bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipidomic Analysis of Media from Castration and Drug Resistant Prostate Cancer Cells
Authors: Mansoura Maryam , Ingram Lishann M. , Cummings Brian S.
Published: June 2021

Castration‐resistant prostate cancer (CRPC) is a severe, metastatic form of prostate cancer (PCa). Several treatment modalities have been approved during the past decade by the FDA for treating CRPC patients. Unfortunately, a high percentage of patients fail to respond to these treatments for several reasons, including developing resistance. One reason for these failures is inadequate methods that allow for accurate staging of PCa. This poses a diagnostic problem that limits our ability to create a patient‐centered treatment plan. We addressed this problem by testing the hypothesis that an association exists between circulating lipids and clinical outcomes of CRPC. This hypothesis is based on recent studies demonstrating a correlation between plasma lipids and CRPC prognosis. To understand this correlation, we used in vitro models of non‐cancerous, hormone‐sensitive, and drug‐resistant CRPC cell lines combined with quantitative lipidomic analysis to study the correlation between CRPC and lipid profiles. An untargeted approach indicated that media from hormone sensitive cells (LNCaP), and CRPC cells (DU145) had lower relative abundance of glycerophospholipids, as compared to a non‐cancer cell model (PNT2). However, drug resistant cells (DU145 DR) and their media had a higher abundance of glycerophospholipids such as phosphatidylinositol (PI), phosphatidic acid (PA), and phosphatidylserine (PS) as compared to parent control (DU145). These data confirm previous in vivo findings and suggest that the lipidomic profiles of PCa cells lines mirror lipidomic profiles in the plasma of PCa patients. Further, these data provide an in vitro model to investigate the molecular mechanisms mediating lipidomic changes during the progression of drug resistant PCa.

Support or Funding Information

National Institute of Biomedical Imaging and Bioengineering NIBIB (EB0160100), Department of Defense Prostate Cancer Research Program Idea Development Award (PC150431 GRANT11996600)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dual Roles of Reactive Oxygen Species in Exacerbated Proinflammatory Effects of Caveolin‐2 Deficiency in Ischemia/Reperfusion and the Anti‐inflammatory Effects of Ethanol Preconditioning
Authors: Wang Meifang , Wang Derek , Liu Yajun , Sowa Grzegorz , Korthuis Ronald
Published: June 2021

Caveolin‐2 (Cav‐2), a member of caveolin protein family, is ubiquitously co‐expressed ubiquitously with caveolin‐1 (Cav‐1) in vascular endothelial cells. Cav‐2 interacts with Cav‐1 to form a hetero‐oligomeric complex within caveolae. In contrast with the extensively studied Cav‐1 including the functional role of Cav‐l expressed in ECs in ischemic injury, much less is known regarding the effects of Cav‐2. We hypothesized that (i) genetic deletion of Cav‐2 (CAV‐2 KO) would promote proinflammatory effects in the absence of I/R in wild‐type (WT) mice; (ii) postischemic inflammation will be exacerbated in CAV‐2 KO mice compared to WT animals; (iii) the proinflammatory effects induced by I/R are due to generation of reactive oxygen species (ROS) in CAV‐2 KO mice; and (iv) ingestion of ethanol at low to moderate levels (EPC) 24 hours prior to ischemia (45 min) followed by reperfusion (60 min) (I/R) will limit postischemic inflammation in CAV‐2 KO mice by a ROS‐dependent mechanism. Intravital fluorescence microscopy was used to visualize leukocyte rolling (LR) and adhesion (LA) in single postcapillary venules of the small intes­tine. Prior to I/R, male CAV‐2 KO mice were treated with EPC, MnTBAP (cell‐permeable SOD mimetic) 1 hour prior to I/R, or MnTBAP 20 minutes prior to EPC, respectively, with postcapillary venular LR and LA quantified during the first hour of reperfusion. Our data shown that LR and LA were markedly elevated in male CAV‐2 KO mice under baseline condition relative to that noted in WT animals. I/R‐induced LR and LA were increased to a greater extent in CAV‐2 KO mice compared to WT animals. These postischemic increases in LR and LA were abolished by treatment with EPC or by exposure to MnTBAP 1 hour prior to I/R. MnTBAP pretreatment 20 minutes prior to EPC also abolished the protective effects of EPC. These results indicate that genetic deletion of CAV‐2 produces proinflammatory effects under baseline conditions and exacerbates these effects after I/R by a ROS‐dependent mechanism. Antecedent EPC prevents the proinflammatory responses to I/R in CAV‐2 KO mice. This protective effect of EPC was initiated by ROS.

Support or Funding Information

NIH: R01‐AA022108, R01‐HL081860; DOD: W81XWH‐15‐1‐0624.

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 effect of maternal high fat diet on offspring muscle regeneration
Authors: Mikovic Jasmine , Islam Rabia , Lamon Séverine
Published: June 2021

Background

Skeletal muscle fibres are post‐mitotic cells. As such, they exclusively rely on regenerative processes to restore muscle structure and function when damage has occurred through direct trauma or everyday tasks. Skeletal muscle quality relies on its development during the pre‐natal period. Foetal muscle development is a highly plastic process that is influenced by the intrauterine environment. Maternal behaviour, such as diet or exercise, can alter this environment. Using a maternal high fat diet model, we fed C57Bl/6 female mice with a low or high fat diet prior to and during gestation and lactation. The offspring were then switched to a low or high fat diet. We assessed whether maternal diet hindered offspring muscle regeneration after injury, and whether post‐natal diet could reverse these effects.

