bluuebunny/crossref_metadata_2025 huggingface dataset:
Using Synthetic Biology methods to construct a dual functional estrogen biosensor
Authors: Yusef Farhiya , Le Kimberly , Meunier Angela , Fontenot Morilyn , Roecklein-Canfield Jennifer
Published: April 2020

The presence of estrogenic compounds (endocrine‐disruptors, EDCs) in the water supply raises concerns about human and aquatic health. Current methods for detecting estrogen contamination require expensive, time‐consuming techniques such as liquid chromatography‐mass spectrometry and high performance liquid chromatography. Previously reported estrogen biosensors required multiple cloning and transformation steps for successful detection in bacteria. As proof of concept we constructed a new dual function estrogen biosensor in bacterial cells. Synthetic Biology demonstrates the integral relationship between biology, chemistry, mathematics and computer science. The field provides innovative approaches to a wide range of applications, such as bioremediation, sustainable energy production and biomedical therapies. Synthetic biology allows for the construction of genetic devises composed of DNA sequences modified to be interchangeable and provide novel functions. New tools and devices are constantly needed to enhance the already extensive list of novel genetic parts. Our estrogen responsive plasmid contains parts that allow for both the constitutive expression of the estrogen receptor followed by an estrogen response element toggled to a reporter gene in a single vector. When the estrogen response element is activated a reporter gene will produce a color change in E. coli. We will construct novel promoter and chromophore reporter genes combinations to optimize estrogen responsiveness. We will present data demonstrating the efficiency of this dual‐functional biosensor and it effectiveness for detection of estrogenic compounds in contaminated water.

Support or Funding Information

NSF‐REU‐1852150, REU Site: A multisite REU in Synthetic Biology, 2019
The Telomerase RNA Stays in the Nucleus in Aspergillus nidulans
Authors: Dejneka Lexi , Palmero Brett , Akins Allison , Day Jessica , Witte Graeme , Kirk Karen
Published: April 2020

Telomeres are protein‐nucleotide sequences at the ends of chromosomes that serve to prevent the loss of genetic information during cell replication. A telomerase ribonucleoprotein complex includes telomerase reverse transcriptase (TERT) and an RNA template (TER) and is responsible for adding telomeric DNA. Our lab has been studying the biogenesis of TER in Aspergillus nidulans, a molecule that is in part structurally similar to that of Saccharomyces cerevisiae and partly to that of mammalian cells. Research in S. cerevisiae has shown that the telomerase RNA appears to leave the nucleus and is potentially assembled in the cytoplasm. Whether TER migrates to the cytoplasm is not known in A. nidulans, but its ability to form a multi‐nucleate state known as a heterokaryon can be used to determine the localization of TER. We assay for the presence of two genetically distinct nuclei via the heterokaryon test, a molecular tool that has allowed us to deduce whether the RNA products for TER and TERT leave the nucleus. We knocked out the genes for TER and TERT, analyzed the results of the heterokaryon test, and determined if assembly of the active enzyme occurs in the nucleus or the cytoplasm. Our results suggest assembly in the nucleus, a scenario that is similar to humans but not to yeast. These results further suggest the potential of A. nidulans as a useful model organism to study the localization of telomerase components.
JAW MORPHOMETRY: AN ANATOMIC STUDY
Authors: Lima Sarah Araújo Santos , Parente Maria Eduarda Cordeiro , Teixeira Claudio Silva , Barreto Joao Erivan Façanha , Cerqueira Gilberto Santos
Published: April 2020

The jaw, an odd bone, being the largest and strongest of the face, serves to receive the lower teeth. It consists of a curved horizontal portion, the body, and two perpendicular portions, the branches, which join the body ends at almost right angles. The jaw is a bone widely used for forensic purpose. Forensic anthropology uses data collected from remains, among which the pelvis and the skull are the most used structures, and through meticulous analysis, enables post‐mortem identification. Thus, The aim of this study was to evaluate the feasibility of measuring morphometric measurements using mandible. Morphometric measurements were performed using a digital caliper, angle, diagonal length, horizontal length, minimum branch width, branch line and jaw line. An exploratory study was performed with 37 jaws of the anatomy laboratory. For data collection, a collection script was used. For statistical analysis, Student’s t‐test and ANOVA onw were used for parametric data. For nonparametric data, the Mann Whitney test was used, being considered significant when presented at p <0.05. A statistically significant difference was observed between the diagonal length of the mandible body (87.43±0.91) compared with the horizontal length (15.22±0.82). We identified the following morphometric measures for anatomical accidents: Jaw branch width (29.39±0.66), jaw line (84.02±0.96). It was found that there are several anatomical differences between the jaws present in the laboratory and that these variations may imply in various clinical situations such as anesthesia for oral procedures. Moreover, these variations are of vital importance for human identification.

Support or Funding Information

CNPQ
Photogrammetric Digitization and labeling of Plastinated Models for Pre‐Clerkship Education
Authors: Oh So‐Young , Dorsainville Gregory A. , Harnik Victoria , Rapkiewicz Amy , Shearer Brian , Ramirez Kristen , Alfaro Veronica , Rosenfeld Mel
Published: April 2020

NYU School of Medicine recently embarked on a re‐design of its anatomy curriculum that decreased the use of cadavers with plastinated specimens. Plastinated models provide an authentic learning experience of the human body, but lack necessary labels outlining important structures. Due to the fragile nature of the specimens, we endeavored to solve the challenge of labeling by developing a digitized supplement and archive of plastinated and pathology specimens.

