bluuebunny/crossref_metadata_2025 huggingface dataset:
Prenatal Exposure to Ethanol and Disturbances in Hepatic One Carbon Metabolism
Authors: Sulistyoningrum Dian C , Innis Sheila M , Weinberg Joanne , Devlin Angela M
Published: June 2021

Disturbances in one carbon metabolism contribute to the pathology of adult alcoholic liver disease. We hypothesize that disturbances in one carbon metabolism may contribute to the pathology associated with prenatal ethanol exposure including fetal alcohol spectrum disorder. We studied offspring at gestational age 21 days (GD 21) from female Sprague‐Dawley rats fed a liquid ethanol diet (36% energy from ethanol), a liquid control diet (maltose‐dextrin isocalorically substituted for ethanol, g/kg body wt/day of gestation), or lab chow throughout pregnancy. Liver methionine levels were higher (P< 0.05) and dimethylglycine levels lower (P<0.01) in pups exposed in utero to ethanol than in pups from lab chow‐fed dams. This was accompanied by higher (P<0.05) levels of methionineadenosyltransferase 2a (Mat2a), methionine synthase (Mtr), methylenetetrahydrofolate reductase (Mthfr), and cysthationine‐β‐synthase (Cbs) mRNA levels in liver from pups exposed in utero to ethanol than in pups from dams fed lab chow. No effects of prenatal ethanol exposure on betaine‐homocysteine methyltransferase, phosphatidylethanolamine methyltransferase, and Mat1a mRNA levels or Cbs protein levels were observed. These finding suggest that prenatal exposure to ethanol affects liver one carbon metabolism in GD21 offspring.
Maternal Green Tea Extract Supplementation Ameliorates Insulin Resistance in Male Offspring from Dams Fed a High‐Fat Diet
Authors: Li Shiying , Li Edmund Tsze Shing
Published: June 2021

Green tea (GT) possesses beneficial effects against obesity and insulin resistance (IR). This study aimed to test the hypothesis that maternal green tea supplementation alleviates metabolic derangements in offspring born from obese dams. Female SD rats were fed a low‐fat (LF), high‐fat (HF), 0.75% or 1.0% GT extract added HF diet (HT1, HT2) from 8 wk before mating until end of lactation. GT dams tended to gain less during gestation but birth weight and litter size were not affected. There were 6 dam/pup diet groups: LF/HF, HF/HF, HF/HT1, HF/HT2, HT1/HF and HT2/HF. On d21 postpartum, GT dams had less visceral fat and lower insulin level than HF dams (n=8, p < 0.05). Body weight of pups from GT dams on d21 were lower (p < 0.05), but not on d91 than from HF dams. On d91, HT1/HF and HT2/HF rats had lower IR index than HF/HF and HF/HT1 rats (n=7–9, p < 0.02). Rats from GT dams had higher expression of uncoupling protein 1 in brown adipose tissue; adiponectin in adipocytes and peroxisome proliferator‐activated receptor‐γ coactivator‐1α in gastrocnemius but lower expression of hepatic glucose‐6‐phosphatase than HF/HF rats (p < 0.05). Hence, offspring of GT dams had better glucose handling and insulin sensitivity, and maternal exposure is more effective than postnatal GT supplementation. These results highlight the potential impact of dietary bioactive compounds on fetal development via metabolic programming. (Supported by a HKU grant)

Grant Funding Source: The University of Hong Kong
Increased homotypic adhesion and resistance to apoptosis of lymphovascular tumor emboli is due to selective proteolytic processing of E‐cadherin rather than its mere overexpression
Authors: Barsky Sanford H , Xiao Yi , Yearsley Kurtis , Ye Yin
Published: June 2021

Previous studies with a human xenograft model of inflammatory breast cancer, MARY‐X, demonstrated that overexpressed E‐cadherin moderated the formation of the lymphovascular embolus in vivo and tumoral spheroids of super‐high density in vitro. Because of the uniqueness of this model we decided to examine E‐cadherin more closely. In both MARY‐X and the MARY‐X spheroids, Western blot revealed 5 E‐cadherin bands: one full length (120 kDa) and four fragments: E‐cad/NFT1 (100 kDa), E‐cad/NTF2 (95 kDa), E‐cad/NTF3 (85 kDa), E‐cad/NTF4 (80kDa). Compared to MARY‐X, NFT1 was dramatically increased in the MARY‐X spheroids whereas NFT2‐4 were significantly decreased. These E‐cadherin fragments were produced by specific proteolytic cleavage based on inhibitor studies: NFT1 was produced by calpain, NFT2 by γ‐secretase, NFT3 by a matrix metalloproteinase and NFT4 by an unknown mechanism. Predictably levels of calpain activity were significantly increased in the MARY‐X spheroids. NFT1 generated by increased calpain activity retained the p120ctn binding site but lost both the β‐catenin and α‐binding sites, facilitating its 360° distribution around the cell. Calpain inhibition decreased NFT1 without any affect on full length E‐cadherin yet induced disadherence of intact spheroids and prevented the assembly of de novo spheroids. This work was supported by the U.S. Army Breast Cancer Research Program grant W81XWH‐06‐1‐0631.
Mice deficient in epidermal PPARγ exhibit loss of sebaceous glands as well as augmented apoptosis and inflammation in response to ultraviolet B
Authors: Konger Raymond Lloyd , Martel Kellie C , DaSilva Sonia C , Sahu Ravi P
Published: June 2021

