bluuebunny/crossref_metadata_2025 huggingface dataset:
Short‐term hindlimb immobilization induces rapid loss of bone strength in a mouse model of disuse atrophy
Authors: Khayrullin Andrew , Parker Emily , Kaiser Helen , Yu Kanglun , McGee-Lawrence Meghan , Hamrick Mark
Published: April 2020

Muscle atrophy and bone loss are known to occur in settings of disuse such as prolonged bedrest, spinal cord injury, or exposure to microgravity with spaceflight. It is also well‐established that crosstalk exists between muscle and bone mediated by myokines such as myostatin and irisin, and that changes in these muscle‐derived factors with exercise, aging, or muscle atrophy may contribute to changes in bone formation and resorption. Muscle loss in many cases often precedes bone loss, and can be more rapid relative to detectable changes in bone mass or density. We investigated short‐term (2 weeks) changes in muscle and bone with disuse atrophy utilizing a single hindlimb immobilization model to identify associated between changes in muscle mass and bone strength. Twelve‐month C57/BL6 mice (8 male, 4 female) were subjected to two weeks of hindlimb (left hindlimb) immobilization, using a casting technique that involved wrapping surgical tape from the pad of the foot to the proximal thigh with a final layer of vetwrap covering the surgical tape. Mice were euthanized following IACUC‐approved procedures, and quadriceps muscles weighed and fiber size measured histologically. Femora were snap frozen in liquid nitrogen and later rehydrated in PBS for 1 hour prior to mechanical testing, after which they were loaded to failure in 3‐point bending (10 mm span length, 10 mm/min) on a Bose ELF mechanical testing system. Results indicate that quadriceps mass and fiber size declined significantly in immobilized (left) hindlimb compared to the non‐immobilized hindlimb, decreasing approximately ~20% and 30%, respectively over the two‐week treatment period. Ultimate force of the femur also declined significantly in the mice over this timeframe, decreasing up to 20% in the male mice. Regression analyses demonstrate that quadriceps mass and fiber size were each significantly correlated with femur ultimate force (P=.01), and that correlation coefficients between muscle mass and fiber size (r=.48) were similar to correlations between muscle mass and femur ultimate force (r=.47) and muscle fiber size and femur ultimate force (r=.47). These findings suggest that even in the setting of short‐term disuse declines in muscle mass and bone strength are tightly coupled, and that therapeutic interventions to mitigate loss of muscle and bone should be considered to reduce fracture risk.
Formulation of CRISPR Edited Cells to Evaluate Glypican‐Targeting in Glioblastoma
Authors: Pitcher Morgan , Zewdu Tehut , Yusuf Diamond , Karmacharya Trishala , Farrell Nicholas , Katner Samantha
Published: April 2020

Heparan sulfate proteoglycans (HSPGs) are ubiquitous protein/carbohydrate complexes that are found on all cell surfaces. They play a role in various biological pathways such as cellular growth and vascularization, processes that are exploited by cancer. Heparanase is an enzyme that modifies HSPGs which can alter the structure of the proteoglycan and change its bioactivity. In glioblastoma, these enzymes are overexpressed and are thought to contribute to the rapid and aggressive growth of cancer. HSPGs, particularly glypicans, are known to be overexpressed in glioblastoma. Here, we generated double and single knockouts of glypican‐3 (GPC3) and glypican‐1 (GPC1) in glioblastoma cells using CRISPR editing with specific sgRNA. Sanger sequencing and western blot determination were utilized to verify the knockout. We then compared these knockouts to unedited glioblastoma cells to understand the importance of glypicans in glioblastoma as well as investigate their sensitivity to platinum‐based chemotherapeutics. The further exploration of targeting HSPGs in general could allow for tailored chemotherapeutic treatments to patients with glioblastoma. This may pave the way for new molecular targets and more effective treatments for glial tumors in the future.

Support or Funding Information

The President's Commission for the Status of Women for funding my research. My collaborates Dr. Karin Forsberg Nilsson at Uppsala University, Sweden for donating the GL261 cells. Dr. Nicholas P. Farrell at the Massey Cancer Center, Virginia Commonwealth University, Richmond, VA for collaborating with me.

Dr. Samantha Katner for her continuous support, encouragement, guidance and great mentorship
Loss of Caveolin‐1 Increases Macromolecule Transport Across Lymphatic Endothelium
Authors: Baranwal Gaurav M. , Upchurch Bradley D. , Cromer Walter E. , Smithhart Matt C. , Vadlamani Suman S. , Clark Mary-Catherine C. , Blais Stephanie N. , Zawieja David C. , Dongaonkar Ranjeet M. , Rutkowski Joseph M.
Published: April 2020

Transport of fluid and macromolecules from the interstitium into lymphatic vessels is necessary for maintaining tissue homeostasis. Lymphatic capillary structure suggests that passive, paracellular transport would be the predominate route of macromolecule entry into lymphatics. Active transcellular transport, however, was recently identified to be equally important to solute flux across lymphatic endothelium in vitro. To specifically test if loss of caveolar transport impacted lymphatic transport, lymphatic conductance and permeability were measured in mice with a global deletion of caveolin‐1 (“CavKO”), the scaffolding protein necessary for caveolae formation, and in mice with a lymphatic endothelial cell‐specific deletion of caveolin‐1 (Lyve1‐Cre x Cav1flox/flox; “LyCav”). In each mouse line, lymphatic capillary architecture was largely unchanged. Using a quantitative microlymphangiography model, lymphatic conductance (mm3 fluid uptake/mm3 tissue/time) was measured to be significantly higher in CavKO mice. Increased lymphatic conductance was also measured in CavKO mice following nitric oxide synthase inhibition and in LyCav mice with lymphatic‐specific loss of caveolae. The permeability to 70kDa dextran was significantly increased in monolayers of lymphatic endothelial cells isolated from CavKO mice. These findings confirm that macromolecule transport is increased with loss of caveolar trafficking. Interestingly, macromolecule transport within the lymphatic system, measured by transit rate of dextran to a sentinel lymph node, was significantly reduced in CavKO and LyCav mice and isolated vessel studies identified increased permeability of CavKO collecting lymphatic vessels resulting in macromolecule loss from the lumen. These studies identify lymphatic caveolar biology as a key regulator of active lymphatic transport functions.