An interdisciplinary team of faculty and multimedia designers at NYU School of Medicine designed and developed electronic resources related to the artistic models and plastinated specimens. Over the course of three months, 60 artistic and plastinated models of different sizes were captured from dozens of angles using a digital camera or an Artec Leo Scanner. The numerous image captures of the plastinated specimens were processed in Agisoft Metashape, a stand‐alone software product, that performs photogrammetric processing of digital images and generates 3D spatial data. After Agisoft Metashape exported a complex 3D mesh with a high‐resolution texture, anatomy faculty added labels to the digitized 3D anatomy specimens using the Sketchfab web platform. The labeled 3D anatomy models were then uploaded into the Living Anatomy site on NYU School of Medicine’s learning management system for students to explore before, during, and after their anatomy lab sessions. Quizzes using these models also were created to help students identify the structures and link them to physiology and clinical scenarios.

The digitized 3D models allow students to zoom in, rotate and explore the specimens in a more interactive way, thereby enhancing the process of just observing fragile plastination models. When asked, 84% of students reported that the 3D models of plastinated specimens contributed “very much so” to their learning of anatomical relationships.

We will continue to find opportunities for the meaningful integration of these 3D models within the anatomy curriculum as well as into other pre‐clerkship and clerkship modules. We will also assess the educational outcomes of the 3D models and, by doing so, will incorporate instructional design into the process.
Investigation of the Role of Manganese Transport Repressor DR2539 in Mn/Fe Homeostasis
Authors: Brown Jordan , Bokretsion Petros , Pattanayek Rekha , Damo Steven
Published: April 2020

Mn and Fe ions are necessary for fundamental cellular processes, where they serve as enzyme cofactors or impart structural stability to proteins. However, Mn and Fe are toxic in excess. For example, dysregulation of Mn/Fe homeostasis results in manganism, a neurological disorder with symptoms similar to Parkinson’s disease. Therefore, tight regulation of intracellular Mn/Fe ratios is critical. Understanding the mechanisms used to regulate Mn/Fe homeostasis has both medical and bioremediation implications. D. radiodurans is particularly successful at establishing maintaining a high Mn/Fe ratio, making it an ideal model to investigate. DR2539, a metal ion dependent transcription factor of the DtxR family, plays a major role in maintaining the intracellular ratio of manganese to iron within D. radiodurans. DR2539 has been shown to bind to the promoter of the Mn importer protein, MntH and repress it in a Mn‐ and Fe‐ dependent manner. 64 other genes are either up‐ or downregulated in the absence of functional DR2539. To further elucidate the role of DR2539 in Mn homeostasis, we recombinantly expressed DR2539 from E. coli and purified it to homogeneity. Preliminary differential light scattering (DLS) and differential scanning fluorimetry (DSF) results suggest DR2539 can bind to Mn, and Mn binding induced oligomerization of the protein. Comparative modeling suggest the Mn‐induced oligomer is either a homodimer or a homotetramer with a similar structure to other members of the DtxR family. Preliminary crystallization screening experiments have identified potential conditions which may lead to samples suitable for high‐resolution structural studies. In total these experiments represent a first step toward elucidating the molecular basis of DR2539 function.

Support or Funding Information

NSF HRD 1547757 Grant
Comparison of Exosomes Secreted by Culture‐Expanded Annulus Fibrosus, Nucleus Pulposus, and Bone Marrow Stromal Cells for Intervertebral Disc Repair
Authors: Linde Peter , Kisiday John , Puttlitz Christian
Published: April 2020

Exosomes have emerged as an alternative to cell therapies in many fields. Bone marrow stromal cells (MSCs) have been almost exclusively used to generate exosomes as progenitor cells are known to rapidly generate exosomes with a high therapeutic potential. Progenitor cells can be obtained from different connective tissues, and it has been shown that the therapeutic potential of exosomal cargo differs among tissues. We postulate the most efficacious exosomes are produced by progenitor cells derived from the tissue that is to be treated. Our long‐term goal is to develop exosome therapies for treatment of intervertebral disc degeneration. The intervertebral disc is made up of two regions that can experience degradation, the outer annulus fibrosus and inner nucleus pulposus. Therefore, the objective of this study was to compare exosomes from MSCs to exosomes from annulus fibrosus and nucleus pulposus cells that adopt progenitor cell properties with culture‐expansion. Bone marrow MSCs, annulus fibrosus, and nucleus pulposus cells were isolated from sheep and culture expanded. Over 20 days of expansion, all cell types grew in a steady exponential fashion for around 28 population doublings and expressed progenitor cell CD markers. Secreted exosomes were purified by ultracentrifugation and identified by transmission electron microscopy and western blot for exosomal proteins. The size and number of exosomes produced was analyzed by nanoparticle tracking analysis. All cell types secreted exosomes that were not significantly different in size or number. MSCs, annulus fibrosus and nucleus pulposus cells all took up exosomes from each cell type. This data confirms that manufacturing of functional exosomes is comparable among these cell types. Exosomal content is currently being analyzed to identify differences among cell types and includes total proteome by protein mass spectrometry, as well as RNA and mi‐RNA content through RNA sequencing.

