bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of Tryptophan‐Nicotinamide (TRP‐NAM) Pathway in Malnutrition Induced Liver Dysfunction
Authors: Hu Guanlan , Ling Catriona , Chi Lijun , Furse Samuel , Koulman Albert , Bandsma Robert
Published: April 2020

Background

Malnutrition contributes to 45% of the death of children under 5 years of age. Mortality in severe malnutrition is often related to metabolic disturbances, including hypoglycemia, indicating hepatic metabolic dysfunction. However, the underlying pathophysiology is poorly understood. Children with complicated severe malnutrition have been found to have significantly lower circulating tryptophan levels in their blood. Disturbances in the tryptophan‐nicotinamide pathway have been implicated in liver and other organ diseases, potentially mediated by NAD‐dependent deacetylase sirtuin‐1 (SIRT1). However, there have been no studies that have evaluated the role of tryptophan‐nicotinamide pathway in severe malnutrition.

Objectives

To evaluate the effect of modulating the tryptophan‐nicotinamide pathway on hepatic metabolism dysfunction in a mouse model of severe malnutrition.

Methods

Weanling male C57BL/6J mice were randomized into different groups fed with a control diet (18% protein), or a malnutrition‐inducing diet (1% protein) with or without supplementation of modulators of the tryptophan‐nicotinamide pathway (nicotinamide, nicotinamide‐riboside and tryptophan). In addition, SIRT1 modulators treatment including resveratrol, EX‐527 and vehicle were tested. Comprehensive metabolic, histological and molecular analyses were performed.

Result

After diet treatment, the mice in the 1% protein diet group had significantly lower body weight and length than in the 18% protein group, without any effect of modulators of the tryptophan‐nicotinamide pathway. Feeding mice with a 1% diet led to severe hepatic steatosis, mitochondrial structural alterations, lower ATP levels, disrupted TRP‐NAM pathway metabolites, and a decrease of phospholipids to triglycerides ratio. Nicotinamide, nicotinamide‐riboside and tryptophan treatment all led to a partial reversal of the hepatic steatosis in the 1% protein‐fed mice. ATP levels, mtDNA copy number, and PPARα target genes in the β‐oxidation pathway were also recovered in treatment group. We postulate that the effect is mediated through changes in levels of nicotinamide adenine dinucleotide (NAD+), which is critical in redox reactions and is a substrate for SIRT1, important for mitochondrial function.

Significance

Modulating TRP‐NAM pathway can partially improve liver metabolic function in a mouse model of severe malnutrition. This study will improve our understanding of the pathophysiology of severe malnutrition. The results of this project could lead to the development of new interventions aimed at improving metabolic disturbances in children with severe malnutrition, which could then be taken to clinical trials.

Support or Funding Information

Bill & Melinda Gates Foundation
MARCH Family E3 Ubiquitin Ligases Selectively Downregulate Cadherin Family Proteins
Authors: Seo Tadahiko , Grimsley-Myers Cynthia M. , Isaacson Robin H. , Kowalczyk Andrew P.
Published: April 2020

VE‐cadherin (VE‐cad) plays a central role in the adherens junctions (AJ) of vascular endothelial cells by regulating endothelial barrier function. Our group previously showed that a viral ubiquitin ligase K5, which is encoded in Human Herpesvirus 8 genome, causes endocytosis and degradation of VE‐cad. K5 is homologous to endogenous human membrane associated RING‐CH‐type ubiquitin ligase (MARCH) proteins. However, the contribution of endogenous MARCH family proteins to cadherin regulation is unclear. We began by performing RT‐PCR analysis to assess which MARCH family proteins are expressed in endothelial cells. Vascular endothelial cells were found to express low levels of MARCH1 and readily detectable levels of MARCH2, MARCH3, MARCH4 and MARCH8. To assess the effect of MARCH family protein expression on VE‐cad and N‐cad, GFP‐tagged MARCH family proteins were expressed in human umbilical endothelial cells (HUVECs) and the effect on VE‐cad and N‐cad was assessed. MARCH1 had no discernable impact on either VE‐cad or N‐cad. MARCH8 slightly downregulated VE‐cad but not N‐cad. In contrast, MARCH2, MARCH3 and MARCH4 transfection markedly downregulated VE‐cad in HUVECs. Interestingly, MARCH2 and MARCH3 did not downregulate N‐cad, but MARCH4 downregulated both VE‐cad and N‐cad. These results suggest that different MARCH proteins have different cadherin specificities. To determine the amino‐acid sequences within VE‐cad that dictate this specificity, we generated VE‐cad/N‐cad chimeras by swapping the transmembrane and juxta‐membrane domains (VE‐cad‐NcadTMDJMD, N‐cad‐VEcadTMDJMD). Interestingly, MARCH2 downregulated N‐cad‐VEcadTMDJMD but not VE‐cad‐NcadTMDJMD. These results indicate that the amino acid sequence within the transmembrane and juxta‐membrane domains is essential for the selectivity of MARCH2‐induced downregulation. These findings indicate that MARCH ubiquitin ligases exhibit selectivity for different members of the classical cadherin family. These findings suggest that MARCH family ligases may differentially regulate cadherins in vascular endothelial cells to modulate endothelial cell migration and barrier function during development and inflammation.
Modeling Blood‐Testis Barrier Characteristics with Novel CRISPR/Cas9 Functional ENT1 and ENT2 Knockout HeLa Cell Lines
Authors: Miller Siennah R. , Zhang Xiaohong Rebecca , Hau Raymond K. , Jennings Erin Q. , Galligan James J. , Wright Stephen H. , Cherrington Nathan J.
Published: April 2020

