bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeted Proteomics‐Driven Computational Modeling of Macrophage Microbial Sensing Pathways
Authors: Nita‐Lazar Aleksandra , Manes Nathan P. , Mann Jessica M. , Kaplan Pauline , Meier‐Schellersheim Martin , Fraser Iain D. C. , Germain Ronald N.
Published: June 2021

Toll‐like receptor (TLR) signaling in macrophages is essential for generating effective innate immune responses. Quantitative differences dependent on the dose and timing of the stimulus critically affect cell function and involve proteins that are not components of widely shared transduction pathways. These features make mathematical modeling an important approach to a better understanding of how these signaling networks function in time and space.

To model the TLR signaling networks, we are using selected reaction monitoring (SRM) to measure the absolute abundance of TLR pathway proteins, with the resulting values used as pathway model parameters. RNA‐seq was performed to identify expressed transcripts, and shotgun mass spectrometry was used to identify proteotypic peptides. SRM assays for the canonical TLR signaling pathway and related proteins and phosphoproteins have been successfully developed. SRM with heavy‐labeled internal peptide standards was used to quantify protein and phosphorylated protein molecule numbers per cell in both untreated and LPS‐stimulated macrophages. A preliminary model of the TLR pathway has been developed using Simmune and the resulting estimated protein abundance values.

Support or Funding Information

This research was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, NIH.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rad5 Prevents the Accumulation of ssDNA Gaps at Stressed DNA Replication Forks
Authors: Brown Grant W. , Gallo David W. , Kim Simon , Zhang Zhaolei , Branzei Dana
Published: June 2021

In S. cerevisiae Rad5 is a component of the post‐replication repair pathway (PRR) and possesses both E3‐ubiquitin ligase and DNA helicase activity. We recently found that Rad5 forms sub‐nuclear foci after treatment with hydroxyurea (HU). Previous studies indicate that Rad5‐dependent poly‐ubiquitination of PCNA is required for error‐free bypass of MMS‐ or UV‐induced DNA lesions but there is little insight into the role of Rad5 in response to HU‐induced replication stress, where base lesions are likely absent. Here we define the function of Rad5 during HU‐induced replication stress. Rad5 foci form predominantly during S phase of the cell cycle and are induced by both chemical and genetic replication stress, suggesting that Rad5 foci correspond to Rad5 action at stressed DNA replication forks. Using chromatin immunoprecipitation followed by deep sequencing (ChIP‐seq) we show that Rad5 is recruited and retained at HU‐stressed forks, and that recruitment depends on mono‐ubiquitination of PCNA. To reveal the function of Rad5 at stressed‐forks we monitored cell cycle progression of WT and rad5 null cells recovering from HU‐induced replication stress. In this scenario rad5 null cells delay in G2/M with an activated DNA damage checkpoint, chromatin bridges, and increased RPA foci, indicating the presence of ssDNA. The ssDNA that accumulates in rad5Δ is not a product of incomplete replication at specific loci, and is not the result of abnormal recombination. In fact, deletion of the anti‐recombinase gene SRS2 rescues the accumulation of chromatin bridges in rad5Δ, suggesting that activating homologous recombination during S phase can compensate for rad5 deficiency during replication stress. We use a modified version of DNA combing to show the presence of ssDNA gaps directly, and to show that these accumulate in rad5Δ. Taken together, our results indicate that Rad5 prevents accumulation of ssDNA and allows for non‐recombinogenic recovery from replication stress.

Support or Funding Information

Funded by the Canadian Cancer Society Research Institute and the Natural Sciences and Engineering Research Council of Canada

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Myeloid‐specific Glut1 Ablation Attenuates Mammary Gland Inflammation and Claudin‐low Breast Cancer Progression
Authors: Fuller Ashley M. , Cozzo Alyssa J. , Freemerman Alex J. , Ezzell J. Ashley , Galanko Joseph A. , Troester Melissa A. , Hursting Stephen D. , Makowski Liza
Published: June 2021

Obesity is a strong risk and progression factor for triple‐negative breast cancer (TNBC), a class of tumors comprised of the basal‐like and claudin‐low (CLBC) molecular subtypes. Macrophage infiltration and chronic inflammation are features of both obese adipose and TNBC microenvironments, and may thus underlie observed associations between obesity and TNBC. We have previously shown that the glucose transporter, GLUT1 (Slc2a1), is a critical mediator of macrophage glucose metabolism and associated pro‐inflammatory responsiveness. Thus, the goal of this study was to evaluate the effect of myeloid lineage Glut1 loss on mammary gland inflammation and obesity‐associated TNBC progression. Lean and obese myeloid‐specific Glut1 knockout mice were orthotopically injected with syngeneic M‐Wnt cells, a model of CLBC that recapitulates genomic and molecular features of human tumors. M‐Wnt tumor growth was impeded in lean Glut1M−/− compared to Glut1MFl/Fl mice; however, a similar protective effect of myeloid Glut1 loss was not observed in obese animals. Among lean mice, no significant genotype‐driven differences in markers of tumor proliferation, apoptosis, angiogenesis, or desmoplasia were observed. However, tumors from lean Glut1M−/− compared to lean Glut1MFl/Fl animals exhibited reduced concentrations of inflammatory mediators, including MCP‐1, TNFα, IL‐1β, and IL‐10, which paralleled in vitro findings in GLUT1‐deficient compared to control macrophages. No change in total myeloid cell or macrophage content was detected in the mammary gland microenvironment. However, mammary tissue macrophages (MTMs) of both lean and obese Glut1M−/−, but not Glut1MFl/Fl, mice were skewed toward an anti‐inflammatory phenotype with reduced iNOS (Nos2) expression, and were associated with reduced expression of pro‐inflammatory and growth‐supportive factors in the mammary gland. Taken together, these results indicate that myeloid Glut1 regulates inflammation and MTM immunophenotypes in both lean and obese models, but attenuates tumor progression only in the lean setting. Changes unique to lean Glut1M−/− tumors may be used to guide development of intervention and treatment strategies for obesity‐associated TNBCs.

