TF Proteins Research Articles

Effects of exposure to nano-plastic drinking during pregnancy on cognitive related proteins in offspring of SD rats

ABSTRACT Nanoplastics, an emerging contaminant, pose risks to fetal neural development, transferring from mother to offspring via placenta and breast milk. Assessing these risks, hippocampal CA3 samples from nanopolystyrene-exposed rat offspring were proteomically analyzed. Findings revealed reduced expression of neural developmental proteins (KIF21A, STMN2, DMTN, DLG1) and increased inhibitory proteins (PZP, α-2M, FN1, SERPINA1, ALOX15) in the hippocampus. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis highlighted ferroptosis enrichment, validated by significantly expressed ALOX15 and TF proteins. These alterations suggest nanopolystyrene’s potential contribution to cognitive deficits and neurodevelopmental disorders, emphasizing its hazardous impact on neural development. This study provides novel insights into nanopolystyrene’s risks.

Biomimetic nanozymes target the heart to suppress ferroptosis for ameliorating doxorubicin-induced cardiotoxicity via synergetic effect of antioxidant stress and Nrf2/Hmox1 inhibition

Abstract Background Doxorubicin (DOX) is the first-line standard treatment of numerous cancer. However, DOX may lead to irreversible degenerative cardiomyopathy, congestive heart failure, and various arrhythmias, known as doxorubicin-induced cardiotoxicity (DIC), which limits the clinical applications. Studies reported that ferroptosis plays an important role on the development of DIC. Targeted intervention of ferroptosis with the specific inhibitors, such as ferrostatin-1 (Fer-1), could effectively prevent DIC. In recent years, catalytic nanozymes, such as cerium oxide (CeO2), have received much attention due to their strong antioxidant properties. Purpose Our previous research found that the transferrin receptor (TfR) protein expression was significantly upregulated in DIC mice (Figure 1) . In this study, we synthesized a biomimetic CeO2-based nanozyme by biomineralization with transferrin (Tf) proteins as the template, and then we assessed its targeted therapy for DIC via Tf-TfR mediated endocytosis. Method Cellular uptake, cytotoxicity test, intracellularmitochondrial membrane potential (MMP), reactive oxygen species (ROS), mitochondrial ferrous ions and lipid peroxidation toxicity were verified with rat cardiomyocytes line H9c2 cells. Afterward, the male C57BL/6N mice were randomly divided into control, DOX, DOX+Fer-1, and DM+NPs groups. Heart structure and function were measured by echocardiographic, electrocardiography and epicardial electrical labeling. Western blot determine mitochondrial homeostasis regulation-related proteins and Nrf2/Hmox1 signalling pathway. Finally, the biodistribution and biocompatibility of CeO2@Tf was assessed. Results Transmission electron microscopy images revealed the encapsulated CeO2@Tf was≈ 5 nm in diameter. The nanozyme significantly reversed cardiac structural and electrical remodeling and reduced myocardial necrosis. Meanwhile, RNA sequencing of ventricular tissue revealed increased cardiac Hmox1 mRNA levels in DIC mice (Figure 2). Further, it demonstrated that nanozyme intervention not only significantly suppressed the oxidative stress, mitochondrial lipid peroxidation and mitochondrial membrane potential damage, but also inhibited activation of Nrf2/Hmox1 signalling pathway to restore mitochondria-dependent ferroptosis. Furthermore, the nanozyme could be cleared by the hepatobiliary systems, and showed a good biocompatibility. Conclusions The current study showed that biomimetic mineralization of hybrid CeO2-based nanozyme against DIC were mediated by effect of antioxidant stress and the inhibition of Nrf2/Hmox1 signalling pathway, thereby maintaining mitochondrial homeostasis and function, and re-storing mitochondria-dependent ferroptosis. Furthermore, the nanozyme could be a promising prevention and treatment candidate for clinical translation as a novel cardiomyocyte ferroptosis protector to mitigate DIC and improve the prognosis and quality of life of cancer patients.

Use of 2D FFT and DTW in Protein Sequence Comparison.