Support or Funding Information

Colorado State University Orthopaedic Bioengineering Research Laboratory
Regulation of Airway Progenitor Homeostasis and Cell Composition by Tight Junction Protein Claudin‐18
Authors: Castaldi Alessandra , Arias Nataly , Pinson-Rose William , Samimi Kayla , Allen Alexa , Horie Masafumi , Li Changgong , Minoo Parviz , Zhou Beiyun , Stripp Barry , Ryan Amy L. , Borok Zea
Published: April 2020

Rationale

Claudin‐18 (Cldn18) is comprised of two isoforms, Cldn 18.1 and Cldn18.2. The lung‐specific isoform, Cldn18.1, regulates distal lung epithelial progenitor cell homeostasis. Its role in airway epithelium has not been elucidated, although Cldn18 expression is decreased in lungs of patients with chronic airway diseases (e.g., asthma and chronic obstructive pulmonary disease (COPD)). The airway epithelium is comprised of three progenitor populations, basal cells (BC), pulmonary neuroendocrine cells (PNEC) and club cells, and two non‐progenitor cell populations, namely goblet and ciliated cells. Club cells are secretory cells that are susceptible to naphthalene (NAP) injury. Club cell depletion occurs by day 3 post‐NAP, with spreading of ciliated cells to cover the denuded epithelial surface, and full reconstitution of the airway epithelium by day 14–21. Here we investigated the role of Cldn18.1 in airway epithelium using a previously developed global Cldn18 knockout (KO) mouse model.

Methods

Wild‐type (WT) and Cldn18 KO mice were injected intraperitoneally with NAP (225–275 mg/kg, corn oil as control). Lungs were harvested at baseline and 3‐days (maximal injury), 7‐days (beginning of regeneration) and 21‐days (full regeneration) post‐NAP, inflated with 4% paraformaldehyde and fixed overnight prior to embedding and sectioning. Hematoxylin and eosin, Periodic Acid‐Schiff (PAS) and immunofluorescence staining were performed.

Results

In WT mice, Cldn18.1 was expressed in a small subset of airway ciliated cells. Cldn18 KO mice showed increased numbers of pulmonary neuroendocrine cells and neuroendocrine bodies, as well as ectopic localization of club cells and localization of keratin 5‐positive cells within the distal parenchyma. At day 3 post‐NAP in WT mice, Cldn18.1 was transiently expressed in all the ciliated cells covering the denuded epithelium, returning to being expressed in only a subset of cells by day 7. Cldn18 KO mice subjected to injury unexpectedly developed marked goblet cell hyperplasia at day 14–21 post‐NAP, with cells expressing markers for both club (CC‐10) and goblet (MUC5AC) cells.

Conclusions

Our findings demonstrate an unexpected role for Cldn18.1 in regulating airway progenitor homeostasis and cell composition, implicating Cldn18.1 in regulation of downstream signaling pathways that inhibit goblet cell differentiation. A transient increase in the number of Cldn18.1‐positive ciliated cells in WT mice in the initial stages post‐injury suggests paracrine regulation of these inhibitory effects. Modulation of Cldn18 expression may be beneficial in the treatment of airway diseases characterized by goblet cell metaplasia.

Support or Funding Information

Funding: American Lung Association, Hastings Foundation, NIH
Optimizing the extraction and purification of maize‐produced Ag2 as a potential vaccine against Valley fever
Authors: Kaur Kamaljeet , Guzman Carlos , Le Carmen , Lagasca Dominique , Esquerra Raymond
Published: April 2020

Valley fever is an infection caused by inhaling spores of the fungal species Coccidioides. A proline rich protein, Ag2, from Coccidioides has been shown to induce an immune response against Valley fever. In this study, Ag2 was expressed in maize to be used as a candidate for a protein subunit vaccine. In order to achieve higher yields, we studied the impact of different detergents coupled with liquid nitrogen during the extraction process. The process was further optimized with downstream purification, resulting in a high‐yielding purified product. Our research showed that the grinding with liquid nitrogen in the presence of tween‐20 led to higher yields after purification.
Induced Pluripotent Stem Cell‐Derived Neuroprogenitor Cells: A Model for Studying Cannabinoid Induced Cell Signaling
Authors: Ryan Gabriella
Published: April 2020

Chronic pain is a growing epidemic and public health issue that continue to affect the aging population in the US. Managing pain is particularly difficult with patients who suffer from cancer‐associated pain, neuropathic pain, as well as central pain states. These conditions are typically treated inadequately with opioids, antidepressants, and anticonvulsive drugs. There is growing evidence that cannabinoids natural products are beneficial for a range of clinical conditions, including pain and inflammations. The use of cannabinoids instead of highly regulated prescription drugs is fast approaching and the development of such cannabinoid drugs requires well planned medical trials as well as very stringent guidelines for contaminants such as heavy metals, pesticides, mycotoxins, etc. However, federal guidelines have not kept up with this emerging industry. Currently, each state is left to manage their own set of regulations, resulting in an industry that has great uncertainty and chaos as it attempts to satisfy the growing demand for its products. A low‐cost cellular model to study the neurological effects of cannabinoids is challenging. Here we show that Neuroprognitor cells (NPs) derived from induced pluripotent stem cells (iPSCs) can be used as a model for studying pathways associated with pain and cannabinoids. NPs derived from iPSCs can be differentiated into multiple lineages of neurons and glial cells. These cells have the Cannabinoid Receptor 1 (CB1) present which, when activated by cannabinoids, inhibits the inflammatory and pain stimulation of Cyclooxygenase 2 (COX2). We show expression and activation of CB1 in correlation to Cannabinoids treatment. Likewise we show how, in iPSC‐derived NPs Cannabinoid treatment leads to the inhibition of Cox‐2, therefore theoretically lessoning the potential for inflammation and pain.

