bluuebunny/crossref_metadata_2025 huggingface dataset:
A Semester Long Teaching Practicum For Graduate Students In Biomedical Sciences
Authors: Towse Theodore Francis , Thomas Derek
Published: April 2020

Introduction

Teaching assistants are often given little or no guidance before receiving their first teaching assignment. As a result they feel underprepared and overwhelmed by the experience, which undoubtedly reduces their effectiveness in the classroom. Therefore we developed a one‐semester teaching practicum to better prepare new graduate students for the rigors of college teaching.

Purpose

To provide students with a mentored experience in college teaching while simultaneously exploring topics in the pedagogy of college teaching.

Methods

We developed a 15‐week course for first year graduate students in the Department of Biomedical Sciences at Grand Valley State University. Student teaching assistants were assigned to shadow a faculty‐mentor teaching an introductory laboratory course in Anatomy and Physiology, or Microbiology. Students performed basic teaching duties including grading and assisting students during the lab. In addition they completed a curriculum, which included weekly readings and writing assignments on topics in college teaching pedagogy. Selected readings, links to external content, and writing reflections were managed through a course website on Blackboard.

Learning Objectives

After completing the course the student should be able to: critically read and apply current concepts in the pedagogy of college teaching, facilitate student‐centered learning activities, make effective use of visual aids in classroom instruction, plan, execute, and evaluate classroom instruction.

Evaluation and Assessment

Weekly assignments included readings on topics in college teaching pedagogy ranging from – setting the right tone in the classroom on day one, managing the classroom, to developing a teaching philosophy. Each assignment required a brief writing reflection and a discussion with the faculty‐teaching mentor. As part of the course students completed two teaching demonstrations. The first teaching demonstration was video taped and later reviewed and evaluated by the student and faculty mentor. Both teaching demonstrations were graded and the grading rubric was provided to the student prior to the demonstration. The course culminated with the student drafting a statement of teaching philosophy.

We developed a 15‐week teaching practicum designed to provide students with a mentored experience in college teaching. The course was initially piloted with two graduate students in the Department of Biomedical Sciences at Grand Valley State University but could easily be scaled up to a larger cohort of students and different academic disciplines.
Mycobacterium tuberculosis novel DNA methyltransferaseenhances mycobacterial infectivity and survival within macrophages and modulates host pro‐inflammatory response via inhibition of phagolysosomal fusion
Authors: Manjunath P. , Samal Jasmine , Rani Anshu , Ahmed Javeed , Ehtesham Nasreen Zafar , Hasnain Seyed Ehtesham
Published: April 2020

DNA methylation is an important epigenetic mechanism that impacts gene expression and helps the mycobacteria to survive inside the host in different microenvironments. About 3% of the genome of Mycobacterium tuberculosis, the tuberculosis (TB) causing bacteria, codes for methyl transferases, many of which are believedto play major role in modulating host‐bacterial pathogenesis. A hypothetical gene coding for a DNA methyl transferase, unique to mycobacterium tuberculosis complex was identified, cloned and transformed into Mycobacterium smegmatisin order to understand the characteristics and function of this gene. Expression of this methyltransferase in M. smegmatis caused growth retardation and morphological changes. In addition, recombinant M. smegmatis showed increased infectivity and intracellular survival in human macrophage cell line by escaping the phagolysosomal maturation. Infection of macrophages with recombinant M. smegmatis was associated with significant necrosis and increased levels of LDH as compared to controls, promoting higher dissemination of bacteria. Surprisingly it also plays role in forming granuloma‐like structures in infected macrophages. Furthermore, macrophages infected with recombinant M. smegmatis showed a significant reduction in the secretion of bactericidal factors such as nitric oxide and major inflammatory cytokines. Co‐culture of infected THP1 cells and T cells induced strong cell mediated immune response by releasing high levels of IFN‐γ and IL‐2, promoting T cell activation and expansion. These findings point to an important role of this DNA methyl transferase in mediating survival of mycobacteria within host cells and regulating pathogenesis.
Impact of Maternal Sleep Deprivation and Caffeine Consumption on Neurodevelopmental and Behavioural Outcomes in Young Rats
Authors: Edem Edem , Okhonmina Uyi , Nebo Kate , Oremosu Ademola
Published: April 2020

Caffeine is a neurostimulant, which has been shown to be both cognitive‐enhancing and sleep‐disrupting. Several reports agree that caffeine and sleep deprivation have opposing effects on behaviours, including cogntive and depressive behaviours. Therefore, this study was designed to evaluate the neurodevelopmental and behavioural outcomes following prenatal and postnatal caffeine treatment and chronic sleep deprivation in rats. Pregnant Long‐Evans rats were sleep‐deprived 8 h/day in the last trimester of gestation using the platform‐over‐water sleep deprivation model; and treated orally with 120 mg/kg of caffeine from gestation day 8 and throughout lactation. Learning and memory, social, anxiety‐like and depressive‐like behaviours were assessed in the prepubescent rats (Postnatal Date 35). The young rats exhibited a higher anxiety level, as revealed by higher open field entry frequencies. They performed poorly in the novel object recognition test, and displayed abnormal sociability and social novelty in the social tests. Additionally, there was decreased hippocampal serotonergic activity and a marked reduction in synaptic protein expression in the CA1 hippocampal region, the prefrontal cortex and amygdala. Taken together, this study suggests that prenatal and postnatal sleep deprivation and caffeine consumption can interfere with brain development and thus impact behavioural outcomes, which could increase susceptibility to neurological disorders in adulthood.
The Role of Innate Immunity in Dystrophic Diaphragm Pathology
Authors: Petrof Basil
Published: April 2020