Protein sequence comparison remains a challenging work for the researchers owing to the computational complexity due to the presence of 20 amino acids compared with only four nucleotides in Genome sequences. Further, protein sequences of different species are of different lengths; it throws additional changes to the researchers to develop methods, specially alignment-free methods, to compare protein sequences. In this work, an efficient technique to compare protein sequences is developed by a graphical representation. First, the classified grouping of 20 amino acids with a cardinality of 4 based on polar class is considered to narrow down the representational range from 20 to 4. Then a unit vector technique based on a two-quadrant Cartesian system is proposed to provide a new two-dimensional graphical representation of the protein sequence. Now, two approaches are proposed to cope with the varying lengths of protein sequences from various species: one uses Dynamic Time Warping (DTW), while the other one uses a two-dimensional Fast Fourier Transform (2D FFT). Next, the effectiveness of these two techniques is analyzed using two evaluation criteria-quantitative measures based on symmetric distance (SD) and computational speed. An analysis is performed on five data sets of 9 ND4, 9 ND5, 9 ND6, 12 Baculovirus, and 24 TF proteins under the two methods. It is found that the FFT-based method produces the same results as DTW but in less computational time. It is found that the result of the proposed method agrees with the known biological reference. Further, the present method produces better clustering than the existing ones.

Integrated analyses of metabolomics and transcriptomics reveal the potential regulatory roles of long non-coding RNAs in gingerol biosynthesis

BackgroundAs the characteristic functional component in ginger, gingerols possess several health-promoting properties. Long non-coding RNAs (lncRNAs) act as crucial regulators of diverse biological processes. However, lncRNAs in ginger are not yet identified so far, and their potential roles in gingerol biosynthesis are still unknown. In this study, metabolomic and transcriptomic analyses were performed in three main ginger cultivars (leshanhuangjiang, tonglingbaijiang, and yujiang 1 hao) in China to understand the potential roles of the specific lncRNAs in gingerol accumulation.ResultsA total of 744 metabolites were monitored by metabolomics analysis, which were divided into eleven categories. Among them, the largest group phenolic acid category contained 143 metabolites, including 21 gingerol derivatives. Of which, three gingerol analogs, [8]-shogaol, [10]-gingerol, and [12]-shogaol, accumulated significantly. Moreover, 16,346 lncRNAs, including 2,513, 1,225, and 2,884 differentially expressed (DE) lncRNA genes (DELs), were identified in all three comparisons by transcriptomic analysis. Gene ontology enrichment (GO) analysis showed that the DELs mainly enriched in the secondary metabolite biosynthetic process, response to plant hormones, and phenol-containing compound metabolic process. Correlation analysis revealed that the expression levels of 11 DE gingerol biosynthesis enzyme genes (GBEGs) and 190 transcription factor genes (TF genes), such as MYB1, ERF100, WRKY40, etc. were strongly correlation coefficient with the contents of the three gingerol analogs. Furthermore, 7 and 111 upstream cis-acting lncRNAs, 1,200 and 2,225 upstream trans-acting lncRNAs corresponding to the GBEGs and TF genes were identified, respectively. Interestingly, 1,184 DELs might function as common upstream regulators to these GBEGs and TFs genes, such as LNC_008452, LNC_006109, LNC_004340, etc. Furthermore, protein–protein interaction networks (PPI) analysis indicated that three TF proteins, MYB4, MYB43, and WRKY70 might interact with four GBEG proteins (PAL1, PAL2, PAL3, and 4CL-4).ConclusionBased on these findings, we for the first time worldwide proposed a putative regulatory cascade of lncRNAs, TFs genes, and GBEGs involved in controlling of gingerol biosynthesis. These results not only provide novel insights into the lncRNAs involved in gingerol metabolism, but also lay a foundation for future in-depth studies of the related molecular mechanism.

Identification of Drug Targets and Agents Associated with Hepatocellular Carcinoma through Integrated Bioinformatics Analysis.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death globally. The mechanisms underlying the development of HCC are mostly unknown till now. The main goal of this study was to identify potential drug target proteins and agents for the treatment of HCC. The publicly available three independent mRNA expression profile datasets were downloaded from the NCBI-GEO database to explore common differentially expressed genes (cDEGs) between HCC and control samples using the Statistical LIMMA approach. Hub-cDEGs as drug targets highlighting their functions, pathways, and regulators were identified by using integrated bioinformatics tools and databases. Finally, Hub-cDEGs-guided top-ranked drug agents were identified by molecular docking study for HCC. We identified 160 common DEGs (cDEGs) from three independent mRNA expression datasets in which ten cDEGs (CDKN3, TK1, NCAPG, CDCA5, RACGAP1, AURKA, PRC1, UBE2T, MELK, and ASPM) were selected as Hub-cDEGs. The GO functional and KEGG pathway enrichment analysis of Hub-cDEGs revealed some crucial cancer-stimulating biological processes, molecular functions, cellular components, and signaling pathways. The interaction network analysis identified three TF proteins and five miRNAs as the key transcriptional and post-transcriptional regulators of HubcDEGs. Then, we detected the proposed Hub-cDEGs guided top-ranked three anti-HCC drug molecules (Dactinomycin, Vincristine, Sirolimus) that were also highly supported by the already published top-ranked HCC-causing Hub-DEGs mediated receptors. The findings of this study would be useful resources for diagnosis, prognosis, and therapies of HCC.

