bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantifying the Genotype to Phenotype Map in Developing Mice
Authors: Richbourg Heather A , Young Nathan , Takenaka Risa , Marcucio Ralph
Published: June 2021

Objective

Quantify phenotypic variance of face shape within mice after ablation of the gene encoding the E3 ubiquitin ligase, nitric oxide synthase‐interacting protein (Nosip).

Methods

Mouse embryos from a nitric oxide synthase‐interacting protein (NOSIP) knock‐out model were collected at e12.5 via cesarean section (n=39). Two‐dimensional images were taken of the head, and DNA was collected from the limb bud for genotyping via PCR. Additionally, skulls were collected post‐natally (23 – 263 days of age (n=25)), and were scanned via microCT (55kV, 0.2 second integration time, 145uA). Anatomical landmarks were manually placed along the skull, creating three‐dimensional coordinates. Data were corrected for rotation and scaling differences through a Procrustes transformation using MorphoJ. Standard geometric morphometric analyses were subsequently performed, and data were compared using principal components analyses (PCA).

Results

All wildtype (WT) embryos appeared to have normal phenotypes (n=8), but there was a large phenotypic variation in mutants (Fig. 1), ranging from normal (n=19) to severe forebrain malformations (n=7) or resorption (n=6). Adult HET mice (n=9) also showed phenotypic variation, including lateral malocclusion (n=1), opaque eyes (n=4), or no eyes present (n=1). After correcting for skull size, there was minimal variation within the WT adults, but there was specific separation between HETs with normal phenotypes and abnormal phenotypes. Animals were separated by PC1 (left‐side of snout width asymmetry; 24.5% of total variation) and PC2 (snout length; 16.3% of total variation) (Fig. 2). Along the PC1 axis, three HETs are clustered within the WTs; however, PC2 separates the two HETs with an abnormal phenotype (no eyes and lateral malocclusion), while the remaining HET within the WT cluster has a normal phenotype. Visualizing the same PCA relative to sex does not distinguish unique clusters, suggesting that phenotype is not related to sex.

Conclusions

This model presents a unique opportunity to understand and study phenotypic variation within a HET genotype, as well as when comparing HETs to WTs, producing variation in both embryos and adults. The malformations present in the HET embryos resembles a sonic‐hedgehog loss‐of‐function phenotype, so more work will be performed to determine the affected signaling pathways and cellular processes.

Support or Funding Information

Travel support was given by the American Association of Anatomists. This project is supported by NIH funding (R01DE01823, R01DE01963,R01DE021708).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Spa47 Oligomerization Plays a Key Role in ATPase Activation and Function in the Shigella Type III Secretion System
Authors: Burgess Jamie Lee , Burgess Robert Alan , Dickenson Nicholas
Published: June 2021

Several Gram‐negative pathogens, including Shigella, utilize a conserved type three secretion system (T3SS) for infection. The type three secretion apparatus (T3SA) provides a unidirectional conduit for injection of effector proteins from the bacteria to the host cell cytoplasm. The conserved T3SS ATPase found at the base of the T3SA is believed to provide the energy for apparatus formation and effector secretion, however, the mechanisms of ATP hydrolysis, effector secretion, and apparatus formation by the T3SS ATPase remain unclear. We have recently shown that Spa47, the T3SS ATPase of Shigella, forms a unique activated oligomeric species during the course of purification. Using our recently published crystal structure of Spa47 as a guide, we modeled an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. To explore the dynamics of Spa47 protomer interaction in vitro and in vivo, we performed a series of ATP hydrolysis, fluorescence microscopy and Shigella invasion experiments. We observe a positive Hill coefficient with the monomeric Spa47 species that is not present with the oligomeric Spa47 species in vitro. Interestingly, we continue to see a positive Hill coefficient even with the addition of engineered ATPase inactive Spa47 point mutants to WT Spa47 monomer species. We have utilized live cell fluorescence microscopy to show that these ATPase inactive Spa47 point mutants localize to the T3SA though they do not support secretion. Furthermore, each of the ATPase inactive Spa47 point mutants as well as an oligomerization deficient Spa47 construct exhibits a dominant negative phenotype. Together, these results suggest that while ATP hydrolysis is not the major driving factor for Spa47 localization to the apparatus base, the Spa47 oligomer may undergo a series of concerted ATP hydrolysis events that are critical for apparatus function, similar to ATP synthase. As we better understand the nuances of T3SS ATPase mechanism and function, the results of this work will provide a strong platform for future work to perhaps target the T3SS ATPase for novel therapeutics.

Support or Funding Information

National Science Foundation Graduate Research Fellowship and National Institute of Health (1R15AI124108‐01A1)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Homarus Gemmarus: Seriated CT‐Scan and Re‐Constructions: Horizontal, Sagittal and 3D
Authors: le floch‐prigent patrice pierre , Verdeille stéphane
Published: June 2021

The european lobster is a large, common, marine, crustacean decapod.

Material

An entire animal was acquired still alive and deeply frozen in extension of the abdomen. It was 26,5 cm long; 7 cm width at its maximal dimension of the cephalo‐thorax. The CT‐scan apparatus was a Siemens somatom sensitive, whole body apparatus with 128 stellar detectors.

