ETC). We also measured the maximum capacity of ETC by uncoupling the electron transport from the oxidative phosphorylation with CCCP. The level of activation of complexes I and II was reported as the OCR for the N or S pathway correspondingly, divided by the maximum capacity (flux control ratio).

Our preliminary results show that, compared to rats, mice have significantly higher ETC maximum capacities in normoxia and hypoxia. Moreover, after 21 days of hypoxia, mice showed an increase of 43% in maximum capacity in comparison to normoxic controls, while no increase in maximum capacity was evidenced in rats. In mice, we observed that complex I is transiently hyperactivated after 1 day of hypoxia and then, its activity reduces down below control levels by day 21 of hypoxia. Contrastingly, complex II activity is reduced after one day of hypoxia, getting back to control levels by day 21 of hypoxia. Furthermore, in rats, an increased activity of complex I occurred after 21 days of hypoxia. Regardless of normoxic or hypoxic exposure, rats showed higher activation levels of complex II than mice.

These results show that mice, but not rats, have the ability to modulate their liver metabolic mitochondrial machinery under chronic hypoxia. The higher and adjustable maximum capacity in mice, suggest a more powerful and plastic liver mitochondrial machinery than in rats. This might explain in part their ability to keep sustained metabolic rates under hypoxic conditions.

Support or Funding Information

Supported by NSERC, the Health Respiratory Network of Québec and CIHR.
Dynamics of nerve dependence during the regeneration of axolotl salamander limbs
Authors: Jeon Eun Kyung , Monaghan James
Published: April 2020

When nerve supply is severed at the brachial plexus, regeneration of Axolotl salamander limbs ceases. Nerves provide specific molecules that help initiate and sustain the process of regeneration. Regenerating limbs show different responses to denervation at each step of the process. Severing nerves right after amputation prevents further growth, but denervation post‐growth of a regenerative tissue yields a miniature limb. Nerves may be providing molecules that maintain cell plasticity, but how regenerative tissue changes in absence of nerves is unknown. In this study, effect of denervation on cell proliferation at each point of regeneration is investigated. Regenerating limbs were denervated one day before collection and labeled with 5‐ethynyl‐2’‐deoxyuridine (EdU, a proliferation marker) in vivo‐3 hours before collection. We show region‐specific depletion of proliferation in limbs 0 (uninjured), 6, 9, 12, 15, 18, and 25 days post amputation. Our results suggest that loss of nerve supply drastically reduces proliferation in both uninjured and regenerating limbs but not in tissue actively undergoing digit formation.

Support or Funding Information

This project was supported by Matz Scholarship in Biotechnology and NSF grants 1558017 and 1656429.
Implication of RAGE Signaling in establishing models of COPD pathogenesis
Authors: Reynolds Paul R.
Published: April 2020

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the United States and it is characterized by inflammation and respiratory tissue loss. Tobacco smoke is the major cause of COPD. Studies identified
receptors for
advanced
glycation
end‐products (RAGE) as a smoke‐induced pattern recognition receptor with potent pro‐inflammatory characteristics and we’ve observed increased pulmonary RAGE following first and secondhand smoke (SHS) exposure. Further, RAGE transgenic mice that conditionally increase RAGE expression manifest COPD characteristics and are possible smokeless models of a smoker’s lung. We evaluated inflammatory effects of SHS with and without semi‐synthetic glycosaminoglycan ethers (SAGEs), a family of anionic, partially lipophilic sulfated polysaccharide derivatives that inhibit RAGE signaling. The current research evaluated the in vivo effects of short‐term smoke exposure in RAGE null, conditional RAGE over‐expressing, and control mice via a nose‐only exposure platform for 4 weeks (Sireq Scientific) and compared them to animals exposed to room air. Groups of mice were also co‐treated with SAGEs via weekly ip injections (a 30mg/kg body weight). Molecular characterization of smoke exposure revealed significant pulmonary inflammation and apoptosis mediated in part by RAGE. Inflammatory cell behaviors were assessed by determining the activation of Ras, intracellular signaling kinases, and cellularity/cytokine secretion in bronchoalveolar lavage fluid (BALF). Inflammatory signaling intermediates and cytokine elaboration were induced by SHS and influenced by the availability of RAGE, as evidenced by RAGE targeted mice and SAGE treatment. We also observed compromised lung mechanics mediated by RAGE expression during exposure. These data reveal captivating information suggesting a role for RAGE signaling in lungs exposed to tobacco smoke and implicates plausible therapeutic modalities that may attenuate lung pathology.

