bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial ROS (mROS) as a possible link between endoplasmic reticulum (ER) ‐stress and inflammatory mediators
Authors: Kozlov Andrey V. , Kehrer Ingeborg , Hartl Romana T , Duvigneau Johanna Catharina , Banerjee Asmita , Dopler Daniela , Redl Heinz
Published: June 2021

The objective of this study was to better understand pathways causing ER stress and mitochondrial dysfunction mediated by inflammatory mediators previously demonstrated in an in vivo model of endotoxic shock. We treated hepatocytes (BRL‐3A) with different additives: media from white blood cells (WBC) (group 1) or from whole blood (group 2) both treated with LPS; or a mixture of TNF‐alpha and IFN‐gamma (group 3). The cells were double stained with mitotracker (MT633) and a ROS‐sensitive fluorescent dye (CM‐H2XROS) and examined by confocal microscopy. Gene expression was analyzed by real time RT‐PCR. We showed a drastic increase in mROS in group 1, a lower but remarkable increase in group 2, and a very weak increase in group 3. Expression analysis showed increased levels of ER‐stress markers in group 1, slightly less in group 2, but no changes in group 3. Detail examination of groups 1 and 2 revealed two types of cell response. While some cells contained mitochondria which displayed increased mROS production without a detectable membrane potential, other cells contained mitochondria showing increased levels of mROS and transmembrane potential. The changes described above were modulated by mitochondria targeted radical scavengers. Our data suggest that during the inflammatory reaction WBC release stimulator(s) (other than TNF‐alpha and IFN‐gamma) mediating enhanced mROS, which modulates ER stress in liver cells.
MNDA regulates a novel nuclear‐mitochondrial circuit critical for progression of apoptosis in human neutrophils
Authors: Fotouhi‐Ardakani Nasser , El Kebir Driss , Pierre‐Charles Natacha , Wang Lili , Ahern Stephane P. , Filep Janos G. , Milot Eric
Published: June 2021

Delayed neutrophil apoptosis perpetuates inflammation and delays the resolution of inflammation. We investigated the role of the nuclear protein MNDA (myeloid nuclear differentiation antigen) in progression of apoptosis of human neutrophils. We found that during constitutive apoptosis, MNDA is cleaved by caspase‐3 and relocated from the nucleus to the cytoplasm. MNDA co‐immunoprecipitates with the anti‐apoptotic protein Mcl‐1, accelerates proteasomal degradation of Mcl‐1, thereby contributing to collapse of mitochondrial transmembrane potential. MNDA knockdown with short hairpin RNA attenuates Mcl‐1 turnover, preserve mitochondrial function and confers resistance to apoptosis in myeloid HL‐60 cells. Culture of neutrophils with inflammatory mediators, including LPS, bacterial DNA, PAF or the acute phase reactants modified C‐reactive protein and serum amyloid A, prevents MNDA cleavage and translocation parallel with suppression of neutrophil apoptosis. Our results identify MNDA as a central mediator of a novel nucleus‐mitochondrion circuit that regulates progression of neutrophil apoptosis. (Grant support: LLSC and CIHR).
Tight Junction Expression Reflects Changes in Intestinal Permeability in the Developing Mouse Intestine
Authors: Patel Ravi , Myers Loren , Kurundkar Ashish , Maheshwari Akhil , Nusrat Asma , Lin Patricia
Published: June 2021

Necrotizing Enterocolitis (NEC) is a devastating disease of preterm infants. Immature intestinal barrier function has been implicated in its pathogenesis. Tight junctions (TJ), comprised of transmembrane Claudins which seal the paracellular space, are a vital component of the intestinal barrier and its regulation of intestinal permeability. However, little is known about how TJ development in the immature intestine influences changes in intestinal permeability. We screened mRNA expression of 29 TJ proteins by qRT‐PCR in the ileum of developing mice over the first 8 weeks of life. Selective TJ proteins were further analyzed by immunoblot analysis and localized by immunofluorescence labeling/confocal microscopy (IF). Temporal changes in expression were correlated with FITC‐dextran intestinal permeability assays. Most TJ proteins demonstrated decreased expression over time. However, Claudin‐3 and Claudin‐7 expression peaked at 3 weeks. IF demonstrated appropriate localization of Claudins in TJ after 3 weeks. Over the same period, intestinal permeability decreased. Changes in Claudin expression likely drive changes in intestinal permeability by tightening the paracellular space. Further studies are needed to elucidate what factors influence temporal changes in TJ expression and intestinal permeability and how they may affect predisposition to NEC.
Increased intestinal permeability results in B/T cell mediated responses that protect from development of colitis
Authors: Khounlotham Manirath , Peatman Eric , Denning Timothy L. , Nava Porfirio , Nusrat Asma , Parkos Charles A.
Published: June 2021

Junctional Adhesion Molecule A (JAM‐A) is a tight junction protein known to regulate intestinal epithelial permeability and leukocyte migration. We have previously shown that JAM‐A−/− mice have increased colonic permeability and B cell lymphoid hyperplasia but do not develop spontaneous colitis. We also observed that JAM‐A−/− mice have increased levels of serum and fecal IgA. To study the role of lymphocytic responses to increased colonic permeability, we generated B/T cell deficient (JAM‐A−/− × RAG1−/−) double knockout mice. JAM‐A−/− × RAG1−/− were more susceptible to dextran sulfate sodium (DSS) induced colitis, manifested by premature mortality and greater body weight loss than JAM‐A+/+ mice. In addition, JAM‐A−/− and JAM‐A−/− × RAG1−/− mice showed increased bacterial translocation to secondary lymphoid organs and colonic submucosa. Broad‐spectrum antibiotic treatment of JAM‐A−/− × RAG1−/− mice significantly reduced disease progression. These data suggest that colonic B cell hyperplasia and increased IgA production serve as protective responses to enhanced translocation of microbial products under conditions of increased mucosal permeability. Furthermore, these findings show that dysfunctional adaptive immunity predisposes to pathologic gut inflammation particularly under conditions of increased colonic permeability. This work is supported by NIH grants DK061379, DK59888 and AA017870.
Cyclin D1 sequestration at tight junctions during mitosis regulates epithelial cell proliferation.
Authors: Capaldo Christopher Todd , Nusrat Asma
Published: June 2021

