bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential effect of white botton and shiitake mushroom‐fortified diets on liver fat content in C57BL/6 female mice
Authors: Christopher Lawrance , Traore Djibril , Kuvibidila Solo
Published: June 2021

Shiitake (SM) are frequently used in Asia for treatment/prevention of chronic diseases and reduction of blood cholesterol. The mechanisms of reduced blood cholesterol are unknown and may involve impaired synthesis or release to blood. We previously observed that 5% SM‐fortified diet increased IL‐6 plasma levels in DBA mice. Considering IL‐6 plasma levels are usually elevated in fatty liver disease, we sought to assess the effect of SM‐fortified diet on liver fat content. Two month‐old C57BL/6 female mice were fed control, SM or button mushrooms (WBM)‐fortified diets (5%) for 6 weeks (n=9). At euthanasia, livers were removed and stained with Hematoxyline & Eosin. Oil O Red, PAS staining and Trichrome staining were done to demonstrate the presence of fat droplet, glycogen content and fibrosis respectively. SM and WBM had no effect on liver weights. Compared to WBM and control groups, mice fed the SM‐fortified diet showed significant increased number of vacuolated hepatocytes suggesting fatty liver (p<0.05). Oil O red staining confirmed the presence of fat droplets. SM did not induce fibrosis or affect glycogen content. WBM increased glycogen but not fat content. Data suggest that one of the mechanisms by which SM decrease blood lipid is likely by blocking liver fat release. The long term health implication of increased liver fat by SM requires further investigation.

Grant Funding Source: Mushroom Council; Oklahoma State University
Calorie restriction increases fatty acid synthesis and whole body fat oxidation rates
Authors: Bruss Matthew Douglas , Khambatta Cyrus F , Aggarwal Ishita , Hellerstein Marc K
Published: June 2021

Calorie restriction (CR) increases longevity and retards the development of many chronic diseases, but the underlying metabolic signals are poorly understood. Increased fatty acid (FA) oxidation and reduced FA synthesis have been hypothesized to be important metabolic adaptations to CR. However at steady state, FA oxidation must match FA intake plus synthesis; FA intake is low, not high, during CR. It is therefore not clear how FA dynamics are altered during CR. Accordingly, we measured food intake patterns, whole‐body fuel selection, endogenous FA synthesis and gene expression in mice on CR. Within two days of starting CR, a shift occurred to a cyclic pattern of whole‐body FA metabolism, with an initial phase of elevated endogenous FA synthesis (respiratory exchange ratio [RER]>1.10, lasting 4–6 hours after food provision), followed by a prolonged phase of FA oxidation (RER=0.70, lasting 18–20 hours). CR mice oxidized four times as much fat per day as ad libitum fed (AL) controls (367 vs 97 mg/d) despite reduced fat intake. This increase in FA oxidation was balanced by a 3‐fold increase in adipose tissue FA synthesis. We conclude that CR induces a surprising metabolic pattern characterized by periods of elevated FA synthesis alternating with periods of FA oxidation disproportionate to dietary FA intake. This pattern may have implications for understanding the reduced oxidative damage and disease risk in CR animals.

Grant Funding Source: NIH
Sodium content of fermented soy foods in the USA
Authors: Daily Jim
Published: June 2021

The sodium content of fermented soy foods is quite high on a weight basis. However, fermented soy foods are typically used as seasonings and soup bases, and are not consumed alone. It is possible that if fermented soy foods are the sole source of added salt, that fermented soy foods may not be excessively high in sodium content. A survey of commonly consumed soy foods found that there is a wide variation in the salt content common foods made with fermented soy products and that the foods may indeed be high in sodium, but they may also have a lower sodium content that their Western counterparts. Therefore, fermented soy foods are not necessarily high in sodium and can be used to prepare healthful foods.
Potential Role of Toll‐like Receptor 4 in Obesity‐Induced Adipose Tissue Remodeling
Authors: Adapala Venkata Jitendra Kumar , Adedokun Sunday Adetayo , Adeola Olayiwola , Ward Meliza G , Ajuwon Kolapo Matthew
Published: June 2021

Obesity is a state of chronic inflammation which contributes to insulin resistance. Toll‐like receptor (TLR) 4 is involved in adipose tissue (AT) inflammation, but its role in the regulation of extracellular matrix (ECM) genes in AT is unknown. We conducted a study on the effect of high fat diet (HFD) and acute TLR4 activation with lipopolysaccharide (LPS) on ECM gene expression. We also investigated the impact of chronic TLR4 activation by HFD on ECM genes in a wild type (WT) C3H/HeOuJ and TLR4 deficient (KO) C3H/HeJ mice. In the subcutaneous (SC) AT, apart from induction of inflammatory cytokines, there was no effect of LPS challenge on the expression of ECM genes. However, HFD results in significant induction of COL1A1 and COL6A1 (P < 0.05). In the epididymal depot, LPS challenge results in the upregulation of biglycan but a down regulation of lumican and COL6A1 (P < 0.05). Higher expression of biglycan, lumican and COL1A1 (P < 0.05) was induced by HFD. The WT mice had higher ECM gene expression than the KO. Limited effect of HFD was observed in the WT. Significant induction of ECM genes by HFD was observed in both the epididymal and the SC depot of the KO mice. Although HFD leads to induction of transcripts of ECM genes, our results suggest a limited role for TLR4 activation in HFD‐induced AT remodeling. However, the WT has a higher basal expression of the ECM mRNA. This indicates a non‐dietary connection to ECM gene regulation by TLR4.