Results

Adiposity levels were higher in C57Bl/6 female mice consuming a high fat diet. There was no difference in offspring weight born to lean or obese dams at weaning; however, in adulthood, offspring born to obese dams were smaller than their control counterparts regardless of post‐natal diet. Markers of muscle regeneration including Pax7, MyoG, Myf5 and MRF4 were downregulated in offspring born to an obese mother or fed a high‐fat diet when compared to offspring experiencing normal growth.

Conclusion

An obesogenic maternal diet negatively affects offspring muscle growth and markers of muscle regeneration. These effects could not be offset by offspring diet, highlighting the importance of the pre‐natal environment in muscle development and function.

Support or Funding Information

Australian Research Council (DE150100538)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanical Characterization of Frozen and Freshly‐Isolated Human Brain Tumors
Authors: Stewart Daniel , Marsh Erica , Simmons Renee , Simmons Chelsey
Published: June 2021

Introduction

Tissue banks are a common human tissue source for oncology researchers, and common pathobiological assays have been optimized to accommodate previously frozen tissue. However, emerging interest in tissue mechanics to motivate tissue‐on‐a‐chip applications necessitate additional optimization for and understanding of mechanical changes with prolonged storage. Here, we seek to compare the mechanical properties of previously frozen to freshly‐isolated human brain tumors.

Methods

All human tumor samples were collected and data analyzed under University of Florida Institutional Review Board‐approve protocols. Meningioma samples were isolated from patients and stored at −80°C for 2 or 5 years before thawing in DMEM at room temperature and characterizing with a custom‐built indentation system within 30 min after thawing. Indentation and subsequent analysis of samples was performed fresh, never frozen tissue (1). Briefly, samples were indented and allowed to undergo stress‐relaxation until a quasi‐static state was reached. The steady‐state modulus was then determined by fitting the data to the Standard Linear Solid viscoelastic model, which represents the quasi‐static properties of the sample. Freshly‐isolated human tumor and normal mouse brain data was originally published in PLoS ONE in 2017 (1). Statistics were performed with a Wilcoxon non‐parametric test with multiple comparisons. Values are reported as median ± median absolute deviation.

Results and Discussion

Freshly‐isolated human meningioma samples were about twice as stiff (2.83±1.26 kPa, Figure 1) as frozen meningiomas stored for 2 or 5 years (1.15±0.26 kPa and 0.97±0.54 kPa, respectively, p<0.0003). These findings are similar to those in the literature comparing showing that frozen tissue is softer than the freshly isolated counterparts (2, 3). The change in the mechanical properties of previously frozen tissue may reflect disruption of the extracellular matrix (ECM) architecture and cellular fidelity due to expansion and contraction of ice crystals during a freeze/thaw cycle (4). Future work will investigate changes in the ECM structure via SEM and immunostaining to correlate ECM structure and mechanical properties, allowing for a better understanding of cryogenically‐stored tissue.

Support or Funding Information

We gratefully acknowledge support from the Florida Center for Brian Tumor Research for human tissue coordination and collection, and funding support from Medtronic and NSF Award No. 1711543

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sensitivity of Trabecular Mapping to Sliding Semilandmark Placement
Authors: Russell C. Kinley , Cunningham Deborah L. , Wescott Daniel J. , Gleiber Devora S. , Sylvester Adam D.
Published: June 2021

Analyses of bone microstructure based on single volumes of interest (VOIs) are limited in their ability to quantify variation in trabecular properties across a joint. Geometric morphometric methods can overcome these limitations by utilizing sliding semilandmarks to locate multiple VOIs within a trabecular structure. Variation in the placement of semilandmarks, however, may change collected bone parameter values. Here we quantify the effect of intraobserver error in delineating the articular surface on sliding semilandmark placement and sampled trabecular properties.

Proximal tibiae of 12 individuals that are part of the Texas State University Donated Skeletal Collection were scanned using micro‐computed tomography at the Forensic Anthropology Center at Texas State. Original grey scale image stacks were converted to binary images, and surface models of the exterior bone surface were created. Medial condyles were trimmed from their full surface models three times over a period of weeks. Sliding semilandmarks (n=141) were distributed on the condyle surfaces and then slid to minimize the bending energy of the thin‐plate spline function relative to the updated Procrustes average. Each sliding semilandmark was then used to locate a single VOI just deep to the cortical shell. To investigate the effect of landmark placement on trabecular bone parameters, each VOI was moved 0.5–1.5 mm to one of 10 neighboring positions. Bone volume fraction (BV/TV) and trabecular thickness (Tb.Th) were calculated for each VOI at each of the 11 positions. VOIs that moved outside the trabecular structure were removed from further analyses. 10,000 randomized combinations of the 11 positions of each VOI were created and correlations between bootstrap samples were calculated.