The pathophysiology of Duchenne muscular dystrophy (DMD) is complex and incompletely understood. However it is clear that the primary defect, lack of dystrophin, is necessary but not sufficient on its own to fully account for the onset of muscle fiber destruction, leading to eventual replacement by fibrotic tissue. Dystrophin restoration strategies are currently under clinical development, but the only treatment clearly shown to slow disease progression thus far in human DMD patients is the use of anti‐inflammatory corticosteroids. This presentation will focus on the role of the innate immune system in promoting the dysregulated inflammatory and aberrant muscle repair responses found in DMD. Using the mdx mouse diaphragm as a preclinical model due to its resemblance to the human DMD phenotype, the importance of monocyte‐derived macrophages in driving early disease progression will be outlined. This is exemplified by the therapeutic benefits (improved force generation and reduced fibrosis) noted when monocyte/macrophage recruitment to the dystrophic diaphragm is prevented through genetic or pharmacologic inhibition of the chemokine receptor CCR2. In addition, preventing activation of Toll‐like receptors (TLRs) is similarly beneficial and suggests significant involvement of damage‐associated molecular patterns (DAMPs) in DMD pathogenesis. Data will also be presented indicating that epigenetic alterations occur at the level of macrophage precursors in the bone marrow, which are likely stimulated by TLR4 recognition of DAMPs, and that subsequently play a role in dictating monocyte/macrophage phenotypic characteristics following their entry into the diseased muscles. Although the ideal treatment for DMD would be restoration of dystrophin to all muscles of the body including the diaphragm, significant hurdles remain for this ultimate therapy. In the meanwhile, there are serious drawbacks to the current practice of treating DMD children with corticosteroids, which have major side effects in DMD such as obesity, diabetes and osteoporosis. Future development of a more targeted therapeutic approach based on innate immune system modulation could represent an important advance in the field.

Support or Funding Information

Canadian Institutes of Health Research
Sequence Elements in Yeast Introns Regulate Translation to Make Functional Proteins
Authors: Hossain Munshi Azad , Codallos Nicholas , Johnson Tracy L.
Published: April 2020

RNA splicing is an important mode of eukaryotic gene regulation. By the process of splicing, introns are excised from the pre‐mRNA and exons are ligated to make a mature message, which is then translated to make protein. In eukaryotes, introns play a key role in alternative splicing whereby alternative exons are ligated to make different mRNAs from the same pre‐RNA transcript. These mRNAs can, in turn, be translated to make different protein isoforms and increase protein diversity.

Here, we explore a unique role for inefficiently‐spliced introns. Previously, we showed that an intron‐retained isoform of GCR1 encodes a protein which is initiated from an in‐frame intronic AUG. We have identified regulatory sequences upstream of the AUG that control translation from the intronic translation start site. Furthermore, we show that Rps25, a small ribosomal protein subunit, regulates utilization of the intronic AUG to facilitate the expression of the protein and control the relative levels of the two Gcr1p isoforms—one from the spliced RNA and one from the unspliced RNA. In light of this result, we next analyzed the introns of all known intron‐containing genes to assess whether other introns contain an AUG that is in‐frame with the same stop codon as the spliced isoform. We have identified and tested several such genes to determine whether the intronic AUGs direct protein expression and find that, indeed, they do. Moreover, when cells deleted of these genes express only the intron‐retained isoform, this restores viability to sick cells, suggesting that the proteins produced by intron retention are functional. These studies demonstrate a unique role for introns in regulating alternative isoform production to generate functionally important protein diversity.

Support or Funding Information

This work was supported by the National Science Foundation (BIO/MCB 1518316 to T.L.J) and National Institute of General Medical Sciences (GM‐085474 to T.L.J)
Developing a molecular age model for oocyte assessment
Authors: Lee Tin Lap , Ng Judy Kin Wing , Qao Qin , Chan David Yiu Leung , Yip Kelvin , Chow King , Chan Wai Yee , Lee Tin Chiu
Published: April 2020

The issue of advanced maternal age (AMA) has become a global issue, the average maternal age increased dramatically through the past decades. In vitro fertilization (IVF) is generally presented as a viable option but the success rate is limited by maternal age. The low success rate is also compound by subjective visual quality assessment, which creates a huge challenge for AMA women.

A novel genetic‐based oocyte assessment platform for age prediction using mouse oocytes had been developed in this study. Based on the transcription profile of both immature (GV) and mature (MII) oocytes in young (6 weeks old) and aged group (12 months old) obtained by single cell RNA sequencing, we were able to identify the differential expression genes (DEGs) that govern the aging process. A total of 1377 DEGs in GV oocyte aging were identified. Of which 745 genes were up‐regulated and 631 genes were down‐regulated. On the other hand, we compared the transcriptome of MII oocytes and identified 1432 DEGs, 706 of them were upregulated and 726 were downregulated DEGs in aging process.

