bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Necrosis Via Necrox‐5 Attenuates The Development Of Hypertension In Male, But Not Female Spontaneously Hypertensive Rats (SHR)
Authors: Abdelbary Mahmoud , Onyilo Chukwuemeka , Crislip G Ryan , Gillis Ellen , Brands Micahel , Sullivan Jennifer C.
Published: June 2021

It is well established that hypertension is accompanied by cell death, however, less is known regarding the role of cell death in blood pressure (BP) control. Necrosis is an uncontrolled pathologic form of cell death that is associated with inflammation. Therefore, the goal of the current study was to test the hypothesis that necrosis contributes to the development of hypertension in SHR. Initial studies measured renal necrosis in 13 wk old male and female SHR (n=9) using flow cytometry. Male SHR had greater renal necrosis compared to female SHR (3.7±0.4% vs. 0.4±0.2% of total renal cells, respectively; p<0.0001). Additional male and female SHR were then randomized to receive vehicle or Necrox‐5 from 6–12 weeks of age (1 mg/kg via IP injection twice weekly; n=4); Necrox‐5 is a cell permeable inhibitor of necrosis that blocks oxidative stress induced necrotic cell death. BP was measured weekly via tail‐cuff analysis and via carotid catheter at sacrifice. Following 6 wks of treatment, kidneys were isolated and necrosis was measured by flow cytometry. Treatment with Necrox‐5 beginning at 6 wks of age attenuated maturation‐induced increases in BP in male SHR (Necrox‐5: 152±11 vs Control: 177±4 mmHg; p=0.07). BP in female SHR was not altered by chronic Necrox‐5 treatment (Necrox‐5: 152±14 vs. Control: 146±11 mmHg; p=0.72). In addition, the sex difference in BP apparent in control rats was abolished by Necrox‐5 treatment. Consistent with these results, Necrox‐5 treatment for 6 wks decreased renal necrosis only in males (Necrox‐5: 1.5±0.5% vs Control: 3±0% of total renal cells). Taken together, our data suggest that greater cell death in male SHR compared to female SHR contributes to sex differences in BP by exacerbating age‐related increases in BP in males.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Senescent Skeletal Muscle Produces a Distinct Cytokine Secretory Profile in Response to Endotoxin Exposure in Vitro
Authors: Mattingly Alex J. , Laitano Orlando J. , Clanton Thomas L.
Published: June 2021

Skeletal muscles are thought to contribute to both pathogen detection and cytokine secretion as a part of the innate immune response. Skeletal muscle fibers contain toll like receptors (TLRs) that bind a wide array of bacterial, viral, and fungal ligands, inducing signal transduction and subsequent cytokine production. Aging populations have significantly greater mortality annually from influenza, incidence of bacterial and fungal infections, and diminished vaccine efficacy. In senescent mice, macrophage TLR expression and cytokine secretion are known to decline in some tissues. It is unknown if skeletal muscle also exhibits age associated changes in innate immune contribution. The objective of this experiment was to determine if there are alterations in cytokine secretory response of aged skeletal muscle to lipopolysaccharide (LPS), a potent TLR2 and TLR4 ligand.

Matched solei muscles from 12–15‐week‐old male C57BL/6 mice (n=8; “young”) and 16‐month‐old (n=10; “old”) were excised in oxygenated (95%O2/5%CO2) Krebs solution and placed under 1 g of tension in 2 ml baths at 35°C. After a 30‐min acclimation period (T0), the buffer was changed and muscles were exposed to 1 μg/ml LPS. Bath samples at T0, 1 hr (T1) and 2 hr (T2) were taken, combined with a protease inhibitor cocktail and frozen at −80°C prior to Luminex multiplex analysis.

In conclusion, solei from old mice generally displayed a diminished cytokine secretory response, with particularly large decrements in IL‐12p70, IL‐6, MCP‐1, KC, and G‐CSF compared to young controls (see Table 1). Interestingly, solei from old mice displayed relatively large increases in IL‐1α, with smaller, yet also significant increases in IL‐10 and TNFα. The drastically lowered IL‐6 secretion in older mice may diminish the normal inhibition on IL‐1α and subsequent TNFα production. The small rise in IL‐10 in the elder mice may serve as a compensatory mechanism to lower IL‐1α due to diminished IL‐6 response. It may also contribute towards the severely lowered KC production, as is seen with the inhibition of human KC homologue IL‐8 by elevated IL‐10. The combination of radically lower IL‐6 and KC secretion, and increased IL‐1α and TNFα may produce an environment favoring excessive inflammatory, yet reduced chemotactic responses to pathogen exposure in older mice.

Support or Funding Information

Supported by NIGMS R01GM118895‐01 and the BK and Betty Stevens Endowment.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PGC‐1α Overexpression Increases Lysosome Abundance and Autophagy in Dystrophic Skeletal Muscle
Authors: Ludwig Amanda , Spaulding Hannah Rose , Hudson Matthew , Selsby Joshua
Published: June 2021