Support or Funding Information

Omni International, Inc

935 Cobb Place Blvd

Kennesaw Ga 30144
A Renal Potassium‐Switch Prioritizes Dietary Potassium Over Sodium, Driving Salt‐Sensitive Hypertension
Authors: Grimm P. Richard , Delpire Eric , Welling Paul A.
Published: April 2020

Reducing dietary salt (NaCl) is well appreciated to lower blood pressure (BP), but a growing body of evidence indicates that increasing dietary potassium (K+) intake plays an equally important role. A ‘renal potassium switch’ that turns on the thiazide‐sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) in response to low dietary potassium intake and off in response to high potassium intake has been implicated.

Here we test the idea in genetically engineered mice, containing a DCT1‐specific, conditionally active SPAK (CA‐SPAK mice) which phospho‐activates NCC, and locks the switch “on.” BP responses to small, physiologic changes in plasma [K+] (P[K+]) in CA‐SPAK mice were compared to control mice. Dietary potassium content was varied to acutely titrate P[K+] over a physiologic range (3.7mM (LK), 4.4mM (MK), and 5.1mM (HK)). Blood pressure was monitored by telemetry at each P[K+] level in mice consuming control or high salt diet (HNa). At the end of each dietary treatment, the BP response to hydrochlorothiazide (HCTZ) was measured to assess the contribution of NCC to BP.

BP decreased by ~10 mmHg when P[K+] increased from 3.7 to 5.1 mM in control mice, coincident with inactivation of NCC. When the switch was on (LK and MK groups), HNa significantly elevated BP but had no effect when the switch was inactivated by HK. HCTZ significantly reduced BP in the LK/HNa and MK/HNa groups but had no effect on BP in the HK/HNa group, further supporting the idea that low potassium‐dependent activation NCC exacerbates the effects of sodium. Studies in CA‐SPAK mice reveal a causal relationship between switch activation and BP responses to sodium and potassium. In contrast to control mice, increasing P[K+] in CA‐SPAK mice had no effect on BP under control salt conditions and failed to blunt the significant hypertensive effects of HNa. HCTZ significantly decreased BP in all CA‐SPAK groups to near control levels, consistent with NCC‐driven salt reabsorption. Thus, locking on the potassium switch prevents the anti‐hypertensive effects of HK. No sex differences were found.

In summary, low K+ consumption, common in modern diets, presses the switch pathway to turn on to conserve K+ at the expense of increasing sodium retention, even in the face of high dietary sodium, and this elevates BP. Thus, switch activation can drive salt‐sensitive hypertension.

Support or Funding Information

NIH
Complete Absence of Oblique Fissure in the Left Lung: A case report on anatomic variation
Authors: Anyadike Kimberly , Cascardo Camilla , Hoevet Mallory , Idris Mohamed , Alakhras Hazem , Barremkala Malli
Published: April 2020

Classically, development of respiratory tissues in humans displays segmentation which produces a left lung with two lobes, separated by the oblique fissure. The bilobar left lung is smaller than the right lung as a standard, allowing space for the heart. However, absence of oblique fissure in the left lung, as is discussed in this abstract, is anomalous. 24 pairs of lungs obtained from routine dissection of adult formalin fixed cadavers used for first‐year regional anatomy course at Oakland University William Beaumont School of Medicine were used in this study. There was a total of 13 female and 11 male and cadavers with an average age of 80 years. The lungs were observed for presence or absence of fissures and lobes. We report a case of absent oblique fissure in the left lung in a 56‐year‐old female cadaver. While hypoplasia of the left lung is apparent, her right lung displayed standard trilobar development. Although no other cadavers within this cohort displayed similar lobar hypoplasia of the lung, our team did find that the left lung from a 74‐year‐old female cadaver was notably small. Developmental lung anomalies in adults are rare, and our findings contribute to prevalence reports of left lung hypoplasia. Recognition of variation in bronchopulmonary fissures may be useful in clinical settings, i.e., in localization of pulmonary lesions, surgical planning, planning for other invasive respiratory procedures, and interpretation of radiographs or computed tomography scans.
Rational design, synthesis and evaluation of “smart”, uncharged bis‐oxime antidotes of OP inhibited human acetylcholinesterase
Authors: Radic Zoran , Gorecki Lukas , Gerlits Oksana , Kong Xiaotian , Cheng Xiaolin , Blumenthal Donald , Taylor Palmer , Ballatore Carlo , Kovalevsky Andrey
Published: April 2020

Organophosphate (OP) intoxications from nerve agent and OP pesticide exposure are treated by outdated, questionably efficient pyridinium aldoxime‐based therapies. Pyridinium cations hamper central nervous system (CNS) uptake, while structural diversity of OPs prevents efficient reactivation of OP inhibited acetylcholinesterase (AChE). Improvements must include much better reactivation of AChE in the CNS. We present here seven novel uncharged bis‐oximes created from our X‐ray structures of an uncharged monoxime, RS194B, reversibly bound to human AChE (hAChE). Bis‐oxime protonation results in equilibration between up to sixteen distinct ionization forms, including uncharged forms capable of diffusing into CNS and multiple zwitterionic forms optimal for reactivation reactions. Conformationally diverse zwitterions acting as structural variants of the compound significantly improved in vitro reactivation of diverse OP‐hAChEs. The enhanced reactivation orientation for one of the bis‐oximes was confirmed by X‐ray structure. Our novel, CNS directed bis‐oximes represent a promising class of “smart”, accelerated antidotes against OP intoxication.