Grant Funding Source: Purdue University
C‐reactive protein (CRP) exclusion cutoffs and serum zinc, ferritin, and copper status in children: a cross sectional study among 6–11 y healthy children in Guatemala
Authors: Bui Vinh Quang , Ramakrishnan Usha , Stein Aryeh D. , Flores‐Ayala Rafael C. , Ramirez‐Zea Manuel , Villalpando Salvador , Martorell Reynaldo , DiGirolamo Ann M.
Published: June 2021

Infections can affect markers of micronutrient status such as serum vitamin A and ferritin but less is known about effects on other micronutrients. C‐Reactive Protein (CRP), a marker of acute inflammation, has been used to correct for the effects of infection but the appropriate cutoffs for excluding subjects are unclear. We assessed the prevalence of low zinc, iron and copper status in children using a range of CRP cutoffs. Serum CRP (by laser nephelometry), Zn, ferritin, and Cu were measured in 720 healthy 6–11 y Guatemalan children. The proportion of samples with CRP ≤0.5, 1, 3, 5, and 10mg/L was 44.8%, 65.5%, 84.9%, 92.5% and 97.6%, respectively. The prevalence of serum Zn <65μg/dL, ferritin <15μg/dL, and Cu <90μg/dL was 21.7%, 2.3%, and 23.8%, respectively. Serum CRP was correlated with serum ferritin (r=0.24, p<0.01) and Cu (r=0.29, p<0.01), but not Zn (r=0.04, p=0.3). Subjects with CRP levels > 1, 3, and 5 mg/L had a reduced risk of low iron and Cu status (all p<0.05). For example, the prevalence of Cu<90μg/dL was 23.8% and 28.7% with and without exclusion of subjects with CRP >1mg/L, respectively. Markers of iron and copper status, but not of zinc, were associated with inflammation.

Funding: National Institute of Health (R01 MH067981).

Grant Funding Source: NIH R01 MH067981
Development and use of a computer model to predict and compare how changing macronutrient composition influences changes in weight and body composition
Authors: Sakita Saori , Davidson Robert T
Published: June 2021

There are many different approaches to weight loss diets. The macronutrient composition of these can vary tremendously for any given energy level. How the body processes and utilizes the varying amounts of macronutrients can have a significant effect with energy balance on the composition of the resultant weight change. We hypothesized that a physiologically‐based mathematical model could be constructed which would accurately describe the changes in body composition seen with a change in energy level or dietary macronutrient composition. With the STELLA modeling software and using dose‐response relationships between macronutrient intake and energy oxidation as identified in the published scientific literature, we developed a multi‐compartment model which was then validated using separately available data. Using this model, weight changes and body composition changes were typically within 10% of reported values when subjects were either at energy balance or underfed (i.e. low calorie diet). This model may be used to identify or compare expected changes in body composition due to macronutrient composition or changes in macronutrient composition.
Epigenetic Placental Programming of Preeclampsia
Authors: Uthus Eric O. , Anderson Cindy M.
Published: June 2021

Preeclampsia (PE) affects 8–10% of women in the US and long‐term consequences include development of maternal hypertension and hypertension in offspring. As methylation patterns are established during fetal life, we focused on epigenetic alterations as a plausible explanation of the heritable development of hypertension resulting from maternal PE. In this proof‐of‐principle study we determined genome‐wide CpG DNA methylation patterns in placental tissue from women with normotensive pregnancy and PE (n=3/group) by using 2.1M microarrays (NimbleGen). These arrays tile 7000 bp upstream and 3000 bp downstream a promoter in 100 bp segments and cover 28000 CpG islands. Criteria for gene selection included methylation +/−1000 bp of the respective gene's transcription start site. We identified 163 genes methylated only in PE and uploaded them to DAVID (http://david.abcc.ncifcrf.gov) where we used a classification stringency of high to determine functional annotation clusters. Cluster 1had an enrichment score of 5.59; this cluster contains the GO Terms of anatomical structure formation, angiogenesis, blood vessel development, vasculature development, and blood vessel morphogenesis. There are 12 genes total in this cluster 10 of which are common to all 5 GO Terms. This work suggests that an epigenetic mechanism may be involved in PE.

Robert Wood Johnson Nurse Faculty Scholar, Sigma Theta Tau International, ARS
PKA prevents mitochondrial pathology induced by loss of PINK1 function
Authors: Dagda Ruben K. , Cherra Salvatore J , Strack Stefan , Green Steven , Chu Charleen T.
Published: June 2021

PTEN‐induced kinase 1 (PINK1) and mitochondrial PKA targeted to mitochondria via AKAP121 are two ser/thr kinases that regulate neuronal survival and mitochondrial function. We have previously shown that stable loss of PINK1 leads to increased generation of mitochondrial ROS, fission, and protective mitophagy (Dagda et al, 2009). Given that these two protective ser/thr kinases have the ability to localize to mitochondria, we hypothesized that PKA and PINK1 regulate common mitochondrial pathways in neurons.

We transiently expressed multiple GFP fusion constructs of PKA in stable PINK1 knockdown SH‐SY5Y cell lines. Confocal analysis of transfected cells showed that treating cells with analogues of cyclic AMP or transient expression of mitochondrially targeted PKA but not of untargeted GFP, reverses mitochondrial fission induced by stable loss of PINK1. Moreover, cell survival assays showed that transient expression of cytosolic PKA or that cyclic AMP treatment significantly increased survival against cell death induced by stable loss of PINK1 in SH‐SY5Y cells or cell death induced by transient expression of PINK1 mutants in primary cortical neurons. Overall, our studies support a PKA‐PINK1 signaling pathway to reverse mitochondrial fission and increase survival of PINK1 deficient cells.

