bluuebunny/crossref_metadata_2025 huggingface dataset:
Seeking Satiety: Dopamine Receptors 1,2 mRNA Expression in Channel Catfish
Authors: Wertz Blaine , Kostner Danica , White Taylor , Moyers Macy , Kobayashi Yass
Published: April 2020

The dopaminergic nervous system regulates pathways of reward, pleasure, and satiety, and is therefore strongly related to food intake and subsequent eating behavior in mammals. Pathologies of this system have been well‐studied in both humans and rodents; however, little is known about this system in fish species, including channel catfish. To increase our understanding in the involvement of the dopaminergic nervous system in regulation of food intake in fish species, the expression of dopamine receptors 1 and 2 (DR1‐2) mRNA, and their modulation in response to changes in food intake was examined. Samples of the brain, hypothalamus, liver, muscle, spleen, kidney, and two segments of intestine were collected from three fish to examine the tissue distribution of DR1 and DR2 mRNA. In addition, the expression of DR1 and DR2 mRNA was examined in four segments of the channel catfish brain. DR1 and DR2 mRNA was detected across all tissues examined, with readily detectable expression in the brain and hypothalamus. Among brain segments, DR2 mRNA was most readily detectable in the hypothalamus and forebrain. Hypothalamic expression of DR1 and DR2 mRNA was unaffected by food exposure when samples were taken at one‐hour prior to feeding, at the time of feeding, and one‐hour post feeding. Currently, we are examining the expression of DR1 and DR2 mRNA in relation to feeding frequency, as well as prolonged fasting to further elucidate the relationship between food intake and dopaminergic neuron function.

Support or Funding Information

K‐INBRE P20GM103418
Long‐term Cardiorespiratory Anomalies in Rat Offspring Following Prenatal Morphine Exposure
Authors: Mayer Catherine A. , MacFarlane Peter M.
Published: April 2020

The incidence of opioid‐related pregnancies is a growing public health concern. Although not all infants born from opioid using mothers require hospitalization, many exhibit a variety of withdrawal symptoms (Neonatal Opioid Withdrawal Syndrome, NOWS) often requiring weeks of neonatal intensive care. There are longer‐term effects of prenatal opioid use including altered CNS function comprising cognitive impairments, neurobehavioral abnormalities and increased risk of Sudden Infant Death Syndrome (SIDS). In fact, abnormal cardiorespiratory regulation in infants born following prenatal opioid exposure includes reduced vagal tone, tachypnea, delayed arousal, and altered ventilatory responses to hypoxia and hypercapnia. In the present study, we used a rat model to characterize long‐term (postnatal (P) 10 days of age) cardiorespiratory responses of the offspring following in utero morphine exposure. Pregnant dams received twice daily (morning and evening) subcutaneous injections of various doses of morphine (1, 5 or 10mg/kg) during the 3rd week of pregnancy to mimic 3rd trimester maternal opioid use. Following birth, rat pups (and the nursing dam) were left untreated and whole‐body plethysmography was used to assess the acute hypoxic (HVR) and hypercapnic (HCVR) ventilatory responses at P10 days of age. Heart rate was also assessed in rats fitted with a custom‐made jacket with built‐in ECG electrodes. Generally, the postnatal effects of morphine were dose‐dependent: 1) increasing doses of in utero morphine exposure caused a progressive attenuation of the HCVR in 10 day old rats; 2) there was a high rate of neonatal (within 24hrs of birth) mortality in the highest (10mg/kg) prenatal dose; 3) however, the rats who survived exhibited both an attenuated HVR and HCVR; and 4) rats also exhibited resting tachycardia and tachypnea (at 5–10 mg/kg) compared to in utero saline treated rats. These data demonstrate a long‐term (10 days) effect of prenatal morphine exposure on resting cardiorespiratory function, including impairments in ventilatory defense responses to acute hypoxia and hypercapnia. These cardiorespiratory anomalies could offer a window of insight into the pathophysiology of the increased risk of SIDS seen in infants born from mothers of substance use.
BAT1 Alters Migration, Invasion and Pro‐Inflammatory Cytokines in Prostate Cancer
Authors: Garcia Aileen Marie , Viquez Olga , Roque Yarelis , Sanchez Maria , Martinez Magaly
Published: April 2020

Prostate cancer (PCa) is the most frequently diagnosed non‐cutaneous cancer and the second cause of cancer related deaths in men. Though PCa is often treated with radical prostatectomy and radiation therapy, 15%–30% of patients may undergo biochemical recurrence. The current biomarkers are inadequate to identify recurrence, thus there is a demand for the development of biomarkers that can identify recurrence. In preliminary data, HLA‐B associated transcript 1 (BAT1) was down‐regulated in patients with PCa recurrence when compared with non‐recurrent PCa patients. BAT1 has been identified as an anti‐inflammatory gene in other types of diseases. The role of BAT1 in PCa has not been identified. We hypothesize that BAT1 expression will affect cell migration, invasion, and inflammatory cytokine expression in vitro and in vivo. Our objective was to identify the role of BAT1 expression in PCa. We down‐regulated and upregulated BAT1 in androgen independent PC3 and androgen dependent 22RV1 prostate cancer cell lines using BAT1 small‐interfering RNA (siRNA) and complementary DNA (cDNA) lentiviral particle infections, respectively. In vitro assays were performed to measure BAT1 protein expression, migration, invasion and gene expression in prostate cancer cells. Western blot analysis showed that BAT1 protein expression was significantly down‐regulated in PC3 and 22RV1 siRNA transfected cells and overexpressed in PC3 and 22RV1 cDNA infected cells when compared to control. BAT1 down‐regulation significantly increased cell migration and invasion when compared to control. In contrast, BAT1 overexpression significantly decreased cell migration and invasion when compared to control. Real time PCR (RT‐PCR) showed changes in expression of pro‐inflammatory cytokines (TNF‐α and IL‐6) and genes that regulate cell adhesion and migration (MMP10, MMP13 and TIMPs) in BAT1 overexpressed cells when compared to BAT1 siRNA cells. In vivo studies were performed using severe combined immunodeficient (SCID) orthotopic mice injected with 22RV1 cells previously transfected with BAT1 short hairpin ribonucleic acid (shRNA) or BAT1 cDNA. The cells were injected directly into the prostate and allowed to form tumors for analysis. Immunohistochemistry analysis showed increases in the expression of TNF‐α, IL‐6 and MMP10 in tumors developed from BAT1 shRNA treated mice when compared to tumors developed from BAT1 cDNA treated mice. These results suggest that BAT1 expression alters inflammation through the modulation of TNF‐α and IL‐6 expression and can eventually promote migration and invasion through the expression of MMPs and TIMPs.