The candidate genes for the age prediction model was selected by logistic regression model classifier with L1 norm penalty from the above sample, the power of prediction was ROC=0.81. Furthermore, we are developing a scoring system of 1–10 to give a clear grading regarding the oocyte quality. Based on the result obtained in mouse oocyte, a similar approach can be applied in human oocyte. Hence, the patients can have better idea about the oocyte quality and may proceed to oocyte treatments before IVF.

Support or Funding Information

Hong Kong Research Grant Council Collaborative Research Fund (C4054‐16G),
Effect of bisphenol Z and its analogs on reproductive health of C. elegans
Authors: Roberts Andrew H. , Bowen John , Zhou Xinrui , Timmons Shannon C. , Kuzmanov Aleksandra
Published: April 2020

Bisphenol A (BPA) is an organic synthetic molecule used in thousands of different plastic‐based products throughout the world. Over the years BPA has been repeatedly linked to neurobehavioral, cardiovascular and reproductive disorders. Because of growing human health concerns, BPA has been increasingly replaced by structurally similar chemicals such as bisphenol S (BPS) and bisphenol Z (BPZ). In recent years, some of these analogs have been under scrutiny by scientists whose research uncovered adverse health effects in model organisms. Whereas numerous studies suggest that BPS may be as toxic as BPA, the impact of BPZ is less understood. To assess its toxicity, we exposed the nematode C. elegans to BPZ at a final concentration of 1 mM throughout their development (from eggs to adulthood). Following exposure, we observed a marked reduction in the brood size compared to the wild type. Moreover, the BPZ‐exposed gonads showed a significant increase in the number of apoptotic nuclei. In the search for potentially safer alternatives, we synthesized three novel analogs of BPZ. Interestingly, these compounds exhibited even stronger effect on the reproductive ability of C. elegans. Our findings suggest that, similar to BPA and BPS, exposure to BPZ and its analogs results in reproductive toxicity.

Support or Funding Information

Department of Natural Sciences, Lawrence Technological University.
CDC20 and TRIB3 Upregulated by TFAP2C Facilitate Cell Proliferation in Non‐small Cell Lung Cancer Cells
Authors: Kim Dain , Seo Danbi , Kim Wanyeon
Published: April 2020

In recent years, non‐small cell lung cancer (NSCLC) is the most serious cause of global cancer‐related mortality. To resolve this circumstance, many studies for identification of novel molecular targets have been conducted. In our previous investigation, a microarray analysis that associated with transcription factor activating enhancer‐binding protein 2C (TFAP2C) was carried out and we found out TFAP2C's oncogenic roles in NSCLC development. In the current study, we intended to reveal novel biomarkers for NSCLC, especially focused on several oncogenes transcriptionally regulated by TFAP2C. With the result of microarray analysis, cell division cycle 20 (CDC20) and tribbles pseudokinase 3 (TRIB3) were selected as promising oncogenes by transcriptionally upregulated by TFAP2C. In the treatment of TFAP2C‐specific siRNA in two NSCLC cell lines (NCI‐H292 and NCI‐H838 cells), we observed that mRNA and protein levels of CDC20 and TRIB3 were down‐regulated. In addition, when CDC20 or TRIB3 was overexpressed, cell viability was increased and reversely apoptosis of these cells was suppressed. Consequently, these results indicated that NSCLC tumorigenesis affected by two promising oncogenes, CDC20 and TRIB3, and they could act as potent markers to prognosis of NSCLC.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2019R1C1C1009423).
Downregulation of GADD45B and PMAIP1 Mediated by TFAP2C Promotes Cell Proliferation in Non‐small Cell Lung Cancer Cells
Authors: Seo Danbi , Kim Dain , Kim Wanyeon
Published: April 2020

Non‐small cell lung cancer (NSCLC) is one of the main causes of death worldwide. At early stage, NSCLC can be treated with positive prognosis, but the limitation of biomarkers makes early diagnosis of lung cancer difficult. To identify lung cancer‐related biomarkers, we focused on the role of transcription factor TFAP2C as oncogene in lung cancer. In this study, microarray data were analyzed to identify tumor suppressor genes and 9 genes were screened for downregulation by TFAP2C. Of the 9 genes, growth arrest and DNA‐damage‐inducible beta (GADD45B) and phorbol‐12‐myristate‐13‐acetate‐induced protein 1 (PMAIP1) were selected as promising candidates for tumor suppressor genes downregulated by TFAP2C. When TFAP2C was overexpressed in NSCLC cells, the expressions of GADD45B and PMAIP1 at mRNA and protein levels were inhibited. In addition, GADD45B and PMAIP1 with downregulated by TFAP2C contributed to induction of cell proliferation and cell motility in NSCLC cells. Altogether, this study showed that GADD45B and PMAIP1 can act as tumor suppressors in NSCLC and can be used as useful markers in NSCLC therapy.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2019R1C1C1009423).
Tyrosine‐based Signals Converge on Daple&[bull]PARD3 Complex to Fine‐tune Polarized Planar Cell Migration
Authors: Ear Jason , Saklecha Anokhi , Rajapakse Navin , Choi Julie , Ghassemian Majid , Kufareva Irina , Ghosh Pradipta
Published: April 2020

Polarized distribution of organelles and molecules inside a cell is vital for a range of cellular processes and its loss is frequently encountered in disease. Polarization during planar cell migration is a special condition in which cellular orientation is triggered by cell‐cell contact. Here, we demonstrate that the multi‐modular signaling scaffold Daple (CCDC88C) is a component of cell junctions in epithelial cells which serves like a cellular ‘compass’ for establishing and maintaining contact‐triggered planar polarity via its interaction with the polarity regulator PARD3. This interaction, mediated by Daple’s PDZ‐binding motif (PBM) and the third PDZ domain of PARD3, is fine‐tuned by two tyrosine phosphoevents on Daple’s PBM that are triggered by a multitude of growth factors. Hypophosphorylation strengthens the interaction, whereas hyperphosphorylation disrupts it. These findings reveal an unexpected role of Daple within the planar cell polarity pathway as a platform for signal integration and gradient sensing for tyrosine‐based signals.