Methods

The entire animal was CT‐scanned all along the rostro‐caudal axis, in axial cross‐sections and reconstructed in the two other planes (horizontal and para‐sagittal) and in 3‐ dimensional ones. The frozen lobster was laid in prone position on the radiological table. It was CT‐scanned with an acquisition (100 kV, 18 mAs) in the axial plane: the axial cross‐sections (every 0,8 mm, 1 mm thick) were joined, and frontal. Two different penetrations were used: B30 for the soft parts and B70 for the hard parts (cartilage and extra‐skeleton).

Results

500 frontal cross‐sections were obtained from the native axial acquisition. 120 para‐sagittal cross‐sections were reconstructed from the preceding ones, and 80 horizontal ones as well. 11, 3D reconstructions were realized with several incidences: superior, inferior, rostral, caudal, postero‐superior, antero‐inferior, antero‐lateral and superior (right and left), lateral (right and left), median sagittal.

Conclusion

The seriated CT‐scan and the reconstructions afforded very numerous morphological elements in Homarus gammarus, and also in the study of their relationships. The muscular masses presented very few differenciation. The rare cartilaginous structures were well vizualized. A voluminous aerial poach at the upper and rostral part of the cephalo‐thorax corresponded to the two parts of the stomach: cardial (rostral) and pyloric (caudal) ones. The reconstructions were very contributive.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prionace glauca (Pole shark): seriated CT‐scan and reconstructions: horizontal, sagittal and 3D
Authors: Le Floch‐Prigent Patrice Pierre , Verdeille Stéphane
Published: June 2021

The pole shark (Prionace glauca, Linnaeus 1758) is a common, pelagian condrichtyan.

Material

The entire, rostral extremity (28,4cm long) of an adult specimen was CT‐scanned all along the rostro‐caudal axis, in axial cross‐sections and reconstructed in the two other planes and in 3‐dimensional ones. The CT‐scan apparatus was a Siemens somatom, whole‐body apparatus with 64 detectors.

Methods

The frozen shark's head was laid in prone position on the radiological table. It was CT‐scanned with an acquisition (100kV, 18mAs) in the axial plane. The frontal cross‐sections (every 0,8 mm, 1 m thick) were joined. Two different penetrations were used: B30 for the soft parts and B70 for the hard parts (cartilage and extra‐skeleton).

Results

355 frontal cross‐sections were obtained from the native, axial acquisition. 100 para‐sagittal cross‐sections were reconstructed from the preceeding ones and 100 horizontal ones as well. 16, 3D reconstructions were realized with several, different incidences and penetrations.

Conclusion

The seriated CT‐scan and the reconstructions gave numerous morphological elements in Prionace glauca, as well as in the study of their relationships. The muscular masses presented very few differenciation. The cartilaginous structures were well vizualized, as the eye and the central nervous system, the teeth were particularly easy to study, on the reconstructions, noticeably the successive raws in the inferior jaw.

We thanks the “Le Venec” Company for the delivery of the animal material and Mr Lionel Feuillassier (Marinarium, Muséum National d'Histoire Naturelle), both in the town of Concarneau (Brittany, France).

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 Warm Ischemic Time on the Viability of Deceased Organ Donor Derived Bone Marrow
Authors: Sherry Aubrey M. , Johnstone Brian H. , LaFontaine Michael , Woods Erik J.
Published: June 2021

It was recognized 60 years ago that deceased donor bone marrow (BM) could be a useful source of viable hematopoietic stem cells (HSCs) for transplantation. Validated procedures have since been published for the recovery of viable deceased donor HSCs and their safe use in immune tolerance clinical trials. It is the goal of Ossium Health to streamline the process of obtaining deceased donor derived BM on an industrial scale. It has been shown by previous studies that BM is tolerant to a wide range of cold and warm ischemic times during collection. CD34+ HSCs prefer a hypoxic environment and remain quiescent in vivo. This observation indicates that BM procured during routine organ and tissue recovery may remain viable for extended periods following cardiac arrest and, thus, will be suitable for transplantation. Ossium is evaluating the range of ischemia times to determine if organ and tissue donor bone marrow recovered using a standardized approach is suitable for transplantation.

Vertebral bodies (VBs) and ilia of 34 deceased organ donors were recovered by Ossium's network of organ procurement organizations (OPOs) over warm ischemia times ranging from 1:03–22:03 (hr:min). Recovered VBs were then cooled and shipped overnight on wet ice to Ossium Health, Indianapolis, IN, for BM recovery using grinding and elution methods. The CD34+ HSC population was enumerated using flow cytometric ISHAGE protocols and a mean viability over the entire ischemia range was determined to be 79.3 ± 3.7% (mean ± SEM) as determined by 7‐Aminoactinomycin D (7‐AAD).

While gross membrane viability is a useful measure, engraftment potential correlates strongly with functional viability as measured through colony forming unit (CFU) assay, and engraftment of transplanted units requires the presence of Granulocyte/Macrophage (GM) colonies specifically. To confirm that the deceased donor marrow would be suitable for engraftment, the CFU assay was performed and CFU‐GM were scored. Over the entire range of ischemic times, an average of 37.05 ±11.16 CFU‐GM (mean ± SEM) per 105 nucleated cells plated was observed.