Duchenne muscular dystrophy is a severe muscle wasting disease caused by the absence of functional dystrophin protein. We have previously established that increased peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α) pathway activity is therapeutic in dystrophic muscle. The purpose of this study was to determine the extent to which increased PGC‐1α pathway activity increased nuclear localization and activity of transcription factor EB (TFEB), a transcription factor that leads to lysosomal biogenesis. We hypothesized that increased PGC‐1α pathway activity would increase TFEB nuclear migration, lysosome abundance, and the restoration of impaired autophagosome degradation. To address this hypothesis, 3‐week‐old mdx mice were injected in one limb with an adeno‐associated virus that drives PGC‐1α gene expression and the other limb was injected with an empty capsid. Animals were sacrificed at 6 weeks of age and the gastrocnemii were harvested. PGC‐1α overexpression was confirmed by qPCR. Total TFEB protein abundance was similar between groups, however, nuclear TFEB was significantly increased in PGC‐1α treated limbs consistent with our hypothesis. We also discovered that Lamp2, a lysosomal marker, was significantly elevated in PGC‐1α treated muscle. Finally, abundance of autophagy‐related proteins were compared between treated and control gastrocnemii of mdx mice as well as to gastrocnemii from adult C57 mice. LC3 II, an indicator of autophagosome formation, was significantly elevated in PGC‐1α‐treated limbs compared to both the C57 and mdx‐control muscles. However, p62, an inverse correlate of autophagosome degradation, was similar between groups suggesting elevated degradation of autophagosomes in PGC‐1α treated limbs. These data suggest increased PGC‐1α pathway activity increased lysosomal degradation of autophagosomes in dystrophic muscle, likely through a TFEB‐mediated mechanism.

Support or Funding Information

Supported by the American Physiological Society.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PETCO2 in Women at Sea‐Level and Upon Acute Exposure to a Normobaric Hypoxic Environment Simulating 3,500 m
Authors: Yasukawa Michio , Degarmo Anna , Quiroga Leonardo , Muza Stephen , Kambis Ken
Published: June 2021

While prior work (Muza, J. Appl. Physiol. 91: 2001) suggests that women and men have similar responses to high altitude, no studies have attempted to develop gender specific predictive values for end‐tidal CO2 (EtCO2) in a normobaric hypoxic (NH) environment simulating acute exposure to an altitude of 3,500 m based on sea‐level (SL) EtCO2 values. As part of a larger study examining individual variability upon acute exposure to NH, EtCO2, heart rate (HR), and respiratory rate (RR) were measured in 107 subjects at sea level (SL) who, no sooner than two days later, entered a normobaric hypoxia chamber at approximating O2 partial pressures equivalent to those found at 3,500 m.

METHODS

Participants in this IRB‐approved study included: 63 informed volunteer female subjects (56 Treatment [NH]: 20.5±1.5 yr.; 165.8±6.9 cm; 63.011.1 kg; 22.93.3 BMI; 7 SHAM: 20.31.1 yr; 167.56.2 cm; 57.26.2 kg; 20.46.4 BMI); 44 informed volunteer male subjects (35 Treatment [NH]: 20.31.3 yr; 178.26.8 cm; 77.414.0 kg; 24.23.2 BMI; and 9 SHAM: 20.71.0 yr; 179.87.8 cm; 73.310.0 kg; 22.73.1 BMI). All subjects were tested at SL then again 2+ days later after entering a NH (FiO2=13.55%) chamber where EtCO2, HR, and RR were measured after 30 minutes of rest.

RESULTS

Bonferroni t‐tests revealed no difference in female or male EtCO2 between the SL and SHAM conditions while, as expected, there was a difference (P≤0.05) in EtCO2 between the SL and NH conditions in the female treatment group (37±3 vs. 36±3 mmHg) and the male treatment group (39±3 vs. 37±2 mmHg). RR and HR remained unchanged in the female and male groups in all conditions.

CONCLUSIONS

Using the following algorithm, we were able to predict EtCO2 in female subjects at the P 0.001 level of confidence [Actual vs. Predictive (EtCO2 at FiO2 = 13.55%) Y = 86.394 – 0.221X1‐ 0.541X2 (Where Y = EtCO2_HYPO, X1 = RR_NH, X2 = SpO2_NH, R2 = 0.520, P<0.001] while the predictive value of the male data was not significant (R2 = 0.099, P = 0.187). Therefore, female responsiveness to acute exposure to simulated altitude may be greater than that of male peers.

Support or Funding Information: The Chuo University Oversea Research Program, The Doug Morton/Marilyn Brown Endowment for Biomedical Research, The Foundation for Aging Studies and Exercise Science Research, and The Borgenicht Program
Swelling of the Subcutaneous Adipose, but not the Dermis, in Burn Wound Edema is Exacerbated by IV Fluids
Authors: Burmeister David M. , Chao Tony , Gomez Belinda I. , Little Joshua S. , Dubick Michael A. , Christy Robert J.
Published: June 2021

