bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization and Pathogenicity of Colletotrichum Species on Philodendron tatei cv. Congo in Gansu Province, China
Authors: Xue Longhai , Zhang Yongwen , Duan Tingyu , Li Mengyuan , White James F. , Liu Yong , Li Chunjie
Published: April 2020

In recent years in China, leaf spot caused by Colletotrichum species has been an emerging disease of Philodendron tatei cv. Congo. From 2016 to 2019, typical symptoms, appearing as circular or ovoid, sunken, and brown lesions with a yellow halo, were commonly observed on P. tatei cv. Congo in and around Lanzhou, Gansu Province, China. Conidiomata were often visible on infected leaf surfaces. Leaf disease incidence was approximately 5 to 20%. A total of 126 single-spored Colletotrichum isolates were obtained from leaf lesions. Multilocus phylogenetic relationships were analyzed based on seven genomic loci (ITS, ACT, GAPDH, HIS3, CAL, CHS-1, and TUB2) and the morphological characters of the isolates determined. These isolates were identified as three Colletotrichum species in this study. A further 93 isolates, accounting for 74% of all Colletotrichum isolates, were described as new species and named as Colletotrichum philodendricola sp. nov. after the host plant genus name, Philodendron; another two isolates were named as C. pseudoboninense sp. nov. based on phylogenetic and morphological relativeness to C. boninense; the other 31 isolates, belonging to the C. orchidearum species complex, were identified as a known species—C. orchidearum. Both novel species C. philodendricola and C. pseudoboninense belong to the C. boninense species complex. Pathogenicity tests by both spray and point inoculations confirmed that all three species could infect leaves of P. tatei cv. Congo. For spray inoculation, the mean infection rate of leaves on the three species was only 4.7% (0 to 12%), and the size on lesions was mostly 1 to 2 mm in length. For point inoculation, 30 days after nonwounding inoculation, the infection rate on leaves was 0 to 35%; in wounding inoculation, the infection rate of leaves was 35 to 65%; wounding in healthy leaves greatly enhanced the pathogenicity of these three species to P. tatei cv. Congo; however, the sizes of lesions among the three species were not significantly different. To our knowledge, this is the first report of Colletotrichum species associated with anthracnose diseases on P. tatei cv. Congo. Results obtained in this study will assist the disease prevention and appropriate management strategies.
The ARTE index: Characterizing free‐living physical activity and indices of cardio‐metabolic health among postmenopausal women
Authors: Altherr Cody A. , Baranauskas Marissa N. , Hunter Gary R. , Martins Catia , Carter Stephen J.
Published: April 2020

Purpose

The doubly‐labeled water (DLW) technique is the gold‐standard used to objectively measure free‐living activity energy expenditure (AEE), however; it does not account for differences in body size or locomotion economy. Whereas the physical activity level (PAL) index, calculated from the ratio of total energy expenditure (TEE) and resting energy expenditure (REE), is employed to adjust for body size – it also does not account for variance in energy expenditure attributed to locomotion economy. Alternatively, the activity‐related time equivalent (ARTE) circumvents the limitations of AEE and PAL. In the present work, DLW was used to compare AEE, PAL, and variations of the ARTE index among a cohort of postmenopausal women dichotomized above/below the 50th percentile of normative data for body fat%. Correlations of AEE, PAL, and ARTE to measures of cardio‐metabolic health including arterial elasticity, body fat% and cardiorespiratory fitness (V̇O2max) were also performed.

Methods

Ninety‐two, healthy postmenopausal women (65+/−4 years) were recruited to measure free‐living AEE over a 14‐day period using DLW. TEE was reduced by 10% to account for the thermogenesis of feeding whereas AEE was calculated by subtracting REE from adjusted‐TEE. PAL was calculated from the ratio of TEE/REE. ARTE4act was calculated by dividing AEE by the average energy expenditure of non‐graded walking, graded walking, stair climbing, and a carrying task. AEE divided by the average energy expenditure of non‐graded walking was used to calculate ARTEwalk. Arterial elasticity was performed using pulse contour analysis from the radial artery. Dual‐energy X‐ray absorptiometry was used to estimate body fat% whereas a modified‐Balke protocol was used to measure V̇O2max. Between‐group comparisons were performed using independent t‐tests and Pearson’s correlation were used to evaluate relationships.

Results

Between‐group differences were not detected for AEE, PAL, or the ARTE4act. However, women with less percent body fat (i.e., < 50th percentile) exhibited greater activity as evidenced by the ARTEwalk (min·day−1) (255+/−154 vs. 193+/−130; p < 0.05). Though AEE and PAL did not correlate with any cardio‐metabolic variables, ARTE4act was related to arterial elasticity (r = 0.24) and V̇O2max (r = 0.27) while ARTEwalk was related to arterial elasticity (r = 0.27), body fat% (r = −0.24), and V̇O2max (r = 0.32).

Conclusions

Consistent with the ARTEwalk, adjustments for energetic cost of non‐graded walking may improve the sensitivity of AEE for detecting differences in free‐living physical activity and parameters of cardio‐metabolic health – known to be critical for overall well‐being.

