bluuebunny/crossref_metadata_2025 huggingface dataset:
The Paradox of Fragile but Dense Bones in Type 2 Diabetes
Authors: Donnelly Eve
Published: April 2020

Epidemiologic evidence from the past decade has demonstrated that increased bone fracture risk is one of the myriad clinical complications of type 2 diabetes mellitus (T2DM). Counterintuitively, the increased fracture risk in T2DM occurs despite greater bone mineral density, and persists after accounting for potential confounders like body mass index, neuropathies, and falls. Therefore, T2DM may alter aspects of bone’s intrinsic resistance to fracture beyond bone mineral density, such as material properties or microarchitecture, thereby increasing bone fragility independently of bone mass. Although the mechanisms that underlie bone fragility in T2DM are not yet well established, a multiplicity of factors, including hyperglycemia, altered bone remodeling, and non‐enzymatic collagen crosslinking in the bone matrix are implicated. In this work we elucidate the factors that influence fragility in T2DM by characterizing the biochemical, material, microarchitectural, and mechanical properties of bone from a clinical population of men with and without T2DM. The cancellous bone from patients with T2DM had greater concentrations of the advanced glycation endproduct pentosidine and sugars bound to the collagen matrix, had greater mineral content, and trended toward a greater bone volume fraction than the non‐DM specimens. Surprisingly, the T2DM specimens had similar strength and toughness as those from non‐DM controls. Regression modeling of the mechanical outcomes revealed competing effects of T2DM on bone mechanical behavior. The trend toward greater bone volume fraction and greater mineral content of the T2DM specimens increased strength, while the greater concentrations of pentosidine in the T2DM group increased brittleness. These data suggest that high concentrations of AGEs can increase fragility by reducing the ability of bone to absorb energy prior to failure, especially for the subset of T2DM patients with low bone volume fraction. Further, our model shows that an increase in bone volume fraction offers a protective effect for T2DM patients, even with the inferior intrinsic quality of the T2DM bone, whereas loss of bone volume fraction substantially increases risk of bone fragility for T2DM patients. The primary clinical implications of these findings are that preservation of bone volume fraction is critical in patients with T2DM.

Support or Funding Information

Supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (K01 AR064314)
Integrated analysis of bioelectric signaling in regulation of proportion.
Authors: Harris Matthew
Published: April 2020

We are proportionate entities: the mechanical and physiological function of our bodies is dependent on allometric scaling between tissues and organs. How such scaling is genetically and developmentally regulated remains a long lasting question and requires knowledge at many levels and across disciplines. Using unbiased forward genetic screens in the zebrafish, we identify mutations in potassium channels that lead to increase in relative fin size. We assess how these mutations affect electrophysiological properties of the encoded potassium channels in Xenopus oocytes. Using the zebrafish fin as an experimental platform we detail key regulation of bioelectric signaling and its control of coordinated growth during development and regeneration. Complementing these experimental approaches, we extend our investigation through a broad scale phylogenomic analysis capitalizing on natural variation in fin length in evolution as well in clinical cases of coordinated overgrowth of extremities. The combination of approaches provides mechanistic insight into bioelectric regulation of coordinated growth during development and regeneration and the prevalence of bioelectric signaling in regulating form.

Support or Funding Information

NIH R01HD084985
Link between miRNA‐19b and mTOR signaling pathway in cancer prognosis: from meta‐analysis to mechanism exploration
Authors: MAMAN SOURAKA TAPARA DRAMANI
Published: April 2020

AIMS

Previous studies came to different conclusions regarding the relationship between miR‐19b and its prognostic value in cancers. Moreover, miR‐19b could affect tumor growth by different pathways mainly targeting PTEN‐PI3K‐AKT which activate mTOR pathway in the downstream. Therefore, we conducted this meta‐analysis to explore the possible correlation between miR‐19b and mTOR in cancers prognosis.

Main methods

We conducted online search and collected a total of 943 articles. According to different authors cross check and our study including/excluding criteria we at end retained 21 articles with 25 studies in this meta‐analysis. Then TCGA data containing miR‐19b level with cancer progression were obtained using OncomiR. Furthermore, Trial Sequential Analysis (TSA) was performed to determine whether the results of our meta‐analysis could be used in clinical applications. After that, articles regarding the mechanism of miR‐19b in various cancers were analyzed and KEGG pathway database was used to find the main regulatory function of miR‐19b in human cancers.

Key Findings

Overall hazard ratio results displayed that higher expression of miR‐19b was correlated with shorter overall survival (HRs=1.54, 95%CIs=1.20–1.98) by promoting distant metastasis, but had no relation with disease‐free survival/progression‐free survival (HRs=0.61, 95%CIs=0.31–1.19). TCGA datasets also revealed the tumorigenesis role of miR‐19b. According to TSA results, more evidence are needed to support that miR‐19b had no correlation with DFS/PFS. Exploration of the mechanism revealed the possible link between miR‐19b and mTOR pathway.

Significance

MiR‐19b might play its pro‐cancer role through mTOR pathway and it is likely to be a therapeutic target for cancers.