Support or Funding Information

This work is supported by the NIGMS‐RISE Program #R25GM061838
Lectins as probes of the human intestinal microbiome
Authors: Isabella Christine R. , Raman Smrithi , Dugan Amanda , Wuo Michael , Kiessling Laura L.
Published: April 2020

The human microbiota is intimately linked to health and disease. However, identifying community members that contribute to a healthy or dysbiotic state remains a challenge. Host factors regulate microbiome composition by promoting colonization or clearance of microbes. One such factor, soluble lectins, can detect microbes at strain‐level resolution by specific recognition of glycans. Several human lectins expressed at mucosal surfaces can bind microbes, but lectin specificity for microbes is under‐characterized. To this end, we have generated and characterized a suite of fluorescently labeled human soluble lectins to elucidate their microbe‐specificity. Using these probes, we analyzed recognition of bacteria from the human intestinal microbiota, and found that different lectins bind distinct populations. These data support the use of lectins as specific probes of microbial glycosylation. Further, analysis of fecal samples using lectins implicated in clearance of pathogens or promotion of colonization could provide a facile method of profiling the microbiota in health and disease.
The Structural and Functional Role of Vertebrate LARP6 N‐Terminal Region
Authors: Norbury Brianna , Lewis Karen
Published: April 2020

La‐related protein 6 (LARP6) belongs to a superfamily of evolutionarily related RNA‐binding proteins. While other LARP families have been characterized more thoroughly, LARP6 is still poorly understood. The majority of the work to date for LARP6 focuses on its RNA binding activity, and has primarily studied the RNA binding domain, the “La Module”, in isolation. However, these studies may not reflect how the full‐length protein behaves in vivo. Previous work in our lab using the Danio rerio (zebrafish) LARP6 protein as a model system demonstrated that a domain adjacent to the La Module, called the “N‐terminal region” (NTR), may play an important role in the full‐length protein. To study the effect of this domain on the RNA binding activity, we created a new protein construct that contained only the NTR and the La Module while omitting the C‐terminal domain, dubbed the “ΔCTD.” The NTR has also been created with an attached His10‐SUMO tag, with the additional creation of the isolated tag to serve as a negative control for biochemical analyses. Several features have been compared between the La Module and our ΔCTD construct, with supporting evidence from the NTR. First, size exclusion chromatography and analytical ultracentrifugation demonstrated that, unlike the La Module, the ΔCTD forms dimers under nonreducing conditions. These dimers were resolved by the addition of reducing agent, suggesting that the dimerization was caused by a disulfide bond. RNA binding activity was measured using electrophoretic mobility shift assays and showed that, unexpectedly, the ΔCTD exhibited distinct binding behavior as compared to the isolated La Module. The ΔCTD appears to exhibit increased affinity to homopolymers poly‐A, poly‐U, and the endogenous stem‐loop ligand HsCOL1A1, but no binding was observed to poly‐C, showing that the NTR not only modulates the RNA binding activity, but also its ligand specificity. Circular dichroism of the ΔCTD and SUMO‐tagged NTR strongly indicates that the NTR is a random coil. Small‐angle X‐ray scattering (SAXS) analysis supports that the NTR is able to interact with the La Module to modulate RNA binding behavior.

Support or Funding Information

National Institutes of Health – National Institute of General Medical Sciences (R15GM119096), National Institute of Health project ALS‐ENABLE (P30 GM124169) and a High‐End Instrumentation Grant (S10OD018483)
Do Action Potentials in Vasopressin Magnocellular Neurons Back‐propagate from Soma to Dendrites to Evoke Neuropeptide Dendritic Release?
Authors: Zhang Meng , Stern Javier E.
Published: April 2020