Support or Funding Information

This paper was supported by NIH CA238042,CA100768 and CA160911 (to P.G). J.E was supported by an NCI/NIH‐funded Cancer Biology, Informatics & Omics (CBIO) Training Program (T32 CA067754) and a Postdoctoral Fellowship from the American Cancer Society (PF‐18‐101‐01‐CSM). I.K. was supported by NIH grants GM071872, AI118985, and GM117424.
Biased allosteric modulation of G protein‐coupled chemoattractant receptor FPR2
Authors: Ye Richard D. , Zhang Shuo , Gong Hao
Published: April 2020

Formyl peptide receptor 2 (FPR2), a G protein‐coupled receptor (GPCR), transduces both proinflammatory and anti‐inflammatory signals in an agonist‐dependent manner. To determine how a GPCR transduces opposing signals, fluorescent biosensors of FPR2 were constructed using single‐molecule Frost resonance energy transfer (FRET). Incubated with Ac2‐26, an annexin AI‐derived peptide ligand with anti‐inflammatory activity, induced decreases in FRET intensity at 10 pM that is far below its Ca2+ flux‐inducing concentrations. WKYMVm (W‐pep) and Aβ42, both proinflammatory agonists of FPR2, stimulated increases in FRET intensity, suggesting that different FPR2 conformational states were induced for pro‐ and anti‐inflammatory signaling. Ac2‐26 and Aβ42 produced biphasic dose curves, but in opposite directions. Neither ligands could compete for W‐pep binding at this concentration, suggesting that they use allosteric sites on FPR2. Preincubation with Ac2‐26 at 10 pM diminished subsequent response to W‐pep in Ca2+ and cAMP inhibition assays, reduced FPR2 binding of W‐pep and ERK phosphorylation, but potentiated W‐pep‐stimulated membrane translocation of β‐arrestin2 and phosphorylation of p38 MAPK. The opposite effects were observed when the FPR2‐expressing cells were first incubated with 10 pM of Aβ42 and then stimulated with W‐pep. These results identified Ac2‐26 and Aβ42 as biased allosteric modulators at the subnanomolar concentrations used, that dose‐dependently alter signaling of the orthosteric agonist WKYMVm. Exploration of this feature may help direct FPR2 to pro‐ or anti‐inflammatory signaling pathways.

Support or Funding Information

This work was supported in part by the National Natural Science Foundation of China grant 31470865.
Inguinal White Adipocytes Differentiate to Alveolar Epithelial Cells that Constitute Mammary Cells in Pregnant Rats
Authors: Albustanji Layla , Dellschaft Neele , Budge Helen , Symonds Michael
Published: April 2020

Introduction

Inguinal adipose tissue has been shown to exhibit high plasticity that exceeds its capacity to differentiate to beige or brown adipocytes (
,
). This has led to the concept of pinking whereby adipocytes differentiate to develop an alveolar structure that forms part of the mammary gland.

Methods

Virgin and pregnant Sprague Dawley rats were used in this study. Food intake for virgin animals (aged 4 weeks) and body weight for virgin and pregnant animals were measured three times a week. Virgin rats were tissue sampled at 10 weeks of age while pregnant rats, mated at 10 weeks of age, were tissue sampled at either 50% and 90% of pregnancy (10 and 19 days of pregnancy, respectively). Gene expression was studied in the interscapular BAT, omental and inguinal adipose tissue using qPCR. Omental and inguinal sections were stained with H&E for examination of histological appearance.

Results

The histological sections showed differentiation of inguinal adipocytes to show an alveoli appearance composed of epithelial cells in pregnant but not virgin rats. Gene expression analysis revealed a higher expression of whey acidic protein (WAP) and E74‐Like ETS Transcription Factor 5 (ELF5) genes during pregnancy in inguinal adipocytes of pregnant rats compared to virgin rats. These two genes were absent in the interscapular and omental fat depots of virgin rats. Continuous loss of PPARG with pregnancy appeared in the inguinal adipose tissue. At the gross level, the inguinal fat appeared more compact in pregnant compared to virgin rats.

Conclusions

Inguinal adipocytes appear to exhibit a pronounced ability to form alveoli which comprises part of the mammary cell structure through gestation. Previous studies have confirmed this from measures of histology and molecular analysis. Further studies designed to examine the mechanism will help to determine factors that may accelerate the formation of neoplastic cells in the mammary glands and possibly breast cancer. Inguinal adipose tissue may no longer be considered a true fat depot in pregnancy.