Support or Funding Information

This research was supported by the CounterACT Program, National Institutes of Health Office of the Director (NIH OD), and the National Institute of Neurological Disorders and Stroke (NINDS), [Grant Numbers U01 NS083451 and R21 NS098998] and by the UCSD Academic Senate grant BG084144.
Localization and changes of tyrosine phosphorylated protein expressions in female reproductive tracts of PCOS rats
Authors: Bunsueb Sudtida , Arun Supatcharee , Iamsaard Sitthichai
Published: April 2020

Polycystic ovary syndrome (PCOS) is a cause of female infertility and its mechanism is still unclear. Tyrosine phosphorylated (TyrPho) proteins have been shown to be important for male infertility. However, the biological functions of TyrPho proteins involved in PCOS condition have never been reported. This study aimed to localize and investigate the TyrPho protein expression in PCOS rats. Twenty female Sprague‐Dawley rats were divided into control and PCOS (induced by letrozole, 1 mg/KgBW, for 21 consecutive days) groups. The estrus cycle of animals was confirmed by vaginal smear daily. At the end of experiment, ovary, oviduct, and uterus tissues of both groups were localized for TyrPho proteins by immunohistochemistry. In addition, the expression of TyrPho proteins in reproductive tissues were investigated and quantified. The results showed that TyrPho proteins were localized in the developing ovarian follicles and epithelial cells of oviduct and uterus. Moreover, TyrPho proteins were significantly overexpressed in ovary (68, 52, 42 and 28 kDas), oviduct (56 and 42 kDa), and uterus (170, 53 and 42 kDas) of PCOS group as compared to control. We concluded that female reproductive tracts have tyrosine phosphorylation essential for female fertility.

Support or Funding Information

Khon Kaen University
Brown adipose tissue mediates cardiac function via release of 12,13‐diHOME
Authors: Stanford Kristin , Pinckard Kelsey , Shettigar Vikram , Baer Lisa , Ziolo Mark
Published: April 2020

Brown adipose tissue (BAT) increases energy expenditure and decreases plasma glucose and lipids, making it a potential target to decrease the risks of obesity, type 2 diabetes, and cardiovascular disease (CVD). BAT functions in an endocrine and autocrine manner to improve whole‐body metabolism, however the role of BAT in the cardiovascular system is unknown. Here, we determined that transplantation of BAT (+BAT) increases in vivo cardiac hemodynamics. Lipidomics analysis revealed that the lipokine most up‐regulated in +BAT mice was 12,13‐diHOME. 12,13‐diHOME acutely increases cardiac hemodynamics via direct effects on the cardiomyocyte. Furthermore, incubation of cardiomyocytes with 12,13‐diHOME increased mitochondrial respiration via the ryanodine receptor. The effects of 12,13‐diHOME were absent in NOS1−/− mice and cardiomyocytes. We found that 12,13‐diHOME is decreased in human patients with heart disease, and this was correlated with decreased ejection fraction. Our results suggest an endocrine role for BAT to enhance cardiac function that is mediated by regulation of calcium cycling via 12,13‐diHOME and NOS1.

Support or Funding Information

R01‐HL138738, R01‐AG060542
Downregulation of HMGB1 is required for the protective role of Nrf2 in EMT‐mediated PF
Authors: Gao Jian
Published: April 2020

Aim

Pulmonary fibrosis (PF) is an interstitial lung disease characterized by the activation of accumulated myofibroblasts and deposition of extracellular matrix (ECM). Epithelial‐mesenchymal transition (EMT) is considered to be one of the major hypotheses in the formation of PF. High mobility group box1 (HMGB1) plays an important biological role in infection, inflammation, and immune responses. Nuclear factor E2‐related factor 2(Nrf2) is an important transcription factor for the regulation of oxidative stress. However, there are no direct evidences regarding the relationship between Nrf2 and HMGB1 in PF.

Methods

PF animal model was established by intratracheal injection of bleomycin. Histological morphology of the lungs were reflected by H&E staining and Masson staining. The expressions of Nrf2, HMGB1 and EMT associated markers were determined by Western blot or immunohistochemistry. In vitro, the effect of Nrf2, HMGB1 and the relationship between them in TGF‐β1‐induced EMT were determined by western blot , coupled using respective pharmacological activators or transfection with siRNA.

Results

EMT was aggravated and the expression of HMGB1 was significantly elevated in Nrf2−/− mice compared with WT mice exposed to BLM. Moreover, the uptake of exogenous Nrf2 or ablation of HMGB1 ameliorated TGF‐β1‐induced EMT in rat type II alveolar epithelial cell line (RLE‐6TN) and human alveolar epithelial cell line(A549). In contrast, Nrf2 deficiency or HMGB1 activation aggravated TGF‐β1‐induced EMT. Furthermore, inhibition of HMGB1 diminished the protective effect of Nrf2 on EMT.

Conclusion

These findings suggest that the inhibitory effect of HMGB1 on EMT in PF is regulated by Nrf2 and provides a new strategy of clinical treatment for PF.