Rapid neutrophil and platelet engraftment in living donor allogeneic grafts has previously been correlated with a dose containing CFU‐GM of 5.6 per 105 TNC in for an average patient of 70kg. The results of this preliminary study indicate deceased donor marrow can easily meet this requirement. These data demonstrate the potential to recover viable and functional HSCs after extended periods of warm ischemia; thus, establishing the feasibility and utility of banking cells from deceased organ and tissue donors.

Support or Funding Information

This work was funded by a Grant from the NIH‐NIAID (1R43AI129444‐01A1, PI: Woods, EJ), and Ossium Health, Inc.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tumor suppressor protein PALB2 has strand exchange activity independent of recombinase Rad51
Authors: Deveryshetty Jaigeeth , Kanikkannan Lakshmi , Korolev Sergey , Doisy E. A.
Published: June 2021

PALB2 (Partner and Localizer of BRCA2) is recombination mediator protein (RMP) involved in homology dependent repair (HDR) of double strand breaks (DSB). PALB2 works synergistically with BRCA2 by promoting Rad51 recombinase filament formation on single‐stranded (ss) DNA to initiate strand exchange essential for HDR. Numerous inherited PLAB2 mutations are reported to predispose to cancers such as breast and pancreatic cancers and rare genetic disease, Fanconi Anemia. However, the molecular mechanism PALB2 function is not well characterized. Here we report the DNA binding properties of PALB2 DNA‐binding domain (DBD), affinity and stoichiometry of interaction with various DNA substrates and identify residues involved in DNA binding. Further, we report an unanticipated activity of PALB2, where PALB2 promoted strand exchange in the absence of recombinase. This activity was enhanced in the presence of recombinase Rad51 and is abolished upon mutations of PALB2 DNA‐binding residues. Several properties also resemble those reported for Rad52. Our study demonstrates that PALB2 has multiple roles in homologous DNA repair.

Support or Funding Information

Siteman Cancer center

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antinociceptive and Gastrointestinal Effects of Mu/Kappa Opioid Mixtures in Rats
Authors: Minervini Vanessa , Osteicoechea Daniela C , France Charles P
Published: June 2021

Prescription opioids (mu receptor agonists) are commonly used to treat moderate to severe pain despite well‐documented adverse effects (e.g., constipation, abuse). Kappa opioid receptor agonists also have antinociceptive and adverse effects but might be useful for treating pain in drug mixtures. The use of smaller doses in mixtures might reduce or avoid adverse effects that occur with larger doses of either drug given alone. This study evaluated the kappa opioid receptor agonist spiradoline, the mu opioid receptor agonist morphine, and spiradoline:morphine mixtures (in 3:1, 1:1, 1:3, and 1:10 ratios) for their effects on antinociception and gastrointestinal (GI) motility to test the hypothesis that mixtures selectively enhance antinociceptive effects. Tail withdrawal latency from 50°C water was used to assay antinociception in one group of 8 male Sprague Dawley rats, and fecal output (count) was used to assay GI motility in another group of 8 male Sprague Dawley rats. Drugs were given i.p., with tests separated by at least 5 days. Spiradoline (3.2–32 mg/kg) and morphine (1.78–17.8 mg/kg) dose‐dependently increased tail withdrawal latency to a maximum of 20 sec; morphine (1–10 mg/kg) dose‐dependently decreased fecal count from 12 to less than 2 pellets. Spiradoline:morphine in 3:1 and 1:1 ratios had additive antinociceptive effects. The potency of morphine for antinociceptive effects was increased by spiradoline:morphine in 1:3 and 1:10 ratios (greater than predicted additivity). Conversely, in no instances was the potency of morphine increased for effects on GI motility. The potency of morphine to inhibit GI motility was either unchanged (e.g., 1:10) or decreased (e.g., 3:1) by spiradoline:morphine mixtures. Overall, spiradoline:morphine mixtures selectively reduce the dose of morphine needed to produce antinociceptive (therapeutic) effects. It remains to be determined whether spiradoline:morphine mixtures have other adverse effects (abuse, respiratory depression, and dysphoria) and whether interactions between spiradoline and morphine on these outcomes might be related to the ratio of each drug in mixtures.

Support or Funding Information

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 effects of intermittent dietary supplementation with fish oil on high fat diet‐induced enhanced sensitivity to the behavioral effects of dopaminergic drugs
Authors: Beltran Nina Marie , Hernandez‐Casner Caroline , Serafine Katherine
Published: June 2021

