bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of N‐linked Glycosylation in Drosophila Development
Authors: Thomas Morgan D. , Selva Erica M. 
Published: November 2023

Asparagine‐linked or N‐linked glycosylation is an important post‐translational modification pathway that adds 14‐sugar oligosaccharide chains to the Asparagine of specific Asn‐Xaa‐Ser/Thr sequences in target proteins on the luminal side of the endoplasmic reticulum. These glycan “tags” are necessary for multiple cell functions including cell‐cell recognition, extracellular signal regulation, and proper protein folding. The focus of this study is to examine the effect of loss of function mutations in the N‐linked pathway during development. A group of human diseases, congenital disorders of glycosylation (CDG), arise from mutations in the genes involved in various steps of this pathway. CDGs display severe and pleotropic phenotypes, which reflect the universal importance of this protein modification, but most cases display phenotypes that arise from impaired neuronal function. The two specific genes under study in this project are alg9 and alg10. Each gene encodes a glycosyltranferase that adds a sugar residue to the growing oligosaccharide chain before it is transferred en masse to a target protein. The Drosophila eye is used as a model organ to study the effects of loss‐of‐function mutations in these genes as it serves as a surrogate for neuronal development. In adult flies, these mutations yield a small rough eye phenotype, which is more severe in alg9, as it acts five steps before alg10 in the pathway. In order to determine the molecular basis of this phenotype, larval eye discs that are homozygous mutate for alg9 and alg10 were dissected, stained for different glycoprotein and neuronal markers, and then imaged using confocal microscopy. Using ELAV, a neuron specific marker, we observed normally differentiated neurons in the eye disc. However, these mutations interrupt proper glycoprotein trafficking, as Chaoptin, a photoreceptor specific cell surface adhesion molecule, accumulates in the cell bodies of alg9 and alg10 mutant photoreceptors. Presence of cleaved Caspase‐3 later in eye development suggests that the accumulation of glycoprotein in the cell body eventually leads to photoreceptor apoptosis. Photoreceptor death likely continues through pupal development resulting in the reduced number of photoreceptors seen in alg10 adult eyes and an almost complete absence in alg9 adult eyes. These results indicate that CDG patients may have normal neuronal specification and differentiation, but experience neuronal deficits due to intracellular accumulation of glycoproteins leading to cell death. These data also suggest induction of the unfolded protein response (UPR) resulting in apoptosis may play a role in this process. Markers of endoplasmic reticulum stress and the unfolded protein response such as XBP1 and BiP will be studied in the future.Support or Funding InformationHoward Hughes Medical Institute Summer Fellowship, 4 University of Delaware Supply and Expense Grants
Downregulating FUK to Get Unstuck: Altered Fucosylation in Melanoma Promotes Tumor Development and Metastasis
Authors: Lau Eric Kirk , Feng Yongmei , Claps Giuseppina , Fukuda Michiko N , Perlina Ally , Donn Dylan , Jilaveanu Lucia , Kluger Harriet , Freeze Hudson H , Ronai Ze'ev A 
Published: November 2023

Melanoma, one of the most lethal skin cancers worldwide, is characterized by a propensity to metastasize. Thus, defining mechanisms that govern metastatic propensity represents an urgent clinical need. Here, we report that Protein Kinase C‐epsilon (PKCe)‐Activating Transcription Factor‐2 (ATF2) signaling controls the motility and invasiveness of melanoma. PKCɛ‐phosphorylated ATF2 transcriptionally represses fucokinase (FUK), a key upstream regulatory kinase of the fucose salvage pathway, which directs the conversion of L‐fucose to GDP‐L‐fucose that is used to fucosylate proteins in cells. Primary melanocytes and early stage melanoma cell lines express low levels of PKCe‐phosphorylated ATF2, which permit the expression of FUK, enabling cellular fucosylation, which promotes cellular adhesion and reduces motility. In contrast, increased levels of PKCɛ and transcriptionally active ATF2, as observed in advanced melanomas, repress FUK transcription. Consequently, reduced levels of cellular fucosylation attenuate adhesion and increase motility. Significantly, increasing tumor fucosylation either by dietary L‐fucose supplementation or by genetic manipulation of murine Fuk expression blunts primary tumor growth, increases intratumoral NK cell populations, and decreases metastatic spread to the lung in murine isograft models. Tumor microarray analysis of > 300 human melanoma specimens confirmed reduced fucosylation in metastatic vs. primary tumors and revealed that high levels of fucosylation in primary melanomas connote increased overall survival rates. Collectively, our findings identify the regulation of fucosylation by ATF2 and its importance in melanoma development and metastasis.Support or Funding InformationWe would like to gratefully acknowledge support from NCI PO1 (CA128814), RO1 (CA179170), Hervey Family Non‐endowment Fund at the San Diego Foundation and from a Melanoma Research Foundation grant to Z.A.R. and K99 (CA172705) grant and T32 (CA121949) fellowship to E.L., and from NIH (R01DK9955) to H.H.F. This work was also supported by the Assistant Secretary of Defense for Health Affairs through the Peer‐Reviewed Cancer Program under Award No. W81XWH‐14‐1‐0127 to Z.A.R.
Transcriptomic and MicroRNA Analyses Identify Gene Networks Regulated by Eicosapentaenoic Acid in Brown Adipose Tissue from Diet‐Induced Obese Mice
Authors: Pahlavani Mandana , Wijayatunga Nadeeja N , Kottapalli Rao , Ramalingam Latha , Gunaratne Preethi H , Coarfa Cristian , Rajapakshe Kimal , Kalupahana Nishan S , Moustaid‐Moussa Naima 
Published: November 2023