Support or Funding Information

This work was supported by a grant from the Flight Attendant’s Medical Research Institute (FAMRI).
Impact of Transcriptional Dysregulation on the DNA Damage Response in Ewing’s Sarcoma
Authors: Rollins Matthew Gregory
Published: April 2020

Ewing’s sarcoma is a soft‐tissue bone malignancy characterized by a translocation event wherein the N‐terminal low complexity domain of EWSR1 is fused with the DNA binding domain of the ETS transcription factor Fli1. The resulting EWS‐Fli1 fusion protein drives expression of oncogenes and knockdown of tumor suppressor proteins. More specifically, EWS‐Fli1 acts as transcriptional regulator by helping recruit RNA Pol II to promoter regions, which is subsequently phosphorylated by CDK9 to escape initiation into elongation. Interestingly, Ewing’s sarcoma is particularly susceptible to chemotherapy compared to other cancers; however, the underlying reason for this sensitivity to DNA damage has not been elucidated. Given EWS‐Fli1’s role in disrupting transcription, it was hypothesized EWS‐Fli1 is concurrently disrupting the DNA damage response. Here, a combination of CDK9 inhibition with DNA damage was found to drastically diminish cell viability in Ewing’s cell lines but not in non‐Ewing’s osteosarcoma or HEK293 cells. Ordinarily, after DNA damage γH2A.X phosphorylation occurs on histones near sites of damaged DNA early during the DNA damage response to signal DNA damage repair initiation. Further work has shown Ewing’s cells undergoing the combination treatment above have a marked decrease in this DNA damage marker γH2A.X as compared to non‐Ewing’s cells. Together, these results suggest Ewing’s sarcoma cells are sensitized to DNA damage due in part to transcriptional dysregulation.

Support or Funding Information

Funding for this research provided by the American Cancer Society and NIH.
Co‐expression of placental RAGE and g‐H2AX during intrauterine growth restriction and preeclampsia in rodents
Authors: Tsai K.Y.F. , Hirschi-Budge K. M. , Davis T. , Llavina S. , Fowers R. , Reynolds Paul R. , Arroyo J.
Published: April 2020

Intrauterine growth restriction (IUGR) and preeclampsia (PE) are obstetrics complications associated with poor outcomes for the both the mother and fetus. Although both characteristically differ, both diseases are associated with placental malfunction that stems in part from increased placental apoptosis. DNA double strand breaks (DNA‐DSBs) represent some of the most destructive forms of genomic damage in which both strands of DNA are ruptured. H2AX is a component of the histone octomer and is phosphorylated to form g‐H2AX in response to the sensing of DNA‐DSBs. The receptor for advanced glycation end‐products (RAGE) is a multi‐ligand receptor primarily expressed on cell membranes, where it functions as a progression factor in inflammatory signaling; however, the nuclear isoform of RAGE (nRAGE) has been described as having a crucial role in DNA‐DSBs repair. The purpose of this study was to identify placental DNA damage, placental nRAGE and g‐H2AX expression in a secondhand smoke exposed rodent model of IUGR and a Gas6 induced PE model. Placental and fetal weights were determined at the time of necropsy in the IUGR model. Blood pressure and proteinuria was assessed in the PE model. Characterizing DNA degradation was used to confirm placental DNA damage during these pregnancy complications. Immunohistochemistry was used to determine localization of both molecules in the placenta. Relative to controls we observed: 1) decreased placental and fetal weights (1.4‐fold and 2.3‐fold; p<0.003) during IUGR; 2) significantly increased systolic and diastolic blood pressure and proteinuria (80%; p<0.05) during PE; 3) increased DNA degradation in the IUGR and PE placentas and 4) Increased staining for RAGE and g‐H2AX in the IUGR and PE placentas. These results may provide insight into the physiological relevance of these molecules, and if so, their modification during gestation may help alleviate placental disease.

