bluuebunny/crossref_metadata_2025 huggingface dataset:
Single-cell nucleic acid profiling in droplets (SNAPD) enables high-throughput analysis of heterogeneous cell populations
Authors: Hyman Leland B , Christopher Clare R , Romero Philip A
Published: June 2021

Experimental methods that capture the individual properties of single cells are revealing the key role of cell-to-cell variability in countless biological processes. These single-cell methods are becoming increasingly important across the life sciences in fields such as immunology, regenerative medicine and cancer biology. In addition to high-dimensional transcriptomic techniques such as single-cell RNA sequencing, there is a need for fast, simple and high-throughput assays to enumerate cell samples based on RNA biomarkers. In this work, we present single-cell nucleic acid profiling in droplets (SNAPD) to analyze sets of transcriptional markers in tens of thousands of single mammalian cells. Individual cells are encapsulated in aqueous droplets on a microfluidic chip and the RNA markers in each cell are amplified. Molecular logic circuits then integrate these amplicons to categorize cells based on the transcriptional markers and produce a detectable fluorescence output. SNAPD is capable of analyzing over 100,000 cells per hour and can be used to quantify distinct cell types within heterogeneous populations, detect rare cells at frequencies down to 0.1% and enrich specific cell types using microfluidic sorting. SNAPD provides a simple, rapid, low cost and scalable approach to study complex phenotypes in heterogeneous cell populations.
Unifying Phylogenetic Birth–Death Models in Epidemiology and Macroevolution
Authors: MacPherson Ailene , Louca Stilianos , McLaughlin Angela , Joy Jeffrey B , Pennell Matthew W
Published: June 2021

Birth–death stochastic processes are the foundations of many phylogenetic models and are widely used to make inferences about epidemiological and macroevolutionary dynamics. There are a large number of birth–death model variants that have been developed; these impose different assumptions about the temporal dynamics of the parameters and about the sampling process. As each of these variants was individually derived, it has been difficult to understand the relationships between them as well as their precise biological and mathematical assumptions. Without a common mathematical foundation, deriving new models is nontrivial. Here, we unify these models into a single framework, prove that many previously developed epidemiological and macroevolutionary models are all special cases of a more general model, and illustrate the connections between these variants. This unification includes both models where the process is the same for all lineages and those in which it varies across types. We also outline a straightforward procedure for deriving likelihood functions for arbitrarily complex birth–death(-sampling) models that will hopefully allow researchers to explore a wider array of scenarios than was previously possible. By rederiving existing single-type birth–death sampling models, we clarify and synthesize the range of explicit and implicit assumptions made by these models. [Birth–death processes; epidemiology; macroevolution; phylogenetics; statistical inference.]
Testing the feasibility and acceptability of a culturally adapted physical activity intervention for adult Somali women
Authors: Murray Kate E , Hellier Villafana Veronica Anne , Sheik Mohamed Amina , Linke Sarah , Bowen Deborah J , Marcus Bess
Published: June 2021

Despite growing numbers in the USA, immigrant populations are underrepresented in existing physical activity (PA) research, in particular Muslim immigrant women. The current study is a pilot evaluation of a culturally adapted evidence-based PA intervention for adult Somali women. Stratified randomization was used to assign participants from a sample of 27 Somali women, aged 18 to 65, to a PA group or a waitlist control group. Bicultural Somali community research team members delivered a 12-week culturally adapted intervention available in English and Somali in a community-based setting. Process and outcome evaluation assessed changes in PA, self-efficacy for PA, access to PA resources, and wellbeing as well as feasibility and satisfaction with the program. Participants in the PA group increased their moderate to vigorous PA significantly more than those from the waitlist group from baseline to post-intervention (2 (SD = 15) to 100 (SD = 53) vs 12 (SD = 21) to 32 (SD = 44) minutes per week). Participants in the PA group had significantly greater scores in wellbeing at post-intervention compared to the waitlist group though there was no significant change from pre- to post-intervention for either group. Participants reported a high level of satisfaction with the program and preliminary evidence supports the general feasibility and acceptability of the program. Findings show that a culturally adapted intervention increased engagement in PA and was feasible and acceptable within a pilot sample of Somali women.
Bisphenol S and Bisphenol F Are Less Disruptive to Cardiac Electrophysiology, as Compared With Bisphenol A
Authors: Prudencio Tomas M , Swift Luther M , Guerrelli Devon , Cooper Blake , Reilly Marissa , Ciccarelli Nina , Sheng Jiansong , Jaimes Rafael , Posnack Nikki Gillum
Published: June 2021