Support or Funding Information

This investigation was supported by the following grants from the National Institutes of Health: R01AG027084‐01, R01AG027084‐S, P30DK056336, P60DK079626, and UL1RR025777.
From Quorum Sensing to Positional Sensing
Authors: Zingg Jean-Marc A. , Daunert Sylvia
Published: April 2020

With Quorum Sensing (QS) bacteria adapt their physiology and metabolism when they reach a certain density and activate genes relevant for their nutrient metabolism, survival and virulence. Genes controlled by QS have relevance for the pathogenic behavior of bacteria including adherence, secretion, toxin production, immune subversion, biofilm formation and resistance to both antimicrobial and biological/chemical stressors. Most studies assess QS by growing bacteria to various densities in liquid culture. Here, we report that when bacteria are grown on agar plates at various densities, in addition to QS, positional sensing (PS) comes into play. Genes controlled by PS can be studied by cloning their promoter in front of an autonomous bioluminescent cassette (luxCDABE) and measuring their time‐ and position‐dependent expression in biofilms by using an in vivo imaging system (IVIS). The Pseudomonas putida XylR/XylS intergenic region controlling genes relevant for the metabolism and degradation of xylene and related organic molecules was cloned in front of the autonomous luciferase cassette from Photorhabdus luminescence, with the sigma 54 controlled Ps1 promoter regulating the a luxCDABE genes and the Pr promoter regulating the XylR gene. Interestingly, when E. coli carrying this construct were grown to various density on agar plates, bioluminescence was not only expressed in a density‐ and time‐dependent, but also position‐dependent manner. Such positional cues may be mediated by signaling molecules in response to cell density, growth stage, and nutrient availability. Overexpression of the sigma factor 54 (NtrA) from Pseudomonas putida modified the density‐ and time‐dependent response suggesting a regulatory role in QS and PS. The use of bioluminescence in QS and PS promises to reveal important regulatory circuits and communication systems relevant for bacterial normal physiology, pathogenicity and virulence.
Mapping of the Contraction‐Regulated Phosphoproteome Identifies TRIM28 as a Key Regulator of Skeletal Muscle Size
Authors: Steinert Nathaniel David , Potts Gregory K. , Wilson Gary M. , Klamen Amelia M. , Lin Kuan-Hung , Hermanson Jake Brenner , McNally Rachel M. , Coons Joshua J. , Hornberger Troy A.
Published: April 2020

Mechanical signals, such as those which are evoked during maximal intensity contractions (MIC), can induce an increase in skeletal muscle size. It has been widely concluded that this process is driven by the activation of rapamycin‐sensitive / mTORC1‐dependent signaling; however, recent studies have revealed that mTORC1‐independent signaling events might also be involved. Thus, in an effort to identify these events, we generated a comprehensive map of the MIC‐regulated, and rapamycin‐sensitive phosphoproteomes. In total, we identified over 2,400 unique MIC‐regulated phosphorylation events, of which, nearly 2,200 were unaffected by rapamycin. Interestingly, one of the most robust MIC‐regulated and rapamycin‐insensitive phosphorylation events was located on the S473 residue on a protein named TRIM28. This was intriguing because TRIM28 is a transcriptional intermediary factor that functions in a variety of biological processes and many of its regulatory effects are dependent on the phosphorylation of the S473 residue. Moreover, a recent study revealed that TRIM28 interacts with several skeletal muscle regulatory factors (e.g., MyoD and Mef2) and their transcriptional co‐repressors and co‐activators. Thus, to investigate the role of TRIM28 in the regulation of muscle size, we generated skeletal muscle specific and inducible TRIM28 knockout mice and then subjected these mice to various forms of mechanical stimuli. Collectively, our results indicated that: i) TRIM28 is not required for the mechanical activation of mTORC1 signaling, ii) TRIM28 plays a key role in the maintenance of normal muscle size, and iii) TRIM28 significantly contributes to the pathway via which mechanical stimuli induce hypertrophy. Taken together, these outcomes establish TRIM28 as a novel, mTORC1‐independent, component of the pathway via which mechanical signals regulate skeletal muscle size.

Support or Funding Information

NIHR01AR057347
Tracking Antigen‐Specific CD8+ T cells Using MHC Class I Pentamers
Authors: Aleksandrovic Emilija , Lazarski Christopher , Bollard Catherine , Stanojevic Maja , Hanley Patrick
Published: April 2020

The treatment of choice for many patients with Acute Myeloid Leukemia (AML) is hematopoietic stem cell transplantation, where the patient’s immune system is replaced with that of a matched donor. Due to the conditioning required, cancer relapse and viral infections such as Cytomegalovirus (CMV) are common complications. One potential treatment for relapse and/or viral infection is adoptive immunotherapy with ex vivo expanded T cells targeting either viral or tumor antigens. Prior to infusion into patients, these T cells are typically characterized using ELISpot assays which do not directly assess the presence of distinct T cell clones. Therefore, without a reliable assay to test T cell specificity, optimizing the culture conditions for increased antigen specificity has been challenging. We hypothesized that we could directly assess the specificity of T cell receptors (TCRs) from expanded T cell populations towards their target antigens over time using MHC Class I Pentamers. To test this, we generated CMV‐specific CD8+ T cells from peripheral blood mononuclear cells (PBMCs) from two consenting CMV+ donors and tumor‐specific T cells from one donor on our IRB approved protocol. After 10 days of culture, the average CMV+ T cells expanded 10‐fold and consisted of 22.2% CD4+ (range 15.9–46.6%) and 68.9% CD8+ (range 32.9–77.9%) T cells. Using flow cytometry, we were able to detect pentamer positive cells by day 7 for three distinct epitopes: RPH (58.1%), TPR (8.45%), and NLV (0.36%) in one of the CMV‐specific T cell lines. In addition, ALY pentamer positive T cells were detected as early as day 6 of culture in the tumor T cell lines stimulated with the entire PRAME antigen and single epitope of the same antigen (0.2 and 0.16% respectively), while Survivin (LML) positive T cells were detected on day 13 for cells stimulated with the PRAME/Survivin/WT‐1 pepmix (0.19%). These results indicate that in two CMV‐specific and one tumor‐specific T cell product, we were successful in tracking T cell populations and their specificity towards their target antigens over time. Additional experiments, however, are required to reach statistical significance and demonstrate reproducibility. Since this method can identify virus‐ and tumor‐specific T cells early in the culturing process, it has the potential to be used for culture optimization and monitoring epitope expansion in real time, as well as sorting T cells according to their specific TCRs.