Eating a high fat diet can lead to obesity, type 2 diabetes, and dopamine system dysfunction. For example, rats eating high fat laboratory chow are more sensitive than rats eating standard chow to the behavioral effects of the dopamine receptor D2/D3 receptor agonist quinpirole. Specifically, quinpirole induces yawning in rats, and quinpirole‐induced yawning is enhanced in rats eating high fat chow as compared to rats eating standard chow. Daily dietary supplementation with 20% (w/w) fish oil prevents this high fat diet‐induced effect; however, for beneficial effects of fish oil in humans, doctors recommend that patients take fish oil only 2–3 times a week. To test the hypothesis that intermittent (e.g., 2/7 days per week) dietary supplementation with fish oil prevents high fat diet‐induced effects (e.g., weight gain and enhanced sensitivity to the behavioral effects of dopaminergic drugs) rats eating standard chow (17% kcal from fat), high fat chow (60% kcal from fat), and rats eating standard or high fat chow with 20% (w/w) intermittent (e.g., 2/7 days per week) dietary fish oil supplementation were tested once weekly with quinpirole (0.0032–0.32 mg/kg, i.p.) using a cumulative dosing procedure. Consistent with previous reports, eating high fat chow enhanced sensitivity of rats to the behavioral effects of quinpirole. That is, the quinpirole‐induced yawning dose‐response curve was shifted significantly to the left for rats eating high fat chow as compared to rats eating standard chow. Intermittent access to fish oil prevented this effect, since quinpirole‐induced yawning was not different among rats eating standard chow and rats eating high fat chow supplemented with fish oil. Future experiments will examine other dopaminergic drugs (e.g., cocaine) and will focus on understanding the mechanism by which fish oil produces these beneficial effects, by examining the specific omega‐3 polyunsaturated fatty acids found in fish oil.

Support or Funding Information

A portion of this project was supported by R25 GM069621.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Attenuation of dementia by AS605240‐PI3K inhibitor in ICV‐STZ induced sporadic Alzheimer's disease rat model
Authors: Alluri Ramesh , Sivamallikarjuna Reddy A , Supriya Reddy G , Banu Nafiza
Published: June 2021

Sporadic Alzheimer's disease is an age allied neurological and psychiatric disorder branded by impaired energy metabolism. Oxidative stress and neuro‐inflammation are associated in pathophysiology of sporadic type of dementia. The quest for chemical entities able to kerb the action of the PI3K/AKT/mTOR pathway is evolving as a potential therapeutic stratagem for its treatment and/or prevention.

The Intra‐cerebroventricular (ICV) administration of streptozotocin prompts behavioural and biochemical changes resembling those in sporadic type of Alzheimer's patients. The current study was intended to investigate the effects of chronic pre‐treatment with Phosphoinositide 3‐kinase(PI3K)inhibitor AS605240 on cognitive dysfunction and oxidative stress markers in intracerebroventricular (ICV) streptozotocin‐treated albino rats.

Treatment with AS605240 orally was done on a daily basis for a period of 21 days, 1 hr prior to ICV‐STZ injection using stereotaxy and evaluated for cognitive function tests and biochemical parameters.

AS605240 when given orally on a daily basis for a period of 21 days significantly improved STZ‐induced cognitive impairment. Besides, improving cognitive dysfunction, AS605240, reduced elevated nitrite levels, and restored reduced glutathione and superoxide dismutase levels, Lipid peroxidation and total Protein content.

Taken together, these results demonstrate that PI3K inhibitor AS605240 can be helpful in treatment of sporadic Alzheimer's Disease.

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 Effect of Chronic Cadmium Exposure on Chemotherapy Sensitivity in Breast Cancer Cells
Authors: Manabat Candice , Bloomfield Mathew , Louie Maggie C
Published: June 2021

Breast cancer is a leading cause of cancer‐related mortality among women in the United States. Exposure to environmental carcinogens, such as cadmium, has been shown to increase the risk of breast cancer. While humans are only exposed to cadmium at minute quantities through diet and cigarette smoke, it tends to bioaccumulate in the body. Epidemiological studies have shown that patients with breast cancer tend to have higher levels of cadmium in comparison to healthy patients. Previous findings from our lab indicate that breast cancer cells chronically exposed to low levels of cadmium (MCF7‐Cd) had altered gene expression profiles and higher rates of cell growth, migration, and invasion compared to the parental cells (MCF7). Despite evidence suggesting chronic cadmium exposure promotes breast cancer progression, its effect on therapeutic response is largely unknown. Here we evaluate the effects of chronic cadmium exposure on the ability of breast cancer cells to response to different classes of cancer therapeutics—antiestrogens (tamoxifen and endoxifen), taxanes (cabazitaxel and docetaxel), an anthracycline (doxorubicin), and an antimetabolite (gemcitabine). Our results suggest that cells chronically exposed to cadmium (MCF7‐Cd7 and ‐Cd12) did not show a differential response to antiestrogens, doxorubicin, or gemcitabine. Interestingly, MCF7‐Cd7 and ‐Cd12 cells did exhibit decreased response to the taxanes in comparison to the parent MCF7 cells (GI50 of 11.34 nM and 15.12 nM, respectively, vs. GI50 of 4.04 nM) suggesting that chronic cadmium exposure contributes to the development of taxane resistance. Analysis at the protein level indicate that 1 nM of cabazitaxel or docetaxel was sufficient to induce PARP cleavage—a signal of apoptosis— in MCF7 cells, while PARP cleavage was not evident in MCF7‐Cd7 and ‐Cd12 cells until 5 and 10 nM, respectively. Consistent with this finding, the expression of p53 and p21 were concordantly increased in response to 1 nM of cabazitaxel or docetaxel in MCF7 cells, but expression did not increase in the cadmium‐adapted cells until a higher taxane concentration. Collectively, this data strongly suggest that chronic cadmium exposure may contribute to the development of resistance to taxane‐induced apoptosis, and does not alter sensitivity to the other cancer drugs. Further work is necessary to understand the mechanism of this drug resistance and the potential implications in the treatment of breast cancer.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lung Fibroblasts Modulate Human Breast Cancer Cell Sensitivity to Chemotherapy and Autophagy Inhibition in a 3‐Dimensional Organotypic Co‐Culture Model
Authors: Regan Daniel , Gustafson Daniel
Published: June 2021