Previous pharmacological studies have provided conflicting results regarding the relevance of PPARγ in photobiology. In these current studies, we utilize a cytokeratin 14 promoter driven CRE‐LoxP strategy to examine how loss of epidermal PPARγ (KO mice) alters ultraviolet B (UVB) responses in mouse skin. We first demonstrate an increase in PPARγ ligand production in lipid extracts of mouse epidermis irradiated with 1500 J/m2 of UVB. We next show that after 24 hrs, KO mice exhibit a marked increase (> 4‐fold) in UVB‐induced apoptosis as measured by epidermal caspase 3 activity. We next assessed UVB‐induced inflammatory responses. At 24 and 72 hrs post‐irradiation, KO mice exhibited increased inflammation as determined by significant increases in skin thickness, myeloperoxidase activity and inflammatory cell infiltrates. Given that inflammatory infiltrates typically occur at 36–48 hrs post‐UVB, these studies suggest an earlier onset of inflammation in KO mice. Finally, in data unrelated to UVB irradiation, adult KO mice also exhibited almost complete loss of sebaceous glands. This data provides compelling evidence that epidermal PPARγ serves an important role in regulating the acute epidermal photoresponse and is necessary for sebaceous gland development and/or maintenance. Supported by funding by the National Institutes of Health and the Prevent Cancer Foundation.
Bile Acids Increase Proinflammatory Gene Expression in Hepatocytes
Authors: Allen Katryn M. , Copple Bryan
Published: June 2021

Inflammation has been shown to exacerbate liver injury during cholestasis. The stimulus for proinflammatory gene expression in cell types, such as hepatocytes, during cholestasis is not known. Bile acid levels are increased during cholestasis and activate several signaling pathways in hepatocytes. Therefore, the hypothesis was tested that bile acids increase proinflammatory gene expression in hepatocytes. Treatment of primary mouse hepatocytes with deoxycholic acid, chenodeoxycholic acid or taurocholic acid increased expression of cytokines (IL‐1β, IL‐6, IL‐10, IL‐17d), chemokines (KC, IP‐10, I‐TAC, MCP‐1, RANTES, MCP‐3, MIP‐1α, MIP‐2α, MIP‐3α, LIX, SR‐PSOX, and BCL), adhesion molecules (ICAM‐1, VCAM‐1), and other proteins (e.g. PAI‐1) that promote inflammation. We previously demonstrated that early growth response factor‐1 (Egr‐1) is required for inflammation in the liver during cholestasis. Therefore, we next determined whether upregulation of proinflammatory mediators in hepatocytes by bile acids and in vivo after bile duct ligation (BDL) required Egr‐1. Upregulation of MCP‐3, PAI‐1, VCAM‐1, and IL‐17d in bile acid‐treated hepatocytes and in the liver after BDL required Egr‐1. These data demonstrate that bile acids are inflammagens and increase proinflammatory gene expression in hepatocytes which may in turn drive inflammation during cholestasis. Supported by NIH grant DK073566.
Induction of Apoptosis by Methanol Extract of Cnidium officinale Makino through Activation of Caspase‐3 and p53 in Human Liver cancer HepG2 cell
Authors: An Jeong‐Cheol , Hong Hee‐Ok , Hwang Seong‐Gu
Published: June 2021

In the present study, we investigated if a methanol extract of Cnidium officinale Makino (CO) can induce the apoptosis against human liver cancer HepG2 cell. HepG2 cells were treated with medium containing 0, 0.1, 0.25, 0.5, 0.75, 1 mg/ml of CO methanol extract. Cell proliferation assay was undertaken after 24hrs of incubation. Caspase‐3 and p53 mRNA expression were measured by RT‐PCR and protein expression by Western Blot. The antioxidant activity of CO was analyzed using DPPH assay. The HepG2 proliferation was inhibited by CO extract in dose dependent manner. The CO promoted activation of apoptotic caspase‐3 and p53 genes. The CO showed free radical scavenging activity dose‐dependently. The results suggested that CO‐induced apoptosis of HepG2 cells may be mediated through the activation of apoptotic pathway involving caspase‐3 and p53. In summary, CO may provide a new therapeutic option as a potential anticancer agent in the treatment of liver cancer.
MG624, an α7‐nicotinic receptor antagonist inhibits growth of human small cell lung cancer by induction of p73
Authors: Brown Kathleen C. , Witte Ted R. , Hardman W. Elaine , Mangiarua Elsa I. , Grover Lawrence M. , Egleton Richard D. , Dasgupta Piyali
Published: June 2021

Small cell lung cancer (SCLC) is characterized by rapid progression, early metastasis and low survival rates. Smoking has been correlated to 90% of all reported SCLC cases suggesting that tobacco components like nicotine contribute to the pathophysiology of this disease. Recent studies showed that SCLC cells have a functional cholinergic loop, and that both acetylcholine and nicotine stimulate SCLC cell growth. We hypothesized that sustained exposure to nicotine stimulates the growth of the human SCLC cell lines via the α7‐nicotinic acetylcholine receptor (nAChR); therefore, α7‐nAChR antagonists should be useful in attenuating the growth of human SCLCs. We tested a panel of α7‐nAChR antagonists for their growth inhibitory effects on DMS114 human SCLC cells. We observed that MG624 displays potent growth‐inhibitory activity on multiple human SCLC cell lines. TUNEL and caspase‐3 cleavage assays showed that MG624 induced robust apoptosis in human SCLC cells. MG624 also suppressed angiogenesis in “Matrigel Assays,” as well as in “Ex vivo rat aortic ring angiogenesis assays.” The apoptotic activity of MG624 was mediated by p73 and the Akt pathway. Our data suggest that MG624 may have potential applications for the treatment of human SCLCs. Funding for our study was supported by the ASPET‐Astellas Foundation, an MU‐ADVANCE Fellowship, Sigma Xi GIAR, and the Flight Attendant Medical Research Institute YCSA Grant.
Identification and Functional Validation of Thymosin Beta4 as a New Tumor Progression Biomarker for Oral Cancer by Tissue MALDI‐Imaging Mass Spectrometry
Authors: Lai Tsung‐ching , Jan Yi‐Hua , Chang Jeng‐Shou , Chiu Tony , Hsiao Michael
Published: June 2021

