bluuebunny/crossref_metadata_2025 huggingface dataset:
Dimensions and Angulations of The Coronary Sinus Tributaries: Implications for Left Ventricular Pacing.
Authors: Tyrak Kamil Wojciech , Mazur Malgorzata , Holda Jakub , Holda Mateusz Krystian , Zabowka Anna , Klimek‐Piotrowska Wieslawa
Published: June 2021

Background

The coronary venous system is represented by the coronary sinus (CS) and its tributaries. High variability of the coronary venous tree challenges electrocardiologists. The aim of this study was to evaluate the morphology of CS tributaries, useful and important for invasive procedures, with regards to their angles of entrance to the CS, cannulation distance and ostium diameter.

Methods

A total of 200 autopsied human hearts form healthy donors (22% females) were investigated. The ostia of coronary veins to the CS were identified and their diameters were measured. Distances between middle cardiac vein (MCV), posterior vein of left ventricle (PVLV) and left marginal vein (LMV) ostium and CS ostium (cannulation distance) and angles between coronary veins and CS long axis were evaluated. To simulate clinical procedures during measurements 8‐ and 7‐French catheters were used. This study has been conducted in conformance with the Basic Principles of the Declaration of Helsinki and their expansion.

Results

Small cardiac vein was absent in 52 examined hearts (26.0%). The MCV was present in all assessed specimens. It's mean distance to CS ostium was 5.3±3.2 mm. All MCVs opened to CS, forming with its long axis mean angle of 82.0±12.8°. The average diameter of MCV ostium was 1.8±0.5 mm. The PVLV was observed in 127 hearts (63.5%), two PVLVs was detected in 54 specimens (27.0%) and three PVLVs in 2 hearts (1.0%). In 5 cases (2.5%) a single main PVLV and small ventricular tributaries were observed. The mean distance between CS ostium and the nearest PVLV (from the CS ostium) was 19.7±9.3 mm; second order vein: 27.5±10.4 mm; third order PVLV 31.3±0.1 mm. The mean diameters of PVLVs ostia were: first: 1.2±0.5 mm; second: 1.1±0.6 mm; third: 1.1±0.2 mm. The PVLV entered to the CS at mean angle of: first: 97.0±14.0°; second: 97.9±11.4° and third vein: 117.5±3.5°. The LMV was present in 94 hearts (47.0%). It was either a tributary of CS (17 specimens) or it entered to the GCV (62 hearts). In 15 cases small ventricular tributaries of LMV were detected (7.5%). The mean distance between CS ostium and LMV was 46.0±12.0 mm. It's mean angle was 92.0±13.4° and average LMV ostium diameter was 0.9±0.5 mm.

Conclusion

In this research we found MCV with diameter never smaller than 0.5 mm, which makes it quite convenient for electrophysiological procedures. The PVLV was highly variable in number, unfortunately presence of more posterior veins makes it less suitable for cannulation due to smaller diameter. The LMV entering to CS would be potentially more accessible for lead implantation, but those were found only in 8.5% of hearts.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Preserving Skeletal Anatomical Structures from Cadaveric Specimens to Enhance the Anatomy Education with Emphasis in Arthrology.
Authors: Rodriguez Carolina , Fossas Arturo , Veras Wilson R
Published: June 2021

INTRODUCTION

The National Board of Medical Education considers the musculoskeletal system to be amongst the three most crucial subjects within an anatomy curriculum, therefore the necessary tools are required to properly instruct students on the subject. Common teaching methods include using bones and dissections to show the origins and insertions of muscles and their related neurovascular bundles, but given the complexity of these structures, some students are not able to visualize important details. Additionally, Universidad Central del Caribe has recently struggled to obtain enough specimens due to increases in student acceptances as well as declines in the supply of bodies to our board of donations. This potentially poses a paucity of bodies for upcoming classes. This study proposes a method that could allow our medical school not only to preserve structures that would otherwise be discarded, but also to broaden their functionality as a tool for learning gross anatomy.

METHODS

The specimens used in this procedure had been previously used to teach dissection‐based anatomy and were in a fragile state that required their preservation in an embalming solution of formaldehyde, phenolic acid, glycerin, and water. These were rinsed and submerged in a solution of Sodium Hypochlorite (NaClO) to soften the tissues. Over the course of 6 weeks, the cadavers were monitored and dissected, either isolating those structures that make up the articulations or the tendons of muscles, depending on the tissues desired to be preserved. Once the desired structures were obtained, these were placed in a bleaching fluid to improve their appearance and then submerged in a preservative fluid preventing further decay.

RESULTS

The approach we took for dealing with the structures proved to be successful. By removing most of the tissues that were susceptible to NaClO, we were able to isolate tendons from their muscles and expose the articulations that through normal dissecting methods would be hard to observe.