Coordinated regulation of cell proliferation is vital for epithelial homeostasis, and uncontrolled proliferation is a hallmark of carcinogenesis. A growing body of evidence indicates that epithelial Tight Junctions (TJs) play a role in these processes, although the mechanisms involved are poorly understood. We now report that the Zonula Occludens (ZO) family of TJ plaque proteins sequesters cyclin D1 at TJs during mitosis, which is facilitated by an evolutionarily conserved class II PDZ‐binding motif within cyclin D1. Live‐cell imaging and photobleaching assays revealed that GFP‐cyclin D1 fusion proteins lacking the PDZ‐binding motif failed to localize to TJ and distributed predominantly in the nucleus due to impaired import/export kinetics. Disruption of the cyclin D1/ZO complex through siRNA‐mediated suppression of ZO‐3 resulted in increased cyclin D1 proteolysis and G0/G1 cell cycle block. We thus identify ZO‐3 as a critical regulator of epithelial cell proliferation and conclude that ZO family TJ plaque proteins regulate epithelial homeostasis through stabilization of cyclin D1 during mitosis.
Wnt/β‐catenin signaling, but not AKT/β‐catenin signaling is required for epithelial restitution following colitis
Authors: Koch Stefan , Nava Porfirio , Parkos Charles A , Nusrat Asma
Published: June 2021

We and others have previously shown that inflammatory cytokine induce the production of the secreted Wnt signaling antagonist Dkk1. Since elevated cytokine levels in the intestinal mucosa are a hallmark of inflammatory disorders of the intestine, we evaluated the contribution of this Wnt inhibitor to the pathogenesis of chronic colitis using Dkk1 hypomorphic doubleridge mice, and anti‐Dkk1 antibody treatment in vivo and in vitro. Dkk1 hypomorphic animals showed a significantly faster recovery from experimental colitis as assessed by a combined disease activity score and histological examination, which coincided with increased LRP6 levels in intestinal epithelial cells and increased PI3K activity. Conversely, AKT protein levels and activity, and subsequent AKT‐mediated β‐catenin phosphorylation were decreased in doubleridge mice during acute colitis. Downregulation of AKT in the absence of Dkk1 was confirmed by extended treatment of model intestinal epithelial cell lines with anti‐Dkk1 antibody. These results suggest that Wnt/β‐catenin, but not AKT/β‐catenin signaling is required for the recovery from inflammatory epithelial injury.

Supported by grants from the National Institutes of Health (DK72564, DK61379 and DK79392 to C.A.P., DK53202, DK55679, DK59888 to A.N.), and the Crohn's & Colitis Foundation of America (to S.K. and P.N.).
Morphogenetic Profiles in Micron Length Scales for Genetics and Systems Biology
Authors: Cheng Keith C , Clark Darin P , Thomas Georgia K , Canada Brian , Peckins Steven , Cheng Anthony M , Barno Eileen , Copper Jean , Tatiparthi Arun , Sledz Timothy , Canfield Victor , Xiao Xianghui , deCarlo Francesco , La Riviere Patrick , Liu Yanxi , Wang James Z , Amsterdam Adam , Hopkins Nancy
Published: June 2021

Information in genomes, together with environment, determine biological structure. Structure distinguishes bacterium, plant, and animal, and defines biological state ‐ adaptive, injured, or cancerous. The power of morphological analysis is illustrated at millimeter and micron length scales in radiology and pathology, respectively. We are exploring the potential of qualitative and quantitative whole‐animal structural analysis to define normal and abnormal biological function using the zebrafish, a vertebrate whose length scale and powerful genetic tools optimize the pursuit of high‐resolution morphogenetic analysis. Whole‐animal, 2D analysis at sub‐micron resolutions using histology, produced morphogenetic profiles that provided functional links between proteins. 3D analysis at micron resolutions using synchrotron‐based x‐rays were used to generate isotropic data sets in which soft tissue discrimination was possible. The potential contributions of 3D morphogenetic profiles in zebrafish using microCT will be discussed.
Proteomic analysis of human pancreas cancers for the identification of targetable biomarkers
Authors: Castronovo Vincent , Wang Ying Hong , Musmeci Davide , Dumont Bruno , Turtoi Andrei
Published: June 2021

Pancreatic ductal adenocarcinoma is characterized with poorest prognoses among all major human cancers. Hence novel diagnostic and treatment modalities, such as targeted antibody based therapy, are required. The purpose of this study is therefore to identify tumor associated antigens in human pancreatic cancer specimens. These should preferentially be abundant in the tumor and accessible via bloodstream deliverable agents. Here, pancreatic tissues obtained from surgical resections of 4 individuals were soaked in EZ‐link Sulfo NHS‐SS‐biotin solution. The biotinylated proteins were captured by streptavidin affinity chromatography and the peptides derived from tryptic digestion were analyzed using the 2D‐HPLC‐MS/MS technique. Twenty four proteins were identified as accessible and differentially expressed in cancer as compared to normal pancreas. Of these, six proteins have been found in previous studies to be associated with pancreatic cancer whereas 14 have been known to be differentially expressed in other types of tumor. The remaining proteins were novel in the context of the pancreas tumor pathology. The differential expression pattern of potential antigens was validated by immunohistochemistry. In conclusion, the present study identified a set of accessible proteins that may be used as antigens for the antibody‐based therapy of pancreatic cancer. Our data have shown the feasibility of using a biotinylated strategy to generate protein expression profiles and identify accessible proteins as potentially therapeutic targets.
Lipidomics in identifying lipid biomarkers of prostate cancer
Authors: Zhou Xinchun , Mao Jinghe , He Zhi , Henegar Jeffrey
Published: June 2021