Support or Funding Information

This project was supported by the Children’s Cancer Foundation grant awarded to Dr. Patrick Hanley.
Total Cellular ATP Production Varies With Primary Substrate in Breast Cancer Cells
Authors: Mookerjee Shona A. , Ton Justin , Brady Maurice L. , Le Diem T. , Mar Jordon N.
Published: April 2020

Cancer cell growth is predicted to require substantial increases in the rates of substrate catabolism and ATP turnover to drive biosynthesis and cell growth. Strategic limitation of substrate supply is therefore a potentially effective way to slow tumor growth. However, the effects of substrate limitation on the rates of ATP production and consumption, especially over the acute timescales relevant to most experimental design, are not straightforward. First, bioenergetic homeostasis will oppose substrate limitation by increasing catabolism of substrates that remain accessible, in order to meet existing ATP demand. Second, ATP demand itself may be different under different substrate conditions. Empirical evaluation of total cellular ATP production is therefore required. Cultured cells remain an important model system for understanding these mechanisms of bioenergetic control.

Using extracellular flux analysis to estimate total cellular ATP production rate, we demonstrate that the MCF7 breast cancer model supports rates of basal ATP production over an approximately 2‐fold range, in the presence of the single substrates glucose, glutamine, and pyruvate, the main substrates commonly present in standard culture media. In comparison, a nontransformed cell culture model shows maintenance of a single ATP production rate across the same single substrates. These novel findings demonstrate that MCF7 cells are highly flexible with respect to total cellular ATP production, over a timeframe that is independent of the slower cellular remodeling caused by altered transcriptional regulation.

Support or Funding Information

NIH 1R15ES025917‐01A1; Touro University California College of Pharmacy
Profiling the Spatial Dynamics of Limb Patterning in Regeneration and Development Using Enhanced V3 HCR‐FISH Probe Design
Authors: Mun Evan , Lovely Alexander , Stein David , Monaghan James
Published: April 2020

The patterning of limbs in development utilizes spatial and temporal signaling dynamics which guide the formation of a complete limb from undefined tissue. Although they may play different roles, a group of key factors guide the development of most species. This process is recapitulated in the limb regeneration of the Mexican axolotl, and the possibility of regeneration in humans will require a precise understanding of how these factors guide this patterning. Previously, observation of limb patterning was restricted by in situ hybridization imaging techniques which had limited resolution and could not reliably compare the collection of essential genes on a single tissue. Now, we have applied V3 Fluorescent in situ Hybridization by Hybridization Chain Reaction (HCR‐FISH) to the model to generate clear, bright, multiplexed images of the expression of these factors. These multiplexed images are the first of their kind in the field, and allow for the overlapping of key factors in both axolotl salamander and murine models to draw essential conclusions regarding each gene’s role in limb patterning within and between species. Development of an efficient probe‐design pipeline and oligonucleotide amplification parameters has improved signal strength, selectivity, and cost effectiveness. Furthermore, we have designed novel V3 HCR‐FISH probes targeting the introns of actively transcribed sequences allows for the identification of actively transcribed RNA via nuclear‐specific signal. Our techniques have not only been applied to confirm predictions derived from single cell RNA‐seq data regarding the roles of Sonic Hedghog (Shh), Fibroblast Growth Factor 8 (Fgf8), and Gremlin 1 (Grem1), in axolotl limb patterning, but also showcased significant differences between the axolotl and murine limb models. These results indicate that application of V3 HCR‐FISH allows for the robust visualization of multi‐gene transcriptional dynamics with a clarity previously impossible in both regenerative and developmental models.

Support or Funding Information

This project was supported by NSF grants 1558017 and 1656429
From Land to the Water: A Comparative Look at the Morphologies of the Auditory Apparatuses of Carnivores
Authors: Koper Lindsey , Rahmat Sulman , Koretsky Irina
Published: April 2020