Oral cancer is the sixth lethal cancer in Taiwan. It mainly occurs in Central and Southeast Asia. Here we used MALDI‐imaging MS and LC MS/MS for the identification of new oral cancer biomarkers. Protein lysates of normal keratinocyte and oral cancer cell lines were subjected to 1D SDS‐PAGE‐based LC‐MS/MS analysis. All proteins were estimated by comparison with software, Scaffold. We also used MALDI‐imaging MS to analyze different clinical stage patient samples. Candidate proteins, recognized by FlexImage software, were compared with the databases from LC MS/MS. Furthermore, we validated candidate proteins by quantitative PCR and immunohistochemistry. Our results of MALDI imaging analysis showed the expression of Thymosin β4 correlated with malignant progression of the oral cancers. The protein expression of Thymosin β4 in oral clinical N/T paired tissue array was determined by immunohistochemistry analysis and showed that Thymosin β4 overexpression was found mainly in malignant stage of oral cancer samples and significantly associated with poor clinical outcome. In addition, mRNA level of Thymosin β4 showed similar trends in different stages of N/T paired oral cancer samples by real‐time semi‐quantitative PCR. Knockdown of Thymosin β4 expression by shRNA inhibited the invasive ability in oral cancer cell lines. In conclusions, we have identified a 5 kDa protein, Thymosin beta4, by MALDI‐Imaging MS analysis. Overexpression of Thymosin beta4 was found to be associated with oral cancer progression. The possible mechanisms were explored.
Activated Farnesoid X Receptor inhibits growth of tamoxifen‐resistant breast cancer cells.
Authors: Giordano Cinzia , Vizza Donatella , Panza Salvatore , Barone Ines , Bonofiglio Daniela , Fuqua Suzanne A , Catalano Stefania , Andò Sebastiano
Published: June 2021

Background

Farnesoid X Receptor (FXR) is mainly expressed in enterohepatic district where regulates lipids metabolism and glucose homeostasis. Recently, new functions of FXR were discovered including its capability to induces apoptosis in breast cancer cells, but the role of FXR in modulating growth of these cells are not fully elucidated.

Results

In this study we evaluated the effects of chenodeoxycholic acid (CDCA), natural ligand of FXR, on growth of breast cancer cell lines sensitive (MCF‐7) and resistant to tamoxifen (MCF‐7TR). Using MTT assay we found that CDCA as well as GW4064, a specific synthetic FXR agonist, reduced cell viability in MCF‐7 and in a higher extent in MCF‐7TR cells. Furthermore, results from anchorage independent growth assay (soft agar) demonstrated that CDCA inhibited tamoxifen‐resistant colonies formation as well as Epidermal Growth Factor (EGF) induced growth in MCF‐7TR cells. Western blot analysis and real‐time RT‐PCR revealed that FXR activation determined a reduction of HER2 expression and consequently an inhibition of EGF‐mediated HER2 and p42/44 MAPK phosphorylation in MCF‐7 TR cells.

Conclusion

These data suggest that FXR ligand‐dependent activity, blocking HER2/MAPK signaling, may reverse antiestrogen resistance in human breast cancer cells and could represent a new therapeutic tool to treat breast cancer patients that develop hormonal resistance.
A New Application for the Drug Tranilast: Effects on Breast Cancer Cell Proliferation, Migration, and Invasion
Authors: Jothy Serge , Ace Olga , Subramaniam Venkateswaran , Prud'homme Gerald
Published: June 2021

Background

Tranilast (Rizaben®) has been used to treat allergic and auto‐immune diseases for the last 26 years. The objective of this study was to investigate whether tranilast also has an effect on breast cancer cell biological features relevant to cancer progression, based on the its mechanism of action which involves TGFβsignaling.

Methods

Cultures of the BT‐474 human breast cancer cell line were used. Cell proliferation, clonogenicity, cell migration, endoglin and MMP‐9 expression were assessed by: MTT assay, soft agar colony formation, wound assay and immunocytochemistry respectively.

Results

Tranilast was found to inhibit breast cancer cell proliferation by 70%. In addition, the drug decreased by 54% the number and size of cell colonies grown in soft agar, consistent with a decrease in clonogenicity. The closure of wounds made in cancer cell monolayers was slowed (55%) by Tranilast, consistent with an inhibitory effect on tumor cell migration. Endoglin, which plays a role in TGFβ signaling, was downregulated by the drug. MMP‐9, which is a metalloproteinase involved in tumor invasion and metastasis was also decreased by tranilast.

Conclusions

These results indicate that tranilast exerts inhibitory effects on key biological features of human breast cancer cells. The results are consistent with an anti‐tumor effect which can be exploited in preclinical and clinical therapeutic trials.
ICAM‐1 modulates expression of interferon‐γ (IFN‐γ) in lung neutrophils during Streptococcus pneumoniae pneumonia
Authors: Gomez John C. , Yamada Mitsuhiro , Doerschuk Claire M.
Published: June 2021

Lung neutrophils produce IFN‐γ in pneumonia induced by S. pneumoniae. Previous studies showed that neutrophil production of IFN‐γ requires the leukocyte integrin CD11/CD18. ICAM‐1 is the ubiquitously expressed cellular counter‐receptor for CD11/CD18. It induces signaling in leukocytes by binding CD11/CD18 on the leukocyte surface or after its ligation by CD11/CD18. This study determined if ICAM‐1 was required for production of IFN‐γ by neutrophils during pneumonia induced by S. pneumoniae. IFN‐γ production by neutrophils in the lungs of wild type (WT) and ICAM‐1−/− mice 24 hours after intratracheal instillation of S. pneumoniae was determined by flow cytometry. The percentage of lung neutrophils that produced intracellular IFN‐γ was less in ICAM‐1−/− mice compared to WT mice (8.1 ± 0.9% vs. 15.2 ± 2%, p<0.005). The total number of lung neutrophils was not different in WT and ICAM‐1 −/− mice (27 ± 3.1 × 10 6 vs. 24 ± 1.7 × 106). ICAM‐1 regulates IFN‐γ production by neutrophils during S. pneumoniae pneumonia, through initiating signaling by either activating CD11/CD18 or by its own ligation, and may contribute to host defense. Funding: HL 048160 and HL 052466.
Constitutive release of a novel free radical scavenger by stellate cells that protects hepatocytes from reperfusion injury
Authors: Gandhi Chandrashekhar R
Published: June 2021