Fibroblasts are one cell type present in the tumor microenvironment (TME) which have been linked to several tumor growth and metastasis promoting activities. Recent data suggest that the molecular mechanisms underlying these metastasis‐promoting effects of the TME share significant overlap with those which mediate therapeutic resistance. Despite this, very few studies have evaluated the contributions of stromal cells on potential extrinsic mechanisms of cancer chemo‐resistance. Periostin (POSTN) is an extracellular matrix protein whose expression by lung fibroblasts has been shown to promote metastatic colonization and induction of a cancer stem cell (CSC) phenotype in a mouse model of breast cancer metastasis. However, the potential effects of lung fibroblasts (LFs) and POSTN in mediating tumor cell survival and chemoresistance in human breast cancer cells (BCCs) has not been evaluated. Therefore, we evaluated the effects of primary human LFs and recombinant POSTN on human BCC sensitivity to doxorubicin (DOX) and pharmacological inhibition of autophagy utilizing a modified 3D organotypic co‐culture model. Initial screening of a panel of six human breast cancer cell lines demonstrated that while rPOSTN induced BCC survival/proliferation under low‐serum conditions, it had no effect on BCC resistance to the autophagy inhibitor chloroquine (CQ). However, compared to standard 2‐D monolayer culture, 3D culture of either human BCCs or LFs alone induced resistance of both LFs and triple‐negative breast cancer (TNBC) cell lines to autophagy inhibition with CQ. Interestingly, the addition of primary human LFs into this 3D co‐culture system re‐sensitized TNBC cell lines to CQ, to a degree similar to that observed in standard 2‐D mono‐culture. In contrast, DOX sensitivity was not altered by 3D mono‐culture of any of the human BCC lines screened, while the addition of LFs induced DOX‐resistance in the luminal, ER+ cell line, T47D. These results suggest that the effect of lung fibroblasts on human BCC sensitivity to chloroquine and doxorubicin may be dependent on breast cancer molecular subtype and provide evidence for lung fibroblast‐mediated regulation of autophagy in human BCCs.

Support or Funding Information

The authors would like to acknowledge the following sources of funding support: NIH‐NCI K01ODO22982 (DP Regan) and NIH‐NCI 1R01CA190170 (DL Gustafson).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural insights into mitochondrial EndoG in response to oxidative stress
Authors: Yuan Hanna S. , Lin Jason L. J. , Chu Woei‐Chyn
Published: June 2021

Endonuclease G (EndoG) is an evolutionarily conserved mitochondrial protein that degrades chromosomal DNA during apoptosis. In mitochondria, EndoG also processes DNA and participates in mitochondrial DNA metabolism. EndoG‐deficient mice have high levels of mitochondria reactive oxygen species (ROS) and develop heart and mental diseases, showing that the EndoG defect is linked to mitochondrial dysfunction and oxidative stress‐linked diseases. However, it remained unclear how EndoG degrades DNA as a homodimer and why EndoG's function is linked to ROS levels in mitochondria. We reported the crystal structures of the C. elegans EndoG homologue, CPS‐6, in complex with single‐stranded DNA at a resolution of 2.3 Å. Two separate DNA strands are bound at the bba‐metal motifs in the homodimer with their nucleobases pointing away from the enzyme, explaining why CPS‐6 degrades DNA without sequence specificity. We also showed that under oxidative stress, homodimeric EndoG becomes oxidized and converts to monomers with a diminished endonuclease activity. High ROS levels are thus linked to mitochondrial dysfunction by repressing EndoG's activity. Modulation of EndoG dimer conformation and endonuclease activity could present an avenue for prevention and treatment of the diseases resulted from oxidative stresses.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intercellular Communication by Gap Junctions in the Cranial Neural Crest
Authors: Taneyhill Lisa
Published: June 2021

Cell‐cell interactions and communication are critical for several fundamental biological processes that underscore vertebrate development, including cell migration and the formation of multicellular tissues. Much of the patterning that occurs during vertebrate embryogenesis is accomplished by neural crest cells, which are initially stationary but acquire the capacity to migrate through the epithelial‐to‐mesenchymal transition (EMT). Once motile, neural crest cells must communicate and interact with one another and with other cells, later differentiating to become sensory neurons and glia, the craniofacial cartilage and skeleton, portions of the heart, and skin pigment cells. The molecular basis by which neural crest cells establish intercellular communication during their initial formation and migration, however, is still not known. One unexplored means of such intercellular communication is through gap junctions that permit the passage of small molecules between connected cells. Gap junctions are comprised of Connexin proteins, of which Connexin 43 (Cx43) is the most abundant, with modulation of Cx43 previously shown to impact the formation of neural crest cell derivatives. Notably, mutations in Cx43 lead to oculodentodigital dysplasia, a disease characterized, in part, by craniofacial defects. To delineate a function for Cx43, and gap junctions, in cranial neural crest cell development, we first characterized the spatio‐temporal expression pattern of Cx43, noting it in premigratory neural crest cells, during EMT, and in fully migratory neural crest cells. Given this expression pattern, we next performed molecular and chemical perturbation assays to respectively alter Cx43 and gap junction function in neural crest cells. Our live imaging experiments reveal the presence of functional gap junctions in premigratory neural crest cells in vivo and in migratory neural crest cells cultured ex vivo. Moreover, gap junctions within the migratory neural crest cell population depend on Cx43 to properly transfer dye between cells. In keeping with this finding, chemical inhibition of general gap junction function, but not formation, abrogates neural crest cell EMT and/or migration in a reversible manner. Depletion of Cx43 from premigratory neural crest cells in vivo alters the expression of some neural crest cell markers but, interestingly, does not overtly affect EMT. These studies will decipher the roles of Cx43 and gap junctions in cranial neural crest cell EMT and migration, providing insight into the significance of intercellular communication during these key processes that are required for the formation of the vertebrate embryo.