Ginsenoside Rg1 Improves Inflammation and Autophagy of the Pancreas and Spleen in Streptozotocin-Induced Type 1 Diabetic Mice.

Ginsenoside Rg1 (Rg1) is one of the key bioactive components of the precious Traditional Chinese Medicine that has been used to treat diabetes in China. Ginsenosides have been reported to protect diabetics from tissue damage, inflammation, and insulin resistance. Type 1 diabetes (T1D) is an organ-specific autoimmune disease that occurred frequently among adolescents over the world, its development was related to inflammation and β-cells immunodeficiency. The aim of this study is to explore the biological mechanism of Rg1 on inflammation and autophagy of β-cells in T1D and its therapeutic potential. The model of T1D mice was established by injecting Streptozotocin (STZ) (55 mg/kg) or citric acids once a day for 5 days and from the fourth day of injection, mice were administered with Rg1 (20 mg/kg) or saline by gavage once a day for 12 days. Hematoxylin-eosin staining, immunofluorescence, ELISA, quantitative real-time PCR, and Western blot were used to observe the histopathological changes, inflammatory factor levels, and autophagy markers after administration of ginsenoside Rg1. Compared to the T1D mice, Rg1 improved the weight (p < 0.05) and blood glucose (p < 0.01) of mice, advanced the injury and apoptosis of β-cells in islets (p < 0.01), and markedly inhibited the protein expression degrees of CD45, CXCL16, ox-LDL, and TF in the pancreas and spleens (p < 0.01), also activated the degrees of insulin in serum (p < 0.01). Besides, in T1D mice' pancreas and spleen, Rg1 markedly repressed the IL-1β, TNF-α, and NOS2 mRNA levels (p < 0.05 or p < 0.01), inhibited the CXCL16, NF-κB, and TF proteins (p < 0.05 or p < 0.01), while elevating the ratio of LC3 II/I (p < 0.01) and P62 (p < 0.05) protein level. This study proved that Rg1 protected mice against T1D possibly by improving islet injury and tissue inflammation, raising serum insulin, and tissue autophagy marker.

Upregulation of SGLT1 and 2 promotes oxidative stress in right atrial appendages of patients with low-grade inflammatory responses: potential role in atrial fibrillation

Abstract Introduction Atrial fibrillation (AF), the most common cardiac arrhythmia, is associated with increased morbidity and mortality. Inflammatory infiltrates have been observed in atrial tissues from AF patients suggesting that low-grade inflammation contributes to the pro-fibrotic, pro-remodeling and pro-senescence responses of the atrial tissue favoring AF. Although sodium glucose co-transporter2 inhibitors (SGLT2i) have shown beneficial effects in heart failure, the expression and role of SGLT2 in cardiac tissues remain unclear. Purpose This study examined the expression of SGLT1 and 2 in right atrial appendages (RAA) of cardiac patients, and, if so, to determine the underling mechanism and their functional role. Methods RAA were harvested from patients undergoing coronary artery bypass surgery or aortic valve replacement at the University Hospital of Strasbourg. Patients with comorbidities such as malignancies and chronic inflammatory diseases were excluded. The expression level of markers was assessed using RT-qPCR, Western blot analysis, and immunofluorescence staining, and the level of reactive oxygen species using dihydroethidium staining. Results Analysis of RAA samples evidenced up-to a 4-fold difference in the NOS3 mRNA level. Those with low levels of NOS3 mRNA showed high levels of ICAM1, F3, MMP9, TGF-β1, TP53, CDKN1A, CDKN2A, SLCA1, SLCA2, IL1B, IL6, TNF-a and CD68 whereas the contrary was observed in those with high levels of NOS3 mRNA. SGLT1/2 mRNA levels were negatively correlated with that of eNOS and positively with those of IL-1, IL-6, TNF- α, CD68, and the senescence marker p53. Western blot analysis of RAA indicated that those expressing low levels of eNOS protein showed high levels of ICAM-1, VCAM-1, TF, MMP-2, TGF-b, SGLT1, SGLT2, p-p65, p53, p21 and p16 proteins whereas the contrary was observed in those with high levels of eNOS protein. Immunofluorescence staining indicated that SGLT1/SGLT2 signals were colocalized with those of CD31, an endothelial cell marker, in RAA, and associated with those of CD68 and TNF-α. Samples with high levels of SGLT1 and 2 displayed higher levels of oxidative stress that were inhibited by the antioxidant NAC (N-acetylcysteine), VAS-2870 (NADPH oxidase inhibitor), perindoprilat (ACE inhibitor), losartan (AT1R antagonist), empagliflozin (selective SGLT2 inhibitor), sotagliflozin (dual SGLT1/2 inhibitor) and by infliximab (TNF-α receptor neutralizing antibody). Conclusion The findings indicate that high expression levels of SGLT1 and 2 are observed in RAA showing low eNOS levels associated with pro-inflammatory, pro-senescent, pro-remodeling and pro-fibrotic responses. They were further associated with increased levels of oxidative stress sensitive to inhibitors of either the local angiotensin system, TNF-alpha or SGLT2. Thus, inhibition of SGLT2 appears to be an interesting approach to reduce the stimulatory pro-oxidant signal in atrium related to low-grade inflammation. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Boehringer Ingelheim Pharma GmbH &amp; Co. KG