Support or Funding Information

This work was funded by the EU‐CASCADE fellowship scheme funded by the EU’s 7th FP PCOFUND‐GA‐2012‐600181 (ND), LA was the recipient of a doctoral scholarship from Mutah University.
Role of B vitamins on Oxidative Stress and Apoptotic Proteins Expression in Experimental Colon Cancer Model
Authors: Taranikanti Varna , Padmanabhan Smitha , Waly Mostafa
Published: April 2020

Background

Azoxymethane is a potent carcinogenic agent commonly used to induce colon cancer and oxidative stress in rats. The endoplasmic reticulum (ER) stress response, represents an adaptive mechanism to support cellular survival in response to adverse microenvironment conditions by accumulating misfolded proteins and leading to apoptosis of cells. GRP78 has the potency of (potential to) inhibiting apoptosis by blocking BAX activity and thereby switching off intrinsic apoptotic pathway. Clinical studies continue to stress that oxidative stress (GRP78) is mediated B‐vitamins (folate and B12) deficiencies, oxidative and hyper homocysteinemia. However, it is relatively unknown how B vitamins affect oxidative stress on colon cancer tissue.

Objective

To study the expression of GRP78 and apoptotic proteins on azoxymethane(AOM) induced colon cancer using B vitamins and also to see any ultrastructure changes using Electron Microscopy.

Methods

Azoxymethane (AOM)‐induced colon cancer model was used in Sprague‐Dawley rats were randomly divided into groups. The control group fed a basal diet with no AOM injection; the AOM‐treated group fed a basal diet with AOM intraperitoneal injections, whereas the other experimental groups received daily doses of folate or vitamin B12 supplementation in the presence or absence of AOM injection. Another 6 groups of rats were tested for the potential synergistic effects of both folate and vitamin B12 supplementation. All animals continuously fed ad‐libitumuntil 16 weeks, then all rats were sacrificed and the colon tissues were examined for the immunohistochemical and ultrastructural changes. Briefly, 2–3mm of colon tissue sections were stored in glutaraldehyde and from these semithin and ultrathin sections were cut and viewed to delineate the cellular organelles architecture in a transmission electron microscope (TEM). Paraffin blocks were cut and the expression of GRP78, Bcl2 and Bax was observed under a light microscope using Immunohistochemistry (IHC) technique.

Results

There was over expression of GRP78 , Bcl2 and Bax proteins on IHC in colon cancer tissues treated with B vitamins however their expression was dose dependent. There were also ultrastructure changes in the cell organelles with dilation of endoplasmic reticulum and increase in the number of mitochondria. This is consistent with our earlier finding that early exposure to AOM causes morphological changes in the colonic mucosa of the assayed groups.

Conclusion

Vitamin B12 and folate play a protective role against oxidative stress at both the cellular and protein levels and might ameliorate the metabolic stress on colon cells caused by cancer. Overexpression of GRP78 and the role of B vitamins will help in future to study the UPR components for understanding the cellular mechanisms involved in cancer progression.

Support or Funding Information

SQU Institutional Fund
Endothelins contribute to early renal insufficiency engendered by acute urinary tract obstruction in neonatal pigs
Authors: Afolabi Jeremiah , Soni Hitesh , Adebiyi Adebowale
Published: April 2020

Urinary tract obstruction reduces kidney functions, which, if left uncorrected, may lead to irreversible renal loss, especially in infants. Despite the removal of obstruction, a decline in steady‐state renal blood flow (RBF) persists, vascular mechanisms of which are poorly understood. Urinary tract obstruction‐induced increase in the levels of vasoactive endothelin (ET)‐1 has been reported in humans and rodents. However, whether obstructive renal vasculopathy is associated with increased renal production of all ET isoforms (ET1‐3) remains unresolved. Also, the mechanisms that underlie ET‐induced renal vasoregulation in neonates are unclear. Here, we show that ET receptor (ETR)‐operated calcium entry via TRPC3 channels increases renal vascular resistance and reduces RBF in neonatal pigs. Acute unilateral urinary tract obstruction (UUO) in neonatal pigs increased peptidase endothelin converting enzyme 1 (ECE1)‐dependent renal production of ET1‐3 and diacylglycerol (DAG). Despite the removal of obstruction, acute UUO reduced kidney perfusion and caused an increase in renal vascular resistance (RVR) and the urinary levels of NGAL, the early predictive biomarker of acute kidney injury. The protein expression levels of ETRs (ETA and ETB) and TRPC3 channels in renal microvessels were unaltered in neonatal pigs subjected to acute UUO. However, pharmacological inhibition of ECE1, ETR, and TRPC3 mitigated acute UUO‐induced increase in RVR and urine NGAL in the pigs. These data suggest that increased ECE1 production and proteolytic processing of all big ETs to their vasoactive isoforms contribute to early neonatal renal insufficiency engendered by UUO. ET‐derived DAG activates renal vascular smooth muscle cell TRPC3 channels, leading to extracellular calcium entry, vasoconstriction, increased RVR and sustained hypoperfusion

Support or Funding Information

NIH/NIDDK: R01DK101668 and R56DK120595
Probing the crosslinking independent roles of tissue transglutaminase in vascular aging using a novel transamidation‐deficient TGM2‐C277S mouse model
Authors: Santhanam Lakshmi , Chen James , Melucci Sean , Jandu Sandeep , Nandakumar Kavitha , Wang Huilei , Barreto-Ortiz Sebastian
Published: April 2020

Introduction

Tissue transglutaminase (TG2) is a multifunctional protein of the transglutaminase family with putative transamidation‐independent functions in aging associated vascular stiffening and dysfunction. The development of preclinical models is critical to fully understand the physiological relevance of transamidation independent functions of TG2 and to identify the specific function of TG2 for therapeutic targeting. Therefore, In this study we harnessed the CRISPR‐Cas9 gene editing technology to introduce a mutation at the active site cysteine (C277) in the mouse TGM2 gene.

Methods and results

We investigated the contributions of the transamidation‐dependent vs. transamidaiton‐independent functions of TG2 in vascular aging using TGM2‐C277S mice, littermate WT mice, and age‐matched TG2−/− mice. Heterozygous and homozygous TGM2‐C277S mice were phenotypically normal and were born at the expected Mendelian frequency. TG2 protein abundance was similar to wild‐type (WT) in the TGM2‐C277S mutant mice in the liver, lung, heart, aorta, and kidney. GTP binding, and interactions with fibronectin and integrin beta1 were preserved in the TG2‐C277S mouse protein. Transglutaminase function was absent in the TG2 protein from TGM2‐C277S mice when compared with wild‐type (WT) littermates. Loss of transamidation function resulted in a marked loss in serotonin induced vasoconstriction and in vitro remodeling of the carotid artery in response to chronic vasoconstriction. On the other hand, cell adhesion and proliferation were comparable to that of WT in the vascular smooth muscle cells from TGM2‐C277S but not in those from TG2−/− mice. Vascular stiffness increased with age in WT mice, as measured by pulse wave velocity, tensile testing, and pressure myography. TGM2‐C277S mice were markedly protected from age‐associated vascular stiffening, but not to the same extent as TG2−/− mice.

Conclusions

Together, these studies show that the TGM2‐C277S mice ubiquitously express a TG2 protein that lacks transamidation function and retains its other functions and can be used a tool for in vivo studies to examine the transamidation‐independent roles of TG2 in physiology and pathophysiology. While TG2‐mediated ECM crosslinking and VSMC dysfunction, both propagate vascular stiffening in aging, the transamidation function is the primary shareholder in aging associated mechanical decline of the large vessels.

Support or Funding Information

This work was funded by a StAAR Investigator Award (to LS) and a MedImmune Research Funds Award (to LS).
Endothelial shear stress in the common carotid artery during boxing training in prehypertension
Authors: Gomez Manuel , Morales-Acuna Francisco , Rodriguez Lisa , Caraveo Paulina , Gurovich Alvaro N.
Published: April 2020

PURPOSE

Endothelial function is highly regulated by the friction between blood flow and the endothelium. Endothelial shear stress (ESS) is defined as the dragging force generated by this interaction and it has been reported that low ESS affects nitric oxide bioavailability which in turn might increase blood pressure. Exercise programs are one of the best suited approaches to treat high blood pressure, however, there are no studies describing changes on ESS in the common carotid artery during upper‐body exercise, such as boxing training. Therefore, the purpose of this study was to quantify ESS in the common carotid artery during 60% and 95% of the maximal boxing exercise capacity in normotensive and pre‐hypertensive subjects.

METHODS

A total of 5 healthy normotensive and 5 pre‐hypertensive subjects matched by age, gender, height, and weight were recruited for this study. All 10 subjects performed two boxing exercise tests. The first was a graded maximal boxing test to estimate their maximal oxygen uptake (VO2max). The second one, performed 48 hours after the first evaluation, was a 2‐workload steady‐state boxing test at 60%VO2max and at 95%VO2max for 3 minutes each. A high‐definition Doppler ultrasound recorded common carotid artery diameters and blood flow velocities throughout each steady‐state condition. ESS was estimated using Womersley’s approximation.

RESULTS

There was a significant increase in antegrade ESS with higher workloads in both groups (p < 0.05 for all). No difference were found in anterograde ESS at baseline (Normotensive: 33.9±13.9 dynes/cm2, Pre‐HTA: 34.7±5.5 dynes/cm2; p = 0.936), at 60%VO2max (Normotensive: 51.3±19.1 dynes/cm2, Pre‐HTA: 49.6±7.6 dynes/cm2; p = 0.894, and at 95%VO2max (Normotensive: 72.9±30.9 dynes/cm2, Pre‐HTA: 85.2±12.5 dynes/cm2; p = 0.560) between both groups. Meanwhile, no retrograde blood flow was present at baseline for either groups, but it was identified at 60%VO2max (Normotensive: 8.1±0.7 dynes/cm2, Pre‐HTA: 7.8±7.8 dynes/cm2; p = 0.971) and 95%VO2max (Normotensive: 22.5±18.9 dynes/cm2, Pre‐HTA: 20.8±5.6 dynes/cm2; p = 0.891).

CONCLUSION

ESS increases in an exercise‐intensity manner during boxing training in normotensive and prehypertensive population. Boxing training might be beneficial in high blood pressure prevention due to increments on ESS.
Investigating the role of the mesentery in the innervation of the gut and enteric nervous system during embryological development ‐ an immunohistochemistry approach
Authors: McDermott Kieran , Cronin Fiona , Coffey Calvin
Published: April 2020

Recent clarification of mesenteric anatomy has indicated that, unlike classical descriptions, the intestines are enveloped in a continuous, rather than a fragmented mesentery. This has prompted a need to re‐examine its development and it anatomical relationships with the developing gastrointestinal tract(GIT). Advances in understanding the development of the mesenteric tissues has also highlighted its potential role as a facilitator of neural crest cell migration during enteric nervous system development. Furthermore spatial and temporal differences in the nature of the mesentery may influence how the different regions of the GIT develop.