Support or Funding Information

This research is supported by NIH R01DE024217 and American Cancer Society RSG‐15‐023‐01‐CSM grants.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Can Cellular Labile Iron Pool be Considered Solely a Pro‐oxidant Species in Cells?
Authors: Damasceno Fernando Cruvinel , Toledo José Carlos , Condeles André Luis , Lopes Angélica Kodama Bueno , Facci Rômulo Rodrigues , Linares Edlaine , Truzzi Daniela Ramos , Augusto Ohara
Published: June 2021

The ubiquitous cellular labile iron pool (LIP) is often associated with the production of the highly reactive species hydroxyl radical, which forms through redox reaction with hydrogen peroxide. Several studies indicate that LIP‐dependent hydroxyl radical production causes cellular damage through oxidation of biomolecules. Although LIP's interaction with hydrogen peroxide and its effects are well established, the interaction of LIP and peroxynitrite, and it's effects are not yet understood. Does peroxynitrite, a potent oxidant species known to interact with transition metals, act as an oxidant species in cells just like hydrogen peroxide? In murine macrophage cells we observed that removal of LIP with membrane‐permeable iron chelators attenuates peroxynitrite‐dependent oxidation and nitrosation of fluorescent probes in both normal and oxidative stress conditions which is in stark contrast to H2O2‐dependent oxidation. These results could not be reproduced in cell‐free systems indicating that the chelator‐enhancing effect on peroxynitrite‐dependent modifications of the probes depend on cells constituents, presumably including LIP, that reacts with these chelators. It is also observed that neither free nor ferrous‐complexed chelators stimulated intracellular or extracellular nitrosative and oxidative processes. These results indicates that LIP is a competitive cellular target for peroxynitrite and its derived oxidants, and thereby reduces oxidative processes, which may change the current view of LIP as a cellular source of pro‐oxidant iron.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilizing live‐cell imaging in Drosophila cells to identify stressors that induce protein misfolding
Authors: Choi Annette , Hrizo Stacy , Buckley Martin
Published: June 2021

Proteins drive many of the biological processes in cells. To do this, they fold into complex 3D structures that are integral for their functionality. Notably, various environmental and chemical stressors can disrupt protein folding and thus disable the functions of proteins, threatening the livelihood of cells. To mitigate this stress, organisms initiate the highly‐conserved heat shock stress response. In eukaryotes, the master heat shock activator heat shock factor (HSF) is rapidly recruited to the Hsp70 heat shock protein genes and triggers the recruitment of additional co‐activator proteins that facilitate gene expression. This leads to the production of heat shock proteins that function as molecular chaperones to promote refolding of proteins, prevent aggregation and increase protein degradation pathways. Notably, activation of the heat shock response pathway, can be visualized by measuring GFP‐tagged HSF binding to the heat shock protein genes in living Drosophila salivary gland nuclei. Our lab is currently using this technique to identify novel compounds that induce the heat shock response pathway. Our pioneering experiments have shown that diamide and hydrogen peroxide, two chemicals known to cause protein misfolding and activation of the heat shock response pathway, trigger the recruitment of GFP‐HSF to the Hsp70 loci in living salivary gland cells to a similar level as HS stress. In addition, to our surprise, high levels of Dithiothreitol (DTT, 100 mM), a chemical known to cause protein misfolding and activation of the unfolded protein response pathway (UPR), results in the recruitment of GFP‐HSF to the Hsp70 gene loci. Here we describe experiments to further explore a possible dose dependent activation of HSF by DTT.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of Microtubule Associated Protein Tau (MAPT) in Saccharomyces cerevisiae to Determine the Effects of N‐terminal Acetylation
Authors: Anderson Jess Lynn , Holmes William
Published: June 2021

Protein aggregates, which can result in cellular toxicity, are found in the brains of people afflicted with neurodegenerative diseases. One of the proteins found to aggregate is the Microtubule Associated Protein Tau, a protein that stabilizes microtubules in neuronal cells. Based on its amino acid sequence, Tau is predicted to be N‐terminally acetylated by the acetyltransferase complex NatA, which is responsible for the acetylation of 50% of the proteome in yeast and humans. Loss of NatA activity results in a variety of pleiotropic detrimental phenotypes, suggesting that acetylation is a crucial modification for many proteins. The first 10 amino acids of Tau have previously been demonstrated to play a role in aggregation, but the specific role of N‐terminal acetylation has yet to be determined. Without the presence of NatA – and therefore without acetylation – Tau may be more prone to aggregation and toxicity. By expressing Tau in yeast strains with and without the presence of NatA, the possible effects of acetylation or lack thereof can be determined by examining protein expression and aggregation within the cells by various methods including SDS‐PAGE, SDD‐AGE, and visualization of GFP‐tagged Tau via microscopy.