Equilibrative nucleoside transporters (ENTs) transport nucleosides across the blood‐testis barrier. ENT1 is located on the basal membrane while ENT2 is located on the apical membrane of human Sertoli cells, creating a transepithelial transport pathway across the blood‐testis barrier. Improving drug disposition to the testis with drugs that use transepithlelial transport pathways may more effectively treat viral infections and cancer within the male genital tract. Here, we characterized uridine transport in two novel ENT cell lines and assessed the impact of nucleoside reverse transcriptase inhibitors (NRTIs) on uridine uptake to gain further insight on ENT function and substrate selectivity. ENT1 or ENT2 functional knockout HeLa cell lines were generated using CRISPR/Cas9 to determine the kinetics and selectivity of each ENT individually. We demonstrate the functional loss of each transporter through quantitation of [3H]uridine uptake in the presence of the ENT specific inhibitor S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR). At low concentrations (100 nM), NBMPR inhibits ENT1; conversely, at high concentrations (100 μM) NBMPR inhibits both ENT1 and ENT2. In ENT2 knockouts, >97% of [3H]uridine uptake was inhibited by 100nM NBMPR, indicating ENT1 function is predominant. In ENT1 knockouts, only 5% of [3H]uridine uptake was inhibited by 100 nM NBMPR and 90% was inhibited by 100 μM NBMPR, indicating ENT2 function is predominant. The Jmax for ENT1 is 23.4 pmol cm−2 min−1 and for ENT2 is 34 pmol cm−2 min−1. The Kt for ENT1 is 10 μM and for ENT2 is 110 μM. We next evaluated the impact of nine NRTIs (abacavir, entecavir, zidovudine, tenofovir, lamivudine, emtricitabine, zalcitabine, didanosine and stavudine) on [3H]uridine uptake, revealing abacavir as the most potent inhibitor of [3H]uridine uptake, with an IC50 of 72 μM for ENT1 and 350 μM for ENT2. Zidovudine had an IC50 of 1.4 mM for ENT1 and 1.2 mM for ENT2. Entecavir had an IC50 of 2.6 mM for ENT1 and 1.08 mM for ENT2. This suggests that abacavir has higher affinity for ENT1, and entecavir has a higher affinity for ENT2. Based on previous results using wild‐type HeLa cells and NBMPR, we believe that abacavir is an inhibitor of the ENTs, entecavir is a substrate, and zidovudine is a low‐affinity substrate. These functional knockout cell lines will allow further screening of therapeutic compounds to establish the substrate selectivity between ENT1 and ENT2, which can aid in the development of future compounds that are able to circumvent the blood‐testis barrier via the ENT1‐ENT2 transepithelial transport pathway.

Support or Funding Information

NIGMS R01GM123643, 5P30ES006694, 2T32ES007091‐36A1
Sequence FEDATHI and TBL (Team Based Learning) improve the learning of human anatomy
Authors: Nunes Bruna Marques Barreto , Neto Josaphat Soares , De Paula Uchôa Francisco Ewerton , Forte Guilherme Aguiar , Pimenta Helder Bindá , Barreto João Erivan Façanha , Teixeira Claudio Silva , Da Silva Ivan Do Nascimento , Cerqueira Gilberto Santos
Published: April 2020

The FEDATHI Sequence is a new teaching methodology designed by researchers from the faculty of Brazilian Education with the aim of improving the teaching and learning process in several areas of knowledge, and its application in the teaching of anatomy requires understanding the educational phenomenon known as mediation. The FEDATHI Sequence is a methodology aimed at improving the pedagogical practice aiming at the proper posture of the teacher in the classroom, whose essence is to contribute for the student to overcome the epistemological and didactic obstacles that occur in the approach of anatomical concepts in the classroom. The main phases of the FEDATHI sequence are: positioning, maturation, solution and proof. Team Basead Learning (TBL) is an active teaching and learning methodology widely used by medical schools in Brazil, which consists of an educational strategy that offers students active learning and can be used with large classes of students divided into small groups. Thus, the objective of this work was to compare learning using two different methodologies: sequence FEDATHI and TBL. A descriptive, cross‐sectional study with a quantitative approach was carried out with 90 students in the discipline of human anatomy. The didactic methodologies sequence Fedathi and TBL were used in the anatomy class of the endocrine and sensory systems. For data collection, a semi‐structured questionnaire with 10 questions was used. For statistical analysis, the Mann‐Whitney test was performed, being considered significant when they presented p <0.05. It was observed that there was no statistically significant difference between learning using FEDATHI and TBL sequences, however when the two methodologies were compared with traditional teaching, both showed statistically significant differences p <0.05. It was found that there were no statistically significant differences between the age of the participants. With regard to methodological preferences, 60% of students stated that the FEDATHI sequence was better for learning, 20% prefer TBL and 20% prefer traditional teaching. It was found that hybrid teaching with diversity of methodologies improves anatomy learning, being an important tool for consolidating anatomical knowledge.
Mutations in GET4 disrupt the transmembrane domain recognition complex pathway
Authors: Tambe Mitali A. , Ng Bobby G. , Shimada Shino , Wolfe Lynne A. , Adams David , Gahl William A. , Malicdan May C.V. , Freeze Hudson H.
Published: April 2020