Morphological characters have been the basis for determining evolutionary relationships among mammalian carnivores and continue to be important in that regard. For the animals that make up the families Phocidae (true seals), Otariidae (sea lions and fur seals), and Odobenidae (walruses), together known as Pinnipedia, the research is very limited on the morphology of their auditory apparatuses. In regard to the few auditory characters that are well studied, they are almost always used for phylogenetic analyses. The reasoning behind their use in these analyses is that there is disagreement as to how pinnipeds evolved: do pinnipeds have a monophyletic or a diphyletic origin? To aide in answering this overall question of phylogeny this study plans to establish the morphology of characters of the auditory region and surrounding basicrania, to determine some of the processes in which auditory characters evolved due to a return to water, and if any of these characters have a phylogenetic signal. Some of the preliminary results include some osteological measurements of both extinct and extant specimens and CT scans of specific semiaquatic carnivoran skulls. One of the CT scans included the extinct seal Devinophoca emryi (early‐middle Miocene, ~16 Ma), which is known to have a mixture of primitive and derived characters in the skull and postcrania. Preliminary observation of the basicranium and auditory region demonstrates a mixture of primitive and derived characters as well. Overall, the comparative morphology of Pinnipedia ossicles and other derived auditory characters can possibly retain high phylogenetic signal even with the adaptive pressures of the aquatic environment. This information will aid in the overall understanding of phocid evolution and hearing adaptation during the transition from land to water.
Antioxidant activity of the ethyl acetate extract of Justicia spicigera in two periods of the year (spring‐summer and autumn‐winter) in the liver of adult diabetic rats
Authors: Murillo-Villicaña Marina , Peña-Montes Donovan J. , Huerta-Cervantes Maribel , Salgado-Garciglia Rafael , Noriega-Cisneros Ruth , Aguilera-Méndez Asdrúbal , Saavedra-Molina Alfredo
Published: April 2020

Background

Diabetes is a chronic disease that occurs either when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces. In studies it was shown that the ethanol extract of Justicia spicigera has a hypoglycemic effect in streptozotocin‐induced rats (STZ), related to the presence of secondary metabolites, such as kaempferitrin. However, the concentration of these compounds presents drastic variations under different environmental conditions.

Objective

The objective of this project is to study the activity of secondary metabolites produced by Justicia spicigeraduring two periods of the year (spring‐summer and autumn‐winter) in a chronic diabetes model on the liver of streptozotocin‐induced rats.

Methods

Male Wistar rats (450–500 g) with 20 weeks of age were used, divided into 6 groups: normoglycemic control (CN) and diabetic (CD), administered with spring‐summer extract (NEPV and DEPV) and autumn‐winter ( NEOI and DEOI) for 60 days, diabetic rats were induced with an intraperitoneal injection of STZ (45 mg / kg). During treatment the weight and blood glucose were determined every 6 days. Subsequently, the rats were submitting euthanasia to obtain weight, homogenate and mitochondria of liver, which were used for the determination of catalase and superoxide dismutase activities and glutathione levels.

Results

The ethyl acetate extract of Justicia Spicigera collected during the spring‐summer season significantly lowers blood glucose levels and prevents excessive loss of body weight and liver growth increases catalase activity and reduced glutathione levels. The ethyl acetate extract of Justicia Spicigera collected during the autumn‐winter season prevents excessive loss of body weight and liver growth, but does not exhibit antioxidant activity when compared to the DEPV group.

In conclusion, the concentration of Justice spicigera compounds related to hypoglycemic and antioxidant activity is affected by environmental factors.

Support or Funding Information

The authors appreciate the partial economic support from CIC‐UMSNH (2.16, to ASM).
Gestational diabetes affects spatial working memory and lipid metabolism but not oxidative stress biomarkers in second generation offspring rats
Authors: Huerta-Cervantes Maribel , Peña-Montes Donovan J. , Manzo-Avalos Salvador , Salgado-Garciglia Rafael , Cortés-Rojo Christian , Montoya-Pérez Rocío , López-Vázquez Miguel Angel , Olvera-Cortés María Esther , Saavedra-Molina Alfredo
Published: April 2020

Gestational diabetes (GD) is an impairment of glucose metabolism during pregnancy, an increment of blood glucose in the intrauterine milieu results in abnormal fetal development and increased risk of developing metabolic disorders as diabetes mellitus 2, neurodegenerative diseases and behavior abnormalities in the offspring. In addition, increased oxidative stress in specific brain regions at early ages has been reported in the offspring of experimental models of GD. However, the consequences in the F2 offspring behavior and oxidative stress in cerebral cortex remains unclear. The aim of this study was to evaluate the effect of gestational diabetes in the spatial working memory and oxidative stress of adult male and female F2 GD offspring. Twelve Wistar female rats were placed mate with a control male. Gestation pregnant rats (day 10) were divided randomly in two groups: Gestation control rats (GC) and gestational diabetic rats (GD). GD rats were injected with a single intraperitoneal injection of a low dose of streptozotocin (STZ), only rats exhibiting blood glucose levels 140–300 mg/dL were considered for the study. Four months female rats (F1) were mated with control male rats to obtain F2 offspring. Six‐months old rats (adult) male and female F2 offspring from GC and GD were employed in this work. We evaluated anxiety in the elevated plus maze and open field and spatial working memory in the eight‐arm radial maze. Oxidative stress biomarkers (reactive oxygen species, lipid‐peroxidation, glutathione status) were evaluated in cerebral cortex. We found an impairment in lipid metabolism and a deficiency in spatial working memory of F2 female GD offspring. On the other hand, we did not find changes in oxidative stress biomarkers. In conclusion, these results suggest a transgenerational effect of gestational diabetes in lipid metabolism and cognitive behavior without the involvement of oxidative stress.

