bluuebunny/crossref_metadata_2025_split huggingface dataset:
Discrete Structural Dynamics of Pseudo‐Palindromic Motifs Control DNA Binding of Bacterial Toxin‐Antitoxin Complexes
Authors: Brodersen Ditlev Egeskov , Bendtsen Kirstine Louise , Xu Kehan , Luckmann Majbritt , Winther Kristoffer , Shah Shiraz A. , Pedersen Christian N. S. 
Published: November 2023

Toxin‐antitoxin (TA) systems have been strongly implicated in bacterial cell survival during antibiotic stress and are likely to play important roles in persister cell formation1,2. Understanding the function and regulation of TA systems will thus be vital for combatting chronic and recurrent human infections in the future. Expression of type II TA systems is regulated at the transcriptional level through direct binding of the antitoxin to pseudo‐palindromic sequences on operator DNA. In this process, the toxin functions as a co‐repressor by stimulating DNA binding through direct interaction with the antitoxin and formation of a higher‐order protein complex3. In the VapBC TA class, the VapC toxin is inhibited by direct protein interaction with the VapB antitoxin, which also contains a DNA binding domain of the AbrB type3. Since TA complexes show a great structural variety and no structures have been determined of the same system both on and off DNA, the structural changes occurring upon DNA binding are not known. Furthermore, details of how the flexible C‐terminus of the antitoxin becomes available for degradation when the toxin is activated, are scarce. Here, we present high‐resolution crystal structures of the complete 90 kDa heterooctameric VapBC1 complex from Caulobacter crescentus CB15 both in isolation and bound to a cognate 27‐bp DNA operator sequence at 1.6 and 2.7 Å resolution, respectively. The structures reveal that DNA binding is associated with a dramatic architectural rearrangement of conserved TA interactions involving VapB antitoxin C‐termini. We find that the C‐terminal tails of the antitoxin VapB1 that inhibit the VapC toxin by directly interacting with its active site, swap positions to interlock the complex in the DNA‐bound state. We further show that 2:1 interaction between toxin and antitoxin depends on a pseudo‐palindromic protein sequence in the antitoxin, which is able to bind and inactivate both molecules of a VapC1 toxin dimer. Sequence analysis of 4,127 orthologous VapB sequences reveals that such palindromic protein sequences are widespread among bacterial and archaeal VapB antitoxins suggesting that we have uncovered a general principle governing regulation of VapBC TA systems. Finally, a structure of C‐terminally truncated VapB1 bound to VapC1 reveals discrete states of the TA interaction that suggest a structural basis for antitoxin degradation and thus toxin activation in vivo.Support or Funding InformationThis work was funded by the Lundbeck Foundation [R173‐2014‐1182 to D.E.B.]; the Danish National Research Foundation's Centre for Bacterial Stress Response and Persistence, BASP [DNRF120 to D.E.B]; and a Chinese Scholarship Council postgraduate [K.X.].
Effect of Maternal Separation on Oxidative and Nitrosative Stress in the Brain of Rat Offspring
Authors: Campos‐Rangel Andrea , Torner‐Aguilar Luz , Saavedra‐Molina Alfredo , Manzo‐Avalos Salvador 
Published: November 2023

The first weeks of life of organisms represent a critical brain development phase, due to the fact that many regions of the brain are not fully formed at birth or are subject to the postnatal maturity. Clinical and laboratory research shows that maternal separation (MS) during critical periods of brain development can affect the cytoarchitecture of the hypothalamus synaptic density, decreasing the length of the dendrites and the activity of the neurons, which contributes to the inability of learning and abnormal behavior. Studies in humans and in animals suggest that the MS may increase the risk of psychiatric disorders, such as depression, anxiety, schizophrenia and disorders of personality, as well as the predisposition to drug abuse and alcohol consumption. In the central nervous system, reactive oxygen and nitrogen species play an important role in the pathophysiology of these disorders, since their accumulation increases the susceptibility of the brain damage caused by oxidative and nitrosative stress generated. Since there are no reports about the effect of MS on the oxidative and nitrosative stress in the hypothalamus of the rat offspring, this investigation is important, which was addressed by subjecting the offspring to the MS daily (3 h/day) during the first fourteen days of life. Lipoperoxidation (LPO) levels were determined by quantifying the reactive species to thiobarbituric acid, and by Western blot were determined the levels of antioxidant enzymes catalase and superoxide dismutase (SOD2), as well as the nitric oxide synthases (NOS1 and NOS2). The results show that acute and/or chronic stress by MS affected levels of LPO, enzymes catalase, SOD2, NOS1 and NOS2, as well as the profile of nitrosylation in the hypothalamus of the rat offspring.Support or Funding InformationAcknowledgements: the authors appreciate the partial economic support of the CIC‐UMSNH (2.16 to ASM; 2.37 to SMA) grant.
Altered Islet Function May Promote a Lean Phenotype in Tafazzin Deficient Mice
Authors: Cole Laura Kathleen , Doucette Christine , Vandel Marilyne , Fonseca Mario , Xiang Bo , Dolinsky Vernon Wayne , Hatch Grant Michael 
Published: November 2023