The transmembrane domain recognition complex (TRC) targets cytoplasmic C‐terminal tail‐anchored (TA) proteins to their respective membranes in the endoplasmic reticulum (ER), Golgi and mitochondria. It is composed of three proteins, GET4, BAG6 and GET5. We identified an individual with compound heterozygous missense changes (p.Arg122His, p.Ile279Met) in GET4 that reduced all three TRC proteins by 70–90% in his fibroblasts, suggesting a possible defect in TA protein targeting. He presented with global developmental delay, intellectual disabilities, seizures, facial dysmorphism and delayed bone age. Indeed, we found the TA protein, syntaxin 5, is poorly targeted to Golgi membranes compared to normal controls. Since GET4 regulates ER to Golgi transport, we hypothesized it would be disrupted in his fibroblasts, and indeed retrograde (but not anterograde) transport is significantly reduced. Despite reduction in these TRC proteins their mRNA levels are unchanged, suggesting increased degradation in patient fibroblasts. Treating fibroblasts with the FDA‐approved proteasome inhibitor Bortezomib (10nM) restored syntaxin 5 localization and nearly normalized the levels of all three TRC proteins. Our study identifies the first individual with GET4 mutations and suggests a potential therapy.

Support or Funding Information

This work was supported by grant R01DK99551 and The Rocket Fund; the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director; and the NHGRI Intramural Research Program of the National Institutes of Health.
So Long Llamas: Structure of Activated Angiotensin Receptor Stabilized by a Synthetic Nanobody
Authors: McMahon Conor , Wingler Laura , Staus Dean , Lefkowitz Robert , Kruse Andrew
Published: April 2020

Nanobodies are single‐domain antibody fragments that are derived from camelids and used extensively as research tools. Despite their usefulness, traditional immunization‐based approaches for generating nanobodies have proven ineffective for producing nanobody binders to some antigen targets. To overcome this, we bypassed the requirement of camelids by creating a synthetic in vitro library of nanobodies. Using this library, we developed a nanobody which stabilizes the active state of a GPCR, angiotensin II type 1 receptor (AT1R). AT1R is a critical regulator of cardiovascular function and our collaborator’s previous nanobody discovery efforts using camelids were unsuccessful. Our synthetic conformational nanobody, Nb.AT110i1, allowed us to determine the crystal structure of active‐state human AT1R bound to an analog of its endogenous hormone angiotensin II. This structure provides detailed insight into hormone binding and may lead to new therapeutics. It also contributes a remarkably complete explanation for receptor activation.
Molecular Mechanisms of P311 on White Adipogenesis
Authors: Badri Kameswara Rao , White Donald A.
Published: April 2020

Background

The obesity epidemic is a constant and chronic detriment to US as well as global health outcomes. Two‐thirds of the US population is either overweight or obese, and obesity can be linked to many chronic diseases, including diabetes mellitus, cardiovascular diseases, stroke and cancer. Diet and exercise remains the primary therapeutic approaches, but still obesity (40% of US adult population) incidence and prevalence continues to rise, prompting the development of new interventions. P311 is an 8 kDa protein shown to induce adipogenesis in an in vitro 3T3‐L1 cell culture model (Nunez et al. 2019). In the current study, we investigated the molecular mechanisms of P311 in white adipocyte biology.

Methods

Both male and female C57BL/6 control mice and P311 knockout (KO) mice were used for this study. Adipocytes were isolated from individual white adipose tissue depots and adipocyte number and size were quantified using LipidTox staining followed by Guava EasyCyte analysis. Additionally adipocyte cellular functions of P311 like apoptosis, lipophagy and adipose tissue macrophage numbers were verified in P311 KOs and compared with wild type control mice. Real‐time PCR, western blot and immunohistology studies were conducted as described by Badri et al. (2013) and Nunez et al. (2019)

Results

P311 was expressed in murine gonadal and subcutaneous white adipose tissue. White adipocytes isolated from 4‐month‐old mice had significantly higher levels of P311 compared to adipocytes isolated from the white subcutaneous adipose tissue of 1‐month‐old mice. However, progenitor cells (stromal vascular fraction) showed no (basal) expression of P311. P311 KO mice showed decreased PPARγ2 expression. P311 KO mice did not differ from wild type mice in subcutaneous or gonadal white adipose tissue weights, but they had fewer and enlarged adipocytes. Further, P311 KOs also showed decreased apoptosis and lipophagy along with decreased adipocyte macrophage numbers and browning of white adipose tissue. In addition, adult human visceral adipose tissue also showed the expression of P311 in adipocytes implicating a translational role for P311 in adipogenesis and obesity.

Conclusions

P311 plays a key role in white adipogenesis and browning of WAT partly through modulating PPARγ2 and also by regulating adipocyte cellular processes including adipocyte apoptosis, lipophagy, browning and macrophage numbers.

Support or Funding Information

• 2019 Medical Student Summer Research and Public Health Experience (Morehouse School of Medicine)

• NIMHD/NIH grant funding through S21MD000101 (to Morehouse School of Medicine)

• U54MD008621 (Hampton University, Hampton, VA)
A QUICK WAY TO DETECT, DISCRIMINATE AND QUANTIFY SIDEROPHORES BY FLUORESCENCE ASSAYS
Authors: Kumar Ashish , Newton Sally , Klebba Phillip
Published: April 2020