Reactive oxygen species (ROS) produced by inflammatory cells cause injury to hepatocytes. Adjacently located hepatic stellate cells (HSCs) can influence the survival and functions of hepatocytes. We investigated role of HSCs in superoxide‐induced death of hepatocytes. Medium conditioned by HSCs prevented superoxide‐induced death of hepatocytes. The free radical scavenger released by HSCs (mw greater than 100,000 kDa) is heat (70ºC)‐stable and pH (4.5–8.5)‐resistant. The scavenger was partially purified by alcohol precipitation, acetate (pH 4.5) treatment and elution from DEAE cellulose (DE52) resin with 100–200 mM sodium chloride. The DE52 fraction did not contain superoxide dismutase (SOD), a major antioxidant produced by several cell types. Further separation by sephadex gel filtration yielded 3 distinct protein peaks; while none of the peaks individually protected hepatocytes from superoxide‐induced death, combination of peaks 2 and 3 reestablished the superoxide‐scavenging activity. The protein purified by gel chromatography was also found to protect the rat liver from warm ischemia/reperfusion injury. These results demonstrate for the first time that HSCs might protect hepatocytes from oxidative damage by producing a novel free radical scavenger, further purification of which may lead to the isolation of a powerful antioxidant with clinical application. Supported by an NIH grant (DK 54411).
Quantitative cytoplasmic TUNEL: the method to measure apoptosis and necrosis coexisting in a single liver or kidney cell
Authors: Basnakian Alexei G. , Apostolov Eugene O. , Braman Nicholas , Cortez Lisa , Hinson Jack , Shah Sudhir V.
Published: June 2021

Apoptosis in solid organs often develops to and overlaps with necrosis, presenting so called “aponecrosis.” Methods for simultaneous quantification of apoptosis and necrosis in a single cell do not exist. We tested whether quantitative fluorescent TUNEL assay that measures 3'OH DNA termini produced by endogenous endonucleases in both apoptosis and necrosis, and in particular, the quantification of the cytoplasmic fraction of TUNEL that is a sign of necrosis, can be applied for apoptosis and necrosis measurement in single cells in two in vivo models: renal ischemia‐reperfusion (IR) in rats and liver acetaminophen (APAP)‐induced injury in mice. Our data showed that renal IR results in full spectrum of tubular epithelial cells undergoing apoptosis conversion to complete necrosis (~50% of total TUNEL leaked to cytoplasm), with the maximum necrosis concentrating in cortico‐medullary junction of the kidney. In centrilobular hepatocytes, the TUNEL‐positive DNA also significantly leaked to cytoplasm, but it reached a maximum of 22% of total TUNEL, indicating less secondary necrosis compared to the kidney. Overall, these data demonstrated that measurement of nuclear and cytoplasmic TUNEL is a useful method for the assessment of coexisting apoptosis and necrosis in individual cells in vivo and a tool to measure progression of kidney or liver injury. The study was supported by NIH/NIDDK grant to A.G.B, and VA Merit Review grants to A.G.B. and S.V.S.
Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation
Authors: Liu Bowen , Paranjpe Shirish , Bowen William , Bell Aaron , Luo Jian‐Hua , Yu Yan‐Ping , Mars Wendy , Michalopoulos George
Published: June 2021

Glypicans are heparan sulfate proteoglycans that are bound to cell surface through a glycosylphosphatidylinositol (GPI) anchor. Our study focused on glypican 3 (GPC3). Loss of function mutations of GPC3 result in the Simpson‐Golabi‐Behmel syndrome, an X‐linked disorder characterized by pre‐ and postnatal liver and other organ overgrowth. GPC3 is over‐expressed in human hepatocellular carcinoma; however, its role in normal liver regeneration and hepatocyte proliferation is unknown. In this study, we investigated the role of GPC3 in hepatocyte proliferation. We found that GPC3 mRNA and protein begin to increase at 2 days after hepatectomy with a peak expression at day 5. In hepatocyte cultures GPC3 reaches plateau level when hepatocyte proliferation decreases. In vitro studies using Morpholino oligonucleotides showed that blocking GPC3 expression promoted hepatocyte growth. Yeast two hybrid assays revealed that GPC3 interacts with CD81, a member of the tetraspanin family. We found that CD81 level also increased from day 2 after PHx and towards the end of regeneration. Immunofluorescence showed that CD81 and GPC3 co‐localize at day 2 and day 6 after hepatectomy. Co‐immunoprecipitation validated the interaction of GPC3 and CD81. Our results indicate that GPC3 may be a negative regulator of liver regeneration and hepatocyte proliferation, and this regulation may involve CD81.
Extracellular matrix changes in melanoma promote tumor cell migration
Authors: Grahovac Jelena , Wu Qian , Conrads Thomas P , Wells Alan
Published: June 2021

In melanoma, invasion through the extracellular matrix (ECM) is a harbinger of dissemination and mortality. We find that changes in ECM composition of invasive human melanoma specimens resemble changes found during skin wound healing. Most highly upregulated in invasive melanoma was Tenascin C (TN‐C). Simultaneously, the matrix proteoglycans decorin and lumican were downregulated and the level of collagen I was decreased as the dermal matrix became ‘less mature’. The cellular origin of these matrix changes are not fully settled; still the melanoma cell lines, derived from tumors of different stages, express varied increased levels of TN‐C and fibronectin and decreased levels of collagen I compared to normal human melanocytes. Cells expressing higher amounts of TN‐C migrate faster in wound healing assays; on TN‐C pre‐coated surfaces both melanoma cells and normal melanocytes increase migration. Our group previously showed that the EGF‐like repeats of TN‐C are able to bind the EGFR with μM affinity and preferentially activate the motogenic signaling cascades. This upregulation of EGFR‐activating TN‐C is concomitant with downregulation of decorin and lumican, which have been reported to dampen EGFR signaling. Therefore, we hypothesize that the increased levels of TN‐C in melanomas promote tumor cell migration and invasion heralding dissemination through enhanced EGFR signaling.