Brown adipose tissue (BAT) dissipates chemical energy as heat and protects against obesity by increasing energy expenditure. In recent years, interest in BAT as an anti‐obesity target has increased. However mechanisms mediating regulation of BAT by dietary factors remain largely unexplored at the mechanistic level. Hence, we used RNA sequencing (RNA‐Seq) and microRNA (miRNA) profiling, as powerful methods to identify novel regulators of BAT transcriptome. These techniques were applied on BAT from C57BL/6J mice fed either a high fat diet (HF, 45% kcal fat) or HF diet supplemented with EPA (HF‐EPA containing 36g EPA/kg diet) for 11 weeks, to characterize differential gene and miRNA expression between HF and HF‐EPA fed mice. RNA sequencing was performed using Illumina Hi‐Seq and pathway analyses were performed using Ingenuity Pathway Analysis software (IPA®). We identified 831 genes that were differentially expressed (95% confidence and p<0.05) in BAT from HF compared to HF‐EPA fed mice. HF‐EPA had significantly higher expression of genes in fatty acid oxidation and thermogenesis such as peroxisome proliferator‐activated receptor‐α (Ppar‐α), retinoid X receptor (Rxr), phosphatase and tensin homolog (Pten) and reduced expression of inflammatory genes such as nuclear factor kappa‐light‐chain‐enhancer of activated B cells (Nf‐κb), high‐mobility group protein B1(Hmgb1) and Tumor necrosis factor receptor 1 (Tnfr1). Moreover, miRNA profiling identified nine miRNAs that were upregulated and six miRNAs that were downregulated by EPA. Few miRNAs including miR‐455‐3 and miR‐150‐5p are key regulators of BAT thermogenic function. These data are currently being further validated using quantitative PCR. In summary, combining RNA‐Seq and miRNA profiling is a powerful approach to identify specific pathways regulated by omega 3 fatty acids, which may help identify novel biomarkers mediating nutritional regulation of thermogenesis.Support or Funding Informationstartup funds from Texas Tech University
Association of serum sex hormone‐binding globulin with features of metabolic syndrome in children
Authors: Al‐Daghri Nasser , Khan Nasiruddin , Al‐Attas Omar , Sabico Shaun , Alokail Majed , Kumar Sudhesh 
Published: November 2023

BackgroundSex hormone binding globulin (SHBG) has been proposed as a biomarker of MetS in children and adults. We aim to determine the association of SHBG with features of MetS in children in the Middle‐East where prevalence of MetS has been documented to be very high.Materials and methodsIn this cross‐sectional observational study, a total of 509 randomly selected school children aged 12–16 years were recruited. Fasting blood glucose and lipid profile were determined using routine laboratory procedures. Serum SHBG is measured with Cobas e411 using an electrochemiluminescence immuno assay. The modified definition of ATP‐III (NHANES III) was used for the diagnosis of MetS.ResultsAmong 509 children, 23.4% had metabolic syndrome. Boys had a significantly higher waist circumference and systolic blood pressure (p<0.032, 0.024, respectively) than girls, while levels of glucose (p<0.029), and SHBG (p<0.003) were significantly higher in girls than in boys. In overall population, a significant negative correlation of SHBG level with age, BMI, systolic blood pressure and triglycerides and a significant direct correlation between SHBG level and HDL‐c was exhibited. Children with the lowest tertile of serum SHBG had significantly higher prevalence of MetS (p<0.05.). ROC curve shows SHBG level as more sensitive marker of MetS in boys (AUC=0.70, p<0.001).ConclusionsSHBG levels not only suggest its promising use as a possible biomarker, but also as a predictor of CVD risk, with more accuracy in boys. Gender dimorphism seems to play a role with higher levels of SHBG in girls than boys.Support or Funding InformationThe authors thank the Deanship of Scientific Research, Prolific Research Group Program (PRG‐1436‐15), Vice Rectorate for Graduate Studies and Scientific Research in King Saud University (KSU), Riyadh, Saudi Arabia for funding the study.
Development and Characterization of Dual‐Site PPARγ Antagonists
Authors: Brust Richard , Asteian Alice , Kamenecka Theodore , Kojetin Douglas 
Published: November 2023