Support or Funding Information

This research was supported by the Texas A&M University College of Medicine and Department of Medical Physiology.
Disentangling The Heterogeneity Of The Midfacial Epithelium To Elucidate The Pathogenesis Of Cleft Lip/Palate
Authors: Llabata Marta Losa , Faure Louis , Chacon Brandon , Adameyko Igor , Selleri Licia
Published: April 2020

Craniofacial development results from a tightly regulated choreography involving patterning, morphogenesis, and growth of multiple embryonic tissues and cell types. Clefts of the lip and/or palate (CL/P) are maiming disruptions of facial shape and represent the most common human craniofacial birth defect. During mammalian facial development, the maxillary prominence extends medially to contact the two nasal prominences (medial and lateral) at a three‐way anatomical seam named lambdoidal junction (λ), wherein the three prominences coalesce. When fusion of the facial prominences is impaired, orofacial clefting ensues. We have previously reported an essential role for the cephalic epithelium at the λ to enable normal fusion of the murine embryonic facial prominences. Using the mouse as a model, I am currently studying the spatial and temporal characteristics of these epithelial subpopulations using single‐cell RNAseq (scRNAseq) on microdissected facial prominences prior and after fusion occurs. By this approach, I have identified the transcriptional profiles that characterize the different epithelial subpopulations at the λ. Employing different bioimaging and fate mapping techniques, I am characterizing the cell behaviors and trajectories of selected λ epithelial subpopulations. In parallel, I am also conducting these analyses directly comparing control and mutant mouse embryos obtained from available genetic mutant lines with orofacial clefting (Pbx1/2 compound mutants). These studies have identified key changes in both transcriptomic signatures and cell behaviors in mutant mouse embryos, providing a deeper understanding of the molecular mechanisms underlying the pathogenesis of CL/P. Altogether, these approaches are helping me to elucidate the spatiotemporal dynamics of the cell subpopulations that comprise the λ epithelium in vivo, and the perturbed landscapes that cause disfiguring anomalies of the facial anatomical features in mammals.

Support or Funding Information

This work was funded by the National Institute of Health [RO1 grant DE024745 to L.S] and by the American Association of Anatomists [Postdoctoral Fellowship to M.L.].
Toxicokinetic of Sublethal Concentrations of Nitrite in Goldfish (Carassius auratus)
Authors: Martinez Melody Anaiz , Mancha Fabiola , Gomes Ana , Huertas Mar
Published: April 2020

Increased agriculture and industrial activity have elevated the concentration of nitrogenous compounds in the terrestrial and aquatic ecosystems. Nitrogenous compounds can naturally occur in aquatic ecosystems, concentrations are controlled and maintained through the nitrogen cycle by bacterial denitrification/nitrification. As large quantities of nitrogenous compounds drain into streams and rivers it can dramatically alter the natural nitrogenous concentrations in aquatic ecosystems. Nitrite is an intermediate compound in the nitrogen cycle, and its concentrations can range from 1 mM to 0.5 mM in freshwater and marine ecosystems. Exposure to elevated nitrite concentrations can disrupt physiological and endocrine processes such as ion and hormone regulation, respiratory, and cardiovascular activity. However, there is a lack of research in the effects of nitrite on sensitive organs such as the olfactory epithelium. Fish olfactory systems is highly sensitive and can detect odors in the picomolar concentration. To detect odors, this specialized sensitive system must be continuously exposed to the environment, making it highly susceptible to environmental toxins. We hypothesize olfactory tissues will be more sensitive to long term exposure to sub lethal concentrations of nitrite. Thus, the goal of this study is to determine the chronic effects of nitrite and its accumulation in the olfactory system and vital organs in goldfish (Carassius auratus). To test our hypothesis, goldfish were exposed to nitrite concentrations (0 mM, 10.0 μM, 100.0 μM, and 1000.0 μM) for 69 days. The nose, brain, gill, liver, kidney, and blood samples were collected on the last day of exposure. Samples were flash frozen (20°C) for biochemical analysis and subsamples were fixed in Bouin or 4% Paraformaldehyde (PFA). After 18hr, subsamples were transferred to ethanol or sucrose, respectively. Sub‐samples of flash frozen brains were dissected, hypothalamus samples were collected for RNA analysis. Notable browning or darkening of blood was seen in goldfish from 100.0 μM, and 1000.0 μM treatments, indicating irreversible oxidation of hemoglobin known as methemoglobinemia. Biochemical analysis showed nitrite accumulated in the nose, brain, and gill tissues (1.0 μM ±0.3, 8.1 μM ±1.4, and 11.1 μM ±0.4, respectively) in 100.0 μM treatment. Nitrite concentrations in these tissues increased as treatment concentration increased. RNA samples were prepared for qPCR analysis to measure gene expression related to appetite. Preliminary histological analysis of nose, gill, liver, and kidney tissues shows structural changes in tissues. The biochemical results indicate environmental concentrations of nitrite accumulate in vital tissues. Furthermore, findings indicate nitrite is entering and accumulating in the brain. These results suggest, nitrite is potentially acting as a neurotoxin.