Tafazzin is a transacylase that maintains mitochondrial membrane integrity and the function of the mitochondrial respiratory chain. Specifically, tafazzin maintains the content and molecular structure of the unique tetra‐acyl phospholipid cardiolipin (CL) located in the inner mitochondrial membrane. Despite abundant evidence that mitochondrial dysfunction is associated with insulin resistance, little is known about the potential role of CL and tafazzin in the trajectory of this disease. To investigate the in vivo effects of tafazzin deficiency, we have utilized a mouse model with a doxycycline‐ inducible tafazzin shRNA knock‐down. We have previously established that tafazzin knock‐down mice are protected against the development of obesity, and insulin resistance compared to control litter mates. Tafazzin deficiency promotes a lean phenotype due to a coordinated elevation in adipose lipolysis and hepatic fatty acid oxidation. We have now determined that glucagon and insulin levels in the blood and whole islets are significantly reduced with tafazzin deficiency. We have also ascertained that the quantity and function of beta‐cells were similar between genotypes. However, despite similar levels of alpha‐cells, glucagon secretion during high‐glucose conditions was elevated from islets isolated from tafazzin knock‐down mice. As a result, tafazzin knock‐down mice exhibited significantly higher circulating ratios of glucagon to insulin during glucose challenges. Our experiments indicate that tafazzin may have a role in regulating islet function. These data also suggest that mice deficient in tafazzin may be protected in part against weight gain by promoting glucagon secretion during fed states. Since, the development of type 2 diabetes is closely related to obesity and pancreatic function altering cardiolipin synthesis may be a novel therapeutic option for patients at risk for type 2 diabetes.Support or Funding InformationHeart and Stroke Foundation of CanadaChildren's Hospital Research Institute of Manitoba
Investigation of Free Fatty Acid on Concerted Trafficking of KATP and Kv2.1 Channels in the Adipoinsular Axis in vivo and in vitro
Authors: Ruan Jing‐Syuan , Chen Pei‐Chun 
Published: November 2023

Type 2 diabetes is the most prevalent metabolic syndrome worldwide and obesity is the major risk factor. The high level of fatty acid accumulation can be observed in the pancreatic β‐cells so called lipotoxicity. The overload of free fatty acid contributes to pancreatic β‐cells apoptosis and dysfunction that are associated with the disease progression. Insulin and leptin form as the adipoinsular axis, a dual regulatory loop maintains nutrient balance. High blood glucose triggers insulin release in pancreatic β‐cells and insulin further stimulates leptin release from adipocytes. Leptin can directly act on pancreatic β‐cells to suppress insulin secretion. Plasma leptin concentration is proportional to body fat mass so it's not surprising that high plasma leptin can be observed in obese individuals. Unfortunately, leptin resistance is easily to develop therefore, chronic leptin treatment is not an effective way to treat obesity. ATP‐sensitive potassium channels (KATP channels) are essential to the connection between membrane excitability and glucose‐stimulated insulin secretion (GSIS). Physiologic role of KATP channels depend on the correct trafficking and cell surface expression. Based on the previous publication, leptin signaling leads to simultaneous increase surface expression of KATP and Kv2.1 channels that is dependent on AMPK, PKA as well as actin depolymerization and this action would cause membrane potential hyperpolarization to reduce insulin secretion. The goal of this study is to understand the effect of fatty acid on trafficking of KATP and Kv2.1 channels in adipoinsular axis. We hypothesize that free fatty acid would induce the change of membrane potentials that results in the defect of KATP and Kv2.1 channels trafficking and this would cause cell malfunction and lead to cell death in obesity‐induced type 2 diabetic β‐cells. The following three specific aims are proposed to comprehensively investigate the above hypothesis: Aim 1: to evaluate the effect of free fatty acid on the adipoinsular axis in vivo using molecular genetic approaches as well as electrophysiological experiments. Aim 2: to understand if concerted trafficking of KATP and Kv2.1 channels plays a role in free fatty acid induced apoptosis using pharmacological approaches. Aim 3: to manipulate whether concerted trafficking of KATP and Kv2.1 channels can recover the adipoinsular axis and enhance cell viability using type 2 diabetes animal model, including diet induced obese mice and leptin receptor knockout mice (db/db). The study provides the new insight by manipulating KATP and Kv2.1 channels trafficking to overcome metabolic diseases related to unbalanced adipoinsular axis.Support or Funding InformationThis work are supported by MoST grants (103‐2320‐B‐006‐005‐MY2 and 105‐2628‐B‐006‐MY3) to P‐C Chen.
Adiponectin receptor 1 resists the decline of serum osteocalcin and GPRC6A expression in ovariectomized mice
Authors: Lin YuanYu , Chen ChingYi , Wu Shinn Chih , Ding ShihTorng 
Published: November 2023