Intravenous (IV) resuscitation of burn patients has greatly improved outcomes and become a cornerstone of modern burn care. However, the large volumes of crystalloid given can lead to various comorbidities such as compartment syndromes and acute respiratory distress syndrome. Simultaneously, it is well established that vascular leak leads to significant edema in the severely burned patient. The objective of this study was to begin to examine the interaction of edema of burn wounds and IV fluids. Anesthetized Yorkshire swine sustained 40% total body surface area (TBSA) full‐thickness contact burns with brass probes heated to 100°C placed on the skin for 30 seconds. Animals were recovered from anesthesia and randomized to one of four treatment groups: no IV fluids (n=6), IV lactated Ringer's (LR) resuscitation with the modified Brooke formula (MB; 2 ml/kg/%TBSA, n=6), 15 mL/kg/d of IV LR (LR: n=6), or 15 mL/kg/d fresh frozen plasma (FFP: n=6). Unburned sham animals (n=4) and injured animals were assessed for 48 hours, at which point animals were humanely euthanized and dorsal and ventral dermis was taken to determine wet to dry weight ratios. Additionally, 3 biopsies to include dermis and the entire subcutaneous fat were taken from both the dorsal and ventral aspects of the animal for histological determination of tissue thickness. Burn wound edema was demonstrated by a significantly higher wet:dry ratio in all groups compared to sham animals for dorsal (P=0.017) and ventral (P=0.044) dermis. However, there was no difference amongst the various IV regimens. Moreover, neither burn nor IV fluids induced a difference in the thickness of the dermis on dorsal or ventral locations. In contrast, the thickness of the subcutaneous fat was significantly elevated in both dorsal (P=0.028) and ventral (P=0.0045) samples, depending on IV fluid regimen. Thickness of the dorsal fat layer was 1865 ± 327 μm, 2170 ± 205 μm, 3466 ± 421 μm, 2452 ± 271 μm, and 2707 ± 302 μm for Sham, no IV, MB, LR, and FFP groups, respectively. Thickness of the ventral fat layer was 1557 ± 164 μm, 2728 ± 184 μm, 2618 ± 172 μm, 2368 ± 236 μm, and 3329 ± 352 μm for Sham, no IV, MB, LR, and FFP groups, respectively. Collectively, these data demonstrate that volumes and types of IV fluids can alter burn‐induced edema in an anatomically‐dependent manner. Elucidating the specific drivers of burn wound edema may lead to personalized medicine approaches that can minimize comorbidities associated with over/under resuscitation of the burn patient.

Support or Funding Information

This study was supported by Military Burn Research Program Award# W81XWH‐16‐2‐0041.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of cervical spinal contusion on the upper airway resistance in the rat
Authors: Xu Ke‐Jun , Lee Kun‐Ze
Published: June 2021

Bronchopulmonary C‐fiber induced pulmonary chemoreflex is a critical component of the respiratory defense reflex and characterized by cessation of inspiratory airflow (i.e., apnea) and increase of upper airway resistance (i.e., laryngeal closure). Our previous reports demonstrated that apneic response was attenuated following cervical spinal cord injury; however, it remained unclear whether the upper airway response of pulmonary chemoreflex was also influenced by spinal injury. Accordingly, the present study was designed to investigate the response of laryngeal function to bronchopulmonary C‐fiber activation following unilateral high‐cervical spinal cord contusion in rats. Adult Sprague–Dawley male rats were received laminectomy or unilateral contusion at C2 spinal cord using the MASCIS Impactor (height: 6.25 mm). The respiratory airflow and subglottal pressure in response to intra‐jugular capsaicin (1.5 μg/kg)‐induced pulmonary chemoreflex were measured under spontaneously breathing condition at 3 days, 2 weeks or 6 weeks following spinal surgery. The result demonstrated that high cervical contusion caused a long‐term reduction in the tidal volume without alteration of laryngeal resistance at three time points after injury. However, capsaicin‐induced increase of subglottal pressure was substantially attenuated in contused animals at 3 days post‐injury, and partially recovered at 2 and 6 weeks post‐injury. These results suggested that cervical spinal cord injury not only influence the breathing pattern but also impact the upper airway function. The attenuated laryngeal closure response may unfavorably reduce the ability to prevent inhaling irritants and cause the pulmonary pathological condition following cervical spinal cord injury.

Support or Funding Information

MOST 105‐2628‐B‐110‐002‐MY3

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence that the preBotzinger complex does not participate in cough rhythmogenesis in the anesthetized cat.
Authors: Shen Tabitha Y. , Poliacek Ivan , Kotmanova Zuzana , Musselwhite M Nicholas , Rose Melanie J. , Bolser Donald C.
Published: June 2021

The nucleus tractus solitarius (NTS) participates in cough phase timing in the anesthetized cat. The preBotzinger complex (preBotC) is key for breathing rhythmogenesis, but the role of this area in regulating cough rhythmogenesis is still unknown. We hypothesized that excitation of neurons in the preBotC would modulate shorten the duration of the inspiratory cough, consistent with the effects during breathing. To test this hypothesis, we unilaterally microinjected 5 mM DL‐Homocysteic acid (DLH) into the preBotC in anesthetized, spontaneously breathing adult cats (n=7). In 3 of these animals, 15 mM DLH also was injected into the preBotC. Criteria for data inclusion were multi‐unit inspiratory neuron recording at the injection site, apneusis or tachypnea as a result of DLH injection, and histological verification of the injection site. We recorded esophageal pressure (EsPr) and electromygrams (EMG) from inspiratory (parasternal and diaphragm) and expiratory (internal oblique) muscles. Tracheobronchial cough was induced via mechanical stimulation of the intrathoracic trachea. DLH microinjection significantly altered the breathing response: breathing frequency increased (18.0±1.4 to 46.9±7.4 bpm) due to changes in both inspiratory time (TI − 1.04±0.5 to 0.75±0.76 s) and expiratory time (TE −2.34±0.19 to 0.63±0.16 s). Diaphragm EMG magnitude did not change significantly. Microinjection of DLH (5 mM) into the preBotC did not alter cough phase timing (cough frequency: 23.3±4.0 to 22.9±4.4 coughs per minute; TI − 1.27±0.13 to 1.31±0.10 s; TE1 −0.26±0.7 to 0.26±0.07 s; TE2 − 1.35±0.24 to 1.29±0.49 s). While the magnitude of cough pressures and diaphragm EMGs were not altered (EsPr – control 49.4±5.8 to DLH 35.7±5.2 ; EMG amplitude ‐ control 70.8±3.5 to DLH 65.1±6.5), cough expiratory motor drive was significantly lower (abdominal – control 62.1±5.5 to DLH 40.0±5.9%, p <0.04). Microinjections of 15 mM DLH into the preBotC eliminated cough. In the 3 animals that had both 5 and 15 mM DLH injections, there was a trend for a longer recovery time for cough with the higher concentration of DLH (5 mM – 33.8±10.4 s; 15 mM – 66.8±27.5 s). Unlike for breathing, excitation of neurons via DLH in the region of the preBotC does not affect phase timing for cough, although DLH appears to exhibit a dose‐dependent cough suppression.