There are no comprehensive studies of lipid species in prostate cancer patients. In this study, global lipid profiles were determined in fresh‐frozen prostate tissues and plasma from prostate cancer patients and controls using electrospray tandem spectrometry (ESI‐MS/MS). In patients' plasma, 78 species increased concentrations >50% and 27 species decreased concentration >50% compared to controls' plasma. In cancer tissues, 56 species increased concentrations >50% and 12 species decreased concentration >50% compared to control tissues. Twenty‐two distinct lipid species in patients' plasma and 15 in cancer tissues were detected, which were absent in controls. Lipid concentrations were significantly different in 22 species between plasma from patients and controls and in 11 species between cancer and control tissues. Compared to controls, the lipid concentrations in patients' plasma increased in 8 of 11 head groups, while that in cancer tissues decreased in 8 of 10 head groups. All of 14 individual lipid species in Lysophosphatidylcholine were increased in both patients' plasma (1.7 times, p=0.025) and cancer tissues (1.33 times). Ceramide phosphorylethanolamine decreased significantly in cancer tissues (0.24 times, p=0.017). The results suggest that alterations in lipid could serve as new biomarkers for studying pathogenesis and monitoring progression of prostate cancer.
Targeted and Pharmacologic Inhibition of DNMT3b Sensitizes MDA‐MB‐453 Breast Cancer Cells to Doxorubicin and Paclitaxel
Authors: Sandhu Rupninder , Rivenbark Ashley G , Coleman William B
Published: June 2021

Basal‐like breast cancers frequently express a hypermethylation defect characterized by DNMT hyperactivity and DNMT3b overexpression, which may contribute to chemotherapy resistance and provides a target for development of new treatment strategies. To determine the effect of DNMT3b status on the sensitivity of hypermethylator MDA‐MB‐453 breast cancer cells to doxorubicin hydrochloride (DOX) and paclitaxel (PAX), we employed RNAi‐mediated DNMT3b knockdown (KD) and/or demethylating agent treatment with 5‐aza‐2′‐deoxycytidine (5‐aza). DNMT3b KD reduces the IC50 for DOX from 0.079 μM to 0.048 μM (39% reduction) and for PAX from 0.497 nM to 0.376 nM (24%). Treatment with 250 nM 5‐aza for 7d did not increase efficacy of DOX or PAX, but 7d treatment with 500 nM 5‐aza sensitized cells, reducing the IC50 for DOX to 0.0349 μM (56%) and the PAX to 0.311 nM (37%). 5‐aza treatment of KD cells reduced the IC50 for DOX to 0.0355 μM (55%) and for PAX to 0.313 nM (37%). Inhibition of DNMT3b produced improved cell killing in response to 0.079 μM DOX and 0.497 nM PAX: DNMT3b KD (19% and 10% respectively), 5‐aza (38% and 18%), and combined KD and 5‐aza treatment (39% and 18%). These results show that effectiveness of DOX and PAX can be increased by targeted and pharmacological inhibition of DNMT3b. These results strongly suggest that DNMT3b can be targeted to improve the efficacy of breast cancer chemotherapy. UNC University Cancer Research Fund
Leukocyte recruitment to the cremaster muscle exhibits circadian oscillations
Authors: Scheiermann Christoph , Kunisaki Yuya , Chow Andrew , Lucas Daniel , Frenette Paul
Published: June 2021

Circulating leukocyte numbers exhibit circadian oscillations with peak values in mice during the day [Zeitgeber time, ZT 5 (hours after the onset of light)], and trough at night [ZT 13]. Here, we report that leukocyte recruitment to the cremaster muscle oscillates in antiphase with blood count patterns, with numbers of adherent and extravasated leukocytes peaking at night and exhibiting a nadir during the day (ZT5/ZT13: 5.3 ± 0.3/7.3 ± 0.3 cells per 100 × 50 μm2 extravascular vessel segment, P<0.001) as assessed by in vivo and ex vivo imaging of the cremasteric microvasculature. Circadian differences depended on ICAM‐1 expression and local innervation, as ICAM‐1 deficiency and surgical denervation of the genitofemoral nerve ablated oscillations. Systemic stimulation of mice with fMLP resulted in increased recruitment of granulocytes during the day compared to night (ZT5/ZT13: 1.3 ± 0.3/0.6 ± 0.3 cells per 100 × 50 μm2 extravascular vessel segment, P<0.05). On the other hand, mice subjected to a 12h jetlag did not exhibit circadian oscillations in leukocyte recruitment and failed to show inflammatory leukocyte infiltration after fMLP treatment. These data show that leukocyte trafficking to the cremaster exhibits circadian oscillations and suggest that circadian time can influence the recruitment of leukocytes during inflammation.