Support or Funding Information

This work was supported by the National Cancer Institute (CA197627) and a UNC University Cancer Research Fund (UCRF)‐UNC LCCC Tier 2 Development Grant to LM and SDH. The UNC Nutrition Obesity Research Center (NORC) is funded by the National Institutes of Health (DK056350). AMF and AJC received support from the UNC Integrated Vascular Biology program (NHLBI HL069768) and the UNC Royster Society of Fellows during the course of this work. AMF was also supported by NCI CA179715 to MT.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biotin deficiency induces Th1 and Th17 mediated inflammatory response in CD4+T lymphocytes via activation of mTOR signaling pathway
Authors: Elahi Asif , Sabui Subrata , Agrawal Anshu , Said Hamid M.
Published: June 2021

Biotin (vitamin B7) is essential for normal human health due to its involvement, as a cofactor, in a variety of critical cellular metabolic reactions. Previous studies have shown that biotin deficiency enhances inflammation, and that certain chronic inflammatory diseases (like inflammatory bowel diseases) are associated with biotin deficiency. However, the mechanisms that mediate the association between biotin status and inflammation are not well understood. Here, we examined the effect of biotin deficiency on human CD4+ T cell responses to determine their role in biotin deficiency associated inflammation. Our investigations revealed that anti‐CD3/CD28 stimulated human CD4+ T cells cultured in biotin deficient media secreted significantly enhanced levels of pro‐inflammatory cytokines, IFN‐γ, TNF‐α, and IL‐17. Expression of the transcription factors, T‐bet and RORγt, was increased along with a decrease in Foxp3 expression in biotin deficient CD4+ T cells. The percentage of T regulatory cells was also decreased under biotin deficient condition. Further investigations indicated that the enhanced inflammatory response was not a consequence of increased T‐cell differentiation towards Th1 and Th17 or increased proliferation. Instead deficiency of biotin enhanced the production of IFN‐γ from already differentiated cells. Moreover, the results indicated that the increased inflammatory response was due to enhanced activation of the mTOR signaling pathway in biotin deficient CD4+ T cells. Similar increase in T‐bet, RORγt and decrease in Foxp3 transcription factors was also observed in inguinal lymph nodes of mice fed with biotin deficient diet relative to pair‐fed controls. Further, the level of IFN‐γ, IL‐17 cytokines and the expression of mTOR were found to be increased significantly in CD4+T cells isolated from inguinal lymph node of diet induced biotin deficient mice. These findings revealed that biotin deficiency leads to the upregulation of IFN‐γ secreting Th1 and IL‐17 secreting Th‐17 cells via activation of mTOR signaling pathway which may contribute to inflammation associated with biotin deficiency

Support or Funding Information

This study supported by grants from the DVA and the NIH (DK58057, DK56061 and AA 018071)

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 Role of the Creatine Kinase Energy System in Intestinal Inflammation
Authors: Lanis Jordi M. , Colgan Sean P.
Published: June 2021

Inflammatory bowel disease (IBD) is a chronic inflammation of the intestine affecting more than 1 million people in the United States. Intestinal epithelia facilitate nutrient transport as well as protect against luminal bacteria and disruption of the intestinal epithelial barrier is a hallmark of IBD. The regulation of intestinal epithelial barrier is made possible by junctional complexes including tight junctions and adherens junctions that are supported by the actin cytoskeleton. Maintenance of the actin cytoskeleton is an energy demanding process and intestinal epithelial cells shuttle energy to the locations of the cellular junctions through the creatine energy circuit. Within the cell, creatine and its associated enzyme creatine kinase (CK) facilitate the shuttling of high energy phosphates in the form of phosphocreatine between sites of ATP generation. Previous work in our lab has shown that CK contributes to apical junction assembly in vitro and that supplementation of creatine in murine colitis models has a protective effect. Further studies have shown that mice lacking the brain and mitochondrial isoforms of CK have worse outcomes in a DSS model of colitis. In addition to impaired barrier function, this may be due in part to exacerbated intestinal bleeding observed in these mice. To that end, we measured platelet counts and aggregation and found that platelet counts are lower in CK double knockout mice compared to wild‐type mice. The platelets from the knockout mice also showed a hyporesponsive phenotype in a collagen adhesion assay. Together, these results provide evidence that the CK energy circuit is essential in maintaining intestinal epithelial barrier function as well as supporting proper platelet function.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of FPR2 Impaired Leukocyte Get‐in Signal and Triggers Non‐Resolving Inflammation in Heart Failure
Authors: Kain Vasundhara , Wright Griffin M. , Halade Ganesh V.
Published: June 2021