Peroxisome proliferator‐activated receptor gamma (PPARγ) has been established as a drug target for type 2 diabetes. Insulin‐sensitizing drugs such as glitazones, which improve plasma glucose maintenance in patients with diabetes, have been shown to target PPARγ. Two commercially available antagonists, T0070907 and GW9662, are used as chemical tools to block ligand binding to PPARγ to show functional specificity of ligands that bind PPARγ. These compounds covalently modify residues Cys285 in the ligand‐binding domain (LBD) of PPARγ. GW9662 and T0070907 share a similar warhead but differ slightly, namely via the presence of a phenyl (GW9662) vs. a pyridyl (T0070907) group. However, in a recent study, we identified a second, alternative ligand‐binding site in PPARγ—one that is not blocked by GW9662 or T0070907. While alternate site ligand binding was not abolished, the phenyl to pyridyl group modification did reduce ligand‐binding affinity to the alternate site, indicating that making further modifications of this scaffold are worth exploring. With this information, we have generated new analogs that target and covalently modify PPARγ. These analogs decrease the binding affinity of PPARγ agonists to the alternate site while also reducing alternate site binding‐induced transactivation in cell based assays. These dual‐site PPARγ antagonists should prove to be a useful chemical tool for the many laboratories that use PPARγ antagonists to demonstrate functional specificity.Support or Funding InformationFunding provided by NIH/NIGMS grant R01‐DK101871.
HMG box‐containing protein 1 (HBP1) modulates matrix metalloproteinase‐13 (MMP‐13)‐mediated cellular migration and invasion in oral cancer
Authors: Chan Chien Yi , Wu Wen‐Chieh , Lin Tzu‐Yuan , Lee Ming‐Fen , Huang Chun‐Yin 
Published: November 2023

Transcription repressor HMG box‐containing protein 1 (HBP1) functions as a potential tumor suppressor in various types of cancer. Previously, we demonstrated that HBP1 modulates cell growth in epidermal growth factor receptor (EGFR)‐overexpressing oral cancer cells; however, whether HBP1 also regulates metastatic potential remains unclear. Analysis of a subset of oral tumor specimens revealed that mean HBP1 expression level in tumors was significantly lower than that of control tissues; moreover, low HBP1/high EGFR status was significantly associated with metastatic potential. When ectopically over‐expressing HBP1 in both HSC‐3 and FaDu oral cancer cells with elevated EGFR levels, we found that HBP1 potently inhibited cell invasion. In contrast, HBP1 knockdown enhanced cell migration and invasion with a concomitant increase in the expression and activity of matrix metalloproteinase (MMP) family members, MMP‐2 and ‐9, as well as a strong induction of MMP‐13. Both chemical inhibitors and MMP‐13 siRNA significantly suppressed MMP‐13 activity and cell invasion in HSC‐3 cells, suggesting a crucial role of MMP‐13 in metastasis. Further, HBP1 was not only a potent repressor of MMP‐13 transcription as shown by the MMP‐13 reporter gene assay, but also a modulator of Akt‐mediated MMP‐13 expression and invasion. Taken together, our study demonstrated that HBP1 is a negative regulator of cell migration and invasion as well as anchorage‐independent growth, supporting HBP1 as a potential predictor of the aggressiveness in the development of oral cancer.Support or Funding InformationThis work was supported by the grant MOST 103‐2320‐B‐039‐031‐MY3 to C‐Y Huang.
Pentoxifylline modulates the AKT/mTOR/AMPK pathway: potential role as an adjunct in cancer chemotherapy
Authors: Le Gloria T , Canatsey Ryan , Tran Kelvin , Monks Terrence J , Lau Serrine 
Published: November 2023