Support or Funding Information

Supported by NIH HL 103415 and 1OT20D001983.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The influence of CO2 on spatiotemporal features of mechanically‐induced coughing in anesthetized cats
Authors: Musselwhite M Nicholas , Shen Tabitha Y. , Rose Melanie J. , Watts Joseph , Bolser Donald C.
Published: June 2021

Cough results in a dramatic increase in ventilation in part due to large increases in tidal volume. This increase in ventilation can cause severe hyperventilation during repetitive cough bouts. The purpose of this study was to quantify how reversal of hyperventilation during repetitive coughing affects the cough motor pattern. Prior studies have shown that increasing CO2 well above eupneic levels can suppress cough in anesthetized humans (Nishino 1989) but the effects of hypocapnia on the behavior are less well understood. Cough was elicited in anesthetized, spontaneously breathing adult cats (n=9) via a flexible cannula inserted into the trachea for 60 seconds. Animals were subjected to both a control condition, where cough‐induced hyperventilation was not perturbed (ETCO2 12±1, arterial CO2 17±1 mm Hg), and an “isocapnic” condition, where inspired CO2 was increased so that ETCO2 during cough approximated eupneic levels (ETCO2 32±1, arterial CO2 28±1 mm Hg). There was no change in cough number between control (9±1) and isocapnic (11±1) conditions. Further, the magnitudes of diaphragm, internal oblique and parasternal EMGs were unchanged under isocapnic relative to control conditions. There also was no significant change in intrathoracic pressures between the two conditions (control 53±12 cm H2O vs isocapnic 47±12 cm H2O). However, there was a decrease in the intensity of the maximal cough effort during the addition of CO2 (control 71±13 cm H2O vs isocapnic 62±13 cm H2O, p<0.05). There was a trend for inspiratory time during cough to be slightly reduced with added CO2 (p=0.07). The E1 phase of cough (control 0.5±0.03 s vs isocapnic 0.5±0.02 s) and E2 phase of cough (control 2±0.5 s vs isocapnic 2±0.3 s) were unchanged under isocapnic conditions. There also was a significantly shorter decrease in the time to first breath following the cessation of coughing in the isocapnic trials (control 16±4 s vs isocapnic 5±1 s, p<0.02). The results indicate that CO2 at or below the eupneic range does not strongly alter the motor pattern of coughing in anesthetized cats. Cough‐induced hyperventilation appears to be solely a consequence of the acceleration of pulmonary gas to accomplish ejection of foreign materials from the airways, rather than an important regulatory feature that modulates the behavior.

Support or Funding Information

Supported by NIH HL 103415 and 1OT20D001983.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RESPIRATORY CHANGES IN OFFSPRING OF HIGH FAT DIET FED DAMS
Authors: Bassi Mirian , Barbosa Rafaela Moreira , de Sa Jessica M. , Menani Jose Vanderlei , Colombari Eduardo , de Almeida Colombari Debora Simoes
Published: June 2021

Several studies have demonstrated that early life over‐nutrition, such as maternal obesity, is associated with increased risk of cardio‐metabolic diseases. In the present study respiratory changes were investigated in offspring of high fat diet (HFD) fed dams. Adult female Holtzman rats (250 g) were fed with standard diet (SD) or HFD during 6 weeks before, during gestation and lactation. After weaning, male offspring of SD dams (O‐SD, n = 7–12) or HFD dams (O‐HFD, n = 7–12) were fed with SD. The results showed that O‐HFD (P28–32) presented an increase in retroperitoneal adipose tissue (0.15 ± 0.01, vs O‐SD: 0.08 ± 0.01 g/100 g of bw, p < 0.05), a higher plasma triglycerides levels (141 ± 11, vs. O‐SD 49 ± 10 mg/dl, p < 0.05) and insulin intolerance (1.28 ± 0.30, vs O‐SD: 2.25 ± 0.15 % of glucose disappearance/min, p <0.05). At P40, baseline ventilation was higher in the O‐HFD group (1.200 ± 48, vs O‐SD: 924 ± 58, ml.kg‐1.min‐1, p <0.05) due to an increase in tidal volume (9.12 ± 0.29, vs O‐SD: 7.43 ± 0.47, ml.kg‐1, p <0.05). An increased response to hypoxia was also observed in O‐HFD (1.634 ± 80, vs O‐SD: 1.325 ± 50 mL.kg‐1.min‐1, p <0.05). The results demonstrated that young offspring of obese dams have metabolic dysfunctions. The data also demonstrated that these offspring have baseline ventilatory changes as well as an exaggerated response to peripheral chemoreflex activation.