Non‐resolving inflammation is a prominent trigger for heart failure (HF)‐related morbidity and mortality following myocardial infarction (MI). Post‐MI activated leukocyte expressed formyl peptide receptor 2 (FPR2) belongs to the essential for inflammation‐resolution mechanisms. However, the role of FPR2 in leukocyte kinetics in acute HF is incompletely understood. Here, we aimed to determine whether pharmacological inhibition of FPR2 would impair leukocyte trafficking to the site of cardiac injury using WRW4 (FPR2 antagonist). For this, male C57BL/6 (8 to 12 week) mice were subjected to acute HF using permanent coronary artery ligation. FPR2 inhibitor WRW4 (1μg/kg/day; subcutaneous) was injected 3 hours post‐MI and saline‐injected‐mice served as MI‐control. Inhibition of FPR2 impaired leukocyte trafficking (“get‐in” signal) that altered post‐MI immune responsive clearance in the infarcted LV and spleen. Leukocyte kinetics, measured using flow cytometry, showed there was an overall decrease in CD45+/CD11b+; 23.3±2% in WRW4‐injected mice when compared with MI‐control (49.1±2%) in the LV. FPR2 inhibitor, WRW4, decreased FPR2 in the LV and spleen tissue, and mice displayed increase F4/80+/Ly6Chi macrophages (14.8±2%) compared to the MI‐control group (10±1%) in LV, this is indicative of amplified proinflammatory profile. Inhibition of WRW4 primes immature neutrophil infiltration Ly6Gint and intensifies the Ccl2 (all p<0.05) expression compared to MI‐control in the infarcted LV post‐MI. WRW4 increased transcripts of pro‐inflammatory markers TNF‐α and IL‐1β, with a decrease in expression of Arg‐1 (p<0.05) in the infarcted LV compared to MI‐controls is suggestive of non‐resolving or immune suppressive inflammation. In summary, FPR2 inhibition using WRW4 altered leukocyte get‐in signal leading to the onset of frustrated resolution signaling in acute HF post‐MI.

Support or Funding Information

HL132989 to GVH and 16POST31000008 to VK

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Cardiomyocyte Cohesion and Gap Junctions via Desmosomal Adhesion
Authors: Schinner Camilla , Erber Bernd M. , Waschke Jens
Published: June 2021

Mutations in desmosomal proteins such as desmoglein‐2, desmoplakin or plakoglobin can induce arrhythmogenic cardiomyopathy, a disease leading to arrhythmia and sudden cardiac death. Previous data demonstrated adrenergic signaling to be important to regulate desmosomal cohesion in cardiomyocytes. Here, we investigated how signaling pathways including adrenergic signaling, PKC and SERCA regulate desmosomal adhesion and how desmosomal adhesion controls gap junctions in cardiomyocytes. As revealed by dissociation and immunostaining experiments in cardiomyocytes, activation of PKC by PMA or adrenergic signaling increased cell cohesion and desmoglein‐2 and desmoplakin localization at cell junctions whereas Trp‐treatment to inhibit cadherin binding or inhibition of SERCA pump by thapsigargin reduced cardiomyocyte cohesion. Cohesion was rescued by both cAMP elevation or PMA. In parallel to increased cohesion elevated phosphorylation of the gap junctions component connexin‐43 was revealed by Western blotting and immunostaining. Using a multi‐electrode array, disruption of cell cohesion by Trp impaired conduction of excitation comparable to the connexin inhibitor carbenoxolone. Elevation of cAMP levels was effective to increase conduction velocity and to improve arrhythmia under baseline conditions as well as after Trp treatment. Immunoprecipitation revealed connexin‐43 to interact with desmoglein‐2 and β1‐adrenergic receptor. Weakened cell cohesion by Trp or siRNA‐mediated depletion of desmoglein‐2 or plakoglobin were sufficient to block signaling via the β1‐adrenergic receptor. Moreover, depletion of desmosomal proteins increased arrhythmia and reduced conduction velocity, both of which was rescued by cAMP elevation. These data demonstrate gap junction and desmosomal proteins to form a complex with the β1‐adrenergic receptor. The function of this complex was regulated by cell cohesion, adrenergic and PKC signaling or inhibition of SERCA. These results support the identification of new targets to treat arrhythmogenic cardiomyopathy.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Uremic toxins are conditional danger‐ or homeostasis‐ associated molecular patterns, which are highly selective increase rather than purely passive accumulation, in chronic kidney disease and coronary arteria disease
Authors: Sun Yu , Lu Yifan , Nanayakkara Gayani , Li Yafeng , Fu Hangfei , Johnson Candice , Shao Ying , Yang William Y. , Wang Hong , Li Rongshan , Yang Xiaofeng
Published: June 2021