Support or Funding Information

The study was supported by Zhongnan Hospital of Wuhan University Science and Technology, Innovation and Education Fund [Project cxpy2018067] and Zhongnan Hospital of Wuhan University Science and Technology, Innovation and Education Fund [Project ZNPY2017054].
Picture and Word Recall Skills as a Measure of Working Memory among Undergraduate Students of Bingham University, Nigeria
Authors: Danborno Angela Member , Yoila Lucky Yusuf
Published: April 2020

Working memory is a form of short‐term memory that is involved in keeping track, manipulation and processing of many bits of information used in cognitive activities such as thinking and decision making. It is also implicated in behavior and personality, and is usually terminated after the task has been completed. This study was designed with the purpose of assessing the working memory in male and female undergraduate students with the view of investigating the differences in memory recall as indicative of evolutionary attributes. Undergraduate students (Male n= 64 and females n= 146) of Bingham University, Karu, Nigeria participated in the study. Questionnaire were administered and the working memory ability of the participants were tested through picture and word recall and placement tests modified from Jordan et al. (2001). Sex differences in working memory performance was examined, and scores relationship between 2D:4D ratio as a measure of memory performance was also examined. Our results showed that females students outperformed the male students in all the memory tasks: picture recall (males=7.11±1.61, females=7.70±1.39, t= −2.702, P=0.007), picture placement (5.77±1.79, 6.77±1.85, t= −3.641, P<0.001), word recall (males=7.42±1.46, females=8.00±1.38, t= −2.754, P=0.006) and word placement (males= 6.34±2.07, females=7.08±1.88, t= −2.545, P=0.012). In conclusion, our findings indicate a high memory performance skill by female more than their male counterparts, this supports the evolutionary theory that women are better in memory recall than men.
Inflammation due to Chronic Alcohol Exposure Promotes Cardiac Atrophy and Dysfunction Resulting from Impaired Fibroblast‐Myocyte Crosstalk
Authors: El Hajj Elia C. , Mouton Alan J. , Ninh Van K. , Gardner Jason D.
Published: April 2020

Alcoholic cardiomyopathy (ACM) is a cardiac disease caused by chronic heavy alcohol abuse. ACM is most notably a type of dilated cardiomyopathy (DCM), accounting for approximately 21–50% of all idiopathic non‐ischemic DCM cases in Western countries. ACM is typically characterized by left ventricular dilation and LV muscle wasting (atrophy), with diastolic and systolic dysfunction (ejection fraction < 40%) occurring in later stages. Previous studies in our lab have shown that chronic alcohol exposure prevents compensatory hypertrophy in both volume‐ and pressure‐overload models of heart failure. However, the mechanisms of alcohol‐induced cardiac atrophy and dysfunction are not clear. The purpose of this study was to investigate mechanisms of chronic alcohol‐induced cardiac remodeling and dysfunction. We also sought to determine the role of cardiac fibroblasts, which play a dynamic role in cardiac remodeling, in mediating these effects.

Adult male Wistar rats were exposed to ethanol (EtOH) vapor inhalation for 16 weeks. Echocardiography was performed to assess terminal cardiac structure and function. Cardiac fibroblasts were isolated from the left ventricle (LV) for both ex vivo and in vitro analysis. Gene expression was determined by real‐time PCR and flow cytometry. Protein expression was determined via Western blot analysis. EtOH led to cardiac wall thinning and impaired systolic function, as evident by decreased LV wall thickness at diastole (LVPWd) and fractional shortening, respectively, Additionally, EtOH decreased mRNA levels of various contractile proteins including Acta1, Actc1, and Myh7. EtOH also increased mRNA and protein levels of LV inflammatory markers, TNF‐α and NF‐κB. Furthermore, EtOH induced increased activation of JNK and decreased mTOR activation, suggestive of decreased hypertrophic signaling and atrophy. EtOH led to myofibroblast activation as assessed by flow cytometry and led to ECM remodeling (increased collagen III content). EtOH increased Tnf and Il6 mRNA in cardiac fibroblasts ex vivo, and increased mRNA levels of several pro‐inflammatory cytokines and chemokines in vitro. Administration of conditioned media from EtOH‐treated fibroblasts decreased contractile protein mRNA levels and impaired Akt and mTOR signaling in differentiated H9C2 cardiomyocytes.

Our results indicate that EtOH‐induced cardiac atrophy and dysfunction is associated with activation of inflammatory pathways. Furthermore, EtOH may induce a pro‐inflammatory cardiac fibroblast phenotype, leading to aberrant fibroblast‐myocyte cross‐talk.

Support or Funding Information

Funding provided by the NIH/NIAAA 1R21AA022690 (JDG) and the Saving tiny Hearts Society (JDG).
In Utero Exposure of Atrazine and its effects on Chronobiology and Gene Expression of Glutathione S‐Transferase
Authors: Bracken Zachary Kenneth
Published: April 2020

Atrazine is a common herbicide that has been implicated in multiple health issues. The enzyme that degrades atrazine is Glutathione S‐Transferase (GST). GST is a detoxifying enzyme present in high concentrations in the liver; the GST isoenzyme pi is specifically responsible for the breakdown of atrazine. Previous research showed decreased expression of GST when mice are exposed to atrazine. Studies have also shown that there is circadian regulation of GST. This study investigated the effect atrazine had on circadian rhythm of GST by utilizing CD‐1 mice exposed in utero to atrazine. The results showed that atrazine inhibited the circadian rhythm of not just GST, but also Period 1, a circadian regulating protein.
Modification of the Endoplasmic Reticulum Translocon Impairs Protein Translocation
Authors: Richards Kyle A. , Runnebohm Avery , Dolan Molly , Vitali Halie , Rubenstein Eric
Published: April 2020