Funding: Internal (AW).
Removal of Integrin Linked Kinase from Hepatocyte Leads to a Prolonged Proliferative Repose to a Mitogenic Stimuli
Authors: Donthamsetty Shashikiran , Michalopoulos George
Published: June 2021

We have recently demonstrated that disruption of ECM/Integrin signaling via elimination of Integrin linked kinase (ILK) interferes with signals leading to termination of regeneration. This study investigates the role of ILK in hepatocyte proliferation induced by Phenobarbital (PB). PB was administered in drinking water for 10 days. Steady supply of PB led to a modest nuclear translocation of CAR in WT and ILK−/ − mice. CAR target like CYP2B6 was also increased in both the groups. We then looked at the hyperplastic response of PB in both the groups. 10 days of phenobarbital administration led to a 3 times increased liver to body weight ratio as compared to 0 day in the ILK−/ − mice while the WT mice show only a 2 times increase. ILK−/ − mice also have increased percent of PCNA positive cells and mitotic cells even at day 5 and 10 of phenobarbital administration suggestion a prolonged proliferative response. Signaling pathways altered in ILK−/ − mice included enhanced HGF‐cMet signaling, increased downstream activation of mitogenic molecules like cyclin D1, increased activation of YAP pathways and downregulation mitoinhibitory molecules like TGF‐β1. In summarize, ECM proteins communicate with the signaling machinery of dividing cells via ILK to regulate hepatocyte proliferation and termination of the proliferative response. Lack of ILK in the hepatocytes have prolonged proliferative response to a mitogenic stimuli suggesting defect in termination of proliferative response.

The work is supported by a R01 grant (5R01CA035373‐26) titled “HGF in Liver Development, Regeneration and Neoplasia”
Genome‐wide DNA methylation profile of pluripotent stem cells
Authors: Sato Shinya , Yagi Shintaro , Shiota Kunio
Published: June 2021

DNA methylation is a vital mark for epigenetic regulation of gene expression. Here, we profiled geneome‐wide DNA methylation status in mouse pluripotent stem cells, focusing on tissue‐dependent and differentially methylated regions (T‐DMRs) of ESCs. By using T‐DMR profiling with restriction tag‐mediated amplification (D‐REAM), we identified thousands of both hypo‐ and hypermethytaed T‐DMRs characterizing the pluripotent cells. The hypomethylated T‐DMRs were found in genes involved in nuclear events as transcriptional regulation network of ESCs, and majority of them are in genes associated with CpG Islands. In addition, hypermethylatied T‐DMRs are found in genes for tissue‐specific function. Based on the T‐DMRs, we further introduce the commonality of DNA methylation profile between pluripotent stem cell lines. Our results indicate that DNA methylation profile contribute to the mechanism of pluripotency, and can make novel criteria for evaluation and standardization of pluripotent stem cells. This work is supported by the grants from the National Institute of Biomedical Innovation and the New Energy and Industrial Technology Development Organization, Japan.
Auscultation simulation system captures/replays diagnostic experiences by synchronizing sound, spatial positioning and anatomic visualizations in real‐time
Authors: Hisley Calvin , Canby Craig , Wattleworth Roberta , Winer Eliot , Foo Jung Leng , Peloquin Catherine , Martinez Marisol , Kopecky Kenneth , Gilbert Stephen
Published: June 2021

Current auscultation training systems reproduce physiologic sounds routinely heard during patient examination; however, they do not capture, model and reproduce auditory/spatial physician‐patient interactions as immersive learning experiences. We hypothesize a schema for the capture and replay of diagnostic sound patterns and spatial coordination as exemplified by stethoscope placement coupled with the synched viewing of 3D CT anatomy that would significantly increase clinical insight and recall. Our simulation system digitally captures actual physician‐patient auscultation experiences as thoracic sound combinations, stethoscope head coordinate positions within a standardized space, and diagnostic signs and impressions as dictated by the examining physician. This is accomplished using an electronic stethoscope with spatial tracking capabilities and a laptop computer. This multimedia experiential “case profile” is then additionally enhanced via 3D CT/MRI visualizations synchronized with stethoscope head placement location patterns. Finally, our training system is able to replay this coordinated experience to students interactively using fellow students registered to the standard anatomic space.
The Center for Translational Neuroscience, 5 years of progress
Authors: Garcia‐Rill Edgar
Published: June 2021

Our aims were to establish 1) a Career Development Program with mentoring/funding for 5 projects and 6 investigators, 2) a multi‐disciplinary Center, a Pilot Study Program, and recruitment of 5 investigators, and 3) two Core Facilities. All aims were exceeded. We developed a Career Development Program and Center so that clinician and basic scientists could be mentored to secure funding. We mentored and supported 10 promising investigators without a history of support. We provided access to Core Facilities to 4 other investigators, and assigned them Mentors. We awarded 5 pilot study grants and assigned them Mentors. We recruited 3 senior and 2 junior faculty and established 6 Core Facilities. We discovered two new cures, two novel treatments and one new mechanism, all with clinical implications. We generated >$13 million in new awards and published >130 articles. The projects produced a novel treatment for tinnitus that relieves the symptoms in a majority of patients, a new use for a drug that relieves the excessive reflexes induced by spinal cord injury, an effective new treatment for spatial neglect due to stroke, and a new mechanism for sleep‐wake control that may lead to the development of new anesthetics and stimulants. Oversight by a dedicated external advisory committee and considerable institutional support were critical to our success, along with selfless mentoring by dedicated senior scientists.
Structural and mechanistic diversity of ABC transporters
Authors: Rees Douglas C.
Published: June 2021