Iron is vital for bacteria, as it plays a central role in energy production, intermediate metabolism, DNA synthesis, nitrogen fixation etc. to survive in host system, Bacteria primarily secrete siderophores that specifically chelate iron with high affinity and actively transport this ferric siderophore complex inside cell. Siderophore production and ferric siderophore acquisition are frequently associated with microbial infections. Moreover, pathogenic bacteria obtain iron with TonB dependent ferric siderophore transport systems. For example, the outer membrane protein IutA actively transports the iron complex ferric aerobactin (FeAero), and the inner membrane protein TonB provides the energy for this uptake reaction. I created fluorescent sensors that monitor high affinity binding reactions, and used them to detect, discriminate and quantify ferric siderophores, as either isolated iron complexes or in complex mixture of metabolites and other biochemicals. By introducing site‐directed Cys residues in bacterial iron transporters and modifying them with maleimide fluorophores, we generated living cells that bind but do not transport target compound. By cloning, genetically engineering and fluoresceinating ferric siderophore transporters, we created specific sensors for the native, degraded and glucosylated forms of the catecholate ferric enterobactin, for the hydroximates ferric aerobactin, ferrichrome and ferrioxamine B, for the porphyrins hemin and vitamin B12and so on. When employed in spectroscopic analysis, these constructs sensitively detected respective ferric siderophores or target compounds and measured their concentrations in solutions. Sensitive Assays of Biochemical specificity, affinity, and capacity are valuable for both basic research and drug discovery. The sensors, which we created monitored production of siderophores by the pathogens in blood samples, food samples or clinical samples, each of which manifested a particular profile of iron chelator production.
The nucleus accumbens core and shell sub‐regions differentially regulate conditioned fear expression and extinction
Authors: Dutta Sohini , Beaver Jasmin , Gilman T. Lee , Jasnow Aaron M.
Published: April 2020

The nucleus accumbens (NAcc), consisting of core (NAcC) and shell (NAcS) sub‐regions, has primarily been studied as a locus mediating the effects of drug reward and addiction. This is due to the region’s role of ascribing valence and salience to internal and external cues. However, there is emerging evidence that this region is also involved in regulating aversive responses, but its exact role is not yet clear. Further, the two sub‐regions differ in their connectivity, neurochemical and physiological properties, and each sub‐regions specific involvement in fear processing has not been determined. In our studies, we sought to elucidate the differential role of the two sub‐regions on cued fear expression and extinction. Using adult male C57BL/6 mice, we stereotaxically implanted bilateral cannulae directed at either the NAcC or NAcS. Twenty‐four hours following fear training to an auditory tone, we infused lidocaine, a sodium channel blocker, into the targeted subregion to temporarily inactivate the subregion 5 mins prior to a fear expression test which also served as extinction training. Within the NAcS, fear expression was not influenced by lidocaine infusions, however, when the mice were tested 24 h later for extinction retention, mice that had their shell subregion inactivated exhibited enhanced fear, indicating that this subregion is important for fear extinction consolidation. Mice that had their NAcC inactivated showed an attenuated fear response during expression testing, suggesting that the core sub‐region is necessary for the expression of fear. This effect was not due to alterations in the ability to produce freezing behavior. In addition, there was no effect on extinction consolidation. We next examined whether activation of ERK signaling, downstream of mGluR1, in the nucleus accumbens shell is necessary for extinction consolidation. Thus, we infused the MEK inhibitor, U0126, prior to extinction training and observed a facilitation of extinction consolidation during the retention test. However, with pre‐extinction infusions we also observed within‐session changes in fear behavior during extinction training. To control for this we conducted post‐extinction infusions. Here, mice that were infused with the MEK inhibitor exhibited enhanced freezing during the retention test suggesting poor extinction consolidation and that this pathway is necessary for appropriate extinction consolidation. These findings suggest that the NAc may be considered a critical region involved in regulating both fear expression and its extinction. It’s dual role in appetitive and aversive regulation likely underlies the high co‐morbidity between anxiety disorders and substance use disorders.

Support or Funding Information

This work was supported by funds from grant NIMH1R15MH118705‐01 to Dr. Aaron M Jasnow.
Repeated remote ischemic preconditioning enhances post‐ischemic myocardial STAT5A and STAT3 but not STAT5B to confer cardioprotection in diabetic rats
Authors: Xiaodong YE , Cai Yin , Wang Tingting , Li Xia , Wang Sheng , Wu Qingping , Liu Danyong , Irwin Michael Garnet , Xia Zhengyuan
Published: April 2020

Background

Remote ischemic preconditioning (RIPC) protects the hearts from ischemia/reperfusion (I/R) injury experimentally. In patients undergoing cardiac surgery, RIPC exhibits cardioprotection, while in studies that included diabetic patients, RIPC lost effectiveness in cardioprotection. In order to confer cardioprotection in diabetes, the threshold for RIPC has to be increased. In diabetic rats, repeated RIPC (rRIPC) for three days reduces myocardial infarction. In a recent study that included diabetic patients, rRIPC conferred beneficial effects (Balin M. et al. Cardiovasc Ther. 2019; doi: 10.1155/2019/9592378), but the mechanism is unclear. RIPC when applied well in advance of cardiac surgery conferred cardioptection that was associated increases of both cardiac STAT3 and STAT5. We hypothesized that both STAT5A and STAT5B are needed for rRIPC cardioprotection.

Methods

Eight‐week STZ‐induced diabetic rats received rRIPC (three episodes of 5 min occlusion of the left femoral artery followed by 5 min of reperfusion for 3 consecutive days) before being subjected to myocardial I/R (30 minutes of coronary artery ligation and 2 hours of reperfusion). In vitro, cardiac myoblast H9c2 cell hypoxia/reoxygenation (H/R) injury model was established with 6 hours of hypoxia followed by 12 hours of normoxia reoxygenation. The cells in the conditioning group were exposed to 3 cycles of 10 minutes of alternating nitrogen‐flushed hypoxia and reoxygenation as stimulated RIPC (sRIPC) 24 hours before inducing prolonged H/R.