The endoplasmic reticulum (ER) is the entry point for most proteins residing and functioning in the eukaryotic endomembrane system. The primary mechanism by which proteins enter the ER is via the translocon complex. Dysfunction in this complex can block access into the ER, which is detrimental to cellular health. The translocon is highly conserved and has been intensely studied in Saccharomyces cerevisiae. Analysis of translocon function in protein trafficking, localization, and interactions in yeast has been facilitated by the use of epitope tags. We have found that a tag on the translocon pore subunit previously suggested not to impair translocon function subtly affects translocation of proteins into the ER in yeast cells. Intriguingly, this tag also suppresses a phenotype associated with defective protein quality control pathways, consistent with a functional link between translocation and quality control. Ongoing work includes characterizing the effects of placing different epitope tags on different translocon subunits, with the goal of identifying tags that affect translocon function the least.

Support or Funding Information

This work was supported by Ball State University ASPiRE research awards to AR and MD, a Ball State University Faculty Advance research award to ER, an Indiana Academy of Science Senior Research Grant to ER, and an NIH grant to ER (GM111713).
Isolation and Screening of Antibiotic‐Producing Bacillus and Pseudomonas Species
Authors: Carrera Esmeralda , Peterson Stacey N.
Published: April 2020

Antibiotic resistance is a major problem in the world today and contributes to many deaths each year, particularly in the immunocompromised. Bacteria use several mechanisms to avoid inhibition and destruction by antibiotics including the formation of biofilms, inactivating the drug, and preventing the antibiotic from reaching its target. Since many antibiotics are no longer effective against certain bacteria, there is a need to find new antibiotics that can inhibit the growth of these bacteria. Many antibiotics currently used have originated from bacteria that live in the soil. The purpose of this project was to isolate bacteria from soil samples collected from Mount Saint Mary’s University and screen them against a variety of bacteria to identify antibiotic‐producers. Ultimately the producers will be further characterized and the antibiotics isolated and identified. Four antibiotic producers were identified whose 16S rRNA genes were amplified by PCR and sent out for sequencing. The producers were identified as gram‐positive Bacillus megaterium and various gram‐negative Pseudomonas species. These bacteria were able to inhibit the growth of Bacillus subtilis and Erwinia carotovora. The Pseudomonas species were chosen for further analysis and isolation of the antibiotics. An ethyl acetate extraction was performed as a first step in isolating the antibiotic from one species most closely related to P. koreensis. The suspected antibiotic was recovered from the organic layer as determined by its activity against B. subtilis. The antibiotic extract was quantified and purified through HPLC. Additionally, biochemical tests such as the citrate utilization test and gelatin hydrolysis test were done to provide more information on the characteristics of the Pseudomonas producers. The biochemical tests showed that producers such as P. koreensis were able to use citrate as a carbon source and produce gelatinase. Ultimately the goal of this research is to identify new antibiotic sources with the long‐term goal of isolating and identifying a new antibiotic.
A New Method for Converting Aptamers to Riboswitches Using the Theophylline Riboswitch
Authors: Schievelbein Mika Jeannette , Lucia Lauren , Dadlani Sonia , Zangrilli Toni , Tilton James , Ouellet Jonathan
Published: April 2020

Fluorescence Activated Cell Sorting, or FACS, is a method that was used to convert the theophylline aptamer into a riboswitch. This method could theoretically be used to convert other discovered aptamers into riboswitches, however it is a costly method and is only available to those with access to these high‐tech, expensive machines. The theophylline riboswitch was previously discovered by implementing the theophylline aptamer, with random sequences linking it to the expression platform, into a specifically‐designed plasmid, and then using a FACS machine to sort the cells.

We can structure a new system that would select only the correct sequence, containing the theophylline riboswitch out of a pool, without the use of a FACS machine. To do so, we place the pool of sequences containing the aptamer, linked to the Shine Dalgarno by eight randomized nucleotides, into a newly designed plasmid, placNEO, and transform the plasmid into bacteria cells. Then, the use of replica plating along with screening will select the cells that only contain the plasmid with the correct riboswitch sequence. By doing so, we confirm that this system is efficient in converting aptamers into riboswitches without the need for a FACS machine.

After an aptamer has been successfully converted into its riboswitch, the system of ratiometric fluorescence will allow for testing of the riboswitch’s function. This is done by designing a plasmid, pTRFlac, that contains genes for red and green fluorescence proteins, mCherry and GFP respectively, on either side of the inserted riboswitch. A PCR product encoding for mCherry, the riboswitch, and GFP will be inserted downstream of the lactose operon in pUC18. Ratios of the fluorescence intensities of the two florescent proteins will provide the ability to measure the riboswitch’s function through fluorescence readings.

Support or Funding Information

Monmouth University Summer Research Program ICFNJ
Investigating the Effects of 1700 FDA Approved Drugs on Proneural Protein Knockdown in Drosophila melanogaster
Authors: Mancini Isabella
Published: April 2020

Basic Helix‐Loop‐Helix (bHLH) proneural proteins comprise a superfamily of transcription factors that are critical to early development. This superfamily includes the Daughterless (Da) gene in Drosophila melanogaster, the fruit fly, and the homologous TCF4 gene in mammals. These genes have many essential functions in sex determination, cell proliferation, muscle development, and neurogenesis. In addition to these roles in development, our laboratory has shown that both Da and TCF4 function in mature, differentiated neurons, where they restrict neurite branching and synapse number. Knockdown of Da thus results in decreased motor function, a readout of proper glutamatergic neurotransmission. These results are important because mutations in TCF4 have been associated with the neurodevelopmental diseases Pitt‐Hopkins syndrome and schizophrenia.