The aim of this research is to describe the development of the mesentery, gut and enteric nervous system independently and subsequently determine how each of these components relates to each other during development. Of particular interest is clarifying the manner in which efferent and afferent autonomic fibres develop, traverse the mesentery and, ultimately innervate the gut and the enteric nervous system. Histological and immunohistochemical approaches are being employed to highlight autonomic axons and mesenteric components of rat tissue at different embryonic ages. Animal procedures were in accordance with Republic of Ireland Department of Health and Children license. Immunohistochemical analysis of serially sectioned rat embryos at embryonic days 12, 14 and 16 is being undertaken, which will allow detailed microscopic confirmation of the continuous mesenteric structure, arising from its primitive mesodermal framework, and will illuminate the process underpinning how autonomic fibres are distributed to the GIT. A further objective is to advance this study of mesenteric development in rodent tissue to human embryonic tissue and, ultimately, to describe key molecular determinants of mesenteric‐directed axonal pathfinding. This will address the paucity of information available on the development and anatomical course of peripheral nerves through the mesentery in the human. An understanding of the developmental innervation of the mesentery and gut has implications for advancing knowledge of normal anatomy as well as relevant pathological states such as Hirschsprung’s Disease.
Analysis of Published PCR Primers to Determine Sex of Industrial Hemp Seedlings
Authors: Nalesnik Allison , Erickson Les
Published: April 2020

Industrial hemp refers to non‐intoxicating strains of Cannabis sativa that produce high levels of cannabidiolic acid (CBDA), the precursor to cannabidiol (CBD). CBD has known health benefits and is being tested for a wide range of potential medicinal uses. When growing hemp for CBD, unfertilized female plants are desired as they produce high amounts of CBDA in their flower organs. Therefore, early identification and elimination of male hemp plants is imperative and allows cultivators to spend fewer resources on plants that won’t produce flower and could possibly pollinate female plants. It has been reported that male cannabis plants can be identified weeks before they show any visual sex features using the polymerase chain reaction (PCR), however the results from reported studies have been inconsistent in identifying male plants. Here, genomic DNA from thirteen hemp seedlings was analyzed using male‐DNA‐specific PCR primer pairs described in the research literature. Three different primer pairs (SCAR323, SCAR119, and MADC2) were used in the analyses. SCAR323 primers have been reported to amplify a male‐specific amplicon of 323 base pairs (bp); SCAR119 have been reported to amplify a male‐associated amplicon of 119 bp; and MADC2 primers have been reported to amplify a male‐associated amplicon of 300 bp and a female‐associated amplicon of 450 bp. PCR analyses showed DNA from all 13 of our tested hemp seedlings produced a single SCAR323 amplicon of around 320 bp, suggesting this primer set might not be useful for sex determination. A SCAR119 amplicon of the expected size was seen using DNA from 6 of the 13 seedlings. The DNA from the same 6 plants produced an amplicon of about 400 bp using the MADC2 primers, while DNA from plants that lacked SCAR119 amplicon showed a MADC2 amplicon of about 550 bp. After seven weeks of growth, the hemp plants revealed the presence of male or female reproductive organs. The presence of male pollen sacs correlated with the SCAR119 amplicon and the MADC2 amplicon of 400 bp, indicating these primer sets could be a useful tools for determining plant sex at the seedling stage.
Malin, an E3 Ubiquitin Ligase, Modulates glycogen metabolism in multiple cellular compartments
Authors: Gentry Matthew , Sun Ramon C. , Dukhande Vikas V. , Zhou Zhengqui , Young Lyndsay E. , Emanuelle Shane , Brainson Christine Fillmore
Published: April 2020

Glycogen is the primary source of storage carbohydrate in mammals. Glycogen synthesis and degradation either consumes or produces glucose‐6‐phosphate (G6P), a key metabolite essential for central carbon metabolism. Several studies have reported glycogen accumulation in specific sub‐cellular organelles, suggesting that glycogen localization is not random. Nuclear glycogen was first reported in the 1890s in hepatocytes, and subsequent reports identified glycogen accumulation near the ER, and mitochondria. Cumulatively, these data suggest compartment‐specific roles for glycogen that have yet to be fully elucidated. The E3 ubiquitin ligase malin is a modulator of glycogen architecture through ubiquitination of multiple glycogen metabolic enzymes. Our previous work and that of others demonstrated that malin activity is required for normal glycogen architecture as somatic loss‐of‐function mutations in malin results in aberrant glycogen. We report that decreased levels of malin in human NSCLC samples results in aberrant accumulation of nuclear and cytoplasmic glycogen followed by glucose metabolism. We employed ultra‐pure nuclear preparations coupled with stable isotope technology to define the origin and biological destiny of nuclear and cytoplasmic glycogen. We show that malin is required to maintain compartmentalized glycogen metabolism, and loss of malin in NSCLC results in 1) the accumulation of nuclear glycogen and reduced histone acetylation in the nucleus, and 2) aberrant accumulation of cytoplasmic glycogen accompanied by increased glucose central carbon metabolism. Re‐expression of malin in model NSCLC cell lines, normalized both nuclear and cytoplasmic glycogen, and decreased glucose metabolism both in vitro and in vivo. Finally, malin expression blunted in vivo xenograft growth of model NSCLC cell lines. These data provide new insights for an alternative signaling pathway that regulates metabolism in NSCLC to drive tumor progression.
Dietary iron modulates fecal microbiota and severity of enterotoxigenic E. coli infection in a weanling pig model
Authors: Ji Peng , Li Chong , Kim Byeonghyeon , Jinno Cynthia Nanako
Published: April 2020

Background

Enterotoxigenic E. coli (ETEC) infection is among the leading causes of diarrhea in young children who live in resource‐limited countries, wherein iron deficiency anemia is usually more prevalent. It is debated whether iron supplementation increases the risk of enteric infection.