Hormonal changes that cause metabolic complications are a common problem in postmenopausal women. Adiponectin and osteocalcin are cytokines associated with glucose regulatory and insulin sensitized function in postmenopausal stages. The current study investigated the role of adiponectin signaling and osteocalcin mediated function in glucose metabolism in ovariectomized mice. In a mouse menopausal‐related metabolic disorder model, overexpression of adiponectin receptor 1 improved glucose tolerance and caused resistance to body weight increase and decline of serum osteocalcin. Furthermore, adiponectin receptor 1 transgenic ovariectomized mice had higher GPRC6A (the putative osteocalcin receptor) expression in muscle tissue. Immunofluorescence indicated that GPRC6A and adiponectin receptor 1 were co‐localized in mouse muscle tissues. The present finding suggested adiponectin receptor 1 can mediate the improvement of glucose metabolism by osteocalcin in ovariectomized mice. Our findings imply the possibility to ameliorate menopause‐induced metabolic disorder by GPRC6A and adiponectin signaling.Support or Funding InformationThis study was supported in part by the Grants NSC 102‐2313‐B‐002‐026‐MY3 and NSC 99‐2628‐B‐002‐067‐MY3 grants from Ministry of Science and Technology in Taiwan.
Use of a three dimensional porcine retinal explant model to detect HIF1α for understanding diabetic retinopathy
Authors: Iwuagwu B. U. , Cruickshank S. F. , Rowe I. , Knott R. M. 
Published: November 2023

IntroductionDiabetic retinopathy is a major microvascular complication of diabetes mellitus and a leading cause of blindness [1]. Hypoxia is suggested to play a crucial pathophysiological role in diabetic retinopathy (DR) [2], but the exact mechanisms are complex and the precise events are still debated. An agarose‐collagen three‐dimensional (3D) porcine retinal explant model is used here to investigate the expression of the hypoxia inducible factor ‐1 alpha (HIF‐1α), a transcription factor critical for cellular response to hypoxia [2].AimTo study the expression and localization of the hypoxia‐inducible factor 1 alpha ((HIF)‐1α, a key target in DR using a 3D porcine retinal explant model.MethodPorcine retinal tissues were extracted from ocular globes, (n ≥ 6) donated by a local abattoir. Extracts were cultured in DMEM within a 3D matrix derived from 0.5% agarose + collagen (0.25mg/ml) co‐gel solution and explants were maintained in a humidified atmosphere with 5% CO2 at 37°C. Explants were harvested at less than 2 h (T0) and 24 h (T1) post culture, fixed overnight in 10% neutral buffered formalin (NBF) and incubated in 30% sucrose. Serial sections (20μm) were cut using a cryostat. To ascertain tissue integrity, sections were stained with Mayer's hematoxylin and Eosin according to protocol (Sigma‐Aldrich). To detect the expression and sub‐cellular location of the hypoxia inducible factor (HIF) 1α at T0 and T1, sections (20μm) were washed 2 × 15 minutes in 50mM Tris buffered saline (TBS) plus 0.1% Triton X‐100, followed with blocking in 10% normal goat serum/1% bovine serum albumin (BSA) (Santa Cruz) for 2 hours at room temperature with final incubation with primary mouse anti‐Hif1α (Thermo Fischer) (1:20) overnight at 4°C. Post‐incubation, specimens were rinsed 1 × 15 minutes with TBS 0.1% Triton. Non‐specific endogenous peroxidase reaction was blocked with 0.3% H2O2 in 0.3% normal serum (goat) in TBS for 15 minutes at RT. Goat anti‐Mouse IgG (H+L) secondary antibody, DyLight 488 (Thermo Fischer scientific) (1:200) was used for detection.Results & ConclusionH & E stain demonstrated preservation of retinal integrity and does indicate that the use of agarose‐based 3D system provided structural and mechanical support which may have prevented early retinal tissue degeneration. Preliminary immunohistochemistry results revealed positive identification of HIF1α expression at less than 2 h (T0) and 24 h (T1). HIF1‐α expression was localized in the photoreceptor and dispersed throughout the outer and inner layers of the retina. Further investigation is needed to establish the precise cell‐specific co‐localization of HIF1α.Support or Funding InformationBenedicta Iwuagwu, the presenting author is a recipient of an academic scholarship from the Robert Gordon University (RGU). This thesis is fully funded by RGU and the author receives a tax‐free monthly personal stipend in addition from the university.
Potential role of hepatic lipase in the accrual of docosahexaenoic acid (DHA) in the brain
Authors: Dhavamani Sugasini , Yang Peng , Ng Dominic , Khetarpal Sumeet , Vitali Cecilia , Rader Daniel , Subbaiah Papasani 
Published: November 2023