The purpose of this research is to screen 1700 FDA approved drugs in our Da knockdown model with the hopes of using this information to better determine future therapy for individuals with Pitt‐Hopkins and schizophrenia.

Support or Funding Information

This research was supported by Drexel University Office of Undergraduate Research through the STAR Scholars program, under the mentorship of Dr. Daniel Marenda.
Emerging mechanistic insights into mTOR‐PASK signaling pathway that connects nutrient signaling to chromatin network in stem cells
Authors: Kikani Chintan , Xiao Michael , Wu Xiaoying
Published: April 2020

Stem cell fate in the tissue niche is intimately connected with intracellular metabolic state and the extra‐cellular hormonal stimulations. We have shown that a sensory kinase, PAS domain Kinase (PASK) phosphorylates Wdr5, a member of COMPASS family of histone methyltransferases, to activate the stem cell differentiation program in multiple differentiation paradigms, in vivo and in vitro (eLife, 2016). PASK is expressed highly and exclusively in stem cells, yet the differentiation signaling cues are required to activate PASK and its downstream functions, suggesting PASK remains inaccessible to bind Wdr5 in proliferating stem cells. Here, we show that the mechanistic Target of Rapamycin (mTOR) phosphorylates PASK to promote Wdr5 recruitment and myogenesis in response to nutrient and hormonal signaling. How mTOR phosphorylation stimulates PASK‐Wdr5 interactions remains unknown but is a key mechanistic question that could help understand how nutrient and metabolic signaling can acutely control stem cell differentiation on demand. By using multi‐disciplinary approaches, we show that the PAS domain of PASK inhibits catalytic activity of the kinase domain. Interestingly, mTOR stimulated phosphorylation induces a conformational change resulting in the increased Wdr5 binding, and catalytic activity of PASK. Thus, our data show how information pertaining to nutrient availability is communicated to epigenetic complexes via sequential activation of the mTORC1‐PASK‐Wdr5 pathway.

Support or Funding Information

NIH R01: Epigenetic Control of muscle stem cell function by PASK‐Wd5 signaling pathway.1R01AR073906‐01A1
A Selective AC1 Inhibitor as Non‐GPCR‐ or Ion Channel‐Dependent Approach for the Treatment of Pain
Authors: Giacoletti Gianna , Price Tatum , Msdi Abdulwhab Shremo , Cook Ryan , Hayes Michael , Vazquez-Falto Katerina , Gonzales Alexandra , Chapman Sumer , Santini Edwin , Nornoo Adwoa , Watts Val , Brust Tarsis
Published: April 2020

Adenylyl cyclases (ACs) are enzymes that catalyze the synthesis of cAMP from ATP. The cAMP signaling cascade is involved in a number of physiological processes spanning from breathing to memory and cardiac function. Among the nine different membranous AC isoforms, a role for adenylyl cyclase 1 (AC1) has been suggested in learning, memory, pain, and opioid dependence. AC1 is one of the calcium/calmodulin‐stimulated ACs and previous studies suggest that inhibition AC1 could relieve inflammatory pain and reduce signs of opioid dependence. However, non‐selective inhibition of AC1 and closely related AC8 could lead to learning and memory impairments. We previously identified ST034307 as a selective inhibitor of AC1 and revealed anti‐allodynic activity in the mouse CFA inflammatory pain model. As part of our ongoing studies to identify selective AC inhibitors we developed a novel cellular model that used CRISPR/Cas 9 editing to remove 95% of the forskolin‐stimulated cAMP signal from wild type HEK293 cells (Soto‐Velasquez et al., 2018). We have now used this cellular model to re‐evaluate the AC isoform selectivity of ST034307. The results confirmed unprecedented selective inhibition of AC1 vs the closely related AC8. The development of AC1‐selective inhibitors like ST034307 now provides an opportunity to better study this protein and determine the physiological effects of inhibiting AC1 in additional animal models. The present work represents a comprehensive pre‐clinical study with the selective AC1 inhibitor ST034307. We have determined ST034307’s biodistribution to mouse blood and brain at different time points after injection. In addition, we have shown that the compound is effective and comparable to ketoprofen to relieve visceral and inflammatory pain in mice, and that it does not disrupt mouse innate behavior (nesting) at analgesic doses. In fear extinction experiments with rats ST034307 had no effects on extinction learning. However, the compound disrupted the consolidation of fear extinction compared to vehicle‐treated rats. These data support previous studies suggesting that AC1 inhibitors may be useful to treat pain and also indicates that normal innate behavior is not disrupted at analgesic doses. Moreover, the effect on consolidation of fear extinction may suggest a novel use for these compounds in anxiety disorders, such as post‐traumatic stress disorders (PTSD).