Objective

the study used a weanling piglet model to investigate the effect of dietary iron on severity of ETEC infection and host microbiota.

Methods

Thirty‐two weaned piglets were blocked by body weight and randomized to the treatments. Pigs were fed 1) an iron‐replete diet (150 mg iron/kg) and received sham inoculation (CON), 2) a low‐iron diet (54 mg iron/kg) and orally inoculated with ETEC (LOI), 3) the iron‐replete diet and orally inoculated with ETEC (COI), or 4) a high‐iron diet (700 mg elemental iron /kg) and orally inoculated with ETEC (HII). Pigs were individually housed with free access to water and diets. After 10 days adaptation to diet, pigs were orally inoculated with a 3‐mL dose of F18+ ETEC (1010 cfu) or sterile saline solution for 3 days. Alertness and fecal consistency were scored daily since day 0 post‐inoculation (d0PI). Fecal samples were collected for 16S RNA sequencing and examining the presence of pathogen. Data was analyzed using PROC MIXED of SAS (v.9.4) with repeated measures for variables measured over time.

Results

Pathogen was positive in fecal samples from inoculated pigs on d3PI, but was absent in most pigs on d6PI. Pigs in LOI had lower hemoglobin and hematocrit compared to those in HII before ETEC infection (P < 0.05), but dietary iron content did not affect growth before infection. Enterotoxigenic E. coli infection reduced hemoglobin and hematocrits compared to CON, but only pigs in LOI had significantly retarded growth after infection (P < 0.05). There were more days of diarrhea (fecal score > 2) in LOI than in CON and COI (P < 0.05). Treatment did not affect alpha‐diversity on d0 or d7PI. Relative abundance of Actinobacteria and Firmicutes were affected by treatment on d7PI. Within the phylum of Actinobacteria, Bifidobacteriaceae was more abundant in LOI (37.3%) than that in CON (2.0%) and HII (5.0%), while the opposite pattern was observed for Coriobacteriaceae. Within the phylum of Firmicutes, Lactobacillaceae had the greatest abundance in LOI (28.8% and 31.7%) in comparison with all the other groups (7.8% – 17.7%) on d0 and d7PI (P < 0.05). Same pattern was observed for Veillonellaceae, while Clostridiaceae had the highest abundance in HII (22.8%) and the lowest in LOI (5.7%) on d7PI (P = 0.07).

Conclusion

Iron deficiency predisposed host to increased vulnerability to ETEC infection in weanling pigs. Both iron‐deficient and iron‐excess diet alters fecal microbiota with more prominent effect post‐ETEC infection.

Support or Funding Information

NIFA hatch project
Manipulating PrP Glycan Structure to Understand Toxic Signaling Pathways Driving Prion‐Induced Neurodegeneration
Authors: Callender Julia A. , Sevillano Alejandro M. , Soldau Katrin , Khuu Helen , Aguilar-Calvo Patricia , Sigurdson Christina J.
Published: April 2020

Prion proteins cause an infectious and rapidly progressive neurodegenerative disease characterized by an exponential increase in prion aggregates as well as spongiform encephalopathy, dystrophic neurites, and neuronal death. These processes depend on the neuronal expression of prion protein (PrPC), which exists on the outer leaflet of the cell membrane as a glycosylphosphatidylinositol (GPI)‐anchored glycoprotein containing two variably occupied N‐linked glycosylation sites on its carboxy terminus. Previous work has shown that glycan modifications may impact PrP aggregation and neuronal toxicity. To further investigate the role of glycans in prion‐induced neurotoxicity, we have engineered a new knockin mouse model that expresses PrP with an additional glycan on its amino terminus. This mouse spontaneously develops neurodegeneration characterized by spongiform encephalopathy. In contrast to other murine models of prion disease, this neurodegenerative pathology develops in the absence of PrP aggregates or infectivity, as shown by RT‐QuIC, ThT fluorescence, and inoculation of wild‐type mice with brain homogenates from the knockin mouse. Therefore, this model provides the opportunity to investigate the neurotoxic role of PrPC, uncoupled from its aggregation. We show that although the extra glycan does not affect PrPC expression, stability, or turnover in cells, the brains of mice display alterations in autophagy signaling proteins. We are currently further defining the N‐linked glycans on PrPC, with the goal of understanding how glycans mediate the neurotoxic functions of prion protein. These studies hold relevance not only to diseases of prion aggregation, but also to neurodegenerative diseases characterized by protein aggregation in general, including Alzheimer’s, Parkinson’s, and Huntington’s Disease.

Support or Funding Information

This work was supported by NIH NS15498 awarded to Christina Sigurdson, and by the National Heart, Lung, and Blood Institute of the National Instituters of Health under K12HL141956. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.