ATP Binding Cassette (ABC) transporters constitute a ubiquitous superfamily of integral membrane proteins responsible for the ATP powered membrane translocation of a wide variety of substrates. The highly conserved ABC domains defining the superfamily provide the nucleotide powered engine that drives transport. In contrast, the transmembrane domains creating the translocation pathway are more variable, with three distinct folds currently recognized. Structural analyses of three bacterial importers, the BtuCD vitamin B12 importer and homologous HI1470/1, and the high affinity methionine MetNI importer, will be discussed within the mechanistic framework of the alternating access model. The interconversion of outward and inward facing conformations of the translocation pathway is coupled to the switching between open and closed interfaces of the ABC subunits that are associated with distinct nucleotide states. As with MetNI, additional domains may be present that can regulate transport activity. An important goal is to develop quantitative models that detail the kinetic and molecular mechanisms by which ABC transporters utilize the binding and hydrolysis of ATP to power substrate translocation.
Transcription Elongation links to Histone Methylation
Authors: Jones Katherine A , Bres Vanessa , Zhang Lirong
Published: June 2021

Many steps in gene expression and mRNA biosynthesis are coupled to transcription elongation. The P‐TEF‐b complex (CycT1:CDK9) overcomes a block to transcription elongation established by the negative‐acting elongation factors, DSIF and NELF. In addition, P‐TEFb phosphorylates the RNAPII CTD, which loads complexes involved in cotranscriptional mRNA processing, surveillance, export, and histone H3K36 methylation. We recently showed that P‐TEFb associates with an alternative splicing factor, SKIP/SNW1, which is required for HIV‐1 Tat transactivation, and will discuss how SKIP stimulates transcription elongation and histone H3K4 methylation at the viral promoter. Thus communication between transcription and splicing occurs in both directions, as the SKIP splicing protein plays a direct role in transcription elongation and H3K4 methylation.

We recently reported that Spt6, a transcription elongation factor and histone H3 chaperone, binds directly to the P‐TEFb‐phosphorylated RNAPII CTD to recruit factors to the elongation complex. This megacomplex includes the Hypb/Setd2 histone methyltransferase, which is a CTD‐interacting enzyme that directs H3K36me3 to the coding region of active genes. The assembly of this complex depends upon P‐TEFb activity and is strongly up‐regulated in activated T cells.
Sus1, A Key Factor Coupling Transcription and mRNA Export
Authors: Rodriguez‐Navarro Susana
Published: June 2021

The transport of mRNA out of the nucleus is an essential step, which is necessary to accomplish gene expression. During their synthesis, mRNAs are packaged into mRNPs and exported through the nuclear pore complex (NPC). Several studies have established links between the factors involved in transcription and export of mRNA. Strong evidence that these processes are coupled was provided by the identification of yeast Sus1 as a subunit of both the SAGA complex and the TREX2 complex; the latter is associated with the NPC, and acts in mRNA export and transcription elongation. Notably, the function of both SAGA and TREX2 are conserved, as a homologous complex—known as SAGA and AMEX—have been characterized. Sus1 participates in different steps during gene expression. Our work is focus on unveiling the mechanisms that coordinate the different steps during gene expression are coupled. Here we will present new data concerning Sus1 role in SAGA and SLIK and a novel link between Sus1 and histones metabolism.
Signaling Pathways that Regulate Skeletal Muscle Atrophy and Hypertrophy
Authors: Glass David J.
Published: June 2021

Skeletal muscle atrophy is a significant co‐morbidity seen in a variety of diseases, including Congestive Heart Failure, renal failure, cancer, and AIDS. Even during simple muscle inactivity, such as when a cast is put on a limb, the affected muscle can decrease by as much as 50% in mass, with a coincident decrease in strength. Two E3 ubiquitin ligases – MuRF1 (Muscle RING Finger 1) and MAFbx (Muscle Atrophy Fbox protein, which is also known as Atrogin), are transcriptionally upregulated in all physiological settings of muscle atrophy that have been analyzed. The pathways which regulate these ligases will be discussed. Also, more recently, potential substrates of MuRF1‐ which comprise part of the skeletal muscle contractile apparatus ‐ have been elucidated. These substrates, including components of the sarcomeric thick filament, will be discussed, as well as conditions that can modulate MuRF1's activity.

An additional important modulator of muscle mass is myostatin, a TGF‐beta family member. We have recently conducted studies demonstrating the ways that myostatin perturbs muscle mass and function, which both confirms other studies showing an inhibitory effect on muscle differentiation, but which also shows several important points of cross‐talk between myostatin signaling and Akt/TORC1 signaling.
Towards light‐induced control of gene expression using RNA
Authors: Sen Dipankar , Lee Hyun‐Wu , Azrahimi Nazanin , Cernak Paul
Published: June 2021

The potential utility of catalytic RNAs and DNAs (ribozymes and deoxyribozymes, respectively) as reagents in molecular biology as well as therapeutic agents for a variety of human diseases, has long been recognized. Although naturally occurring RNA‐cleaving ribozymes are typically not subject to feedback control, rational methodologies for the creation of allosteric ribozymes, by functional combination of ribozyme and ligand‐responsive aptamer RNA elements, have existed for some years. I will describe the in vitro selection of RNA aptamers specific for binding one but not the other of two light‐induced isomers of a dihydrobenzo[e]pyrene “photochromic switch” compound, and the utilization of such an aptamer for the construction of the UG‐dihydropyrene ribozyme, an allosteric hammerhead ribozyme whose catalysis is controllable by irradiation with visible versus ultraviolet light. In the presence of micromolar concentrations of the photo‐switch compound, the ribozyme behaves as a two‐state switch, exhibiting a > 900‐fold difference in catalytic rates between the two irradiation regimes. I will then describe our efforts to use this RNA‐based system to control gene expression within yeast cells.
A Potential Role of Mevalonate Pathway in Adrenocortical Tumors
Authors: Lin Chin Jia , Lima‐Valassi Helena Panteliou , Lerario Antonio Marcondes , Mendonca Berenice Bilharinho
Published: June 2021