CONCLUSION

This approach provides a potential means for medical institutions to lengthen the use of aged cadavers and proposes an alternate method to enhance the study of hard to observe anatomical structures that are oftentimes concealed by an abundance of blood vessels, nerves and muscle tissue. The effectiveness of this method in studying anatomy have yet to be compared to traditional methods of cadaveric studies. However, it provides a simple alternative that is reliable for medical schools facing a cadaver shortage.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing the Role of the Gene, Pebbled, in D. melanogaster ASP Invasive Behavior
Authors: Salim Hasan , Srivastava Ajay
Published: June 2021

The Drosophila Air Sac Primordia (ASP) can be used to study invasive behavior as it mimics tumor behavior when it invades into the Wing Imaginal Disc (WID) during larval development. A protein expressed in the ASP was found to be coded by the gene pebbled (peb). This study was conducted to see whether pebbled influences the invasive behavior of the ASP. Using Drosophila genetics and RNA interference, the pebbled gene was knocked down and less‐developed and less‐invaded ASP's were seen as the result. The data presented provides information on the possible role of pebbled in tumor invasion. The downregulation of pebbled also lead to a decrease in filopodia (actin projections protruding from the ASP) supporting the claim that there is a direct positive correlation between quantity of filopodia and the invasive capabilities of the ASP.

Support or Funding Information

KBRIN AREA and IDeA Grants funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health Grant # P20GM103436

WKU Biotechnology Center

Gatton Academy Research Internship Grant (RIG)

WKU Faculty Undergraduate Student Engagement (FUSE) Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differentiation of adipose‐derived stem cells into urothelial and smooth muscle cell lines within the structure of collagen/hyaluronan scaffold
Authors: Galambosova Martina , Ziaran Stanislav , Novakova Zuzana Varchulova , Nicodemou Andreas , Varga Ivan , Danisovic Lubos
Published: June 2021

Many urethral pathologies require surgical interventions. Although the commonly used surgical techniques are still being developed, complications such as the urethral strictures often occur and repeated surgery is needed. Tissue engineering of the urethral tissue, using autologous stem cells and appropriate scaffolds, represents promising technique to overcome these complications. In our experiment, we focused on the differentiation of adipose tissue‐derived stem cells seeded on the collagen/hyaluronan scaffolds into urothelial and smooth muscle cells. Differentiation culture media containing keratinocyte serum‐free medium with 2 % fetal bovine serum (FBS) and 30 ng/mL EGF, and D‐MEM with 10 % FBS, 2.5 ng/mL TGF‐β and 5 ng/mL PDGF were used to induce urothelial and smooth muscle differentiation, respectively. After two weeks of cultivation, cells were analyzed by scanning electron microscope (JEOL 7500F) for morphological features. Expression of the uroplakin 2 (UPK2) gene, keratin 2 gene (KRT2), ACTA 2 gene, and calponin 1 gene (CNN1) was tested in order to determine successful differentiation by quantitative Real‐Time PCR. Obtained results showed attachment of the cells on scaffold surface. The increased levels of UPK2 and KRT2 confirmed urothelial differentiation and expression of ACTA 2 and CNN1 confirmed smooth muscle differentiation. On the basis of obtained results, it can be emphasized that collagen/ hyaluronan scaffolds colonized by adipose tissue‐derived stem cells after performing further tests dealing with biosafety may have promising potential for tissue engineering of the urethra.

Support or Funding Information

Supported by grant APVV no. 15‐0111.

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 artificial tumor‐derived monolayers from long‐term MCF‐7 breast adenocarcinoma constructs using flow cytometry and immunocytological analysis.
Authors: Lyden Timothy , Topel Samantha , Thueson Hanna
Published: June 2021

For the past decade our laboratory has been engaged in developing and testing a variety of 3D artificial tissues, both normal and pathologic, using several types of natural de‐cellularized extracellular matrix materials. Throughout this work, one notable observation stood out with all of the tissues generated, specifically, 3D constructs transitioned through a series of “developmental” stages reflective of in‐vivo tissue development. These stages involve the early colonization and establishment of the basic tissue structure followed by a period of maturation and a final relatively long term stable mature tissue. In this case, we employed de‐cellularized and de‐calcified bovine trabicular bone matrix and MCF‐7 cells to develop artificial tumor tissues which were maintained for periods of 1, 2, 3 and 4 years respectively. During these culture periods, each tissue displayed the stages listed above and following the initial colonization period of one to two weeks each also began to shed cells and later spheres which, in turn, established monolayers of “post‐scaffold” cells. These monolayers were mainly established as multiple individual colonies by shed and explanted spheres or small tissue fragments. These various colonies eventually coalesced to form a single monolayer. Flow cytometric analysis of monolayers from each of the four culture periods demonstrate enhanced populations of cells with immunophenotypes for breast cancer stem cells based on CD44/CD24 labeling. In addition, individual cellular morphology as well as culture growth patterns were consistently changed from the baseline standard MCF‐7 cultures. These cells also displayed a substantially reduced cell‐cell adhesion pattern leading to loose patches in place of the relatively tight colonies seen with original MCF‐7 cultures. Taken together, these cellular modifications resulting from long‐term culture of artificial tissues support our interpretation that such constructs reflect the process of tumor progression in‐vivo and result in enhanced metastatic cell populations.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lysophosphatidic Acid Regulates the Vitamin D Receptor and its Metabolism‐Related Genes in Human Gingival Fibroblasts
Authors: Cerutis D. Roselyn , Weston Michael D. , Nunn Martha E. , Williams David E. , Ogunleye Afolabi O. , Miyamoto Takanari
Published: June 2021