Support or Funding Information

This work was supported by the National Natural Science Foundation of China( No.: 8197141254, 81274172, 81473267, 81503330 and 81503129 ).
Extract of Adenopus breviflorus Fruit Alters the Expressions of Hepatic Cytochrome C, Caspase 3 and Sperm Parameters in Rats
Authors: Oyedeji Tolulope Adejoke , Olorunsogo Olufunso O.
Published: April 2020

Adenopus breviflorus fruit is used in traditional medicine against the outbreak of measles and the treatment of gonorrhoea in men. The fruit has been shown to have anti‐implantation activity. However, the safety of the aqueous extract of Adenopus breviflorus (AEAB) fruit on the function of the liver and male reproduction has not been studied. Therefore, this study investigated the effect of the oral administration of the AEAB fruit on rat liver function enzymes, cytochrome c release, caspase 3 expression and male reproductive functions. Adult male rats (15) were randomly divided into three groups of 5 animals per group. Animals in group 1 received distilled water while animals in groups 2 and 3 received an oral dose of 200 mg/kg and 400 mg/kg of AEAB fruit for 30 days, respectively. Alanine transaminase, alkaline phosphatase and aspartate transaminase enzyme activities were assayed spectrophotometrically. Cytochrome c and caspase 3 protein expressions in rat liver tissues were determined using immunohistochemistry. Sperm abnormality, sperm count and sperm motility were also determined. The result obtained showed that there was no significant (p < 0.05) difference in serum alanine transaminase, alkaline phosphatase and aspartate transaminase between the treated groups and the control. Immunohistochemistry showed that cytochrome c release and caspase 3 proteins were significantly (p < 0.05) expressed in the liver of animals in the treated groups when compared to the control. Furthermore, sperm abnormality significantly (p < 0.05) increased while sperm count of rats orally exposed to AEAB fruit were significantly (p < 0.05) reduced in the treated groups when compared to the control group. Specifically, sperm motility was significantly (p < 0.05) reduced only in group 3 when compared with the control. This study show that the aqueous extract of Adenopus breviflorus fruit may not be hepatotoxic. However, this extract may have the potential to activate mitochondria cell death through cytochrome c release and activation of caspase 3 in rat liver. Furthermore, our study revealed that the extract may cause reproductive dysfunction in men. Therefore, the use of the extract in the treatment of gonorrhoea in traditional medicine should be carried out with utmost care.
Analyzing Nitrogenase’s Regulator ‐ NifA’s Interaction with DNA
Authors: Standke Heidi , Kim Lois , Medina Michael , Owens Cedric
Published: April 2020

The bacterium Gluconacetobacter diazotrophicus contains an enzyme called nitrogenase which converts dinitrogen gas to ammonia ‐ an essential plant nutrient. The breakdown of dinitrogen has a high energy demand, utilizing 16 ATPs and 16 reducing equivalents. For this reason, nitrogenase is highly regulated. Regulation of nitrogenase is controlled by the protein NifA, a sigma‐54‐activator. NifA is a transcriptional activator that binds to DNA through its DNA binding domain (DBD) to activate nitrogen fixation genes when G. diazotrophicus needs ammonia. NifA also regulates nitrogen fixation depending on whether sufficient redox equivalents are present to support nitrogenase. However, while the role of NifA is known, its mechanism is not fully understood. It is not known if NifA senses oxygen or if it directly senses reducing equivalents. There are two cysteines at the start of NifA’s DNA binding domain. Cysteines are redox active and oxidize to form disulfide bonds which are known to affect protein activity. It is unknown if these cysteines sense oxygen and/or redox conditions and if this potential sensing mechanism affects NifA’s DNA binding. Here, we determine the functional importance of the two cysteines by investigating their redox and O2 sensing activity, and how the redox state of the cysteines influences NifA’s interaction with DNA. To test the hypothesis that NifA’s DNA binding is regulated by the redox state of the cysteines, two constructs of NifA’s DBD were made: one truncated version containing neither cysteine (No‐Cys) and another containing both cysteines (2‐Cys). Both the No‐Cys and 2‐Cys constructs were cloned into expression plasmids, expressed heterologously in E. coli, purified, and functionally examined through DNA binding assays and biophysical methods. We hypothesized that if NifA is a redox/O2 sensor, it will only be active when both cysteines are reduced. Our current results indicate that oxidized conditions may be required for proper DNA binding, resulting in Kd values in the lower micromolar range. Experiments are currently underway to conclusively determine the redox state of the cysteines and the structure of NifA’s DNA binding domain.

Support or Funding Information

Chapman University's Undergraduate Student Scholarly Research/Creative Grant

Chapman University's Summer Undergraduate Research Fellowship (SURF)
Calcium Homeostasis Modulator (CALHM1/2) and Pulmonary Arterial Hypertension
Authors: Rodriguez Marisela , Babicheva Aleksandra , Xiong Mingmei , Jain Pritesh P. , Zhao Tengteng , Lai Ning , Rahimi Shamin , Balistrieri Francesca , Hernandez Moises , Thistlethwaite Patricia A. , Makino Ayako , Yuan Jason X.-J.
Published: April 2020

RATIONALE

Concentric pulmonary vascular remodeling is a major cause for the elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in patients with pulmonary arterial hypertension (PAH). Excessive PASMC proliferation and inhibited PASMC apoptosis have been implicated in the development and progression of pulmonary vascular wall thickening in PAH. Transition from the contractile phenotype to the synthetic or proliferative phenotype of PASMC is thus an important pathogenic process that promotes vascular remodeling. An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in PASMC is not only a trigger for PASMC contraction (and pulmonary vasoconstriction), but also an important stimulus for PASMC proliferation. Calcium homeostasis modulators (CALHM), CALHM1/2, are membrane Ca2+ channels that are allosterically regulated by voltage and extracellular Ca2+. CALHM1/2 are closed at the resting membrane potential but can be opened by strong membrane depolarization. Reduction of extracellular [Ca2+] increases CALHM open probability, which allows the channel to be activated at negative potential. We aimed to study whether CALHM1/2 are involved in the phenotypical transition of PASMC and whether CALHM1/2 are upregulated in highly proliferative PASMC from patients with PAH.