Support or Funding Information

The authors appreciate the partial economic support from CIC‐UMSNH (2.16 to ASM).
Kinetic Effects of Inside‐Positive Membrane Potential on the Lactose Efflux of LacY
Authors: Jiang Xiaoxu , Ermolova Natalia , Lim John , Choi Seo Woo , Kaback H. Ronald
Published: April 2020

LacY, the lactose permease of E. coli., utilizes the free energy released from the downhill movement of H+ in response of an electrochemical H+ gradient (Δμ̃H+, interior negative and/or alkaline) to catalyze the accumulation of lactose against a concentration gradient. In the absence of Δμ̃H+, the downhill transport of lactose in response of a concentration gradient drives uphill translocation of H+ to generate a H+ gradient, and the polarity of the H+ gradient depends on the direction of lactose gradient. In other words, LacY is a lactose/H+ symporter, in which the lactose transport and H+ translocation are obligatorily coupled. LacY functions as a monomer with a total of 12 transmembrane helices arranged into two 6‐helix bundles tethered by a long loop between helix VI and VII. The sugar‐ and H+‐binding sites are located at approximate the center of the permease and can be accessed from either side of the membrane via an alternating access conformational change driven by the binding and dissociation of sugar. In LacY, Glu325 plays an essential role in H+ translocation since replacing Glu325 with neutral amino residues abolishes transport activities involving H+ translocation, but the mutants still bind sugar with normal affinity and catalyze equilibrium exchange. Previous studies have shown that the presence of Δμ̃H+ (interior negative) significantly accelerates lactose influx by decreasing the Km 50‐100‐fold with no change in Kd. The goal of this study is to investigate kinetic effects of Δμ̃H+ on lactose efflux in right‐side‐out membrane vesicles. We measured the initial rate of efflux at different pHs in the absence or presence of an imposed membrane potential (ΔΨ, generated by K+ influx in response of a concentration gradient in the presence of valinomycin, interior positive). The results revealed that the lactose efflux of WT LacY exhibits an apparent pK value near 7.2, which is not affected by the imposition of ΔΨ (interior positive). The initial rate of lactose efflux increases in the presence of imposed ΔΨ (interior positive) at all tested pHs, although enhancement is about two orders of magnitude less than that observed in lactose influx. Moreover, the effect of ΔΨ (interior positive) on the Km of lactose efflux is almost negligible compared to the dramatic change of the Km of lactose influx caused by ΔΨ (interior negative). In contrast to WT LacY, the E325A mutant does not carry out lactose efflux in the absence or presence of imposed ΔΨ (interior positive) at all tested pHs.

Support or Funding Information

NSF Eager Grant MCB‐1547801 to H.R.K., NIH Grant 1R01 GM120043 to H.R.K., and a grant from Ruth and Bucky Stein to H.R.K..
Utilizing Knockdowns, Overexpression and Tagging to Study the SUMO Proteases Ulp1 and Ulp2 in Tetrahymena thermophila
Authors: Lillian Jenna , Tessema Liya , Gessner Rachel , Wilson Cierra , Kandl Kim
Published: April 2020

SUMO, small ubiquitin like modifier, plays a role in post‐translational modification of proteins, altering protein‐protein interactions and regulating cellular processes. Much of what is known about the SUMO pathway and SUMOylation comes from studies conducted in yeast and vertebrates. As a unicellular ciliated eukaryote, Tetrahymena thermophila exhibit complexity and diversity of cellular processes that are not present in yeast, and they possess the advantages of other single celled model organisms, such as short cell division times and ease of growth and genetic manipulation. We aimed to develop a better understanding of the function of the two SUMO proteases, Ulp1 and Ulp2, by knocking out the genes, ULP1 and ULP2, that encode these proteins. We designed knockout plasmid constructs by cloning the 5′ and 3′ flanking sequences of each gene into a vector with a neo resistance marker. The plasmid construct was then introduced into Tetrahymena, and cells were passaged into increasingly higher concentrations of paromomycin to promote phenotypic assortment. Quantitative RT‐PCR suggests that we were able to knockdown ULP2 but not ULP1, despite high paromomycin drug resistance, suggesting that ULP1 is an essential gene in Tetrahymena. We have also created plasmid constructs using a Gateway cloning method that enable us to both overexpress and YFP tag ULP1 and ULP2. Preliminary results show that Ulp1 localizes to the nuclear envelope, and Ulp2 localizes to the nucleus. Future work will focus on characterizing the overexpression phenotypes of ULP1 and ULP2 and the knockdown phenotype of ULP2 in Tetrahymena.

Support or Funding Information

This research was supported by the St. Olaf College Office of Collaborative Undergraduate Research, St. Olaf TRIO McNair Scholars Program (L.T.) and the Beta Beta Beta National Biological Honor Society (J.L.).
Lafora Disease: Differential Metabolic Disturbances in Neurons and Astrocytes
Authors: Markussen Kia Hjulmand , Andersen Jens V. , Sun Ramon C. , Waagepetersen Helle S. , Aldana Blanca I. , Gentry Matthew S.
Published: April 2020