Funded by the German Academic Exchange Service (DAAD) and the NIH.
Enhanced retinal degeneration induced by sodium iodate in alphaB‐crystallin knockout mice
Authors: Hinton David R , Zhou Peng , Kannan Ram
Published: June 2021

The aim of this study was to determine the effect of αB‐crystallin knockout on sodium iodate (NaIO3)‐induced retinal degeneration in mice. Time and dose dependent effects of intravenous NaIO3 (20–70 mg/kg for 3–21 days) on visual function, retinal pigment epithelium (RPE) and retinal neuronal loss, and signaling pathways involved were investigated. NaIO3 caused a time‐dependent decrease in a and b waves in the electroretinogram and progressive retinal degeneration. Absence of αB‐crystallin accelerated and augmented the retinal degeneration in NaIO3‐treated mice and was associated with increased retinal cell apoptosis. In vitro studies revealed increased accumulation of reactive oxygen species (ROS) in cultured human RPE cells after NaIO3 treatment, and ROS were significantly increased in RPE after αB‐crystallin siRNA knock down. Increased RPE apoptosis was found in low dose NaIO3‐treated RPE after αB‐crystallin siRNA knock down; however, necrosis was minimal. Upregulation of AKT phosphorylation and peroxisome proliferator‐activated receptor‐γ (PPARγ) was found in NaIO3‐treated human RPE which was suppressed in αB‐crystallin siRNA knock down RPE cells. Our data suggest that αB‐crystallin plays a critical role in protection of NaIO3 induced oxidative stress and retinal degeneration in part through upregulation of AKT phosphorylation and PPARγ expression.
β‐catenin knockout mice are protected from TNF‐α mediated apoptosis
Authors: Nejak‐Bowen Kari Nichole , Tan Xinping , Monga Satdarshan
Published: June 2021

β‐catenin plays multiple roles in liver health and disease through regulation of processes such as proliferation, differentiation and adhesion. However its role in hepatic injury remains unexplored. Here, we demonstrate that loss of β‐catenin protects against TNF‐α mediated apoptosis. Analysis of mRNA expression levels in wild‐type (WT) and β‐catenin knockout (KO) mice reveals that KO livers have an increased expression of TNF‐α induced genes, as determined by gene array. Additionally, baseline levels of caspase‐3 and caspase‐8 are increased in KOs over WTs. WT and KO mice injected with either galactosamine (GalN) or actinomycin D (ActD) and lipopolysaccharide (LPS) (which activates the TNF‐α pathway) were monitored for morbidity. Surprisingly, KO mice are refractory to both GalN/LPS and ActD/LPS treatment: the mice show decreased morbidity, and examination of WT and KO livers morphologically and histologically upon sacrifice reveals that KO mice are protected from damage and apoptosis. Analysis of liver enzymes and TUNEL staining confirms the presence of massive injury and apoptosis in WT but not in KO mice. Expression of NF‐κB p50 and p65, as well as their downstream targets Fas and Traf‐1, are unchanged between WT and KO under unstimulated conditions. However, levels of the anti‐apoptotic transcription factors GSK‐3β and NF‐κB are elevated in KO mice as compared to WT after insult. In conclusion, our results demonstrate that deletion of β‐catenin from hepatocytes confers protection from TNF‐α induced injury and apoptosis, perhaps through a compensatory upregulation of the anti‐apoptotic NF‐κB pathway.
Mechanisms of 7β‐hydroxycholesterol‐induced apoptosis in mouse vascular smooth muscle cells
Authors: Park Hye‐Jin , Rhee Dong‐Kwon , Pyo Suhkneung
Published: June 2021

Oxidized low‐density lipoprotein (oxLDL) exhibits various biological properties and it has been suggested that oxLDL plays a critical role in the pathogenesis of atherosclerosis and destabilization of the atherosclerotic plaque. Especially, apoptosis induced by oxLDL is considered as a major factor in atherosclerosis and vascular dysfunction. In this study, we examined the mechanism of apoptosis in mouse vascular smooth muscle cells (VSMCs) induced by 7β‐hydroxycholesterol (7β‐OH), one of oxysterols that are common components of oxLDL. Cells were cultured and treated with different concentration of 7β‐OH for various times. 7β‐OH induced apoptosis in VSMCs in a dose‐ and time‐dependent manner. Western blot analysis revealed that 7β‐OH did not up‐regulate p53 expression but its phosphorylation, and reduced expression of both p21 and p16. The levels of Bcl2, BclxL, and AKT were decreased in 7β‐OH treated cells, while Bax was increased. Furthermore, 7β‐OH attenuated the level of KLF5, a transcription factor that is important in regulation of the cardiovascular response to external stress. These findings collectively suggest that 7β‐OH activates p53 and down‐regulates the expression of Bcl2 and BclxL while up‐regulates Bax expression to induce apoptosis in VSMCs. In addition, it is believed that KLF5 is involved in 7β‐OH ‐induced apoptosis in VSMCs.
Migfilin contributes to cell‐matrix adhesion‐mediated survival signaling through direct interaction with Src
Authors: Zhao Jianping , Zhang Yongjun , Ithychanda Sujay Subbayya , Tu Yizeng , Chen Ka , Qin Jun , Wu Chuanyue
Published: June 2021

Integrin‐mediated cell‐extracellular matrix (ECM) adhesion is essential for protection of epithelial cells against apoptosis, but the underlying mechanism is incompletely understood. Here we show that migfilin, an integrin‐proximal adaptor protein, interacts with Src and contributes to cell‐ECM‐mediated survival signaling. Loss of cell‐ECM adhesion markedly reduces the migfilin level in untransformed epithelial cells and concomitantly induces apoptosis(anoikis). Overexpression of migfilin substantially desensitizes cell detachment‐induced apoptosis. Conversely, depletion of migfilin promotes apoptosis despite the presence of cell‐ECM adhesion. At the molecular level, migfilin directly interacts with Src and the migfilin binding surface overlaps with the inhibitory intramolecular interaction sites in Src. Consequently, the binding of migfilin activates Src, resulting in suppression of apoptosis. Our results reveal a novel mechanism by which cell‐ECM adhesion regulates Src activation and survival signaling. This migfilin‐mediated signaling pathway is dysfunctional in multiple types of carcinoma cells, which likely contributes to aberrant Src activation and anoikis resistance in the cancerous cells.
Compression‐induced cell distension stimulates coordinated migration of mammary carcinoma cells
Authors: Tse Janet , Cheng Gang , Tyrrell James Alex , Wilcox‐Adelman Sarah A. , Jain Rakesh K , Munn Lance L
Published: June 2021