Bisphenol A (BPA) is a high-production volume chemical used to manufacture consumer and medical-grade plastic products. Due to its ubiquity, the general population can incur daily environmental exposure to BPA, whereas heightened exposure has been reported in intensive care patients and industrial workers. Due to health concerns, structural analogs are being explored as replacements for BPA. This study aimed to examine the direct effects of BPA on cardiac electrophysiology compared with recently developed alternatives, including BPS (bisphenol S) and BPF (bisphenol F). Whole-cell voltage-clamp recordings were performed on cell lines transfected to express the voltage-gated sodium channel (Nav1.5), L-type voltage-gated calcium channel (Cav1.2), or the rapidly activating delayed rectifier potassium channel (hERG). Cardiac electrophysiology parameters were measured using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and intact, whole rat heart preparations. BPA was the most potent inhibitor of fast/peak (INa-P) and late (INa-L) sodium channel (IC50 = 55.3, 23.6 µM, respectively), L-type calcium channel (IC50 = 30.8 µM), and hERG channel current (IC50 = 127 µM). Inhibitory effects on L-type calcium channels were supported by microelectrode array recordings, which revealed a shortening of the extracellular field potential (akin to QT interval). BPA and BPF exposures slowed atrioventricular (AV) conduction and increased AV node refractoriness in isolated rat heart preparations, in a dose-dependent manner (BPA: +9.2% 0.001 µM, +95.7% 100 µM; BPF: +20.7% 100 µM). BPS did not alter any of the cardiac electrophysiology parameters tested. Results of this study demonstrate that BPA and BPF exert an immediate inhibitory effect on cardiac ion channels, whereas BPS is markedly less potent. Additional studies are necessary to fully elucidate the safety profile of bisphenol analogs on the heart.
Exploring the Role of Calmodulin and Calcium Signaling in Leukocyte Transmigration
Authors: Dalal Prarthana J. , Sullivan David P. , Muller William A.
Published: June 2021

Inflammation results in the dilation of local blood vessels, the activation of endothelium by cytokines and the recruitment of circulating leukocytes into areas of tissue damage. A transient increase in endothelial cytosolic free calcium (Ca2+) is required for leukocyte transendothelial migration (TEM) and transient receptor potential canonical 6 (TRPC6) has been found to mediate this required Ca2+ influx. Furthermore, we have also found that IQ‐domain GTPase‐activating protein 1 (IQGAP1) is essential for TEM. Specifically, the actin‐binding domain and the calmodulin‐binding isoleucine‐glutamine (IQ) domain of IQGAP1 are critical for its function during TEM. In endothelial cells re‐expression of IQGAP1 truncation mutants lacking the N‐terminal actin‐binding domain did not localize to endothelial borders and did not support TEM. Re‐expression of truncated IQGAP1 constructs containing the actin‐binding domain and the IQ domain localized to cell borders and supported TEM while constructs containing the actin‐binding domain but lacking the IQ domain localized to the cell borders but did not restore TEM.

Calmodulin (CaM) is a small, highly conserved intracellular calcium‐binding protein, which has been shown to interact with IQGAP1 through the IQ domain. It has also been previously found to regulate TRPC channel activation; however, not much is known regarding its role in TEM. Therefore, we hypothesized that it may be involved in the calcium signaling cascade within endothelial cells essential for supporting leukocyte transmigration.

Human endothelial cells treated with calmodulin inhibitor trifluoperazine (TFP) demonstrated a marked reduction in leukocyte TEM. Endothelial monolayers pretreated with 100 mM TFP for 1 hour drastically reduced leukocyte TEM. Only about 13% of leukocytes were able to transmigrate compared to 84% TEM in control, untreated monolayers (p=0.0025).

After identifying the potential role of calmodulin, we generated CaM shRNA to deplete endogenous CaM within endothelial cells, wildtype Flag‐tagged CaM re‐expression constructs and CaM calcium‐binding mutants that we are currently in the process of testing. Identifying the specific mechanism by which endothelial Ca2+ influx through TRPC6 mediates the events of TEM will provide novel insight into the signaling involved in TEM and also provide potential therapeutic targets for anti‐inflammatory therapy.

Support or Funding Information

F30HL134202, R01HL046849, R01HL064774

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CD99L2 as a Major Regulator in Human Transendothelial Migration
Authors: Rutledge Nakisha S. , Muller William A.
Published: June 2021

Transendothelial Migration (TEM) is a crucial step in the inflammatory response as it is the process in which leukocytes leave the bloodstream to enter the inflamed tissues. Adhesion/signaling molecules such as PECAM and CD99 are important for the initiation and completion of TEM. We and others have shown that CD99‐like 2 (L2), a highly glycosylated 52 kD type‐1 membrane protein that is the only molecule in the genome related to CD99 is also important for TEM. However, L2 is different enough from CD99 (60% larger and with only 38% sequence identity) that it is not clear whether it regulates TEM by the same mechanism as CD99. Furthermore, all of the published studies on L2 have been performed in mice. The role of L2 in human cells is not known. In order to study the mechanism used by L2 and the relevance to human inflammation, we studied the role of L2 on human cells in vitro.