In addition to releasing their peptidergic cargo into the systemic circulation from their axons in the posterior pituitary, magnocellular neurosecretory vasopressin (VP) and oxytocin (OT) neurons in the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei also release these neuropeptides from their dendrites. Dendritic release of VP and OT play important functional roles, including recruitment and coordination of neurohumoral responses to an osmotic stimulation. Dendritic release of neuropeptides involves Ca2+‐dependent exocytosis, which in other neuronal types, including substantia nigra dopaminergic neurons, has been shown to be triggered by backpropagating action potentials (bAPs). However, it is at present unknown whether action potentials in VP neurons can back‐propagate from soma to dendrites, and whether they participate in dendritic release of VP. To directly test this, we performed simultaneous dual patch‐clamp electrophysiological recordings from somata (whole‐cell, current‐clamp) and dendrites (loose patch, voltage clamp) in identified VP neurons of eGFP‐VP rats. Various degrees and patters of somatic firing activity were evoked by direct current injection through the somatic patch pipette. Surprisingly, we found evidence for bAPs only in a very small proportion of recorded VP neurons (~20%, 91/435 recordings). When present, bAPs appeared as fast inward deflections that occurred within less than 1 millisecond from the evoked somatic AP. bAPs displayed high fidelity, even at relatively high somatic firing frequencies (up to 10 Hz), with almost no propagation failures observed within the measured distance from the soma. The presence or absence of bAPs was not dependent on neuronal input resistance, dendritic access resistance or dendritic distance from the soma (15–100 μM) Similarly, neither the number, frequency nor pattern (continuous or bursting) of somatic action potential had an influence on the incidence or properties of bAPs (p> 0.5 in all cases). Blockade of K+ channels with 4AP (5 mM n=11) or TEA (30 mM, n=7) did not enabled somatic action potentials to back‐propagate to the patched dendrite. Ongoing morphometric analysis of patched neurons aim at determining whether back‐propagation of action potential correlates with specific morphological features of these neurons. Taken together, our results indicate that despite being highly electrotonically compact, the vast majority of neurosecretory VP neurons do not show evidence for bAPS. These findings raise important questions regarding mechanisms underlying activity‐dependent dendritic release of neuropeptides in hypothalamic neurons.

Support or Funding Information

NIH R01NS094640 to JES
Optimal Chemotherapeutic Combination of 9 Putative Natural Compounds from Traditional Chinese Medicine
Authors: Berlin Ian Geddes , Kenealey Jason
Published: April 2020

Prostate cancer accounts for 9.9% of all new cancer cases in the United States annually. Despite its high average 5‐year survival rate of 98%, if the cancer metastases to a distant site the 5‐year survival rate drops to just 30.5%. Natural compounds are often studied for their potential chemotherapeutic effects or their sensitizing effects to decrease cancer progression. Traditional Chinese medicine is a common source for finding bioactive small molecules which may have chemotherapeutic effects. This study focused on 9 putative natural compounds and their effectiveness of treating PC‐3 human prostate cancer cells. PC‐3 cancer cells were allowed to adhere for 24h incubated at 37°C with 5% CO2 then treated for 48h. Cell viability was analyzed after an additional 6h incubation with AlamarBlue and tested for relative cell viability. IC50s for each compound were determined and then used in Mixture Design Response Surface Methodology (MDRSM) to determine the optimal mixture of compounds. Chou Talalay statistical analysis was used to determine if combination effects were synergistic, antagonistic or additive. The compounds used include; Triptolide, .01819uM (Ttd), Shikonin, .6002uM (Shk), Curcumin 20.83uM (Cur), Emodin, 57.38uM (Em), Wogonin, 97.87uM (Wo) Berberine, 101.4uM (BB), Silibinin, 106.2uM (or Silybin) (Sy), Epigallocatechin gallate, 272.6uM (EGCG), and β‐Elemene, 304.3uM (β‐E). Emodin, Silibinin and EGCG all appeared to act primarily via cell cycle inhibition and their effectiveness was found to increase in combination with other small molecules. The ideal combination provided a multi‐faced approach reduce cell viability which suggests it may help treat prostate cancer cells in vivo either in tandem with classical chemo‐therapeutics or alone.

Support or Funding Information

Simmons Center for Cancer Research
P2Y2 Receptor Modulates Leukocyte‐Endothelium Interaction
Authors: Wang Jianjie , Thomas Spencer , Ulbricht Randi , Marino Hailee , Sheravina Sharon , Huxley Virginia , Erb Laurie , Weisman Gary
Published: April 2020

Nucleotide P2Y2 receptor (P2Y2R) plays important roles in inflammation. The leukocyte‐endothelium interaction is essential for leukocyte recruitment at the site of tissue damage or injury. Little is known about the role of P2Y2R in the regulation of in vivo leukocyte‐endothelium interaction. This study tested the hypothesis that P2Y2R mediates leukocyte‐endothelium interaction using the cremaster muscle. Leukocytes were labeled with Rhodamine 6G injected via tail vein. Leukocyte rolling and stable adhesion were analyzed using a semi‐automatic leukocyte tracking method. P2Y2R activation by UTP increased leukocyte rolling (EC50 = 4.7 ± 1.4 x 10−7 M) and adhesion (EC50= 4.3 ± 1.3 x 10−6 M) in a concentration‐dependent manner in C57BL/6 wild‐type (WT) mice (n=3). UTP (10−5 M) induced sustained increases in both leukocyte rolling and adhesion in WT mice (n=7–19, P<0.05) over 60 min. P2Y2R knockout (P2Y2R−/−) mice exhibited a 2‐fold increase in baseline leukocyte rolling (rolling fraction=37.8%, n=8, p < 0.001) compared to WT mice (rolling fraction=16.2%, n=12) and stayed at the high basal level without changes in rolling following stimulation of UTP (10−5 M) over 60 min. Baseline leukocyte adhesion in P2Y2R−/− mice was 1.5‐fold increase (15 ± 4 adherent cells.100 μm−1, n=12, p < 0.001) compared with WT mice (9 ± 4 adherent cells·100 μm−1; n=19). There was no change in leukocyte adhesion from the baseline in response to UPT (10−5 M) stimulation in P2Y2R−/− mice (n=5–12). We conclude that P2Y2R prevents in vivo leukocyte rolling and adhesion under resting condition while activation P2Y2R by UTP increases both leukocyte rolling and adhesion.

Support or Funding Information

Missouri State University
Potential of Aerobic Physical Training to Minimize the Effects of Leptin Deficiency in Female Mice
Authors: Azevedo-Martins Anna Karenina , Boschetti Daniela , Muller Cynthia R. , Américo Anna Laura , Vecchiatto Bruno , Martucci Luiz Felipe , Pereira Renata , Oliveira Cláudia , Evangelista Fabiana
Published: April 2020

Aerobic physical training (APT) is an important strategy for prevention and treatment of comorbidities such as type 2 diabetes and obesity. Leptin and adiponectin play an important role in energy metabolism, whose imbalance is strongly related to obesity and insulin resistance (IR). Although women have higher levels of leptin and adiponectin compared to men, in conditions such as obesity and/or type 2 diabetes these levels decrease, abolishing the protective effects of these adipokines in females. This study investigated the effects of APT on the metabolic parameters in leptin‐deficient female mice. For this, 8‐week‐old wild C57BL/J6 (C57) or leptin‐deficient ob/ob (Lepob) female mice were separated into the groups sedentary (S) (C57S, n = 6 and LepobS, n = 5) and trained (T) (LepobT, n = 8). The APT was performed during 1 h/day at 60% of maximal speed achieved in the maximal physical exertion test, 5 times/week for eight weeks. The procedures were approved by the Ethics Committee of the School of Arts, Sciences and Humanities of University of Sao Paulo (#001/2017). LepobS and LepobT groups increased body weight, the weight of retroperitoneal (WAT‐RP) and subcutaneous (WAT‐SC) fat pads compared to C57S. Adipocyte area and diameter of WAT‐RP and WAT‐SC were higher in both LepobS and LepobT compared to C57S, however the APT decreased the WAT‐SC adipocyte area and diameter in LepobT group compared to LepobS. LepobS and LepobT groups showed lower weight of pancreas, gastrocnemius and plantar muscles compared to C57S, however APT prevented the mass reduction of gastrocnemius and plantar muscles in LepobT. Food intake was higher in LepobS compared to C57S, which was counteracted by APT in the LepobT group. The LepobS had fasting hyperglycemia (131 ± 1.02 mg/dL) and glucose intolerance (AUC: 31522 ± 1148 mg/dL/120min) compared to C57S (92 ± 2.51 mg/dL and AUC: 20339 ± 1857 mg/dL/120min). Despite of LepobT group had shown glucose intolerance (AUC: 30085 ± 547 mg/dL/120min), the APT decreased the fasting glycemia (114 ± 6.11 mg/dL) compared to LepobS group. Adiponectin level did not change among groups, as well as the relative area of islets in the pancreatic tissue. During the maximal physical exertion test, LepobT showed higher maximal speed, time to exhaustion and speed on the VO2máx compared to LepobS group. In conclusion, APT prevented the loss of gastrocnemius and plantar muscles mass, reduced WAT‐SC adipocyte hypertrophy and food intake, improved fasting glycemia and aerobic capacity in leptin deficient female mice.

Support or Funding Information

FAPESP (#2018/22361‐9), CAPES, CNPq
A Screen for Protein Encoding Genes for Air Sac Primordium Invasion of the Wing Imaginal Disc in Drosophila melanogaster
Authors: VanZant Elisha , Srivastava Ajay
Published: April 2020

The Air Sac Primordia (ASP) in Drosophila melanogaster develops outside of the Drosophila’s Wing Imaginal Disc (WID) and grows into and within it, similar to the behavior of metastatic tumors developing into human lungs. Genes involved in Basement Membrane defects have been found to influence tumor metastasis and ASP invasion of the Wing Imaginal Disc. By utilizing protein traps and finding the genes responsible for Basement Membrane degradation in Drosophila, the genes responsible for tumor metastasis can be identified. In this study, several lines of Drosophila were dissected and screened for positive ASP expression in the Wing Imaginal Disc. Out of these, some lines appear to have positive expression in the Wing Imaginal Disc and few appears to have positive ASP expression. This will be followed up by knock down of the gene with positive ASP expression to note the effects on ASP invasion when the gene is not present as well as the screening of several more Drosophila stocks. The genes identified with positive ASP expression could possibly parallel genes found in tumor invasion in humans. This result will provide further reason for this gene to be studied and analyzed in the context of human cancer.
Student Use of Outside Study Resources
Authors: Ikonne Uzoma S. , Brodie Adrienne , Bay Curt , Campbell Anna
Published: April 2020

Understanding student resource use and selection can be informative to instructors and may guide curricular development and student advising. The purpose of this study is to evaluate the specific study resources students rely on in their first‐year academic courses, to determine the degree of influence various sources have on resource selection, and to determine the frequency of use of outside resources for specific disciplines. Further, we will investigate if resource use changes over time, and we hypothesize that resource use may vary based on level of academic performance. A survey was developed and sent to first‐year medical students in the fall semester. Preliminary survey responses were summarized and will be linked to academic and library records. First year students reported using instructor slides, self‐generated resources, and practice questions most frequently. Outside resources such as
Youtube.com and Wikipedia were also reported as being frequently used. Survey responses indicated that students find recommendations for outside resources identified by other students most influential compared to recommendations from the internet, faculty, and the library. Students reported more frequent use of outside resources for anatomy compared to other disciplines, especially clinical science and osteopathic principles and practice. Additional analyses will be performed to determine if survey responses vary with academic performance and will be expanded to include additional student cohorts.
Doxorubicin Cardiotoxicity in Young Tumor‐Bearing Mice
Authors: Abdelgawad Ibrahim Y. , Grant Marianne K.O. , Lewis Christine , Popescu Flavia , Largaespada David , Zordoky Beshay N.
Published: April 2020

Background

Childhood cancer survivors have about 15‐times higher risk of heart failure than their siblings mainly due to the cardiotoxicity of chemotherapy. Nearly 50% of pediatric cancer patients receive doxorubicin (DOX) which is known to be cardiotoxic. DOX‐induced cardiotoxicity had been extensively studied in tumor‐free mouse models. The objective of the current study is to characterize a juvenile tumor‐bearing immunocompetent mouse model of DOX‐induced cardiotoxicity.