Cardiovascular morbidity and mortality in patients with advanced CKD are 10‐ to 30‐ folds greater than that in the general population. In the U.S. and worldwide, the incidence of CKD is increasing, and atherosclerosis‐ related CVD is a major cause of mortality in these patients. We performed novel metabolomics/gene database mining, where we identified protein‐bound uremic toxin (UT) receptors as well as the enzymes for UT generation, and analyzed their gene expression changes in chronic kidney disease (CKD), vascular and metabolic diseases. We made the following findings: 1) In CKD, UTs represent a very small fraction of the total human serum small‐molecule metabolome, roughly 1/80th; 2) The serum concentrations of some UTs are increased not only in CKD but in other diseases; 3) Protein‐bound UTs either induce or suppress the expression of pro‐inflammatory molecules; 4) The expression of UT genes is significantly modulated in the tubules of CKD patients, and adipose tissue of coronary artery disease (CAD) patients, compared with those of metabolic syndrome and type 2 diabetes patients; 5) The expression of UT genes is upregulated by caspase‐1 and TNF‐α pathways, more than toll‐like receptors (TLRs), IL‐1β and IFN‐g pathways; and 6) The expression of UT genes is inhibited by regulatory T cells. Our results have demonstrated that UTs are highly selectively accumulated, and serve as danger signal‐associated molecular patterns (DAMPs) and homeostasis‐associated molecular patterns (HAMPs) that modulate inflammation. In addition, our results show that some UT genes are upregulated in CKD and CAD, presumably via caspase‐1/inflammatory cytokine pathways, rather than by purely passive accumulation.

Support or Funding Information

This work is partially supported by NIH grants to Drs. XF. Yang, H. Wang.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aberrant Retinal Angiogenesis, Structure, and Synaptic Morphology in an in vivo Mouse Model of ROP
Authors: Mezu‐Ndubuisi Olachi Joy , Macke Erica , Kalavacherla Raja , Nwaba Amanda , Ikeda Akhiro
Published: June 2021

Objective

Retinopathy of prematurity (ROP) is a condition of aberrant retinal vascularization in premature infants. Vascular endothelial growth factor (VEGF) is a major angiogenic factor in both physiologic and pathologic retinal angiogenesis including ROP. However, cellular changes that take place in ROP are not fully understood. The aim of this study is to understand changes in retinal morphology, vasculature, and structure in mice with oxygen‐induced retinopathy (OIR), a model for ROP.

Methods

Twenty‐two mice underwent hyperoxia exposure at 77% to induce OIR, while twenty‐three age‐matched control mice were raised in room air (RA). After anesthesia, pupils were dilated and fluorescein was injected intraperitoneally. After in vivo imaging at P19, P24, P32, and P47, eyes were isolated and fixed in Bouin's fixative for morphological analysis by H&E staining, and paraformaldehyde for immunohistochemistry. For immunohistochemistry, markers for photoreceptor presynapses (PSD95), bipolar cells (PKCalpha), microglia (iba1), and vascularization (VEGF) were used to investigate synaptic abnormalities, inflammation, and vascularization. To detect apoptosis, ApopTag Fluorescein In Situ Apoptosis Detection Kit (Millipore) was used.

Results

OIR mice had a vascular‐avascular retinal phenotype, with more dilated veins and more tortuous arteries than RA mice in early ages. The OIR retinas showed disorganized synaptic interactions between photoreceptor cells and bipolar cells, with significant thinning in the ganglion cell, inner nuclear, inner plexiform, and outer plexiform layers. Retinal cross‐sections showed altered structure in OIR mice, with dilated blood vessels and neovascularization in the inner retina. There was significantly increased microglia activity in ganglion cell, inner nuclear, and inner plexiform layers. There was increased apoptosis in OIR mice, which decreased over time, but persisted until P47. There was increased VEGF expression in bipolar cells. TUNEL staining showed increased apoptosis in OIR mice than RA mice at P19 (P<0.001), P24 (P<0.001), P32 (P<0.001), and P47 (P<0.001). Apoptosis decreased over time with developmental maturity of the OIR mice, but persisted at P47.

Conclusion

: Our results demonstrate patchy vascular–avascular phenotype, defective synaptic interactions and persistent apoptosis in the OIR retina, which affected retinal vascularization, and structure in the long term. Our study aids the understanding of the cellular and molecular complexities of the retina following oxidative stress, and could lead to potential new therapeutic targets.

Support or Funding Information

Department of Pediatrics, University of Wisconsin, Madison (OJM), National Institutes of Health (R01 EY022086) (AI), and Timothy William Trout Professorship in Eye Research (AI)
VEGF is required for the initiation of Cerebral Cavernous Malformations
Authors: Glading Angela , DiStefano Peter , Chen Tiehua , Su Hua , Whitehead Kevin
Published: June 2021

There is a narrow developmental window of susceptibility shortly after birth in which genetic deletion of CCM genes is sufficient to lead to spontaneous vascular malformations. Using injection of viral vectors, we show that CCM lesion formation not only requires loss of CCM genes but also active angiogenesis. Furthermore, VEGFR2 activation is increased following the depletion of KRIT1, CCM2 or CCM3 gene expression in endothelial cells (although increased VEGFa production is only seen with the loss of KRIT1). Subsequently, we show that inhibition of VEGF with the kinase inhibitor SU5416 reduces CCM lesion formation and vascular permeability in Krit1ecKO mice, with a decreased likelihood of lesion hemorrhage as shown by peri‐lesion iron deposits. This mechanism is not specific to KRIT1, as inhibition of VEGF using bevacizumab also reduces lesion formation in Ccm3ecKO mice.