Our data show that similar to PECAM and CD99, human L2 is constitutively expressed at the borders of endothelial cells and on the surface of leukocytes; treatment with proinflammatory cytokines changes neither its surface expression nor its distribution. Blocking L2 using antibodies significantly reduces transmigration of human neutrophils and monocytes across human endothelial cells. Blockade of either leukocytes or endothelial cells blocks equivalently to blocking both, consistent with a homophilic mechanism of interaction. Furthermore, knockdown of L2 using shRNAs dose‐dependently reduces TEM of neutrophils and monocytes across endothelial cells. TEM is restored by re‐expression of L2. Our data also show that L2 regulates a step in TEM after PECAM and before CD99. Consistent with this, preliminary data suggest that L2 can promote TEM by engaging either the signaling pathway used by PECAM or the one used by CD99. Similar to PECAM and CD99, L2 promotes transmigration by recruiting the lateral border recycling compartment to the site of TEM. Ongoing studies are focused on identifying unique signaling pathways (if any) used by L2 to promote TEM.

Support or Funding Information

T32‐5T32AI007476‐19

R37‐HL064774

F31‐HL131355‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Whole transcriptomic evaluation of SK‐MEL‐3 melanoma cells in response to histone deacetylase inhibitor trichostatin A.
Authors: Mazzio Elizabeth A. , Soliman Karam F.
Published: June 2021

Malignant melanoma has a proclivity for resistance to radiation and chemotherapy drugs such as dacarbazine or kinase inhibitors that target RAS‐independent auto‐activated serine/threonine kinase, a consequence of somatic missense mutant BRAF v 599E. An underlying controlling component of aggressive melanoma is a change to the epigenetic histone landscape to which overexpression of histone deacetylases (HDACs) has been attributed. The full effects of direct inhibition of HDAC on the entire transcriptome are not well understood, yet there is growing number of synthetic drugs classified as HDAC inhibitors (HDACis). In this work, we evaluated the whole transcriptome response in SK‐MEL‐3 human melanoma to trichostatin A using both Affymetrix 2.1 Human Arrays and 4.1 miRNA chips. TSA was confirmed as a potent HDAC inhibitor in nuclear lysates, and in cells trichostatin, A (TSA) [10nM] at sublethal concentrations were used for to evaluate transcriptomic shifts at 24 hours exposure. Of interest, the data show no changes to the expression of 12 HDAC transcripts (HDACs subtypes 1–12) or B‐raf protooncogene V599E by TSA. In contrast, TSA evoked differential expressed changes with 833 up‐regulated and 810 down‐regulated genes. The top 10 up‐regulated genes were TSPAN13, SERPINI1, ATP1B2, NMNAT2, PDGFRL, CYP1A1, PARM1, SCG2, SYT11, ROPN1L and top 10 down‐regulated genes GALNT3, CA14, BCL2A1, TRPM1, PYCARD, UBAP1L, GPX8, IL‐6, TP53, SERPINH1. Functional pathway analysis shows TSA effects to negatively impact cell survival systems, cell cycle progression, migration and biological pH buffering specifically that of carbonic anhydrase 14. These changes occurred concomitant to an elevation of miRs 185‐3p, 4783‐3p, 6716‐3p and reduction of miR‐6795‐5P. In conclusion, data from this work provide an overall effect of TSA on the whole transcriptome of coding and non‐coding RNA in malignant melanoma

Support or Funding Information

This study was supported by the National Institute on Minority Health and Health Disparities (NIMHD) awards G12MD007582 and P20 MD 00738.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rapid flow‐induced Gαq/11 activation occurs upstream of Piezo1 activation
Authors: dela Paz Nathaniel G. , Frangos John A.
Published: June 2021

Endothelial mechanotransduction is the process by which mechanical stimuli are sensed by endothelial cells (ECs) and transduced into biochemical signals and ultimately into physiological responses. Identifying the primary mechanosensor(s) and describing the mechanism(s) by which they receive and transmit the signals has remained a central focus within the field. The heterotrimeric G protein, Gαq/11, is proposed to be part of a macromolecular complex together with PECAM‐1 at the cell‐cell junction of ECs and may serve as a primary mechanosensor as it is rapidly activated within seconds of flow onset. The mechanically‐activated cation channel, Piezo1, has recently been implicated due in part to its involvement in mediating early responses, such as calcium influx and ATP release. In this study, we used in situ proximity ligation assay (PLA) to investigate the role of Piezo1 in the rapid shear stress‐induced activation of Gαq/11. We show that the flow‐induced dissociation of Gαq/11 from PECAM‐1 in ECs at 15 sec is abrogated by BIM‐46187, a specific inhibitor of Gαq/11 activation, suggesting that Gαq/11 activation is a prerequisite for PECAM‐1/Gαq/11 dissociation. Although siRNA knockdown of Piezo1 caused a dramatic decrease in PECAM‐1/Gαq/11 association in the basal condition, it had no effect on their flow‐induced dissociation. Interestingly, siRNA knockdown of Piezo1 caused a marked decrease in PECAM‐1 expression. Selective blockade of Piezo1 with ion channel inhibitors also had no effect on flow‐induced PECAM‐1/Gαq/11 dissociations despite having an inhibitory effect on Akt phosphorylation. Rapid flow (15 sec) also led to increased association of Gβ1 with Piezo1 as well as with the p101 subunit of phosphoinositide 3‐kinase (PI3K), which were both blocked in the presence of the Gβγ inhibitor, gallein. Taken together, our results indicate that flow‐induced activation of Piezo1 is downstream of G protein activation.