Uncontrolled cell proliferation of a solid tumor in a confined space not only creates oxidative stress (hypoxia), but also generates mechanical compressive stress, which can influence the tumor cells and modify their interactions with neighboring cells and the extracellular matrix. Little is known about how such compressive stress affects cancer progression. Here we show, for the first time, that externally‐applied compressive stress results in faster migration of mammary carcinoma cells. Compression induces migration of mammary carcinoma cells in a coordinated sheet, initiated by “leader cells” – single cells at the leading edge of the sheet, extending long filopodia. Accompanied by redistribution of fibronectin deposition, applied compression enhances cell‐matrix adhesion and stabilizes cell distension independent of actomyosin contractile machinery. Our results suggest that mechanical stress accumulated during tumor growth is sufficient to enhance cell‐substrate adhesion and stimulate cancer cell migration. This mechanism opens the door to a new class of targets for blocking mechanical stress pathways. Funding for this work was provided by NIH grant R01CA085140.
Requirement of a4b1 and a5b1 Integrin Expression in Bone‐Marrow Derived Progenitor Cells in Preventing Endotoxin‐Induced Lung Vascular Injury and Edema in Mice
Authors: Wary Kishore K , Malik Asrar B
Published: June 2021

The goal of this study was to address the hypothesis that integrin‐mediated cell adhesion is a critical requirement for the endothelial barrier protection induced by bone marrow derived progenitor cells (BMPCs). Bone marrow (BM)‐derived mononuclear cells (MNCs) from donor C57BL mice were used as a source for BMPCs and characterized for BMPCs marker and integrin expression. To test the efficacy of BMPCs treatment and to address the mechanisms of the benefit, we used a mouse model of endotoxin‐induced ALI. We noted robust expression of α4 and α5 integrins in donor BMPCs. The adhesion of BMPCs onto purified extracellular matrix not only induced Fak phosphorylation, but also the formation of branching point structures in an α4 and α5 integrin‐dependent manner. BMPCs expressing red fluorescent protein (RFP) were administered via the retro‐orbital venous route in mice treated intraperitonially with LPS. These mice received either control endothelial cells (ECs) or BMPC or BMPCs incubated with specific anti‐integrin monoclonal antibodies, or BMPCs tranduced with a specific shRNA integrin construct. BMPCs afforded a 50% survival and reduced extravascular fluid content. Blocking or silencing the α4 and α5 integrins in donor BMPCs failed to protect these mice from injury or to reduce extravascular fluid content. In these mice, we also noted an increased retention of RFP+, Flk1+ and CD34+ BMPCs (20%) for the next 8 weeks, as well as overall improved lung architecture. Collectively, our data showed that BMPCs play a critical role in mounting lung microvessel repair through the expression of α4β1 and α5β1 integrins, causing restoration of lung fluid balance and increased survival rates in a mouse model of ALI.

This study was supported by NIH‐RO1, HL079356 and R01HL090152.
The Scientists' Perspective on Kennewick Man
Authors: Owsley Douglas W.
Published: June 2021

Kennewick Man is one of the oldest and most complete skeletons ever found in North America. Findings from this 9,300 year old skeleton are providing new insights into the ongoing mystery of who colonized the New World, a process more complicated than previously envisioned. Recent evidence suggests multiple migrations by different peoples entering the Americas from different homelands at a time earlier than originally thought. Securing the right to study important discoveries found on federal lands, such as was the case with Kennewick Man, has helped us learn more about what life was like for the earliest Americans. It helps secure the right to ask questions of the past, and to challenge long held assumptions about a time period for which we have much to learn.

The Kennewick skeleton was discovered after it had eroded out of the south bank of the Columbia River in the state of Washington. Although not found in situ, analysis by a team of scientists has determined the original stratigraphic placement and positioning of the remains in the bank. Other observations provide detailed information about his body size and shape, health, injuries, habitual activities, diet, and origin.

Investigation of the skeleton was enhanced by the use of computer aided high technology systems. In order to reconstruct the skull and right os coxa, industrial computed tomography imaging and replication was necessary. This work produced dimensionally accurate copies of the broken and separated bones, which could be reassembled for study and measurement. CT visualization and rapid prototyping also made it possible to digitally remove for examination a projectile point embedded in the right os coxa. This presentation will highlight what has been revealed through careful scientific analysis.
Oxygen tension and reactive oxygen species affect an age‐related response in human keratinocytes
Authors: Aykin‐Burns Nukhet , Cloud Caitlin , Tomanek‐Chalkley Ann , Wiechert Susan , Bickenbach Jackie R.
Published: June 2021