METHODS

Freshly isolated pulmonary artery with the endothelium removed (as contractile PASMC) and primary cultured PASMC (as synthetic or proliferative PASMC) from rats were used for this study. Western blot analysis was used to compare protein expression levels of CALHM1 and CALHM2 in fresh PA tissue and cultured PASMC derived from the same rats. In addition, we compared protein expression levels of CALHM1/2 in PASMC from normal subjects and PAH patients.

RESULTS

The protein expression level of CALHM2 in proliferative PASMC (the primary cultured PASMC in growth factor‐containing medium) was significantly higher than in contractile PASMC (the freshly isolated PA tissue). The increased CALHM2 in proliferative PASMC was indeed associated with a significantly increased expression of PCNA, a cell proliferation marker, and a significantly decreased expression of MHC and TGLN4, markers for contractile phenotype of SMC. Furthermore, we also observed that mRNA expression level of CALHM2 was significantly higher in PASMC isolated from PAH patients than in PASMC from normal subjects.

CONCLUSION

Upregulation of CALHM2 may play an important role in the phenotypical transition of PASMC from the contractile phenotype to the synthetic or proliferative phenotype. Ca2+ influx through voltage‐ and extracellular Ca2+‐regulated CALHM2 in the plasma membrane of PASMC is an important trigger for PASMC proliferation and may play an important role in stimulating PASMC proliferation in patients with PAH.

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NIH
Deciphering the role of a unique methyltransferase in Mycobacterial infection
Authors: Rani Anshu , Tripathi Prajna , Manjunath P. , Hasnain Seyed Ehtesham , Ehtesham Nasreen Z.
Published: April 2020

Tuberculosis is a life threatening disease caused by the intracellular pathogen Mycobacterium tuberculosis (Mtb), resulting in around two million deaths annually worldwide. Mtb is known to have acquired certain features that help in establishing infection and persisting for longer durations in the host. Recent studies have revealed the acquisition of certain unique methyltransferases by Mtb as a function of phylogenetic evolution which points towards their potential involvement in mycobacterial pathogenesis. In the present study, we identified one such hypothetical methyltransferase (Rvxxxx) gene unique to Mtb complex i.e., absent in non‐pathogenic Mycobacteria. Our in‐silico analysis revealed that Rvxxxx gene possesses an S‐adenosylmethionine binding site and a methyltransferase domain, which was further confirmed through in vitro studies. The introduction of Rvxxxx in the genome of Mycobacterium smegmatis lead to significant perturbations in the physiological and morphological features of the bacilli as well as markedly enhanced its ability to resist and adapt to hostile conditions. Furthermore, recombinant M. smegmatis showed increase in infectivity and survival within host macrophages ex vivo. In addition, Rvxxxx was also found to modulate the proteome profile of recombinant M. smegmatis. Cumulatively, the results of this study indicate that the identified hypothetical methyltransferase Rvxxxx may play a significant role in Mtb pathogenesis and niche adaptation.

Support or Funding Information

The Council of Scientific and Industrial Research, Govt. of India
Mechanisms of Transcriptional Regulation by an ER Stress‐Responsive Enhancer
Authors: He Yanfeng , Tomomori-Sato Chieri , Sato Shigeo , Conaway Joan W , Conaway Ronald C
Published: April 2020

ATF6α is a transcription factor that functions as a master regulator of many ER stress‐responsive target genes. HSPA5 (also known as Grp78) is an ATF6α‐targeted ER‐stress responsive genes. It contains 3 tandemly repeated ER stress response elements (ERSE). The ERSE enhancer‐like sites are very close to the core promoter, making it an excellent model for studies of enhancer function using a natural promoter with its regulatory regions. Previous studies have shown that a collection of transcription factors contribute to transcriptional regulation via the HSPA5 ERSE region; however, little is known about how these transcription factors collaborate to drive activation. To gain insight into this question, we have established an in vitro system that recapitulates ATF6α‐dependent transcriptional activation with purified RNA polymerase II, Mediator, and transcription factors. An unexpected finding from this work is our discovery that Elongin, best known as an RNA polymerase II elongation factor, plays an important role in ATF6α and NF‐Y dependent activation of the HSPA5 promoter. Mechanistic studies suggest that Elongin functions with TFIID and Mediator at an early stage in assembly of active initiation complexes. These results provide a foundation for future efforts to dissect the interplay of ERSE‐binding transcription factors and coregulators in HSPA5 activation.

Support or Funding Information

This work was supported in part by a grant to the Stowers Institute for Medical Research from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation.
Nanoscale‐Organization of Key Molecular Complexes Differentially Alter Vascular Smooth Muscle Function in Male and Females
Authors: Matsumoto Collin , Tajada Sendoa , Santana Luis Fernando
Published: April 2020

Arterial smooth muscle contraction is the result of both electro‐mechanical and pharmaco‐mechanical coupling. During electro‐mechanical coupling, membrane depolarization activates dihydropyridine‐sensitive CaV1.2 channels, raising intracellular calcium concentrations ([Ca2+]i) and results in contraction of arterial smooth muscle. Pharmaco‐mechanical coupling activation of G‐protein coupled receptors (GPRCs) by angiotensin II (AngII) produces inositol 1,4,5‐triphosphate (IP3) and protein kinase C (PKC) activated diacylglycerol (DAG). This leads to an increase in [Ca2+]i via sarcoplasmic reticulum IP3 receptors and PKC dependent Ca2+ channels in the sarcolemma. It is known that male and females respond differently to antihypertensive drugs. However, it is unknown whether the nano‐scale organization of key molecular complexes between sexes play a role in smooth muscle physiology. Our data show that Ca2+ influx through CaV1.2 channels is larger in female than in male arterial myocytes. Furthermore, male smooth muscle responds greater to GPCR activation by AngII than female. We observed sex‐specific nano‐organization of CaV1.2 and AngII related signaling proteins angiotensin II receptor type 1 (AngIIR1), AKAP150 and PKC in arterial smooth muscle. We propose that there are sex‐based differences in smooth muscle physiology that are a result of different nano‐organization of signaling complexes.