DHA is an essential fatty acid that is required for the normal development and function of the brain. Because of its inability to synthesize adequate amounts of DHA from the precursors, the brain has to acquire DHA through the blood brain barrier (BBB). Recent studies demonstrated the presence of a transporter at the BBB that specifically transports DHA into the brain in the form of lysophosphatidylcholine (LPC‐DHA). However, the mechanism by which LPC‐DHA is generated in the plasma is not known. Our previous studies showed that there are three different enzymes in plasma‐ lecithin cholesterol acyltransferase (LCAT), endothelial lipase (EL), and hepatic lipase (HL), which can generate LPC‐DHA from sn‐2 DHA PC of lipoproteins. In this study we determined the potential roles of these enzymes in the delivery of DHA to the brain by examining the brain DHA levels in the mice genetically deficient in each of these enzymes. The results show that the brain DHA levels of LCAT‐deficient mice (16.3%) or EL‐deficient mice (15.2%) were not significantly lower than those of their littermates. However, the concentration of brain DHA was significantly lower in HL knockout (KO) mice (13.5%) compared to their littermates (17.1%) (p<0.002). The DHA levels were also lower in the plasma but not in the liver, or heart of HL KO mice. Furthermore, the concentration of LPC‐DHA was significantly decreased in the plasma of HL KO mice. These results suggest that HL may be important for the supply of DHA to the brain by generating LPC‐DHA either in the plasma itself, or in the liver, followed by its secretion into plasma.Support or Funding InformationSupported by NIH R01 HL 68685 (PVS)
Crystal structure and activation mechanism of human neutral sphingomyelinase 2
Authors: Shanbhogue Prajna , Airola Michael , Shamseddine Achraf , Guja Kip , Maini Rohan , Bartke Nana , Wu Bill , Obeid Lina , Garcia‐Diaz Miguel , Hannun Yusuf 
Published: November 2023

Neutral sphingomyelinase 2 (nSMase2) is a magnesium‐dependent phophodiesterase that generates the bioactive sphingolipid ceramide. Ceramide is a critically important secondary messenger that acts in a variety of cellular pathways such as apoptosis, inflammation, cell growth and differentiation. Accordingly, mutations in nSMase2 can affect ceramide production and have been implicated in cancers of the breast, urinary bladder and colon. Obtaining a detailed understanding of the structure and function of this enzyme is of significant interest and has the potential to uncover new avenues for cancer therapeutics. We have obtained the first high‐resolution X‐ray crystallographic structure of human nSMase2 and developed a yeast two‐hybrid system to analyze its inter‐domain interactions, which likely play a role in activation of the enzyme by anionic phospholipids (APLs). Previous work in our laboratory identified amino acid residues in the N‐terminal hydrophobic domain of nSMase2 that interact with APLs in the inner membrane. Our current results suggest that these residues also interact with the cytoplasmic C‐terminal domain of nSMase2 and may help regulate activation of the enzyme by APLs. Furthermore, we have identified additional amino acid residues in the juxtamembrane region of nSMase2 that may also contribute to regulation of enzyme activity. In sum, our results provide a model for activation of nSMase2, and reveal a novel structure that paves the way for rational drug design endeavors.Support or Funding InformationThis work was supported by grants from the National Institutes of Health R37GM043825 (to Y.A.H.), R01GM100021 (to M.G.D.), F32GM100679 (to M.V.A.), and F30ES022930 (to K.E.G.).
Phosphorylation of the Nem1‐Spo7/Pah1 Phosphatase Cascade by Pkc1 Protein Kinase C
Authors: Dey Prabuddha , Su Wen‐Min , Carman George M 
Published: November 2023