Targeting specific pathways involved in the growth and death of cells, such as AKT (signal transduction pathway promoting growth), mTOR (intracellular signaling pathway regulating cell cycle), and AMPK (coordination of growth, autophagy, and metabolism), is an established, effective strategy to inhibit cancer cell growth. Previously, we have shown that pentoxifylline (PTX), a clinically‐utilized non‐specific phosphodiesterase inhibitor, can inhibit growth and induce G1 arrest in prostate cancer cells. In the present studies, we examined the mechanism of PTX induced growth inhibition via modulation of growth/metabolic pathways, and the potential use of PTX as an adjunct in cancer chemotherapy. In prostate adenocarcinoma cells (LNCaP), we show PTX modulates the AKT pathway, as evidenced by the decreased phosphorylation of AKT. Downstream modulation of the mTOR pathway is indicated by decreased phosphorylation of mTOR, ribosomal S6K, and 4EBP1, and total cyclin D1. P‐mTOR levels return to baseline by 24h, while p‐S6K, p‐4EBP1, and cyclin D1 levels are suppressed for at least 48h, indicating a sustained decrease in the activity of cell growth pathway proteins. PTX also modulates the AMPK pathway, as revealed by modulation of downstream p‐RAPTOR, p‐ACC and p‐ULK. Both the AKT and AMPK pathways regulate the activation of cellular autophagy, an intracellular degradation system and determinant of cell survival. Treatment of LNCaP cells with PTX alone, and in combination with bafilomycin, a lysosomal degradation inhibitor, increased LC3‐II expression, indicating an induction of autophagic flux. Overall, PTX modulates both AKT and AMPK pathways, decreasing cell growth and increasing autophagy. Moreover, LNCaP cells treated with PTX were more susceptible to docetaxel (DOC; first‐line chemotherapy) induced cytotoxicity than DOC alone. Interestingly, the PTX/DOC combination decreased the expression of LC3 relative to PTX alone, suggesting activation of an alternate cell death pathway. Indeed, assessment of caspase cleavage, pBcl‐2, and annexin staining revealed a synergistic effect of PTX/DOC treatment on the induction of apoptosis. In summary, the findings provide a mechanistic basis for the potential use of PTX as an adjunct in the chemotherapy of prostate cancer. The ability of PTX to potentiate the effects of DOC on tumor growth inhibition in a mouse xenograft model is currently being assessed.Support or Funding InformationASPET SURF, P30ES006694, T32ES006694
B‐Raf is required for normal cardiomyocyte growth
Authors: Rees Katherina , Chattergoon Natasha 
Published: November 2023

ObjectivesThe postnatal heart does not retain the proliferative capacity it had during fetal life. Unlike large mammals, murine cardiomyocytes (CM) continue to divide into the first week of life before terminal differentiation and binucleation. This postnatal proliferation and maturation window offers the ability to study the mechanisms of CM terminal differentiation. We hypothesize that B‐Raf regulates ERK (extracellular‐regulated kinase) activation in newborn CM and that loss of B‐Raf suppresses cyclin levels and reduces CM proliferation. To test this, we determined whether the loss of B‐Raf disrupts the ERK cascade and impairs CM growth.MethodsCM specific knockout (KO) of B‐Raf was generated using CRE/lox (floxed B‐Raf x αMHC CRE) resulting in a truncated, unstable B‐Raf and a null phenotype. KO mice (α‐MHC‐CRE/B‐Raf lox/lox) were compared to CRE negative/B‐Raf lox/lox mice (wild type; WT). Hearts from 1d, 3d, 8d and 14d old pups were harvested for molecular analysis of B‐Raf signaling and cell cycle markers (p21, p27, p53, cyclins A1, B1, D1 and E). Mid‐wall sections of the heart were stained with Masson's trichrome to determine changes in tissue composition.ResultsHeart weight to body weight (HW/BW) ratio was less in 3d KO versus 3d WT (n=50, p<0.05). HW/BW ratio became greater in 8d KO; there was no difference in 3d and 8d HW/BW in WT animals. B‐Raf and phosphorylated ERK levels were reduced in KO hearts (*p<0.05). Cell cycle inhibitors p21 and p53 were increased in 3d KO hearts with decreased levels of all cyclins (Cyclin A1 and B1, p<0.05). In 8d KO hearts, increased p21, p27, and p53 expression was accompanied with decreased cyclin levels (p<0.05). In 14d KO hearts, gene expression of p21, p27, and p53 are increased (p<0.05), along with cyclins D1 and E. Tissue composition of cardiomyocytes decreased and collagen increased in 3d and 8d hearts (p<0.05).ConclusionsERK activation was suppressed in KO hearts resulting in smaller newborn hearts but which exceeded normal HW/BW by 8d. This is may represent premature hypertrophy as the proliferative period of CM development had ended. Cell cycle analysis supports reduced CM mitosis among 8d CM. Trichrome analysis supports proliferation has ended prematurely by the reduction of the CM fraction. Such early disturbances in normal CM growth may increase susceptibility for reduced cardiac function in the face of increased postnatal load stress.
Proteomic Analysis of the Mammalian Katanin Family of Microtubule‐severing Enzymes Defines KATNBL1 as a Regulator of Mammalian Katanin Microtubule‐severing
Authors: Cheung Keith C. , Torres Jorge Z. 
Published: November 2023