Support or Funding Information

This work was supported by the American Association of Colleges of Pharmacy (New Investigator Award), Palm Beach Atlantic University (Quality Initiative Grant), Lloyd L. Gregory School of Pharmacy (Integra Connect Grant), Purdue College of Pharmacy, and Richard and Anne Borch Award.
The First‐Year Research Advancement Program (FRAP): A research‐centered experience to foster discipline identity, persistence, and agency in under‐represented students in chemistry and biology.
Authors: Mills Kenneth V. , Paxson Julia
Published: April 2020

The First‐Year Research Advancement Program (FRAP) was started at Holy Cross in 2014 to increase persistence in STEM of academically promising students who belong to groups that traditionally do not persist at the same rate as majority students, including first‐generation students, PELL‐eligible students, and underrepresented minority students. We cofounded FRAP based on evidence that early experiences in research laboratories can increase discipline identity and persistence. We are now in the sixth year of the program, with 58 total first‐year participants, and six former participants serving as mentors. Two first‐year students are paired with both a faculty mentor and a senior student in the lab, and all students are paid for eight hours per week such that their participation replaces campus employment rather than adding an extra burden. Students work in the lab six hours per week, and participate in one‐hour meetings with their lab group and in a one‐hour meeting with all participants in order to create two cohorts of support: one among the research lab who typically are of all class years, and another among their first‐year peers. We have assessed the program in collaboration with the Office of Assessment and Research, and have found that participants show growth in aspects of their attitudes toward science and in their agreement with statements that students like them can succeed in science and that even if they forget facts they can still use thinking skills they learned in science. They did not show as much growth in their confidence to do well in classes. As our total number of participants has grown, we are analyzing persistence data in STEM disciplines. Nine of the 12 students who started in the Mills lab as FRAP students are either still in the lab or stayed in the lab for their four years at Holy Cross.

Support or Funding Information

This work was supported by the National Science Foundation (MCB‐1517138, KVM) and a Henry Dreyfus Teacher‐Scholar Award (KVM).
Investigation of mutants with aberrant transcriptional regulation of OLE1
Authors: Cribbs Lauren , Filippelli Sara , McDonough Virginia
Published: April 2020

The OLE1 gene in Saccharomyces cerevisiae encodes the Δ9 desaturase. Previously, we isolated mutants that affect the expression of OLE1. In this work, we describe the phenotypes of two of these mutants, arv1 and nro (“no regulation of OLE1”). OLE1 promoter‐URA3 ORF reporter gene assays have shown that both mutants express OLE1 in the presence of the normally repressive unsaturated fatty acid 18:2Δ9,12. Analysis of OLE1 RNA amounts by qPCR, indicate that the nro mutant has similar amounts of mRNA present to the wildtype control, however, the level of OLE1 mRNA is significantly lower in arv1, and confirmed that it is not regulating expression in response to 18:2 feeding. OLE1 promoter deletion analysis resulted in decreased OLE1 transcription in arv1 compared to the wildtype, and did not regulate in response to 18:2 feeding. Surprisingly, OLE1 expression regulation by 18:2 is somewhat recovered with a OLE1 promoter deletion between −488 and −471 in arv1 cells, although this regulation is lost at −255 (same as wildtype). This assay also indicated that the OLE1 promoter region between the −225 and −142 may be important for nro regulation of OLE1 transcription. These results indicate that the arv1 and nro mutants do have an effect on the transcriptional regulation of the OLE1 gene.

Support or Funding Information

Research reported was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM132853
Rapid formation of ER‐PM contacts during early stages of phagocytosis
Authors: Lee Minhyoung , Fairn Greg
Published: April 2020

Membrane contact sites (MCS) are specialized structures where the endoplasmic reticulum (ER) and other organelle form come in close proximity (~10–30 nm). Functionally, these regions are thought to serve as hubs for cellular processes such as inter‐organellar exchange of lipids, calcium homeostasis, and the access of phosphatases to substrates. Consequently, the spatial‐temporal organization of these structures will impact numerous cellular functions. The formation of ER plasma membrane (PM) contact sites are controlled by a variety of membrane tether proteins as well as the morphology of the ER. We and others have found that the sub‐plasmalemmal cortical actin cytoskeleton, a mesh‐like network that occupies 100–200 nm below the PM, can also limit the formation of ER‐PM contact sites. Yet, how ER‐PM junctions are regulated in circumstances where cells have extensive cortical actin remodeling is not understood. An example of such cellular process involving dynamic cortical actin re‐arrangement is phagocytosis. Phagocytosis is an actin‐dependent processes used to internalize particulate material (≥0.5 μm) that serves both antimicrobial and homeostatic functions. The role of the ER in phagocytosis has been a matter of much controversy. Electron micrographs of macrophages undergoing phagocytosis revealed the presence of ER in extensive, close contact with the forming phagosome. It is speculated that the role for ER in this context is to form ER‐PM contact sites with the PM. My studies have revealed that during phagocytosis, the disassembly of F‐actin from the base of the phagocytic cup allows for the formation of new ER‐PM contact sites. ER‐PM contacts formed promptly and specifically where the polymerized actin has been cleared. As such I have found that the spatial occupancy of ER‐PM junction increased ~3 fold during phagocytosis. Finally, I identified PTP1B, a tyrosine phosphatase, as one of the ER‐PM contact proteins that may be functionally important during phagocytosis.