Sliding semilandmark placement differed by 0.27 mm on average. Landmarks most affected by intraobserver error were those placed along the edge of the condylar surface (mean = 0.38 mm), while those on the interior portion of the condyle were affected less (mean = 0.21 mm). Sampled BV/TV and Tb.Th vary with VOI position, although there is a strong correlation among the 11 sampled values for BV/TV (r=0.83) and Tb.Th (r=0.80). These results suggest that VOI positions calculated from sliding semilandmarks provide consistent information about bone microstructure even when VOI placement is forced to deviate beyond average variation in sliding landmark position.

Support or Funding Information

This work was supported in part by instrumentation funded by the NSF under grant NSF: MRI 1338044.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neanderthal Eustachian tube morphology lowered population‐wide fitness and contributed to extinction
Authors: Pagano Anthony Santino , Marquez Samuel , Laitman Jeffrey T.
Published: June 2021

Among modern humans, otitis media (benign otitis media with effusion and acute otitis media, AOM) is the second most common pediatric clinical conditions. Acute otitis media is a fatal disease that can result in meningitis and pneumonia if untreated. Osteological signs of this disease may be found in erosion patterns on the middle ear ossicles, ossification of the tympanic membrane, and a lack of mastoid pneumatization. Recent studies report that nearly all known Neanderthal ossicles exhibit erosion patterns consistent with chronic AOM. To account for the anatomical basis of this apparent disease susceptibility, we assessed the morphological configuration of the cartilaginous Eustachian tube (CET) and dilator tubae (DT) musculature among a modern human growth series ranging from newborns to adults (n=442) and a sample of Neanderthals (La Chapelle aux Saints 1, La Ferrassie 1, Forbes Quarry 1, Saccopastore 1, Guattari 1) and mid‐Pleistocene pre‐Neanderthals (Atapuerca 5 and Steinheim 1). We performed morphometric analysis of the bony upper respiratory tract boundaries. A total of 31 bilateral and midline 3D landmarks were used and coordinate data were subjected to Procrustes‐fitting for geometric morphometric analysis and calculation of univariate measures.

Neanderthals exhibited tall narrow choanae and greater overall robusticity. La Ferrassie 1 and Forbes Quarry 1 possessed extremely infant‐like CET orientation relative to the Frankfurt Horizontal Plane while other specimens had adult‐like or intermediate orientation. Choanal orientation among all Neanderthals was horizontal as among human infants. This trait occurred alongside a relatively adult‐like elongation and vertical orientation of the DT whereas the pre‐Neanderthals were distinguished primarily by an extremely short DT relative to Neanderthals and modern humans.

The morphological configuration of Neanderthals was unlike any modern human population or growth stage. Differences between these groups appear to be species‐level with Neanderthals retaining infant‐like horizontal orientation of both the choanae and (among some) CET into adulthood. Neanderthal children were probably susceptible to AOM for longer periods of development. While modern human children experience steep reduction in disease occurrence at approximately six years of age with changes in both CET length and orientation, Neanderthal children would have remained susceptible as there was no such growth change in CET orientation. This could have impacted population‐wide fitness when sympatric modern humans invaded Neanderthal habitats and competed for limited resources. AOM should thus be considered among the many potential factors involved in the extinction of the Neanderthals.

Support or Funding Information

National Science Foundation, Grant number 1128901

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RNA helicase homolog and its association with the JAMM2 metalloprotease required for targeted protein turnover in Archaea
Authors: De Jesus Ramirez Cuper , Hauserman Matthew , Cao Shiyun , Maupin‐Furlow Julie A
Published: June 2021

A functional analogue of the eukaryotic ubiquitin‐proteasome system (UPS) is found in archaea. This archaeal system, termed the SAMP‐proteasome system (SPS), operates in targeted protein turnover; however, many of its protein components [E1‐like UbaA, Cdc48/p97‐/Rpt‐ type AAA ATPases, and JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) homologs] remain to be fully characterized. To dissect the role of the JAMM metalloprotease homologs within the SPS, we previously expressed and purified the two Haloferax volcanii JAMM/MPN+ homologs (HvJAMM1/2) from recombinant Escherichia coli. Only HvJAMM1 was demonstrated to be active as an Ubl isopeptidase. By contrast, HvJAMM2 was found to be required in vivo for protein turnover by the SPS. Here we focused on identifying and characterizing the protein partners and/or biochemical conditions which may be required in vitro for HvJAMM2 activity. For this objective, His6‐HvJAMM2 was expressed in H. volcanii and purified by Ni2+‐ chromatography to overcome factors that may be absent from recombinant E. coli. Co‐purifying protein partners were identified by LC‐MS/MS analysis, including HVO_2955 which is predicted to be a structural homolog of the JAMM/MPN− interacting RNA helicase Brr2 of the eukaryal spliceosome. While hvo_2955 was not essential for growth, we found an Δhvo_2955 mutant to have decreased growth under standard conditions, with the genetic defect trans complemented by expression of His6‐HVO_2955. Current work is focused on determining the role of HVO_2955 during stress as well as its potential binding to JAMM‐domain proteins in archaea. Assessing the role of this Brr2 structural homolog in RNA processing in conjunction with SPS components such as HvJAMM2 is anticipated to show a clearer picture of archaeal physiology.