Support or Funding Information

This work was supported by NIH grant HL117885 to A.J.G, NIH grant NS075168 to K.J.W., and AHA pre‐doctoral fellowship 14PRE2048009 to P.V.D.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Creating a Unique Graduate Training Program for Educator Scholars in the Anatomical Sciences
Authors: Notebaert Andrew
Published: June 2021

Healthcare education has made large leaps to catch up to new educational theories and best practices. Teaching roles in healthcare have traditionally fell to basic science researchers and clinicians with content area expertise but possibly little educational training. Recently, there has been a push to educate future teachers through graduate programs, most notably in anatomical education. While most of these education track PhD programs devote a large amount of training in the anatomical sciences and pedagogy skills, other educational facets are taught sparingly. Using other graduate programs in both science education and anatomy education as curricular models, the University of Mississippi Medical Center has restructured its PhD in Clinical Anatomy which itself was a restructuring of a traditional PhD in Anatomy. The original Clinical Anatomy PhD had students taking the medical anatomy courses (gross anatomy, histology, developmental, and neuroscience) and supplementing with four total credit hours of education training over three courses. Additionally, students would serve as a teaching assistant for each of the medical anatomy classes, earning additional credit hours before progressing to the dissertation phase of the program. The new Clinical Anatomy curriculum was designed to focus on three equal aspects of training; anatomical content, educational content, and educational research. The program gives equal focus to each of these important aspects as well as the integration of these areas with each other to train educator scholars capable of contributing to the education and research mission of healthcare educational programs. The anatomical content courses were restructured to focus on a foundational level of graduate anatomy content rather than limiting the training to only medical anatomy. The educational courses were expanded and focus on three areas; learning theory from both an educational and neuroscience perspective, pedagogical theory and methods, and curricular development and design. The research experiences were restructured so that the students get early exposure to research projects and statistical analysis, leading into abstract and manuscript work much earlier in their training that in previous renditions of the program. However, as with any graduate program, the graduates have to be able to fill positions for which they were trained upon graduation. Based on a recent pilot analysis of job openings seeking teachers of the various anatomy disciplines, there are a variety of open anatomy education vacancies but it is unknown if these vacancies would value students trained under these new graduate programs over the traditionally trained anatomists and basic science researchers.

Support or Funding Information

This project was not funded

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Integration, Evolvability, and Constraint within the Primate Functional Shoulder Complex
Authors: Agosto Elizabeth R. , Auerbach Benjamin M.
Published: June 2021

In vertebrates, especially primates, the morphology of the scapula is closely associated with locomotion, and individual traits of the shoulder are hypothesized to be under directional selection to meet these behavioral demands. By ascribing individual traits to variation in locomotor behaviors, however, researchers implicitly assume traits evolve independently. Traits that share mechanical function may covary genetically, and could therefore evolve through correlated responses to selection. The scapula, for example, is part of a complex of functionally related traits, linked both developmentally and mechanically, which includes dimensions of the clavicle, humerus, and basicranium. This study investigates whether the functional relationships among these skeletal regions relate to evolutionary covariance by comparing patterns of integration and evolvability among homologous traits in humans and tamarins.

Three‐dimensional landmarks were taken from the scapula, humerus, clavicle and basicranium of 109 humans (Homo sapiens) and 100 tamarins (Saguinus sp.). Twelve interlandmark distances were calculated from these landmarks for analysis, representing a complex of traits relating to mechanical function from both the basicranium and the shoulder girdle. Analyses aimed at examining evolutionary covariance were applied using both raw and data scaled by the geometric mean. The strength of integration was quantified using the analysis of eigenvalues and a measure of trait variational autonomy. Evolvability was estimated by applying 1000 simulated random selection gradients to the phenotypic variance/covariance matrices and comparing evolutionary responses among taxa. Statistical significance of differences among taxa was determined by recomputing each value of integration and evolvability for 1000 iterations of the bootstrapped sample. A t‐test was used for pairwise comparisons of the bootstrapped values among taxa (α = 0.05).

Results indicate when traits from the basicranium and shoulder girdle are evaluated as a complex, the ability of these regions to respond to directional selection is decreased compared to their responses were they independent. Evolutionary independence among these traits is not supported, and analyses instead indicate evolutionary constraint among these regions. Morphological differences in the nuchal region and shoulder complex between humans and tamarins further reflect differences in the ability to respond to directional selection in these two genera, which may have had functional implications for evolution of the shoulder in apes and monkeys. Namely, the traits in humans are more tightly integrated, resulting in weaker evolvability.