Support or Funding Information

This work was supported by a grant from the Flight Attendant’s Medical Research Institute (FAMRI, PRR and JAA) and a BYU Mentoring Environment Grant (JAA and PRR).
Cardiac‐specific Deletion of Filamin C Decouples Myocyte Contractility and Calcium Handling in Adult Mice
Authors: Powers Joseph D. , Liu Canzhao , Fang Xi , Omens Jeffrey H. , Chen Ju , McCulloch Andrew D.
Published: April 2020

Costameres are sub‐membranous protein complexes in striated muscle that play roles in mechanical signal transduction and force transmission between the Z‐disc of the sarcomere and the extracellular matrix. In cardiac myocytes (CMs), the cytoskeletal protein Filamin C (FLNC) interacts with multiple proteins in the Z‐disc and the costamere, suggesting that it contributes to costamere‐based mechanotransduction in the heart. Moreover, mutations in the gene that encodes FLNC have been associated with multiple forms of human cardiomyopathies. To investigate the role of FLNC in cardiac mechanosignaling and pathological remodeling, we employed a recently developed murine model of inducible and cardiac‐specific FLNC knockout (FLNC‐icKO). Previous studies with this mouse model used echocardiographic measurements of adult FLNC‐icKO mice to reveal a severe dilated cardiomyopathy (DCM) phenotype by 2 weeks post‐induction, including significantly reduced ventricular fractional shortening. Correspondingly, we found that intact papillary muscles dissected from adult FLNC‐icKO hearts have significantly reduced twitch force compared to those dissected from wild‐type (WT) hearts. Interestingly, while the fractional shortening of isolated CMs from FLNC‐icKO hearts is also significantly reduced compared to WT CMs, the calcium transient (peak amplitude and duration) during a twitch is not different between groups. These results suggest that contractility and calcium handling in FLNC‐null hearts are decoupled. Potential mechanisms that may be responsible for this apparent decoupling are a disruption in force transmission between the sarcomere and the cell membrane, an increased myofilament lattice spacing, or altered calcium sensitivity of the myofilaments upon the deletion of FLNC in CMs.

Support or Funding Information

Funding: HL007444‐36 to JDP, HL144872 to JC, and HL137100 to ADM.
Integrated Machine Learning Approach to Identify Metabolome Fingerprints of Pathological Stages Following Heart Failure Treatment
Authors: Choi Howard , Mirza Bilal , Liem David A. , Wang Ding , Deng Mario C. , Wang Wei , Ping Peipei
Published: April 2020

Introduction

Pathologic cardiac remodeling from a healthy to diseased state is characterized by an intricate and coordinated multitude of alterations among various biomolecules over time. Conversely, mechanical circulatory support devices (MCSD) are a promising strategy for unloading the heart and reversing this process. Understanding the molecular drivers of remodeling and reverse remodeling would inform new mechanistic insights and more nuanced diagnostics, prognostics, and therapeutics for hypertrophy and/or heart failure (HF). Plasma metabolomics holds great promise for the temporal profiling and characterization of healthy and diseased phenotypes, but large‐scale technological platforms with machine learning (ML) pipelines for clinical temporal metabolomics profiling are still lacking.

Methods

In this study, we established a Multiple Reaction Monitoring (MRM)‐based MS quantitative platform and a corresponding ML‐based computational workflow to unravel the metabolomics fingerprints of healthy humans, HF patients, and a mouse model. Accordingly, we quantified the plasma levels of 610 metabolites, and identified those exhibiting high correlation to phenotypic changes using feature selection algorithms following random forest‐based missing data imputation.

Results

In summary, a total of 30 metabolites were significantly altered during the cardiac remodeling: 12 metabolites were continuously upregulated during ISO‐treatment, belonging to the biological categories of amino acids and sphingomyelin (SM). In parallel, 18 metabolites were downregulated, consisting of 10 free fatty acids (FFA), 6 ceramides, and 2 phosphatidylcholine (PC). Among these FFAs, we identified 3 saturated FFAs, 6 monounsaturated FFAs (with 1 double bond) and 5 polyunsaturated fatty acids (PUFA). Importantly, we have identified 4 driver metabolites that are significantly associated with changes in HW/BW ratio, all of which are FFAs: C14:0 (Myristic acid), cis‐C16:1w7 (Palmitoleic acid), cis‐C18:1w9 (Oleic acid), and cis‐C18:3w3 (α‐Linolenic acid, ALA).

Our study highlights a novel ML workflow that, in conjunction with benchmark datasets, will facilitate future biomarker discovery.