Results

rRIPC reduced post‐ischemic myocardial infarct size and apoptotic cardiomyocyte death in diabetic rats (p <0.05) that was concomitant with significantly increases of myocardial p‐STAT3, p‐STAT5A, but not p‐STAT5B. In cultured H9C2 cells, sRIPC significantly attenuated the H/R‐induced increases in LDH release and the reduction in cell viability (all p<0.05, sRIPC+H/R vs. H/R). However, sRIPC mediated cellular protection was cancelled respectively by the Janus Kinase 2 Inhibitor AG490, by gene knock‐down of either STAT3 or STAT5A, but not by STAT5B gene knockdown.

Conclusions

Our findings suggest that STAT5A and STAT3 but not STAT5B are critical for repeated RIPC to confer cardioprotection.

Support or Funding Information

This study is supported by Health and Medical Research Fund (05161826) and NSFC grants(81770824 and 81670770)
Histone H1 Remains Bound to the Nucleosome Dyad During TF Binding within a Nucleosome
Authors: Burge Nathaniel L. , Thuma Jenna L. , Poirier Michael G.
Published: April 2020

H1 is a lysine rich histone that binds nucleosomes and alters nucleosome/chromatin dynamics. Chromatin dynamics, such as chromatin compaction and nucleosome unwrapping, are integral for regulation of fundamental cellular processes including transcription, DNA replication, and DNA repair. Nucleosomal DNA in the entry/exit region spontaneously dissociates from the histone octamer allowing transcription factors (TFs) to bind to DNA in the region when the nucleosome is in an unwrapped state. Instead of blocking wrapping, H1 shifts this equilibrium towards a wrapped state which represses TF binding without fully occluding it. Details of this TF binding mechanism remain unknown, including the structural dynamics of H1 domains during TF binding and which domains affect wrapping and TF binding. Using in vitro fluorescence‐based assays, we have determined the structural dynamics of H1 during TF binding to a nucleosome. H1 binds nucleosomes via electrostatic interactions with DNA and is composed of 3 domains: The winged helix domain that binds the nucleosome dyad and linker DNA, and the intrinsically disordered C terminal and N terminal domains that bind linker DNA. Previous work implied that H1 remains bound to a nucleosome during TF binding, but the details of H1 domain locations were unexplored. We have attached fluorophores and fluorophore quenchers to all 3 H1 domains and either the DNA or histone octamer to measure FRET and fluorophore quenching. This allows us to monitor the movement of the H1 domains relative to the DNA and histone octamer during TF binding. Our results indicate that H1 remains bound to the nucleosome dyad during a TF binding event, and the H1 C terminal domain dissociates from the linker DNA to remain at the dyad. These data further understanding of the mechanistic details of H1 suppression of TF binding to nucleosomes. Future experiments will focus on which H1 domains affect DNA wrapping and TF binding to a nucleosome to further understanding of how H1 regulates TF binding and the implications for transcription.

Support or Funding Information

NIH R01‐GM121966

NIH T32‐GM086252

OSU University Fellowship
Sigma‐1 Receptor Agonists Are Renoprotective in Experimental Kidney Transplantation
Authors: Hosszu Adam , Lakat Tamas , Balogh Dora Bianka , Lenart Lilla , Rimaszombati Fruzsina , Saeed Adar , Toth Akos , Hodrea Judit , Wagner Laszlo Jozsef , Szabo Attila Jozsef , Fekete Andrea
Published: April 2020

Introduction and aims

Kidney transplantation (Tx) is associated with better quality of life and reduced costs compared to dialysis, but the shortage in donor organs is a limiting factor. Graft survival is highly dependent on the extent of ischemia/reperfusion injury (IRI) during Tx. We recently described the renoprotective effects of Sigma‐1 receptor (S1R) agonist treatment in renal IRI. Thus, our aim was to develop a preservation solution which minimizes ischemic graft damage in order to improve Tx outcomes and to increase the number of organs suitable for Tx.

Methods

Kidneys of male Wistar rats were perfused and placed in ice cold (i) Custodiol preservation solution; Custodiol containing S1R agonists (ii) fluvoxamine or (iii) SA‐4503 for 2 hours, then autotransplanted and sacrificed 24 hours after reperfusion. Sham‐operated rats served as controls. In a second experiment kidneys of rats as well as wild‐type and S1R knockout mice were perfused and placed in ice cold Custodiol or Custodiol containing various selective S1R agonists for 2/3/8/24 hours and tissue samples were collected.

Results

S1R agonists mitigated renal functional impairment and tubular dilatation following Tx. Expression of early and sensitive tubular injury markers Kim1 and Ngal were markedly less elevated in S1R agonist‐treated kidneys. S1R agonists alleviated renal apoptosis as shown on TUNEL‐stained kidney sections, decreased apoptotic Bax expression, while increased anti‐apoptotic Bcl2 expression. Reduced number of CD45+ leukocytes and reduced inflammatory cytokine (Mcp1, Il1a, Il6, Tnf) expressions confirmed the anti‐inflammatory effect of S1R agonists. All S1R agonists mitigated cold ischemic structural kidney damage at all time points.

Conclusion

The addition of S1R agonists to the preservation solution during Tx improves graft function and alleviates structural damage, thus improving long‐term outcomes. S1R agonists reduce graft injury during cold storage, therefore the number of transplantable donor organs can be increased.