Support or Funding Information

Funds for this project were awarded to JM‐F through the Bilateral NSF/BIO‐BBSRC program (NSF 1642283), the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Program (DOE DEFG02‐05ER15650) and the National Institutes of Health (NIH R01 GM57498). Additional support is provided by the University of Florida's Preeminence Fellowship awarded to CR.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Conditional Protein Splicing of Inteins from Extremophiles
Authors: Mills Kenneth V
Published: June 2021

Protein splicing is a post‐translational process facilitated by an intein, by which the intein catalyzes its own excision from flanking polypeptides (the exteins), concomitant with ligation of the exteins. Many inteins are themselves interrupted by homing endonuclease domains that can facilitate homing into intein‐less alleles. We have discovered inteins from extremophiles that can be expressed in E. coli, purified as unspliced precursors, and induced to splice by cues such as incubation under reducing conditions, at high temperature, or at high ionic strength. We have shown that the activity and folding of inteins from the extreme halophiles Halobacterium salinarum and Haloquadratum walsbyi demonstrate variable dependence on salt concentration, and we have designed transgenic organisms lacking inteins to test the physiological role of splicing. We have solved the structure of an intein from the extreme thermophile Pyrococcus abyssi, and studied the influence of structural elements on its stability at high temperature and pressure. We also have discovered that the splicing and nuclease activity of inteins from extreme thermophiles can be influenced by pressures consistent with those found at the seafloor.

Support or Funding Information

This work was supported by grants from the National Science Foundation (grants MCB1244089 and MCB1517138) and the Camille and Henry Dreyfus Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using fluazifop‐p‐butyl for low cost increases in lipid accumulation for the generation of algal biofuels from Chlorella vulgaris
Authors: Pauley Annabella Grace , Smythers Amanda , Perry Nicole , Kolling Derrick
Published: June 2021

The demand for sustainable, low‐emission fuels is increasing with the global expansion of industrialization. Biofuels have the potential to meet this need without the problematic addition of carbon into the earth's atmosphere. Algae, a potential biofuels feedstock, have the capability to quickly accumulate biomass under various conditions, including low‐nutritional growth medium and a reduced need for arable land. Current research is seeking to increase organismal efficiency in order to increase economic feasibility. Previous work has shown that exposing algae to environmental stressors or limitations, such as nitrogen deprivation, can increase lipid production in the cultures, though this is typically accompanied by a decrease in overall biomass. Nitrogen deprivation is also expensive to facilitate, so finding more economical methods is paramount to increasing algal biofuel feasibility. In this study, Chlorella vulgaris, a unicellular green alga, was exposed to a formulation of an inexpensive and readily available mass‐produced herbicide that targets acetyl Co‐A carboxylase with its active ingredient, fluazifop‐p‐butyl. The compositional changes of the algae cultures were monitored as well as the photosynthetic efficiency. Preliminary results have shown an increase in percent lipids in cultures exposed to the herbicide formulation, though the overall biomass decreased. Current results in this study will be presented.

Support or Funding Information

This research was made possible by NASA West Virginia Space Grant Consortium, Training Grant # NNX15AI01H

This material is based upon work supported by the National Science Foundation under Cooperative Agreement No. OIA‐1458952

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cytotoxic Interactions between Pseudomonas aeruginosa Virulence Factors and Metal‐Based Antimicrobials In Vitro
Authors: Mawhinney Thomas , Mossine Valeri V. , Chance Deborah L. , Waters James K.
Published: June 2021

Emergence of multidrug‐resistant (MDR) bacterial pathogens has triggered a search for alternatives to current antibiotic‐based treatments. Toxic metal salts are widely used as anticeptics, but gallium(III) may act systemically, by disrupting iron metabolism in MDR pathogens like Pseudomonas aeruginosa. As P. aeruginosa infections release a range of toxins on their own, we asked whether bacterial toxins may interact with metal‐based drugs against the host.

Using stress‐inducible transcription factor (Nrf2, HIF‐1α, ATF‐6, and MTF‐1) reporter constructs, we have developed a cell‐based protocol for simultaneous evaluation of the cellular metabolic activity, transcriptional/translational activity, and activation of intracellular stress responses in NIH‐3T3 fibroblasts and BEAS‐2B bronchial epithelial cells. The cytotoxicities of four P. aeruginosa cytotoxic virulence factors phenazine‐1‐carboxylate, phenazine‐1‐carboxamide, pyocyanin (Pyo) and its main metabolite, 1‐hydroxyphenazine (1HP), alone and in combinations with five clinically approved antimicrobial metal salts (Ag, Zn, Ce, Bi, and Ga) were evaluated in vitro.