Support or Funding Information

Research support: Texas State University start‐up funding to Dr. Mar Huertas.
Effects of Neem Extracts on Formed Biofilm of Pseudomonas aeruginosa
Authors: Kaverimanian Vishnu , Heuertz Rita M.
Published: April 2020

Over time, bacteria have been developing increasing resistance to commonly used antibiotics in medicine. To compound this issue, certain surface adherent bacteria can undergo a community response to form biofilms, a protective layer consisting of extracellular polymeric matrix that enhances resistance to antibiotics and host defenses. Biofilm association can confer bacteria with up to 1,000 times more antibiotic‐resistance. According to the NIH, up to 65% of all human infections and 80% of chronic infections are biofilm‐associated. P. aeruginosa, a common nosocomial pathogen exhibiting antibiotic resistance, is a known biofilm producer. Due to the prevalence of biofilms and their role in the antibiotic resistance of common nosocomial pathogens, it is important to look for antibiofilm agents. One understudied source of antibiofilm agents is plant derived compounds (phytochemicals). Of note, neem leaf extracts have been shown to inhibit biofilm formation in scientific literature. The purpose of my project was to identify the effect of neem extracts (bark, fresh leaf, dried leaf) on eradicating pre‐formed biofilm of P. aeruginosa (ATCC 27853) via a microplate biofilm quantitative assay. Bacteria were first inoculated into tryptic soy broth (TSB) and incubated (37°C, overnight). Afterwards, bacteria were density standardized (A590=0.2), transferred to microplates (polystyrene U‐bottom 24‐well), and incubated (37°C, overnight) to form biofilm. To isolate and treat the pre‐formed biofilm, microplates were washed (3 times, TSB), TSB was replaced, neem extracts were placed in the appropriate wells (a final concentration of 10% for each neem extract), and plates were incubated (37°C, 5 days). Afterwards, biofilm associated bacteria were killed with glutaraldehyde (10%) and microplates were washed again (3 times, dH2O). Subsequently, crystal violet (0.1% aqueous) was added to stain the biofilm. Excess dye was removed by washing (3 times, dH2O) and stained biofilm was solubilized (8% acetic acid). Solubilized biofilm was quantitated by A590 readings. Results indicated that all the neem extracts had no significant (p<0.05) effect on biofilm eradication for P. aeruginosa 27853. This suggests that neem extracts may be more effective on developing biofilm rather than formed biofilm. Further strains will be tested for neem effect on biofilm eradication.

Support or Funding Information

Financial support for VK was received from the DeNardo Education and Research Foundation.
High‐fat diet increases p62 and mTOR phosphorylation in diabetic eNOS deficient mice
Authors: George Jasmine , Chen Xiaoming , Zhao Xueying
Published: April 2020

Diabetic nephropathy is the leading cause of chronic kidney disease and end stage renal disease. Emerging evidence has suggested that dysregulated renal autophagy may contribute to both glomerular and tubulointerstitial damage under diabetic conditions. This study was designed to elucidate the effect of a high‐fat diet on autophagy in eNOS deficient animals with and without hyperglycemia. Six‐week old wild‐type (WT) C57BL/6J and eNOS deficient mice were placed on either a normal diet (ND, 4.5% from fat) or high‐fat diet (HFD, 45% from fat). Two weeks later, half of them were rendered diabetic with alloxan. Animals were maintained on ND or HFD for 5 months post diabetes induction. Kidney damage and autophagy activity were evaluated in diabetic WT and eNOS deficient mice. HFD and diabetes induction in eNOS deficient mice resulted in significantly increased urinary albumin to creatinine ratio, matrix metalloproteinase‐9, and kidney injury molecule‐1. Western blot further indicated a significant increase in autophagy substrate p62 in kidney cortex of diabetic eNOS deficient mice fed with HFD, whereas there was no notable change in p62 expression in WT mice. In addition, mammalian target of rapamycin (mTOR) phosphorylation was increased in diabetic eNOS deficient mice. Taken together, our results suggest that HFD may impair renal autophagy by activating mTOR in diabetic eNOS deficient mice.

Support or Funding Information

NIH (SC1DK112151), NIH/NCRR /RCMI (8G12MD007602) and (8U54MD007588), and R25GM058268
The Role of FSH in Osteoporosis and Subsequent Oral Disease
Authors: Fitz Emily , Cohen Brian D.
Published: April 2020

Osteoporosis is an age‐related chronic disease in which osteoclasts degrade minerals of the bone faster than osteoblasts can replace them. In addition to increased risk of fracture, osteoporosis patients experience oral, dental and prosthodontic complications due to degradation of the alveolar and jaw bone. Elderly women are disproportionately impacted by this disease. One potential culprit for the gender‐based disparity is the increased levels of follicle stimulating hormone (FSH) during menopause. An isoform of the FSH receptor can be found on osteoclasts; when FSH binds to this receptor, osteoclast activity and differentiation are increased. Murine monocytes were differentiated into osteoclasts using RANK ligand, and FSH dependent signaling was measured through western blotting. Protein phosphorylation was detected in response to FSH in osteoclasts. A better understanding of the osteoclast FSH receptor could allow for pharmaceutical intervention to interrupt the pathway. Negative allosteric modulators (NAMs) that inhibit FSH signalling on granulosa cells could be used for inhibiting FSH signalling on osteoclasts.
Effects of Ambient Temperature and Social Housing on Locomotor Activity, Thermoregulation, and Weight Loss Following “Binge” Administration of Structurally‐Related Abused Psychostimulants
Authors: Russell Lauren , Hyatt William , Urquhart Kyle , Fantegrossi William
Published: April 2020

3,4‐methylenedioxypyrovalerone (MDPV) and α‐pyrrolidinopentiophenone (α‐PVP) are common active constituents of abused “bath salts” products. The structures of these synthetic cathinone analogues differ only in that MDPV contains a methylenedioxy ring. Additionally, MDPV is structurally similar to the amphetamine analogue 3,4‐methylenedioxymethamphetamine (MDMA), which differs fromthe structure of methamphetamine (METH) only by the addition of a methylenedioxy ring. MDPV, α‐PVP, MDMA, and METH are four psychostimulants often used interchangeably by humans in crowded, hot environments. The effects of MDMA are known to be dramatically impacted by crowding and ambient temperature and because of the structural similarities and use patterns in humans, it was of interest to investigate the modulatory effects of such environmental variables on the in vivo pharmacology of the four compounds in mice. In the present studies, adult male NIH Swiss mice were housed singly or in groups of 3 or 6, at 20°C, then administered a “binge” regimen of 1.0, 3.0, or 10.0 mg/kg MDPV, α‐PVP, MDMA or METH, or repeated saline, via intraperitoneal injection, every 2 hours for a total of 4 injections. Locomotor activity and core temperature were continuously collected throughout drug administration, continuing 2 hours after the 4th injection. The lowest dose per social housing condition different from saline was repeated at an elevated temperature of 28°C. Doses were limited by lethal effects which were potentiated both by the increased ambient temperature and social housing conditions. Increased ambient temperature resulted in increased locomotor activity for all compounds, and an increased hyperthermic effect occurred in animals administered MDPV or MDMA, both methylenedioxy ring containing compounds. No pronounced differences regarding body weight and core temperature as a function of social housing were observed. Brain regions (including hippocampus and striatum) were collected for neurochemical analysis 10 days after the last injection. Regions from one hemisphere will be processed and analyzed for tissue content of monoamines and their primary metabolites by HPLC, while samples from the other hemisphere will be utilized to determine GLUT2 concentration via western blotting. These studies suggest that the addition of specific moieties, in this case a methylenedioxy ring, to structurally similiar cathinones and amphetamines can result in same effect compared to the parent compound.

Support or Funding Information

These studies supported by DA039195, DA022981 and the UAMS Center for Translational Neuroscience.
Developmental Analysis of GABAA Receptor Gene Expression in Gallus gallus Chick Brain
Authors: Nason Taylor , Kubera Cathryn
Published: April 2020

Fetal Alcohol Spectrum Disorders (FASD) are a class of neurological disorders that arise from both ethanol‐related toxicity and alterations in a variety of molecular pathways during fetal development. One result of embryonic ethanol exposure is overactivation of the GABAA receptors within neuronal cells. Due to the varying subunit composition of these pentameric ionotropic receptors in different brain regions and timepoints during development, this experiment characterized the expression levels of nine GABAA receptor subunits (α1–6, β1–3) in an embryonic chick model. RNA was isolated from three brain tissue regions (cerebellum, forebrain, optic tectum). After, the RNA was converted to cDNA by reverse transcription and the gene transcripts were assessed using RT‐qPCR with specifically designed primers for each GABAA subunit type. With the exception of the α6 subunit, expression of the GABAA receptor genes were consistent throughout all brain tissue types and only showed a slight variation in level of expression when it came to the different developmental time points of E7–E17. α6 was absent in forebrain, and not detected until E15 in cerebellum and optic tectum samples. Based on the data collected, it can be concluded that the nine GABAA subunit genes tested are mostly expressed throughout the chicken embryonic timeline. There is currently limited data as to how their gene expression levels change as the embryo develops. Further research is needed to determine the distinct differences between the different subunit types. These subtle differences between the expression of each GABAA receptor subunit gene will point to a better understanding of the influence ethanol has on the developing embryo in the chick model. This evidence may hold meaning to why alcohol exposure during certain time periods in fetal development for humans prove to have more severe effects on the fetus, like FASD, than during other time periods during embryonic growth.
Nicotinamide mononucleotide (NMN) supplementation rescues cerebromicrovascular endothelial function and neurovascular coupling responses and improves cognitive function in aged mice.
Authors: Tarantini Stefano , Valcarcel-Ares Marta Noa , Toth Peter , Yabluchanskiy Andriy , Kiss Tamas , Ballabh Praveen , Farkas Eszter , Baur Joseph , Sinclair David , Csiszar Anna , Ungvari Zoltan
Published: April 2020

Adjustment of cerebral blood flow (CBF) to neuronal activity via neurovascular coupling (NVC) has an essential role in maintenance of healthy cognitive function. In aging increased oxidative stress and cerebromicrovascular endothelial dysfunction impair NVC, contributing to cognitive decline. There is increasing evidence showing that a decrease in NAD+ availability with age plays a critical role in a range of age‐related cellular impairments but its role in impaired NVC responses remains unexplored. The present study was designed to test the hypothesis that restoring NAD+ concentration may exert beneficial effects on NVC responses in aging. To test this hypothesis 24‐month‐old C57BL/6 mice were treated with nicotinamide mononucleotide (NMN), a key NAD+ intermediate, for 2 weeks. NVC was assessed by measuring CBF responses (laser Doppler flowmetry) evoked by contralateral whisker stimulation. We found that NVC responses were significantly impaired in aged mice. NMN supplementation rescued NVC responses by increasing endothelial NO‐mediated vasodilation, which was associated with significantly improved spatial working memory and gait coordination. These findings are paralleled by the sirtuin‐dependent protective effects of NMN on mitochondrial production of reactive oxygen species and mitochondrial bioenergetics in cultured cerebromicrovascular endothelial cells derived from aged animals. Thus, a decrease in NAD+ availability contributes to age‐related cerebromicrovascular dysfunction, exacerbating cognitive decline. The cerebromicrovascular protective effects of NMN highlight the preventive and therapeutic potential of NAD+ intermediates as effective interventions in patients at risk for vascular cognitive impairment (VCI).