Support or Funding Information

NSERC PGS‐DCIHR Project Grant
Heparan Sulfate Reduction to Enhance Wound Induced Neogenesis
Authors: Zurich Raymond , Phan Anne , Esko Jeffrey
Published: April 2020

Heparan sulfate regulates cell signaling important in wound healing and regeneration. In normal mammalian wound healing, injuries that penetrate the dermis result in a scar lacking hair follicles and subcutaneous fat because hair and fat do not regenerate. However, if the dermal wound is larger than a critical size (≥20 mm in adult mice) a process termed ‘Wound Induced Neogenesis’ occurs and some neogenic hair and fat is regenerated in the center of the wound. Based on previous observation that a deficiency in heparan sulfate accelerates cutaneous wound healing in mice, we hypothesized that heparan sulfate deficiency can enhance wound induced neogenesis of hair and fat in dermal wounds. Using systemic genetic models of heparan sulfate deficiency (Ext1+/−Ext2+/−), we tested whether mice with less heparan sulfate had enhanced wound induced neogenesis of hair and fat after a subcritical size (15 mm) full‐thickness dorsal skin excisional wound. Although we observed no significant difference in the rate or extent of wound closure, we found increased neogenesis of hair follicles and fat in heparan sulfate deficient mice. Our experiments suggest that inducing heparan sulfate deficiency can be a therapeutic avenue to enhance mammalian regeneration.

Support or Funding Information

This work was supported by the National Institute of Arthritis, Muscular, and Skin Diseases (R61 AR073031).
Reverse transcription and amplification of 2′F DNA by commercially available DNA polymerases
Authors: Thompson Arianna , Barrett Susanna , Weiden Aurora , Leconte Aaron
Published: April 2020

Modified‐DNA polymerases have been evolved that can synthesize long strands of modified oligonucleotides. The resulting modified DNA (M‐DNA) has many high‐value potential applications, such as clinical diagnostics and therapeutics. To fully apply M‐DNA, it must be reverse transcribed back into natural DNA. Previously, error prone reverse transcriptases have been required for this step, and then the resulting DNA has to be amplified in a separate amplification reaction. Using a synthetic template with 2′F modified nucleotides, we tested a panel of commercially available DNA‐dependent DNA polymerases for their ability to reverse transcribe (RT) and amplify M‐DNA in a one‐pot reaction. We found that four of the six polymerases chosen (Phusion, Q5, Deep Vent, and Vent) were able to RT and amplify synthetic 2′F modified DNA in a single reaction. These products were sent in for high‐throughput sequencing and it was determined that Q5, Deep Vent and Phusion had between 90 and 95% matched reads, while Vent had less than 85%. The conditions of this process (reaction buffer and PCR cycling conditions) were also shown to affect the accuracy, but to a lesser degree. These polymerases were also able to RT and amplify enzymatically synthesized 2′F templates, allowing us to use this as a tool to quantitatively determine error rates of M‐DNA polymerases using high‐throughput sequencing. This demonstrated that P1, one of our labs best modified‐DNA polymerases, was almost four times more accurate than SFM4‐6, the fields previous best enzyme. These discoveries will enable the wider use of 2′F M‐DNA, as well as add to the. evidence supporting the use of commercially available DNA polymerases for reverse transcription reactions.

Support or Funding Information

National Science Foundation
Characterization of Effect of Hydrogen Peroxide on Hydrolysis of Collagen
Authors: Patel Silkey , Chew Charmaine , Keenan Kelly
Published: April 2020

Americans spend billions of dollars a year on teeth whitening and the active ingredient is usually hydrogen peroxide. The outermost layer of teeth is the enamel and the dentin is underneath: the major protein found in the dentin of teeth is Type I collagen. The goal of this project was to characterize the effect of hydrogen peroxide at concentrations similar to those used in over‐the‐counter‐whitening strips on collagen. Type I collagen was mixed either with hydrogen peroxide, collagenase, both or neither and the amount of protein released was characterized in several ways: dialysis (with a molecular weight cutoff of 3.5 kDa) and gel filtration with an exclusion limit of 10 kDa. The amount of protein or peptide released was measured with a modified Lowry assay, which has been adapted to measure collagen accurately. Collagenase is a metalloprotease that hydrolyzes the peptide bond. When treated with collagenase, no protein was released but 17 mg of protein was released when treated with 7.5% hydrogen peroxide and 18 mg when treated with both. Similar results were obtained with collagen isolated from human teeth. The amounts increased as hydrogen peroxide concentrations increased. For the gel filtration column, untreated collagen emerged in the first fraction after the void volume but when pre‐treated with hydrogen peroxide, there were proteins in later fractions. Type I collagen shows a molecular weight (MW) of 129 kDa when run on 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) that was silver‐stained. This amount matches the expected value based on the subunits of the protein. The addition of hydrogen peroxide caused this band to disappear from the gel and no smaller MW bands were visible. When treated with collagenase, the original band as well as several smaller proteins of 80, 66 and 47 kDa are observed as collagenase produces the occasional hydrolysis with large protein fragments that result. No bands were observed when collagen was treated with both collagenase and hydrogen peroxide. These results suggest that hydrogen peroxide is capable of hydrolyzing type I collagen and produces peptides that are too small to be detected on the gels but have a size of 3.5 kDa or less.