Support or Funding Information

This work was supported by NIH MERIT Award R37 HL040696 to J.A.F.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insulin and ketones elicit disparate effects on mitochondrial uncoupling in adipose tissue
Authors: Bikman Benjamin T.
Published: June 2021

Objective

Insulin is a powerful regulator of metabolic function, promoting a general storage of nutrients. In contrast, ketones, inversely regulated by insulin, lead to enhanced energy use and wasting. The purpose of this study was to explore the effects of insulin and ketones on mitochondrial coupling status in adipose tissue.

Methods

We used cell, rodent, and human models, to varying degrees. 3T3‐L1 adipocytes were treated at various times with insulin or ketones. Similarly, rodents were chronically injected with insulin or placed on ketogenic diets. Finally, human fat biopsies were obtained from in states of ketosis. In all models, mitochondrial respiration was determined and, where available, uncoupling protein (UCP) 1 was measured.

Results

In cells and rodents, insulin reduced mitochondrial respiration and, in rodents, reduced indirect calorimetry. In all models, including human adipose, ketones increased respiration rates and UCP1 levels.

Conclusions

Insulin and ketones elicit disparate effects on mitochondrial uncoupling in adipose tissue. Whereas insulin reduces uncoupling, resulting in more tightly coupled electron transport, ketones enhanced uncoupling, resulting in energy wasting.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Misregulation of Polycomb Repressive Complexes by Oncohistones
Authors: Lewis Peter W.
Published: June 2021

Covalent modifications to DNA and the histone packaging proteins allow chromatin to act as a dynamic signaling platform to regulate genomic DNA access and ultimately establish and maintain cellular phenotypes. Moreover, there is increasing appreciation that chromatin alterations per se, including DNA and histone modifications, are involved in the pathogenesis of cancer. Nowhere is this better supported than with the groundbreaking discoveries of high‐frequency, somatic mutations in histones that are drivers of oncogenesis. These mutations (collectively called ‘oncohistones') cause amino acid substitutions that localize to conserved residues in the N‐terminal tail of histone H3 and all seem to be linked, either directly or indirectly, to disruption of normal levels and distribution of histone H3 lysine 27 methylation and thus transcription regulation. We are currently using a combination of biochemical, chromatin and transcriptomic approaches, along with defined cell lines to understand how histone H3 lead to altered chromatin states that profoundly influence gene expression patterns. I will discuss some of our recent work on how histone mutations perturb cellular differentiation through inhibition of specific chromatin‐modifying enzymes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glutamine Synthetase Serves as an Accurate Immunohistological Marker for Perivenular Hepatocytes in the Liver
Authors: Mak Ki M.
Published: June 2021

In the mouse liver, Axin2 (axis inhibition protein 2) is selectively expressed in hepatocytes that form a single layer surrounding the central vein, defined as perivenular hepatocytes. Axin2 hepatocytes display hepatocyte stem cell‐like properties. These cells self‐renew and their progenies migrate from the centrilobular zone towards the periportal area, replacing lobular uninjured or senescent hepatoytes and contributing to physiological turnover of parenchymal cells. In one year, Axin2 cells replenish on average 40% of hepatocytes in the liver under normal conditions. Axin2 cells also express glutamine synthetase (GS), which detoxifies the ammonia entering the liver circulation. However, both Axin2 and GS expression is maintained by the Wnt/β‐catenin pathway derived from the central vein endothelium. Because no suitable antibody that detects perivenular Axin2 expression by immunohistochemistry is available to date, GS has been used as a surrogate marker for Axin2 to stain perivenular hepatocytes. The present study tested whether GS serves as a marker for perivenular hepatocytes and evaluated the effects of fibrogenesis on the lobular distribution of GS‐positive cells in the human liver using cadaveric liver samples. Immunoperoxidase staining with a polyclonal GS antibody revealed that in the liver with minimal fibrosis (N = 8), GS selectively stained perivenular hepatocytes generally arranged in 1–3 layers surrounding the central veins. GS‐positive cells were also seen as a single layer along the larger collecting veins. There was a variable presence of small aggregates of GS‐positive non‐perivenular hepatocytes in the centrilobular zone. The GS immunoreactivity in hepatocytes was cytoplasmic. No GS staining of periportal hepatocytes was seen. A similar distribution of GS‐positive hepatocytes was observed in the liver showing advanced stages of septal fibrosis (N = 8), bridging fibrosis (N = 8) and incomplete cirrhosis (N = 6). In end‐stage cirrhosis (N = 4), GS expression could not be observed around the central veins in the nodular parenchyma.