Methods

Juvenile five‐week old male C57Bl/6 mice were injected subcutaneously with EL4 lymphoma cells (5X104 cells/mouse) in the flank region suspended in sterile PBS, while tumorfree mice were only injected with sterile PBS. One week after tumor implantation, both tumorfree and tumor‐bearing mice were injected intraperitoneally with either a clinically relevant dose of DOX (4 mg/kg/week) or saline for 3 weeks. One week after the last DOX injection, mice were euthanized and the hearts and tumors were harvested and weighed. Gene expression of molecular markers of cardiotoxicity and inflammation was quantified using real‐time PCR.

Results

DOX 4 mg/kg/week for 3 weeks significantly reduced tumor weights in EL4 tumor‐bearing mice. DOX administration caused significant cardiac atrophy in tumor‐free and tumor‐bearing mice. Moreover, tumor itself caused a significant reduction in heart weight compared to tumor‐free mice. Additionally, DOX administration induced the expression of markers of cardiotoxicity, ANP and BNP, in both tumor‐free and tumor‐bearing mice. Saline‐treated tumor‐bearing mice demonstrated higher expression of TNF‐alpha, an inflammatory marker, compared to tumor‐free mice.

Conclusion

We have established a juvenile tumor‐bearing immunocompetent mouse model to investigate DOX‐induced cardiotoxicity. Our tumor‐bearing mouse model will optimize our understanding of the interplay between tumor and DOX‐induced cardiotoxicity. Additionally, this clinically relevant model will be used for investigation of cardioprotective strategies that mitigate DOX cardiotoxicity without compromising its chemotherapeutic efficacy before translating to the clinical settings.

Support or Funding Information

Research was supported by the National Institutes of Health’s National Center for Advancing Translational Sciences, grant UL1TR002494, and by a grant from the St. Baldrick’s Foundation [B.N.Z.]. B.N.Z. is a Masonic Cancer Center Women’s Health Scholar, sponsored by the Masonic Cancer Center, and administered by the University of Minnesota Women’s Health Research Program.
Optimization of Cas9 nuclear localization in a tilapia (Oreochromis mossambicus) cell line
Authors: Villapando Angelika Marie , Hamar Jens Carlton , Kültz Dietmar
Published: April 2020

CRISPR gene targeting studies in cell lines require reliable expression of Cas9 along with appropriate target‐specific guide RNAs. Effective CRISPR/Cas9 gene editing requires Cas9 localization in the nucleus. Previous attempts to implement CRISPR/Cas9 gene editing in a tilapia (Oreochromis mossambicus) cell line (OmB cells) were unsuccessful. A possible contribution to these results could be inefficient nuclear localization of Cas9. Conventionally, the mammalian SV40 nuclear localization signal (SV40 NLS) is used to localize proteins to the nucleus. Our previous studies have shown that the SV40 promoter is poorly recognized in OmB cells. Therefore, we hypothesized that the SV40 NLS will also be poorly recognized in OmB cells. To test this hypothesis we fused the SV40 NLS to the C‐terminus of GFP (green fluorescent protein), transfected it into the tilapia OmB cell line, and determined the efficiency of nuclear GFP localization using fluorescence imaging (Leica Dmi8). Our imaging results show that the SV40 NLS moderately induced nuclear GFP localization. An incomplete but significant increase in nuclear GFP localization relative to the control (no NLS, just GFP) was observed using Leica image analysis software (LASX 2.0, Leica). Nevertheless, a large amount of GFP still remained in the cytosol when using SV40 NLS. Current work is comparing the efficiency of SV40 NLS mediated nuclear localization of GFP in fish cell lines to that mediated by an endogenous tilapia NLS. We have cloned an endogenous tilapia NLS and are currently testing whether this endogenous NLS improves nuclear localization of ectopic GFP in tilapia OmB cells. Optimization of nuclear localization of Cas9 will aid future gene editing studies in the OmB cell line and in cells from related fish species.

Support or Funding Information

This study was funded by a grant from NSF (IOS‐1656371).
Lysosome‐MVB Encountering and Inflammatory Exosome Release Controlled by Acid Sphingomyelinase in Podocytes during Hyperhomocysteinemia
Authors: Li Guangbi , Huang Dandan , Camus Sarah , Zou Yao , Ritter Joseph K. , Li Pin-Lan
Published: April 2020