The brain is an incredible energy‐demanding organ and even small disturbances in brain energetics may have fatal consequences. Lafora disease (LD) is a rare metabolic and neurodegenerative disorder clinically presenting with tonic‐clonic epileptic seizures and myoclonic jerks. LD patients experience rapid neurodegeneration starting in early adolescence and the disease is indisputable fatal typically within 10 years of onset. LD is caused by mutations in the genes EPM2A or EPM2B encoding the proteins Laforin and Malin, both implicated in glycogen metabolism. However, the mechanism of disease is poorly understood, though emerging evidence suggests that neurons and glial cells are affected differently in LD. This study aims to elucidate the effect of laforin on central carbon metabolism in different cellular compartments. We employed a systematic characterization of glucose metabolism in cerebral brain slices from 3‐months old Epm2a−/− and wildtype animals in addition to primary cultures of neurons and astrocytes utilizing [U‐13C]glucose and subsequent analysis by gas chromatography‐mass spectroscopy. Furthermore, slices were incubated with [U‐13C]glutamine and [U‐13C]glutamate to assess neuronal and astrocytic metabolic compartmentation, since glutamine is predominantly taken up and metabolized by neurons, and glutamate by astrocytes. The results of this study revealed complex disturbances of central carbon metabolism in the LD brain with clear metabolic distinctions between cerebral cell types. Disturbed oxidative glucose metabolism was observed in both primary cultures of neurons and astrocytes and in acutely isolated brain slices. Interestingly, the results indicate a compartmentalized glucose metabolism where neuronal glucose metabolism is downregulated, and astrocytic glucose metabolism upregulated in Epm2A−/− mice. Furthermore, neuronal glutamine metabolism was increased whereas glial glutamate metabolism was unaffected. Overall, this study provides evidence of altered glucose and neurotransmitter metabolism in LD related to loss of laforin and hereby dysfunctional glycogen metabolism. Further investigation of metabolic alterations in cerebral cell types may provide important insights into the mechanism of disease and targeted treatment.
Nox4‐related miR‐92a Mimic Improves Alcohol‐Induced Derangements in Alveolar Macrophage Immunometabolism
Authors: Morris Niya L. , Yeligar Samantha M.
Published: April 2020

Objective

Individuals with a history of alcohol use disorders (AUD) are at increased risk of developing respiratory infections due in part to alveolar macrophage (AM) phagocytic dysfunction. Cellular metabolism and bioenergetics are known to play key roles in immune cell functions. Chronic alcohol exposure increases NADPH oxidase (Nox)4, which increases AM oxidative stress, and may contribute to derangements in AM immunometabolism. Nox4 expression may be post‐transcriptionally regulated by microRNA (miR)‐92a, leading to Nox4 downregulation. Therefore, we hypothesized that chronic alcohol exposure decreases mir‐92a to upregulate Nox4, which increases mitochondrial oxidative stress and dysfunction in immunometabolism, resulting in increased susceptibility to respiratory infections.

Methods

MH‐S, a mouse alveolar macrophage cell line, were treated ± 0.08% ethanol (EtOH) for 3 d ± miR‐92a mimic (50 μM, last 1 d). Expression of miR‐92a and Nox4 were measured by qRT‐PCR. Cellular reactive oxygen species (ROS) generation and H2O2 were measured using 2′,7′‐dichlorofluorescein‐diacetate fluorescence and Amplex Red assay, respectively. Co‐localization of Nox4 and mitochondria were assessed by confocal microscopy. Mitochondrial respiration and bioenergetics were measured using an extracellular flux bioanalyzer. In vitro phagocytic capacity was measured using pHrodo Staphylococcus aureus BioParticles conjugate.

Results

In vitro EtOH: 1) decreased miR‐92a and increased Nox4 expression, 2) increased ROS and H2O2, 3) increased Nox4 co‐localization with mitochondria, 4) decreased mitochondrial respiration and bioenergetics, and 5) decreased AM phagocytic ability. Treatment with miR‐92a mimic reversed these EtOH‐induced impairments.

Conclusions

Chronic alcohol consumption increases alveolar macrophage oxidative stress, while reducing mitochondrial respiration and phagocytic capacity. Treatment with miR‐92a mimic reduced EtOH‐induced oxidative stress and improved AM immunometabolism and phagocytic function. Our studies reveal that miR‐92a may be a novel therapeutic target in the treatment of pulmonary infections in individuals with a history of AUD.

Support or Funding Information

This work was supported by the National Institute of Alcohol Abuse and Alcoholism R01‐AA026086 (SMY) and by the Emory Acute Lung Injury Training Program T32‐HL116271‐05 (David M. Guidot).
Differential Cardiac Adaptation to Free‐Wheel Running in Old and Young Mice: Impact of the Cardiac Circadian Clock
Authors: Yusifova Musharraf , McNair Benjamin , Cook Ross F. , Schmitt Emily E. , Bruns Danielle R.
Published: April 2020

Critical cardiovascular processes are under control of the central circadian clock. Disruption of this clock (i.e. during aging) results in increased risk for development of chronic disease such as cardiovascular disease. Exercise is a protective intervention implicit in both age and circadian related pathologies, yet its role in regulating circadian gene expression in the heart remains untested. We hypothesized that circadian rhythm would be disrupted in aged mice and rescued by free‐wheel running. We analyzed wheel running patterns in male and female C57BL/6J mice at young (3–6 months) and old (18–20 months) ages exposed to standard 12h light/dark cycle. As expected, young female mice ran further than male mice, and old mice ran significantly less than young mice. Older mice of both sexes had a delayed start time in activity and early cessation of free‐wheel running, which likely contributes to shorter total diurnal running distances. Free‐wheel running resulted in an expected left ventricular (LV) hypertrophy in young animals, however older animals demonstrated a regression of LV mass. Old mice demonstrated dysregulated cardiac circadian clock expression (BMAL, Per2) compared to young animals, an effect which was not rescued by free‐wheel running. Together our data suggest that the aged heart undergoes a unique adaptation to running compared to the young heart. While voluntary running was not sufficient to rescue age‐related changes in circadian rhythm, ongoing studies will determine if forced exercise and/or chronotimed exercise can improve age‐related cardiac and circadian dysfunction.
Receptor Mechanisms in Nicotinic Enhancement of Opioid Antinociception
Authors: de Moura Fernando Barreto , Bergman Jack
Published: April 2020