Conclusions

GS serves as an accurate immunohistological marker for perivenular hepatocytes that are considered as hepatocyte stem cells in the mouse. GS expression in non‐perivenular hepatocytes in the centrilobular zone suggests an expansion of perivenular hepatocytes in response to hepatic fibrogenesis. Loss of perivenular GS expression in cirrhosis may reflect a disruption of contact between the central vein endothelium and perivenular hepatocytes. Understanding the histogenesis of GS expressing perivenular hepatocytes may help in therapeutic planning for enhancing hepatic regeneration in liver fibrosis disease.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protective Effect of Beta vulgaris on Carbon tetrachloride induced Hepatotoxicity in adult Wistar Rat
Authors: EGEONU SOLOMON , IHENTUGE CHURCHILL , OKECHUKWU HOPE , ANIBEZE CHIKE , AKPUAKA FRANK
Published: June 2021

Beta vulgaris also known as beet root has been used to treat a wide range of symptoms and diseases, such as fever, constipation, cancer, diabetes, liver and heart diseases. The aim of this study was to investigate the protective ability of beta vulgaris on the liver following Carbon tetrachloride (CCl4) intoxication. Twenty five adult albino wistar weighing between 150g and 200g were used in this study. The animals were divided into five groups A, B, C, D, and E. Groups A and B served as control were injected with paraffin oil for two consecutive days and group B fed with Beta vulgaris extract alone. Groups C and D were injected with CCl4 (1.0ml/kg b.w i.p) for two consecutive days and group D fed with BE throughout the experimental days. Group E was fed with Beta vulgaris extract throughout the experiment and injected CCl4 on the last two days of the experimental period (28 days).

At the end of the experimental period animals were reweighed and sacrificed using chloroform vapour. Blood was collected for serum enzyme analysis and liver was harvested and trimmed for histological studies. The result showed that, there was a decrease in mean body weight of 5% in group C animals while there was an increase in other groups. The liver weight were significantly higher in group C (p<0.05) when compared with control groups and other experimental groups. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in group C were significantly higher (p<0.05) when compared with the control group while groups B, D and E were normal.

Histological examination of liver (H&E) showed numerous fatty changes, distortion of hepatocytes, focal necrosis, loss of nuclei and cytoplasmic shrinkage in group C animals, groups A, B and E appear normal, while group D showed mild focal inflammation and areas of regeneration of tissues

These results shows that Beta vulgaris extract has the ability to prevent and also restore histological and biochemical changes in the liver following CCl4 injection.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Immunolocalization of Hsc70 at Bacterially‐Generated Actin‐Rich Structures
Authors: Walker Brittany Dominique , Chua Michael Dominic , Guttman Julian Andrew
Published: June 2021

The actin cytoskeleton is commonly hijacked by bacterial pathogens. Listeria commandeers this filament system during their internalization, and for actin‐rich comet tail formation that enable the microbes to move within the infected cells as well as transfer from cell to cell. Enteropathogenic Escherichia coli (EPEC), an extracellular microbe, controls this cytoskeletal system when they generate E. coli pedestals as part of their firm docking to the plasma membrane of their host's cells and as they “surf” atop the infected cells. Finally, Salmonella exploit the host actin cytoskeleton to enter eukaryotic cells by forcing the generation of actin‐based membrane ruffling. We recently identified Hsc70 in a proteomics screen of EPEC pedestal components. This protein is normally involved with chaperoning functions within the cell. Despite there being a lack of evidence for an association of Hsc70 and actin, due to our proteomics identification of Hsc70 at EPEC pedestals we hypothesized that Hsc70 could be a common target of bacterial pathogens that control the actin cytoskeleton during their disease processes. To test this hypothesis we immunolocalized Hsc70 during Listeria, EPEC and Salmonella infections and found it concentrated at all actin structures generated by the microbes, demonstrating that this protein is a universal component for bacterial actin‐based structures.

Support or Funding Information

Funding provided through NSERC. (Grant No. 355316 to J. A. G.)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Plant PCR inhibitor release as a function of sample dissociation method
Authors: Easparro Brandon , Morehouse Zachary , Proctor Caleb , Atwood James
Published: June 2021

The polymerase chain reaction (PCR) is an invaluable technique used in a wide range of molecular biology applications across multiple disciplines ranging from medical diagnosis and forensic analysis to botanical research. Since PCR is an enzymatic reaction, it is sensitive to inhibitors. PCR inhibitors may interfere with multiple steps in the PCR process leading to reduced sensitivity or even false negatives through inhibition of the amplification of nucleic acids by enzyme binding, nucleic acid cross‐linking/interaction, or through mimicking the nucleic acid structure. Inhibitors result from the original sample or during the extraction process. For plant studies, PCR inhibition may result from contamination by acidic polysaccharides and phenols including xylan, dextran sulfate, and flavonol; among others. While PCR inhibition has been demonstrated for multiple plant‐based compounds and procedural approaches have been recommended for removal of these inhibitors, to our knowledge, there has not been a study to compare the release of these compounds and the subsequent inhibitory effect based on different methods of sample dissociation.

Herein, we evaluate three different dissociation techniques, including bead milling, cryo‐grinding and rotor‐stator dissociation for the extraction of genomic DNA and total RNA from sweet basil (Ocimum basilicum) and blueberries (Vaccinium corymbosum) and the release of plant PCR inhibitors. Polysaccharide, phenol, nucleic acid, and protein content was quantified and compared between each approach. qPCR was then employed to monitor the impact of PCR inhibitor content for both classic PCR and RT‐PCR

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Diet Restriction Induces Heat Shock Gene Expression While Tempering Heat Stress Response in Drosophila melanogaster
Authors: Tresser Jason W. , Bliss Megan , Mayweather Hailey , Rustand Emerson , Sears Alex , Varamini Behzad
Published: June 2021

Diet restriction (DR) in fruit flies mimics the longevity effects that calorie reduction has in other organisms. Long‐lived flies are smaller, have global down regulation of protein levels, and lower fecundity. We predicted that flies on DR food would have a limited ability to respond to heat stress. We used qPCR to measure expression levels of heat shock proteins in Drosophila melanogaster fed control food or diet restricted food at standard or high temperatures. HSP22, HSP70 and HSP83 are known to be upregulated in flies in response to various stressors. We show here that flies under diet restriction have elevated HSP22 and HSP70 expression at standard temperatures and upregulate HSP 83 in response to heat stress.

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 subcellular distribution of the human Fic protein, HYPE/FicD, using a high resolution, tomography compatible electron microscopic method
Authors: Mattoo Seema , Sengupta Ranjan
Published: June 2021

Localization of membrane proteins via electron microscopy (EM) at high resolution is dependent on a robust detection technology and a sample preparation method that confers superior ultrastructural preservation of membranes. Current methods for precise localization rely on chemical fixation of the sample followed by alcohol dehydration at room temperature. The combination of these traditional methods exhibit poor preservation of membrane, induces membrane artifacts and affects resolution. High pressure freezing (HPF) of cells followed by heavy metal staining via freeze substitution (FS) in acetone at sub‐zero temperatures are techniques that exhibit the best possible membrane preservation. Therefore, we have developed a hybrid method that combines high pressure freezing with an existing peroxide based detection technique typically used with chemical fixation methods. As proof of concept, we assessed the localization of a fusion construct, consisting of the N‐terminus of HMG CO‐A protein fused with a peroxidase tag (that faces the cytosol). We exploited the induction of OSERs (organized smooth ER) in cells by the over‐expression of this N‐terminal fragment, as a metric for specific staining and membrane preservation. We next extended our studies to assess the localization of the sole human Fic (filament induced by cyclic AMP) protein, HYPE. We previously showed that HYPE resides in the ER lumen, but various global transcriptomic and proteomic studies suggest the existence of cytosolic and extracellular substrates for HYPE. Using our hybrid methodology, here we provide unequivocal evidence that the HYPE is compartmentalized within the various subdomains of the ER. We further demonstrate via serial sectioning, that our technique enables the localization of HYPE through a large volume of the cell. Our work thus aligns well with the three‐dimensional mapping of membrane proteins in large organelles using EM‐tomography in a near‐native cellular context.

Support or Funding Information

This work was supported by funding from 1) an Indiana CTSI (Clinical and Translational Sciences Institute) Project Development Team Award and 2) the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under Award Number R01GM100092.

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 polypeptide exit tunnel of the ribosomal large subunit requires assembly factors for proper construction and function
Authors: Wilson Daniel , LaPeruta Amber , Woolford John
Published: June 2021

The final product of ribosome assembly consists of two ribosomal subunits capable of functioning with high fidelity. In eukaryotes, this process is facilitated by protein assembly factors that can promote proper formation of functional centers through structural probing, functional test‐driving, acting as a scaffold, or protecting the site from premature activity. The polypeptide exit tunnel (PET) of the ribosomal large subunit (LSU) is the conduit through which nascent polypeptide chains are passed as they are synthesized. Protein and RNA components of the PET lumen actively participate in translation by interacting with the nascent chain in order to regulate ribosome stalling events, thus regulating translation speed and gene expression. Recent cryo‐EM structures of immature LSU particles from different stages of assembly spanning the nucleoplasm to the cytoplasm have shown that the PET is sequentially probed by the C‐termini of three assembly factors: first, Nog1, then Rei1, and finally Reh1. The C‐termini of these three assembly factors mimic nascent chains, plugging the PET for almost the entirety of LSU assembly. No biological function, however, has been assigned to this redundant occupation of the PET. Here, we show that truncating PET‐inclusive residues from the C‐terminus of Nog1 causes LSU assembly defects that are exacerbated by analogous truncations to one or both of the Rei1 and Reh1 C‐termini. These defects manifest in processing of late immature ribosomal RNA intermediates. Interestingly, these assembly defects are further exacerbated at lower temperatures. Despite these mutants displaying slow growth at permissive temperatures, however, mature ribosomes are produced that may be defective in translation. Our data supports the model that Nog1 is necessary for efficient construction of the PET during early stages of assembly while Rei1 and Reh1 carry out checkpoints during later stages.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Repeat Problems: Combinatorial Effect of C9orf72‐Derived Dipeptide Repeat Proteins
Authors: Darling April , Braydo Leonid , Blair Laura , Koren John , Uversky Vladimir
Published: June 2021

The most common genetic cause of both Amyotrophic Lateral Sclerosis and Frontotemporal Dementia is a microsatellite expansion mutation in the 5′ UTR region of C9orf72. Expansion of the hexanucleotide repeat region of C9orf72 leads to loss of function, RNA foci, and five species of non‐AUG RAN translated dipeptide repeat proteins (GA, GP, GR, PA, & PR). It has been observed that the sense and antisense dipeptide repeat proteins can be translated simultaneously in one cell, however the interplay between the different species has not been heavily investigated. Here we determined that the antisense repeat protein PR is localized to the nucleus, causes cytotoxicity, disrupts nuclear transport, and triggers PERK phosphorylation and p62 puncta accumulation. However, when PR and its sense partner GA were co‐expressed, PR's localization and cytotoxic outputs were significantly altered. In addition, we showed that GA and PR interact through direct binding that leads to structural and morphological changes. We also demonstrate the ability of dipeptide repeat protein species to be released from one cell and taken up by another. Importantly, we demonstrate that co‐expression of distinct DPRs enhances the release of individual peptides. Thus, the combined expression of distinct C9orf72 derived dipeptide repeat species produces cellular outcomes and structural differences that are unique compared to the expression of a single species.

Support or Funding Information

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.
Dietary Glycine Supplementation Extends Lifespan of Genetically Heterogeneous Mice
Authors: Brind Joel , Miller Richard A. , Strong Randy , Harrison David E. , Macchiarini Francesca
Published: June 2021

Preliminary experiments suggested lifespan extension by glycine supplementation (GlyS) without the growth inhibition seen with methionine restriction in Fisher 344 rats. This suggested that the benefit GlyS, rather than owing to increased methionine clearance (as was originally envisioned), was attributable to increased levels of glycine itself, acting via the glycine receptor. The glycine receptor is a glycine‐gated chloride channel that is widespread among cell types, including macrophages, and GlyS has been shown by others to exhibit anti‐inflammatory and anti‐cancer properties in several rodent models, and even to reverse Type 2 diabetes in a human trial. GlyS has also recently been found by others to restore the youthful mitochondrial phenotype in senescent cultured human fibroblasts, whose senescence was linked to epigenetic down‐regulation of the principal pathways of endogenous glycine synthesis. The present study set out to replicate the life extension previously observed in rats, in large, reproducible populations of UM‐HET‐3, 4‐way cross mice, in a Phase I Interventions Testing Program (ITP) trial of the National Institute on Aging (NIA). Beginning at 9 months of age, GlyS mice were fed a diet containing 8% glycine (dry weight, compared to the level of 2.3% in the control diet) simultaneously at 3 sites (The Jackson Labs, Bar Harbor, ME; University of Michigan, Ann Arbor, MI; U.T. Health San Antonio, TX), employing approximately 50 each of male and female GlyS and 100 each of male and female controls at each site. Data were compiled after 90% of control mice had died at all 3 sites. Pooling data from all 3 sites (shown in Figure below), median lifespan was increased among GlyS mice by 3.7% among females and 1.3% among males, and the 90th percentile lifespan was increased by 1.8% among females and 3.6% among males (p = 0.00004 by log‐rank test and p = 0.002 by Wang‐Allison test for pooled males and females at all 3 sites combined). The observed lifespan extension is statistically robust, in that significant log‐rank p‐values were seen for each sex (pooling across site) and for each site (pooling across sexes). The Stage I ITP protocol measures only lifespan, and it is anticipated that Stage II trials will provide the opportunity for biochemical and other cross‐sectional measures to elucidate the mechanism(s) involved in the life extension observed in our system.

Support or Funding Information

Interventions Testing Program, National Institute on Aging

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulatory mechanisms controlling the subcellular localization and activity of the RhoA GEF Net1 in breast cancer
Authors: Frost Jeffrey A. , Zuo Yan , Ulu Arzu
Published: June 2021

Net1 is a RhoA/B‐specific guanine nucleotide exchange factor that is overexpressed in human breast cancer and is required for efficient cancer cell motility and invasion in vitro. Net1 knockout mice are viable but exhibit defects in tumor initiation and lung metastasis in a genetic model of breast cancer. Net1 is unusual among RhoGEFs in that it localizes to the nucleus in quiescent cells, but relocalizes to the cytoplasm in response to motility stimuli. However, regulatory mechanisms controlling its cytoplasmic accumulation are not well understood. In the present work we show that accumulation of the Net1A isoform in the cytosol requires CRM1‐dependent nuclear export and also requires phosphorylation by stress activated MAP kinases. Overexpression of constitutively active MKK7 or MKK3, which stimulate the Jnk and p38 MAPKs respectively, stimulates Net1A cytosolic localization in a ligand independent manner. Mutation of the main Jnk phosphorylation site, Ser52, to alanine prevents ligand stimulated cytosolic localization. Mechanistically, Net1A phosphorylation by Jnk prevents nuclear re‐import of cytosolic Net1A. Glutamic acid substitution of Ser52 promotes constitutive cytosolic localization and results in elevated RhoA activation and signaling. Expression of Net1A S52E promotes increased motility, extracellular matrix invasion, and invadopodia formation in breast cancer cells that are normally not invasive. Taken together, these data identify a novel regulatory mechanism controlling the subcellular localization and proinvasive functions of Net1A, and suggest novel therapeutic avenues to target Net1‐dependent cancers.

Support or Funding Information

This work was supported by NCI grant to CA172129 J.A.F. and a CPRIT fellowship RP160015 to A.U.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Acid Phosphatase Adsorption to Montmorillonite
Authors: McTaggart William Stuart
Published: June 2021

Phosphorus is important in agriculture because it limits the growth of plants and can influence the size of crop yields. The phosphorus cycle takes significant time in replenishing phosphorus lost to runoff and human activities — in the short‐term, phosphorus is non‐renewable. This project aims to better understand how naturally‐occurring enzymes influence the phosphorus cycle, particularly how these enzymes adsorb to soil. The project is taking advantage of microplate assays to analyze how acid phosphatase (AP) from wheat interacts with montmorillonite, a clay. Key experiments investigate the time it takes for the binding of AP to montmorillonite to equilibrate, the effect of AP concentration on adsorption and the total amount of binding. The data showed that binding came to equilibrium in approximately two hours, that greater binding occurs with more AP, and that the percentage of AP adsorbed increases significantly with the amount of montmorillonite. A fuller understanding of phosphorus cycling may allow for manipulation of the cycle to make phosphorus use more efficient.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Composition and viability of the microbial flora of the sigmoid colon obtained from embalmed human cadavers
Authors: Fracassi Rebeca Elizabeth , Holz Jonathan , Olivieri Marion Pace , Davie Jeremiah
Published: June 2021

The gut microbiome contributes to proper digestion and nutrition. Abnormalities affecting the normal gut physiology can be responsible for disrupting the gut microbiome. Disruptions to the gut microbiome can be the cause of several pathologies. This research sought to determine the efficacy of utilizing cadavers for studying the gut microbiome. Previous studies utilizing DNA from fixed tissues for enzyme based assays, such as PCR were constrained by the chemicals in the fixative that researchers found it was difficult to eliminate these chemicals. The aim of this study was to determine if there were viable bacteria present in the sigmoid colon of embalmed cadavers. If present, these bacteria could be grown and expanded for further molecular based studies. The sigmoid colons of three male cadavers were isolated. The contents of the colon were assayed for the presence of culturable bacteria. Additionally, the sigmoid samples were sectioned for analysis of tissue morphology. These preliminary baseline histological studies suggest that the implemented embalming procedure of normal tissue architecture in the sigmoid colon resulted in elimination of all culturable bacteria. These results further suggest that, if viable bacteria are present, other methods are needed to fully access the presence of bacteria and microbiome in the sigmoid colon of embalmed cadavers.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A network approach to vertical transfer and articulation for student success in biology: A fourth workshop hosted by the Northwest Biosciences Consortium RCN‐UBE
Authors: Kruchten Anne , Baumgartner Erin , Beadles‐Bohling Amy , Brown Jeffrey , Duncan Jason , Kayes Lori , Kiser Stacey , Seidel Shannon , Shriner Walter , Stavrianeas Stasinos , Tillberg Chad
Published: June 2021

The Northwest Bioscience Consortium (NWBC) is an NSF funded RCN‐UBE group working to align introductory biology curricula in the Pacific Northwest with the concepts and competencies of Vision and Change. During the last four years, the NWBC has hosted three workshops providing faculty development opportunities for colleagues across the region focused on classroom inclusivity, designing curricula for majors' introductory biology classes, and designing curricula for non‐majors introductory biology courses. We now report on our fourth winter workshop, entitled “A network approach to vertical transfer and articulation for student success in biology.” Held February 8–9, 2018, this workshop brought together faculty and administrative participants to collaborate on the NWBC model that all students are transfer students, whether they are transitioning from one institution to another or from introductory courses to advanced courses within the same institution. Day one of the workshop addressed issues affecting faculty, advisors, and administrators working with students in introductory biology courses, including state level initiatives mandating course topic coverage. The second day facilitated faculty groups working on common curricular outcomes to help students be successful in introductory biology courses and in vertical transfer to advanced biology courses. Here we present an overview of our workshop accomplishments and future plans for the NWBC and its RCN‐UBE funding.

Support or Funding Information

This work is funded by NSF RCN‐UBE 1248121.

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