High levels of reactive oxygen species (ROS) can damage key genetic and cellular processes, but low levels are required for redox cell signaling. Accumulation of ROS is directly linked to genetic damage in aging tissues and implicated in delayed wound healing. In this study, we examined the effect of age and oxygen tension in primary human skin keratinocytes using cell migration and level of ROS as the affected functions. Baseline levels of ROS were higher in young keratinocytes (from 22–35 year old patients) grown at 21% oxygen as compared to 4% oxygen. However, we found the opposite with older keratinocytes (from 78–90 year old patients)– higher levels of ROS at 4% oxygen than at 21% oxygen. Both young and old keratinocytes showed decreased rates of cell migration in response to increased ROS. The in vitro scratch assay demonstrated that cells located at the edge of the scratch greatly increased their ROS levels immediately upon scratching and had declined to near baseline after 24 hours. While young cells showed higher levels of ROS at scratch margins than old cells did, the young cells were less effective at removing the ROS. Furthermore the old keratinocytes were most successful in reducing their ROS levels at the scratch margin when grown at 21% oxygen. Recovery of the cells from increased levels of ROS at their scratch margins correlated with their migration rates. These findings indicate that keratinocytes show an age‐related differential response to both oxygen tension and ROS levels (Supported by a McCord Foundation Research Grant, RO1AR053619, and R21AR053936).
Detection of vascular oxidative stress in atherosclerotic lesions by electrochemical impedance spectroscopy
Authors: YU FEI , Ai Lisong , Edington Collin David , Kim Eun Sok , Hsiai Tzung
Published: June 2021

Rupture of atherosclerotic plaques leads to acute coronary syndromes and stroke. However, detecting mechanically unstable plaque remains an unmet clinical need. We proposed that arterial walls harboring oxidative stress exhibit distinct electrochemical properties as measured by electrochemical impedance spectroscopy (EIS). Using concentric microelectrodes, we developed an EIS sensor to assess vascular oxidative stress (defined as foam cells and oxizided low density lipoprotein [oxLDL]) in explants of New Zealand White rabbit aortas. EIS sensor was made in contact with the endoluminal surface of aortas isolated from rabbits fed on a high‐fat diet for 8 weeks. Statistical correlation between EIS and oxidative stress was established by immunohistochemical analysis for oxLDL and foam cell infiltration. The increase in impedance measurements were statistically significant in atherosclerotic lesions harboring oxidative stress at 100kHz (lesions: 26.7kΩ±2.7kΩ; control: 15.8kΩ±2.4kΩ, n=10, P<0.001) and at 10kHz (lesions: 49.2kΩ±7.3kΩ; control: 27.6 kΩ±2.7kΩ, n=10, P<0.001). Characterization of 2mm × 2mm lesions harboring 10% endoluminal stenosis was reproducible. Hence, our data provides a basis to further develop intravascular oxidative stress sensors for the assessment of mechanically unstable plaque. Research supported by NHLB 083015 and NHLBI 068689.

Grant Funding Source: NHLBI
Epigenetic Lineage Barriers ensure normal Trophoblast Differentiation in Early Development
Authors: Hemberger Myriam C
Published: June 2021

Cells that give rise to the trophoblast lineage are set aside in the earliest differentiation event after fertilization to mediate implantation, placenta formation and fetal nutrition. We are interested in genetic and epigenetic factors that contribute to normal trophoblast differentiation and formation of a functional placenta. Correlating with the global asymmetry in DNA methylation levels between cells of the embryonic and trophoblast lineage, we have found that DNA methylation is critical to ensure the stability of early cell lineage determination. In a genome‐wide screen for promoter DNA methylation differences, we have identified a gatekeeper gene, Elf5, that is unmethylated and expressed in the trophoblast lineage, but methylated and stably repressed in the embryonic lineage. We show that this gatekeeper acts downstream of initial lineage determination to either reinforce commitment to the trophoblast lineage, or to abort this pathway in the embryonic lineage. This finding is the first example of a major epigenetic restriction of cell lineage fate and provides a molecular mechanism for the concept of canalization of developmental pathways during embryogenesis.

This work was supported by the MRC, BBSRC and the Centre for Trophoblast Research.
Manipulating cell signaling by exploiting modularity and localization
Authors: Pryciak Peter
Published: June 2021

Our efforts to understand natural signal transduction pathways provide lessons for designing new signaling proteins and circuits. In particular, localization and modularity provide straightforward and powerful ways to manipulate signaling. For example, the role of scaffolds in fostering signaling interactions between bound proteins can be mimicked by co‐targeting the proteins to a restricted subcellular locale or by directly fusing them together. Signaling responses often begin at the plasma membrane, and proper membrane localization often requires the concerted action of multiple weak‐affinity binding motifs. We found that multiple signaling proteins contain short cryptic membrane‐binding domains that control normal localization and functional engagement with membrane‐bound activators. These independent modular domains can be exchanged to alter the target membrane and/or recruiting target protein. In addition, membrane interactions can be regulated via simple electrostatic effects by surrounding membrane‐binding domains with phosphorylation sites, and the number of phosphorylation sites can quantitatively tune the strength of regulation. Thus, analogous to synthetic gene regulatory circuits, structurally and functionally autonomous protein domains can be linked together to yield signaling proteins whose properties reflect the additive properties of the individual modules.
The Design Logic of Cell Signaling Systems
Authors: Lim Wendell A
Published: June 2021

Cells use complex networks of signaling proteins to accurately transmit information and make decisions. Despite their complexity, many of these systems appear to be built from a finite set of relatively simple modular catalytic and regulatory components. We are studying the logic of how such modules are used to build complex signaling systems. We are tracking how signaling modules originated and how they are used as building blocks in evolution. We are also exploring how these modules can be used in synthetic biology to engineer cells with new behaviors and potential therapeutic or biotechnological applications.
Defects in mitochondrial DNA replication and human disease
Authors: Copeland William C
Published: June 2021