Our laboratory has established that lysophosphatidic acid (LPA) regulates the biology of human oral fibroblasts, and that gingival fibroblasts (GF) express the LPA1–5 receptor subtypes. We have also shown that LPA 18:1 controls immediate‐early GF gene transcription of inflammatory cytokines, and of their receptors and regulators. Vitamin D3 is a key hormone in decreasing inflammation, and has been shown by Nastri et al. (2018) to decrease pro‐inflammatory and increase anti‐inflammatory cytokine production by P. gingivalis‐ and S. pyogenes‐infected human GF. Therefore, we hypothesized that LPA would also modulate transcription of its receptor, VDR, since chronic periodontal disease (PD) is an inflammatory condition in which we have reported that LPA is elevated in human gingival crevicular fluid and saliva (Bathena et al., 2011). After GF treatment with 1 × 10−5 M LPA 18:1 for 2h or 8h, mRNA was extracted. Agilent Whole Human Genome Oligonucleotide Microarray analyses [n=3; each n= pooled mRNA derived from 3 healthy young donors; (9 total donors; the study conformed to the Declaration of Helsinki guidelines, was approved by the Creighton University IRB, and informed written consent was obtained from all donors)] revealed that LPA consistently and significantly down‐regulated the VDR vs. control (≤ −2.0 ± 0.5‐fold at 2h, and −3.1 ± 1.1 fold at 8h, respectively). Of great interest, the receptor cubilin (CUBN), which has been shown in other systems to bind vitamin D binding protein (DBP), was also down‐regulated in groups 1 (−2.0) and 2 (−4.1) at 8h; it may be involved in the secretion of vitamin D by GF, as postulated by Liu et al., (2012). LPA treatment also significantly affected transcription of the cytochromes (CYP) CYP27A1 [−3.0 ± 0.6 (2h); −3.2 ± 1.4 (8h)] and CYP27B1 (induction or repression varied by group) which is involved in the production of metabolically active metabolites and which catalyzes the hydroxylation at the C‐1α position of 25(OH)D3 and 24R, 25(OH)2D3, respectively. These results for the VDR (minimum level of significance, p < 1×10−7) provide preliminary evidence that LPA likely exerts complex actions during periodontal inflammation by regulating it, along with other key receptors and enzymes in the vitamin D pathway.

Support or Funding Information

Health Future Foundation (D.R.C).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altering Gut Microbiota to Enhance Therapeutic Efficacy in Resistant Colon Cancer by Natural Agents
Authors: Majumdar Adhip P.N. , Farhana Lulu , Sarkar Sarah , Nangia‐Makker Pratima , Yu Yingjie
Published: June 2021

Tumor recurrence, a common phenomenon in allmalignancies, is observed in nearly 50% of patients with colorectal cancer(CRC) and is thought to be due to re‐emergence of chemotherapy‐resistant cancerstem cells (CSCs), accompanied by dysfunction of the gut microbiota, resultingin alterations in microbial metabolites. Thus,development of preventive/therapeutic strategies that specifically target CSCs and are non‐toxic should be effective in reducing the risk of relapse. We have examinedthe effectiveness of the combination of ETO‐Curcumin (ETO‐Cur; curcumincomplexed with essential turmeric oil) and TRF, tocotrienol‐rich fractionof palm oil in inhibiting the growth of SCID micexenograft of chemo‐resistant (CR) colon cancer cells and whether thisinhibition could be attributed to alterations in gut and tumor microbiome andtheir metabolites. Indeed, feeding of ETO‐Cur (5 mg/kg) together with TRF (2mg/kg) or the vehicle (control) in SCID mice, initiated after 20 days of inoculationof CR colon cancer cells and continued for 45 days resulted in inhibition of growth of colon tumor that became apparent after about 30 days of treatment. At 40‐ and 45 days, differences in tumor growth between the two groups were highly significant. This inhibition of growth was associated with a significant down‐regulation of β‐catenin and TNF‐α in the tumor and alterations in gutmicrobiota. The latter was evidenced by an overlap of 340 bacterial species between the control and ETO‐Cur/TRF‐treated mice, and 216 and 120 species being different in ETO‐Cur/TRF treated and control mice, respectively. ETO‐Cur/TRF treatment also caused a significant reduction in the expression of 7‐α‐dehydroxylase, an enzyme that catalyzes the conversion of primary to secondary bile acids. Since secondary bile acids, specifically deoxycholic acid (DCA) and lithocholic acid(LCA) are known for their co‐carcinogenic activity and to induce CSCs in the colon, we postulate that the decreased conversion of primary bile acids in the gut to secondary bile acids DCA and/or LCA by ETO‐Cur/TRF may have contributed to the suppression of growth of colon tumors.