Bacterial phenazines Pyo and 1HP displayed cytotoxicities (IC50 30–90 μM) at concentrations comparable with those detected in sites of P. aeruginosa infections. Silver nitrate was the most cytotoxic metal salt (IC50 < 10 μM), while citrates of cerium(III) and bismuth(III) were not affecting cell proliferation even at 1 mM. In combinations, Bi and Ce inhibited cytotoxicity of Pyo and 1HP, increasing the IC50 2‐fold. In contrast, zinc(II) and gallium(III) strongly synergized with the phenazines, with a decrease in the IC50 reaching over 100‐fold in some experiments. Both Pyo and 1HP strongly (200–500‐fold) activated Nrf2 in the reporter cells, suggesting oxidative stress is a significant contributor to their cytotoxicity mechanism. Both Zn and Ga induced activity of MTF‐1, at subtoxic concentrations, thus implying that interaction of these metals with cytotoxic phenazines occurs along pathways maintaining intracellular redox homeostasis.

This is the first report documenting cytotoxic interactions between clinically approved antimicrobial metal salts and bacterial phenazines at physiologically relevant concentrations. It provides a rationale for further mechanistic and in vivo studies of the phenomenon.

Support or Funding Information

This research was supported by the University of Missouri Agricultural Station Chemical Laboratories, the Cystic Fibrosis Association of Missouri (CFAM), the Leda J Sears Trust Fund for Child Health Research; and National Institute of Food and Agriculture (NIFA) grant No. MO‐HABC0002.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Macrophage migration inhibitory factor (MIF):A novel therapeutic target against aggressive breast cancer
Authors: Das Subhadip , Chatterjee Nabanita , Ahirwar Dinesh K , Mishra Sanjay , Varikuti Sanjay , Kaul Kirti , Satoskar Abhay R , Ganju Ramesh K
Published: June 2021

Triple negative breast cancer (TNBC), defined as loss of estrogen, progesterone and Her2 receptors, is a subtype of breast cancer with worse prognosis and poor survival rate. Increased inflammation further reduces the TNBC patient survival. Macrophage migration inhibitory factor (MIF) is a pro‐inflammatory molecule known to enhance breast cancer progression and metastasis. However, not much is known about the role of MIF in TNBC progression and metastasis. MIF interacts with various oncogenic molecules and signaling pathways. Therefore, targeting MIF might be a promising strategy to inhibit TNBC progression and metastasis. Here we report that CPSI‐1306, a small molecule inhibitor specific to MIF, possesses the ability to target TNBC cell in vitro by inducing apoptosis. The mechanistic studies revealed that CPSI‐1306 induces apoptosis by enhancing reactive oxygen species (ROS), mitochondria membrane potential alteration, cytochrome C release and activation of Caspases. In addition, CPSI‐1306 also inhibits cell survival and apoptosis signaling molecules, including AKT, PDK and RAF expression. Next, we analyzed the clinical utility of CPSI‐1306 using mouse MVT‐1 cells derived orthotopic and human MDA‐MB‐231 cells derived xenograft TNBC mouse models. The oral treatment of tumor bearing mice with CPSI‐1306 significantly inhibited TNBC growth and metastasis to the lungs in a dose dependent manner. The histological analysis of tumors showed higher number of apoptotic cells in CPSI‐1306 treated tumors compared to vehicle controls. We also observed an association of higher MIF mRNA expression with poor patient survival, including TNBC, by using publically available datasets. These results suggest that CPSI‐1306 induces cell death in TNBC cells via activating ROS and mitochondria‐mediated apoptosis and significantly inhibits TNBC growth and metastasis in pre‐clinical TNBC mouse models.

Support or Funding Information

DOD, Breakthrough Award

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interrelationship between sex, age, blood volume and VO2max
Authors: Convertino Victor , Koons Natalie , Schlotman Taylor , Suresh Mithun
Published: June 2021

Background

Circulating blood volume (BV) and maximal oxygen uptake (VO2max) are physiological characteristics important for optimal human performance in aerospace and military operational environments. Addressing the interrelationships between BV, VO2max, sex, and age together is relevant to identifying those individuals with greatest risk of poor outcomes from hemorrhage, as well as assessing the levels of performance competence of individuals deployed in operational environments. In the present investigation, we report and compare for the first time BV and VO2max in a cohort of males and females together to test the hypothesis that BV and VO2max is lower in older people independent of sex.

Methods

To accomplish this, a ‘data mining’ effort of an historic database generated from NASA and U.S. Air Force experiments was conducted. BV, red cell volume, plasma volume, hematocrit, and VO2max were measured in 94 healthy individuals (28 females, 66 males) across an age range of 23 to 65 years to assess the interrelationship between sex, age, BV, and VO2max. Subjects were classified in age groups by < 40 years and ≥ 40 years; these groups identified females as pre‐ vs. post‐menopausal.

Results

Consistent with our hypothesis, comparisons revealed that males had higher (p < 0.01) BV, red cell volume, hematocrit, and VO2max than females when standardized for body size. Against expectations, BV was not different in older compared with younger males and females. VO2max was not different in older compared with younger females, while VO2max was lower in older males (p < 0.01).

Conclusion

We conclude that physiological mechanisms other than BV associated with aging appear to be responsible for a decline in VO2max of our older male subjects. Furthermore, factors other than menopause may also influence the control of BV in the female population. Our results provide evidence that aging may not compromise males or females in scenarios where BV can affect performance in aerospace and military environments.