Human mitochondrial DNA is replicated and repaired by DNA polymerase gamma, composed of a 140kDa catalytic subunit and a 55kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3′–5′ exonuclease activity, and a 5′dRP lyase activity while the accessory subunit is critical for highly processive DNA synthesis and imparts tighter DNA binding to the complex. Mutations in the gene for the catalytic subunit, POLG, are linked to several mitochondrial disorders including progressive external ophthalmoplegia (PEO), sensory and ataxic neuropathy, and Alpers syndrome, a fatal early childhood disease causing brain and liver failure. The carrier frequency of POLG disease mutations is ~2% in the population and is a major cause of mitochondrial disease. Over 150 disease mutations have been identified in POLG (see Human DNA Polymerase Gamma Mutation Database (http://tools.niehs.nih.gov/polg/). Using biochemical analysis, yeast genetics and mouse models, we are determining the consequences of disease mutations in the POLG protein as well as disease mutations in the accessory subunit, POLG2, and the Twinkle helicase, PEO1. These results will be discussed in terms of their role in the mechanism leading to defects in mtDNA replication.
Cause and consequences of genome instability in Huntington's Disease
Authors: Lang Walter , Majka Jerzy , Owen Barbara , Klugland Arne , Bjoras Magnar , McMurray Cynthia T.
Published: June 2021

Mammalian cells have evolved sophisticated DNA repair systems to correct mispaired or damaged bases and extra helical loops. However, we find that two DNA repair systems, the mismatch recognition system (MMR) and the base excision repair, are turned into a causative factor rather than a safeguard of genomic integrity. We have followed CAG expansion in germ cells and in somatic tissues of transgenic mice harboring a fragment of the human Huntington's disease (HD) gene. CAG expansion of the hHD allele is abrogated in both somatic cells and in germ cells of transgenic mice that lack Msh2. A complex involving Msh2 is thought to cause expansions by binding to, but failing to correct at the site of DNA breaks. Base excision repair may cause the break that needs to be repaired. Surprisingly, expansion in somatic cells only occurs at midlife of the mouse. These data indicate that some process associated with aging causes expansion and DNA breaks only in midlife. We have purified the MSH2/MSH3 enzymes and examined their biochemical properties. We find that key steps of the MMR system are impaired and that other and repair pathways may be called into play to repair the site.
CtBP, an NADH‐sensing transcriptional co‐repressor
Authors: Zhang Qinghong , Deng Yu , Liu Jing
Published: June 2021

Cancer cells are well known for their high glycolysis, which produces NADH in the cells. Otto Warburg postulated that this change in metabolism is the fundamental cause of cancer. Many substances have been developed to inhibit glycolysis, and such glycolytic inhibitors are currently the subject of intense research as anticancer agents. Carboxyl‐terminal binding protein (CtBP) was initially identified by virtue of its ability to interact with E1A, the major transforming protein of adenovirus. Our work has uncovered CtBP being the first NADH‐sensing transcriptional regulator in mammals. We found that NADH binding to CtBP stimulates the CtBP interaction with transcription repressors and cellular perturbations that affect the ratio of free NAD+/NADH regulate the ability of CtBP to interact with its binding partners. As a foe of multiple tumor suppressors, CtBP promotes EMT and functions as apoptosis antagonist. The unique function of this protein as a redox‐sensor in transcription control allows cells to couple the metabolic status with gene expression, contributing to tumor progression. This work was supported by NIH grant R01CA115468.
Engineering of Monovalent, Multivalent and Bispecific Antibodies for New Therapeutic Applications
Authors: Lugovskoy Alexey Alexandrovich , Farrington Graham , Miller Brian , Glaser Scott
Published: June 2021

Engineered bispecific and tetravalent IgG‐like antibodies that engage multiple target epitopes represent promising new classes of therapeutic agents. To address their intrinsic low stability and high aggregation propensity, we applied rational design to increase the thermostability of single chain Fv fragments without compromising their affinity towards antigen. Incorporation of these stabilized modules into full‐length therapeutic bispecific and tetravalent molecules led to molecules with markedly improved properties. We additionally describe the development and characterization of new monovalent and heteromeric antibody platforms that allow us to interrogate the classes of targets, such as receptor tyrosine kinases and cytokines, which may be difficult to inhibit by bivalent antibodies.
β‐Catenin loss promotes DEN‐induced liver cancer via oxidative stress & PDGFRα/PI3K signaling
Authors: Zhang Xufeng , Monga Satdarshan Paul
Published: June 2021

β‐Catenin, the central effector of canonical Wnt pathway is a recognized ‘oncogene?for its role in tumors, including hepatocellular carcinoma (HCC). We investigated whether β‐catenin liver conditional knockout mice β‐cat KO) were protected from chemically induced HCC. Intriguingly, β‐cat KO mice were more susceptible to N‐nitrosodiethylamine (DEN)‐induced hepatocarcinogenesis than controls Wildtype animals. This susceptibility correlated to unrestricted oxidative stress, higher apoptosis and compensatory hepatocyte proliferation, secondary to increased platelet‐derived growth factor receptor‐α; (PDGFRα) and Phosphoinositide 3‐kinase (PIK3CA)/Akt activation. By in vivo and in vitro study, we found activation of nuclear factor κB (NF‐κB) in absence of β‐catenin to be accounting for the upregulation of PDGFRα. Short‐term treatment with PDGFRα inhibitor or long‐term feeding of N‐acetyl‐L cysteine (NAC)‐ an antioxidant and NF‐κB inhibitor, blocked PDGFRα/PIK3CA/Akt pathway, significantly impacting hepatocarcinogenesis. These results demonstrate an important role of β‐catenin as a regulator of oxidative stress in liver and highlights novel compensatory role of PDGFRα signaling in promoting hepatocarcinogenesis in absence of β‐catenin.
Dysregulation of Multiple MicroRNAs Accompanies DNMT3b Overexpression and Expression of a Hypermethylation Defect in Breast Cancer Cell Lines
Authors: Sandhu Rupninder , Coleman William B
Published: June 2021