Support or Funding Information

NIH and Department of Veterans Affairs

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural dynamics of blue light sensitive bacterial photoreceptor blsA
Authors: Chitrakar Iva , French Jarrod
Published: June 2021

Blue light sensing A (blsA) is a blue light using flavin (BLUF) domain isolated from opportunistic pathogen Acinetobacter baumanii. BlsA is vital for A. baumanii’s motility, biofilm formation and virulence. When blue light activates blsA [in the N‐terminus], the protein interacts with its effector domain(s) [in the C‐terminus] with implications in downstream signaling events. Photo absorption modifies the hydrogen bonds and the electrostatic interactions between flavin and the BLUF domain with consequent structural changes that are integral in the signal transduction. Spectroscopic data highlight involvement of residues glutamine (51) and tryptophan (92) in the photoactivated signaling state of blsA. My goal is to solve blsA's structure using x‐ray crystallography to determine the specific residues involved in the signaling state. Comparison of crystal structures of blsA (~1.8 Å) in the dark state and the photoactivated light state does not show movement of the residues glutamine and tryptophan in the two states. However, we detect a distinct conformational change of an alpha‐helix in the C‐terminus‐ the effector protein binding terminus‐ of blsA.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of limonene on tracheal and vascular reactivity in a mouse model of asthma
Authors: Rajalingam Sahith , Bhavsar Riya , Patel Mehaben , Siddiquee Armaan , Kurade Mangesh , Ponnoth Dovenia S
Published: June 2021

Animal models of asthma have shown that limonene (terpene in citrus fruits) reduces inflammation and airway reactivity. We have shown a role for A2A adenosine receptors (A2AAR) in mediating these effects in asthma. We investigated the effects of limonene (in vivo administration) on isolated tracheal and aortic rings to determine tissue‐specific reactivity using organ bath experiments. Mice were divided into control (CON) and allergen sensitized‐challenged (SEN) groups, and were sensitized i.p. on days 1, 6 with 20μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13 (n=4–6 mice per group). Limonene was administered as an inhalation prior to allergen challenges in one group of allergic (SEN+LIM). Response (contraction) to methacholine (MCh; 0.1μM) in isolated trachea was significantly higher in SEN tissues (225.99±67.34% vs 5.57±19.74% in CON). Treatment with limonene significantly reduced the tracheal reactivity (80.68±20% in SEN+LIM vs SEN; p<0.05). For isolated aorta, we found a significant difference in endothelial‐mediated relaxation as measured by response to acetylcholine in SEN (12.74±13%) compared to controls (53.94±.26). Limonene treated groups showed significant increase in endothelial response compared to non‐treated groups. No difference was observed in aortic smooth muscle responses (measured against sodium nitroprusside) for any group. These data suggest that limonene reduces tracheal reactivity and improves endothelial response in asthmatic mice. Further experiments are needed to identify the exact mechanisms of these effects.

Support or Funding Information

Institutional Research Grant, LIU (DSP)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anti‐Cancer Effect of 2‐Cyano‐3, 12‐dioxoolean‐1,9‐dien‐28‐oic acid (CDDO) Derivatives on Multiple Myeloma Cells
Authors: Rogers Lanerica Jamese , Wang Xinyu
Published: June 2021

Multiple myeloma (MM) is a blood cancer that affects 30,000 people yearly in the U.S. It stems from monoclonal plasma cells and can cause symptoms such as bone loss and fatigue. Recent studies have shown that the bone marrow microenvironment may mediate tumor proliferation, drug resistance, and migration of the multiple myeloma cells. Synthetic oleanane triterpenoids have been readily used in research due to their anti‐proliferative and anti‐inflammatory effects. The objective of this study is to examine the anti‐cancer effect of 2‐cyano‐3, 12 dioxoolean‐1,9‐dien‐28‐oic acid (CDDO) derivatives on multiple myeloma cells. Three CDDO derivatives (CDDO‐Methyl Ester (Me), CDDO‐Trifluoethyl Amide (TFEA), and CDDO‐Imidazolide (Im)) with a dose range from 0.1 to 0.5 μM were tested on the growth of MM RPMI 8226 cells within 72 hours. Our current results show that all CDDO derivatives decrease the cell viability and proliferation of the MM 8226 cells in a dose and time‐dependent manner. Based on the potency, CDDO‐Im was selected to study whether its inhibitory effect on MM cell growth is due to the induction of apoptosis. Western blotting was used to check the protein expression of apoptosis‐related factors such as caspase 3, caspase 8, caspase 9 and poly‐(ADP‐ribose) polymerase (PARP). The result indicates that CDDO‐Im may induce the intrinsic apoptotic pathway. To evaluate the effect of CDDO‐Im on MM 8226 cells in the presence of the microenvironment, a Transwell (TW) model using MM cells co‐culturing with bone marrow stromal cells will be applied to mimic the bone marrow microenvironment in vivo. When treating the HS‐5 stromal cells with CDDO‐Im, no significant decrease in proliferation was seen after 24 hours. We expect that after co‐culturing the MM 8226 cells with HS‐5 stromal cells using the TW model, the growth of MM cells will still be prohibited by CDDO‐Im to some extent if not the same as MM cells being treated alone. In conclusion, our data suggest that CDDO derivatives inhibit the growth of MM cells through induction of apoptosis.