This study was supported by NIH (AG026389 and DC009120, CTC)and RKD was supported by F32 AG030821.
Potential hepatoprotective role for CXC Chemokine Receptor‐1 after Liver Ischemia/Reperfusion in Mice
Authors: Clarke Callisia N , Sakai Nozomu , Tevar Amit D , Edwards Michael J , Lentsch Alex B
Published: June 2021

BACKGROUND

CXC chemokines and their receptors, CXCR1 and CXCR2, have been implicated as key mediators of inflammation following hepatic I/R. We have recently shown that signaling through CXCR2 is detrimental to liver recovery and regeneration after hepatic I/R. However, the role of CXCR1 signaling is unknown. Here we investigated the function of CXCR1 after liver I/R.

METHODS

Male wild‐type and CXCR2−/− mice underwent partial hepatic ischemia for 90 minutes and were reperfused up to 96 hours. Sham operations used the same protocol without vascular occlusion. Repertaxin (CXCR1 and 2 antagonist) was used to inhibit CXCR1 in CXCR2−/− mice.

RESULTS

Liver CXCR1 expression was increased in all mice after 24 hrs of reperfusion and was maximal after 96 hrs of reperfusion. Expression was hepatocyte‐specific by Western blot and immunohistochemistry. CXCR1 signaling blockade with repertaxin in CXCR2−/− mice at the time of reperfusion showed no difference in hepatocellular injury or inflammation when compared to control. However, when repertaxin was administered 24 hrs post‐reperfusion there was significant increase in hepatocellular injury and delayed recovery. There was no difference in hepatocyte proliferation between control‐ and repertaxin‐treated mice.

CONCLUSION

CXCR1 has divergent effects from CXCR2 and may have hepatoprotective effects during liver recovery and regeneration after I/R.
β‐Catenin and PDGFRα signaling: Two Divergent mechanisms of hepatocellular carcinoma
Authors: Zhang Xufeng , Monga Satdarshan P
Published: June 2021

We have identified accelerated DEN‐induced hepatocarcinogenesis in conditional liver‐specific β‐catenin knockout (β‐cat KO) mice as compared to littermate controls. The susceptibility tumorigenesis appeared to be the result of PDGFRα‐driven PI3K/Akt pathway activation. To investigate the significance of this inverse relationship between β‐catenin and PDGFRα; signaling, we conducted further studies in hepatoma cell lines and hepatocellular carcinoma (HCC) patient samples. Hep3B and HepG2 cells transfected with either CTNNB1 siRNA or antisense reduced β‐catenin levels along with concomitant increase in PDGFRα levels when compared to respective controls. Treatment with lithium chloride or transfection of Hep3B cells with constitutively active FLAG‐Δβ‐catenin expression plasmid, led to a notable decrease of PDGFRα. Next, we asked if this inverse relation of β‐catenin and PDGFRα is evident in HCC patients. In the 23 frozen and 24 paraffin samples, we identified a significant inverse relationship of β‐catenin and PDGFRα; protein levels. Based on these studies it appears that activation of β‐catenin suppresses PDGFRα through yet unidentified mechanisms. On the other hand activation of PDGFRα appears to occur in HCC patients without β‐catenin mutations. Hence, we report the identification of PDGFRα and β‐catenin as two divergent mechanisms of HCC.
Detecting phenotypical subtypes of breast cancer with multiplexed immunohistochemistry
Authors: Mansfield James , Hoyt Clifford
Published: June 2021

Treatment for breast cancer has benefited significantly from advances in molecular biology. IHC tests for protein receptors ER, PR, and Her2 have lead to a new patient classification system. Traditional approaches to assessing multiple proteins use serial sections, staining for one protein per serial section. Multiple proteins can be assessed in the same tissue section, determining phenotype on a per‐cell basis, possibly revealing significant subtypes and leading to more targeted and more effective treatments and therapies.

Multispectral imaging (MSI) was performed on two sets of a 712‐core TMA (356 patients, in duplicate). One set was stained for ER and ki67, the second for ER, PR and Her2 (both plus counterstain). IHC signals were spectrally unmixed from each other and counterstain. Machine‐learning‐based automated image analysis was performed to locate cancer cells, segment subcellular compartments, and extract IHC signals on a per‐cell basis. Per‐cell co‐expression subtypes were detected using flow‐cytometry data analysis software. Percent double and triple positivity were determined, revealing subtypes. Correlation between subtypes and clinical outcomes will be the topic of future publications.

MSI and analysis software, coupled with flow‐cytometry analysis tools, can be used to reveal molecular subtypes, which may lead to new targeted strategies for breast cancer research and clinical care.
Expression Profiles of Metastasis‐Related Genes in a Cohort of Childhood and Adult Adrenocortical Tumors
Authors: Lin Chin Jia , Lerario Antonio Marcondes , Fragoso Maria Candida , Mendonca Berenice Bilharinho
Published: June 2021

Backgroud

Adrenocortical carcinoma (ACC) is a rare neoplasm with a poor prognosis. Although molecular studies have uncovered many aspects of ACC tumorigenesis, little is known about molecular pathways involved in metastatic spread. The objective of our study is to analyze the expression profile of metastasis‐related genes in a cohort of metastatic and non‐metastatic adrenocortical tumors in order to identify genes involved in the metastatic spread.