Support or Funding Information

FAPESP (2015/234677), CNPq (425586/2016‐2).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intakes of vegetables and red meat in relation to circulating nitrate levels
Authors: Wu Tianying , Sonoda Samantha , Fan Guochang
Published: June 2021

Objective

Our study aimed to determine the associations between intakes of vegetables and red meat (processed and unprocessed) and circulating nitrate levels.

Design

Using a cross‐sectional study design.

Setting and Participants

A secondary analysis using data from the National Health and Nutrition Examination Survey (NHANES) and from the Health Professionals Follow‐up Study (HPFS). 5,058 adults (men and women) in the 2005–2006 NHANES and 1,260 men from HPFS were analyzed.

Measurements

The non‐quantitative food frequency questionnaire (NFFQ) and two 24‐hr recalls were collected and urinary nitrate was measured in the NHANES. Plasma nitrate was measured and a semi‐quantitative FFQ (SFFQ) was collected in the HPFS.

Results

Using three dietary assessment instruments (NFFQ, SFFQ and average 24‐hr recalls), we found a similar trend when assessing the associations of vegetables and red meat with circulating (plasma and urinary) nitrate. Across instruments, circulating nitrate levels were 15–33% higher for participants in the highest, when compared to the lowest, quintile of vegetable intake (p<0.001). Circulating nitrate levels were 8–16% lower for participants in the highest, when compared to the lowest, quintile of red meat intake (p<0.001).

Conclusion

Our study has demonstrated a positive association between vegetable and circulating nitrate and an inverse association between red meat and circulating nitrate. As nitrate has been demonstrated to be beneficial for preventing several chronic diseases, our results provide valuable information for studying dietary etiologic factors associated with nitrate and chronic diseases.

Support or Funding Information

NCI KO7 award

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Multiscale Analysis of Early Cardiogenesis Following VEGF Perturbations
Authors: Rongish Brenda J. , Aleksandrova Anastasiia , Filla Michael B. , Kosa Edina , Little Charles D. , Petersen Alan , Czirok Andras , Rugonyi Sandra , Rykiel Graham , Lansford Rusty
Published: June 2021

Congenital Heart Defects (CHDs) are multi‐factorial in origin, and there is a critical need to identify other contributing factors in the development of CHDs. Endogenous factors that regulate development, such as altered levels of cardiovascular growth factors in the embryo/fetus, may initiate or contribute to CHDs. Our objective was to determine if alteration of Vascular Endothelial Growth Factor (VEGF) levels at key developmental periods leads to reproducible cardiovascular defects, which may be correlated with underlying altered cell behavior, physiological parameters and/or molecular signaling events. We used ex ovo culture and imaging systems that capitalize on the experimental accessibility of quail embryos, which, like humans, have four‐chambered hearts. In order to alter VEGF signaling, the following were introduced into quail embryos: recombinant human VEGF, exogenous VEGF‐GFP fusion protein, and inhibitors of VEGF receptor tyrosine kinase signaling and downstream effectors of VEGF. Time‐lapse imaging (KUMC) and optical coherence tomography (OHSU) revealed abnormal phenotypes between Hamburger and Hamilton (1951) stages 4 to HH38 – encompassing vasculogenic (pre‐circulatory) stages through primordial valve formation. Hemodynamic parameters were measured and related to structural defects at circulation stages. These studies showed the effects of altered VEGF signaling on cardiac and endothelial cellular behavior, organogenesis of four‐chambered hearts and great vessels, and cardiac function. Further, we identified potential molecular pathways involved in VEGF signaling. The ability to observe the behavior of endothelial, endocardial, and myocardial precursors, gross cardiac phenotypes and hemodynamic changes following experimental manipulation of VEGF allowed a careful analysis of the role of VEGF at multiple length scales, and over time, during cardiovascular morphogenesis. We conclude that VEGF signaling, when dysregulated, leads to detrimental cardiovascular malformations and altered cardiac cellular behavior and function. This work has helped to determine the underlying mechanisms of VEGF's role and actions in cardiac and vessel development, as well as identified structural and functional cardiovascular defects that are attributable to aberrant VEGF signaling; which are similar to CHDs observed in humans.

Support or Funding Information

American Heart Association 16GRNT29840002, the Molecular Regulation of Cell Development and Differentiation COBRE, P30 GM122731, and the University of Kansas School of Medicine Bridging Award.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Professional Identity: What is it, when is it formed, and what is the role of anatomy in this developmental process?
Authors: Byram Jessica N.
Published: June 2021

Professional identity formation (PIF) has been described as an essential process by which medical students internalize knowledge, skills, and values of the profession to ultimately become physicians. This session will introduce the developmental process of PIF in medical students and will discuss its relationship to professionalism. The described model and its role in anatomy education is grounded in the results of a thematic analysis of nine medical students who shared how their training and anatomy education experiences shaped their professional identities and perceptions of professionalism. Overall, the evidence suggests anatomy educators can foster PIF by promoting a complex and reflective understanding of professionalism and by encouraging students to enact those professional values as components of their professional identities during team‐based experiences in both laboratory and didactic settings. Specifically, the values of respect, both for the patient (donor) and the team, duty, and good communication were found to be fostered through team‐based cadaveric dissection.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nonsense‐mediated mRNA decay and human disease: Genome guardian and executor
Authors: Maquat Lynne E.
Published: June 2021