The use of prescription opioids for pain management can be limited by their adverse behavioral effects, which usually are not measured in the initial preclinical assessment of established or novel candidate analgesics. Recently, we have developed a protocol for concurrently assessing the antinociceptive effects of drugs (an indicator of analgesic promise) and their ability to decrease rates of operant behavior (an indicator of unwanted behavioral effects). Under these procedures, nicotine has been shown to augment the antinociceptive, but not the rate‐decreasing, effects of opioid analgesics with an inverse relationship to efficacy at the m‐opioid receptor‐ e.g., nicotine‐induced enhancement of nalbuphine>oxycodone>fentanyl. The present study was designed to further elucidate nicotinic‐opioid interactions by exploring the role of nicotinic receptor efficacy in the nicotinic augmentation of opioid antinociception. Male squirrel monkeys (n=4) lever‐pressed under a fixed ratio (FR) 10 schedule of 20% (v/v) condensed milk reinforcement. A 30‐s timeout (TO) period followed either the completion of the FR 10 or the elapse of 20‐s (limited hold (LH) 20). During the 30‐s TO, the distal portion of the monkey’s tail was immersed in 35, 50, 52, or 55°C water, and the latency to remove the tail was recorded. Dose response functions for tail‐withdrawal latency and decreases in response rate were generated for the high efficacy m‐opioid agonist fentanyl and the low efficacy m‐agonist nalbuphine alone and in the presence of nicotinic agonists reported to have high or intermediate efficacy‐‐epibatidine (0.00056 mg/kg) or varenicline (0.032 mg/kg), respectively. Fentanyl dose‐dependently increased tail‐withdrawal latencies at all water temperatures and, concomitantly, decreased rates of responding. Nalbuphine did not significantly decrease response rate, and increased tail‐withdrawal latencies at 50 and 52°C, but not at 55°C. Neither epibatidine nor varenicline significantly modified the rate decreasing effects of any of the opioids or the antinociceptive effects of fentanyl. However, the antinociceptive potency of nalbuphine was markedly enhanced by epibatidine (up to ~70‐fold) and only slightly increased by varenicline. The present results add to previous findings with nicotine, showing that nicotinic enhancement of opioid antinociception is inversely related to m‐opioid efficacy. They also extend those findings to suggest that such adjuvant effects are more robustly produced by nicotinic full agonists (nicotine, epibatidine) than a partial agonist (varenicline). In conjunction with previous reports, these findings support the idea that nicotinic agonists may be clinically useful as adjuvants for opioid analgesia.

Support or Funding Information

DA035857 and Harvard Medical School Livingston Fellowship
Smooth Muscle Tone Contributes to Bladder Wall Stiffness during Filling
Authors: Saxena Pragya , Tykocki Nathan , Roccabianca Sara
Published: April 2020

Sensations of bladder fullness are conveyed to the central nervous system by sensory nerves as a response to distention of the urinary bladder. This distention is accompanied by non‐voiding transient pressure events and contractions called “micromotions” that are responsible for the majority of sensory outflow during bladder filling. Changes in smooth muscle contractility along with alterations in wall stiffness and sensory feedback are observed in most UB pathologies. Thus, the mechanical properties of bladder wall may play a crucial role in communicating a sense of bladder fullness. We hypothesize that the contractile apparatus of the urinary bladder is actively involved in maintaining bladder wall stiffness during filling.

Whole mouse bladders were dissected and mounted in a custom designed pentaplanar reflected image macroscopy (PRIM) system, for simultaneous measurement of bladder wall micromotions, bladder volume, intravesical pressure, bladder wall stress, and bladder wall strain during ex vivo bladder filling. Bladders were filled at a constant rate (30 μl/min) until intravesical pressure reached 25 mmHg. During filling, bladder wall micromotions led to transient pressure events (0–2 mmHg in amplitude). These micromotions began on the lateral sides of the bladder wall near the trigone and expanded upwards as the bladder filled. Baseline bladder pressure, however, remained relatively low over a large infused volume (~350 μL). Next, we derived the stress versus strain relationship of the bladder wall from the pressure‐volume recordings during filling. At baseline pressures >15 mmHg, intravesical pressure rapidly increased and transient pressure events could no longer be distinguished implying that wall compliance is maintained until larger infused volumes, after which bladder wall stress with respect to strain increases rapidly. To determine if smooth muscle tone altered bladder wall stress during filling, bladder was exposed to myosin inhibitor blebbistatin (10 μM) or PI3 kinase inhibitor wortmannin (10 μM). Both blebbistatin and wortmannin effectively diminished the micromotions and shifted the stress vs strain curve to the right, indicating that the smooth muscle contractile apparatus and smooth muscle tone contribute to bladder wall stiffness, but predominantly only at higher filling volumes. These findings suggest that smooth muscle tone directly increases bladder wall stiffness and that changes in this tone could alter sensory feedback during bladder filling.