Support or Funding Information

T32 HL086350
Role of Dopamine D2 Receptor in the Leptin Receptor‐expressing Neurons in Modulation of Compulsive‐like Eating Behavior
Authors: Go Bok Soon , Kim Wanyeon
Published: April 2020

Leptin, a hormone secreted by adipose tissue, is known to modulate satiety, caloric intake and body weight. There are six alternative spliced isoforms of leptin receptors (LepRa‐f). LepRb is highly expressed in the hypothalamic arcuate nucleus (ARC) which is involved in the regulation of food intake and body weight. Leptin activates pro‐opiomelanocortin‐expressing (POMC) neurons and inhibits agouti‐related peptide (AgRP)‐expressing neurons in the ARC.A recent study shows that dopamine modulates neuronal activities of arcuate POMC neurons and AgRP neurons. Dopamine inhibits arcuate POMC neurons via dopamine D2 receptor but activates AgRP neurons through dopamine D1 receptor. Dopamine is a neurotransmitter that plays a key role in mood, motivation and satiety. Dysregulation of dopamine neurotransmission is involved in the drug addiction. Food addiction is a recently proposed concept and compulsive eating behavior is shown in food addiction as well as an obesity. Negative reinforcement in drug addiction is a concept that negative affect associated with drug withdrawal promotes drug taking to ‘self‐medicate’ or remove those negative states. In the present study, we generated mice lacking D2 receptor in cells expressing LepRb (D2R flox; LepRb‐Cre; D2R cKO). Mice were provided palatable food for more than two weeks to induce food‐addiction like phenotypes in mice. To examine the role of D2 receptor in the leptin receptor‐expressing neurons in modulation of compulsive‐like eating behavior, light‐dark conflict test (LD test) was performed during the palatable food withdrawal period. In addition, palatable food withdrawal‐mediated anxiety/depression‐like behavior were measured during marble burying test (MBT), elevated plus maze (EPM), open filed test (OFT), and forced swim test (FST). No significant changes were found in palatable food (PF) intake in the light box during LD test, suggesting that D2 receptor in cells expressing LepRb would not be involved in the regulation of compulsive eating behavior. Furthermore, there was no significant changes in anxiety/depression‐like behavior measured by MBT, EPM, OPT and FST during the PF withdrawal period.

Support or Funding Information

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2019R1I1A1A01060007).
Expansion of NK cells by co‐crosslinking of 4‐1BBL and IL15R
Authors: Kim Jongil
Published: April 2020

Adaptive transfer therapy using NK cell has been widely applied for cancer treatment. However, previous strategies on expansion and activation of NK cells is unsuitable for clinical application, because previous expansion strategies require feeder cells. Here, we established a method based on enhancement of NK cell function using removable magnetic protein G beads. We developed the fusion proteins such as 4‐1BB/igG1Fc and IL‐15Rα/IgG1Fc, then attached to magnetic protein G beads. The effect of immobilized 4‐1BB/IgG1Fc and IL‐15Rα/IgG1Fc on NK cell was assessed. We could confirm that fusion protein titration, proliferation, cytotoxicity, expression of activating receptors and IFN‐γ in NK cells were increased by immobilized fusion proteins on day 12. In summary, these results suggest that NK cells stimulated with bead platform can be efficiently without feeder cell, and appropriate for clinical safety. These bead platforms can be applied to a variety of diseases and cell types by changing the molecules to attach to the beads

Support or Funding Information

Prof. KyuBum KwackDepartment of Biomedical Science, College of Life Science, CHA University, Seongnam‐si, Gyeonggi‐do, Republic of Korea.
Glutathione peroxidase‐1 regulates adhesion and metastasis of triple‐negative breast cancer cells via FAK signaling
Authors: Lee Eun Kyung
Published: April 2020

Triple‐negative breast cancer (TNBC) cells, which do not express genes for estrogen receptor (ER), progesterone receptor (PR), and Her2/neu, develop highly aggressive and metastatic tumors resistant to chemo‐ and hormonal therapies. We found that expression of glutathione peroxidase‐1 (Gpx1) is silenced in the non‐TNBC cells but resumed in the TNBC cell lines. Such restored Gpx1 expression plays a vital role in the metastasis of TNBC cells by regulating cell adhesion. Transcriptomic and signaling pathway analyses demonstrate that depletion of Gpx1 essentially impairs cell adhesion/spreading by down‐regulating FAK/c‐Src activation. Mechanistically, the substrate of Gpx1, H2O2, directly inhibits the FAK kinase activity. As a result, depletion of Gpx1 greatly suppresses lung metastasis of TNBC cells in vivo. Overall, our study identifies that Gpx1 is a redox safeguard of FAK kinase and allows for a therapeutic process to effectively control the metastasis of deadly malignant TNBC.

Support or Funding Information

National Research Foundation of Korea (2018R1A2B3006323 and 2017M3A9B6073098)

National R&D Program for Cancer Control (1420280).