Support or Funding Information

This work was supported by grants the Oklahoma Center for the Advancement of Science and Technology, the Oklahoma Shared Clinical and Translational Resources (OSCTR) program funded by the National Institute of General Medical Sciences (GM104938), the NIA‐supported Geroscience Training Program in Oklahoma (T32AG052363), the Oklahoma Nathan Shock Center (P30AG050911), the Cellular and Molecular GeroScience CoBRE (1P20GM125528, sub#5337), and the NIGMS supported Center of Biomedical Research Excellence (CoBRE).
Myology of the Reptilia
Authors: Holliday Casey M , Wilken Alec , Verhulst Conner , Bales London , Sellers Kaleb , Lessner Emily , Middleton Kevin
Published: April 2020

Sauropsid vertebrates (lepidosaurs, turtles, crocodilians and birds) have evolved a diversity of head shapes and feeding behaviors during their history. A key to understanding this great radiation of reptiles is the physiology of the jaw musculature which powers the feeding apparatus. However we still know little about jaw muscle mechanics within lineages of reptiles or how this complicated musculoskeletal system has evolved to employ a variety of behaviors. New imaging and computational methods are now enabling an extraordinary view into the 3D anatomy and biomechanics of reptiles and other vertebrates. Here we illustrate several approaches to analyzing jaw muscle morphology and architecture using contrast imaging, 3D fiber tracking, biomechanical analysis, and data visualization methods that offer enormous potential for exploring the anatomy, function and evolution of jaw muscles. We first illustrate basic workflow of 3D jaw muscle visualization, morphometrics and interpretation using crocodilian jaw muscle anatomy. Second, we show how homologous jaw muscle bellies evolve among lineages of different reptiles and birds to elicit different functional demands. Third, we show how the 3D architecture of hard‐to‐dissect, small, deep protractor muscles correlate with different types of cranial kinesis among a sample of lizards and birds. These new imaging and analytical approaches offer incredible potential for the quantification of soft tissue morphology like muscle architecture and their applications to comparative biomechanics, ecomorphology, and evolutionary biology should prove remarkable.

Support or Funding Information

NSF IOS 1457319, NSF EAR 1631684, University of Missouri Life Sciences Fellowship, University of Missouri Integrative Anatomy Fellowship, IMSD‐Express
Identification of Putative RNA Methyltransferases in Trypanosoma brucei
Authors: Williams Laura G , Smith Xiane , Taber Cassandra C , Ellis Lauren , Militello Kevin T
Published: April 2020

Human African Trypanosomiasis (African sleeping sickness) is a potentially fatal disease in sub‐Saharan Africa that is caused by the protozoan parasite Trypanosoma brucei. This vector‐borne disease afflicts tens of thousands of individuals every year, and without a vaccine, it is a prominent threat to the health and safety of many African communities. RNA methylation may be an important mechanism for the regulation of gene expression in T. brucei, as this organism lacks regulatory DNA sequences. The presence of 5‐methylcytosine has been detected in the RNA of T. brucei through sodium bisulfite sequencing. Seven putative RNA methyltransferases were identified in the genome of the parasite via bioinformatics and were named TbCRMTs (
T.
brucei
cytosine
RNA
methyl
transferases). The levels of TbCRMT RNA in both procyclic and bloodstream forms were measured via RT‐qPCR and demonstrated the expression of all seven genes in both life cycle stages. TbCRMT4 is 103
kDa, and contains the conserved SAM binding domain and catalytic cysteines found
inbona fide RNA methyltransferases. TbCRMT4 is required for maximum parasite growth as determined by RNAi knockdowns. In an attempt to determine whether TbCRMT4 functions as an RNA methyltransferase, the gene was expressed in E. coli with an N‐terminal 6x‐His tag. SDS‐PAGE results suggested that the full‐length protein was fragmented due to the presence of multiple bands. In light of this, the 6x‐His tag was moved to the C‐terminus and fragments of the gene containing putative SAM‐binding and catalytic sites were then amplified by PCR. The PCR products were then inserted into the pET101/D vector and transformed into competent E. coli. Expression of each construct in E. coli was induced under different conditions, with varying degrees of success. The construct which
contains amino acids 87‐852, when expressed at 37°C, is most reliably produced, as confirmed via SDS‐PAGE/Coomassie blue staining and Western Blot analysis. Isolation of this protein was attempted using denaturing protocols and His‐affinity chromatography. Solubility of the protein has proven difficult, thus optimization of protein isolation is still ongoing. Ultimately, we plan to isolate the TbCRMT4 87‐852 protein and conduct a series of methyltransferase assays, which will determine whether or not this protein catalyzes RNA methylation. By determining if TbCRMT4 functions as an RNA methyltransferase, we can further understand the T. brucei epitranscriptome.