Previous studies have demonstrated that overexpression of acid sphingomyelinase (ASM) enhances NLRP3 inflammasome activation and podocyte‐derived inflammatory exosome release during hyperhomocysteinemia (hHcy). However, the mechanism underlying the enhancement of inflammatory exosome release remains unclear. The present study tested a hypothesis that overexpression of ASM blocks lysosome‐MVB encountering via inhibition of lysosome trafficking, leading to enhanced inflammatory exosome release from podocytes during hHcy. Nanoparticle tracking analysis showed a largely increased urinary exosomes in WT/WT mice with hHcy, but not in Smpd1−/− (ASM gene knockout) mice. In Smpd1trg/Podocre (podocyte‐specific ASM gene overexpression) mice, this increase in urinary exosomes was not only during hHcy, but also on the ND. Increased exosomes were podocytes‐derived by detection of podocin as podocyte marker and CD63 as exosome marker. Biochemical analyses showed that NLRP3 products, IL‐1β and IL‐18 significantly increased in hHcy in WT/WT and Smpd1trg/Podocre mice, but not in Smpd1−/− mice. In vitro, we found that Hcy stimulation enhanced release of exosomes out of podocytes from WT/WT and Smpd1trg/Podocre mice, but it had no effect on exosome release from podocytes of Smpd1−/− mice. Exosomes were isolated with ultracentrifugation and purified with exosome purification kit from podocyte culture medium. It was found that Hcy increased CD63 in purified exosomes from WT/WT and Smpd1trg/Podocre podocytes, but it had no effect in Smpd1−/− podocytes. ELISA analysis showed that these increased exosomes contain a lot of IL‐1β and IL‐18 when podocytes were stimulated by Hcy. Super‐resolution microscopy showed the interaction of lysosomes with MVBs. Lysosomes encountered and fused to MVBs much less in podocytes from WT/WT and Smpd1trg/Podocre mice when they were treated with Hcy. In Smpd1−/− podocytes, Hcy failed to change the lysosome‐MVB encounters and interactions. ML‐SA1 as a cell‐permeable lysosome trafficking stimulator via activation of lysosomal TRPML1 channel markedly increased lysosomes movement toward nucleus. Hcy significantly reduced lysosomes movements to nucleus and overexpression of Smpd1 gene enhanced the effect of Hcy. In Smpd1−/− podocytes, ML‐SA1‐induced lysosome movement toward nucleus remained unchanged during Hcy treatment. These results suggest that ASM‐mediated sphingolipid signaling controls lysosome function determining inflammatory exosome release from podocytes during hHcy.

Support or Funding Information

This study was supported by NIH grants DK54927 and DK102539.
Four weeks of environmentally relevant second hand smoke exposure decreased neuronal excitability of cardiac vagal neurons in the dorsal motor nucleus of vagus
Authors: Pan Shi-Yue , Sun Jun-Qing , Chen Chao
Published: April 2020

BACKGROUND

Second hand smoke (SHS), a main indoor air pollutant, is a significant risk factor for cardiovascular morbidity and mortality. We previously showed that exposure to SHS, for as short as four weeks, significantly reduced heart rate variability (HRV), suggesting a decreased vagal regulation of the heart. We further showed that, in cardiac vagal neurons in the nucleus ambiguus (NA), SHS exposure significantly reduced spiking response to depolarizing current injections and increased current‐ and voltage‐threshold for action potential generation. However, we have no information on the effects of SHS exposure on cardiac vagal neurons in the dorsal motor nucleus of vagus (DMNV). Here, we sought to test the hypothesis that SHS exposure decreases neuronal excitability of cardiac vagal neurons in the DMNV.

METHODS

Seven‐week old male C57BL/6J mice underwent surgeries to retrograde label cardiac vagal neurons by applying a fluorescent dye (DiI) to the sinoatrial node. Two weeks after surgery, mice were randomly assigned to either filtered air‐ (FA, n = 14) or SHS‐exposed (n = 16) group. The SHS exposure (6 hr/d, 5 d/wk) was set at a level that is similar to that of a smoky bar (3 mg/m3). After four weeks of FA or SHS exposure, whole‐cell current clamp recordings in brainstem slices were performed on anatomically identified cardiac vagal neurons in the DMNV with fluorescent labeling.

RESULTS

DMNV cardiac vagal neurons from the SHS‐exposed group were significantly more hyperpolarized than the FA control group (−53.3 ± 0.9 mV vs. −55.7 ± 0.7 mV, FA vs. SHS respectively, p < 0.05). Four weeks of SHS exposure also significantly decreased neuronal spontaneous spiking activity at a holding potential of −45 mV (1.6 ± 0.4 Hz vs. 0.8 ± 0.2 Hz, FA vs. SHS respectively, p < 0.05). The decreased spontaneous activity was not associated with the spiking response to depolarizing current injections from a holding potential of −60 mV, current and voltage threshold for action potential generation, action potential peak and afterhyperpolarization. Furthermore, there was no significant difference in the neuronal input‐output relationship of DMNV cardiac vagal neurons between FA control and SHS‐exposed groups, suggesting an unchanged dynamic responsiveness to stimuli.

CONCLUSIONS

Four weeks exposure to environmentally relevant level of SHS decreased neuronal excitability of cardiac vagal neurons in a region‐dependent manner – reduced resting membrane potential and spontaneous spiking activity in the DMNV and reduced spiking response to depolarizing current injections and increased threshold for action potential generation in the NA.

Support or Funding Information

Funding: R01 ES025229
De Novo Walleye Transcriptomic Database Pairing and Genetic Market Identification
Authors: Landgraf Austin E. , Stewart Nicholas , Kobayashi Yass
Published: April 2020

Fish species such as yellow perch (Perca flavescens), Atlantic salmon (Salmo salar) are major sport fish and commercially important in the United States. This has led to extensive research on their genome and transcriptome. Between sport and commercial fishing, the walleye (Sander vitreus) has been established as one of the most popular fish in North America. Despite their value, no annotated transcriptome database is currently available to the public. In order to expand our understanding of walleye transcriptome, we generated a transcriptome using the Next Generation Sequencing technology. The de novo transcriptome assembly from walleye brain tissue was constructed using RNA samples harvested from sexually mature male and female walleye (n=5 per sex). Sequence from the walleye transcriptome was aligned to the yellow perch transcriptome. Over 8400 fully assembled transcripts from the walleye transcriptome were identified. We were able to recover over 90% of the BUSCO genes associated with bony fish, indicating a quality assembly. Currently, we are expanding upon our original list of 8400 annotated transcripts by using possible open reading frames as well as further identifying partially assembled transcripts in our assembly. Our annotated transcriptome should provide tools for further research in physiology, evolutionary history, and ecology.
Skin and Kidney Contribute to Salt‐Sensitive Hypertension in the SAME rat
Authors: Mullins Linda Jane , Ivy Jessica , Rakova Natalia , Mueller Dominik , Mullins John
Published: April 2020

Loss of Hsd11b2 causes the syndrome of apparent mineralocorticoid excess (SAME) and salt‐sensitive hypertension, which has been demonstrated in the SAME rat
1. We aimed to determine how salt‐sensitive hypertension developed in young SAME rats.

Hsd11b2−/− rats were hypertensive (158 versus 119mmHg controls; measured under anaesthesia) and plasma volume contracted (haematocrit 0.49 versus 0.42 control) by 5 weeks of age and showed profound hypokalemia (2mM plasma K+ versus 4.5mM control) the latter being evident from 15 days of age.

Adult kidney RNA‐seq analysis suggested a coordinate down‐regulation of electrolyte and water transporters, while a‐ENaC (Scnn1a; 1.66‐fold), the flow‐induced BK channel (Kcnma1; 2.65‐fold) and the HKA2 channel (Atp12a; 4.29‐fold) were significantly up‐regulated. Young Hsd11b2−/− animals exhibited polydipsia and polyuria from weaning age (~20 days old) and, as with adults, they excreted a higher proportion of ingested Na+ (56%) through the kidney, compared to controls (38%), suggesting that the coordinate changes in nephron transporters are already in place around weaning age.

Notably, we observed increased skin electrolytes in neonatal animals, (by drying and ashing pelts and determining electrolytes using atomic absorption spectroscopy). Neonatal Hsd11b2−/− animals retained higher [Na+] in their pelts than the controls.

Our data suggest that increased Na+ intake [from mothers’ milk] results in increased Na+ storage in skin at the expense of K+. The ensuing hypokalemia primes the developing nephron to deliver increased Na+ and water to the connecting tubule and cortical collecting duct by weaning age. Glucocorticoid‐induced Na+ uptake in the kidney, and reduced Na+ storage capacity in the pelt, both then contribute to progressive salt‐sensitive hypertension.

Support or Funding Information

British Heart Foundation Centre of Research Excellence Award (RE/08/001/23904); Kidney Research UK project grant
Investigating the Protein Kinase C Polarized Growth‐Related Protein Complex in Aspergillus nidulans
Authors: Jackson-Hayes Loretta , Craft Alexis , Hameed Muhammad , Hines Zariah , Freyaldenhoven W. Toler , Hill Terry
Published: April 2020

The long‐term goal of our research is to advance understanding of the mechanisms involved in hyphal growth, as well as maintenance of cell walls in filamentous fungi. Recently our work has focused on proteins that localize to sites of cell wall synthesis, namely growing cell apices (hyphal tips) and forming septa. This project focusses on Protein kinase C (PkcA). In previous work, we identified a mutation in the gene that encodes an orthologue of protein kinase C in our model organism Aspergillus nidulans, which results in reduced cell wall integrity. We showed that wild type PkcA localizes to growing hyphal apices and the contractile actomyosin ring at forming septa, and we identified amino acid sequences within PkcA that are required for PkcA to localize to these sites of cell wall synthesis. This project seeks to uncover the protein‐protein interactions involving PkcA and determine the PkcA‐complexed proteins it phosphorylates and those that it does not. Previously we reported that PkcA forms a complex with the formin SepA, and that this interaction requires the FH2 domain of SepA. Published results from another group suggested that the Rho‐type GTPase Rho4 plays a role in SepA function and that Bud3 acts as a GEF for Rho4. Here we show that the putative Rho binding domain of PkcA (HR1A) is sufficient for its interaction with SepA. Fluorescence microscopy of a dually labeled A. nidulans strain illustrate that although PkcA‐mRFP and Rho4‐GFP often reside at the contractile actomyosin concurrently, PkcA precedes Rho4 in ring constriction and yeast two‐hybrid assays failed to show an interaction between PkcA and Rho4. Further, neither of 4 other Rho homologues tested interacted with PkcA in yeast two‐hybrid experiments. Sequence analysis of the FH2 domain of SepA revealed a Rho‐like motif encompassing amino acids 377–408 having 43.3% sequence identity with RhoA. This result suggests that the sufficiency of the HR1A domain for PkcA’s interaction with SepA is due to its affinity for the Rho‐like motif within FH2. Co‐immunoprecipitation of GFP‐PkcA followed by tandem mass spectrometry and proteomics analysis of complexed proteins identified chitin synthase B and the glucan synthase FksA within the PkcA complex. Ongoing studies will seek to determine if these proteins are substrates for PkcA.