Much progress has been made on how nonsense‐mediated mRNA decay (NMD), which we first described for humans in 1981, controls the quality of gene expression by detecting and rapidly degrading aberrant mRNAs that contain a premature termination codon1. Our studies of NMD have led to the discovery of the pioneer round of translation, the post‐splicing “mark” on newly synthesized mRNAs – later named the exon‐junction complex (EJC) with Melissa Moore – the mechanistically related and competing Staufen‐mediated mRNA decay pathway2,3, including new roles for SINEs4, and most recently a microRNA decay pathway5,6. We tracked individual cellular transcripts in collaboration with Rob Singer to confirm our results from the mid‐1990's indicating that NMD for a number of mRNAs occurs on the cytoplasmic side of the nuclear envelop7. Our data provide explicit evidence that proteins acquired by newly synthesized mRNAs in the nucleus, including the cap‐binding protein CBP80 and constituents of the EJC, are critical for mRNA quality control via translation in the cytoplasm. We have also described the molecular mechanism for how NMD targets are discriminated from other transcripts: the central NMD factor – the ATP‐dependent RNA helicase UPF1 – preferentially associates with mRNA 3′‐untranslated regions (3′ UTRs) in a way that correlates with 3′ UTR length and the presence of a 3′ UTR EJC8,9. Importantly, NMD also targets ~10% physiologic mRNAs that are key to maintaining cellular homeostasis in a changing environmental milieu. For example, we reported that a sufficient level of DNA damage induced by commonly used frontline chemotherapeutics inhibits NMD by triggering the caspase‐mediated cleavage of sub‐stochiometric amounts of UPF1, thereby upregulating the half‐lives of mRNAs that include those encoding proteins promoting apoptosis10. Notably, the modest inhibition of NMD promotes but is not sufficient for programmed cell death. These and other results will be discussed.

Support or Funding Information

Work on nonsense‐mediated mRNA decay in the Maquat lab is supported by NIH R01 GM 059614 to L.E.M.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chemoinformatics Profiling of the Bioactive Fraction of Bizzy Nut
Authors: Gray wesley , Philips Orville , Telles Caroline
Published: June 2021

Bizzy Nut status as a traditional natural medicine resulted from its reputation as a stimulant, increasing energy and strength, staving off hunger and a multitude of biological activities such as anti‐oxidation, anti‐cancer, and prevention of certain diseases. We have developed a method for fingerprinting and quantifying the anti‐prostatic activity existing in Bizzy Nut using liquid chromatography (LC) with a diode array detector and LC‐MS. Using this method, the chemical fingerprint of Biz‐2Fr.3 extract was established, in which the separation of more than five analytes were realized in about 30 mins, and 6–8 distinctive peaks were identified by LC/MS analysis. The linearity, precision, stability, repeatability, and accuracy of the method will clearly reveal any variations of Bizzy Nut samples produced in Jamaica in different years. To provide clues to the chemical identity of bioactive peaks (Biz‐2Fr.3), we applied LC‐MS because of its sensitivity, reproducibility, speed, and versatility. Analysis of Biz‐2Fr.3 using an Agilent 6210 Quadrupole LC‐MS with an Agilent Ultra‐2 fused silica capillary MS column in the negative ion mode resulted in three major peaks with retention times of 1.1, 5.8 and 7.5 minutes. Characterization of the molecular ions in peak one revealed the presence of the analyte, with m/z ranging from 120 m/z to 445 m/z. Peaks two and three, on the other hand, contained a single molecular ion with m/z of 181. The 1H NMR spectrum of Biz‐2Fr.3 contained peaks with the following chemical shifts, in ppm, and assignments: a stretch of H between 3.5–4ppm indicating the presence of straight chains CH, CH2 or CH3. Methyl hydrogens that are bonded to oxygen (eg. ROCH3) were observed between chemical shifts between 2.9–3.6 ppm. There is a possibility of the presence of C=C (eg. R2C=CHR) due to the shift between 4.0–4.5 ppm. The absence of the chemical shift between 6–8ppm indicates the absence of any aromatic hydrogen. The LC‐MS and NMR data suggested that we have three groups of compounds in the Biz‐2Fr.3 fraction.

Support or Funding Information

Grant support: U.S. Public Health Service grants R15AT006821, awarded by the National Center for Complementary and Integrative Health.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Connection between antimicrobial venom peptides and bacterial ATP Synthase
Authors: Syed Hiba , Ahmad Zulfiqar
Published: June 2021

Background

Antibiotic resistance is becoming a huge threat with devastating effects on public health and the global economy. Therefore, finding new alternatives to combat antibiotic resistant microbes is of paramount importance. Selective inhibition of bacterial ATP synthase provides a fascinating opportunity to deal with this ongoing problem. ATP synthase is vital for cellular energy production in almost all organisms from bacteria to man. Inhibition of ATP synthase can deprive cells of ATP resulting in cell death. Many peptides are known to have antimicrobial and antitumor properties. Earlier some peptide were shown to inhibit Escherichia coli ATP synthase establishing that the antimicrobial effects of peptides can be connected to the blocking of ATP generation in bacterial cells. Currently, our lab is exploring the connection between a variety of antimicrobial venom peptides and E. coli ATP synthase.

Method

The growth properties of wild‐type and mutant E. coli strains along with null strains were verified on fermentable and non‐fermentable carbon sources before harvesting cells in minimal media to isolate ATP synthase. Membrane bound F1Fo ATP synthase inhibitory studies and E. coli cell growth assays were performed in presence of varied concentrations of natural and modified venom peptides. A null control with deleted ATPase gene was also used in all the studies.