Support or Funding Information

NIH grant 1R15AI133428‐01 to KTM
Characterization of the Dual Pathways for Bacillus halodurans Asparaginyl‐tRNA Formation
Authors: Bile Jon Matthew , Sheppard Kelly
Published: April 2020

Protein synthesis requires the attachment of an amino acid to its cognate transfer RNA (tRNA). Two distinct pathways for attaching asparagine (Asn) to tRNAAsn are known: the direct pathway, in which Asn is directly attached to tRNAAsn by the enzyme AsnRS; and the indirect pathway, in which Asn is attached to tRNA by a non‐discriminating AspRS and GatCAB. Bacillus halodurans uses both routes for Asn‐tRNAAsn formation. To better understand why B. halodurans possesses both pathways, we are purifying its AsnRS and AspRS in order to test and compare their activities under different chemical conditions such as reactive oxygen species and pH. This research will provide insight into how B. halodurans has adapted to survive in different environmental conditions.

Support or Funding Information

National Science Foundation (MCB‐1615770)
Quantifying the Stability of Human Cytomegalovirus RNA5.0
Authors: Tsingine Hannah P.
Published: April 2020

HCMV is the largest of the human herpesviruses containing a DNA genome of approximately 240 kilobases encoding 225 open reading frames (ORFs). Its large and complex genome also encodes long noncoding RNAs (lncRNA) which are known for not being translated, but rather function as RNA. In HCMV, one of the lncRNA, known as RNA5.0, is a stable intron that is believed to play an important role in viral pathogenesis. Recent studies have shown that RNA5.0 is a nuclear localized stable intron that is transcribed by RNA polymerase II, and that it's also enriched with high adenine and thymine residues. The true role of RNA5.0 is still unknown. It could activate transcription, regulate gene silencing, or play a role in HCMV latency. To begin to understand why an intron that should be degraded within minutes is stable for hours, we are developing an assay to quantify the stability of these unusual lncRNA using transfected cultured 293T cells.

Support or Funding Information

NIH MARC U*STAR recipient T34GM092711
Detecting and quantifying oligomerization of ARF PB1 Domains
Authors: Davis Cory Bishop , Johnston Shelby Bishop , Ellis Jamie P.
Published: April 2020

Auxin response factors (ARFs) are a family of transcription factors within plants that serve to regulate the plant’s response to auxin, a growth initiating hormone. ARFs can either serve as transcription activators or repressors to auxin regulation in the presence of auxin. The majority ARFs have 3 domains: a PB1 protein interaction domain, the low‐complexity middle region, and a DNA binding domain. Major differences between ARF activators and repressors lay in large sequence differences in the middle region, but sequence analysis of the PB1s domains show key differences with implications for their role in transcription regulation. PB1 domains are responsible the interactions necessary for auxin regulation. All ARF PB1 domains are type I/II meaning they contain both a positive and negative residue on opposite sides for electrostatic interactions with copies of themselves or other PB1 domains. The potential for bidentate interaction interfaces complicates the model of auxin response by requiring us to consider oligomerization through the PB1 domain including the potential for hetero‐oligomerizatin between ARF activators and repressors. The work examines the degree of oligomerization and quantifies dimerization affinity in both the wild‐type ARF5 PB1, a transcription activator, and the ARF1 PB1, a transcription repressor. More specifically, to answer these questions crosslinking, gel filtration, and fluorescence anisotropy were used to compare a broad range of concentrations for analysis as well as to improve quantification. Both wild‐type ARF5 and ARF1 self‐associated as expected under varying conditions, forming small oligomers, but ARF5 remained primarily in the dimer state. This work suggests a more nuanced model of transcriptional regulation depending on the specific population of regulatory PB1s present.
Differential Regulation of Hepatic UDP‐glucuronosyltransferase (UGT) 1A1 by Toll‐like Receptors during Irinotecan‐induced Steatosis
Authors: Tao Gabriel , Chityala Pavan Kumar , Ghose Romi
Published: April 2020

Irinotecan (CAMPTOSAR®) has been widely used in preoperative chemotherapy for colorectal cancer liver metastases. However, irinotecan‐induced steatosis and steatohepatitis increases mortality of liver surgery. Up to date the mechanism underlying this hepatoxicity remains largely unknown. It is known that in human irinotecan is hydrolyzed to SN‐38, a reactive metabolite, by carboxylesterases in liver. SN‐38 is then rapidly detoxified by enzyme, UDP‐glucuronosyltransferase (UGT1A1) to generate its glucuronide form. Our preliminary study indicated that irinotecan‐induced steatosis is associated with activation of Toll‐like receptors (TLRs) signaling. Previously we reported that activation of TLRs decreased multiple drug metabolism enzymes in liver, including UGT1A1. Hereby, we hypothesize that TLRs are involved in the regulation of irinotecan detoxification. TLR2wt(C57BL/6J), TLR2−/−( B6.129‐Tlr2tm1Kir/J), TLR4wt(C3HeB/FeJ) and TLR4mutant (C3H/HeJ) mice (all male, 5‐weeks old) were treated with 50mg/kg irinotecan via intraperitoneal injection once daily for nine days. Wildtype mice were sacrificed on day 2 and day 9, while TLR‐deficient mice were sacrificed on day 9. Blood and liver were collected for further analysis. We found that in C57BL/6J mice, after two days irinotecan did not cause any changes in liver. However, after nine days constitutive injection in wildtype mice, irinotecan increased proinflammatory cytokines and reduced ugt1a1 expression. Also, it decreased UGT1A1 activity in liver. Compared to wildtype mice, deficiency of either TLR2 or TLR4 signaling alleviated irinotecan‐induced proinflammatory response. However, histology evidences showed that only in TLR2−/− mice but not TLR4mutant mice, irinotecan‐induced intracellular vacuoles in liver became less and smaller than in wildtype mice. Additionally, ugt1a1 downregulation by irinotecan was attenuated in TLR2−/− mice. Pharmacokinetics study indicated that knockout of TLR2 significantly decreased the systemic exposure of SN‐38, the toxic metabolite of irinotecan; no such effect was observed in TLR4mutant mice. Based on the results, we confirm that TLRs are involved in irinotecan metabolism and associated steatosis. Although both TLR2 and TLR4 share the same adaptors to regulate similar downstream pathways, TLR2 probably plays more important role in reducing ugt1a1 during irinotecan‐induced steatosis. Hereby, we conclude that TLR2 is preferentially activated during irinotecan hepatotoxicity and such activation suppress irinotecan detoxification. We characterize a novel feature of TLRs signaling in irinotecan‐induced liver injury. It provides valuable information to address drug safety issues for public health.
Investigating the Potential of NHE1 Inhibitors as Potential Adjuvant Therapies in the Treatment of Ovarian Cancer
Authors: Corradi Anna , Wallert Mark A.
Published: April 2020