Support or Funding Information

NIH/NHLBI T32 Postdoctoral Fellowship, T32HL139450
Modulation of GPCR Function by Membrane Lipids and Water
Authors: Weerasinghe Nipuna , Fried Steven D.E. , Eitel Anna R. , Mann Helen F. , Cosgriff Emily L. , Struts Andrey V. , Perera Suchithranga M.D.C. , Brown Michael F.
Published: April 2020

G‐protein‐coupled receptors (GPCRs) are integral membrane proteins that regulate a wide range of normal and disease‐related physiological processes. Our understanding of the role of soft matter such as membrane lipids and cellular water on activation and regulation of the GPCR function remains incomplete. In the standard biochemical model, GPCRs behave as primarily agonist dependent bimodal switches, with little influence of the surrounding medium. However, using the visual receptor rhodopsin as a model GPCR, we show that water drives rhodopsin to a partially disordered, solvent‐swollen conformational ensemble upon light absorption, rendering the standard model obsolete
[1],
[2]. We placed rhodopsin under varying degrees of osmotic stress using polyethylene glycol solutes and investigated the activation equilibrium response using UV‐Visible spectroscopy. Our results show a flood of ~ 80 water molecules into the rhodopsin interior during photoactivation, a result supported by atomistic molecular dynamics simulations
[3]. Furthermore, the osmolyte effects on rhodopsin activation are size dependent: large osmolytes back shift the equilibrium to inactive metarhodopsin‐I (MI), while small osmolytes forward‐shift the equilibrium to active metarhodopsin‐II (MII). We attribute these size effects to varying degrees of osmolyte penetration into the rhodopsin core. Large polymers behave similarly to ideal osmolytes and dehydrate rhodopsin, while smaller polymers wriggle into the rhodopsin interior and stabilize the open MII conformation of rhodopsin. Besides osmotic pressure, the application of hydrostatic pressure also back shifts the metarhodopsin equilibrium but for fundamentally different reasons. Integrating the two force‐based methods together with neutron scattering experiments indicates that the active state of rhodopsin is more hydrated yet simultaneously more structurally collapsed
[4]. At the same time, the active GPCR undergoes dynamic volume fluctuations and solvent coupling, which give rise to greater thermal volume. Our results necessitate a new understanding of GPCR activation in which the surrounding soft matter is paramount in governing conformational energy landscapes.

Support or Funding Information

NSF (MCB 11817862 and CHE 1904125) and US NIH (EY012049 and EY02604)
Mitochondrial ROS production in lung microvascular endothelial cells sensitizes the transient receptor potential vanilloid‐4 (TRPV4) channel to physical and chemical stimuli.
Authors: Philip Nicolas , Servinsky Laura , Jiang Haiyang , Huetsch John , Damarla Mahendra , Shimoda Larissa , Suresh Karthik
Published: April 2020

Rationale

Increased proliferation of lung microvascular endothelial cells (MVECs) is a known feature of pulmonary arterial hypertension (PAH). We have previously shown that increased mitochondrial reactive oxygen species (mtROS) and basal intracellular calcium ([Ca2+]i) are associated with increased proliferation and migration in MVECs isolated from animals undergoing experimental PAH (Sugen Hypoxia model; SuHx‐MVECs) compared to MVECs isolated from normoxic controls (N‐MVECs). In these studies, we observed that quenching mtROS in SuHx‐MVECs attenuated TRPV4 activity, basal [Ca2+]i, migration and proliferation in SuHx‐MVECs. However, whether induction of mtROS production alone, in N‐MVECs, was sufficient to activate TRPV4 was unclear.

Methods

To measure [Ca2+]i in MVECs, cells were incubated on glass coverslips with the calcium‐sensing dye FURA‐2 AM and transferred to a temperature‐controlled flow chamber set at baseline to 37 degrees and flow rate of 0.1 dynes/cm2. Cells were perfused with freshly‐prepared Krebs buffer. TRPV4 inhibition was achieved with HC‐067047 (HC) treatment. mtROS production was induced using treatment with antimycin A (AA) or Mito‐Paraquat (MPQ). TRPV4 was activated using either the chemical agonist GSK1016790A (GSK) or by increasing chamber and perfusate temperature to 40 degrees (since heat is a known activator of this channel).