Support or Funding Information

Authors thankfully recognize support by research and professional development funds from Stockton University.
Characterization of Peptides Released from Treatment of Type I Collagen with Hydrogen Peroxide
Authors: Amoah Akua , Ailes Isaiah , Keenan Kelly
Published: April 2020

Previous research has demonstrated that hydrogen peroxide is capable of hydrolyzing the peptide bond in proteins including collagen. Proteases, like collagenase, are also capable of hydrolyzing peptide bonds at specific amino acid residues producing fragments. The goal of this project was to characterize the size and nature of the peptides produced by treating proteins with hydrogen peroxide. In order to characterize the response of proteins in general to hydrogen peroxide, various albumin proteins were treated with varying concentrations, precipitated using trichloroacetic acid and centrifuged. The amount of peptide small enough to not be precipitated was measured in the supernatant using the Lowry assay. The results demonstrated that increasing concentrations of hydrogen peroxide resulted in increased amounts of peptides too small to be precipitated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was used to examine the size of these proteins following treatment with hydrogen peroxide treatment or the protease trypsin, which hydrolyzes following the amino acid residues arginine or lysine. Chick albumin showed a band at the expected MW of 43 kDa and treatment with hydrogen peroxide (5.0%) reduced the size slightly. When treated with trypsin, the original band as well and other bands were visible on SDS PAGE. These results suggest that hydrogen peroxide works differently than proteases to hydrolyze proteins. Since trichloroacetic acid cannot precipitate collagen, other methods were used to characterize the peptides released by hydrogen peroxide. Glycine, proline, and hydroxyproline, which are overrepresented in the collagen primary structure, were detectable using thin layer chromatography (TLC). Silica plates were spotted with 1 mg/ml solutions with 70%/30% (v/v) n‐propanol/water as mobile phase. Glycine produced a red spot with an retention factor (Rf ) of 0.37 while proline resulted in a yellow spot (Rf 0.37) and hydroxyproline was pink (Rf 0.46). When the tripeptide Gly‐Pro‐Ala was used, the spot was yellow with an Rf value of 0.18. This result suggests that amino acids and small peptides can be resolved using this method. Type I collagen was mixed with 6.7% hydrogen peroxide or collagenase or both or neither in dialysis tubing and the contents of the fluid outside the tubing was tested using this TLC method as well as by measuring hydroxyproline in a colorimetric test. Reverse phase high pressure liquid chromatography were used to measure Glycine and Proline,

Support or Funding Information

Authors gratefully acknowledge research and professional development funds from Stockton University.
Enzyme polymerization in nucleotide biosynthesis
Authors: Peterson Jeff , Akizu Naiara , Simonet Jacqueline , Foster Maya , O’Reilly Alana
Published: April 2020

Polymerization of metabolic enzymes into micron‐scale assemblies is an emerging phenomenon. However the role assembly plays in regulating metabolic flux differs between enzymes and in many cases is unknown. Furthermore, the signaling pathways that regulate assembly are often not well understood. Our work focuses on enzymes mediating pyrimidine and purine biosynthetic pathways that undergo regulated assembly. To address the biological function of these assemblies we have developed point mutants of CTP synthase and inosine monophosphate dehydrogenase (IMPDH), that prevent their assembly without eliminating their catalytic activity. We will present new data using these novel tools in mice and Drosophila to elucidate the role of polymerization of these enzymes in native contexts in which they assemble. Our findings demonstrate a critical role for assembly of CTPS and IMPDH in supporting the growth and proliferation of cells with elevated nucleotide demands. Thus, we propose that assembly of these nucleotide biosynthetic enzymes has been harnessed in metazoans as a homeostatic mechanism to ensure that metabolic biosynthetic flux meets demand.

Support or Funding Information

NIH GM083025
Insecticides resistance status in Aedes aegypti and Aedes albopictus from Lahore, Pakistan
Authors: Rahman Rafi ur , Costa Monique , Carrara Luana Silva , Lima Josè Bento Pereira , Martins Ademir Jesus
Published: April 2020

Aedes‐borne disease in Pakistan drew the attention after the dengue outbreak in Lahore in 2011, leaving thousands hospitalized and around 300 deaths. Since then many small and big outbreaks have been reported from various parts of the country. The only available and mostly used mosquito control strategy in Pakistan is the use of neurotoxic chemicals. Pyrethroid deltamethrin and permethrin were extensively used for adults’ control along with organophosphate temephos as a larvicide during and after the outbreaks. This study was designed to find the efficacy of the insecticides in use for Aedes control as well as other insecticides of the same or different classes. Preliminary results after WHO‐like tube tests or CDC‐bottle tests showed that both Aedes aegypti and Aedes albopictus are susceptible to larvicide OP temephos, IGR pyriproxifen and adulticide OP malathion while they are resistant to all classes of pyrethroids tested (deltamethrin, permethrin, etofenprox, alphacypermethrin). Genotyping assays showed low kdr frequencies while assays to find the expression of P450s and other enzymes related to resistance are underway. Sequencing of the voltage gated sodium channel gene will give us a better picture about the mutations in the IIS6 and IIIS6 regions which plays an important role in pyrethroids sensitivity. This study will assist the authorities to design future control strategies.

Support or Funding Information

The study is part of PhD thesis of the presenting author Rafi ur Rahman, who receives monthly scholarship from TWAS and CNPq. No additional funding is provided to the laboratory or the student.
Histone H3 K14 acetylation and H2B SUMOylation help regulate binding of the RSC remodeler complex to the nucleosome
Authors: Wilkins Bryan J. , Jain Neha , Evans Brian , Chaudhry Shereen , Neumann Heinz
Published: April 2020