Each year in the United States over 200,000 women are diagnosed with ovarian cancer leading to more than 14,000 deaths. Increases in the expression and activity Na+‐H+ Exchanger Isoform 1 (NHE1) was recently demonstrated to regulate cell proliferation in ovarian cancer patients. NHE1 is an 815 amino acid transmembrane transport protein that regulates intracellular pH. NHE1 is hyperactivated in an array of cancer types leading to a pH inversion that supports both cell proliferation and invasion. One mechanism for this NHE1 hyperactivation is phosphorylation of the regulatory domain by a range of protein kinases. A significant issue in ovarian cancer therapy is the development of drug resistance and disease recurrence. In an effort to combat these outcomes, it is hypothesized that combining common ovarian cancer therapeutic agents along with compounds that indirectly inhibit NHE1 activation will enhance the ability of the therapeutic agents to slow ovarian cancer progression. Two ovarian adenocarcinoma cell lines; CAOV‐3 and SKOV‐3 are used in this research. Utilizing a proliferation assay, I assessed the impact of different ovarian cancer therapeutics, Rucaparib, Olaparib, Carboplatin and Cisplatin, in these two cell lines in the presence and absence of inhibitors that decrease NHE1 activity directly. This work has also been expanded to a three‐dimensional environment, investigating the impact of these combination therapies in a spheroid assay. To begin to evaluate mechanisms of NHE1 involvement changes, a steady‐state intracellular pH was measured. It is believed that through the pairing of chemotherapeutics with the NHE1 inhibitors, a synergistic effect will ensue in which the combination is more proficient at reducing cancer growth than either compound can alone. This relationship would allow for a reduction in the required drug dosage and harmful side effects in patients. These data indicate that the direct inhibition of NHE1 may serve as a potential therapeutic target in Ovarian Cancer treatment.
Angiotensin‐(1‐7) Improves Integrated Cardiometabolic Function in Aged Mice
Authors: Miller Amanda Joy , Bingaman Sarah , Mehay Daren , Medina Daniela , Arnold Amy Christine
Published: April 2020

Background

Aging is associated with impaired cardiometabolic function including insulin resistance, elevated blood pressure, and cardiac hypertrophy. Angiotensin (Ang)‐(1‐7) is a beneficial hormone of the renin‐angiotensin system that improves control of blood pressure and glucose homeostasis in models of cardiovascular and metabolic diseases in young mice. To our knowledge, no studies have evaluated the importance of Ang‐(1‐7) to integrated cardiometabolic function in aging. We hypothesized that Ang‐(1‐7) would improve insulin sensitivity as well as decrease blood pressure and cardiac hypertrophy in healthy aged mice.

Methods

Sixteen month‐old male C57BL/6J mice received 6‐week Ang‐(1‐7) [400 ng/kg/min, n=9] or saline infusion (n=9) via subcutaneous osmotic mini‐pumps. All mice were fed standard chow diet (18% kcal from fat). During the last week of treatment, intraperitoneal insulin and glucose tolerance tests were performed and blood pressure was measured via a carotid artery catheter. Heart weight was measured at the end of experiments. Gene expression of tyrosine hydroxylase (index of sympathetic activity), inflammatory markers (IL10, IL1β, IL6, TNFα) and Ang II AT1 and Ang‐(1‐7) Mas receptors were measured in myocardial tissue by quantitative real‐time PCR and quantified with 2‐ΔΔCT methods.

Results

We found that Ang‐(1‐7) decreased systolic [Ang‐(1‐7): 105±2 mmHg, saline: 116±4 mmHg, P=0.031], diastolic [Ang‐(1‐7): 77±2 mmHg, saline: 83±2 mmHg, P=0.041], and mean [Ang‐(1‐7): 86±2 mmHg, saline: 94±3 mmHg, P = 0.023] blood pressure. Furthermore, Ang‐(1‐7) reduced heart weight [Ang‐(1‐7): 0.18±0.01 g; saline: 0.21±0.01 g, P=0.042]. The improvement in cardiac hypertrophy with Ang‐(1‐7) was associated with decreased tyrosine hydroxylase gene expression in the heart [Ang‐(1‐7): 0.4±0.1, saline: 0.9±0.1, p=0.014], with no effect on gene expression of inflammatory markers or angiotensin receptors (p>0.3). Ang‐(1‐7) did not alter body mass [Ang‐(1‐7): 46.5±2.0 g, saline: 45.9±1.5 g, p=0.792] or body composition (p>0.2). Ang‐(1‐7) also improved insulin sensitivity [Ang‐(1‐7): −2896±593 AUC, saline:‐1171 ± 624 AUC, p=0.033] but did not affect fasting glucose levels [Ang‐(1‐7): 180±5 mg/dL, saline: 169±8 mg/dL, p=0.225] or glucose tolerance (p=0.202).