Results

Treatment of N‐MVECs with antimycin A (AA) increased basal [Ca2+]i. GSK‐induced Ca2+ influx was also significantly higher in AA‐treated N‐MVECs. Both increased basal Ca2+ and increased GSK‐induced Ca2+ influx were attenuated with HC pre‐treatment (i.e. TRPV4 inhibition). Using temperature as a TRPV4 stimulus, we observed greater heat‐induced Ca2+ in AA‐treated N‐MVECs. Since AA may have off‐target effects, we repeated our GSK‐induced Ca2+ experiments following treatment with MitoPQ, a redox cycler also known to increase mtROS. Similarly to AA, Mito‐PQ treatment increased basal [Ca2+]i and GSK‐induced calcium influx.

Conclusion

These data suggest that, in N‐MVECs, mitochondrial ROS elevate basal intracellular calcium levels and both activate TRPV4 and sensitize the channel to subsequent activation by either chemical or physical stimuli.

Support or Funding Information

This work was supported by by the National Heart, Lung and Blood Institute Grants F32HL124930 and K08HL132055 (KS), R01HL073859, R25HL084762 and R01HL126514 (LAS), F32HL124727, and K08HL133475 (JH) and T32HL007534 (KS and JH).
Understanding the Role of the G Protein‐Coupled Estrogen Receptor (GPR30/GPER‐1) in Neurodevelopment
Authors: DeLeon Chelsea , Pemberton Kyle , Xu Fenglian , Arnatt Christopher
Published: April 2020

Several studies have commented on the effects of 17β‐estradiol(E2) on neurodevelopment. These studies have primarily focused on the classical nuclear estrogen receptors, ERα and ERβ. It has been established that E2 has the ability to activate rapid, non‐genomic signaling through the G Protein‐Coupled Estrogen Receptor (GPR30/GPER‐1). Few studies have examined the neurodevelopmental effects of selectively targeting GPER‐1. Previously, our group synthesized novel, selective GPER‐1 agonists. During biological evaluation, the pharmacology associated with the novel agonists appeared to differ from known GPER‐1 agonist, G‐1. Treatment with the beta‐gamma subunit inhibitor, galein, revealed a significant attenuation in signal for the synthesized agonists but not for G‐1 at concentrations above 1μM. Additionally, the efficacy of eliciting neurite outgrowth in rat hippocampal E18 neurons was greater for the synthesized agonists than G‐1. To further understand the observed differences in signaling between the synthesized compounds and G‐1, rat hippocampal E18 neurons and neural progenitor cells (NPCs) originating from healthy humans were studied. RNA sequencing was utilized to reveal the presence of changes in gene expression in response to GPER‐1 compounds in rat hippocampal E18 neurons, excitatory NPCs, and inhibitory NPCs. These results provide information regarding opportunities associated with targeting GPER‐1 in neurodevelopment as well as information related to the cell‐signaling pathway that may be leveraged in the design of new therapeutic lead compounds.

Support or Funding Information

Saint Louis University
Optogenetic control of the Sphingosine‐1‐phosphate receptor 1 enables light‐mediated regulation of endothelial permeability and angiogenesis
Authors: Kang Hojin , Matsche Jacob , Karginov Andrei
Published: April 2020

Sphingosine‐1‐phosphate receptor 1 (S1PR1) is critical regulator of endothelial cell (EC) migration, cytoskeletal structure, capillary‐network formation, sprouting angiogenesis, and vascular maturation. Here we developed a strategy for controlling endothelial barrier function and sprouting angiogenesis using an engineered photo activatable (PA)‐S1PR1 receptor. Regulation of signaling and cell function by PA‐S1PR1 was demonstrated in human pulmonary arterial endothelial cells (HPAECs). Following illumination with blue light, PA‐S1PR1 induced activation of known downstream signaling pathways (AKT, Erk1/2 and FAK). The kinetic and the extent of activation correlated with what was observed in HPAECs treated with 1μM S1P. Furthermore, activation of PA‐S1PR1 lead to enhancement of endothelial barrier and stimulation of sprouting angiogenesis in vitro. Thus, these findings suggest that engineered PA‐S1PR1 can be used to mimic its function in living cells and dissect S1P signaling with precise temporal and spatial resolution.

Support or Funding Information

This work was supported in part by American Heart Association Scientist Development Grant 17SDG33700146 to H. K., and R21CA159179 and CBC Pilot Grant to A.V.K.