bluuebunny/crossref_metadata_2025_split huggingface dataset:
Probing the Molecular Mechanism of ProQ‐RNA Interactions using a Bacterial Three‐hybrid Assay
Authors: Gravel Chandra 
Published: April 2020

In bacteria, small RNAs (sRNAs) are known to play important roles in gene regulation. The interactions between sRNAs and mRNAs are often facilitated by protein chaperones. Recently, ProQ has been shown to bind to dozens of E. coli sRNAs and mRNAs, and to facilitate base pairing between the mRNA hupA and sRNA RaiZ with the effect of regulating hupA. These observations have led to the proposal that ProQ may act as a widespread regulator of bacterial gene expression. Our goal has been to understand the molecular mechanisms of ProQ’s interaction with RNAs, mapping the amino acids on ProQ’s surface and nucleotides of RNAs that contribute to binding and regulation. To do this, we have used a bacterial three‐hybrid (B3H) assay to detect ProQ‐RNA interactions genetically. In the B3H assay, ProQ is fused to RNA polymerase (RNAP) and a hybrid RNA containing an sRNA or mRNA of interest is tethered to a DNA sequence upstream of a test promoter. Interaction of ProQ with the RNA stabilizes the binding of RNAP to the test promoter and activates transcription of a reporter gene, lacZ. Using domain truncations, site‐directed mutagenesis, and an unbiased forward genetic screen, we have identified a surface on ProQ’s NTD that we believe to be the primary surface for recognition of these SibB and cspE RNAs in vivo, and have created a genetically guided model for ProQ binding to an RNA duplex. In addition, we are working to probe whether ProQ possesses unique surfaces that contribute to binding of certain sRNAs and/or mRNAs and to determine what aspects of RNA sequence and structure contribute to recognition by ProQ.Support or Funding InformationThis work was supported by National Institutes of Health [R15GM135878], Mount Holyoke College and the Henry Luce foundation.
Chemogenetic Activation of Parasubthalamic nucleus Corticotropin‐Releasing Factor Neurons recapitulates Alcohol Withdrawal‐Related Behavioral Symptomatology
Authors: Okhuarobo Agbonlahor , Kreifeldt Max , de Macedo Giovana Camila , Igbe Ighodaro , Contet Candice 
Published: April 2020

Negative reinforcing effects of alcohol, driven by negative affect during withdrawal, are thought to be crucial to drinking escalation in alcohol use disorders. While the brain regions and neurotransmitter signaling affected by chronic alcohol use are well characterized, our understanding of the specific neural circuits driving drinking escalation and negative affect during withdrawal remains limited. Animal studies have highlighted a critical role of corticotropin‐releasing factor (CRF) signaling in the behavioral symptomatology of alcohol dependence. Hence, the present study sought to examine the role of a cluster of CRF neurons located in the parasubthalamic nucleus (PSTN), a small region of the posterior hypothalamus, which has received little attention in behaviors related to alcohol dependence. Adult male Crh‐IRES‐Cre mice were bilaterally injected in the PSTN with a viral vector encoding the excitatory designer receptor hM3D(Gq) fused to the fluorescent reporter mCherry upon Cre recombination. Injection of the hM3D(Gq) agonist clozapine‐N‐oxide (CNO, 1 mg/kg) significantly increased alcohol and saccharin consumption but did not change food and water consumption compared to vehicle. CNO had no effect in mice expressing mCherry only. CNO also increased locomotor and digging activity but did not alter open arm exploration in the elevated plus maze. CNO also decreased grooming duration in the splash test and immobility duration in the tail suspension test. Interestingly, the increased alcohol consumption, digging activity, active coping and reduced grooming all recapitulate phenotypes observed in mice withdrawn from chronic intermittent ethanol vapor inhalation, a model of alcohol dependence. However, CNO injection caused a significant increase in nociceptive thresholds in both the tail immersion and tail pressure tests, which is at odds with the hyperalgesia typically associated with alcohol withdrawal.The PSTN sends a strong projection to the central amygdala (CeA), a region that plays a critical role in the transition to alcohol dependence. We therefore tested whether the effects of PSTN CRF neuron chemogenetic activation may be mediated by the activation of CeA neurons carrying CRF1 receptors. However, CNO did not affect alcohol drinking, locomotion and digging in Crhr1‐IRES‐Cre mice expressing hM3D(Gq) in the CeA. Combined with our recent finding that PSTN neurons become activated during alcohol withdrawal, our results suggest that PSTN CRF neurons contribute to the behavioral symptomatology of ethanol withdrawal, but CRF1 signaling in the CeA may not be relevant to their mechanism of action.Support or Funding InformationThis work was funded by the following grants from the National Institutes of Health: AA026685 (CC), P60 AA006420 (CC), and University of Benin, Benin City, Nigeria, (AO).
The Role of Vascular Smooth Muscle RhoBTB1 in Hypertension
Authors: Fang Shi , Wu Jing , Silva Sebastiao Donato , Lu Ko-ting , Sigmund Curt D. 
Published: April 2020

Rho‐related BTB domain containing protein 1 (RhoBTB1) is a novel transcriptional target of peroxisome proliferator activated receptor gamma (PPARγ). In a GWAS study of over 1 million subjects, RhoBTB1 was identified as an interacting locus for hypertension. However, the physiological and molecular function of RhoBTB1 is not well‐understood. Previous studies suggest that RhoBTB1 in vascular smooth muscle cells (VSMC) mediates the anti‐hypertensive effect of PPARγ. RhoBTB1 expression was decreased in aortas from mice expressing smooth muscle specific dominant negative PPARγ mutation. Genetic complementation of smooth muscle RhoBTB1 reverses the hypertension, vascular dysfunction and arterial stiffness caused by dominant negative PPARγ mutation. In HEK293 cells, RhoBTB1 interacts with phosphodiesterase 5 (PDE5) and increases PDE5 ubiquitination in a Cullin‐3 dependent manner, suggesting that RhoBTB1 might function as a substrate adaptor for Cullin‐3‐RING ubiquitin ligase and mediate PDE5 turn over.RhoBTB1 expression was also decreased in aortas from mice treated with angiotensin II (Ang‐II, 490ng/kg/min, two weeks). We hypothesized that restoration of VSMC RhoBTB1 will reverse Ang‐II mediated hypertension, vascular dysfunction and arterial stiffness. We treated transgenic mice expressing inducible VSMC specific RhoBTB1 (S‐RhoBTB1 mice) with Ang‐II (490ng/kg/min, six weeks) and activated the transgene with Tamoxifen injections during the third week of Ang‐II infusion. ISM‐Cre mice expressing smooth muscle specific Cre‐recombinase were also treated with Ang‐II and Tamoxifen to serve as control. Blood pressure was measured by radiotelemetry. Ang‐II increased blood pressure in both ISM‐Cre and S‐RhoBTB1 mice to a similar extent. Thus, Ang‐II induced hypertension was not affected by RhoBTB1 transgene expression in VSMC. Carotid artery vasodilation response to acetylcholine and sodium nitroprusside, as well as vasoconstriction response to KCl, serotonin and endothelin‐1 were tested using wire myography. No differences were found in these measures of vascular function between S‐RhoBTB1 mice and ISM‐Cre mice. Arterial stiffness was assessed by pulse wave velocity (PWV). Ang‐II infusion increased PWV to a similar level in ISM‐Cre and S‐RhoBTB1 mice before the transgene induction. PWV of S‐RhoBTB1 mice started to decrease after Tamoxifen whereas PWV of ISM‐Cre mice continued to increase. By the second week after Tamoxifen, RhoBTB1 transgene expression in VSMC significantly attenuated Ang‐II mediated arterial stiffness (ISM‐Cre vs. S‐RhoBTB1 4.21±0.37 vs. 3.17±0.16 mm/ms, p=0.02, n=7–9). This data suggests a potential mechanism where RhoBTB1 improves vascular plasticity independently from blood pressure and is protective against arterial stiffness.Support or Funding InformationNIH R35 HL144807AHA 20PRE35120137
Increasing Versatility of Small Angle X‐Ray Scattering
Authors: Chakravarthy Srinivas , Hopkins Jesse , Bilsel Osman , Irving Thomas 
Published: April 2020

Small Angle X‐ray Scattering (SAXS), a technique initially popular in the bio‐medical community largely due to the relative logistical ease compared to other structural biological techniques has in recent years emerged as a much more versatile biophysical tool that gives unprecedented access to a real time view of structural alterations occurring during critical processes such as protein and nucleic acid folding, enzyme‐substrate binding and other ligand induced conformational changes in biological macromolecules. Developmental efforts spanning the last decade at the Biophysics Collaborative Access Team (BioCAT) located at The Advanced Photon Source (APS), Chicago, have resulted in a time‐resolved SAXS instrument which combines a state‐of‐the‐art synchrotron beamline capable of delivering up to 1013 photons/s with microfluidic mixers that afford access to time regimes ranging from 10s of microseconds to 1.5 seconds. After being made available to the general SAXS user community the time‐resolved SAXS setup has been used to study several interesting systems of biomedical interest, which in turn have led to unprecedented insights. For instance, key aspects of protein‐folding were elucidated using the chaotic flow mixer which gives access to a time regime from ~80μs to ~95ms and mechanistic details of the Insulin Degrading Enzyme (IDE) were revealed using the laminar flow mixer which allows measurement of time‐points from ~5ms to 1.5s. Improved operational efficiency and minimal sample consumption have made these heretofore challenging experiments feasible for a wider user community.Furthermore, the equilibrium SAXS instrument, which already had an integrated liquid chromatography setup to ensure optimal sample quality, now also has in‐line MALS and DLS detectors making BioCAT a unique facility for comprehensive biophysical characterization of biological macromolecules. Software tools developed in‐house have ensured a high degree of user‐friendliness and we hope to introduce this powerful biophysical tool to a larger community of non‐expert users.Support or Funding InformationNIGMS ‐ P41 GM103622
Synergistic Correlation between Dietary Patterns, Lifestyle and Hypertension among Medical School Population
Authors: Mhamankar Suyog , Kotak Vikas , Yadav Prashant , Pandey Rajeev , Murthy Guru 
Published: April 2020

Hypertension (High blood pressure) is responsible for 7.6 million deaths per annum worldwide (13.5% of the total), more than any other risk factors. Around 54% of stroke and 47% of coronary heart disease are attributable to high BP. The present study was conducted to find out how dietary patterns and lifestyle can affect or cause hypertension and its prevalence among the population on medical school campus. The cross‐sectional study involved a survey of 90 respondents aged 16 to 57 years using the random sampling technique. The results were based majorly on two factors i.e. diet and lifestyle. Considering the analysis on these factors, only 4.5% participants had high cholesterol intake, whereas 16.6% participants take heavy amounts of sugars,3.3% participants had high caffeine intake and 32% of population had high salt intake. Prevalence of smoking was 11.1% and heavy alcohol consumption was 4.07%. Contradictory to the expected results, only 6.7% population was found to be diagnosed by hypertension while 40–45% of people among the selected population were at risk of developing hypertension. 30.7% population did not have any exercise habits which is a risk factor leading to hypertension.Support or Funding InformationCommunity Health and Research of Spartan (CHARCOS), Spartan Health Science University School of Medicine, St. Lucia, West Indies
Transcriptomic landscape of lenalidomide‐treated NK cells: Functional enhancement by down‐regulation of CD161
Authors: Sim JeongMin , Ahn JuWon , Park YoungJoon , Kim JongIl , Kang SuJung , Choi Seo-Hyeon , Lee SeLin , Hwang Min , Cho JooYeon , Lim Jaejoon , Oh Soo-Yeon , Kwack KyuBum 
Published: April 2020

Lenalidomide is a potential immunomodulator that enhances the anti‐tumor effect of natural killer (NK) cells. However, the underlying molecular mechanism of anti‐tumor effects and long‐term activating effect on NK cells are unclear. Here, we report that lenalidomide in the long stimulation with interleukin‐2 (IL‐2) enhances the proliferation and specific anti‐tumor function of NK cells by modulating transcriptomic patterns, including downregulation of KLRB1 (CD161). In conclusion, the results suggest that lenalidomide may be more applicable in the field of cancer immunotherapy by exhibiting specific cytotoxicity ability enhancement of NK cells against cancer cells as well as long‐term activating effect on NK cells.Support or Funding InformationThe research fund was supported by Ministry of Science, Technology and Information, Republic of Korea (Grant no. 2019R1F1A1054920)
Autologous Haematopoietic Stem Cell Transplantation Followed by Low‐dose Cyclophosphamide Ameliorates the Disease Course and Influences the Microglia Phenotype in Rats With Experimental Allergic Encephalomyelitis
Authors: Kowara Michał Krzysztof , Kasarełło Kaja , Seta Martyna , Sulejczak Dorota , Wrzesień Robert , Snarski Emilian , Cudnoch-Jędrzejewska Agnieszka 
Published: April 2020

Experimental allergic encephalomyelitis (EAE) is the animal model of multiple sclerosis (MS), the human autoimmune disease, which leads to neurodegeneration. Immune response is developed by autoreactive lymphocytes infiltrating the CNS, but further maintained by resident immune cells, such as microglia. Microglia, until recently was recognized as pro‐inflammatory cells. Lately, it was discovered, that microglia can also differentiate into an anti‐inflammatory phenotype involved in the regression of inflammatory reaction and regenerative processes. Therapy based on the autologous haematopoietic stem cell transplantation (HSCT) was recently approved for treatment of highly‐active or progressive MS. In some regimens the additional conditioning is used after the HSCT to eliminate the autoreactive cells, which may persist after the first dose of chemotherapy.In our experiment, we aimed to investigate the effect of HSCT preceded by high‐dose of cyclophosphamide (CP) on the disease course in rats with evoked EAE, and if the additional, low‐dose of CP following the high‐dose of CP and HSCT may improve the observed effect.Rats with evoked EAE (day 0) were treated with (i) high‐dose (125mg/kg) CP (day +6), followed by HSCT (day +7), or (ii) high‐dose (125mg/kg) CP, followed by HSCT, followed by low‐dose (20mg/kg) CP (day +10, +11). Control animals were (iii) healthy non‐treated and (iv) EAE non‐treated. Clinical symptoms of EAE (0–5 scale) were observed during the disease course and after the resolution of symptoms rats were euthanized. Spinal cords were collected for immunofluorohistochemical analysis to exmine the microglia phenotype in the CNS.Results showed that the applied therapy in the EAE rats, including only the high‐dose CP followed by HSCT did neither improve clinical symptoms observed in the rats nor influenced the microglia phenotype in comparison to the EAE non‐treated animals. However, the additional low‐dose CP after HSCT resulted in the significant reduction of clinical symptoms and increase in the number of anti‐inflammatory microglia in the CNS. There was no effect observed of applied therapies on the numer of pro‐inflammatory microglia.Our results demonstrated, that the appliction of additional low‐dose CP after HSCT led to the increase in the number of anti‐inflammatory microglia in the CNS. This shift into the anti‐inflammatory phenotype might be engaged in the reduction of clinical symptoms in the EAE rats and accentuates the role of microglia in MS pathomechanism.Support or Funding InformationSupported by grant no. 2018/02/X/NZ5/01487 financed by the National Science Centre
Sigma‐1 Receptor May Participate in the Regulation of Epigenetic Modifications Related to Neuropathic Pain
Authors: Goguadze Nino , Wu Hsiang-En , Su Tsung-Ping 
Published: April 2020

Nerve injury causes abnormal hyperactivity of primary sensory nerves and causes changes in the expression of pro‐ and antinociceptive genes in the DRG.The ER chaperone sigma‐1 receptor (Sig‐1R) was demonstrated to play a role in neuropathic pain and is known to exist in the dorsal root ganglia (DRG), notably, at the nuclear envelope. According to the published data, in Spared Nerve Injury (SNI) model of neuropathic pain, Sig‐1R knockout mice did not develop cold allodynia and showed significantly less mechanical allodynia. However, the molecular mechanisms whereby Sig‐1Rs modulate neuropathic pain at the DRG are largely unknown.Recently, it was shown that the inhibition of histone methyltransferase at the DRG attenuates the neuropathic pain by correcting the dysfunctional potassium channels. Inasmuch as Sig‐1Rs exist at the nuclear envelope, we hypothesized that Sig‐1Rs may participate in the regulation of epigenetic modifications related to neuropathic pain.Immunoblotting of extracted histones from Sig‐1R knockout DRGs, when compared to wild type DRGs, showed a decrease in the level of H3K9me2, whereas total H3 methylation/acetylation, H3K9ac, H3K9me3, H3K27me3 and H3K4me3 histone marks were unchanged. However, the protein level or mRNA expression of euchromatic histone‐lysine N‐methyltransferase‐2 (G9a) which is an enzyme responsible for H3K9me2 modification was not altered in wild type and Sig‐1R knockout mice. Those results suggest that the Sig‐1R plays a role on the maintenance of the level of H3K9me2 downstream of the G9a.It is known that H3K9me2 preferentially marks chromatin at the nuclear periphery that interestingly is where Sig‐1Rs are located. Thus, we performed peptide pulldown experiments using unmodified and modified histone peptides to pull down Sig‐1Rs and found that indeed the H3K9me2 peptide can bind the Sig‐1R. When taken together, those results suggest that the Sig‐1R relates to the maintenance of the H3K9me2 level in the DRG and that the Sig‐1R may affect neuropathic pain by regulating the level of potassium channels by the epigenetic modification on histone 3.Support or Funding InformationIRP/NIDA/NIH
Transcriptional Regulation of the Proteasome: Implications for Cancer Therapy
Authors: Radhakrishnan Senthil , Vangala Janakiram 
Published: April 2020

While proteasome inhibitors are being effectively used in the clinic to treat multiple myeloma and mantle cell lymphoma, the reasons for why this strategy is not generally applicable to other tumor types is poorly understood. A detailed characterization of the cellular events triggered in response to these agents could offer vital clues to target the protein degradation pathway(s) more effectively in a broad range of tumors.Our previous studies have established the transcription factor Nrf1 as a key player in the stress‐response pathway invoked by inhibition of cellular proteasome. Nrf1, by its ability to induce de novo synthesis of proteasome subunit genes in response to proteasome inhibitors, promotes the recovery of proteasome activity, thus mitigating proteotoxic stress and enhancing cellular survival. From a therapeutic stand‐point, this suggests that disabling the Nrf1‐mediated proteasome recovery pathway could potentiate the action of proteasome inhibitor treatments. However, effective means to curb the Nrf1 pathway remain scarce. Here, using an in silico chemical biology approach, we uncovered a class of therapeutic agents that can effectively inhibit the Nrf1 pathway. Mechanistic dissection of this phenomenon provided further novel insight into the way by which Nrf1 maintains its transcriptional activity. Using triple negative breast cancer (TNBC) cell lines and patient‐derived xenograft (PDX) models, we further validated our hypothesis that attenuating the Nrf1 pathway could increase the efficacy of proteasome inhibitor therapeutics. Thus, our study sheds light on the unique biology of Nrf1 along with validating this pathway as a therapeutic target in TNBC. This could serve as a motivation for similar future efforts where Nrf1 pathway inhibitors can be tested as adjuvants for proteasome inhibitor treatments in other cancer types.Support or Funding InformationNIH grant R01GM132396
Cytotoxic Effects of Digested Edible Insects on Intestinal Epithelia
Authors: Scheers Nathalie , Dobermann Darja 
Published: April 2020

Insects have been proposed as a substitute for livestock in future foods as a source of protein and micronutrients. In an attempt to compare iron bioaccessibility between different insect species to human intestinal epithelia, we discovered unexpected cytotoxic effects using standard procedures for estimating iron bioaccessibility. Pre‐confluent and confluent, differentiated epithelia of human intestinal Caco‐2 cells were exposed to simulated gastrointestinal digests of three edible insect species (meal worm, desert locust and black cricket) at varying concentrations. Cell survival and metabolic assays indicated cell death already at low levels of insect digests. Proteomic analyses revealed increased levels of cellular stress markers. Taken together, the data indicate that the investigated insects are cytotoxic to cultured human intestinal cell epithelia.Support or Funding InformationThis work was supported by the UK Biotechnology and Biological Sciences Research Council Nottingham‐Rothamsted Doctoral Training Partnership [grant number BB/J014508/1] and by the Swedish Research council FORMAS (grant # 2014‐417).
Effects of Dexamethasone on HDAC4 Expression and Cellular Localization in C2C12 Cell‐Derived Myotubes: Implications for Skeletal Muscle Atrophy
Authors: Alves Paula Ketilly Nascimento , de Oliveira André Cruz , Moriscot Anselmo Sigari 
Published: April 2020

Skeletal muscle can rapidly decrease its mass and force to various conditions such as denervation, mechanical unload, kidney disease, sepsis and hormonal stimuli such as corticoids. Although it is well known that such conditions can trigger a set of genes related to atrophy (mainly Atrogin‐1 and MuRF1), the specific cellular processes that control atrophy under different conditions are not well understood. Accordingly, histone deacetylases (HDACs) can be important modulators of skeletal muscle atrophy and it has been shown that HDAC4 can trigger Atrogin‐1, and MuRF‐1 expression in denervation model. Nonetheless, it is not known whether HDCA4 is modulated in models involving hormonal stimulation such as corticoids. Thus, our objective was to investigate HDAC4 expression and localization in C2C12 cell‐derived myotubes submitted to dexamethasone treatment. We examined HDAC4 localization, gene expression and protein content on a in vivo, and in vitro models. C2C12 cells, treated with dexamethasone (10 nM) for 12h, 24h and 48h and the results are expressed by means ± SD, and the statistical analysis was performed using Student’s t test (p ≤ 0.05). Initially we confirmed the responsiveness of HDCA4 to atrophy mediated mechanical unload. As expected, we found a marked increase in HDCA4 mRNA expression (~3 fold, p<0.05) after 7 days of hind limb immobilization. Other marker of atrophy was also induced (Atrogin‐1 1.7 fold; p <0.05). In C2C12 cell‐derived myotubes, we confirmed decreased in myotube diameter induced by dexamethasone (~25% p<0.05). In addition, we observed increased HDAC4 mRNA levels after 24 h of dexamethasone treatment (~1.8 fold, p<0.05). Atrogin‐1 mRNA level, as expected, was also elevated (~1.6 fold, p<0.05). Concerning cell location, our preliminary findings suggest a nuclear accumulation of HDAC4 in myotubes treated with dexamethasone. As time course and dose response experiments are ongoing, overall, these results provide further insight into molecular mechanisms involving HDAC4 responses in skeletal muscle. This project was approved by the Ethics Committee on the Use of Animals by the University of São Paulo (Protocol No. 2034200718).Support or Funding InformationFAPESP; CAPES; CNPq