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 Warburg Effect and Lactate Signaling Augment Fgf Signaling to Promote Sensory‐neural Development in the Otic Vesicle
Authors: Riley Bruce , Kantarci Husniye
Published: June 2021

In a forward ENU screen in zebrafish, we recovered a novel mutation that specifically impairs development of sensory hair cells and neurons in the otic vesicle. Whole‐genome sequencing identified a null mutation in the gene encoding the glycolytic enzyme Phosphoglycerate Kinase‐1 (Pgk1). Targeted disruption of Pgk1 using CRISPR/Cas9 yields an identical phenotype. Surprisingly, analysis of genetic mosaics showed that Pgk1 acts non‐autonomously to promote efficient Fgf signaling at a distance, as required for specification of neurons and hair cells of the inner ear. Expression of known Fgf ligands appears normal but Fgf‐reporters Pea3 and Erm are dramatically under‐expressed in mutant embryos. Moreover, transient overexpression of Fgf8 from an inducible transgene does not rescue the phenotype, suggesting a problem with signal transduction. These and other data led us to the discovery that otic cells require metabolic reprogramming to enhance glycolysis and “aerobic fermentation” – similar to the “Warburg Effect” exhibited by tumor cells – to promote synthesis and secretion of lactate. Wild‐type embryos treated with drugs that block synthesis or secretion of lactate mimic the phenotype of Pgk1 mutants, whereas Pgk1 mutants are fully rescued by treatment with exogenous lactate. Lactate treatment of wild‐type embryos elevates expression of Pea3 and Erm even when Fgf signaling is blocked. Thus, by raising steady‐state levels of Pea3 and Erm (critical effectors of the Fgf‐MAPK pathway), lactate signaling primes the pathway and renders cells more responsive to dynamic changes in Fgf signaling required for sensory‐neural development in the inner ear.

Support or Funding Information

NIH‐NIDCD grant R01‐DC03806

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SMRT‐Cappable‐seq reveals complex operon variants in bacteria
Authors: Ettwiller Laurence , Yan Bo
Published: June 2021

Gene expression in bacteria is organized into operons, functional units of genomic DNA containing clusters of genes under the control of single promoters. The definition of an operon relies on the precise identification of the start of transcription (TSS), the end of transcription and (TTS) and the relation between the TSS and the TTS for each transcript molecule. Yet current experimental methods for sequencing RNA in bacteria rely on the necessary fragmentation of transcripts for short read sequencing. Consequently the larger context by which genes are expressed and the phasing between the start and the end of primary transcripts are lost. As a result, and despite overwhelming mass of transcriptome information, accurate operon structure have so far been solved only for a handful of cases. Here we describe SMRT‐cappable‐seq as the first experimental methodology to identify operons genome‐wide in prokaryotes. It combines the isolation of full‐length primary transcripts with long read SMRT sequencing. Applied to E.coli, SMRT‐cappable‐seq identifies a total of 2300 operons from which around 900 are novel. Importantly, our result reveals a pervasive read‐through of previous experimentally validated transcription termination sites. Termination read‐through represents a powerful strategy to control gene expression and redefines operons with thousand of new operons containing more genes than previously thought. Taken together this data provides a first glance at the complexity of the ‘operome’ in bacteria and presents an invaluable resource for understanding gene regulation and function in bacteria.

Support or Funding Information

New England Biolabs Inc. and Pacific Biosciences of California, Inc supported this research.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Describing the molecular mechanism by which the A2 protein improves survival in mice with endotoxemia.
Authors: Martinez Marina , Da Qi , Fisipe Titilope A. , Cruz Miguel A.
Published: June 2021

Von Willebrand factor (VWF) is an essential protein that mediates the platelet adhesion to the exposed subendothelium surface and significantly contributes to arterial thrombosis and stroke. Secreted VWF from the stimulated endothelium as long strings remain anchored to the surface of the endothelial cells and interact with circulating platelets, leading to vessel occlusion. We have shown that the isolated A2 domain of VWF (A2 protein) contains bind activity for the A1 domain, vimentin, and fibrin(ogen). In fact, as a result of those interactions, the A2 protein impairs platelet adhesion to VWF and fibrin(ogen), formation of VWF strings on the endothelium, and fibrin polymerization. The A2 protein was effective in preventing fibrin‐rich microvascular thrombosis and improving survival (100%) of mice with endotoxemia (LPS)‐induced disseminated intravascular coagulation (DIC). Thus, a goal was to identify the binding sites of A1 domain, vimentin, and fibrin in the A2 protein and dissect the mode of action by which the A2 protein recuperates the sick animals. To determine the amino acid residues that are important in the recognition for vimentin, A1 and fibrin, I used the crystal structure of the A2 domain as a guide for point mutations. Two mutations, E1523A and E1567A were introduced in the A2 protein. The purified mutants were tested for their binding capacity for the ligands using ELISA. Moreover, the A2 mutants were tested in mice with endotoxemia (LPS). We are now demonstrating that E1523A mutation had a reduced binding affinity for Vimentin and A1 domain. Strikingly, E1567A and E1523A mutations significantly abolished the binding to fibrin(ogen). Importantly, both mutations failed to replicate the beneficial effect of the wild type A2 protein in improving survival of mice with LPS‐induced DIC. Together, these findings suggest that the A2 protein impairs microvascular thrombosis in mice with endotoxemia mainly via its interaction with fibrin.

Support or Funding Information

This research was supported by funding provided by the following NIH Diversity Supplement: R01GM112806‐0351.

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 metabolomic signature of Giardia sp.
Authors: Allain Thibault , Groves Ryan , Lewis Ian A. , Buret Andre G.
Published: June 2021

BACKGROUND

Giardia duodenalis is a protozoan parasite responsible for giardiasis, a parasitic disease characterized by intestinal malabsorption, diarrhea, weight loss and abdominal pain. The pathophysiological processes occurring during giardiasis involve epithelial abnormalities, mucus depletion and microbiota disruption, however the mechanisms are poorly understood. Upon infection, Giardia, which has a minimal biosynthetic capacity, compete locally with the commensal microbiome for nutrients and ecological niches in the duodenum.

AIM

In this context, we performed a metabolomic study to characterize the nutritional requirements and the secretome of several Giardia strains, isolated from human patients (assemblages A and B), outbreaks, as well as a variety of mammals.

METHODS

The metabolomic profiles of Giardia sp. isolates were determined using mass spectrometry. Trophozoites were grown to confluence in MTYI media and supernatants were collected at different time points (3, 6, 9, 12 and 24 hours) and mixed with ice cold methanol. Methanolic extracts were analyzed by Ultra High Performance Liquid Chromatography mass spectrometry and resolved by Hydrophilic interaction liquid chromatography column.

RESULTS

We identified a multitude of produced and consumed compounds such as amino acids, nucleic acid precursors, protein catabolism precursors, as well as carbohydrates and energy metabolites. Interestingly, nutrition requirements of Giardia trophozoites are significantly different between isolates, both intra and inter‐assemblages. For instance, isolate WB6 (assemblage A) is more auxotrophic for arginine than GSM strain (assemblage B), while GSM strain requires more inosine, uridine and as paragine than other isolates.

CONCLUSION

This study gives insight into how Giardia survives the host's intestinal tract and shows strain specific metabolomic signatures. A metabolomic approach may further help to understand the impact of nutritional modulation by Giardia trophozoites on the gut microbiome and host physiology.

Support or Funding Information

Grant support: University of Calgary Eyes High postdoctoral fellowship

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Shp2/Ptpn11 Deletion Suppresses Liver Tumorigenesis Driven by MET, β‐Catenin and PIK3CA
Authors: LIU Jacey
Published: June 2021

Most recent experimental data demonstrated a liver tumor‐suppressing effect for Shp2/Ptpn11, as ablating Shp2 in hepatocytes triggered spontaneous development of hepatocellular adenoma (HCA) in aged mice and also aggravated hepatocellular carcinoma (HCC) induced by chemical carcinogen or Pten deletion. Herein we report surprisingly that despite the induction of hepatic injuries, inflammation, and fibrosis, Shp2 deletion in hepatocytes suppressed liver tumorigenesis driven by overexpression of oncogenic proteins MET and ΔN90‐β‐catenin (MET/CAT), or MET and PIK3CAH1047R (MET/PIK). Shp2 loss inhibited proliferative signaling from MET, Wnt/β‐catenin, Erk and PI3K/Akt pathways, but induced cell senescence following exogenous expression of the oncogenes. These results clearly distinguish a requirement for Shp2 in mediating and amplifying the oncogenic signals of these oncoproteins from a tumor‐promoting hepatic microenvironment resulting from Shp2 deficiency. Based on these data, we propose a new molecular targeted therapy by inhibiting Shp2 for HCCs driven by oncogenic signals emanating from RTKs and other upstream molecules. However, to achieve optimal and lasting therapeutic effect, it is necessary to suppress the tumor‐enhancing environmental factors produced secondary to Shp2 inhibition. This simultaneous targeting approach may be developed into a widely used clinical recipe, as long as the cell‐intrinsic drivers and the stromal factors are identified for a given liver cancer patient in the precision medicine era.

Support or Funding Information

This project was supported by NIHR01CA176012 and R01CA188506 (to Dr. Gen‐Sheng Feng).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Upregulation of Stromal‐derived Factor‐1 by Hypoxia Requires Hypoxia‐Inducible Factor‐1α and Transforming Growth Factor‐1
Authors: Strickland Jenna D. , Garrison Dominique , Copple Bryan
Published: June 2021

Following both acute and chronic liver injury, expression of the chemokine stromal‐derived factor‐1 (SDF‐1), or CxCl12, is upregulated and plays a vital role in the activation of down‐stream signaling pathways implicated in either driving hepatic injury progression or leading to its repair. For example, while SDF‐1 has been implicated in promoting tumor growth and metastasis in patients with hepatocellular carcinoma, in acute injuries it may play an important role in liver regeneration. This suggests SDF‐1 may represent a novel target for therapeutic intervention in both acute and chronic liver diseases. However, the mechanism by which SDF‐1 is upregulated following hepatic injury is not fully known. The present study determined whether the transcription factor, hypoxia‐inducible factor‐1α (HIF‐1α), regulates SDF‐1 in primary mouse hepatocytes. To activate HIF‐1α in vitro, primary hepatocytes were incubated for 72 hrs in either room air or hypoxic (1% O2) conditions. Under hypoxic conditions, SDF‐1 mRNA levels were upregulated after 72 hr in WT mouse hepatocytes, an effect that was prevented in hepatocytes isolated from either HIF‐1α or HIF‐1β knockout mice. We demonstrated previously that upregulation of several genes in hepatocytes during hypoxia requires autocrine release and activation of transforming growth factor‐1 (TGF‐β1). To determine if TGF‐β1 is required for the upregulation of SDF‐1 during hypoxia, primary mouse hepatocytes were pretreated with a TGF‐β1 receptor antagonist (SB‐431542) and placed in hypoxic conditions for 72 hrs. Pretreatment with SB‐431542 completely inhibited upregulation of SDF‐1 by hypoxia, suggesting TGF‐β is required for upregulation of SDF‐1 in hypoxic hepatocytes. Additionally, while treatment of hepatocytes with TGF‐β1 upregulated SDF‐1, this effect did not require HIF‐1α, suggesting that TGF‐β1 is downstream of HIF‐1α activation. We previously demonstrated that hypoxia upregulates thrombospondin‐1 in hepatocytes, which could be responsible for activation of latent TGF‐β1. Upregulation of SDF‐1 by hypoxia, however was not different between hepatocytes isolated from wild‐type or thrombospondin‐1 knockout mice. The results indicate that hypoxia activates HIF‐1α in hepatocytes, which leads to activation of latent‐TGF‐β1. TGF‐β1 then acts in an autocrine fashion to upregulate SDF‐1. However, the mediator responsible for converting latent TGF‐β to its active form remains unknown.