Support or Funding Information

PCOM Division of Research Funding

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 production of an antimicrobial compound by plant‐mediated bioreduction of benzofuranyl methyl ketone
Authors: Cheatwood Nicholas Young , Harris Michele Young
Published: June 2021

Benzofuran‐2‐yl methyl ketone (BMK) is a prochiral ketone that is reduced to benzofuran‐2‐yl ethanol (BMA) when exposed to tissues of certain plants. The process of using biological tissue to convert one compound to another, typically with high enantiomeric excess, is called a biotransformation reaction. Because different plants catalyze this conversion, the primary goals of this research were to identify plants that are successful in conversion, determine an approximate time necessary for each plant to perform the conversion, and then analyze the product of each plant to determine the enantiomeric excess. Optical purity of BMA was determined using polarimetry. The BMA produced by carrots had a [α] = −16.6 which agrees with the literature value for the S‐isomer of BMA. However, the BMA produced by potatoes had a [α] = −.74 which may indicate a mixture that is nearly racemic. To explore the reaction further, antimicrobial studies were carried out adding either BMK or BMA to YPD agar plates growing with Baker's yeast. In these preliminary studies, S‐BMA, produced by carrots, consistently demonstrated a greater growth inhibition of the Baker's yeast than BMK. The growth inhibition of the yeast by BMA was dose dependent. The antimicrobial studies were also expanded to BL21 E. coli grown on LB agar, showing similar results to those of yeast. The antimicrobial activity of S‐BMA against yeast and bacteria was also compared to that of the racemic mixture produced by potato, which showed no significant differences between the two. The antifungal activity of BMA against yeast was then compared to the common over‐the‐counter antifungal compounds miconazole, clotrimazole, terbinafine and tolnaftate. The latter two had no effect on growth, but miconazole and clotrimazole inhibited yeast growth substantially. The antibacterial activity of BMA against E. coli was compared to ampicillin. In these preliminary antimicrobial studies, BMA has shown comparable activity to common compounds.

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 and Evaluation of ROS‐Containing Photosystem I Light‐Harvesting Complex I (PSI‐LHCI) Isolated from the Green Microalga Botryococcus braunii as a Potential Anticancer Drug
Authors: Ovalle Freisa Milagros Joaquín , Guihurt Grace , Barceló‐Bovea Vanessa Celeste , Ramirez‐Paz Josell , Doble Katerina , Saba Andraous Hani , Griebenow Kai
Published: June 2021

Reactive oxygen species (ROS) are generated during normal metabolism; however, at high levels, they can promote cell damage and death. In green microalgae, such as Botryococcus braunii (B. braunii), the thylakoid membrane (TM) and particularly photosystem I (PSI) are the main contributors to the overall production of ROS. B. braunii PSI‐LHCI was purified by differential centrifugation in a 15–35% sucrose gradient for 16.5 hours, 4 ºC at 37,500 rpm and characterized by visible absorption spectroscopy, low temperature (77K) fluorescence emission spectroscopy and LC‐MS/MS. PSI can act as a light‐sensitive molecule called photosensitizer; that upon photoactivation by exposure to LED light (λmax=660 ± 10 nm) at a distance of 10 cm and a fluence rate of 50 mW/cm2 for 40 minutes triggered the formation of ROS species. ROS generation in TM and PSI, specifically singlet oxygen was monitored spectrophotometrically using the p‐nitrosodimethylaniline (RNO) assay at 440 nm, which produced RNO dye bleaching upon singlet oxygen generation. To test PSI‐LHCI use as a prospective anticancer drug, two cancer cell lines, HeLa and MDA‐MB‐231, and one normal cell line, NIH‐3T3 were utilized for in vitro cell viability studies. Each cell line was incubated for 24 h with various concentrations of PSI‐LHCI (3.125, 6.25, 12.5, 25, and 50 μg/ml) which demonstrated a concentration‐dependent reduction in cell viability. At a 12.5 μg/ml PSI‐LHCI concentration, cell viability experienced a steep drop to less than 5% in HeLa cells, and a 47% and 53% decreased cell viability in NIH‐3T3 and MDA‐MB‐231 cell lines. Confocal microscopy demonstrated PSI‐LHCI internalization, and apoptosis induction studies confirmed cellular damage due to morphological change hallmarks such as condensed nuclear chromatin, decreased and fragmented nucleus after DAPI staining and PI counterstaining positively identified dead or necrotic cells represented by the intense, condensed nucleus staining.