HMGCR (EC ) is the rate‐limiting enzyme of the mevalonate pathway, providing substrates for both protein prenylation and biosynthesis of cholesterol. HMGCR is overexpressed in several forms of human neoplasms, but its importance for adrenocortical tumors (ACTs) is unknown. Expression of HMCGR, StAR, TSPO and CYP11B1 in ACT [Adult: 17 adenomas (ACA) and 10 carcinomas (ACC); Pediatric: 13 adenomas (PAD) and 8 carcinomas (PCA)] were assessed by real‐time RT‐PCR. The viability of NCI‐H295 adrenal carcinoma cells upon mevalonate pathway inhibition was tested using a commercial assay and phosphorylation of ERK1/2 was evaluated by immunobloting. In most of ACT, expression of StAR, TSPO, and CYP11B1 was decreased while the expression of HMGCR was increased. Expression of StAR, CYP11B1 and TSPO were significantly lower in ACC than ACA (P<0.05). No differences in the expression of these genes were found between PAD and PCA. The expression of HMGCR and TSPO correlated negatively while a positive correlation between StAR, TSPO and CYP11B1 was observed. Lovastatin decreased the viability of NCI‐H295A cells. Such an effect was not seen upon treatment of zaragozic acid. Treatment with lovastatin produced a progressive decrease in ERK phosphorylation. Conclusions: HMGCR is overexpressed in ACTs and relevant for survival of neoplastic adrenocortical cells. This effect of HMGCR is probably mediated by protein prenylation rather than cholesterol synthesis.
IQGAP1 is a Novel HER2 Binding Partner and Regulates HER2‐Mediated Cell Proliferation
Authors: White Colin David , Li Zhigang , Niepel Mario , Sacks David Barry
Published: June 2021

The receptor tyrosine kinase HER2 is overexpressed in 25–30% of breast carcinomas. These tumors often have an increased rate of proliferation and give rise to more frequent metastases than HER2(−) neoplasms. HER2(+) breast cancer is treated with Trastuzumab, but ~60% of patients do not respond. Of those who do, ~15% subsequently develop metastatic disease. An understanding of the molecular mechanisms of HER2 signaling is necessary to develop novel therapeutics. IQGAP1 is a ubiquitously expressed 190 kDa scaffold protein. Through interaction with multiple binding partners, IQGAP1 regulates processes such as cell proliferation and migration, cell‐cell adhesion and cytoskeletal remodeling. IQGAP1 plays defined roles in several human malignancies, including those of the breast. Here, we investigate the interaction between IQGAP1 and HER2. In vitro assays using purified IQGAP1 and the GST‐tagged intracellular domain of HER2 (GST‐HER2) revealed that IQGAP1 binds HER2. GST‐HER2 binds IQGAP1 in Sk‐BR‐3 cells, and endogenous HER2 co‐immunoprecipitates with endogenous IQGAP1 from cell lysates. Analogous to other IQGAP1 binding partners, calmodulin abrogates the association of IQGAP1 with HER2. Overexpression of IQGAP1 significantly increases Sk‐BR‐3 cell proliferation, while siRNA‐mediated knockdown of IQGAP1 significantly reduces proliferation. These data suggest that IQGAP1 may be a potential target for HER2(+) breast cancer therapy.

Work in the authors' laboratory is funded by the NIH (to D.B.S.) and the DoD BCRP (to C.D.W).
Laminin 332 expression in mammary cell lines
Authors: Carpenter Philip M , Ohanian Gary K , Zhang Zesong , Shanazarian Meghedy , Madatovian Harut
Published: June 2021

Laminin 332 (LN332), produced by normal mammary epithelium, can induce motility and invasion in breast cancer cells. Although down regulated in most breast carcinomas, LN332 is present in triple negative (TN) carcinomas, those without estrogen (ER) and progesterone receptors (PR) and no HER2 gene amplification. Thus, we sought to determine LN332 expression patterns in breast carcinoma lines, including the ER positive (MCF‐7), HER2 gene amplified (SKBR3), and TN (BT‐20, MDA‐MB‐231) carcinomas, and immortalized normal breast epithelium (184A1). By immunoperoxidase and Western blotting, 184A1, MDA‐MB‐231 and to a lesser extent, BT‐20 showed LN332 β3 chain staining. No expression was detected for SKBR3 or MCF‐7. By RT‐PCR, MDA‐MB‐231 expression of all LN332 chains was higher than in MCF‐7 and BT‐20, but less than the values for 184A1. Finally, to determine if the cell lines' LN332 was sufficient to induce motility, serum‐free conditioned medium (CM) from all lines was incubated with MCF‐7, and scattering was measured. Only 184‐A1 and MDA‐MB‐231 CM induced motility, but antibodies against α3 integrin, which mediates LN332 motility in MCF‐7, only inhibited 184‐A1 induced motility, indicating that other motility factors may be produced by MDA‐MB‐231. In summary, LN332 protein was only measurable in BT‐20 and MDA‐MB‐231, both of which are TN tumors, and thus may prove useful for the study of LN332 in breast cancer.
The proliferative effects of sympathetic nerves and neuropeptide Y in a 4T1 cell breast cancer model
Authors: Medeiros Philip J , Jackson Dwayne N
Published: June 2021