Support or Funding Information

Grants: VKE‐2017‐00006; OTKA PD‐131637; FK‐124491; K‐116928; NN‐114607; FIKP; STIA_18_KF
Characterization of a Human ALDH2 Mutant that Causes Alcohol Flushing in Non‐east Asians
Authors: Rwere Freeborn , White Joseph R. , Chen Che-Hong , Mochly-Rosen Daria , Gross Eric R.
Published: April 2020

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a ~55 kDa protein responsible for the metabolism of the alcohol derived intermediate, acetaldehyde, in addition to a variety of toxic aldehydes including 4‐hydroxynonenal (4HNE) and acrolein. A single nucleotide polymorphism (SNP) of the ALDH2 gene (rs671) is highly prevalent among East Asians with ~540 million people in the world with the ALDH2 genetic variant (ALDH2*2 or E504K) that causes facial flushing and tachycardia after alcohol consumption due inefficient acetaldehyde metabolism. However, anecdotal reports exist of non‐East Asians who flush after alcohol consumption. Therefore, we hypothesize that other dysfunctional human genetic variants exist, besides ALDH2*2, that cause inefficient acetaldehyde metabolism.

To address our hypothesis, after receiving IRB approval, we recruited human volunteers with self‐reported alcohol‐induced facial flushing. Volunteers were genotyped in addition to being subjected to an alcohol challenge (0.25g/kg) where levels of acetaldehyde in the breath were quantified by gas mass spectrometry for the 2 hours after an alcohol challenge. In addition, to support these findings, we carried out site‐directed mutagenesis, expressed, and purified an ALDH2 mutant we identified in humans using a nickel affinity column for histidine tagged protein. We measured enzymatic activity of the ADLH2 mutant using wild type ALDH2 and ALDH2*2 as comparators. All data are presented as mean ± SEM. Statistical analysis was performed by ANOVA and Tukey’s post hoc with significance set at P<0.05.

We discovered a novel non‐East Asian ALDH2 mutation (R114W) that also causes alcohol‐induced facial flushing and acetaldehyde accumulation. Acetaldehyde levels measured in human breath showed that a human heterozygous for ALDH2 R114W had ~30% more acetaldehyde accumulation compared to homozygote wild‐type ALDH2 human subjects. The heart rate of ALDH2 R114W was not affected by alcohol intake, whereas skin temperature increased after alcohol consumption. To confirm these findings in humans, enzymatic activity of the purified proteins showed that E504K and R114W had 49±3 and 603±11 nmol/min/mg protein, respectively compared to 1209±15 nmol/min/mg protein for wild‐type ALDH2 (n=3, p<0.05). When mixed in the 1:1 ratio with wild‐type enzyme to mimic the heterozygous condition, the activity of E504K and R114W increased to 683±6 and 838±16 nmol/min/mg of pure protein, respectively, but still significantly below the activity of the wild‐type ALDH2. The results presented here demonstrate that a newly found ALDH2 variant, R114W, is also responsible for alcohol‐induced acetaldehyde accumulation and facial flushing. In conclusion, individuals carrying this mutation are more potentially also more susceptible to esophageal cancer similar to those heterozygous for the ALDH2*2 genetic variant.

Support or Funding Information

NIGMS HL119522 (ERG) and a Stanford ChEM‐H seed grant (ERG, DMR).
Role of Sucrose Synthase in the Metabolism of Indican
Authors: Inoue Shintaro , Morita Rihito , Minami Yoshiko
Published: April 2020

Indigo,a blue dye, is obtained from the cultivated plant species Polygonum tinctorium. It is derived from indican (indoxyl β‐D‐glucoside), one of the secondary metabolites stored in the vacuole of the leaf cells. In the cells, indican is biosynthesized by the catalysis of indoxyl b‐D‐glucoside synthase (IGS) that uses indoxyl and UDP‐glucose as its substrates. Indoxyl is possibly synthesized from indole by the catalysis of a monooxygenase, such as cytochrome P450 (CYP), whereas, sucrose synthase (SUS) or UDP‐glucose pyrophosphatase catalyzes the synthesis of UDP‐glucose. In the metabolism of indican, except IGS, the related proteins have not been identified to date; therefore, this study aimed to identify the specific proteins involved in the metabolism of the compound indican. Indoxyl is usually a very unstable substrate and is easily oxidized to indigo; due to its instability, it must be allowed to complete its indican synthesis reaction as fast as possible. In the plant secondary metabolism, the ER membrane‐associated CYPs that hydroxylate metabolites are well known. Furthermore, these CYPs may function as a scaffold for a temporary complex, known as metabolon, on the ER membrane. We assumed that the IGS also associates with some ER membrane proteins, because a part of IGS localizes on the microsome. We then performed protein–protein interaction analysis (coimmunoprecipitation and pull‐down assay) and detected some candidate proteins. Some proteins including CYPs and SUS were identified by the MS/MS analysis. We then examined the involvement of SUS in the metabolism of indican by performing protein–protein interaction analysis and cell fractionation using recombinant SUS.

The full‐length cDNA of SUS gene obtained using the RACE‐PCR encodes a soluble cytosol protein composed of 806 amino acids. The recombinant SUS expressed in Escherichia coli was used to perform the experiments, as shown below. Firstly, we examined the intracellular localization of SUS using the cytosol and microsomal fractions prepared from the crude extract of leaf cells. Similar to IGS, SUS was also detected in both the fractions. The interaction between IGS and SUS was examined by coimmunoprecipitation using specific antibodies. We found that both proteins interacted with one another, suggesting that SUS might be a supply source for UDP‐glucose in the reaction involving the synthesis of indican. We are going to further discuss the data required to elucidate the entire indican metabolic pathway and to confirm the presence of its metabolon.
Notch‐1 signaling through MCL‐1 modulates macrophage polarization and immune defense against Mycobacterium avium paratuberculosis infection in autoimmune disease
Authors: Keewan Esra’a , Naser Saleh A.
Published: April 2020

We previously reported that IL‐6 plays a major role in the survival of Mycobacterium avium paratuberculosis (MAP) in infected macrophages. MAP has been associated with Crohn’s disease (CD) and Rheumatoid Arthritis (RA) pathogenesis. The role of Notch‐1 signaling in macrophages during intracellular infection such as MAP is not clearly understood. We hypothesis that IL‐6 expression in macrophages is induced by Notch‐1 signaling which inhibits apoptosis through Myeloid cell leukemia sequence‐1 (MCL‐1), which ultimately induce persistence of chronic infection. In this study, we measured the in vitro effect of MAP infection on Notch‐1 signaling and downstream influence on IL‐6 and MCL‐1. Specifically, we evaluated the role of Notch‐1 signaling in apoptosis and consequent MAP viability in infected macrophages and its effect on macrophage polarization and inflammatory response. Overall, the data showed that MAP infection upregulated Notch‐1, IL‐6, and MCL‐1 by (1.2, 1.8 and 4.2 fold expression, respectively) in activated THP‐1 compared to uninfected macrophages. Interestingly, treatment of MAP‐infected macrophages with IL‐6 showed upregulation of Notch‐1 and MCL‐1, which suggest a role for Notch‐1 and IL‐6 in the upregulation of MCL‐1 and possibly regulation of apoptosis in MAP infected macrophages. Targeting Notch signaling by a gamma‐secretase inhibitor resulted in decreased expression of IL‐6 and MCL‐1 (1.38 ± 0.32 and 0.63 ± 0.034, respectively) in infected macrophages compared to untreated macrophages (2.7 ± 0.25 and 2.0 ± 0.21, respectively). Moreover, gamma‐secretase inhibitor decreased MAP viability by 1.3 log CFU/ml. On the other hand, MAP modulates the macrophage polarization toward M1 macrophage polarization (M1/M2=2.20) compared uninfected macrophages (M1/M2=1.27), whereas the Notch inhibitor promotes M2 and diminish M1 macrophage polarization (M1/M2=0.76). Collectively, targeting Notch‐1 signaling will regulates the inflammatory response and enhances bacterial clearance in MAP infected macrophages. The study provides significant data that support the mechanistic role of Notch‐1 signaling in chronic infection in autoimmune disease.

Support or Funding Information

This work was funded, in part, by the Florida Legislative Grant.
Effect of enhancement of hepatic metabolic and glucose homeostasis by gut microbiome regulation of DP in obese mice
Authors: Lee Jung-Eun , Jung Jeeyoun
Published: April 2020

The traditional medicine DP is generally used to treat obesity‐related disease and plays central role in the improvement of high‐fat and high‐cholesterol‐induced obesity. In order to reveal the systematic mechanisms of DP on obesity‐related disease, we induced the HFHC diet for 12 weeks in C57BL/6 mice and administrated DP for 6 weeks. After that, we performed non‐targeted lipidomics analysis using mass spectrometry and integrated analysis with various clinical marker of liver. The DP prescription altered liver inflammation status and ameliorated insulin resistance (IR) by regulating GLP‐1 secretion. Additionally, the DP alleviated non‐alcoholic fatty liver disease (NAFLD) through reduce of CD1d‐restricted lipid antigen coupled with γδ‐T cells. More importantly, we have found using microbiome analysis that these mechanisms were regulated by gut microbiota which has been changed by DP treatment. Comprehensively, DP is considered to be an effective herbal decoctions for obesity‐induced systemic hepatic inflammation, glucose homeostasis, and hepatic metabolic homeostasis by regulating microbiome. Therefore, due to the potential therapeutic effects of DP, it is expected to be widely used for the treatment of NAFLD in Korean and Chinese medicine.
Cutaneous microvessel circulation and vasomotion responses to local skin heating in women with HIV.
Authors: Wang Dan , Ma Yuchi , Guendsechadze Shirin Claudia Nouri , Yang Xiaoru , Sun Philena , Kalakurthy Samantha , Carter Gabriela H. , Kummar Princy , Kassaye Seble , Wilcox Christopher S.
Published: April 2020

Background

Patients infected with the human immunodeficiency virus (HIV) who are well treated by ART still suffer from many complications of microvascular dysfunction. Our previous ex vivo study reported that subcutaneous microvessels from women living with HIV had severe endothelial dysfunction. However, it is unknown whether the microvascular defect is more widespread. We tested the hypothesis that microvascular endothelial, neural and myogenic functions are impaired in women living with HIV compared to control women (control).

Methods

Women with HIV (n = 35) were compared to matched controls (n = 24). HIV infection was virally suppressed on antiretroviral therapy and both groups were enrolled in the DC Women’s Interagency HIV Study (DC – WIHS). Cutaneous blood flow (CBF) was measured by laser Doppler flowmetry (LDF) during an increase of local skin temperature from 33°C to 42°C. Vasomotion responses were analyzed using a wavelet transform to calculate the power of the frequency intervals associated with endothelial (0.0095 ‐0.012Hz), neural (0.021‐0.05Hz) and myogenic (0.051 ‐0.15Hz) activities of the Laser‐Doppler signal. Data are present as mean ± SEM.

Result

CBF was increased in both groups with LH at 42°C, but the increases of CBF to heat was lower in HIV infected women than controls (13.9 ± 1.1 vs 15.6 ± 1.2 fold, p<0.05). Vasomotion activities were similar between groups at baseline (33°C) and increased in both groups with LH at 42°C, but the increase was less in HIV compared to controls (13.9 ± 1.1 vs 15.6 ± 1.2 fold change, P <0.05). Moreover, compared to the control group, HIV had lower wavelet amplitude during LH in endothelial (100±3.8 vs 149± 15 PU/Hz/min, P<0.05), neural (92 ± 5 vs 132 ±15 PU/Hz/min, P<0.05) and myogenic (46±3 vs 60 ±7 PU/Hz/min, P=NS) frequency intervals.

