bluuebunny/crossref_metadata_2025 huggingface dataset:
Computed Cell‐Cell Interactions Correlate with Physical Location of Cells in C. elegans
Authors: Armingol Erick , Joshi Chintan , Baghdassarian Hratch Matthew , Shamie Isaac , Lewis Nathan
Published: April 2020

Behaviors, functions and phenotypes of multicellular organisms are shaped by the coordination of cellular activities and properties. Those cellular activities and properties depend on activation of cell‐cell communication pathways and proper intercellular interactions amongst cells. The most important factors that influence how cells communicate and interact are localization of cells in the organism, expression levels of membrane proteins and secreted proteins. We sought to understand how intercellular interactions may be shaped by cell proximity, by analyzing protein‐protein interactions that could lead cell‐cell communication and interaction. We evaluated cell‐cell communication in C. elegans by integrating single‐cell RNA‐seq data and a list of ligand‐receptor pairs. To assess the potential of interaction, we computed a cell‐cell interaction (CCI) score that captures the number of ligand‐receptor pairs that are active between two cells versus the active ligands or receptors in each of those cells. We used this approach for every cell pair in the transcriptomics data. To associate the CCI score to the physical location of cells we used a published 3D‐map of cells to create a distance matrix among cell pairs. Afterwards, we calculated a correlation score between CCI score and physical distance of cell pairs. The score was used as an objective function to be maximized as ligand‐receptor pairs were randomly dropped from the list by a genetic algorithm (GA). Subsequently, a permutation analysis was run to evaluate the extent of significance of the ligand‐receptor pairs that were selected to maximize the correlation. To understand the role of those protein‐protein interactions, we performed a functional analysis on the list obtained by the GA. From these analyses, we obtained a negative spearman correlation between CCI scores and physical distances, showing that the protein‐protein interactions obtained by the GA capture properties of 3D‐location of cells and their potential of interaction given their proximity to certain cells. In addition, functional analysis of enriched ligand‐receptor pairs gave us more insights about the role that they could have given the cell locations. Our approach of computing CCI scores will facilitate data‐driven discoveries such as detecting key ligand‐receptor pairs for given properties or phenotypes of cells, and in the case of C. elegans, demonstrates that physical location of cells may be predicted by molecular signatures associated with the proteins mediating the interactions.

Support or Funding Information

This work was supported by the Keck Foundation, and scholarship awards from Becas Chile (CONICYT) and the Fulbright Commission.
Enzymes expression of antioxidant protective function of microorganisms in process of develop antibiotic resistance
Authors: Shabunin Sergey , Vostroilova Galina , Pasko Nadezhda , Ermakova Tatiana , Achilov Vadim , Plemyashov Kirill
Published: April 2020

Antibiotic resistance is one of the biggest threats in veterinary medicine that prevents effective treat animals with bacterial etiology diseases. The aim of the study was to research E.coli resistance to antimicrobial therapy. A different strain of E. coli 866 which is stable to complex antibiotic was used in the research. Primary cell cultures and cell cultures after 40 and 60 passages were used. Bacterial strains were cultured in pepted meat broth with growing concentration of bacteriostatic agents of Apramycin, Cefotaksim and .drug K‐2 is based on mixing of this two antibiotics. Threshold cycle is defined with software Rotor‐Gene 6000 (Australia). We have analyzed the relative level of gene expression with 2‐ΔΔCt method. Control group is primary cell cultures. Using of Cefotaksim after 40 passages gives results of insignificant decrease of peroxidase transcripts (0,33), small rise of catalase level (1,92) and iron superoxide dismutase (SOD Fe) (1,93) and strong growth of copper‐zinc superoxide dismutase (SOD) level (141,04). But after 60 passages we can see regress of peroxidase level (0,000019), catalase (0,0065) and copper‐zinc superoxide dismutase (SOD) level (0,0002). While level iron superoxide dismutase (SOD Fe) was high (3,61).Using of Apramycin for E. coli 866 after 40 passages gives results of notable fall of peroxidase level (0,000021), growth of catalase level (3,081), iron superoxide dismutase (SOD Fe) (1,14) and copper‐zinc superoxide dismutase (SOD) level (1,8). But after 60 passages we can see stable level of peroxidase (0,87) in relative to 40 passages. Decrease of catalase level (0,59), rise of copper‐zinc superoxide dismutase (SOD) level (2,58) and strong rise of iron superoxide dismutase (SOD Fe) (155,42).Study results of using K‐2 (complex of Apramycin and Cefotaksim) for E.coli 866 after 40 passages is significant growth of iron superoxide dismutase (SOD Fe) (113,77) and not so high rise of copper‐zinc superoxide dismutase (SOD) level (1,8).In the study of gene expression patterns of E.coli in process of develop antibiotic resistance by bacterial culture in MPB with growing concentration of bacteriostatic drugs during 60 passages appears changes of catalytic antioxidants in transcript representation. In conclusion superoxide dismutase (SOD) has the most visible changes. Relative level of gene expression of copper‐zinc superoxide dismutase is 141,04 with Cefotaksim after 40 passages and 155,42 with Apramycin after 60 passages. Relative level of iron superoxide dismutase (SOD Fe) is 113,77 and zinc superoxide dismutase (SOD Zn) – 88,035.Gene’s changes of catalytic antioxidant occur due to high survival by enzymes on antibiotic effects because of strong SOD antioxidant defense. Using of K‐2 leads to decrease the level of antibiotic resistance of E.coli 866.
Protective effects of Gallic acid against COPD linked inflammation and emphysema
Authors: Singla Esha , Naura Amarjit Singh
Published: April 2020