The functional impact of sympathetically released neuropeptide Y (NPY) on breast cancer has been a question of growing interest due to the expression of NPY receptors in several cancer cell lines. Recently, our group reported that the 4T1 cell line expresses NPY receptors (Y1 &Y2) and mammary tumors grown from this cell line are sympathetically innervated. In the current study we evaluated the effect of NPY on 4T1 cell proliferation using 96‐hr treatment with NPY (10−12 to 10−6M) and observed an increase in proliferation (MTS‐based assay) in treated cells compared to controls (10−8: 55%, 10−7:122%, 10−6: 157%, P < 0.05). Additionally, we examined the effect of chemical sympathectomy (via 6‐hydroxydopamine bromide, 6OHDA) on in vivo tumor growth. 4T1 cells (105)were injected into the sympathectomized inguinal mammary fat pad (200 μg 6OHDA × 2 localized injections, 1 week prior to 4T1 inoculation) and intact fat pad of female BALB/c mice (n = 5/group). Tumor growth was monitored by caliper measures over 21 days and animals were sacrificed and tumors were harvested and weighed. Tumor volume was lower in sympathectomized animals compared to intact animals at all time points (wk1: 70%, wk2: 44% & wk3: 45%, P < 0.05), and final tumor mass was 52% of intact tumors (P < 0.05). These findings suggest that NPY and sympathetic nerves impact cellular proliferation in this model. NSERC & Canadian Breast Cancer Foundation.
Single v dual knockdowns of the chromatin remodeling ATPase, SNF2L and its isoform, SNF2LT have similar effects on DNA damage but opposite effects on the DNA damage response, cell cycle arrest and apoptosis
Authors: Barsky Sanford H , Xiao Yi , Yearsley Kurtis , Ye Yin
Published: June 2021

SNF2L, a chromatin‐remodeling ATPase, is nearly ubiquitously expressed in most malignant and normal cell lines, but our previous studies have shown that only malignant cell lines are sensitive to its knockdown. We have recently observed the presence of a truncated isoform of SNF2L, termed SNF2LT also ubiquitously expressed. Single knockdown of either SNF2L or SNF2LT caused DNA damage, a DNA damage response, cell cycle arrest and apoptosis in a number of highly malignant cell lines including Hela, the MDA‐MB‐468 and ‐231. Both single and dual knockdown similarly caused DNA damage measured by COMET as well as by increased p‐H2AX. However single v dual knockdown had opposite effects on the DNA damage response, cell cycle arrest and apoptosis. Single knockdown of either SNF2L or SNF2LT increased phosphorylated p53 and its downstream genes, GADD45A, 14‐3‐3σ and p21 whereas double knockdown had no effect. Single knockdown caused a G2/M arrest with upregulation of p‐cdc2 whereas double knockdown had no effect. Single knockdown caused profound apoptosis with increased Apaf‐1/caspase 9 whereas double knockdown actually increased cellular proliferation. The results suggest that the ratio of SNF2L/SNF2LT determines how a cancer cell responds to DNA damage, either by initiating a damage response which leads to apoptosis or ignoring the DNA damage and continuing to proliferate. This work was supported by the U.S. Army Breast Cancer Research Program grant W81XWH‐06‐1‐0631.
Optional splicing of T‐Cell Intercellular antigen‐1 (TIA‐1), a RNA‐binding Protein Regulates VEGF Expression in Colorectal Cancer
Authors: Hamdollah‐Zadeh Maryam , Hoareau‐Aveilla Coralie , Varey Alexander H , Amin Ellianna Mohamed , Ladomery Michael R , Harper Steve J , Bates Dave O
Published: June 2021

To determine the role of post‐transcriptional regulation of Vascular Endothelial Growth Factor (VEGF) in colorectal cancer we investigated the relationship between VEGF and the RNA binding protein TIA‐1. An endogenous splice variant of TIA‐1 was expressed in 66% of colon cancer samples and in cancer cells with high angiogenic VEGFxxx expression. This splice variant resulted in a truncated protein termed short TIA‐1 (sTIA‐1). Whereas full length TIA‐1 bound to the VEGFxxx isoform (RNA immunoprecipitation, MS2‐Maltose Binding Protein assay) there was no TIA‐1 binding in cells expressing sTIA‐1. siRNA knockdown of TIA‐1 resulted in higher expression of total VEGF. Using VEGF terminal exon luciferase reporter constructs, TIA‐1 reduced translation of the angiogenic (exon 8a) but not the anti‐angiogenic (exon 8b) isoforms.

In summary, we show that over expression of the wild type TIA‐1 in adenocarcinoma cells expressing sTIA‐1 results in restoration of the anti‐angiogenic VEGF isoforms. This suggest that TIA‐1 can act as both a splicing and splice specific translational regulator of VEGF and alternative splicing of TIA‐1 could lead to enhanced angiogenesis in colorectal cancer. Supported by AICR.
Detecting protein unfolding with a UV light pen
Authors: West Lori , Sherwood Laura , Bergeron Sean , DeLaLuz Paul
Published: June 2021

Green fluorescent protein (GFP) has become increasingly useful in both research and educational settings because of its ease of detection. Our goal was to develop a low tech way in which to use GFP to demonstrate the effects of denaturants on protein folding. This technique would be especially practical for high schools and small colleges where access to specialized equipment is limited. Crude lysate from E. coli cells expressing GFP was used to monitor the effects of SDS and urea on fluorescence and hence folding of GFP. We first confirmed that GFP fluorescence could be observed visually by excitation with a UV light pen (Bio‐Rad®). We then denatured the protein with 5M urea or 1% SDS and again monitored fluorescence. We were unable to detect fluorescence as expected for denatured GFP. We confirmed these results by performing an unfolding curve with concentrations of urea between 1M and 5M and SDS between 0.25% and 1%. Although we were unable to visually detect a significant change in fluorescence at the intermediate concentrations of urea and SDS, we were able to observe differences between fully folded GFP and unfolded GFP using a UV light pen.