Methods

The expression profiles of 27 adrenocortical tumors from 15 adults (8 ACC and 7 adenomas) and from 12 children (5 metastatic and 7 non‐metastatic) were evaluated by an array of 113 genes known to be involved in human metastasis.

Results

The comparison of adult adenoma and ACC identified 13 differentially expressed genes. In the pediatric group, 6 genes were found to be differentially expressed between metastatic and non‐metastatic tumors. Most of the differentially expressed genes are involved in processes of cell cycle control and extracellular matrix remodeling.

Conclusion

the expression profiles of metastasis‐relatad genes identified important aspects of involved in metastatic spread of adrenocortical tumors. Distinctive patterns of gene expression between metastatic and non‐metastatic tumors may be useful in prognosis prediction.
O‐Glycosylation sites on CD44v6 modulate PMN transepithelial migration
Authors: Brazil Jennifer Catherine , Lee Winston , Parkos Charles A , Louis Nancy A
Published: June 2021

The migration of large numbers of neutrophils (PMN) across the apical epithelial surface is a histopathological hallmark of many mucosal inflammatory diseases. However the precise signals governing this process remain poorly defined. Previous studies from our lab have identified an antibody‐GM35 that binds to an apical epithelial ligand and blocks PMN transepithelial migration. Preliminary data identified the GM35 antigen as a v6 exon‐containing variant form of the glycoprotein CD44. In the current study, immunoblotting and confocal microscopic analysis of protein expression and localization in T84 cells confirmed GM35 binding to CD44v6 and further demonstrated binding of GM35 to apically expressed CD44v6. Gene silencing experiments confirmed the identification of the GM35 antigen as epithelial CD44 and demonstrated for the first time a direct functional effect for CD44 in GM35 function. Further work demonstrates that both GM35 ligand binding and the GM35 mediated decrease in PMN detachment/migration are blocked by specific inhibition of O‐Glycosylation. These studies identify epithelial CD44v6 as an important mediator of PMN clearance and suggest that O‐Glycosylation sites on this protein may serve as potential targets for therapeutic intervention. This work is supported by R01 funding (Parkos), an NIH Career Development Award (Louis) and an Emory University Research Council seed grant.
Polio Virus Receptor and DNAM1 functionally interact in Transendothelial Migration at a step between PECAM and CD99
Authors: Sullivan David P , Seidman Michael A , Muller William A
Published: June 2021

The movement of leukocytes across the endothelium (referred to as Transendothelial Migration or TEM) is a critical step in the inflammatory process and known to be mediated by PECAM and CD99. Recently Reymond and coworkers showed that Polio Virus Receptor (PVR) on endothelial cells and DNAX‐associated molecule 1 (DNAM1) on monocytes functionally interact to facilitate TEM (J Exp Med, 2004, 199, 1331–41). Having already established that PECAM and CD99 function sequentially in TEM, we sought to determine where PVR and DNAM1 function relative to the activities of PECAM and CD99. We assayed TEM using human endothelial cells and monocytes that had been differentially and sequentially treated with antibodies directed against PVR, DNAM1, PECAM or CD99 that reversibly block TEM. Our findings indicate that endothelial PVR and monocyte DNAM1 interact at a step in between PECAM and CD99. Interestingly, endothelial PVR is protected from extracellular reagents at 4°C, a finding that is consistent with the functional pool of PVR being localized to the lateral border recycling compartment (LBRC) in a fashion similar to PECAM and CD99. This research was supported by F32 AI084454 to DPS and RO1 HL046849 to WAM.
Primary role for human neutrophil FcγRIIA and C5aR in the development of inflammatory rheumatoid arthritis
Authors: Tsuboi Naotake , Ernandez Thomas , Koehl Joerg , Lee David M , Mayadas Tanya N
Published: June 2021

Deposition of antigen‐antibody complexes in tissues is a hallmark of autoimmune diseases such as rheumatoid arthritis. Mice lacking FcγRs for complexed IgG (γ‐chain deficient, γ−/−) or complement components are protected from developing arthritis and mast cells and vascular permeability are required for disease. However, these studies may not fully reflect human inflammation as mouse and human FcγRs structurally differ. Here we show that neutrophil‐selective transgenic expression of the uniquely human FcγRIIA in γ−/− mice was sufficient to restore susceptibility to K/BxN serum induced arthritis. FcγRIIAtg/γ−/− mice exhibited robust neutrophil accumulation, ankle swelling and bone erosion while γ−/− mice remained resistant. Arthritis in FcγRIIAtg/γ−/− was observed in the absence of joint edema and was also evident in the context of mast cell deficiency. Thus mast cells and joint permeability were not essential for disease development. C5aR antagonist or C3 depletion significantly reduced disease indices in transgenic animals. In vitro, complement C5a stimulation of neutrophils enhanced FcγRIIA mediated binding of complexed IgG. Thus human neutrophil FcγRIIA coupled with C5aR are sufficient to promote joint injury in a model that has relevance for human disease. In addition, our results provide a possible mechanism of cross‐talk between FcγRs and complement, two important immune effector pathways.
Lack of TGF‐beta signaling in dendritic cells leads to systemic autoimmunity
Authors: Ramalingam Rajalakshmy , Larmonier Claire B , Bommireddy Ramireddy , Ghishan Fayez K , Kiela Pawel R
Published: June 2021

Objective

TGF‐β is an immunoregulatory cytokine that has a pivotal function in maintenance of tolerance via lymphocyte proliferation, differentiation and survival. Defects in TGF‐β1 expression or its signaling in T cells correlate with the onset of several autoimmune diseases. However, the early effects of the cytokine on the innate component of the immune system, particularly the dendritic cells (DCs) which play an equally important role in development of immune tolerance, are not well documented.