Conclusion

Women living with HIV have impaired microvascular dysfunction with a complex derangement of microvascular circulatory regulation involving the endothelial, neural and myogenic components. Thus, fully preventing microvascular disease in HIV will require correction of widespread microvascular defects.

Support or Funding Information

National Heart, Lung, and Blood Institute (NHLBI), R01 HL134511
Mitomycin C Induces Immunogenic Cell Death Biomarkers In Human Bladder Tumors
Authors: Sun Yajing , Lu Ze , Chao Fangching , Taylor John A. , Au Jessie L.-S.
Published: April 2020

Introduction

Standard‐of‐care for non‐muscle invasive bladder cancer is complete transurethral tumor resection followed by intravesical therapy with BCG, mitomycin C (MMC), gemcitabine, or other agents. Tumor recurrence is common and occurs in 40% to 80% of patients, with 10–20% progressing to muscle‐invasive disease. Earlier studies have shown selected cytotoxic agents induce immunogenic cell death (ICD) that promotes tumor response to immunotherapy and thereby offers the opportunity of developing synergistic combinations of ICD‐inducing cytotoxics with immune checkpoint blockers. The current qualifications for an ICD‐inducing cytotoxic include induction of DAMPs (danger‐associated molecular patterns) in vitro and vaccination with cytotoxic‐treated tumor cells in immunocompetent mice. Under these criteria, MMC is considered a poor ICD inducer. The present study tested the hypothesis that MMC can induce ICD in a more clinically relevant model, i.e., 3D histocultures of bladder tumors obtained from patients.

Methods

We evaluated the ability of MMC to induce two ICD biomarkers (i.e., ecto‐CRT (calreticulin) and release of HMGB1 from nucleus) and/or dendritic cell (DC) maturation in 3 bladder cancer cell lines with different differentiation status (RT4, T24, and TCCSUP), and 21 MMC‐treated human bladder tumor histocultures including 11 archived paraffin‐embedded tumors and 10 freshly obtained surgical tumor specimens. The methods included immunocytochemical staining flow/immune‐cytometry, immunoblotting, and immunohistochemical staining and quantitative imaging. Co‐cultures of human peripheral blood DC with MMC‐treated tumor cells (48 h) were used to detect DC maturation markers (CD83 and HLA‐DR).

Results

Over 85% of human bladder tumors (7/10 fresh tumors and 11/11 archived tumors) showed ecto‐CRT and/or HMGB1 release upon treatments with MMC at clinically achievable concentrations (1 and 3 μM). Induction of ICD markers by MMC was time‐dependent (peaked at 18 h) and increased with tumor grade (TCCSUP (grade 4) >T24 (grade 3)>RT4 (grade 1) (3 experiments, P<0.01, 2‐way ANOVA). HMGB1 release was increased in three cell lines with a dose‐dependent manner after 48 h MMC treatment (2 experiments, p value<0.05, 2‐way ANOVA). MMC further induced DC maturation in TCCSUP cells but not RT4 cells.

Conclusion

Our results indicate MMC induced ICD biomarkers and/or DC maturation in cultured human tumor cells and histocultures of human bladder tumors; the MMC effects were dependent on time, tumor grade and culture conditions.

Support or Funding Information

Supported in part by Mosier Endowed Chair in Pharmaceutical Sciences at University of Oklahoma, Albert Charitable Trust, 1R01CA163015 from National Cancer Institute, and University of Kansas Cancer Center P30‐CA168524‐03.
Expression, purification and initial characterization of LysE membrane exporter from Mycobacterium tuberculosis: Towards comprehensive functional and structural study
Authors: Georgieva Elka R. , Fanouraki Christina , Borbat Peter P.
Published: April 2020

Mycobacterium tuberculosis (Mtb) causes the very severe disease tuberculosis. To fight Mtb infection, detailed understanding of the pathogen molecular mechanisms in the host is needed. To this end, comprehensive knowledge about Mtb membrane transport system, which is currently not well researched, is a high priority. Our focus is on the Mtb‐encoded L‐lysine exporter (LysE), which removes the excess of metabolically produced basic amino acids from the bacterial cytosol. LysE is a target for inhibition, since its deficiency leads to toxic levels of L‐lysine and suppression of bacterial growth. The most studied H37Rv strain of Mtb encodes two LysEs, but one of then, the Rv1986 protein, is considered to be the primary exporter. Also, Rv1986 expression is increased during the early stages of Mtb infection under conditions of hypoxia, thus, the protein could be a vaccine target. Therefore, we selected to study this protein. To do so, we developed custom protocols for its cloning, expression, purification, and initial characterization. To overcome the low expression levels in E. coli, we fused LysE at its C‐terminus to a combination of FLAG and 8xHistidine tags. Through these tags, we employed a dual‐affinity purification. We purified the protein in lipodiscs made of native E. coli membranes and in detergent. We obtained highly pure protein as confirmed by SDS‐PAGE and Western blotting. Both techniques visualized multiple bands at LysE suggesting protein oligomerization. We further created and purified a single cysteine mutant N175C for spin labeling. Pulse EPR study on it also points to multimeric protein organization. These studies as well as electron microscopy on LysE‐nanogold particle complexes are ongoing. To the best of our knowledge, this is the first highly‐pure LysE produced, which will facilitate further thorough investigations of the functional mechanisms of this protein.

Support or Funding Information

NIAID R03 AI137735