Abstract 3623: Targetable cofactor landscape of transcription factors in human cancers

Abstract Transcription factors (TF) are proteins that bind to DNA to regulate target gene expression and play a crucial role in maintaining tumor initiating and maintenance programs. However, except for a small fraction, it is difficult to directly target TF proteins due to lack of defined pockets and inherent protein-DNA, protein-protein interactions. In many cases, TF activity is tuned via cofactors, which e.g. dimerize to bind DNA (MYC/MAX) or maintain active TF protein turnover (HIF1A/EGLN1, MYC/HDAC2). Identifying such targetable cofactors of TFs will open new opportunities to target oncogenic TFs in cancers. Genome-scale CRISPR knock-out screens have been proven to be a valuable resource for identifying targets and biomarkers in cancers. In this work, we developed a framework called CONTEXT (COfactor depeNdencies in high Transcription factor EXpression phenoType) utilizing functional genomic screens and matched TF expression data. In CONTEXT, we aim to identify cofactor dependencies associated with TF activities using 859 cell lines spanning 28 cancer types. We further utilized patient cancer gene expression data from the TCGA project, to prioritize clinically relevant TF-cofactor associations. In total, we identified 1,003 TF-cofactor associations which involve 196 and 503 unique TFs and cofactors, respectively. Our approach reliably identified previously known TF-cofactor associations e.g. HIF1A/EGLN1, HIF1A/VHL, TEAD1/JUN but also previously unknown associations such as TEAD1/RAC1 or TEAD1/PTK2. Our analysis and validations provide a framework for the identification of potential cofactors of TFs which are crucial for the development and progression of cancer. We anticipate the cofactor dependencies from this resource to guide early stage drug development and inform on potential combination therapies in resistant phenotypes. Citation Format: Venu Thatikonda, Jesse Lipp, Andreas Schlattl, Daniel Gerlach, Alexandra Popa. Targetable cofactor landscape of transcription factors in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3623.

TAMI-54. SEXUAL DIMORPHISM IN IRON ACQUISITION IN GLIOBLASTOMA

Abstract Sexual dimorphism in incidence and the clinical outcomes of Glioblastoma (GBM) has been reported, however, our knowledge of contributing biological mechanisms is limited. Iron acquisition is key to robust tumor growth. Upregulation of Transferrin (TF, iron transport protein)/Transferrin receptor (TFR) is critical for found in multiple different cancers, specifically, we have identified H-ferritin (FTH1) as a contributor to iron transport and protection in cancer stem cells. To interrogate brain tumor iron uptake mechanisms,we performed binding studies on homogenized samples of human male and female GBM tissue samples using 125I labeled TF and FTH1. Tumors from males had a ̴ 3.8-fold increased binding of both proteins compared to tumors from females. We interrogated iron uptake in a syngeneic orthotopic mouse model (GL261 cells) using male and female mice. After the tumors were established, radioactive 125I labeled TF and FTH1 proteins were injected retro-orbitally in the mice. After 24 hours, tumors wereremoved, and analyzed for TF and FTH1 uptake. Male tumors showed an increased uptake, of ̴ 3.2-fold, as compared to female tumors. There was no significant difference in TF uptake between male and female tumors nor between tumor and matched non-tumor brain tissue. We next queried role of FTH1 in the context of sexual dimorphism in GBM in a FTH1+/- mouse strain developed in our laboratory. Survival was monitored in the mice which were injected with GL261 cells at 3 months. Male mice that had reduced expression of FTH1 had poorer survival as compared to the male wild type controls whereas wild type and FTH+/- females had no major differences in survival outcomes. In summary, this study demonstrates sexual dimorphism in iron acquisition in GBM and animal models further suggesting a pathophysiological role of iron metabolism in GBM development and its possible role in prognosis.