Support or Funding Information

RISE Program: 5R25GM061151‐16

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RNA‐mediated Mechanisms of Translation Control
Authors: Dunham Christine M. , Hoffer Eric D. , Nguyen Ha An , Subramanian Sunita , Hong Samuel , Maehigashi Tatsuya
Published: June 2021

Gene expression relies on the interplay of dozens of proteins and large nucleic acid molecules that assemble into complex macromolecular machines such as the ribosome. These machines are regulated by posttranscriptional and posttranslational modifications. Numerous tRNA modifications, for example, are known to influence the fidelity of mRNA decoding by the ribosome. In the absence of this translational fidelity, nonsense protein products are expressed or premature termination occurs resulting in toxic protein products that overwhelm the cellular proteolysis machinery resulting in cell death. Posttranscriptional modifications of tRNAs have been known for decades but most studies have focused on how anticodon modifications expand the genetic code by allowing non Watson‐Crick base‐pairing at the third position of the codon‐anticodon interaction on the ribosome. However, most tRNAs are also extensively modified outside their anticodon stem‐loop. These modifications affect other aspects of translational fidelity such as the ability of the ribosome to maintain the three‐nucleotide codon of the mRNA as it moves through the ribosome. The absolute requirement for precise correlation between the mRNA frame and the correct protein sequence to be expressed underlies an fundamental question in molecular biology: what regulates the mRNA reading frame? To address this question, we study two examples of defined biological systems that subvert the three‐nucleotide mRNA reading frame resulting in high levels of programmed frameshifting. Our biochemical and structural results reveal that tRNA distortion and conformational changes of the small ribosomal subunit are induced by frameshift‐prone tRNAs and highly structured mRNAs. These programmed gene expression events cause changes in the dynamic motions of the ribosome required for productive translation. This dysregulation causes the ribosome to lose its grip on the mRNA, allowing the tRNA to shift into a new reading frame. Together these studies reveal how the ribosome can be programmed for to encode for alternative mRNA frames and in addition suggest how dysregulation of the mRNA frame may occur by the disruption of key interactions between tRNAs, mRNA, and translation factors with the ribosome.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bacterial lipid trafficking and outer membrane homeostasis
Authors: Chng Shu Sin
Published: June 2021

Gram‐negative bacteria can survive in harsh environments in part because of the presence of the outer membrane (OM), which comprises lipopolysaccharides (LPS) and phospholipids (PLs) in the outer and inner leaflets, respectively. This asymmetric distribution of lipids renders the OM a very effective permeability barrier against toxic compounds, including bile salts and hydrophobic antibiotics. To build a stable OM with the requisite lipid asymmetry, the transport and assembly of LPS, PLs and OM proteins into the OM must be coordinated. While assembly pathways for LPS and OM proteins have been well‐characterized, the processes by which PLs are transported to and from the OM, and their coordination with these other systems, are much less understood. In this talk, I will describe our work in understanding and characterizing PL transport systems in Escherichia coli, and discuss how these systems function in maintaining lipid homeostasis in the OM.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adenosine controls tissue fluid and pH homeostasis through transcriptional regulation of SLC26A3
Authors: Cartwright Ian Michael , Curtis Valerie F , Lanis Jordi M , Lee Joseph S , Alexeev Erica E , Onyiah Joseph C , Welch Nichole , Schaefer Rachael E.M , Colgan Sean P
Published: June 2021

Inflammatory bowel disease (IBD) affects 1.5 million individuals and their families in the United States, with rates of diagnosis on the rise and no effective cures. A hallmark of IBD is the presence of crypt abscesses, accumulation of neutrophils in the lumen of intestinal crypts. It has been well established that activated neutrophils release high levels of adenine nucleotides, which is quickly converted to adenosine. In the setting of acute inflammation, such adenosine is made available to can activate epithelial adenosine receptors to regulate multiple cellular functions. As a starting point, we performed an unbiased microarray profiling of epithelial transcripts in response to adenosine stimulation. This profiling identified to prominent induction of SLC26A3, a member of the solute carrier family of genes that serves as a chloride – bicarbonate exchanger. Using a combination of RT‐qPCR, western blots, and a promoter assay in T83 IEC, we show that SLC26A3 is a cAMP/CREB‐regulated gene. We also investigated the role of SLC26A3 in the adenosine response. It has been well established that in IECs adenosine elevates cAMP levels through the adenosine A2B receptor. These elevated cAMP levels trigger the activation of the CFTR and apical efflux of chloride. To investigate the role of SLC26A3 in functional adenosine responses, we utilized CRISPR to knockdown SLC26A3 and an ORF‐lentiviral vector to overexpress SLC26A3. Elevated levels of SLC26A3 prevented adenosine‐induced fluid transport while knockdown of SLC26A3 increased fluid transport. Moreover, intracellular pH changes in response to adenosine correlated with loss and gain of SLC26A3 expression, likely related to chloride‐bicarbonate exchange with elevated intracellular cAMP. Finally, we compared the expression of SLC26A3 in colon biopsies from healthy and patients with active ulcerative colitis. This analysis revealed that SLC26A3 is repressed with active inflammation and residual protein expression is abnormally localized within colonic tissues. These results identify adenosine‐regulated SLC26A3 as a relevant epithelial protein that contributes to tissue pH homeostasis. Targeted regulation of SLC26A3 may serve as a new therapeutic approach in mucosal inflammation.