Chromatin remodeler (CR) complexes play crucial roles in regulating chromosomal architectural and act to modify nucleosomal DNA contacts by repositioning nucleosomes. The mechanistic details of the CR family of proteins are of importance because each remodeler complex contributes to unique chromatin structural maintenance. Here, we study the RSC complex of yeast to better characterize its dynamics in the living nucleus. Utilizing an expanded genetic code, we incorporate the photo‐crosslinking amino acid, p‐benzoyl‐L‐phenylalanine (pBPA), site‐specifically into histone proteins in order to monitor non‐histone protein interactions at the nucleosomal surface, in vivo. We map the interface between the RSC motor protein, Sth1, and the nucleosome core particle, revealing that crosslinking efficiency to the H3 N‐terminal tail is dependent upon the acetylation of H3 K14. This modification is also a known binding site of the RSC subunit, Rsc4, suggesting that the remodeler favors binding symmetrical H3 K14ac marks on the same nucleosome. Additionally, we reveal that RSC binding is dependent on SUMOylation of histone H2B. Pairing crosslinking with temporal control of the cell cycle we observe that RSC has differential binding modes as the chromatin architectures alter during compaction and passage into mitosis. All together, this data provides greater mechanistic insight to the functional dynamics of the RSC remodeler under true physiological conditions.

Support or Funding Information

National Institute Of General Medical Sciences of the National Institutes of Health under [Award Number R15GM124600] German Research Foundation (DFG) [Grants NE1589/5‐1 and 6‐1]
Epinephrine Responsiveness is Reduced in Livers from Trained Mice
Authors: Dibe Hana A. , Townsend Logan K. , McKie Greg L. , Wright David C.
Published: April 2020

Catecholamines increase liver glucose production at least in part through increases in glycogenolysis and gluconeogenesis. To date, the effects of prior exercise training on the liver’s response to epinephrine, in vivo, have not been fully elucidated. To examine the role of epinephrine signaling on indices of liver glucose production in trained mice, male C57BL/6 mice were subject to either 12 days of voluntary wheel running (TR) or remained sedentary (SED). Epinephrine (0.5 mg/kg body weight), or vehicle were injected intraperitoneally following 12 days of training and tissues harvested 15 minutes post‐injection. The rise in blood glucose following epinephrine injection was significantly blunted in trained compared to sedentary mice and this was paralleled by a blunted reduction in liver glycogen in trained mice. There was a main effect of epinephrine to increase the phosphorylation of protein kinase‐A substrates (pPKAs), which was driven by increases in the sedentary, but not trained, mice. Similarly, epinephrine‐induced increases in the mRNA expression of α‐ and β‐adrenergic receptor 1 and 2 and glucose‐6‐phosphatase were greater in SED compared to TR mice. Exercise and epinephrine decreased serum insulin levels and increased glucagon levels, however, there were no differences within epinephrine treated groups. Changes in phosphorylated AKT, AMPK, and phosphodiesterase 4B protein content, negative regulators of PKA signaling, do not explain the protective effects of exercise training against epinephrine induced increases in blood glucose and liver glycogen breakdown. Taken together our data suggest that prior exercise training reduces the liver’s response to epinephrine. This could be beneficial in the context of training‐induced sparing of glycogen during exercise.

Support or Funding Information

This work was funded, in part, through a National Sciences and Engineering Research Council of Canada (NSERC) grant to D.C.W., who is a Tier II Canada Research Chair in Lipids, Metabolism, and Health. H.A.D was supported by an Ontario Graduate Scholarship. G.L.M. was supported by an Ontario Graduate Scholarship and subsequently an NSERC Canada Graduate Scholarship. L.K.T. was supported by a Dairy Farmers of Ontario Doctoral Research Assistantship and an NSERC Postgraduate Scholarship.
INTERACTION OF DEXTRIN CAPPED CADMIUM SULFIDE QUANTUM DOTS WITH BLOOD COMPONENTS: COAGULATION AND HEMOLYSIS STUDIES
Authors: Rodríguez-López Yaneth Anahi , Rodriguez-Fragoso Patricia , de la Cruz Gerardo Gonzalez , Reyes-Esparza Jorge , Rodriguez-Fragoso Lourdes
Published: April 2020

Evaluate at interaction of dextrin capped cadmium sulfide quantum dots with blood components. The intravenous administration of quantum dots (QDs) has been widely reported as a promising alternative for delivery of drugs to specific cells. Therefore, previous studies have pointed out the importance of studying of, at least, factors like coagulation effect; and their hemolytic character when nanoparticles are intended to be administered for intravenous injection. The hemolytic activity and platelets aggregation of the nanoparticles was tested on isolated rat erythrocytes and platelets. Atomic force microscopy (AFM) facilitates the real‐time studies of blood cells and the morphological, structural, optical properties of the CdS‐dex QDs. In this work a methodology for erythrocytes and platelets study by platelet aggregation, hemolysis and AFM was developed. The results show that CdS‐Dex/QDs caused platelet aggregation at concentration‐dependent manner, this effect being greater at 1000 ug/mL, where the increase was up to 57% (p<0.05). The AFM measurements establish a more direct correlation between the surface topography of platelets with the surface nanostructure and reveal that modified the morphology of the platelets from the concentration of 0.01 ug/mL. The CdS‐Dex/QDs do not cause lysis of erythrocytes significantly, although they modified their morphology, suggesting that they can cause eriptosis. The CdS‐Dex/QDs, caused effects in oval and biconcave morphology of the erythrocytes, making it round and of edges not defined, semi‐flat and with cracks in its surface and lacking central depression in a concentration‐dependent manner and observed in its surface some artifacts that may correspond to agglomerated NPs. We found that CdS‐dex/QDs caused aggregation of rats platelets in vitro; however; there were both quantitative and qualitative differences depends to various concentrations.