Methods

To address the role of TGF‐β signaling in DCs, we have generated two lines of conditional knockout mice with TGF‐β receptor deleted specifically in DCs using Cre‐Lox recombination system.

Results

Preliminary observations of DC‐TGFβRII knock‐out mice indicate development of systemic autoimmunity, spontaneous DC and T cell activation and premature death. The TGFβRII‐deficient DCs also demonstrated an impaired ability to drive tolerogenic reponses and in vitro conversion of regulatory T cells.

Conclusions

The phenotype of this novel mouse model clearly indicates the importance of TGF‐β signaling in DCs in the maintenance of immune homeostasis and prevention of autoimmunity and provides an opportunity to study the pathogenesis of complex disorders such as autoimmune gastritis, hepatitis and inflammatory bowel diseases.
Nutrition, Epigenetics and Complex Birth Defects
Authors: Finnell Richard
Published: June 2021

Birth defects are a global problem that affects ~6% of births worldwide. In the U.S., birth defects are the leading cause of pediatric hospitalizations, medical expenditures, and death in the first year of life. Globally, at least 3.3 million children <5 years die each year because of a birth defect. Indeed, birth defects are one of, if not the leading healthcare concern for the youngest members of society. Neural tube defects (NTDs) are the 2nd most common of all human birth defects, annually affecting approximately 324,000 births worldwide and 3,000 pregnancies in the U.S.

We have explored the potential role of environmental factors that alter maternal epigenetic homeostasis and contributes to birth defects. We believe that environmental exposures or nutritional status can impact production of reactive oxidative molecules, the availability of methyl groups, methyltransferase activity, inflammation and endocrine disruption which all have epigenetic implications during the process of neural tube closure. Such exposures may modify multiple biological processes that affect epigenetic mechanisms, including DNA methylation, histone codes, and miRNA expression. These changes may, in turn, modify chromatin organization and condensation, gene expression, and adversely affect pathogenetic processes in the developing embryo. Such factors may interact and act synergistically, such as a nutritional folate deficiency may create altered immune responses that compromise neural tube closure. An in depth exploration of the epigenetic relationship between environmental toxicants, nutritional status and the developmental impact on the embryo will be the focus of this presentation.

Supported by NIH grants DE016315 and NS050249
Development of gender‐specific cardiac hypertrophy: role of the nitric oxide synthase system
Authors: Wong Philip G , Brander Emily PA , Tse M Yat , Armstrong David WJ , Pang Stephen C.
Published: June 2021

Introduction

Gender‐specific differences in hormonal regulation may influence susceptibility to cardiac hypertrophy (CH). The nitric oxide synthase (NOS) and natriuretic peptide (NP) systems are homeostatic regulators of blood pressure; ablation of either system increases predisposition to hypertension and CH.

Objective

To elucidate how the NOS and NP systems work in concert, as well as the impact of estrogen on these systems using the atrial natriuretic peptide gene‐disrupted (ANP−/−) mouse model.

Methods

At 13–16 weeks, female ANP+/+ and ANP−/− mice were euthanized during either the estrus (high estrogen) or proestrus (low estrogen) phases of their estrus cycle. Organs were harvested and left ventricle tissue gene expression of NOS system components were assessed using real‐time quantitative RT‐PCR.

Results

Female ANP+/+ at estrus had a decreased expression of iNOS, eNOS, sGCα1 and sGCβ1 as compared to female ANP+/+ at proestrus. Female ANP−/− at estrus had an increased expression of iNOS, eNOS, sGCα1 and sGCβ1 as compared to female ANP−/− at proestrus.

Conclusions

High estrogen levels led to a downregulation of the NOS system in ANP+/+ mice, and an upregulation of the NOS system in ANP−/− mice. Whether alteration in NOS system gene expression is a result of hypertension or reduction in circulating ANP levels remains to be determined. (Support from the Heart & Stroke Foundation of Ontario)

Grant Funding Source: Heart and Stroke Foundation of Ontario
Temporospatial expression of reactive nitroxidative species in supraspinal regions during the induction and maintenance of central sensitization
Authors: Little Joshua W , Salvemini Daniela
Published: June 2021

Reactive nitroxidative species are implicated in the development and maintenance of central sensitization in pain states. The purpose of this pilot study was to describe the temporospatial and cellular expressions of the neuronal nitric oxide synthase (nNOS) isoform in supraspinal regions during pain states and therefore determine if these sites are acceptable for future investigations of related nitroxidative species. An inflammatory pain model was used to evaluate changes in nNOS expression in the rostral ventromedial medulla (RVM) and periaqueductal gray (PAG), sites that modulate nociception. Eight (n=8) Sprague Dawley rats received subcutaneous hindpaw injections of either carrageenan or saline and were evaluated for hyperalgesia and immunofluorescence. Our findings revealed a bilateral increase in nNOS positive neurons before the onset of thermal hyperalgesia (3 hours post carrageenan, n=2) compared to the saline group in the RVM (Fold change: Ipsilateral: 3; Contralateral: 3.5) and dorsolateral PAG (Fold change: Ipsilateral: 1.7; Contralateral: 1.7). These changes persisted during hyperalgesia (6 hours post carrageenan, n=2). Our preliminary results lead us to hypothesize that formation of nitroxidative species at supraspinal sites, specifically the RVM and PAG, may contribute to central sensitization. Support: Saint Louis University Center for Anatomical Science and Education.
Maximizing Team Mentoring for Empowering Success in Multidisciplinary Research and Career Development
Authors: Lakoski Joan Marie
Published: June 2021