Support or Funding Information

NIH R01‐DK095491

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 regulation and consequences of lipid droplet catabolism
Authors: Mashek Doug
Published: June 2021

Lipid droplets are recognized to be the primary storage form of energy in most cell types. While this important function is critical to supply cells or organisms with energy during times of nutrient deprivation, their role beyond energy storage has not been sufficiently explored. Our laboratory has focused on elucidating the mechanisms through which lipid droplets communicate within cells to coordinate energy storage with cell signaling and metabolism. This presentation will focus on the regulation of lipid droplet catabolism through cytosolic lipases and autophagic pathways. We will present data showing the role of perilipin 5, lipid droplet protein highly expressed in fasting liver, in mediating lipid droplet degradation. We will also explore the role of perilipin 5 as a key node that links lipolysis to downstream signaling networks that govern mitochondrial biogenesis, dynamics and function. In summary, this presentation will highlight novel roles for lipid droplets and resident lipid droplet proteins in cellular and organismal biology and their importance in human health.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Single Cell RNA‐sequencing (scRNA‐seq) Reveals Reprogramming and Functional Compensation Preceding Cellular Recovery in Multiple Models of Acute Liver Injury
Authors: Walesky Chad , Kolb Kellie , Winston Carolyn , Henderson Jake , Apte Udayan , Mueller Florian , Shalek Alex , Goessling Wolfram
Published: June 2021

The liver is an essential organ critical for several homeostatic functions. Because of its vital role in detoxificaion, the liver is regularly exposed to toxic insults leading to injury, cell death, and a loss of functional mass. Consequently, the liver has evolved a unique capacity to regenerate in order to maintain function. While numerous studies have focused on the proliferative capacity of the liver to regenerate tissue mass after injury, the ability of the liver to maintain function after massive cell loss has not been explored in detail. Here, we deploy single cell RNA‐sequencing (scRNA‐seq) to define dynamic changes in the transcriptional profile of the liver following different forms of acute injury.

Hepatocytes, the major parenchymal cell of the liver, are heterogeneous and arranged in a pattern consistent with their specific function, known as hepatic zonation. Select toxic insults will injure particular zones of the liver depending on zonation‐specific metabolism. One example is acetaminophen (APAP) toxicity, the most common cause of acute liver failure in the U.S. APAP affects a specific group of hepatocytes within an area known as the pericentral zone. In order to elucidate the immediate transcriptional responses of the entire liver after acute injury, we performed scRNA‐seq analysis in murine livers following APAP overdose compared to injury of the periportal zone (allyl alcohol) as well as physical injury that is not specific to a particular zone (partial liver resection). We discover that hepatocytes exhibit the ability to alter their transcriptional identity in order to maintain essential, zone‐dependent liver function, such as glucose metabolism, coagulation, and detoxification, prior to and during proliferative regeneration of liver mass. These findings are further validated and quantified by single‐molecule FISH. Further, our analysis reveals that zonal reprogramming is dependent on the Wnt pathway, which also regulates liver zonation and cellular regeneration. Therefore, we have identified a novel regenerative response that is focused on the preservation of function prior to the onset of cellular regeneration.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Examination of Molecular Dynamic Simulations for Glucokinase (GCK) Mutations in Type 2 Diabetes
Authors: Rios‐Rosales Anapaula , Jacus Isabella G. , Shor Audrey C , Prokop Jeremy
Published: June 2021

An essential aspect of undergraduate biology includes visualization of the relationship between protein structure and function. Glucokinase (GCK) is significant to glucose homeostasis. When mutated, GCK has been demonstrated to play a role in Type II Diabetes, leading to diminished sensitivity to insulin detection. The focus of this project was to create a molecular dynamic simulation that could be used to visualize mutations at residues L25Q, L25R, R36P, V33E, E40K, G44D, M41T, and T49N on GCK and, in turn, illustrate how amino acid substitutions alter the structure and function of GCK. Glucokinase was selected because it is a common protein demonstrated to have mutations suspected of the Type II Diabetes phenotype when screened through Clinvar, Polyphen, Provean, and SIFT. Specific residues were determined as most prevalent and classifiable as “damageable” or “pathogenic” in the impact of affected cellular function upon comparing information from the aforementioned databases. Yet Another Scientific Alternate Reality Application (YASARA) and cloud computing were used to model selected mutations to asses structural changes compared to wild type. Such research and specific use of bioinformatics is important because it has the capacity to consolidate large masses of information from multiple sources and specify the focus for groups working on treatments of particular diseases. Modelling is further useful in communication of disease and amino acid level mutations to patients as it provides visuals to compare wild type and mutated phenotypes of affected proteins. Further research could be conducted on specific perturbations caused by selected GCK variants.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Post‐translational Modification of Poly (A)‐Specific Ribonuclease Regulates its Enzymatic Activity
Authors: Dejene Eden Alemneh , Li Yixuan , Seto Edward
Published: June 2021