Chronic Obstructive Pulmonary Disorder (COPD) is a chronic inflammatory disease, currently affecting around 328 million people worldwide. Gallic acid (GA), a naturally occurring phenolic compound is known to possess anti‐oxidant and anti‐inflammatory activities. The aim of the present work was to investigate the beneficial effects of Gallic acid against COPD linked inflammation and emphysema by utilizing Elastase (ET) and Cigarette smoke (CS) based mouse model(s) of the disease. Male Balb/c mice were treated with ET (1U/mouse) or exposed to CS (9 cigarettes/day for 4 days). GA administration was started 7 days (daily) prior to induction of injury with ET or CS exposure. Broncho‐alveolar lavage was analysed for inflammatory cells and pro‐inflammatory cytokines. Lung homogenate was assessed for oxidative stress related biochemical parameters. Further, lung tissue was subjected to semi‐quantitative RT‐PCR, Immunoblotting and histological analysis. Our data showed that GA treatment suppressed the ET induced recruitment of inflammatory cells particularly neutrophils as well as myeloperoxidase activity in lungs at 24 hrs post injury. Reduction in the inflammatory cells was associated with subdued production of pro‐inflammatory cytokines namely IL‐1β, TNF‐α and IL‐6. Additionally, GA normalized the ET induced redox imbalance in the lungs towards normal as reflected by levels of ROS, MDA, GSH and protein carbonyls. Further, GA treatment blunted the ET induced phosphorylation of p65NF‐kB and its inhibitor IκBα along with down‐regulation of mRNA expression of NF‐kB dependent pro‐inflammatory factors including IL‐1β, TNF‐α, MIP‐2, KC and GCSF. Moreover, GA suppressed the CS induced influx of neutrophils and macrophages along with blunted gene expression of pro‐inflammatory cytokines TNF‐α, MIP‐2 and KC. Finally, histological analysis of lungs at 21 days after elastase administration showed that the phytochemical blocks the development of emphysema by restoring the balance between matrix metallo‐proteinases and their inhibitor(s). Overall, our data shows that GA effectively modulates pulmonary inflammation and emphysema associated with COPD pathogenesis in mice and thus may offer a new/alternative treatment strategies in the area.

Support or Funding Information

Financial support from Department of Biotechnology, Government of India (BT/PR17968/MED/122/33/2016 and BT/RLF/Re‐entry/36/2012), UGC‐SAP and Senior Research Fellowship from Indian Council of Medical Research (ICMR), India
Bitter taste receptor agonists induced chronotropic effects on rat heart
Authors: Yuan Guanyin , Jing Yang , Wang Ting , Fernandes Vitor Samuel Leite , Xin Wenkuan
Published: April 2020

Background

Bitter taste receptors (Tas2rs), the members of the G‐protein‐coupled receptors, mediate the bitter taste and express in extra‐oral tissues. Previous studies have shown that Tas2r mRNAs are expressed in the whole heart and cultured cardiomyocytes of neonatal rat.

Aim

This study investigates the expression of Tas2rs in the sinoatrial node (SA node) and left ventricle of rat heart, and the mechanism of bitter compounds regulation of the heart rate.

Methods

Rats were excised by thoracotomy and the sinoatrial node and left ventricle tissues were isolated. These tissues were processed to study the mRNA expression levels of Tas2rs and their coupled G‐protein subunits by real time quantitative PCR (qPCR). Langendorff isolated heart models were used to record the heart rate and electrocardiogram. SA node beating recordings were used to measure the beating rate in combination with bitter compounds, quinine and chloroquine. The statistic dada are expressed as mean ± SEM.

Results

We detected the mRNA expression of 7 subtypes of Tas2rs (Tas2r108, 120, 121, 126, 135, 137, 143) in the left ventricle and 8 subtypes in the SA node including Tas2r38 in addition to the 7 subtypes in the left ventricle, in which the mRNA level of Tas2r120 is the highest among them followed by Tas2r143 and 121. In addition, Tas2r coupled G‐protein subunits also expressed in the SA node and left ventricle.

Tas2r agonists, quinine (1–50 μM) and chloroquine (1–20 μM), decreased the heart rate and increased the RR interval and QRS duration in Langendoff‐perfused isolated rat hearts; they reduced the spontaneous beating rate of isolated SA nodes with pEC50 values of 4.907 ± 0.045 and 4.968 ± 0.030, respectively.

The blockade of Tas2r108 with abscisic acid (500 μM), the inhibition of PDEs with IBMX (50 μM), or the selective inhibition of PDE3 and PDE4 with a cocktail of cilostamide (0.5 μM) and rolipram (10 μM), attenuated the negative chronotropic effects of quinine and chloroquine on the SA node. Furthermore, quinine (20 μM) and chloroquine (10 μM) suppressed the tachycardia effect of isoprenaline on the SA node and shifted the concentration‐response curve of isoprenaline rightward.

Conclusion

Tas2r agonists, quinine and chloroquine, induce negative ionotropic effects in rat hearts. Our data provide mechanistic evidence that Tas2r agonists decreased the heart rate by prolonging ventricular depolarization, and by attenuating the SA node pace in a PDE‐dependent manner. Tas2r agonists can counteract with β‐adrenergic receptor activation and eliminate isoprenaline‐induced tachycardia, suggesting Tas2rs could be the potential targets of drugs for treating sinus or ventricular tachycardia and elevated heart rate related to heart failure.

Support or Funding Information

This work was supported by Funding from Southwest University [Grant number SWU 115086, 104290/22300503] and Administration of Foreign Experts Affairs of China [Grant number BC2018042].
Differential phosphorylation modulation of GluN2B at tyrosine 1336 in two types of chemical‐induced LTP in hippocampal slices
Authors: Zhang Bin , Lu Wen , Ma Wu , Xue Fusheng , Zhang Yongchao , Fang Weiqing
Published: April 2020

Synaptic plasticity, i.e., long‐term potentiation (LTP) and long‐term depression (LTD), has been extensively studied as a cellular mechanism of learning and memory. Chemcal‐induced LTP, which is dependent on activation of NMDA receptor, was widely accepted as neuronal model for exploring synaptic plasticity in brain slice or neuronal cultures. However, the phosphorylation regulation of GluN2B, a critical modulatory subunit of NMDA receptor chemical‐induced LTP remains poorly understood. In the present study, we examined the regulation of GluN2B at tyrosine 1336 in two types of chemical‐induced LTP, either by forskolin & rolipram or by glycine‐treated acute hippocampal slices. Elevated phosphorylation level of tyrosine 1336 at GluN2B in forskolin & rolipram‐induced LTP rather than glycine‐induced LTP was observed. Moreover, a similar increment of Src activity was detected, evidenced by higher phosphorylation level of tyrosine 416 at Src. Taken together, our results reveal a diassociated regulation of tyrosine 1336 at GluN2B and tyrosine 416 at Src in forskolin & rolipram‐induced LTP, uncovering phosphorylation site specific modulation in two types of chemical‐induced LTP, thus broadening our understanding of the role of GluN2B in learning and memory.

Support or Funding Information

This work was funded by National Natural Science Foundation of China Grant No. 31700882 (to B.Z.) and 31600863 (to W.L.), Zhejiang Provincial Natural Science Foundation of China LY20C090004 ( to B.Z) and LY18H090010 to (H.A.), the start‐up fund of Hainan Medical University (to W.L.).
The uterine contractile activity in the usage of «Endometramag» drugs containing propranolol hydrochloride
Authors: Minin Alexander , Filatov Andrey , Khlopitsky Vasily , Plemyashova Anna , Nikitin Georgy , Stekol'nikov Anatolii
Published: April 2020

The malfunction of the contractile activity of the uterus plays a significant role in mechanisms of the development inflammatory diseases of the sows ’uterus and mammary glands. This material is intended to research the stimulating impact of propranolol hydrochloride on the contractile activity of the sows’ uterus with endometritis when applying intrauterine insertion of «Endometramag» containing propranolol hydrochloride, and determine the duration of its biological action. The research conducted was based on results of five sows with acute postpartum catarrhal purulent endometritis. The uterine contractile activity was recorded by internal hysterographie method using the device «Hysterograph». «Endometramag» series preparations containing 15 mg / ml propranolol hydrochloride were inserted intrauterine in a dose of 75 ml. Uterine activity was recorded at baseline and 1, 3, and 6 hours after intrauterine insertion. The analysis of the received hysterograms showed that the contractility of the uterus of sows with acute postpartum endometritis was characterized by frequent contractions with low amplitude and duration. The frequency of uterine contractions for 30 minutes is on average 6.67 ± 0.67, the amplitude of the contractions − 6.04 ± 0.58 mm Hg., the duration of the contraction was 2.02 ± 0.18 minutes, the contractional index ‐ the integral indicator of functional activity of the myometrium was 85.13 ± 22.69 units, it follows from the records of the hysterograms. Intrauterine insertion of preparation «Endometramag» containing propranolol hydrochloride had a positive effect on the myometrium manifested in an increase in the amplitude of muscle contractions and their duration. Beyond intrauterine usage of the complex drug «Endometramag» after 1 hour, the average height of the reductions was 1.72 times higher and their duration was 1.88 times, after 3 hours ‐ 2.02 and 1.71 times, and after 6 hours ‐ 1.55 and 1.42 times, respectively. At the same time, the frequency of reductions decreased by 20.09–25.03% with an increase in pauses between them. The contractional index after 1,3 and 6 hours was higher at 2.22, 2.65 and 1.63 times compared to the baseline. The local effect of «Endometramag» containing propranolol hydrochloride for the contractile activity of the smooth musculature of the uterus begins to manifest within 1 hour after its insertion and lasts more than 6 hours. The increase in the total contractile activity of the uterus is manifested by the energization of the amplitude and duration of the contractions. Restoration of excitability and contractility of the sows' uterus with endometritis facilitates faster evacuation of its contents and a favorable flow of postpartum involutional processes.
Hell's Gate globin as a model for the functionality of human neuroglobin
Authors: Ales Ruby , Edmunds Mary , Schimnowski Ava , Torenvliet Alyne , Lane Jim
Published: April 2020

Methylacidiphilum infernorum (M.infernorum) is an extremely acidophilic, aerobic, and methanotrophic bacteria in the Verrucomicrobia phylum. A unique aspect of M. Infernorum is the presence of a special globin called Hell’s Gate globin I (HGbI). This globin has a ferric heme group and the unusual ability to bind acetate as well as oxygen with a high affinity. Discovered in geothermal vents in New Zealand, the bacteria belongs to the only known group of aerobic methanotrophs outside of proteobacteria, making M. infernorum a novelty. The bacteria has acquired numerous genes, including those for enzymes of methylotrophic pathways via horizontal gene transfer, in particular, from proteobacteria. M. Infernorum is the most acidophilic bacteria capable of methane oxidation. The low pH conditions favored by M. Infernorum have interesting effects on HGbI’s ligand bonds. HGbI also facilitates fast oxygen binding and slow release due to distal residues gln and tyr. Although not everything about HGbl is known, there have been multiple functions proposed. We used these proposed functions to speculate on a structurally similar protein, human neuroglobin (Ngb). Ngb is a recently discovered protein in the human body that is found in the central nervous system. Its proposed functions include transporting oxygen, raising hypoxia tolerance, or scavenging damaging reactive oxygen/nitrogen species. Because HGbl and Ngb have been proven to be structurally similar, we compared their structures in JMol and isolated the main differences, including a shorter CD loop and the absence of a D‐helix in HGbI, and the presence of a distal Gln50(E7) in HGbI compared to the His(E7) of Ngb. We cross‐examined these differences in order to speculate on the likelihood of the previously proposed functions of both HGbI and Ngb and gain a deeper understanding of both proteins. The Mahtomedi MSOE Center for BioMolecular MAPS Team used 3‐D modeling and printing technology to examine structure‐function relationships of HGbI and Ngb. The visual model was a valuable tool in developing our story.
Astrocyte Regional Heterogeneity Revealed Through Machine Learning‐based Glial Neuroanatomical Assays
Authors: Blackburn Jessica , Alves Michele Joana , Zhao Jing , Czeisler Catherine M. , Otero José Javier
Published: April 2020

Astrocytes undergo activation during brain injury through an ill‐defined process known as gliosis. Recent work focused on elucidating the molecular underpinnings of glial response to injury. However, astrocytes exist in a unique three‐dimensional space within the brain, and this location is highly relevant to the astrocyte’s function. Therefore, generating neuroanatomical assays, which maintain CNS positional information of astrocytes relative to their neural networks, represent optimal modalities to assess astrocyte function and reactivity. The majority of astrocytic neuroanatomical assays in the scientific literature measure mean fluorescence intensity of GFAP or ALDH1L1. Modern advances in computer vision and machine learning algorithms have now given us the tools with which we can extract a multitude of features from microscopic imaging data. By segmenting astrocytes from an image into numerous GFAP or ALDH1L1 positive “objects”, extracted numerical values indicate object location, object intensities, object shapes, as well as object textures from the original image. We tested the hypothesis that morphological changes in astrocyte reaction to injury represents a heterogeneous response in different brain regions. To test this hypothesis, we developed an automated segmentation algorithm for both GFAP and ALDH1L1 immunostained mouse brain sections treated with vehicle or systemic lipopolysaccharide to induce neuroinflammation. These images were then captured on a laser scanning confocal microscope and features characterizing shape, fluorescent intensity, and fluorescent texture. We then utilized a covariance matrix, random forest‐based boruta algorithm, cluster analysis and principal component analysis to evaluate the differences between reactive astrocytes and baseline astrocytes. By implementing a support vector machine algorithm, we show classification identification methods of reactive versus baseline astrocytes. Machine learning models developed to classify neuroanatomical images provides a highly insightful methodology to discover new processes by which astrocytes react to injury.

Support or Funding Information

3R01HL132355
Molecular Mechanisms of Stem Cell Reprogramming by CD47 Antagonists in Primary Human Cells
Authors: Cicalese Kyle V. , Kaur Sukhbir , Roberts David D.
Published: April 2020

CD47 is a ubiquitously expressed transmembrane receptor whose interaction with signal regulatory protein alpha on macrophages initiates anti‐phagocytic signaling. Additionally, thrombospondin‐1 (THBS1) binding to CD47 induces cell‐autonomous signaling that regulates cell proliferation, differentiation, and recovery from genotoxic stress. Previous studies showed that CD47‐ or THBS1‐null primary lung endothelial cells exposed to ionizing radiation have higher viability than primary wildtype cells. The mechanisms that enhance the viability of CD47‐null primary cells include enhanced autophagy, anabolic metabolism, and increased expression of c‐Myc and other stem cell transcription factors. The latter enable null cells to spontaneously form embryoid bodies. These results suggest that loss of CD47 may generally facilitate cell reprogramming and self‐renewal. Induced pluripotent stem cells (iPS) can be induced by introducing Yamanaka stem cell factors (MYC, KLF4, SOX2, and OCT4) into primary somatic cells, but iPS cells can form teratomas and thus pose risk of cancer when used for regenerative medicine. Therefore, alternative approaches are needed to overcome these limitations. We propose that CD47 gene knock out (KO) via CRISPR or treating with CD47 antagonists from primary human cells could be a new, simpler, and more effective way to develop multipotent stem cells. To investigate this, we have studied the effects of CD47 knockout and antagonists on embryoid body formation and mRNA expression of stem cell factors using fibroblasts, adipocytes, and human umbilical vein cells (HUVEC). Reprogramming was measured by quantifying embryoid body formation, cell viability, and mRNA expression using real‐time PCR. Preliminary results with HUVEC, fibroblast, and adipocyte primary cells treated with CD47 knockout or antagonists have shown enhanced cell proliferation and expression of stem cell factors similar to murine cd47‐null lung endothelial cells. These results suggest that CD47 plays a key regulatory role in stem cell reprogramming of human primary cells.

Support or Funding Information

This work was supported by the Intramural Research Program of the NIH/National Cancer Institute.
Competition and Identification of Anaerobic Organisms Isolated from Apis mellifera Gut Microbiota
Authors: Knier Rochelle Helen
Published: April 2020

Apis mellifera, the most common species of honeybee, has been suffering the epidemic of colony collapse disorder (CCD). This is defined by the sudden and unexplainable death of an entire colony of bees, and populations worldwide dwindle further every year. Although research continues, there exists no lasting or effective treatment. One underdeveloped area of study has been the option to treat bees with probiotics (
Audisio, 2017; Baffoni et al., 2015). There have been numerous studies within other organisms and honeybees that show that a well‐maintained gut microbiome is a key component of health, especially when the pathogen is endemic to the intestines – as in the case of Nosema pathogen, which has been implicated in CCD (
Raymann, 2018; Shreiner, 2015). The hypothesis being investigated is that microbes cultivated from the gut of normal bees will suppress the growth of known honey bee pathogens. Identification of such normal flora may serve as a probiotic for treatment of bees experiencing CCD due to infectious agents.

Support or Funding Information

Research Grant from the University of Wisconsin ‐ Stout
Skeletal Muscle ULK1 and ULK2 Jointly Couple Muscle Mass with Force and Are Required for Survival Under Low Nutrient Availability
Authors: Fuqua Jordan D. , Hughes Julia , Kuennen Dylan , Harris Matthew , Turner Kristen , Kronemberger Ana , Ebert Scott , Dierdorff Jason , Zhu Zhiyong , Zingman Leonid , Adams Christopher , Lira Vitor A.
Published: April 2020

The protein kinases Unc‐51 like autophagy activating kinase 1 and 2 (ULK1 and ULK2) have evolved from a single yeast gene, autophagy related 1 (Atg1). Although ULK1 and ULK2 are thought to redundantly stimulate autophagy, other functions are emerging. Interestingly, skeletal muscle expresses both ULK1 and ULK2 at high levels (i.e., >2‐fold the median expression of 91 mouse tissues and cell lines) suggesting an important, yet unknown role for their combined elevated expression in muscle. Therefore, to investigate the joint role of ULK1 and ULK2 on muscle homeostasis and whole‐body metabolism we generated mice with skeletal muscle‐specific knockout of these genes (ULK2/1 mKO). Here, we demonstrate that ULK2/1 mKO mice have larger muscles (~12%) that are actually weaker (~21% decrease in relative force) in comparison with wild type littermates. Loss of ULK2/1 in skeletal muscle also affects whole‐body metabolic homeostasis as indicated by decreased body fat mass (~20% at 7wk of age), attenuated fat mass gains with aging (~40% at 21mo. of age), and life‐threatening inability to maintain euglycemia under fasting conditions (~32% reduction of blood glucose compared to WT). Mechanistically, loss of ULK 2/1 not only impairs skeletal muscle autophagy but also AMPK function as indicated by accumulation of LC3, p62, NBR1, and ubiquitinated proteins, as well as reduced phosphorylation of ACC (a surrogate of AMPK activity), respectively. These results identify a previously unknown role for ULK2 and ULK1 in jointly coupling skeletal muscle size with force. They also reveal skeletal muscle ULK2 and ULK1 as fundamental proteins for survival under conditions of reduced nutrient availability.

Support or Funding Information

This work is funded by:AHA 16SDG30360001 (V.A.L.). and NASA Iowa Space Grant Graduate Fellowship Program
Neuroprotective effect of PEA‐OXA on oxaliplatin‐Induced neuropathic pain.
Authors: Cuzzocrea Salvatore , Lanza Marika , Campolo Michela , Casili Giovanna , Paterniti Irene , Filippone Alessia , Esposito Emanuela
Published: April 2020

Chemotherapy‐induced neuropathy is a common, dose‐dependent adverse effect of several antineoplastics, like oxaliplatin. It can lead to detrimental dose reductions and discontinuation of treatment, and severely affects the quality of life of cancer survivors. N‐Acylethanolamines (NAEs) involve a family of lipid molecules existent in animal and plant, of which is N‐palmitoylethanolamide (PEA) that arouses great attention owing to its anti‐inflammatory, analgesic, and neuroprotective activities. The modulation of specific amidases for NAEs (and in particular NAE‐hydrolyzing acid amidase NAAA, which is more selective for PEA) could be a condition to preserve its levels.

So, the aim of the present work was to evaluate the potential beneficial effects of 2‐pentadecyl‐2‐oxazoline (PEA‐OXA) the oxazoline of PEA that reportedly modulates activity of NAAA, in a model of oxaliplatin‐induced peripheral neuropathy. Chemotherapic pain was induced by an intraperitoneally injection of oxaliplatin (oxa) in rats on 5 consecutive days (D0–4) at an injection volume of 0.2 ml for a final cumulative dose of 10 mg/kg. In prophylactic paradigms, PEA‐OXA was given orally 15–20 min prior oxa. Sacrifice was made on day 25.

Our results demonstrated that oxaliplatin developed mechano‐hypersensitivity (allodynia and hyperalgesia) in rats; this was associated with the hyperactivation of glia cells and an increased production of pro‐inflammatory cytokines ( IL‐1β and TNF) in the dorsal horn of the spinal cord. Moreover, we showed that oxaliplatin activated NF‐κB pathway and modulated Nrf‐2 ones. The oral injection of PEA‐OXA attenuated inflammatory and oxidative process, decreasing neuropathic pain.

Our findings identify PEA‐OXA as a therapeutic target in chemotherapy‐induced painful neuropathy, throughout the biomolecular signaling NF‐κB/Nrf‐2 axis. Therefore, we can consider PEA‐OXA as a promising adjunct to chemoterapy to reduce chronic pain in patients.
Measuring T‐cell avidity and enrichment using acoustic force‐based technology
Authors: Harris Michael James , Anderson Leif , Raja Ali , Candelli Andrea
Published: April 2020

A key metric in the development of effective immunotherapies is the overall interaction strength between an immune cell and a target cell. The parameter describing this cell‐cell interaction strength is termed avidity; and refers to the measurement of the cumulative non‐covalent bond affinities between an immune cell and a target cell. Studies in adoptive cell transfer and antibody‐based therapeutics have shown that cell binding avidity of an immune cell to its target is a better predictor of its function than single molecule affinity measurements and that this parameter is integral to the development of long‐lasting and potent immune responses. The z‐Movi™ technology uses acoustic forces to effectively discriminate immune cells based on their avidity to target cells (e.g. tumor cells) in a high‐throughput manner. We have applied our technology to characterize bispecific antibody interactions, as well as TCR‐T and CAR‐T adoptive cell therapies. Here, we present the workflow of an example study in which we were able to distinguish cell binding avidity of tumor‐specific and non‐specific T cells. T cells engineered with a melanoma antigen‐specific T cell receptor showed a six‐fold increase in avidity relative to non‐specific T cells. In conclusion, we demonstrate a novel method to measure cell binding avidity with a high degree of specificity through the application of acoustic forces.
Autophagy contributes to the development of salt‐induced hypertension and kidney injury in Dahl S rats
Authors: Kumar Vikash , Kurth Theresa , Cowley Allen W.
Published: April 2020

Studies were carried to determine the role of autophagy in the development of renal injury in the hypertensive Dahl salt‐sensitive (SS) rat fed a high‐salt (HS; 4.0% NaCl) diet. Autophagy is an essential process (autophagosome formation, maturation, fusion with lysosome, release of macromolecular back into cytosol) required to remove misfolded proteins and damaged organelles (mitochondria, endoplasmic reticulum and peroxisomes) from all cells. It is a dynamic process (“autophagic flux”) and challenged in cells faced with excessive metabolic workloads such as the renal nephrons in kidneys of SS rats fed a HS diet. The tubular mitochondria of SS rats fed a HS produce excess reactive oxygen species (ROS) which we have found can activate mammalian target of rapamycin complex 1 (mTORC1). We have reported that the elevation of renal tissue H2O2 in kidneys of SS rats fed a HS diet can activate mTORC1 and that inhibition of this pathway with rapamycin attenuated salt‐induced hypertension and kidney injury. It is also known that mTORC1 plays a crucial role in regulating autophagy but the extent to which these events contribute to the renal injury observed in the SS rat has not been examined.

Goal

Studies were carried out in male SS rats to determine the effects of a HS diet (4.0% NaCl) upon the “autophagic flux” of the kidneys. SS rats (n=6 each group) were studied at days 3 or 21 of the HS diet and compared to those receiving a 0.4% NaCl diet. Rat received either the HS diet treated with vehicle, or HS plus daily rapamycin (IP, 1.5 mg/kg per day); Western blot analysis of renal cortical and medullary tissue LCII (microtubule‐associated protein light chain 2) and BECLIN1 protein levels was carried out as an index of autophagic flux.

Results

While no changes in the LCII levels were observed at day 3 of HS, a significant reduction was found in both the cortex and medulla after 21 days of HS compared to rats fed 0.4% NaCl. In rapamycin treated SS rats, both the renal cortex and medulla showed significantly greater increases of LCII and BECLIN1 compared with vehicle treated rats suggested an increase of autophagic index. Together, our observations suggest that the attenuation of salt‐induced hypertension and kidney injury in rapamycin treated SS rats may be in part the consequence of increased autophagic influx.

Support or Funding Information

HL‐116264
Frequency of Skulls with Lambdoidal Suture Wormian Bones in Two English Historical Ossuaries
Authors: Schmeidler Katherine T.
Published: April 2020

Wormian or sutural bones vary in number, size, and arrangement in humans, and are thus sometimes described as “odd”. Currently, there are not reliable data to estimate the frequency at which they are found. Some anthropologists have attempted to assess Wormian bone frequency in different, mostly non‐European human populations hoping to find whether there are differences among peoples of different genetic lineage. However, the sample sizes in these studies are too small to be able to compare such highly variable structures. Wormian bone incidence in skulls in the Paris Catacombs (les catacombes de Paris) [Schmeidler, 2018] and in three Italian anthropological collections [Schmeidler, 2019] have been previously presented, demonstrating Wormian bone occurrence in about 78% of 1100 skulls examined. This contrasts with most published studies that report incidences in the 10–20% range. The Parisian and Italian collections represent European populations, presumably from recent centuries, circa 1700–1900 C.E. These studies have been extended to analysis of mostly medieval, presumed to be mostly circa 1300–1400 C.E. skulls in the only two known extant English church ossuaries, at Hythe and Rothwell. The incidence of Wormian bones in the lambdoidal suture at these two sites is consistent with the other, more recent western European sites in this series. The photographic evidence collected in this project may be used to support further research in human anatomical variation. These findings expand the data set for comparison of Wormian bones from different parts of Europe and to peoples from other regions of the world, serving the greater community by providing a baseline against which smaller sample groups may be compared.
Development of FRET Biosensors to Detect Kinase Activity in Living Cells
Authors: Okáľová Jennifer , Burton Jeremy , Grimsey Neil J.
Published: April 2020

G‐protein‐coupled receptors (GPCRs) are a group of over 800 membrane receptors essential for pathological proinflammatory signaling. The p38 mitogen‐activated protein kinase (MAPK) pathway plays a crucial role in inflammatory responses found in many diseases. Anti‐inflammatory therapeutics targeting p38 have been widely underwhelming in clinical trials, mostly due to the broad physiological and pathophysiological pathways controlled by p38. Our group discovered proinflammatory GPCRs that are associated with an atypical pathway for p38 activation in vascular cells. This atypical pathway requires transforming growth factor‐β (TGFβ) activated kinase‐1 binding protein‐1 (TAB1) to bind directly to p38, ultimately inducing p38 auto‐activation via autophosphorylation. Atypical p38 signaling provides a mechanism to selectively target pathological p38 signaling. We are developing a fluorescent biosensor platform to assess the spatiotemporal activation of atypical p38 signaling using subcellular targeted fluorescence resonance energy transfer (FRET) biosensors, which consist of two fluorescent proteins, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), joined by a linker peptide that changes conformation upon phosphorylation by p38. These FRET biosensors enable real‐time detection of p38 kinase activity at specific organelles by measuring changes in fluorescence ratios. When in close proximity, CFP will donate energy to YFP, changing the mean fluorescence from 485 to 527 nm. Through the use of endosomal, nuclear, plasma membrane, or diffuse/cytosolic targeting motifs, we have characterized specific variances in spatiotemporal GPCR dependent activation of p38. These studies represent a fundamental advancement to our knowledge of p38 activation. Additionally, future expansion of this platform into stable cell lines will enable the screening of small‐molecule inhibitors of the pathway to regulate the progression of vascular inflammation.
DNA clamp loader subunit DnaX gamma plays a role in the response to DNA damage
Authors: Tashjian Tommy Fay , Chien Peter
Published: April 2020

The bacterial DNA damage response (DDR) is an important cellular system used to maintain a balance between maintenance of genomic integrity and mutagenesis that drives adaptation in the face of stress. An essential part of this response is the AAA+ protease ClpXP, which degrades several key DDR proteins. Our lab discovered a novel ClpXP substrate in Caulobacter crescentus, DnaX, a conserved component of the DNA replication clamp loader complex. DnaX has two forms: the essential full‐length DnaX tau and a shorter DnaX gamma. DnaX gamma is conserved in bacterial species, yet three distinct mechanism have been found to create it: ribosomal frameshift, transcriptional slippage, and partial ClpXP proteolysis in Caulobacter. Despite being discovered over 30 years ago, the function of DnaX gamma remains unclear.

We find that a strain encoding nonproteolyzable version of DnaX (tau‐only), which does not contain any DnaX gamma has no observable growth or morphological defects, but is sensitive to certain types of DNA damage. While the DnaX tau‐only strain is not more sensitive than the wild type to ultra‐violet light, which primarily causes intra‐strand crosslinks, it is significantly more sensitive than wild type to mitomycin C and zeocin, which primarily cause inter‐ and intra‐strand crosslinks and double strand breaks, respectively. To better define the pathways that are affected in the absence of DnaX gamma, we performed transposon (Tn) sequencing analysis and found synthetically lethal and conditionally dispensable genetic interactions with the dnaX‐tau‐only allele. Even in normal growth conditions, we find several synthetic lethal interactions with this allele and DNA damage response genes, especially those involved in double‐strand break repair. We also find that the global transcriptional repressor of the DDR, LexA, is conditionally dispensable in our DnaX tau‐only strain. Together, this suggests a role for DnaX gamma in DNA damage prevention or repair under normal growth conditions and addresses the long‐standing question of why the shorter forms of DnaX are so broadly conserved in bacteria.
Using C. elegans Genetics to Identify Regulators of μ‐Opioid Receptor Signaling
Authors: Wang Dandan , Stoveken Hannah M. , Zucca Stefano , Dao Maria , Orlandi Cesare , Song Chenghui , Masuho Ikuo , Johnston Caitlin , Opperman Karla J. , Giles Andrew C. , Gill Matthew S. , Lundquist Erik A. , Grill Brock , Martemyanov Kirill A.
Published: April 2020

Opioids exert their analgesic effects by activating the μ‐opioid receptor (MOR), a G protein‐coupled receptor expressed in the nervous system. In addition to clinical pain relief, activation of MOR produces euphoria and leads to dependence. Despite significant process in understanding molecular mechanisms of MOR signaling, the regulatory mechanisms of MOR function are not fully understood. To better understand MOR regulation and to uncover genes influencing MOR signaling, we developed a genetic behavioral platform utilizing nematode C. elegans. Mammalian MOR was transgenically expressed in the nervous system of C. elegans endowing the resulting model (tgMOR) with opioid sensitivity. We validated tgMOR platform showing conservation of pharmacological properties and regulatory mechanisms of MOR mediated behavioral responsiveness. We then performed a large scale forward genetic screen isolating a population of mutants with altered opioid sensitivity. Integrating whole genome sequencing, CRISPR/Cas9 gene editing and transgenic rescue, we identified a set of candidate negative modulators of MOR signaling. One of these genes encoded an orphan GPCR, FRPR‐13. Our phylogenic and functional analysis revealed that mammalian ortholog of FRPR‐13 is GPR139. We found GPR139 to be extensively co‐expressed with MOR in the mammalian nervous system. Knockout of GPR139 in mice enhanced opioid analgesia and reward, but diminished withdrawal. These observations suggest the existence of an “anti‐opioid” system that regulates the extent of MOR signaling in vivo. Our findings further showcase the utility of C. elegans as a scalable platform for genetic discovery of novel GPCR signaling principles.