Support or Funding Information

NSF RUI 1615192
Calorie Restriction without Restricting Vitamins and Minerals Does Not Deteriorate Bones
Authors: Rahman Md Mizanur , Sankaralingam Sowndramalingam
Published: April 2020

Calorie restriction has a number of health benefits, however is associated with reduction of bone mineral density (BMD). As calcium and vitamin D are considered to be the most essential nutrients for bone health, this study aimed to determine if restriction of vitamins and minerals is the cause of this bone deterioration in calorie‐restricted diet. We hypothesize that calorie restriction without restricting the vitamins and minerals will not reduce bone mineral density in calorie‐restricted mice. To determine the effect of calorie restriction (CR) on bone mineral density, 6‐month‐old C57BL/6 female mice were fed AIN93 diet ad libitum (AL) or 20% CR for 8 months. Mice were divided into 2 groups: 1) Mice fed AL and 2) Mice fed 20% complete calorie restriction (CCR) where vitamins and minerals are also restricted. Mice were scanned with dual energy x‐ray absorptiometry (DXA) at the age of 6 month (baseline), and 14 month (after 8 months of experimental diet) and analyzed for the bone mass. Calorie restricted mice showed a noticeable reduction in BMD as compared to AL fed mice. To determine if long‐term calorie restriction without restricting vitamins and minerals can attenuate this bone deterioration in CCR mice after 8 months of experimental diet. Six‐month‐old C57BL/6 mice were divided into 2 groups: 1) Mice fed AL and 2) Mice fed 20% CCR for 8 months to induce bone loss. Then 20% CCR diet was replaced with 20% CR diet without restricting vitamins and minerals for another 8 months. Interestingly, reduction of BMD seen in CCR fed mice at the age of 14 month was attenuated at the age of 22 month when this group was fed for 8 months with 20% CR diet without restricting vitamins and minerals. In either case (vitamins and mineral restricted or not restricted), muscle mass, and strength were improved in 20% calorie restricted group as compared to AL group as determined by DXA, and wet weight at sacrifice for muscle mass and by rotarod performance test and endurance stress test for muscle strength. These data indicate that 20% CR without compromising vitamin and minerals could be a natural, cost effective and safe optimal dietary regimen that would improve muscle mass and strength without any deleterious effect on bone.

Support or Funding Information

NIH NIA K01 KAG034233A and QNRF UREP23‐097‐1‐026
The Cryptococcus neoformans capsule polysaccharide glucuronoxylomannan induces endothelial barrier permeability via the phosphorylation and internalization of VE‐cadherin
Authors: Gandhi Jaani , Jahan Ishrat , Rooklin Rikki , Martinez Luis , Dores Michael Robert
Published: April 2020

Cryptococcus neoformans is a pathogenic yeast that has been shown to induce meningoencephalitis within immunocompromised individuals. This pathogen contributes to over 220,000 cases of fungal meningitis annually and is a major cause of mortality in patients with HIV in developing countries. C. neoformans sheds the polysaccharide glucuronoxylomannan (GXM) from its cell capsule, allowing the pathogen to evade the host immune systems and enter neuronal tissues. We hypothesize that C. neoformans GXM induces the disruption of the blood‐brain barrier by stimulating the breakdown of the adherens junctions that hold human brain endothelial cells together. Here, we demonstrate that GXM stimulates endothelial barrier permeability. We found that stimulation of human brain endothelial cells with GXM induces the internalization of VE‐cadherin from the plasma membrane, but not lysosomal sorting and degradation of VE‐cadherin. Instead, VE‐cadherin appears to be transiently phosphorylated and sorted to endosomes, suggesting that GXM‐mediated endothelial barrier disruption is reversible. Our current research is focused on identifying the intracellular signaling pathways that regulate VE‐cadherin phosphorylation and internalization in response to GXM exposure.

Support or Funding Information

NIH NIAID: 1R01AI145559‐01A1
(E, Z, E)‐GGPP: A Potential Novel Bactericidal Compound
Authors: Poulos Leah , Mann Francis
Published: April 2020

Mycobacterium tuberculosis (Mtb), a pathogenic Actinobacterium, has infected one quarter of the world’s population, according to the World Health Organization. The standard treatment of Mtb is a regimen of antibiotics, which has resulted in a mutated strain, XDRTb, that is resistant to every known antibiotic. The key to this antibiotic resistance is thought to be the fastidious cell wall. Most bacteria, including Mtb, utilize an isoprenoid glycosyl carrier for transfer of proteoglycan structures to the extracellular side of the membrane; however, unique to Mtb is the flexibility of its isoprenoid metabolism. Mtb synthesizes a carrier composed of 10 stereospecific isoprene units that is produced by elongation of the two‐unit (E)‐GPP by the (Z)‐FPP synthase, Rv1086, to produce (Z,E)‐FPP which then has 35 carbons added in a cis‐orientation by Rv2361c, resulting in (Z8,E)‐decaprenyl diphosphate (DPP). Although this pathway is well characterized, multiple additional isoprenoid synthases are encoded in the Mtb genome, and previous in vitro studies have indicated Rv2361c can utilize multiple alternate substrates. While attempting to understand the apparent flexibility of DPP metabolism, it was observed that heterologous co‐expression of either of the encoded (E,E,E)‐GGPP synthases, Rv0562 or Rv3383c, with Rv1086 in the heterologous host, Escherichia coli (E. coli), results in complete cell death. Computational modeling of both Rv0562 and Rv3383c suggest that both active sites may accept (Z,E)‐FPP as a substrate, and if this is true, the product would be the novel compound (E,Z,E)‐GGPP. Protein purifications of Rv0562 and Rv3383c individually combined with synthetic (Z,E)‐FPP have resulted in the synthesis of the potentially novel terpenoid product, (E,Z,E)‐GGPP. Subsequent coupled assays of Rv0562 or Rv3383c and Rv1086 resulted in higher product formation than the synthetic substrate, suggesting complex formation. Purification of the proposed novel product will be confirmed spectroscopically and has implications for synthesis of a new antibiotic.

Support or Funding Information

University of Wisconsin ‐ Parkside College of Natural and Health Sciences’ Summer Undergraduate Research Fellowship and the SC Johnson Integrated Sciences Laboratory