Results

Our preliminary results show that the natural and modified venom peptides affected degree of inhibition. It seems that inhibitory potency of venom peptides can be enhanced by addition of a c‐terminal NH2 groups. We also observed that venom peptides follow a differential pattern of inhibition between wild type and mutant ATP synthase. Our initial results also indicate that incremental additions of positive charges can augment the extent of inhibition. Our early results suggest a feasible connection between the antimicrobial properties of venom peptides and bacterial ATP synthase. We are finalizing our preliminary results and we are investigating the synergetic effects of venom peptides on ATP synthase.

Conclusions

Apparently, antimicrobial venom peptides inhibit E. coli ATP synthase and bind at the βDELSEED‐motif of ATP synthase. Our preliminary data suggests that the inhibitory potency of venom peptides can be augmented through structural modification of venom peptides. Also, selective inhibition of ATP synthase by venom peptides offers a valuable alternative to combat antibiotic resistant bacterial infection.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genetic correction of structural variations in patient‐derived iPSCs using CRISPR/Cas9
Authors: Park Chul‐Yong , Kim Dong‐Wook
Published: June 2021

Genome engineering technology using engineered nucleases has been rapidly developed, enabling the efficient correction of simple mutations. However, the precise correction of structural variations such as large inversions remains limited. Here, we describe a detailed procedure for the modeling or correction of large chromosomal rearrangements and short nucleotide repeat expansions using engineered nucleases in human induced pluripotent stem cells (hiPSCs) from a healthy donor and patients with structural variations. This protocol enables the correction of large inverted segments and short nucleotide repeat expansions in diseases such as hemophilia A, fragile X syndrome, Hunter syndrome, and Friedreich's ataxia.

Support or Funding Information

C.Y. P. was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2016R1C1B1008742). D.W.K. was supported by grants from the National Research Foundation of Korea funded by the Ministry of Science & ICT (the Bio & Medical Technology Development Program, 2017M3A9B4042580) and from Korean Ministry of Health & Welfare (HI15C0916).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mass Spectrometry based Proteomics Investigation of Induced Obstructive Sleep Apnea (OSA) in Rat Atria
Authors: Darie Costel C , Channaveerappa Devika , Wormwood Kelly L. , Lux Jacob , Panama Brian
Published: June 2021

Introduction

Obstructive sleep apnea (OSA) affects up to 24% of the adult population and is associated with several atrial diseases. OSA is characterized by transient cessations in respiration lasting >10 seconds as a result of narrowing of the upper airway during sleep. An estimated 20% of adults have mild OSA and 7% of adults have moderate to severe OSA, with 85% of patients remaining undiagnosed. Although clinical evidence linking OSA to proarrhythmaic atrial changes is well known, the specific molecular mechanisms by which OSA causes atrial disease remain elusive. To study the OSA‐induced cardiac changes, we have initiated a recently developed rat model which closely recapitulates the characteristics of OSA.

Methods

Male Sprague Dawley rats, aged 50–70 days, received surgically implanted tracheal balloons which were inflated to cause transient airway obstructions. Apnea groups experienced 60 apneas per hour of either 13 seconds (moderate apnea) or 23 seconds (severe apnea) for 2 weeks and 8 hours per day. Control rats received surgeries but no inflations. Proteomics analysis was done on the rat atria homogenates to identify dysregulated proteins in moderate and severe apnea when compared to control. SDS‐PAGE was performed to separate the proteins and the peptide mixtures after trypsin digestion were analyzed by a Nano Acquity UPLC coupled with Xevo G2 Mass Spectrometer. Data analysis was done using ProteinLynx Global Server (PLGS 2.4), Mascot server and Scaffold 4.1 software.

Results

Rats were given transient apneas for two durations: 13 and 23 s, we termed ‘moderate’ and ‘severe,’ respectively. These two apnea durations are within the range of those experienced by people with OSA. ECG recordings showed that the P wave durations (associated with left and right atrial depolarization) and T wave amplitudes (associated with ventricular repolarization) were increased by 2 weeks of chronic OSA. This was important since OSA patients show atrial electrical remodeling. The apnea model recapitulated the pathophysiological characteristics of OSA. The proteomics analysis revealed that 3 of the 9 enzymes in glycolysis and 2 proteins related to oxidative phosphorylation were down‐regulated in the severe apnea group. In contrast, several structural and pro‐hypertrophic proteins were up‐regulated with chronic OSA. The data suggests the chronic OSA causes proteins changes which lead to cessation of glycolysis, a diminished capacity to generate reducing equivalents (i.e. NADH) as well as promotion of cardiac hypertrophy.

Support or Funding Information

CAMP matching funds

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the intersection of cellular zinc and kinase activity with single cell imaging
Authors: Anson Kelsie J , Palmer Amy
Published: June 2021

Zinc is an essential metal in biology, playing a structural or functional role in up to 10% of the human proteome. Certain cell types experience fluctuations in labile zinc, and these zinc dynamics have been shown to play a role in cellular signaling. Neurons in the hippocampus concentrate synaptic zinc, but its function in signal transduction and neuronal development is not fully understood. We use and develop fluorescent tools to explore how physiologically‐relevant changes in labile zinc impact intracellular kinase signaling in single cells and in the context of neuronal signaling. In this work, we demonstrate that increases in labile cellular zinc lead to greater activation of ERK kinase and explore the mechanism of this activation. The impact of zinc on other signaling pathways are also explored using fluorescent biosensors and kinase activity profiling. Understanding how zinc impacts cell signaling pathways is fundamental to characterizing the effects of zinc fluctuations in a variety of cell types.

Support or Funding Information

NSF Graduate Research Fellowship and NIH T32 Biophysics Training Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Double‐stranded RNAs Attenuate Interferon Response via Parkin‐mediated MAVS Ubiquitination
Authors: Kim Seong‐Jun , Ahn Dae‐Gyun , Syed Gulam Hussain , McMillan Aimee , Thomas Sunil , Gale Michael , Kim Bum‐Tae , Siddiqui Aleem
Published: June 2021

Mitochondria play a central role in innate immune signaling and are primarily targeted by many viruses which have evolved elaborate mechanisms to evade host antiviral response. Recent studies demonstrate that viruses explicitly alter mitochondrial dynamics and mitophagy to evade innate immunity and maintain viral persistence. Here we report that Parkin and an upstream regulator, PTEN‐induced putative kinase 1 (PINK1), beyond their recognized role in mitochondrial quality control and Parkinson's disease, also serve as modulators of mitochondrial antiviral signaling protein (MAVS)‐mediated host innate immune response. MAVS serves as a mitochondrial platform for transduction of signal from various RIG‐I‐like receptors (RLRs) that sense pathogen‐associated molecular patterns (PAMPs) to orchestrate type I interferon (IFN)‐α/β production in response to viral infection. We show that the synthetic double‐stranded (ds) RNA mimics, poly(I:C) and 5′ppp‐dsRNA, potently induce Parkin translocation to the mitochondria, leading to Parkin‐dependent K63‐linked polyubiquitination of MAVS, eventually resulting in the attenuation of type I IFN production. Interference of Parkin and PINK1 expression led to a distinct elevation of type I IFN production on stimulation with viral RNA mimics or infection with RNA viruses. In agreement, primary mouse embryonic fibroblasts (MEFs) derived from PINK1 knockout mice (PINK1−/−) staged more robust type 1 IFN response compared to wild type PINK1 MEFs on stimulation with viral RNA mimics. This study unravels the yet unidentified function of Parkin and PINK1 in the modulation of MAVS‐dependent innate immune signaling and depicts how the RNA viruses exploit them to evade innate immunity.

Support or Funding Information

This work was supported in whole or in part by the National Institute of Health Grants DK077704 (to A.S.), AI085087 (to A.S.), AI104002 (to M.G.), AI100625 (to M.G.), Michael J Fox Foundation grants (to A.S. and S.J.K.), and National Research Council of Science & Technology (NST) grant by the Korea government (MSIP) CRC‐16‐01‐KRICT (to S.J.K. and B.T.K.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phenotype analysis of Myo10 knockout mice lacking the motorized full‐length, but not the brain‐specific headless isoform
Authors: Bachg Anne Christina , Horsthemke Markus , Skryabin Boris V. , Klasen Tim , Nagelmann Nina , Faber Cornelius , Woodham Emma F. , Machesky Laura M. , Bachg Sandra , Stange Richard , Jeong Hyun‐Woo , Adams Ralf H. , Bähler Martin , Hanley Peter J.
Published: June 2021

Unconventional myosin‐X (Myo10) is a MyTH4‐FERM and PH domain containing actin‐based motor protein which is widely expressed among vertebrate tissues. MyTH4‐FERM myosins are associated with protrusions enriched in bundled actin filaments. Full‐length (fl) Myo10 is best known for its localization to the tips of filopodia and for its ability to induce and regulate the formation of these dynamic finger‐like structures. At the cellular level, Myo10 has been implicated in diverse functions, including oriented cell migration, axon guidance, melanosome transfer, spindle assembly and phagocytosis. However, the physiological functions at the organismal level of this filopodial cargo carrier are not clear. We studied the phenotypes of Myo10 reporter knockout mice (Myo10tm2), to elucidate Myo10 function in vivo. Using Western blot analysis, we showed that in homozygous knockout Myo10tm2/tm2 mice, the expression of motorized fl Myo10 was deleted, but the brain‐specific headless (hdl) isoform was not abolished. Macroscopic examination of Myo10‐deficient mice revealed ventral coat pigmentation defects (100% penetrance) and a limb malformation, syndactyly, with a high penetrance of >95%. 24% of homozygous KO embryos developed exencephaly, a severe neural tube closure defect that is lethal. Magnetic resonance imaging (MRI) of isolated brains revealed, in contrast to mice lacking the Myo10 cargo protein DCC, intact cerebral commissures. Furthermore, Myo10 KO mice consistently displayed bilateral persistence of the hyaloid vasculature, visualized by MRI and retinal whole mount preparations, whereas retinal vascularization, phagocytosis and chemotactic behavior were not impaired. Confocal spinning disk microscopy confirmed that the hdl isoform of Myo10 does not induce filopodia and we found out that hdl Myo10 strongly localizes to the plasma membrane in dependency of its PH domains, supporting the auto‐inhibition model in which head‐tail interactions of fl Myo10 abolishes inactive monomer binding to PIP3‐rich membranes. In conclusion, Myo10 is important for normal vertebrate development, both prenatal (neural tube closure and digit separation) and postnatal (hyaloid regression).

Support or Funding Information

Deutsche Forschungsgemeinschaft (DFG)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.