The integration of basic, clinical and translational research has required new approaches to complex problem solving in neurosciences and other discipline. Often colleagues and trainees with very diverse expertise are needed to come together and share their unique vantage points to develop unique solutions to complex questions – what is now known as “team science”. Yet few basic and clinical investigators have been prepared in their training to work in this collaborative manner – and one that will be essential toward a wide distribution of new knowledge and improved clinical practice via translational research approaches. With support from the NIH Roadmap Initiative and the Clinical Science and Translational Award at the Clinical and Translational Science Institute at the University of Pittsburgh, we have developed a comprehensive program for mentoring trainees, including graduate students, postdocs, clinical fellows and junior faculty to help maximize their unique group of mentors who bring the necessary multidisciplinary perspectives to the research investigation at hand. This presentation will provide an overview of some of the elements that contribute to this new model of preparing individuals for success with team mentoring including 1) definitions of the roles of mentee and mentors, 2) the use of annual contracts to affirm commitment to the process, 3) the approaches and strategies used to train both mentees and mentors to ensure a successful launch of a new team, 4) approaches for monitoring, advising, adjusting and/or terminating mentoring relationships, if needed, and 5) strategies to keep the process focused on the mentee's career goals in clinical and translational research – typically a unique path that will differ from the path of the more experienced mentors.
Cytokines in Alzheimer pathogenesis
Authors: Griffin Sue T.
Published: June 2021

Our discovery that overexpression of cytokines such as interleukin‐1 (IL‐1) and S100B accompanies the glial activation, noted by Alzheimer himself, led to our proposed cytokine hypothesis to explain the progressive nature of the clinical and neuropathological changes of Alzheimer's disease (AD). This hypothesis states that in response to neuronal insults of known and unknown origin, e.g., genetic inheritance and environmental factors, microglia and astrocytes are activated and release excess IL‐1 and S100B, respectively. As these glial events occur in fetuses and neonates with Down's syndrome (DS) decades before the inevitable neuropathological changes of AD in DS, glial changes must precede rather than follow AD‐related neuropathologies such as the neuritic Abeta plaques diagnostic of AD. As IL‐1 induces neuronal expression of Abeta precursor protein (APP) and astrocytic expression of S100B in vivo, the excess IL‐1 and S100B in AD and DS likely act as drivers of Abeta deposition and dystrophic neurite proliferation necessary for formation of neuritic Abeta plaques. In this way chronic excesses of IL‐1 and S100B stress neurons to relentless production of APP and nonsensical neurite growth, creating a cycle of neuronal dysfunction and loss and further glial activation and cytokine overexpression. Support: NIA AG12411 and the Donald W. Reynolds Foundation
Regulation of phosphatidic acid phosphatase
Authors: Carman George M
Published: June 2021

Phosphatidic acid phosphatase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) to yield diacylglycerol (DAG) and Pi. The DAG generated in the reaction is used for the synthesis of triacylglycerol (TAG), and for the synthesis of phosphatidylethanolamine and phosphatidylcholine via the Kennedy pathway. By the nature of its reaction, PAP also controls the cellular content of PA, which is the precursor of phospholipids synthesized via the CDP‐DAG pathway. PA is also a signaling molecule that triggers phospholipid synthesis gene expression, membrane expansion, vesicular trafficking, secretion, and endocytosis. Biochemical and genetic studies to establish the roles of PAP in lipid metabolism became possible by the discoveries of the PAP‐encoding genes from the yeast Saccharomyces cerevisiae (e.g., PAH1) and mammals (e.g., LPIN1). The importance PAP in lipid metabolism is exemplified by its mutant phenotypes. In yeast, PAP mutants exhibit defects in the transcriptional regulation of phospholipid synthesis genes, the anomalous expansion of the nuclear/ER membrane, and a reduction in TAG content in stationary phase cells. Studies with mice and humans have shown that genetic defects in lipin 1 and lipin 2 are manifested in several metabolic diseases that include lipodystrophy, obesity, and peripheral neuropathy.
Combination Therapies to Treat Hypertensive Patients with CKD
Authors: Wright Jackson T.
Published: June 2021

The need for combination therapy in hypertensive patients with chronic kidney disease (CKD) should require no elaboration. This is particularly true when considering the need to achieve the lower blood pressure goals (<130/80 mmHg) recommended by most guidelines for patients with CKD. Mean number of antihypertensives required to achieve this blood pressure goal in renal outcome trials is between 3.5 and 4 agents. However, the benefit of inhibitors of renin angiotensin system (RAS) shown in multiple renal outcome trials makes their inclusion in the antihypertensive regimen in hypertensive patients with CKD obvious. Adequate diuresis is required to achieve blood pressure goal. In addition, in the ALLHAT trial, the thiazide‐type diuretic, chlorthalidone, was found to be at least as effective as the ACE inhibitor, lisinopril, in slowing progression of renal disease regardless of diabetic status (including the metabolic syndrome) and stage of kidney disease. Although calcium channel blockers in renal outcome trials have been shown to be less effective than RAS inhibitors in slowing the progression of CKD, their efficacy in blood pressure lowering generally requires their inclusion in the regimen. In addition, no loss of renal protection is seen when they are combined with RAS inhibitors. In those with estimated GFR < 40 ml/min/1.73m2, loop diuretics should replace thiazide‐type diuretics in antihypertensive drug combinations, though indapamide and metolazone may retain some efficacy if volume status is not an issue. While some earlier studies suggested additive benefit of multiple RAS inhibitors on renal outcomes, recent large renal outcome trials have failed to confirm this benefit.
The role of ATM signalling in DNA double strand break repair.
Authors: Jeggo Penelope Ann , Goodarzi Aaron A , Shibata Atsushi , Lobrich Markus
Published: June 2021