The Katanin family of microtubule‐severing enzymes are critical for remodeling microtubule‐based structures that influence cell division, motility, morphogenesis and signaling. In lower eukaryotes, Katanin is composed of a catalytic p60 subunit (A subunit, KATNA1) and a regulatory p80 subunit (B subunit, KATNB1). However, the mammalian genome encodes two additional A‐like subunits (KATNAL1 and KATNAL2) and one additional B‐like subunit (KATNBL1) that have remained poorly characterized. To better understand the factors and mechanisms controlling mammalian microtubule‐severing, we have taken a mass proteomic approach to define the protein interaction module for each mammalian Katanin subunit and to generate the mammalian Katanin family interaction network (Katan‐ome). Further, we have analyzed the function of the KATNBL1 subunit and determined that it associates with KATNA1 and KATNAL1, it localizes to the spindle poles only during mitosis and it regulates Katanin A subunit microtubule‐severing activity in vitro. Interestingly, during interphase, KATNBL1 is actively sequestered in the nucleus through an N‐terminal nuclear localization signal. Finally KATNB1 was able to compete the interaction of KATNBL1 with KATNA1 and KATNAL1. These data indicate that KATNBL1 functions as a regulator of Katanin A subunit microtubule‐severing activity during mitosis and that it likely coordinates with KATNB1 to perform this function.Support or Funding InformationNational Science Foundation and UCLA Whitcome fellowship
AKAP7 and Calcineurin Control CaM Kinases and Cell Growth
Authors: Papenfuse Lael , McFarland Hannah M , Dodge‐Kafka Kimberly , Schmitt John M 
Published: November 2023

Estrogen (E2) and the agonist ionomycin elevate intracellular calcium and activate the calcium/calmodulin‐dependent protein kinases (CaM Kinases) in breast cancer cells. CaM Kinases regulate ERK leading to transcription and cell growth. Inhibition of CaM KK blocks breast cancer cell growth and protein phosphatases such as Calcineurin (CN) may negatively regulate CaM KK pathways. CN regulates CaM Kinases and associates with A‐kinase‐anchoring‐proteins (AKAPs). AKAPs are used to link together and regulate enzymes within in a signaling pathway. One specific AKAP, AKAP7, is involved in calcium signaling and may regulate CaM Kinase control of ERK activation in cancer cells. Our goal was to determine if CN and AKAP7 associated in breast cancer cells, and to examine the role of CN in regulating E2 and calcium activation of CaM KK/KI, ERK and cell growth in MCF‐7 and MDA‐MB‐231 cells. Our results suggest that E2 treatment of MCF‐7 cells activates CaM KI and ERK, an effect that is blocked by the selective CaM KK‐inhibitor STO‐609. Immunoprecipitation with purified STag‐AKAP7 but not STag‐AKAP5 revealed CaM KK and CaM KI association from E2‐treated MCF‐7 extracts. Interestingly, purified AKAP7 directly binds purified CaM KK and endogenous AKAP7 co‐localizes with CaM KK in MCF‐7 and MDA‐MB‐231 cells. Ionomycin stimulation of MCF‐7 cells also increased CaM KI and ERK activation an effect that was enhanced by treating cells with the CN inhibitor cyclosporin A (CsA). Similarly, ionomycin treatment enhanced the binding of endogenous CaM KK‐AKAP7‐CN as determined by both immunoprecipitation and confocal microscopy. Interestingly, siRNA knockdown of AKAP7 disrupted CaM KK association with CN and increased basal ERK phosphorylation in MCF‐7 cells. Finally, E2‐stimulated cell growth was inhibited by pre‐treatment with STO‐609 but not the CN inhibitor CsA. Taken together, these results suggest that AKAP7 directly associates with CaM KK/KI and forms a complex with CN that may function to negatively regulate CaM KK and CaM KI activation of ERK and cell growth.Support or Funding InformationThe Paul K. and Evalyn E. C. Richter Memorial Fund.This work was supported through a Life Science grant from the M.J. Murdock Charitable Trust to J.M.S. grant #2011267.