Support or Funding Information

Supported by NIH K01DK103840 (NRT) and R01DK119615 (NRT).
Long noncoding RNA LINC00844‐mediated molecular network regulates expression of drug metabolizing enzymes and nuclear receptors in HepaRG cells
Authors: Ning Baitang , Li Dongying , Wu Leihong , Knox Bridgett , Chen Si , Tolleson William , Liu Fang , Guo Lei , Tong Weida
Published: April 2020

Noncoding RNAs, such as long noncoding RNAs (lncRNAs) and microRNAs (miRNAs), regulate gene expression in many physiological and pathological processes, including drug metabolism. Drug metabolizing enzymes (DMES) are critical components in drug‐induced liver toxicity. In this study, we used human hepatic HepaRG cells treated with 5 or 10 mM acetaminophen (APAP) as our model system and identified LINC00844 as a toxicity‐associated lncRNA. We analyzed the expression profiles and the correlations between the expression of long noncoding RNAs and key enzymes for acetaminophen metabolism in humans. Our results showed that lncRNA LINC00844 is enriched in the liver and its expression correlates positively with that of CYP3A4, CYP2E1, SULT2A1, NR1I2, and HNF4A mRNA transcripts. We demonstrated that LINC00844 regulates the expression of these five genes in HepaRG cells using gain‐ and loss‐of‐function assays. Further, we discovered that LINC00844 was predominantly localized in the cytoplasm and acted as a hsa‐miR‐486‐5p sponge, via direct binding, to protect SULT2A1 from miRNA‐mediated gene silencing. Our data also showed that hsa‐miR‐486‐5p upregulated the expression of CYP3A4, SULT2A1, NR1I2, and HNF4A by positively regulating LINC00844. We depict a LINC00844‐mediated regulatory network that involves miRNA and NRs and influences the expression of DMEs.

Support or Funding Information

FDA E0753201
Eukaryotic Initiation Factor 3 Interactions with 5′ and 3′ RNA structures of the Barley Yellow Dwarf Virus Facilitate a Non‐Canonical Translation Mechanism
Authors: Powell Paul , Bhardwaj Usha , Goss Dixie
Published: April 2020

Barley Yellow Dwarf Virus (BYDV) is a positive strand RNA plant virus that translates without using a 5′ 7‐methylguanosine cap or a 3′ poly‐adenosine tail that are required for the canonical translation mechanism used by most mRNA. BYDV’s non‐canonical translation relies on RNA structures in the 5′ and 3′ untranslated regions (UTRs) that recruit eukaryotic initiation factors (eIFs) and ribosomes. Using a combination of biophysical, biochemical and chemical probing techniques, we have measured direct binding of eukaryotic initiation factors (eIFs) to BYVD 5′ and 3′ UTRs and probed binding sites. Our data show that one of the initiation factors, eIF3, can bind tightly, specifically, and simultaneously to structures in both 5′ and 3′ UTRs. This suggests that eIF3’s mechanistic role includes stabilization of the long‐distance RNA‐RNA interaction and the transfer of 3′‐bound factors/complexes to the 5′ UTR. Further work with RNA chemical probing has identified regions of the BYDV UTRs that interact with eIF3. This data highlights commonalities between BYDV structures and structures found in other viruses (e.g. Hepatitis C virus) and cellular mRNAs (e.g. c_JUN mRNA).

Support or Funding Information

NSF MCB‐1902054 to Dixie J. Goss, NSF IGERT‐0965983 to Hunter College
Molecular Basis of Takotsubo Syndrome
Authors: Chandler Selena , Joshi Hirva , Hoff Ellianna , Patel Karishma , Ohanyan Vahagn , Kiedrowski Mathew , Chilian William M. , Dong Feng
Published: April 2020

Introduction

Takotsubo syndrome (TTS), also known as the “Broken Heart Syndrome” or “Apical Ballooning Syndrome is defined by its characteristic anomaly: when the heart contracts during systole, the apex of the heart dilates as the base of the heart contracts. Severe TTS can lead to cardiogenic shock and death in 3–4% of patients. There is no standard medical therapy for TTS because the mechanism underlying the development of the syndrome is unknown. Our goal is to determine the molecular mechanisms of TTS as a first step towards better treatment plans and outcomes.

Methods

Our model of TTS is a Kv1.5 null mouse, with compromised coronary metabolic dilation, subjected to transaortic constriction (TAC). Two weeks after TAC, when Kv1.5 null mouse showed profound apical ballooning during ventricular contraction (echocardiography), myocardial blood flow (MBF) was measured by contrast echocardiography in the base and apex of the left ventricle of mice under control conditions and during acute administration of norepinephrine to increase cardiac work. Hearts were collected and gene expression (RNA deep sequencing) in both the apex and the base were performed and followed by bioinformatic analysis. Wild type (WT) and unstressed Kv1.5 null mice were used as controls. To increase the scientific rigor, we purposefully analyzed RNA expression from different animals with Real‐Time‐PCR, to confirm the sequencing data.

Results

A total of 3875 genes were identified by differentially expressed between TTS vs unstressed Kv1.5 null hearts. Gene Set Enrichment Analysis shows many families of genes downregulated (metabolism) and upregulated (inflammation, hypoxia, apoptosis) in the apical (ballooning) area of the heart in TTS (compared to the base of the heart). RT‐qPCR revealed significant upregulation of the genes for Postn, Lox, and C920009B18Rik (p<0.01) and down‐regulation of the genes Ucp3, Acaa2, Pfkb1, Mir133a‐2 (p<0.05). These significant changes in expression were also shown in the apex of TTS hearts as compared to the apex of control Kv1.5 null hearts (p<0.01). We also found that in the mice with TTS, MBF was lower in the apex than in the base.

Conclusion

Our results suggest that the apex of the heart in TTS is receiving insufficient perfusion compared to the base, which may be one of the root problems in TTS. There is significant upregulation of structural genes in the apex of TTS hearts versus their bases and the apexes of normal hearts, whereas there is a significant downregulation of genes playing roles in metabolism. This confirms the importance of these specific genes in the development of the anomaly shown in TTS.