Upregulation of Transferrin and Major Royal Jelly Proteins in the Spermathecal Fluid of Mated Honeybee (Apis mellifera) Queens.

Simple SummaryTo understand the mechanisms underlying long-term storage and survival of sperm in honeybee Apis mellifera queens, previous studies have elucidated the components of honeybee spermathecal fluid. However, the expression profiles of transferrin (Tf) and major royal jelly proteins 1–9 (MRJPs 1–9) in the spermatheca and spermathecal fluid of mated honeybee queens have still not been characterized. In this study, we confirmed upregulation of Tf and MRJPs in the spermatheca and spermathecal fluid of mated honeybee queens by using RNA sequencing, reverse transcription-polymerase chain reaction, and Western blot analyses. The levels of Tf and antioxidant enzymes were elevated in the spermathecal fluid of the mated queens, paralleling the levels of reactive oxygen species, H2O2, and iron. The increased levels of MRJPs, especially MRJP1, MRJP4, and MRJP6, in the spermathecal fluid of mated queens may be responsible for energy provision during sperm storage in honeybee queens. Overall, our findings indicate that Tf and MRJPs are upregulated in the spermatheca and spermathecal fluid of mated honeybee queens, providing a novel insight into antioxidant defense and energy metabolism for stored sperm in honeybee queens.Sperm storage in the spermathecae of honeybee (Apis mellifera) queens is vital for reproduction of honeybees. However, the molecular mechanisms whereby queens store sperm in a viable state over prolonged periods in the spermatheca are not fully understood. Here, we conducted RNA sequencing analysis of the spermathecae in both virgin and mated A. mellifera queens 24 h after mating and observed that the genes encoding transferrin (Tf) and major royal jelly proteins (MRJPs) were differentially expressed in the spermathecae of mated queens. The concentrations of Tf and antioxidant proteins such as superoxide dismutase 1, catalase, and glutathione peroxidase as well as the levels of reactive oxygen species, H2O2, and iron were higher in the spermathecal fluid of the mated queens than in virgin queens. Tf upregulation is likely to perform a protective role against the Fenton reaction occurring between iron and H2O2 in the antioxidant pathway in the mated queen’s spermathecal fluid. Furthermore, MRJPs—especially MRJP1, MRJP4, and MRJP6—were upregulated in the mated queen’s spermathecal fluid, indicating that they may serve as antimicrobial and antioxidant agents as well as an energy source for stored sperm in the spermathecal fluid of honeybee queens. Together, our findings show that Tf and MRJPs are upregulated in the spermatheca and spermathecal fluid of mated honeybee queens.

Label‐free subcellular mapping of iron‐bound proteins in breast cancer cells

Dysregulation of iron transport, storage and utilization is at the basis of a variety of human diseases, since free iron can participate in redox reactions that are deleterious to lipid membranes and signaling pathways. As intracellular iron transport occurs in a pH-dependent manner, changes in endosomal pH, often associated with cancer, could lead to alterations in iron homeostasis. Essential to iron transport is transferrin (Tf), which binds iron at a neutral pH for safe transport throughout the body and releases it upon uptake and delivery to a mildly acidified endosome. Excess iron can be stored in cytoplasmic protein ferritin (Ft) or delivered into mitochondria to be incorporated into proteins with heme or iron-sulfur structures. Recently, we have developed a novel label-free Raman hyperspectral microscopy methodology to identify the subcellular distributions and relative quantities of iron-bound Tf, Ft and heme proteins within heterogenous cell populations. Previously, we have shown that Raman hyperspectral imaging could identify endocytic distributions of iron-bound Tf using a specific Raman peak at 1300 cm-1 in intact breast cancer cells. Moreover, we have shown that alterations in the subcellular localization of iron-bound Tf populations correlated with altered endocytic regulation of iron-bound Tf in two distinct breast cancer cell lines. