bluuebunny/crossref_metadata_2025 huggingface dataset:
Adiponectin Inhibits LPS‐Induced HMGB1 Release through Heme Oxygenase‐1 Dependent Pathway in RAW 264 Cells
Authors: Elfeky Mohamed , Yoneshiro Takeshi , Okamatsu‐Ogura Yuko , Kimura Kazuhiro
Published: November 2023

Introduction

High mobility group protein B1 (HMGB1) is an important late inflammatory mediator in sepsis. Adiponectin, an adipokine predominantly secreted from adipose tissue, has potent anti‐inflammatory properties. However, the possible anti‐inflammatory effects of adiponectin on lipopolysaccharides (LPS)‐induced HMGB1 release are unknown. The aim of this study was to investigate whether adiponectin could inhibit HMGB1 release in LPS‐stimulated RAW 264 cells.

Materials and methods

Murine macrophage RAW 264 cells were treated with or without LPS (200 ng/ml) in the presence or absence of adiponectin, interleukin (IL)‐10, HO‐1 inhibitor (ZnPP) and p38 MAPK inhibitor (SB203580). HMGB1 translocation and release were measured by immunofluorescence staining of the cells and western blotting analysis of the medium, respectively. Expression levels of mRNA for toll like receptor (TLR) 4, TLR2, myeloid differentiation factor‐2 (MD‐2), HMGB1, IL‐10, heme oxygenase‐1(HO‐1) and nuclear factor erythoid‐derived 2 related factor 2 (Nrf2) mRNA were assessed by real‐time PCR.

Results and discussion

Treatment of the cells with LPS induced significant HMGB1 release and obvious translocation from nucleus to cytosol. However, prior treatment with either adiponectin or IL‐10 suppressed the LPS‐induced HMGB1 release and translocation. Adiponectin treatment decreased TLR4 mRNA and induced expression of HO‐1 mRNA, but not IL‐10 mRNA, while IL‐10 treatment decreased TLR2 and HMGB1 mRNA and enhanced both IL‐10 and HO‐1 gene expressions. Treatment of RAW 264 cells with ZnPP before adiponectin and IL‐10 treatment obviated the suppression of HMGB1 release by adiponectin completely, but partially by IL‐10 while treatment with SB203580 didn't had a significant effect. In conclusion, our results indicate that adiponectin suppresses HMGB1 release by LPS through a HO‐1‐dependent while neither HO‐1 nor p38 MAPK was involved in IL‐10 anti‐inflammatory effect. Therefore, both adiponectin and IL‐10 may play a role as an anti‐inflammatory molecule in the late inflammatory process by preventing release of a septic exaggeration factor, HMGB1.
Interlukin‐17A Promotes Aortic Endothelial Activation Via Transcriptionally and Post Transcriptionally Activating p38 MAPK Pathway
Authors: Mai Jietang , Nanayakkara Gayani , Lopez‐Pastrana Jahaira , Li Xinyuan , Li Ya‐Feng , Wang Xin , Virtue Anthony , Shao Ying , Shan Huimin , Liu Fang , Autieri Michael , Iwakura Yoichiro , Jiang Xiaohua , Wang Hong , Yang Xiao‐Feng
Published: November 2023

Interleukin‐17A (IL‐17) is a potent mediator of various inflammatory and autoimmune diseases, and is known to be induced by hyperlipidemia. However, the roles of IL‐17A in atherosclerosis and aortic endothelial cell activation is not clearly defined and remains controversial. Nevertheless, its role as a pro‐atherogenic stimuli prevails, thus we formulated the hypothesis that IL‐17A promotes endothelial cell activation under hyperlipidemic stress. The objective of this study was to elucidate the mechanism of how IL‐17A promote endothelial activation that leads to atherosclerosis under hyperlipidemia. We utilized biochemical, immunological, microarray, flow cytometry, intra‐vital microscopy, data mining analysis techniques and ApoE−/−/ IL‐17A−/− null mice to test our hypothesis. Our data revealed that hyperlipidemic stimuli can induce IL‐17A receptor expression in human/ mouse aortic endothelial cells (HAEC/ MAEC). In addition, IL‐17A treatment significantly induced monocyte adhesion to HAEC. Also, by gene expression studies, we implicated that IL‐17A induces pro‐inflammatory chemokines C‐X‐C motif ligand 1 and 2 (CXCL 1 and 2) expression in HAEC. Interestingly, our mouse aortic microarray data revealed that IL‐17A upregulates the endothelial cell adhesion molecules and the p38 component of the mitogen activated protein kinase (MAPK) pathway. We have demonstrated that the pharmacological inhibition of p38 MAPK can significantly impair IL‐17A induced endothelial adhesion molecules and CXCL1/ 2 expression, suggesting that p38 MAPK is a mediator of IL‐17A induced endothelial cell activation. Moreover, we observed that IL‐17A depletion in ApoE mice had significantly decreased levels of phosphorylated p38 MAPK relative to controls. We further consolidated these finding in‐vivo by utilizing intra‐vital microscopy to demonstrate that IL‐17A knockout in ApoE−/− mice reduced leukocyte rolling and adhesion to the endothelium. Based on our findings, we conclude that IL‐17A mediates the early phase of hyperlipidemia induced endothelial cell activation by activating the p38 MAPK pathway.

Support or Funding Information

NIH R01, AHA predoctoral and postdoctoral grants
HSP70 and HSP90 Inhibitors Repress Expression of TSC1 in T24 Bladder Cancer Cells and UOK139, UOK 140 Kidney Cancer Cells
Authors: Andolino Chaylen , Bresticker Julia , Berg Adrian , Cox Avery , Williams Heinric , Chernin Mitchell
Published: November 2023

Normal cells are able to survive in stressful conditions by increased expression of heat shock protein (HSP) genes. Both HSP90 and HSP70 are up regulated in cancers of the bladder and kidney. Next generation sequencing studies have identified pathways involved in kidney and bladder cancer. The objective of this study was to elucidate the impact of HSP70 and HSP90 inhibition on the signal transduction pathways driving their tumorigenic process. The TSC1‐TSC2 complex has emerged as a central signal‐integrating node within the cell.

Methods

We analyzed the expression of key mediators of the TSC1‐TSC2 pathway when treated with the HSP90 inhibitor (STA9090) or HSP70 inhibitors (VER155008, MAL3‐101) in bladder cancer (T24) and kidney cancer (UOK139 and UOK140) cell lines. The impact of treatment on cell growth was analyzed using a MTT assay and on signal protein expression by Western blot analyses.

Results

Growth inhibition was seen in T24 bladder cancer cells when treated with 1 μM STA9090 (STA) and 50 μM VER155008 (VER) but not with 30 μM MAL3‐101 (MAL) for 72 h. Synergistic growth inhibition was seen with combination STA+VER, STA+MAL and MAL+VER in T24 cell lines. We tested the effect of these inhibitors on the expression of TSC pathway mediators in both bladder and kidney cancer cell lysates and noted that TSC1 was down regulated in all cell lines tested in the presence STA and VER. In T24 cells, only the combination VER + MAL led to TSC1 down regulation. Neither STA, VER nor MAL had any impact on S6 kinase (a downstream mediator) expression in T24 cells. Rather, only STA and not VER or MAL downregulated the expression of the active form of S6 kinase (pS6kinase).

Conclusion

The inhibition of HSP70 and HSP90 appears to cause a down regulation of TSC1. TSC1/2 functions as a GTPase‐activating protein (GAP) for the small Ras‐related GTPase Rheb. The active, GTP‐bound form of Rheb directly interacts with mTORC1 to stimulate its activity. mTORC1 promotes protein synthesis by phosphorylating S6 kinase. Together, these data suggest a role for HSP90 and HSP70 inhibitors in kidney and bladder cancer management.

Support or Funding Information

Bucknell‐Geisinger Research Initiative
Investigating the impact of Doublecortin X (DCX) pathogenic mutants on microtubule polymerization and bundling
Authors: Moslehi Maryam , Ng Dominic C.H. , Bogoyevitch Marie A.
Published: November 2023

Doublecortin X (DCX), a neuron‐specific microtubule‐associated protein, plays vital roles in microtubule organization and microtubule‐related developmental events. DCX directly binds to microtubules via its two evolutionarily conserved, structured doublecortin (DC) domains, N‐DC and C‐DC, and this interaction has been proposed to be regulated by flanking regions of the DCX N‐terminus and the C‐terminus. Although DCX is known to regulate microtubule function, its precise mechanism of action is yet to be determined. In our study, we are investigating the mechanism of cytoskeletal arrangement by DCX through identification of the impact of DCX pathogenic mutations within the N‐DC domain and DCX N‐terminus on microtubule network organization. A recently identified pathogenic mutation in the DCX N‐terminus, E2K (that resulted in epilepsy), and a mutant within the N‐DC domain, R102S (that resulted in X‐linked lissencephaly) have been studied to define the effects of these missense mutations on microtubule polymerization and bundling. Specifically, we are interested in defining if there are common features shared by these two mutants that may explain their pathological impacts. Our results so far have indicated that DCX wildtype and the DCX E2K mutant induce comparable levels of microtubule bundling while the DCX R102S mutant showed reduced bundling ability. Our studies on the pathogenic mutant DCX R102S showed its decreased microtubule bundling ability as well as its attenuated actions in microtubule polymerization of the cells. However, these results have not been observed for DCX E2K mutant. Ongoing studies will continue to explore shared actions of DCX E2K and R102S mutants, particularly their phospho‐dependent regulation. Taken together, these results emphasize the critical regulation of microtubule organization by DCX but the need for further investigations of the role of DCX and DCX pathogenic mutants in the modulation of microtubule organization to improve our understanding of the pathogenesis of neuronal migration disorders.
Comparative Analysis of ELISA and MALDI‐TOF Mass Spectrometry Methods for Microcystins in Freshwater Samples
Authors: Kadlec Kimberly Michele , Turczyn Andrew , Grant Jennifer
Published: November 2023

Health risks associated with freshwater cyanobacterial blooms have led to the development of a variety of analytical methods to detect the presence of microcystins, which are monocyclic heptapeptides that can cause acute and chronic hepatotoxicity when ingested by animals and humans. In this study of soluble microcystin levels in lake water, the sensitivity of detection of Matrix‐Assisted Laser Desorption/Ionization Time‐of‐Flight mass spectrometry (MALDI‐TOF MS) was compared to that of Enzyme Linked Immunosorbent Assay (ELISA) methods.

Lake water samples were collected monthly during the first week of each month, May through August in 2012, 2013 and 2014 and samples were frozen at −20 °C. Prior to analysis, lake water was centrifuged (5 min @ 10,000 × g) to remove suspended solids. ELISA was performed using Microcystins (ADDA)‐DM ELISA Microtiter Plate, following the manufacturer's protocol (Abraxis Inc.). Absorbance was measured at 450 nm on a BioTek Epoch Microplate spectrophotometer. MALDI‐TOF MS was performed by applying a small volume of sample to a stainless steel MALDI plate, followed by an equal volume of α‐cyano‐4‐hydroxycinnamic acid, using the dried droplet method. Relative quantitation of microcystin content was performed using angiotensin II to create standards ranging from 0.06 μM to 5.0 μM in concentration. Data were acquired using a Bruker Microflex Linear Time of Flight MALDI‐TOF Mass Spectrometer. Spectra were acquired over the mass range of 0.6 to 4 kDa using 50 laser shots in the positive ion mode at a laser power of 25%. Calibration of the mass spectra was achieved using a calibration solution comprised of 1 μM bradykinin and 1 μM neurotensin.

Semi‐quantitation with the ELISA kit showed seasonal fluctuations of microcystins peaking in July or August with concentrations up to 10‐fold greater than detected using the MALDI‐TOF MS method. The ELISA kit indicated microcystin levels less than 1.0 μg/L, the maximum concentration for drinking water set by the WHO, in 50% of the samples. MALDI‐TOF MS analysis of the same samples detected seasonal fluctuations of microcystins and indicated the presence of 18 different congeners of microcystin over the course of the entire study. The congener that was visualized most consistently was MC‐LAba (m/z 923), recorded at both locations at concentrations ranging from 0.25 – 0.47 μg/L in 70% of the samples. The congener present in the greatest concentration was found to be MC‐RR (m/z 1037), at 3.42 μg/L in August 2013.

Our findings showed general agreement between the ELISA and MALDI‐TOF MS data. The primary drawback in the ELISA methodology is that ELISA cross‐reacts with non‐microcystin targets (e.g. nodularins, N‐hemi‐ADDA and ADDA), often causing an overestimation of the actual concentration of the target microcystins in the sample. MALDI‐TOF MS, in addition to requiring far less sample preparation, identified and quantified specific congeners.
Pneumolysin induces cellular senescence in microglial cells: involvement of MAPK and NF‐κB pathways
Authors: Kwon Ii‐Seul , Pyo Suhkneung
Published: November 2023

Senescence, which is an irreversible proliferation arrest, is induced by various stress stimuli such as genotoxic stress and oxidative stress. Pneumolysin (Ply) that is important pathogenicity factor unique to S. pneumoniae plays a pathogenic role in pneumococcal‐induced meningitis. Brain cells exposed to PLY undergo cell‐cycle arrest or apoptosis. However, the cell fate response to toxin is mechanistically unclear. In the present study, we investigated the effect of PLY on cellular senescence in BV‐2 microglial cells. Exposure to PLY resulted in changes in the expression of genes associated with senescence and increased senescence‐associated β‐galactosidase (SA‐β‐gal) activity. PLY‐treatment also showed a concentration‐ and time‐dependent increase in PAI‐1 expression and G1/G0 phase arrest. PLY induced NF‐κB activation through the suppression of SIRT‐1 expression and phosphorylation of ERK, JNK and p38 MAP kinase. Additionally, PLY increased the production of reactive oxygen species. Overall, our results suggest that PLY induces cellular senescence by enhancing ROS production, activation of MAPK and NF‐κB, and the inhibition of the SIRT‐1 expression, resulting in regulation of cellular senescence in microglial cells.

Senescence, which is an irreversible proliferation arrest, is induced by various stress stimuli such as genotoxic stress and oxidative stress. Pneumolysin (Ply) that is important pathogenicity factor unique to S. pneumoniae plays a pathogenic role in pneumococcal‐induced meningitis. Brain cells exposed to PLY undergo cell‐cycle arrest or apoptosis. However, the cell fate response to toxin is mechanistically unclear. In the present study, we investigated the effect of PLY on cellular senescence in BV‐2 microglial cells. Exposure to PLY resulted in changes in the expression of genes associated with senescence and increased senescence‐associated β‐galactosidase (SA‐β‐gal) activity. PLY‐treatment also showed a concentration‐ and time‐dependent increase in PAI‐1 expression and G1/G0 phase arrest. PLY induced NF‐κB activation through the suppression of SIRT‐1 expression and phosphorylation of ERK, JNK and p38 MAP kinase. Additionally, PLY increased the production of reactive oxygen species. Overall, our results suggest that PLY induces cellular senescence by enhancing ROS production, activation of MAPK and NF‐κB, and the inhibition of the SIRT‐1 expression, resulting in regulation of cellular senescence in microglial cells.
Obesity intensifies hepatotoxicity by asparaginase in mice deleted for GCN2 but not ATF4
Authors: Nikonorova Inna A. , Mirek Emily T. , Wang Yongping , Dixon Joseph L. , Anthony Tracy G.
Published: November 2023

Liver dysfunction is a serious adverse effect of asparaginase treatment in patients with acute lymphoblastic leukemia, reducing overall survival. Asparaginase depletes asparagine and glutamine levels in the blood causing rapid tumor regression. Depletion of these amino acids also reduces liver protein synthesis via phosphorylation of eukaryotic initiation factor 2 (eIF2) by the GCN2 (aka eIF2AK4) kinase. Phosphorylation of eIF2 promotes synthesis of activating transcription factor 4 (ATF4) which reconfigures the cellular transcriptome to alleviate the amino acid stress. Failure of this integrated stress response (ISR) leads to severe liver dysfunction. A potent clinical predictor of hepatotoxicity by asparaginase is pre‐existing obesity. Molecular mechanisms of this clinical observation remain to be determined. The focus of our study was to examine the role of the ISR pathway on the background of obesity in the development of hepatotoxicity by asparaginase. Wild type, Gcn2−/− and liver specific‐Atf4−/−(ls‐Atf4−/−) mice of C57Bl/6J background were fed a high fat diet for 12 weeks. The onset of obesity was monitored by assessing body composition and glucose metabolism. Mice were randomly assigned to receive daily injections of Elspar L‐asparaginase at 0 or 3 IU per gram body weight for 8 days. Liver tissues were collected and analyzed by immunoblotting for activation of the ISR pathway and its crosstalk to usual suspects of liver dysfunction, mTORC1 and unfolded protein response (UPR) pathways. Our data showed that asparaginase treatment of obese mice resulted in hepatic steatosis that intensified significantly in Gcn2−/− but not ls‐Atf4−/− mice. Liver pathology in Gcn2−/− mice corresponded with induction of the UPR pathway that was assessed by PERK (aka eIF2AK3) phosphorylation. PERK phosphorylation was moderate in the livers of asparaginase‐treated wild type and ls‐ATF4−/− mice (5‐fold) and very intense in the livers of Gcn2−/− mice (20‐fold). Asparaginase treatment corresponded with reduced mTORC1 activity (3‐fold reduction in p70S6K phosphorylation and 2‐fold reduction in 4EBP1 phosphorylation) in wild type and ls‐Atf4−/− livers, whereas Gcn2−/− group demonstrated strong activation of mTORC1 pathway (2‐fold increase in mTOR‐Ser2448 and 4EBP1 phosphorylation, 3‐fold increase in p70S6K phosphorylation). Downregulation of mTORC1 pathway in the livers of asparaginase‐treated wild type mice and the absence of such downregulation in asparaginase‐treated Gcn2−/− mice suggest that GCN2 signals to mTORC1 pathway. Furthermore, downregulation of mTORC1 in asparaginase‐treated ls‐Atf4−/− livers suggest that the point of coordination of GCN2 pathway to mTORC1 pathway is not through ATF4. Overall our study provides important molecular insights into the mechanism of acute liver damage by asparaginase and identifies a novel role for the ISR in coordinating mTORC1 signal transduction.

Support or Funding Information

NIH HD070487
Hepatic ATGL manipulation regulates lipid metabolism through altered autophagy
Authors: Sathyanarayan Aishwarya , Mashek Mara T , Mashek Douglas G
Published: November 2023

Adipose triglyceride lipase (ATGL) catalyzes the rate‐limiting step of triacylglycerol (TAG) hydrolysis in multiple tissues. Our laboratory has previously shown that hepatic ATGL preferentially channels hydrolyzed fatty acids (FAs) to β‐oxidation with a concomitant induction of peroxisome proliferator activated receptor‐α (PPAR‐α), independent of a FA carrier. Additionally, our work describes a novel signaling axis involving cAMP/PKA, ATGL‐catalyzed lipolysis and sirtuin1 (SIRT1) activation that governs transcriptional regulation of oxidative metabolism and mitochondrial biogenesis. Recent studies have also shown SIRT1 promotes autophagy, which also contributes to lipid droplet catabolism (lipophagy). Thus, the objective of these studies is to determine the effects of ATGL on autophagy in the liver. Hepatic ATGL overexpression promotes the expression of autophagy genes, whereas ATGL knockdown reduces the expression of the same genes. ATGL regulates autophagosome biogenesis and lysosomal levels through alterations in the microtubule‐associated protein 1A/1B‐light chain 3 (LC3), sequestome1 (p62) and lysosomal‐associated membrane protein 1 (LAMP1). Use of a chemical inhibitor of ATGL, ATGListatin (Astat) in hepatocytes shows a marked suppression of autophagy, similar to the effects observed with ATGL knockdown in vivo. Finally, ATGL requires SIRT1 to govern its effects on autophagy as evidenced by studies showing that ATGL overexpression in liver‐specific SIRT1 KO mice is unable to induce autophagy. Thus, our findings identify ATGL‐mediated lipolysis as an important and potent regulator of autophagy that could have important implications for NAFLD etiology and its comorbidities.

Support or Funding Information

Funding sources include the NIDDK (DK085008 to DGM) and the Minnesota Obesity Center (DK050456).
Intact Autophagy is Requisite for Shear‐Stress‐Induced Nitric Oxide Generation by Endothelial Cells via a Novel Glucose‐Dependent Purinergic Signaling Pathway
Authors: Bharath Leena P , Ruan Ting , Babu PVA , Boudina Sihem , Graham Timothy E , Symons J David
Published: November 2023

Impaired intracellular autophagy has been implicated as a cause of vascular dysfunction in aging human arteries. Earlier we reported that shear‐stress (180‐min × 20 dyne•cm2) induces autophagy that is concurrent with phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) at serine (S) 1177 (p‐eNOSS1177) and NO generation in bovine aortic endothelial cells (ECs). Genetic inhibition of autophagy via Atg3 or Atg5 siRNA suppresses shear‐induced p‐eNOSS1177 and NO generation, increases reactive oxygen species generation, and unleashes inflammatory/adhesion responses, suggesting strongly that autophagy is critical for maintaining EC function. Here we report one mechanism through which autophagy regulates shear‐induced p‐eNOSS1177 and NO production in ECs. Tumor suppressor and metabolism regulator, p53, is strongly upregulated by Atg3 siRNA, causing reduced shear‐induced Glut‐1 expression, glucose transport, glycolysis, and ATP production in ECs. As expected, AMPK is activated by the energy deficit, but does not restore p‐eNOSS1177 and NO generation in this setting. However, shear‐evoked p‐eNOSS1177 and NO generation can be restored in autophagy‐impaired ECs by (i) adding extracellular ADP or by (ii) overexpressing Glut‐1. These findings prompted a further investigation of purinergic signaling to eNOS in the context of suppressed EC autophagy. As predicted, P2Y1 receptor blockade (MRS2179) and P2Y1 siRNA can recapitulate the Atg3 phenotype i.e., shear‐induced p‐eNOSS1177 and NO generation are prevented. Furthermore, shear‐induced ATP/ADP release increases p‐PKCδThr505, a downstream effector of P2Y1 and activator of p‐eNOSS1177. As would be anticipated by these findings, shear‐induced p‐eNOSS1177 and NO generation can be restored in autophagy‐impaired ECs by (i) the PKCδ activator bryostatin or by (ii) PKCδ overexpression, whereas shear‐induced p‐eNOSS1177 and NO generation are prevented when autophagy‐intact ECs are transfected with PKCδ siRNA. Ongoing experiments are determining the extent to which this mechanism is responsible for impaired endothelial function that we have observed in arteries from old mice, that is concurrent with lower Atg3 protein expression, compromised autophagy, and attenuated p‐eNOSS1177 : total eNOS, vs. arteries from adult mice. Collectively, targeted activation of purinergic signaling and/or PKCδ may present a new strategy for treating arterial dysfunction that exists with impaired EC autophagy in the context of healthy aging.

Support or Funding Information

UU Diabetes and Metabolism Center; UU Center on Aging; UU College of Health
Evidence for the interplay between post‐translational modifications in regulating mycobacterial sirtuin activity
Authors: Yadav Ghanshyam Singh , Ravala Sandeep K , Malhotra Neha , Chakraborti Pradip K
Published: November 2023

Regulation of cellular processes by post‐translational modification of proteins is well known throughout the phylogeny. Phosphorylation/dephosphorylation of a given protein in bacteria is often controlled by Eukaryotic‐type serine/threonine kinases and cognate phosphatases. Similarly bacterial proteins are also governed by acetylation/deacetylation events. In this context, we focussed on only sirtuin, the NAD+‐dependent deacetylase present in the Mycobacterium tuberculosis genome, involved in removal of acetyl group from Nɛ‐acetyl‐lysine of a protein. Sirtuin family of proteins are conserved throughout the phylogeny and in eukaryotes they are also regulated by different kinases affecting diverse cellular functions. This interplay of kinase(s) and sirtuins is not elucidated in prokaryotes. Utilizing in vitro and in vivo transphosphorylation assays we found PknA, an essential mycobacterial eukaryotic‐type serine/threonine kinase is able to phosphorylate only sirtuin (mDAC) of M. tuberculosis. Compared to unphosphorylated mDAC, phosphorylation of mDAC causes decrease in deacetylase activity. Utilizing mass spectrometry approach, we observed that different serine and threonine residues were phosphorylated. In vitro kinase assays with different phospho‐site mutants of mDAC identified Thr‐214 as the major phosphosite. Over‐expression of mDAC or T214A in Mycobacterium smegmatis, where endogenous kinases are present, yielded phosphorylated proteins. Expectedly, magnitude of phosphorylation, as assessed by monitoring band intensity in western blot using anti‐phosphothreonine antibody, was considerably low in T214A protein compared to that of the mDAC. We further noted that sirtuin knock‐out E. coli strain was compromised in its growth profile in nutrient deprived condition (acetate medium). This phenotype, however, was reversed upon mDAC expression. Interestingly, expression of PknA in the mDAC complemented E. coli strain exhibited growth inhibition in acetate medium, indicating phosphorylation mediated regulation of mycobacterial sirtuin. In consonance with this finding, a phosphomimic, T214E also exhibited growth compromised behaviour compared to that of the wild‐type. Our study therefore, draws its logical inspiration on the possibility of interactions of two distinct post‐translational events in successful pathogens like M. tuberculosis for their versatile metabolic adaptability.

Support or Funding Information

This study was supported by institutional (CSIR‐Institute of Microbial Technology, Chandigarh) research grants (OLP‐97) and also from Department of Science and Technology (JC Bose National Fellowship to PKC, No. SR/S2/JCB‐87/2012, Science and Engineering Research Board), New Delhi, India.
Quantitative Analysis of Cross‐Talk Between RET and Other Growth Factor Receptors
Authors: Huestis Mikayla Joy , Chow Jennifer
Published: November 2023

RET (Rearranged During Transfection) is a receptor tyrosine kinase, involved in neuronal and kidney development, that activates multiple signaling cascades, including the Raf/MEK/ERK MAP Kinase and Akt pathways. We developed a luciferase reporter gene assay that is responsive to MAP Kinase pathway activation, and used it to investigate RET signaling dynamics from receptor activation to gene transcription and translation, and to quantitatively characterize crosstalk between RET and other receptor signaling pathways. The results showed that the first committed step in RET‐activated protein expression is gene transcription. Previous studies from the Hynes and Tufro laboratories have shown that crosstalk between RET and other receptors has biological importance. However, quantitative analysis of crosstalk between growth factor receptors has rarely been investigated. We used our luciferase assay, in conjunction with ELISAs measuring the amplitude and dynamics of ERK and Akt signaling, and assays measuring functional cellular outcomes, to screen for growth factors and corresponding receptors involved in cross‐talk with RET, and to achieve quantitative insights into the interaction between the pathways and its functional significance.

Support or Funding Information

Arnold and Mabel Beckman Foundation, National Institutes of Health, Biogen Idec
Characterizing the Domains of Perilipin 5 Using Proteolytic Mapping
Authors: Bailey Hannah M , Huggins Pearson N , Tansey John T
Published: November 2023

Cellular lipid storage occurs in lipid droplets, subcellular organelles coated with perilipin proteins. Members of the perilipin family have been found to play key roles in the regulation and metabolism of cellular lipid stores. Perilipin 5 is known to interact with adipocyte triacylglycerol lipase (ATGL) and hormone sensitive lipase as well as the ATGL activator CGI‐58, yet little is known about the structure of perilipin 5. Based on comparisons of perilipin 5 sequence to other family members we hypothesized that there would be discrete domains of perilipin 5 which would account for the different observed interactions. Chinese hamster ovary (CHO) cell lines stably expressing perilipin 5 with a carboxy terminal 3X FLAG epitope tag were used as a source of protein. Cells were treated with oleic acid to promote triacylglycerol storage. To identify perilipin domains, partial proteolysis was performed using trypsin and samples were analyzed by western blotting using either an amino terminal perilipin 5 specific antibody or an anti 3XFLAG antibody. Proteolytic digestion patterns differed based on oleic acid treatment. In oleic acid treated cells, a breakdown product of approximately 40 kDa was observed indicating two potential domains, an amino terminal one of ~20 kDa and a carboxy terminal of ~40 kDa. These data indicate that the perilipin proteins may fold into at least two domains with an exposed linker between them.
Involvement of Stromal Interaction Molecule‐1 in Angiotensin‐II‐Induced Expression of The Early Growth Response Protein‐1 in Vascular Smooth Muscle Cells
Authors: SIMO CHEYOU ESTELLE R , TAN JU JING , GRYGORCZYK RYSZARD , SRIVASTAVA ASHOK K
Published: November 2023

The early growth response protein 1 (Egr‐1) is a zinc finger transcription factor that has been suggested to regulate the expression of genes linked with inflammation and cell cycle regulation. An up‐regulation of Egr‐1 expression has been reported in models of atherosclerosis and intimal hyperplasia. Various vasoactive peptides and growth promoting stimuli have been shown to induce the expression of Egr‐1 in vascular smooth muscle cells (VSMC). Angiotensin‐II (Ang‐II) is a critical vasoactive peptide implicated in the pathogenesis of vascular diseases. Ang‐II elevates the intracellular level of Ca2+ through activation of store‐operated Ca2+ entry involving inositol‐3‐phosphate receptor (IP3R)‐coupled depletion of endoplasmic reticular Ca2+ and stromal interaction molecule 1 (STIM‐1). However, an involvement of IP3R/STIM‐1‐induced Ca2+ signaling pathway in Ang‐II‐induced Egr‐1 expression remains unexplored. Therefore in the present studies we have examined the role of Ang‐II‐induced Ca2+ release in Egr‐1 expression in VSMC and investigated the contribution of STIM‐1 in this process. Ca2+ chelation with BAPTA‐AM as well as pharmacological blockade of IP3R with 2‐aminoethoxydiphenyl borate (2‐APB) reduced Ang‐II‐induced Ca2+ release in VSMC loaded with Fura‐2 AM. Consistent with this, both BAPTA‐AM and 2‐APB attenuated Ang‐II‐induced enhanced expression of Egr‐1 protein levels. In addition, pharmacological blockade of both the epidermal growth factor receptor and the insulin‐like growth factor‐1 receptor attenuated Ca2+ release and inhibited the Egr‐1 up‐regulation induced by Ang‐II. Furthermore, silencing of STIM‐1 via RNA interference significantly abrogated STIM‐1 protein and mRNA expression and resulted in an attenuation of Ang‐II‐induced Egr‐1 expression as well as ERK1/2 phosphorylation. In summary, our data demonstrate that Ang‐II‐induced Egr‐1 expression is mediated by STIM‐1 and Ca2+ release in A‐10 VSMC and suggest an implication of STIM‐1 in the pathogenesis of vascular proliferative diseases.

Support or Funding Information

Supported by a grant from the Canadian Institutes of Health Research
Lipidomics In Disease And Drug Discovery
Authors: Dennis Edward A.
Published: November 2023

The largest number of distinct molecular species in cellular metabolism are the lipids where tens of thousands of distinct molecular species exist in cells/tissues. We have developed novel liquid chromatographic‐mass spectrometric based lipidomics techniques termed “CLASS” [1] to solve lipidomics problems, often in the context of an overall omics analysis of immunologically‐activated macrophages integrating transcriptomics, proteomics, and metabolomics of lipid metabolites [2]. As part of the LIPID MAPS Consortium [www.lipidmaps.org], our laboratory has developed a robust and comprehensive approach to the lipidomics analysis of hundreds of fatty acids, acylethanolamines and inflammatory eicosanoids [3]. We have built on our previous application of lipidomic analysis to characterize “synergistic” cellular lipid signaling of Toll‐like (TLR) and purinergic receptors in stimulated macrophages as models of bacterial infection and inflammation. This has recently led to an elucidation of the dual role of aspirin in enhancing lipoxin formation during inflammasome formation [4] and the dual role cytosolic PLA2 [5] plays in lipoxin synthesis. We have also elucidated the details of how cytosolic and calcium‐independent PLA2s associate allosterically with a membrane and extract the phospholipid substrate so as to release arachidonic acid for eicosanoid formation [6]. We will also discuss recent results on the precise nature and molecular dynamics of the interaction of these enzymes with novel inhibitors and how new potent and selective inhibitors block substrate phospholipid binding [Supported by LIPID MAPS Glue Grant U54 GM069338 and R01 GM020501]
Histopathology for the Influence of CMV Infection on Tomato Cellular Structures
Authors: Montasser Magdy S , Al‐Ajmy Afrah Moshare
Published: November 2023

Cucumber mosaic virus (CMV) causes wide‐spread diseases that damage some crops worldwide. One strain of the virus was isolated from tomato plants grown in Kuwait designated CMV‐KU1. This isolate was found to be associated with a benign viral satellite RNA, causing mild symptoms on infected tomato plants. Another isolate, CMV‐16, a severe stunting strain and lacking viral satellite RNA was used in a comparative study with the isolated mild strain of the virus CMV‐KU1. Both CMV strains were used as antigens to prepare murine monoclonal and polyclonal antibodies for serological confirmation using enzyme linked immunosorbent assay (ELISA) to confirm the identity of viral strains in infected plant tissues. The objectives of this study were to investigate the effects of the viral infection on tissues, cells and cellular organelles in test plants using Transmission Electron Microscopy (TEM) to determine the extent of infection and host‐virus interactions and damage caused by virus isolates on tomato. Electron micrographs of ultra‐thin sections proved the presence of the viral particles, the formation of viral crystalline bodies, chloroplast damage and malformation due to the infection caused by the virus in infected plant tissues. The viral infection caused a reduction in chloroplast number and size. As the infection progressed, chloroplasts were completely destroyed and the grana were disorganized and scattered into the cytoplasm that causes reduction in chlorophyll contents in plant tissues.

Support or Funding Information

This work was supported and funded by Kuwait University Research Grant No. [SL 01/13 & YS 07/10]
Early Life Methyl Donor Supplementation Attenuates Obesity‐Risk
Authors: McKee Sarah E , Reyes Teresa M
Published: November 2023

Excessive gestational weight gain is defined when women with a normal pre‐pregnancy body mass index gain more weight than recommended during pregnancy. Excessive gestational weight gain (GWG) has a prevalence rate of nearly 50% and it significantly increases the risk for the development of obesity in the offspring. Excessive GWG can be modeled in animals by feeding pregnant dams a high fat diet, leading to increased maternal weight gain and obesity‐promoting phenotypes in the offspring. Using a mouse model of excessive GWG, we have documented in the offspring an increased preference for sucrose and fat, increased expression of genes that underlie reward‐related behaviors, and both global and gene specific decreases in DNA methylation. These changes in reward‐related neural circuitry may contribute to the increased risk for the development of obesity in the offspring by altering the animal's response to highly palatable, energy dense foods. Methyl donor supplementation (MDS) during pregnancy can reverse some of these obesity‐promoting phenotypes in offspring, yet it is unknown whether postnatal MDS can reverse these phenotypes. To determine this, offspring from dams fed either a high fat diet (HFD) or control diet during gestation were fed a methyl donor supplemented diet during early life (3–6 weeks). We find that postnatal MDS significantly decreased body weight in both male and female adult HFD offspring, and does not alter body weights of control diet offspring. Further, postnatal MDS can rescue adult male fat preference and contributes to region specific normalization of global DNA hypomethylation. We also find that MDS can increase motivation to work for palatable reinforcer in adult female offspring. Continued assessment of obesity development, preference for palatable foods, metabolic phenotypes, and expression changes of genes underlying homeostatic and hedonic feeding will elucidate the mechanism by which early postnatal MDS offsets adult obesity risk.

Support or Funding Information

NIH MH087978, MH100828, MH106330 to TMR and ES019851 to SEM
Weight Loss Success Among Obese Women of Mexican‐origin in Mexico and the US
Authors: Kaufer‐Horwitz Martha , Weintraub Miranda Ritterman , Guendelman Sylvia
Published: November 2023

Background

We assessed variations in and correlates of weight‐loss success among overweight/obese women in Mexico (WIMX) and Mexican‐American women (MA).

Methods

We used cross‐national data from 2006 ENSANUT (Mexico) and NHANES (2001–2008) to compare 5,061 WIMX with 550 MA's without known metabolic conditions. WLS was defined as losing ≥ 5% of body weight over one year.

Results

MA's were more likely to attain weight‐loss success (OR=1.31; 95%CI=1.01–1.70). Weight‐loss success among WIMX was higher in those with at least high school, a provider screen of overweight and a lower BMI. Among MA's, an incomplete high school versus primary education reduced the odds of weight‐loss success. Among women who lost ≥10lbs, weight‐loss strategies such as eating less were higher among MA's.

Discussion

MA women were more likely than WIMX to attain weight loss success. Understanding these disparities can help design customized public health interventions that curb the obesity epidemic in these women in both countries.

Support or Funding Information

Programa de Investigación en Migración y Salud (PIMSA).
Child Dietary Patterns and Diversity in Rural and Semi‐Rural Ghanaian Communities
Authors: Gyenes Natalie , Agbemafle Isaac , Zotor Francis , Duggan Christopher , Fawzi Wafaie , Willett Walter , Ellahi Basma
Published: November 2023

Background

Poor child feeding practices contribute to childhood malnutrition and have long‐term consequences for non‐communicable diseases (NCDs) in later life, but little is known about these patterns in rural Ghana. This study assessed dietary patterns and diversity among children under five years in rural and semi‐rural areas in Ghana.

Methods

A cross‐sectional survey was undertaken among 1185 households in Eastern, Volta and Brong Ahafo regions of Ghana. Households with children under age 5 years were sampled. Children from the households were randomly selected and information on liquid/medicine, supplement use, breast feeding history and special foods consumed were obtained from their caregivers using a pretested semi‐structured questionnaire. Dietary intake patterns and dietary diversity of the children were assessed using a 24‐hour recall. Pearson's chi‐square was used to determine associations between region and dietary patterns and diversity.

Results

873 children were recruited of whom 52% were females. Breastfeeding frequency (at least 6 times) was highest within each of the three geographical regions. Multivitamins and oral rehydration solutions were the commonly reported medications. In increasing order of consumption, the main liquids consumed were soup, porridge and plain water. The main ingredients used for porridge were maize flour, water and sugar. Food items used to enrich the porridge varied by region. Groundnut paste and milk were commonly used to enrich porridge in all the three regions. Special foods such as commercially prepared fortified cereal‐legume blends and locally prepared wheat/corn‐soy blend were reported to be eaten occasionally. The commonly consumed food groups were cereals (14.9%), vegetables (14.9%), fish and sea foods (12.2%) and roots and tubers (10.4%) and these differed significantly by region (p<0.0001). Dietary diversity was low for 91% of the children and this trend was similar in all the three regions. There were significant differences in dietary diversity across the three regions (p=0.001).

Conclusion

Child feeding practices in rural and semi‐rural communities in Ghana show low dietary diversity. This has implications for childhood malnutrition and non communicable diseases in adult life, highlighting the importance of prioritizing nutrition interventions in order to address childhood malnutrition.

Support or Funding Information

This Research was supported in part by a grant from the Department of Global Health and Population, Harvard T.H. Chan School of Public Health, as well as the Department of Nutrition, Harvard T.H.Chan School of Public Health.
Pumping Breast Milk on Campus: University‐Based Needs for Nursing Mothers
Authors: Dinour Lauren Melissa , Bai Yeon
Published: November 2023

The purpose of this study is to examine the factors associated with pumping breast milk on a university campus under the Theory of Planned Behavior (TBP). In January 2015, all employees and students of one New Jersey university were invited via email to participate in a 20‐minute online survey measuring pumping behaviors, psychosocial variables framed by the TPB, and demographic factors. Using multiple regression analysis, the most influential predictors (attitudes, subjective norms, and perceived behavioral control) of the intention to pump breast milk on campus were identified. One hundred twenty‐five women completed the survey. The average age of respondents was 30.05 years (SD 8.83), and the majority of respondents was White (53.6%) and married (63.2%). Half of the women were employees (24.8% faculty, 24.8% staff), while the other half were students (50.4%). Together, attitudes, subjective norms, and perceived behavioral control explained 21.2% of the total variance of the intention to pump breast milk on campus (P<0.01, R=0.46). The most influential factor for pumping intention was subjective norm (β=0.36, P<0.01), followed by perceived behavioral control (β=0.18, P=0.04). Attitudes did not significantly influence the intention to pump breast milk on campus (β=0.15, P=0.11). Specific beliefs that were significantly correlated with the intention to pump on campus included experiencing approval from other women in general (β=0.39, P<0.01), experiencing superiors’ approval (β=0.30, P<0.01), experiencing approval from other women with breastfeeding experience (β=0.29, P<0.01), and feeling supported by the university (β=0.22, P<0.01). Based on the findings, continued efforts are needed to improve breastfeeding awareness, as well as to provide pump loan and milk storage options. Since the culture of lactation accommodation may impact breastfeeding exclusivity and duration, universities must do more to support all women who wish to continue a breastfeeding relationship while at school or work.
Normal Plasma Volume Expansion and Micronutrient Status in Pregnancy
Authors: Aguree Sixtus , Roy Aditi , Gernand Alison D.
Published: November 2023

Plasma volume expansion is widely known as an important physiologic change in pregnancy, yet normal expansion in healthy pregnancies has not been well described. This expansion influences plasma micronutrient concentrations and may impact interpretation of biomarkers. Our objective was to review studies that measured plasma volume longitudinally across gestation to describe normal plasma volume expansion; secondarily, we examined the impact of plasma volume expansion on micronutrient biomarkers. We systematically searched PubMed for original articles that directly measured plasma volume in pregnant women at more than one time across gestation. We also searched reference lists from these publications for additional papers. We found 15 studies published from 1963 to 2006 that directly measured plasma volume in pregnancy more than one time. We excluded 3 studies because the measurements were not longitudinal and one study because it used an unconventional measurement technique (iron dextran) and some values appeared extreme. Of the 11 longitudinal studies included, 8 studies were conducted in the UK and one each was conducted in India, Chile, and Nigeria. All studies used the Evans blue dye (T‐1824) indicator dilution method to measure the plasma volume. The total number of women in all studies was 524; study size ranged from 11 to 95. Plasma volume was measured 3 to 7 times in women across the studies. Two studies measured plasma volume before conception and 5 studies measured postpartum values (6–8 weeks after birth) as a proxy for pre‐conception. Plasma volume increases varied across the studies. Plasma volume increased by 8% between preconception and 12 weeks, 18% by 16 weeks, 27% by 20 weeks and 36% by 25 weeks gestation. The maximum mean plasma volume increase was 52% (1267 ± 38 ml) at 33–34 weeks gestation. This peak mean expansion ranged from 48% to 59% (1218 to 1323 ml) across studies, while reports of expansion for individual women ranged from 25% to 80%. Plasma volume slightly declined from late third trimester to term. Only one plasma volume study measured micronutrient status. In that study, plasma zinc concentrations declined across gestation to 30 weeks but intravascular zinc mass (the product of zinc concentration and plasma volume) increased across pregnancy. Plasma copper concentration increased up to gestational week 35 but intravascular copper mass was not reported. Few studies have measured plasma volume longitudinally in pregnancy. Studies of healthy pregnancies similarly show plasma volume increases progressively to approximately 52% above the non‐pregnant state in the third trimester with a slight decline to term. Plasma zinc concentration changes in pregnancy did not reflect actual changes in zinc status. Future studies in pregnancy should measure plasma volume to have a valid estimation of micronutrient status and to understand underlying physiologic changes that may be related to adverse outcomes.

Support or Funding Information

NIH BIRCWH 12HD055882 (Dr. Gernand)
Metformin to Augment Low Milk Supply: A Protocol Summary
Authors: Nommsen‐Rivers Laurie A , Riddle Sarah W , Thompson Amy A , Ward Laura P , Wagner Erin A
Published: November 2023

Maternal obesity is associated with shortened breastfeeding duration, but the mechanism remains unclear. A progression of research is pointing to waning insulin secretion in the context of insulin resistance as a strong contributor to low milk supply in mothers attempting to exclusively breastfeed. Metformin enhances insulin action, but it is not known if it improves milk production. Our primary aim is to enact a phase I/II double‐masked randomized placebo‐controlled trial (RCT) of metformin versus placebo to treat low milk supply in women with signs of insulin resistance. At baseline, all referred women receive standard guidance for increasing milk supply with breast expression, record 24‐hours of milk production, and are evaluated for RCT eligibility. If eligible, participants are randomly assigned to metformin or placebo. Specimen collection includes milk fat RNA at baseline and post‐treatment. Baseline measurements are ongoing until N=30 RCT‐eligible participants complete the trial. The study is powered to demonstrate feasibility, obtain variance estimates, and test for a trend in the primary hypothesis that the metformin group will experience a greater increase in milk output as compared to the placebo group. Trial completion expected July, 2016.

Support or Funding Information

NIH 5 K12 HD051953 (Tsevat, PI), Bridging Interdisciplinary Research Careers in Women's Health (BIRCWH award to LN‐R)