DNA double strand breaks (DSBs) induced by ionizing radiation are repaired with fast and slow kinetics. The slow component of DSB repair represents the repair of DSBs located close to or within regions of heterochromatin (HC), demonstrating that HC provides a barrier to DSB repair. These DSBs specifically require ATM, the nuclease, Artemis, and mediator proteins involved in ATM signaling. ATM phosphorylates the heterochromatic building factor, Kap1, and phosphorylated Kap1 changes its chromatin binding capacity. The available evidence suggests that ATM modifies Kap1 to promote chromatin relaxation in the vicinity of the DSB to allow DSB repair. The mediator protein, 53BP1, is dispensable for pan nuclear Kap1 phosphorylation but is specifically required for p‐Kap1 foci formation, which specifically occurs at DSBs located at HC regions. We propose that this allows concentrated changes in Kap1 at the site of the DSB. Following these chromatin modifications, the DSBs are repaired by a process involving the DNA non‐homologous end‐joining proteins in G1 phase but by homologous recombination in G2 phase.
Active Site Purines and Catalysis of RNA Self‐cleavage
Authors: Fedor Martha J. , Liu Lu , Cottrell Joseph W. , Scott Lincoln G.
Published: June 2021

The hairpin ribozyme mediates catalysis through nucleotide functional groups, without metal cation cofactors. The positions of G8 and A38 in the ribozyme active site resemble the orientation of two histidines in the active site of ribonuclease A, leading to the model that G8 and A38 mediate catalysis through a similar general acid base mechanism. However, adenosine and guanosine undergo ionization only at pH extremes, at least in solution, which seems to make them poor acid base catalysts. We used 8‐azapurine fluorescence to learn whether purine ionization equilibria change in the active site relative to pKa values in solution. 8‐azapurines display high fluorescence emission intensity when N1 is deprotonated and low intensity when N1 is protonated. A ribozyme with 8azaG8 exhibits full catalytic activity and cleaves with an apparent pKa value in the neutral range, similar to an unmodified ribozyme. Microscopic pKa values for deprotonation of 8azaG in the active site were about 3 units higher than apparent pKa values determined from the pH dependence of self‐cleavage kinetics. Thus, the increase in self‐cleavage activity with increasing pH does not reflect G8 deprotonation and G8 is mostly protonated at neutral pH. A simple interpretation of these results is that G8 functions in the protonated form, perhaps by donating hydrogen bonds that stabilize the transition state.
Flexizymes that enable to reprogram the genetic code
Authors: Suga Hiroaki
Published: June 2021

The lecture describes a new means to reprogram the genetic code, which enables us to express “non‐standard” peptides containing multiple non‐proteinogenic amino acids. To execute the genetic code reprogramming, we developed an artificial RNA enzyme (ribozyme), referred to as “flexizyme” capable of aminoacylating tRNAs. The most notable feature of flexizyme is its versatility; it is able to charge virtually any amino acids onto tRNAs bearing various anticodons. Moreover, the flexizyme also charges amino acids with non‐proteinogenic sidechain, D‐amino acids, N‐alkyl‐amino acids, and hydroxy acids onto tRNA. Thus, the reassignment of such non‐proteinogenic amino acids to We also integrated this unique enzyme system with a specially reconstituted E. coli cell‐free transcription‐translation coupled system, from which certain amino acids (occasionally their cognate aminoacyl‐tRNA synthetases) were withdrawn to vacant the corresponding codons. By the integration of these two systems, any desired amino and hydroxy acids can be reassigned to the vacant codons, and we have recently showed that a wide variety of non‐standard peptides can be expressed from mRNAs under the reprogrammed genetic code. The lecture also describes applications of the methodology to the ribosomal generation of cyclic peptides closed by a linkage between the N‐terminus and Cys sidechain or a linkage between the N‐terminus and C‐terminus (backbone‐cyclic peptides).
The epigenome landscape in pluripotent and lineage‐committed human cells
Authors: Ren Bing
Published: June 2021

Human embryonic stem cells share identical genomic sequences with other lineage‐committed cells yet possess the remarkable properties of self‐renewal and pluripotency. It has been proposed that epigenetic regulatory mechanisms, involving DNA methylation and various chromatin modifications, are at least partly responsible for the distinct cellular properties between different cell types. Using next generation sequencing technologies, we have determined the profiles of a dozen chromatin modification marks and the state of DNA methylation at high resolution throughout the genome in the human embryonic stem cells and primary fetal lung fibroblasts. Analysis of the epigenomic profiles in these cells revealed a new set of relationships between chromatin modifications and DNA methylation. Additionally, we defined two types of chromatin domains: one that forms large blocks and the other that occupies small and punctuated regions. We found that epigenomic landscapes are drastically different between the ES cells and fibroblasts: over a third of the human genome differs in their chromatin structure between the two cell types, and that chromatin dynamics at these sequences are frequently associated with change in DNA methylation. Our results provide new insights into epigenetic regulatory mechanisms underlying properties of pluripotency and cell fate commitment.
Genomic approaches for enhancer identification
Authors: Rubin Edward M.
Published: June 2021