A subset of breast cancer cell lines express a hypermethylation defect associated with DNMT hyperactivity and DNMT3b overexpression. To determine the mechanism of DNMT3b overexpression in breast cancer cell lines, we examined the role of regulatory microRNAs (miRs) that have been implicated in the regulation of DNMT3b (miR‐29a, 29b, 29c, 148a, 148b) and miRs that are predicted to regulate DNMT3b (26a, 26b, 203, 222). miR expression levels were determined by qPCR (normalized to RNU66) in non‐neoplastic MCF12A cells and a panel of 16 breast cancer cell lines previously characterized as hypermethylators (n=10) or non‐hypermethylators (n=6). 90% of hypermethylator cell lines express low levels of ≥4 miRs and 100% of non‐hypermethylator cell lines express ≥4 miRs at normal levels. Average expression of miRs 29c, 148a, 148b, 26a, 26b, and 203 in hypermethylator cell lines was reduced 60%–85% compared to non‐hypermethylator cell lines. Further, miR expression correlated with methylation‐sensitive gene expression (R=0.68, p=0.0035) and with gene promoter methylation status (R=0.55, p=0.026) among these cell lines. These findings suggest that post‐transcriptional regulation of DNMT3b involves multiple miR species and that loss of regulation of miR expression contributes to the DNMT3b overexpression in hypermethylator cell lines.

UNC University Cancer Research Fund
Hemodynamic and renal microcirculatory dysfunction during sepsis‐induced acute kidney injury (AKI) in mice
Authors: Wang Zhen , Mayeux Philip R
Published: June 2021

Understanding the sequence of events leading to sepsis‐induced renal injury, especially at the early stages of sepsis, is critical for developing new therapy to reduce the high mortality in patients with severe sepsis. The cecal ligation and puncture (CLP) model of sepsis was used to study the development of AKI in male 40‐week C57BL/6 mice. Mean artery blood pressure (MAP) and heart rate (HR) was recorded by telemetry for 24 h post CLP. Intravital video microscopy (IVVM) was used to quantitate changes in the peritubular microcirculation at 2, 4, or 6 h following CLP (or Sham). Mitosox fluorescence was used as a measured of superoxide generation by tubular cells in situ in the same field of view as capillary perfusion. Serum nitrite/nitrate level was measured to indicate systemic NO generation. Using detailed time course studies, we found that MAP began to decrease 1.5h following CLP and reached its lowest level by 4h, the time of peak increase in serum NO. HR decreased at 3 h and reached it lowest level by 8h. Peritubular capillary perfusion fell 2 h post CLP and was followed by superoxide generation at 4h. This study shows that hemodynamic and renal microcirculatory failures are extremely early events during sepsis that may contribute to renal oxidative stress and renal injury. Supported by AHA Grant In Aid #850227Z.
Lung‐kidney interactions during pneumonia and acute kidney injury
Authors: Singbartl Kai , Bishop Jeffrey , Kellum John
Published: June 2021

Sepsis is a major contributor to organ failure in critically‐ill patients. Mortality drastically increases with the number of failing organs. Both lung and kidney injury are very common in severe sepsis. The aim of this study was to investigate kidney‐lung interactions during pneumonia‐induced sepsis and acute kidney injury (AKI).

We have sequentially combined murine models of folic‐acid (FA)‐induced AKI and P. aeruginosa‐induced pneumonia (PNA). A subgroup of mice received a neutrophil (PMN)‐depleting antibody or respective control before induction of PNA. 24h after induction of PNA, we measured oxygen saturation (SpO2), plasma creatinine (pCrea), and P. aeruginosa colony‐forming units (CFUs) in lung homogenates. Statistical analysis included one‐way ANOVA, Kruskal‐Wallis one‐way analysis of variance, post‐hoc Student‐Newman‐Keuls test, and t‐test. All data is given as median (inter‐quartile range).

PNA leads to severe AKI in mice, which seems to be independent of PMNs. In a PMN‐dependent model of PNA, AKI worsens PNA. As AKI is known to impair pulmonary recruitment of PMNs [1], one can hypothesize that the effects of AKI on pneumonia are mediated by PMNs. In summary, we show functionally relevant kidney‐lung interactions during PNA and AKI, which are partially PMN‐dependent.
Learner assessment using interactive web‐based instructional techniques enhances preclinical education
Authors: Klinkhachorn Penprapa S. , Allen Edwin , Reilly Frank D.
Published: June 2021

Retrospective data from 9 years of instruction involving 856 students were used to determine (1) the effect of web‐based interactive instructional techniques on written exam item performance in a web‐based peripheral nervous system component of a first‐year human anatomy course, and (2) differences between student opinions of the benefit level of the interactive learning objects used for assessment, i.e., patient case studies, review games, simulated interactive patients, flashcards, and unit quizzes. Exam item analysis scores were found to be significantly higher (p < .05) for students receiving the instructional treatment incorporating web‐based interactive learning objects than for students not receiving this treatment. Questionnaires using a five‐point Likert scale were analyzed to determine student opinion ratings of the interactive learning objects. Students reported favorably on the benefit level of all learning objects. Students rated the benefit level of the simulated interactive patients highest, and this rating was significantly higher (p < .05) than all other interactive learning objects. This study suggests that web‐based interactive instructional techniques improve student exam performance. Students indicated a strong acceptance of simulated interactive patient learning objects used for assessment. (Supported in part by WVU acct#:491030050, fund#:12305495.)