In the yeast Saccharomyces cerevisiae, the Pah1 phosphatidate phosphatase is responsible for catalyzing the penultimate step in the synthesis of triacylglycerol. Phosphatidate phosphatase dephosphorylates phosphatidate to form diacylglycerol, the precursor of triacylglycerol. Pah1 exists in the cytosol as a phosphoprotein, but it must be dephosphorylated by the Nem1‐Spo7 protein phosphatase for its interaction with the ER membrane to associate with the substrate phosphatidate. In this work, we examined the regulation of the Nem1‐Spo7/Pah1 phosphatase cascade by its phosphorylation by Pkc1 protein kinase C (PKC). Pah1, Nem1, and Spo7 were expressed and purified from Escherichia coli to utilize non‐phosphorylated pristine substrates for PKC, which was purified from yeast. PKC phosphorylated each component of the phosphatase cascade on a serine residue, and the reactions were dependent on phosphatidylserine (0.5 mM) and diacylglycerol (0.15 mM) for the maximum phosphorylations. This was a surprising result given the fact that previous data indicated that yeast PKC does not require lipids for its activity, at least in the absence of GTP‐bound Rho1. Owing that prior work on yeast PKC utilized a peptide substrate, we examined the PKC activity using peptide substrates with sequences derived from Nem1, Spo7, or Pah1. With these substrates, PKC did not require phosphatidylserine and/or diacylglycerol for its activity. We found that mammalian PKC, using Pah1 or the Pah1‐derived peptide as substrates, were dependent on phosphatidylserine and diacylglycerol for activity. These results indicated a fundamental difference in the mechanisms of the reactions catalyzed by the yeast and mammalian PKC enzymes, and that yeast PKC is likely to require lipids for activity in vivo.Support or Funding InformationSupported by NIH grant GM050679
ANGPTL8 Promotes the Ability of ANGPTL3 to Inhibit Lipoprotein Lipase
Authors: Davies Brandon S.J. , Chi Xun , Britt Emily C. , Shows Hannah W. , Hjelmaas Alexander J. , Shetty Shwetha K. , Cushing Emily M. , Zhang Ren 
Published: November 2023

Lipoprotein lipase (LPL) is critically important for the delivery of plasma lipoprotein–derived triglycerides to peripheral tissues. Activity of LPL is carefully regulated and misregulation of LPL can contribute to metabolic diseases including obesity, metabolic syndrome, and cardiovascular disease. Several members of the angiopoietin‐like (ANGPTL) family of proteins, including ANGPTL3 and ANGPTL8, regulate LPL activity. Deficiency in either ANGPTL3 or ANGPTL8 reduces plasma triglyceride levels and increases LPL activity, whereas overexpression of either protein does the opposite. Recent studies showed that ANGPTL8 overexpression only raises plasma triglycerides when ANGPTL3 is present suggesting that ANGPTL3 and ANGPTL8 work together to alter clearance of plasma triglycerides, however the nature of this interaction has remained elusive. We tested the hypothesis that ANGPTL8 forms a complex with ANGPTL3 and that this complex is necessary for efficient inhibition of LPL. We found that ANGPTL3 or ANGPTL8 alone could inhibit LPL only at concentrations that far exceeded physiological levels, especially when LPL was bound to its endothelial cell receptor/transporter GPIHBP1 (glycosylphosphatidylinositol‐anchored high‐density lipoprotein binding protein 1). Physical interaction was observed between ANGPTL3 and ANGPTL8 when the proteins were co‐expressed, and co‐expression with ANGPTL3 greatly enhanced the secretion of ANGPTL8. Importantly, the ability of ANGPTL3‐ANGPTL8 complexes to inhibit LPL was dramatically increased compared to either protein alone. Part of this improvement could be ascribed to an improved ability to bind LPL as protein interaction assays showed that ANGPTL8 increased the ability of ANGPTL3 to bind LPL. Together, these data suggest that ANGPTL8 binds to ANGPTL3, forming a complex that has an increased ability to bind and inhibit LPL.Support or Funding InformationThis work was supported by grants from the NHLBI (HL130146) and the Carver Medical Research Initiative