Support or Funding Information

This work was supported by an NIH Cutting Edge Basic Research Award (R21DA040406) to B.G. and K.A.M., DA036596 to K.A.M., and an NIH Center of Biomedical Research Excellence Grant (P20GM103638) to University of Kansas Genome Sequencing Core.
Dissection of anterograde trafficking and sorting of GPCRs in primary neurons
Authors: Wei Zhe , Naughton Sean X. , Terry Alvin V. , Wu Guangyu
Published: April 2020

G protein‐coupled receptors (GPCRs) play an important role in regulating the function of the nervous system. However, the molecular mechanisms underlying the targeting and sorting of GPCRs to the functional destination in neurons are not well understood. Here we investigated the role of small GTPase Rab43, via dominant negative inhibition and CRISPR‐Cas9‐mediated knockout, in the transport of several prototypic GPCRs to dendrites and spines and elucidated the possible mechanisms. We demonstrated that inhibition of Rab43 function significantly reduced the surface numbers of α2‐adrenergic receptors (ARs) and β2‐ARs, but not M3‐muscarinic receptors (MRs) in primary cultures of neurons as measured by live neuron radioligand binding. Confocal microscopy revealed that three receptors utilized similar structural determinants, including the highly conserved LL motif in the C‐termini, for their anterograde transport, and that Rab43 affected the expression of α2‐ARs, but not M3‐MRs and vesicular stomatitis virus glycoprotein, at both dendrites and spines. Interestingly, Rab43 only altered β2‐AR expression at spines, but not dendrites. We also found that Rab43 directly and activation‐dependently interacted with α2‐ARs, but not M3‐MRs, and the Rab43‐binding domain was located in the third intracellular loop of the receptor. The chimeric M3‐MRs containing the Rab43‐binding domain of α2‐ARs were subject to Rab43 regulation. These data demonstrate distinct, as yet unidentified pathways that transport different GPCRs to dendrites and spines, in which Rab43 plays a crucial role in the sorting and trafficking of the receptors likely via direct interaction.

Support or Funding Information

NIH R01 GM118915
Direct Observation of APT Protein Dynamics Using Tryptophan Fluorescence
Authors: Zeheralis Chris Louis , Johnson R. Jeremy
Published: April 2020

Human acyl‐protein thioesterases (APTs) catalyze the depalmitoylation of key signaling proteins attached to the plasma membrane. Catalysis was hypothesized to be regulated by the large‐scale movement of an essential dynamic loop, which transitioned the protein between active and inactive states. However, the causes for the movement of this loop are unknown. Herein, we investigated several potential factors that trigger loop movement in APTs. Specifically, we measured the effect of two irreversible serine hydrolase inhibitors and various membrane lipid models on this dynamic loop movement using intrinsic tryptophan fluorescence. Using a library of combinatorial tryptophan variants of human APT1 and APT2, we followed dynamic changes in their structure in response to shifting ligand and membrane incubations. Amongst these various conditions, the APT specific inhibitor Palmostatin B showed the largest specific effect on loop dynamics. Relating these loop dynamics to sites of modification allowed us to identify distinct regions of APTs interacting with inhibitors and with membranes.

Support or Funding Information

Funded by NSF MCB‐1812971
The Roles of Inducible Nitric Oxide Synthase in Mediating the Effects of Embryotoxic Cytokines on Mouse Embryonic Cells
Authors: Guo Yanlin , Chen Bohan , Fendereski Mona , Hargis McKenzie
Published: April 2020

An early embryo is the most vulnerable stage in the life cycle of mammals, and it faces various immunologic challenges during the course of pregnancy. In particular, the blastocyst (the pre‐implantation embryo) is exposed to inflammatory cytokines resulting from the implantation process. Embryonic cells in the blastocyst have a dedicated task of rapid cell proliferation; how they deal with inflammatory cytokines known to negatively affect cell viability and proliferation is an important yet poorly understood question. We have investigated the effects of TNFα and IFNγ, two “embryotoxic cytokines” that impair blastocyst development and the process of implantation, on embryonic cells. Our results suggest that embryonic stem cells (ESCs), the pluripotent stem cells derived from the inner cell mass of the blastocyst, are insensitive to the cytotoxicity of TNFα and IFNγ. However, when ESCs are differentiated to fibroblasts (ESC‐FBs), they become susceptible to the cytotoxicity of TNFα and IFNγ. In particular, the combination of TNFα and IFNγ (TNFα/IFNγ) significantly reduced cell proliferation and induced apoptosis of ESC‐FBs. The cytotoxic effect of TNFα/IFNγ parallels the induction of nitric oxide synthase (iNOS) in ESC‐FBs, which has been known to cause cellular damage by producing nitric oxide (NO) as a free radical. In contrast, TNFα/IFNγ does not induce the expression of iNOS in ESCs or exert any detectable negative effects on cell proliferation and pluripotency of ESCs. However, ESCs are sensitive to the cytotoxicity of sodium nitroprusside, which can chemically generate NO in cell culture. Together, our results suggest that iNOS is a key effector molecule that mediates the cytotoxicity of TNFα/IFNγ in ESC‐FBs, but not in ESCs. Therefore, the lack of iNOS induction by TNFα/IFNγ in ESCs may be a protective mechanism that allows ESCs to avoid the cytotoxicity of the two cytokines. We are currently investigating the underlying molecular mechanisms and the biological implications of this finding.

Support or Funding Information

This work was in part supported by the National Institute of General Medical Sciences (R15GM128196‐01). We thank Mississippi‐IDeA Network of Biomedical Research Excellence for the use of the imaging facility (funded by the National Institute of General Medical Sciences P20 GM103476‐11).