Support or Funding Information

This work was made possible by an Early Career Development Award through RI‐INBRE (2P20GM103430) and by Dr. William Holmes.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression and Purification of Soluble, Glycosylated T‐cell Receptors in Chinese Hamster Ovary Cells
Authors: Pearson Amanda Jo , Liedig Chance , Gallagher Elyssia S
Published: June 2021

T‐cell receptors (TCRs) are glycoproteins that are expressed on the surface of T cells, which are a major component of the adaptive immune response. The glycans on the constant domain of this protein are involved in interactions with other proteins, such as galectin‐3, which mediates the immune response1. Additionally, altering the glycosylation of this protein has been associated with diseases such as Multiple Sclerosis1. Herein, we aim to express a soluble, glycosylated TCR in Chinese hamster ovary (CHO) suspension cells and purify it with the goal of characterizing the effects of glycosylation on TCR structure and function. To date, a soluble, unglycosylated TCR was expressed once, in 1990,2 but has not been replicated since. This project aims to express a glycosylated, soluble TCR with more current technology, which should result in more efficient expression.

This project began with the design of a solubilized glycoprotein. We selected a starting sequence that has known binding affinity to ovalbumin and proceeded with modifications for optimized expression. The transmembrane sequence was removed after the last cysteine and replaced with a glycosylphosphatidylinositol (GPI) ‐ anchored sequence. This targets the protein to the endoplasmic reticulum (ER) and Golgi apparatus to ensure glycosylation. Following expression and glycosylation, the TCR should be localized to the surface of the cell, where the GPI sequence can be enzymatically cleaved using phosphatidylinositol‐phospholipase C (PI‐PLC), resulting in a soluble released protein. The α and β chains of the heterodimer were combined into a single plasmid using a 2A peptide. The use of 2A peptides instead of a more traditional technique, such as internal ribosome entry site (IRES), results in more efficient folding due to expression of stoichiometric ratios of both chains of the protein, as compared to a higher concentration of a single chain.

The protein is transfected in CHO‐S cells and the expressed protein is harvested after 24 hours. The protein of interest is purified by affinity chromatography using a nickel column, followed by analysis with SDS‐PAGE. Imaging of the gels after surface cleavage with PI‐PLC and cell lysis confirmed that the protein was not localized to the plasma membrane as it was not recovered after enzymatic cleavage. However, the protein is purified from the inside of the cell after cell lysis. To determine the cellular localization of the protein we are using immunofluorescence using fluorescently labeled antibodies to target the His tag on the protein, as well as the ER, Golgi, and plasma membrane. Currently we are addressing the incorrect localization and analyzing methods to redirect the protein to the cell surface.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Alpha‐Arrestin Adaptor Protein Regulation of YOR1 in Yeast
Authors: Cheranda Nina Kiran , Wang Yuqi
Published: June 2021

ATP‐binding cassette (ABC) proteins are membrane‐bound transporters that utilize ATP to pump various substrates across biological membranes. Serious pathological conditions can result when these transporters are defective, as exemplified by cystic fibrosis caused by a defective transporter CFTR. Yor1 is an ABC transporter in budding yeast, Saccharomyces cerevisiae. In this study, we investigated the potential regulation of Yor1 by alpha‐arrestins. Using immunoblotting techniques to check the effects of the overexpression of nine different alpha arrestin protein on Yor1 abundance in yeast cells, we found that upregulating the activity of specifically one alpha‐arrestin protein leads to a marked elevation of the level of Yor1. This observation is exciting because it provides a novel strategy for regulating and stabilizing Yor1, which could have valuable implications for the modulation of dysfunctional CFTR transporters in humans with cystic fibrosis.

Support or Funding Information

Knoedler Undergraduate Fund from Saint Louis University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of βγ‐Mediated Membrane Localization on Growth Signaling by Overexpressed Gα13
Authors: Brown Katherine M. , White Nicholas F. , Tagliatela Alicia C. , Meigs Thomas E.
Published: June 2021

The G12/13 class of heterotrimeric G proteins is unique in its ability to stimulate cellular transformation via overexpression of the wildtype α subunit, Gα12 or Gα13, absent of activating mutations. We have studied the mechanism of wildtype Gα12/13 signaling to the transcriptional activator Serum Response Factor (SRF), a pathway implicated in multiple cancer types. When overexpressed in cultured human embryonic kidney cells, wildtype Gα13 showed robust stimulation of this transcriptional response. This signaling by Gα13 was blunted by overexpression of G protein β1 and γ2 subunits, suggesting that aberrant signaling by the overexpressed α subunit results from stoichiometric imbalance between the trimeric G protein subunits. Next, using an epitope‐tagged Gα13 to track its subcellular location, we discovered the overexpressed α subunit shifted from a membrane‐associated fraction to a soluble fraction, coincident with its sharp increase in SRF signaling. Overexpression of the β1γ2 dimer caused re‐localization of wildtype Gα13 to the membrane‐associated fraction, coincident with its diminished signaling. Interestingly, our preliminary data utilizing a non‐prenylated γ2 subunit suggest the ability of the βγ dimer to suppress signaling by overexpressed, wildtype Gα13 is independent of βγ association with the cell membrane. We currently are examining effects of this non‐membrane bound dimer on subcellular localization of wildtype Gα13. These findings add to our understanding of the mechanism utilized by overexpressed Gα13 to drive aberrant growth signaling.