Conclusions

Chronic Ang‐(1‐7) treatment decreases blood pressure, cardiac hypertrophy, and markers of cardiac sympathetic activity as well as increases insulin sensitivity in aged mice. These beneficial cardiometabolic effects of Ang‐(1‐7) occurred in the absence of significant changes in body weight or composition. Overall, these data suggest that Ang‐(1‐7) may provide a novel pharmacological target to improve age‐related cardiovascular and metabolic risk.

Support or Funding Information

NIH Pathway to Independence Award: R00 HL122507 to A.C. ArnoldAHA Postdoctoral Fellowship: 18POST33960087 to A.J. Miller
Progesterone induced blocking factor improves blood pressure, mitochondrial dysfunction and reactive oxygen species in response to sFlt‐1 induced hypertension during pregnancy
Authors: Deer Evangeline , Jones Jalisa , Comely Kyliegh , Cornelius Denise C. , Ibrahim Tarek , Vaka Venkata Ramana , Franks Michael , LaMarca Babbette , Amaral Lorena M.
Published: April 2020

Preeclampsia (PE) is characterized by new onset hypertension in association with placental ischemia, reduced fetal weight, elevated soluble fms‐like tyrosine kinase‐1 (sFlt‐1) and placental mitochondrial (mt) dysfunction and oxidative stress (ROS). Infusion of sFlt‐1 causes hypertension and endothelial and renal dysfunction in pregnant rodent models of PE. However a role for sFlt‐1 in causing mt dysfunction and ROS is unknown. Progesterone induced blocking factor (PIBF), is a product of progesterone signaling in early pregnant which controls pro‐inflammatory processes and thus markers of endothelial dysfunction. We have previously shown that progesterone supplementation improves markers of endothelial dysfunction in the RUPP rat. Moreover, we have more recently shown that PIBF lowers blood pressure in the RUPP rat model of PE. This study was designed to test two hypothesis. We first wanted to determine a role for sFlt‐1 to cause mt dysfunction and ROS during pregnancy. We next hypothesized the PIBF supplementation would lower in sFlt‐1 induced hypertension during pregnancy and improve mt function in response to sFlt‐1 during pregnancy. sFlt‐1 was infused into normal pregnant (NP) Sprague‐Dawley rats (3.7 μg·kg−1·day−1 for 6 days, gestation days 13–19) in the presence or absence of PIBF (2.0 μg/mL) administered intraperitoneal on GD 15 to normal pregnant (NP) rats. Mean arterial blood pressure (MAP) and placental mt ROS were measured on GD 19. Infusion of sFlt‐1 into NP rats increased MAP 112 ±2 (n=11) compared with control NP rats 98±2 mmHg (n=15, p<0.05). Administration of PIBF reduced MAP to 100±1 mmHg in the presence of sFlt‐1 (n=5, p<0.05). Mt ROS in placenta was 108±6 in NP (n=4), 429±32 in NP+ sFlt‐1 (n=3, p<0.05), and significantly reduced to 234±15 in NP+ sFlt‐1+ PIBF (n=3, p<0.05). State 3 respiration, which is indicative of ATP production, was reduced in placentas of sFlt‐1 infused rats versus NP, but was improved with PIBF. Overall, our study implicates a role for sFlt‐1 to reduce placental mt function during pregnancy and that supplementation of PIBF improved placental mt function which was associated with improved blood pressures. Our data indicate the importance of improved progesterone signaling to restore placental function in the presence of sFlt‐1 which led to improved hypertension and therefore should be considered a potential therapeutic for PE.

Support or Funding Information

NIH Grants: RO1HD067541‐06 and P20GM121334; T32HL105324
Sensitivity of Mouse Small Intestinal Immune Tissue Architecture
Authors: Wang Wei , Cromer Walter , Zawieja David
Published: April 2020

The different regions of the small intestinal lymphoid tissues may have developed a different immuno‐cytohistological structures with a different sensitivity in response to different intralumenal contents. However, the distribution and density of immune cell subsets, lymphatics, and their spatiotemporal relationship within or among Peyer’s Patches (PP), the major lymphoid immune tissues from proximal to distal ends of the small intestine, have not been clearly documented. The jejunal (Jej), mid‐segmental (Mid, Btw. Jej & Ile), and ileal (Ile) PPs in C57BL/6 mice were isolated, frozen‐sectioned, and immunohistochemistrico‐stained for the imaging analysis and comparison on the distribution, density, area, and size of immune cells, lymphatics, and their spatiotemporal relations between the peripheral and interfollicular regions within or among the different PPs.

Results

1. Density of CD4+cells in the interfollicular regions (IFR) were significantly higher than those in the peripheral regions (PR) within each PP. 2. Total density of CD4+ or MHCII+ cells in MidPP were significantly higher than those in the JejPP. 3. The total density of CD8+cells in MidPP or CD68+cells in IlePP were significantly higher than those in the JejPP. 4. Total lymphatic area, density, or size in MidPP were significantly larger than those in JejPP. 5. Lymphatic area in IFR of MidPP or its density in IFR of MidPPs or IlePPs were significantly larger than those in the PR of the corresponding PPs. 6. Branched villi‐lacteals & their subsequent submucous lymphatic network wrapped by a higher density of CD4+ & MHCII+ cells directly form a specialized histo‐architecture, branched villi‐Lv‐PP complex for immune recognition/differentiation in mouse small intestine. The histo‐morphological architectures of mouse small intestinal immune tissues infrastructure its sensitivity as an immune‐surveillance sensor, facilitating its sensibility. This study provides a necessary foundation for understanding immune‐related intestinal dysfunctions and diseases.