Support or Funding Information

NIH grant 2 R01 DK073566NIH Training Grant T32 ES007255

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding the interplay between amino acid and lipid metabolism in tumor growth
Authors: Metallo Christian
Published: June 2021

Metabolism is central to virtually all cellular functions and contributes to a range of diseases. A quantitative understanding of how biochemical pathways are dysregulated in the context of diseases such as cancer and metabolic syndrome is necessary to identify new therapeutic targets. To this end we apply stable isotope tracers, mass spectrometry, and metabolic flux analysis (MFA) to study metabolism in mammalian cells, animal models, and human patients. Using these approaches we have characterized how proliferating and differentiated cells regulate flux of glucose and amino acids into lipid biosynthetic pathways. We are particularly interested in understanding how amino acid and lipid metabolism are coordinated to meet these needs. Transport of pyruvate into mitochondria is a critical step in these processes, and activity of the mitochondrial pyruvate carrier (MPC) strongly influences cancer growth in vivo and as tumor spheroids. Using metabolomics and lipidomics approaches we have identified mechanisms through which MPC flux and amino acid availability impact sphingolipid metabolism. Modulation of these pathways influences tumor growth, providing mechanistic insights into how amino acid metabolism can influence lipids and mitochondrial function.

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 struggle for metal at the host‐pathogen interface
Authors: Skaar Eric
Published: June 2021

Hospital and community‐acquired infections caused by bacterial pathogens represent an increasing threat to global public health. This threat is compounded by the fact that bacterial pathogens are rapidly becoming resistant to all existing antimicrobial. Research in my laboratory is focused on identifying novel targets for therapeutic intervention against bacterial pathogens with a particular emphasis on systems involved in metal trafficking and metabolism. Metals are essential for all life because approximately 40% of all proteins in nature require metals to carry out their biological function. Bacterial pathogens must acquire metals inside their hosts in order to successfully mediate infection. However, vertebrates have evolved strategies to sequester nutrient metal from invading bacteria in a process known as “nutritional immunity”. Based on the strict requirement of bacteria for metal, and the fact that the bacterial and eukaryotic machinery involved in metal acquisition and homeostasis are remarkably different, targeting bacterial machinery involved in metal metabolism has excellent therapeutic potential. This presentation will describe research in my laboratory that is focused on identifying factors and processes involved in the battle for metal between bacterial pathogens and their hosts.

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 Education Track PhD Program in Anatomy at Indiana University School of Medicine: A Decade Producing Anatomy Educator‐Scholars
Authors: Brokaw James J. , Husmann Polly R. , O'Loughlin Valerie D.
Published: June 2021

In 2008, the Indiana University School of Medicine (IUSM) admitted its first student to a newly approved PhD program focusing on anatomical education rather than biomedical research. The goal of the Education Track PhD program is twofold: (1) to provide students with extensive training in all of the anatomical sub‐disciplines coupled with sufficient teaching experience to be fully prepared to assume major educational responsibilities upon graduation and (2) to train students to conduct rigorous, medical education research and other educational scholarship necessary for promotion and tenure. The 90‐credit hour curriculum includes coursework in anatomy and other biomedical subjects, education, statistics, and electives. For their dissertation work, the students complete a research project about some aspect of medical education. As of December 2017, the Education Track program had admitted 23 students and produced 10 graduates. Two more students are anticipated to graduate by June 2018. All of the graduates were offered faculty appointments (8 tenure track and 2 non‐tenure track) immediately upon graduation at major universities and medical schools. Four of the graduates were offered positions at IUSM. Eight graduates have appointments in medical school anatomy departments, 1 in a physical therapy department, and 1 in a physician assistant department. None of the graduates have been in their faculty positions long enough yet to have achieved tenure. During the period from 2009 through 2017, the students and graduates of the program gave 77 conference presentations and authored 40 peer‐reviewed publications about anatomy or other aspects of medical education. Thus, in the 10 years since its inception, the Education Track program has successfully produced a small but stable supply of doctoral‐level anatomy educator‐scholars for a growing academic market.

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