Support or Funding Information

We acknowledge funding from the North Carolina Biotechnology Center, NC GlaxoSmithKline and CD Spangler Foundations, UNC Lineberger Comprehensive Cancer Center, and UNC‐Asheville Undergraduate Research Program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The yeast Nem1‐Spo7 phosphatase complex, which dephosphorylates and regulates Pah1 phosphatidate phosphatase, is phosphorylated by protein kinase C
Authors: Dey Prabuddha , Mirheydari Mona M , Han Gil‐Soo , Carman George M
Published: June 2021

Phosphatidic acid (PA) phosphatase, which catalyzes the conversion of PA to diacylglycerol, is a key regulatory enzyme in lipid metabolism; it plays a major role in controlling the balance between the synthesis of membrane phospholipids and triacylglycerol. Loss of PA phosphatase in yeast, mouse, and human results in a variety of cellular defects and lipid‐based diseases. The yeast PA phosphatase, Pah1, is regulated by its cellular location (cytosolic versus membrane), which is governed by its phosphorylation and dephosphorylation. The dephosphorylation is catalyzed by the Nem1 (catalytic subunit)‐Spo7 (regulatory subunit) phosphatase complex, and this modification facilitates Pah1 interaction with PA at the membrane for its reaction. Like Pah1, Nem1 and Spo7 are subject to phosphorylation, but the protein kinases involved are largely unknown. In this work, we examined the premise that both subunits are substrates for protein kinase C. Using purified reagents from yeast, we showed that protein kinase C phosphorylated Nem1 or Spo7 on multiple serine residues. Using Nem1 or Spo7 as a substrate, protein kinase C activity was dependent on time, the protein kinase, ATP, and substrate, as well as on phosphatidylserine and diacylglycerol. A multifaceted approach using synthetic peptide substrates, site‐specific mutagenesis, phosphopeptide mapping, and mass spectrometry lead to identification of Ser‐201 and Ser‐210 in Nem1 and Ser‐22 and Ser‐28 in Spo7 as the major sites phosphorylated by protein kinase C. Supported by NIH grant GM05679.

Support or Funding Information

Supported by NIH grant GM05679.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The lipid acyltransferase LYCAT controls phosphatidylinositol‐3,4,5‐trisphosphate (PIP3) signaling
Authors: Antonescu Costin N , Bone Leslie N , Khuffash Saba , Awadeh Yasmin , Genc Gizem E , Defferrari Marina , Botelho Roberto J
Published: June 2021

Phosphatidylinositol‐3,4,5‐trisphospate (PIP3) is a potent signaling lipid that exerts key control over cell growth, survival, adhesion and migration. PIP3 signaling is often disrupted in various cancers, making understanding the regulation of PIP3 signaling an important priority. Much has been learned from the study of phosphorylation of the inositol headgroup of phosphoinositides, such as the phosphorylation of phosphatidylinositol‐4,5‐bisphospate (PIP2) to produce PIP3, as well as negative regulation of PIP3 by phosphoinositide phosphatases such as PTEN. However, much less is known about another key dimension of control of phosphoinositide action, the regulation of the fatty acyl profile of these lipids. Indeed, phosphatidylinositol and phosphoinositides exhibit remarkable selectivity of acyl chains (>50% harboring 1‐stearoyl, 2‐arachidonoyl), an acyl profile quite distinct from other phospholipids. This suggests that control of fatty acyl profile of phosphoinositides may be an important determinant of the function of these lipids that has to date remained largely unexplored. We recently uncovered that the acyltransferase LYCAT is a key regulator of the acyl profile of specific phosphoinositides such as PIP2, thus exerting control over membrane traffic phenomena dependent on these phosphoinositides (Bone LN et al 2017 Mol Biol Cell. 28:161–172). We now examine how LYCAT controls PIP3 signaling and PIP3‐dependent control of cell physiology, including the activation of Akt and control of actin dynamics. We find that LYCAT perturbation impairs the activation of Akt and the activation of a number of Akt substrates, which in turn impacts cell growth and survival. Furthermore, LYCAT perturbation elicits dramatic changes in actin filament morphology, cell migration and cancer cell invasion. These results indicate that control of phosphoinositide acyl chain profile is an important novel dimension of regulation of PIP3 signaling, impacting proliferative, growth and migration signaling, and as such may be a novel dimension to control of cancer cell growth and progression.

Support or Funding Information

This work was supported by an Ontario Early Researcher Award, a Canada Research Chair Award, and a Natural Sciences and Engineering Research Council Grant to R.J.B, and a Project Grant and New Investigator Salary Award from the Canadian Institutes of Health Research to C.N.A.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.