Support or Funding Information

This research was supported in part by appointment of Ms. Koons to the Postgraduate Research Fellowship Program at the US Army Institute of Surgical Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and USAMRMC. Study funding was provided by a grant from the US Army Combat Casualty Care Research Program (D‐009‐2014‐USAISR).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Distribution and Expression of the FMRFamide‐gated Sodium Channel in the CNS of Biomphalaria glabrata Under Parasitic Infection
Authors: Vicente‐Rodriguez Laura Cristina , Méndez‐De Jesús Paola , Rosa‐Casillas Mariela , Hernández Anthony , Ramírez Cayra , Rosenthal Joshua , Miller Mark
Published: June 2021

Schistosomiasis is a disease of major global concern for health and socioeconomic development. This neglected tropical disease represents a significant health burden for over 80 countries in Africa, Asia, and South America. Is estimated that more than 240 million of people required preventive treatment in 2016, and thousands are at risk of death annually. Schistosoma mansoni, the trematode specie that causes the most widespread form of intestinal schistosomiasis, requires two host organisms: snails from the genus Biomphalaria as its intermediary host, and humans or other mammals as its definitive host. Snails undergo major physiological and behavioral changes upon parasitic infection, but the neural contribution to these changes is poorly understood. As FMRFamide‐related neuropeptides regulate diverse physiological processes in the snail, such as respiration, reproduction and feeding, and these vital processes are known to be affected by parasitosis, we hypothesized that this peptide system participates in the behavioral and physiological modifications that occur in the snail following the parasitic infection. The FMRFamide peptides have two types of receptors, a G‐coupled receptor and a peptide gated sodium channel (FaNaC) that is unique to this phyla and is the sole sodium channel gated by a peptide. We performed immunohistochemistry and western blot experiments in snail CNS samples to determine the FaNaC receptor distribution and expression. We found a change in the neuronal distribution of the receptor in the visceral and left parietal ganglia from infected snails. Our overarching goal is to better understand the neural circuits involved in the physiological changes observed in B. glabrata during parasitic infection. This knowledge could lead to effective and selective strategies to control B. glabrata proliferation.

Support or Funding Information

NIH (USA): RCMI MD007600, NIGMS‐RISE R25 GM061838NSF (USA): CREST HRD‐1137725, PIRE OISE 1545803 National Academy of Sciences (NAS, USA): U.S.‐Egypt Science and Technology (S&T) Joint Fund 2000007152; Science and Technology Development Fund (STDF, Egypt): USC17‐188

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of a Membrane‐Bound Insect Transferrin
Authors: Najera Diana G , Coca Michelle , Nutsch Kayla E , Gorman Maureen J
Published: June 2021

Transferrins are extracellular proteins that bind iron. Vertebrate transferrins have well‐characterized roles in iron transport and immunity, but the functions of transferrins in most other animals are poorly understood. The goals of this study are to identify the functions of transferrin‐3 (Tsf3) from Drosophila melanogaster (fruit fly), and to determine whether Tsf3 is conserved in other species of insects. Our experimental approach has been to predict important features of the protein, analyze gene expression, perform RNAi‐mediated knockdown in cultured cells, and use phylogenetic analysis to identify Tsf3 orthologs. An analysis of the Tsf3 amino acid sequence predicts that Tsf3 is extracellular and anchored to the plasma membrane. Putative iron‐binding residues are present in the carboxyl‐lobe, but iron‐binding residues appear to be lacking in the amino‐lobe; therefore, we predict that only the carboxyl‐lobe binds iron. Tsf3 is expressed at a low level throughout development in many tissues, but it is very highly expressed in prepupal salivary glands. Cells of the prepupal salivary glands are exceptional because of their extensive DNA replication and because they synthesize large amounts of glue proteins that are used by the insect to attach itself to a solid surface prior to pupation. High expression of Tsf3 in the prepupal salivary glands suggests that Tsf3 may participate in the uptake of iron in these glands, possibly to be used as a cofactor for enzymes required for DNA synthesis, or perhaps to be used in the glue biosynthesis process. To test the hypothesis that Tsf3 is used for iron uptake by insect cells, we are in the process of evaluating the effect of RNAi‐mediated knockdown of Tsf3 on cellular iron content. Toward this goal, we verified that Tsf3 is expressed in cultured insect cells, synthesized dsRNA that targets Tsf3, and optimized a method to measure cellular iron content. Finally, we used a reciprocal BLAST method to search for possible orthologs of Tsf3 in insects from 12 different orders. We found that all 13 species analyzed have putative Tsf3 orthologs. Our findings demonstrate that Tsf3 is likely to be an extracellular, membrane‐bound, iron‐binding protein that may be involved in cellular iron uptake in diverse species of insects.

Support or Funding Information

NSF IOS 1656388

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Uptake of Sialic Acids by Macrophage Cell Lines under Inflammatory Conditions
Authors: Zhao Yu , Sun Xue‐Long , Wang Dan
Published: June 2021

Sialic acids (SAs) are the terminal sugars of cell surface glycoconjugates and are the critical determinants in many physiological and pathological processes. Macrophage cell surface expresses a dense layer of glycans often terminated with SAs which are involved in many biological processes of macrophages such as host‐pathogen recognition, migration, and antigen presentation. Notably, humans make Neu5Ac but are incapable of synthesizing Neu5Gc. However, humans can incorporate Neu5Gc into glycoconjugates through dietary acquisition. Neu5Gc‐containing glycoconjugates can be immunogenic and associated with many diseases and disease progressions such as cancer and cancer development. Moreover, the Neu5Gc‐containing glycoconjugates contribute to infectious diseases as they can serve as receptors for pathogens or toxin. Because the incorporation of Neu5Gc carries negative consequences for humans, we investigated if human macrophage cells may intake Neu5Gc under different condition and have some effects on its functions. Here, we utilized THP‐1 macrophages as a model to examine its Neu5Ac and Neu5Gc intake under the normal and inflammatory condition which was induced by lipopolysaccharide (LPS). LC‐MS/MS was used to quantify the total Neu5Ac and Neu5Gc changes, and cell surface SAs lectin bindings confirmed by flow cytometry analysis. Also, their phagocytosis capacity was evaluated upon Neu5Ac and Neu5Gc under inflammatory conditions. Overall, we found that THP‐1 macrophages has a different intake of Neu5Ac and Neu5Gc under different conditions and affect its phagocytosis as well.

Support or Funding Information

This work was supported by Faculty Research Development Fund and the research fund from the Center for Gene Regulation in Health and Disease (GRHD) at Cleveland State University supported by Ohio Department of Development (ODOD). The authors acknowledge the National Science Foundation Major Research Instrumentation Grants for supporting NMR and QTrap 5500 mass spectrometer instrument requisition at Cleveland State 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.
Conformational Dynamics of the PH Domain in Phospholipases Cɛ and β may Contribute to Subfamily‐Specific Roles in Regulation
Authors: Garland‐Kuntz Elisabeth E , Vago Frank S. , Sieng Monita , Van Camp Michelle M. , Blaine Arryn T. , Corpstein Clairissa , Chakravarthy Srinivas , Jiang Wen , Lyon Angeline M.
Published: June 2021

Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce second messengers, including inositol‐1,4,5‐triphosphate (IP3) and diacylgycerol (DAG), which increase intracellular calcium and activate protein kinase C (PKC), respectively. PLCɛ contributes to cardiac hypertrophy and contractility, as well as to oncogenic and inflammatory signaling pathways following activation of G protein‐coupled receptors and receptor tyrosine kinases. PLCɛ shares a conserved core with the PLC superfamily, but the roles of individual domains in regulation of activity and membrane binding have not been established. We used functional assays to show that the PLCɛ PH domain significantly increases basal lipase activity, but is dispensable for stability. We provide the first structural insights into domain organization of PLCɛ using small‐angle X‐ray scattering (SAXS) and electron microscopy (EM) to reveal that the PH domain is conformationally heterogeneous in solution. Comparisons of the PLCɛ solution structure to that of the closely‐related PLCβ enzyme demonstrate that the PLCβ PH domain is also mobile in solution, in contrast to previously reported crystal structures. We propose that the dynamic nature of the PLC PH domain and resulting conformational heterogeneity contributes to subfamily‐specific differences in activity and regulation by G proteins. We are now using cryo‐EM to expand on these findings and obtain higher resolution structures.

Support or Funding Information

This work is supported by the Purdue Center for Cancer Research, AHA grant 16SDG29930017, NIH NLHBI 1R01HL141076‐01 to A.M.L., and Purdue College of Science Staff and Administrative & Professional Staff Advisory Council Professional Development awards to E.E.G. SAXS experiments used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357. This project was supported by grant 9 P41 GM103622 from the National Institute of General Medical Sciences of the National Institutes of Health. Use of the Pilatus 3 1M detector was provided by grant 1S10OD018090‐01 from NIGMS.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unraveling signaling mechanisms regulating cluster‐based metastasis
Authors: Cheung Kevin , Wrenn Emma D , Moore Breanna , Yamamoto Ami , McBirney Margaux , Greenwood Erin , Thomas Aaron
Published: June 2021

There is mounting evidence that tumor cells can migrate and metastasize as cell clusters. The presence of circulating tumor cell clusters is associated with early relapse and death in many cancer types. Experimentally, clustering of tumor cells greatly increases colonizing potential, but the molecular mechanisms explaining this phenomenon are largely unknown. We performed time‐lapse imaging and genome wide transcription studies in mouse and human breast tumor organoids. This revealed that clustering of tumor cells rapidly induces expression of multiple EGFR pathway genes and increased auto‐phosphorylation of EGFR. Interestingly, pEGFR colocalized with multiple EGFR ligands at cell‐cell contacts, suggesting that clusters' EGFR activation may be due to increased ligand binding. Consistent with this hypothesis, RNAi knockdowns of multiple individual ligands reduced tumor organoid growth by 40 to 95% of control. Downstream of receptor activation, expression of EGFR effector pERK was increased in clusters. The most significantly cluster‐upregulated transcription factor was the ERK‐activated oncogene Fra1. Fra1 knockdown reduced growth as did ERK inhibition. Using patient breast cancer samples, including primary and metastatic tumors, we found that Fra1 expression was highly upregulated in human tumor cell clusters, supporting the human disease relevance of this pathway. Taken together, our studies show that tumor cell clusters activate EGFR‐ERK‐Fra1 growth signaling at cell‐cell contacts. We propose that disrupting this pathway could reduce metastasis and extend survival in cancer patients.

Support or Funding Information

I am grateful for funding support from the Burroughs Wellcome Fund, Department of Defense, Susan G. Komen, Breast Cancer Research Foundation, and the V Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of inhibitors on oxygen permeability of wild type and knockout mouse red blood cells
Authors: Zhao Pan , Occhipinti Rossana , Boron Walter F
Published: June 2021

We now recognize that the paradigm that all gases cross red blood cell (RBC) membranes by dissolving in and diffusing through the lipid phase is not correct. Work on human RBCs genetically deficient in aquaporin‐1 (AQP1) or the Rh complex (mainly RhAG) showed that these two proteins account for ~90% of membrane CO2 permeability (PM,CO2), and that 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS) reduces PM,CO2. We used stopped‐flow (SF) analysis of hemoglobin (Hb) absorbance spectra as we mixed oxygenated RBCs with an extracellular O2 scavenger, and determined the rate constant of Hb deoxygenation (kHbO2). Because kHbO2 depends on Hb kinetics as well as diffusion of O2 to the extracellular space, we used a mathematical model to estimate effects on membrane O2 permeability (PM,O2). We examined the effects of DIDS and chloromercuribenzenesulfonate (pCMBS), agents that decrease PM,CO2. We find that, in RBCs from wild type (WT) mice, pCMBS reduces kHbO2 by 64% (est. PM,O2 by ~84%) and DIDS, by 41% (est. PM,O2 by ~67%). Because pCMBS and DIDS likely act by targeting membrane proteins (rather than lipids), we hypothesized that O2, at least in part, crosses RBC membranes via the same membrane proteins that conduct CO2. Therefore, we examined RBCs from mice genetically deficient in AQP1, RhAG, or both, ± inhibitors. Deletion of AQP1 lowers kHbO2 by 11% (est. PM,O2 by ~27%); RhAG, by 16% (est. PM,O2 by ~35%); the double knockout (dKO), by 33% (est. PM,O2 by ~59%). The combination dKO + pCMBS lowers kHbO2 (vs. WT without inhibitors) by 77% (est. PM,O2 by ~91%), whereas dKO + DIDS lowers kHbO2 by 52% (est. PM,O2 by ~76%). The effects of pCMBS on estimated PM,O2 are very similar when we compare dKO vs. RhAG−/−, or when we compare WT vs. AQP1−/−. Thus, the deletion of AQP1 may have little effect on the sensitivity of PM,O2 to pCMBS—consistent with the hypothesis that AQP1 conducts O2 only via pathways other than the mercury‐sensitive monomeric pores (e.g., via the hydrophobic central pore). In summary, our data suggest that AQP1 and RhAG account for nearly 60% of RBC O2 permeability, and point towards the existence of other O2 channels that account for at least 30%.

Support or Funding Information

Supported by ONR N00014‐15‐1‐2060, ONR N00014‐16‐1‐2535 to WFB and NIH K01‐DK107787 to RO

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ketamine Blunts Sympathetic Nerve Activity Responses to a Cold Pressor test
Authors: Moralez Gilbert , Huang Mu , Cramer Matthew N , Hendrix Joseph M , Hinojosa‐Laborde Carmen , Ryan Kathy L , Fu Qi , Crandall Craig G
Published: June 2021

Pain evokes increases in blood pressure, heart rate, and central sympathetic outflow, and the perception of pain increases sympathetic nerve activity in an intensity‐dependent manner. That said, the mechanisms underlying the perception of pain and the effects on sympathetic nerve activity are not well established. The purpose of this experiment was to examine the effects of the analgesic ketamine, an NMDA receptor antagonist that reduces pain perception, on pain perception and muscle sympathetic nerve activity (MSNA) during a painful stimulus. In seven healthy participants (2 males; age 30 ± 6 yrs., mass of 83 ± 5 kg) MSNA and pain perception (Visual Analog Scale for Pain) were assessed during a 2‐min cold pressor test with and without intravenous infusion of 20 mg of ketamine, a dose consistent with guidelines from the US Army's Committee on Tactical Combat Casualty Care for pain management on the battlefield. Prior to the cold pressor test, ketamine alone increased heart rate (P=0.020), mean arterial blood pressure (P <0.001), and MSNA from 22 ± 6 burst/min to 32 ± 7 bust/min (P<0.01). During the subsequent cold pressor test, ketamine both decreased pain perception (Visual Analog Scale for Painscore ‐ control: 69 ± 22; ketamine: 22 ± 23, P<0.001) and blunted the peak increase in MSNA (control: Δ22 ± 4 burst/min; ketamine: Δ8 ± 7 burst/min). The increases in blood pressure, heart rate and sympathetic nerve activity elicited by the administration of ketamine suggest that ketamine is acting centrally within the cardiovascular control centers. Furthermore, these data support previous findings demonstrating that sympathetic outflow to a painful stimulus is graded with the pain perception.

Support or Funding Information

Funding: Department of Defense – US Army, W81XWH1820012

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