Despite their anticipated importance in human biology and disease, transcriptional enhancers remain challenging to both identify as well as predict their associated in vivo gene regulatory activities. I will describe two whole genome strategies for the identification of Mendelian enhancers, comparative genomics and Chromatin Immunoparticipation coupled with massively parallel next generation sequencing (ChiP‐Seq). Both of these enhancer prediction approaches have been linked to large scale in vivo validation where ~1500 putative enhancers have been tested in a transgenic mouse enhancer assay system. Summaries of the results and insights will be presented.
How enzymes adapt: lessons from directed evolution
Authors: Arnold Frances Hamilton
Published: June 2021

When making his case for the key role of natural selection in evolution, Darwin pointed to the enormous phenotypic variation that could be achieved in relatively few generations of artificial selection. Today, artificial selection (or ‘directed evolution’) applied to proteins allows us to observe how readily the functional molecules of life adapt in the face of defined selection pressures. Circumventing our profound ignorance of how sequence encodes function, directed evolution is a powerful approach to generating useful new biological molecules. I will describe our efforts to engineer a cytochrome P450 (CYP102A1 from Bacillus megaterium) by directed evolution. Properties such as catalytic activity or stability can frequently be enhanced by single amino acid substitutions, and accumulating relatively few beneficial mutations (as little as 1–2% of the sequence) can make very significant changes to enzyme function. I will show how a P450 fatty acid hydroxylase has been converted into a whole family of catalysts for oxidation of small alkanes to carbohydrate synthesis and drug lead diversification. Where natural evolution has gone (e.g. impressive diversification of function in the P450 enzyme superfamily), directed evolution can follow. Even more interesting are the catalysts nature may not care about, but chemists dream of. While yielding useful biocatalysts for chemical synthesis, these studies provide new insights into the mechanisms underlying evolution of natural enzymes.
IGF‐1R Regulation of Colon Cancer Stem Cells
Authors: Majumdar Adhip PN , Yu Yingie , Du Jianhua , Patel Bhaumik B
Published: June 2021

Despite recent advances in therapeutics, recurrence of colon cancer occurs in 50% of the cases, which could partly be due to the presence of self renewing cancer stem‐like cells (CSCs) which are resistant to chemotherapy. We have observed that chemosurviving colon cancer cells are enriched in CSCs as evidenced by increased number of CD44+ cells, associated with activated growth factor receptors especially IGF‐1R (4.5‐fold). Depletion of IGF‐1R by si‐RNA results in growth inhibition of chemosurviving cells accompanied by a reduction in CD44 suggesting that IGF‐1R might target CSCs. Indeed, we observed a significant inhibition of colonosphere formation, a CSC property, in IGF‐1R si‐RNA transfected cells in p53 wt HCT‐116 cells accompanied by decrease in CD44 and CD166 expression and induction in miRNA which post transcriptional regulate gene expression. We identified miRNA‐363, a non‐p53 regulated miRNA, to be highly overexpressed (18.9‐fold) following IGF‐1R inhibition. In addition, miR‐215, a p53 regulated miRNA, which is normally downregulated in colon cancer, was upregulated by 2‐fold following IGF‐1R inhibition resulting in induction of its downstream effector p21waf1/cip1 only in p53 wild type colon cancer cells. We conclude that Inhibition of IGF‐1R results in reduction of CSC phenotype in colorectal cancer cells which could partly be due to induction of miRNAs ‐363 and ‐215.
Loss of nNOS Signaling Exacerbates Dystrophic Pathology, but not Skeletal Muscle Fatigue in the mdx Mouse Model of Duchenne Muscular Dystrophy
Authors: Percival Justin , Reed Sarah , Bible Ken , Froehner Stanley
Published: June 2021

Debilitating fatigue is common in muscle wasting diseases such as Duchenne Muscular Dystrophy (DMD). NO produced by nNOSμ and nNOSβ is a critical regulator of skeletal muscle fatigue. Levels of NOSμ are depleted in the skeletal muscles of DMD patients and may contribute to their fatigue. Increased nitric oxide (NO) levels in the mdx mouse (a model for DMD) significantly reduced dystrophic pathology; however, it is unclear whether the anti‐dystrophic effects of exogenous NO are due to activation of downstream targets of nNOSμ or nNOSβ or both. To address nNOS function in mdx muscle, mdx mice were engineered to lack residual nNOSμ and all nNOSβ nNOS‐deficient mdx muscles were smaller in size and showed evidence of increased inflammation. Tibialis anterior (TA) muscles from nNOS‐deficient mdx mice exhibited reduced maximal tetanic and specific force capacity. Unexpectedly, nNOS‐deficient mdx muscle exhibited increased susceptibility to injury by eccentric contractions. Also surprisingly, absence of both nNOS isozymes from mdx muscle did not affect muscle fatigue. In conclusion, the absence of fatigue in nNOS‐deficient mdx muscle suggests the existence of mechanisms that compensate for absent nNOS signaling. This novel compensatory mechanism may contribute to the mild phenotype of the mdx mouse compared to its DMD homologue. These data support the hypothesis that nNOS, particularly nNOSβ, modulates clinically relevant features of dystrophin‐deficient muscle.

Funding sources: PPMD, MDA, NIH.