Post‐translational protein modifications (PTMs), such as acetylation, play an important role in mediating the function, expression, localization, and interactions of proteins. Protein acetylation is a reversible process that is tightly regulated by lysine acetyltransferases (KATs) and lysine deacetylases (KDACs). Lysine acetylation of non‐histone proteins has been reported to affect their enzymatic activity, stability, and complex formation. However, the exact role of acetylation on non‐histone substrates is protein‐specific. Examination of a published dataset identifying acetylated lysine enriched substrates revealed that Poly (A)‐specific ribonuclease (PARN) may be post‐translationally modified by acetylation. PARN is a 3′ exoribonuclease; thus, its enzymatic activity allows it to cleave RNA bases at the 3′ end, one nucleotide at a time. PARN has the highest substrate affinity for adenosines; therefore it is also referred to as a deadenylase. Adenosine repeats (poly (A)) on the 3′ end of RNA prevent mature transcripts from degradation by nucleases, especially during transport from the nucleus to the cytoplasm for translation. Thus, removal of poly (A) tails by deadenylase enzymes such as PARN promotes mRNA decay. PARN's exoribonuclease activity plays an important role in many biological processes, such as embryogenesis, telomere maintenance, and DNA damage response. However, very little is known about how PARN is regulated in humans. The objective of this research is to investigate whether PARN is post‐translationally regulated by acetylation and whether this modification affects its biological function. Our findings revealed that PARN's activity is tightly regulated by acetylation in humans, and that acetylation plays a key role in mediating PARN's enzymatic activity.

Support or Funding Information

This research was supported by the National Cancer Institute of the National Institutes of Health under award number [3R01CA187040‐05S1]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the N‐ and C‐terminal Extensions of the Yeast DEAD‐box Protein Dbp6
Authors: DeMarzo Payton R. , Young‐Erdos Crystal L.
Published: June 2021

DEAD‐box proteins are RNA‐dependent ATPases that are involved in all aspects of RNA metabolism. Also known as DEAD‐box helicases, these enzymes bind RNA and ATP and unwind short RNA duplexes via a highly conserved helicase core. N‐ and C‐terminal extensions that flank this core differ in both length and composition between DEAD‐box proteins and have been shown to contribute to RNA specificity and binding, thereby conferring the diverse cellular roles of DEAD‐box proteins. Here, we are investigating the 192 amino acid N‐terminal and 63 amino acid C‐terminal extensions of Dbp6, a yeast DEAD‐box protein that is required for 60S ribosome biogenesis. To find the minimum functional length of the flanking regions, we have systematically designed truncation constructs and observed their effects on the growth of yeast cells in which endogenous Dbp6 has been depleted. Our results indicate that deletion of up to 168 amino acids at the N‐terminus (ΔN168) or 27 amino acids at the C‐terminus (ΔC27) give either wild‐type level or slow growth. However, removal of five more residues at the N‐terminus or two more at the C‐terminus results in a lethal phenotype. Interestingly, even though Dbp6 ΔN143 and Dbp6 ΔC21 each have near normal growth, the double truncation construct Dbp6 ΔN143ΔC21 has no growth. We are currently conducting Western blot analysis to confirm that Dbp6 is indeed being expressed in these yeast cells with growth phenotypes. Follow‐up experiments include measuring the ATP and/or RNA binding and RNA duplex unwinding activities of Dbp6 truncation constructs in vitro. Since most of the yeast DEAD‐box proteins have orthologs in humans and many of those proteins have been implicated in cancer development or progression, we hope that these insights into the molecular workings of Dbp6 can contribute to our broader understanding of DEAD‐box proteins and their uses as potential cancer therapeutic targets.

Support or Funding Information

Eckerd College Natural Sciences Summer Research Program (NSSRP)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing Cell Growth in a Microtiter Plate Reader with Selective Pressure Incorporation of Unnatural Selenium and Tellurium Containing Amino Acids
Authors: Brown Amy , Hill Austin R. , Boles Jeffrey O.
Published: June 2021

Metalloid‐containing unnatural amino acids, when incorporated into target proteins, provide a means of solving for three‐dimensional structure by MAD or SAD techniques. The use of Tellurium‐containing amino acids are of special interest since they can also be exploited by more traditional MIR techniques, which don't require travel to synchrotron centers. Tellurium, when incorporated into protein as telluromethionine, generally induces toxicity to E. coli. Selenium, when incorporated as selenomethionine, induces much lower toxic effects to cell culture but remains problematic, at times. Altering growth conditions can potentially reduce such toxicity. This study demonstrates a rapid screening method for biosynthetic incorporation of unnatural amino acids by various forms of selective pressure.

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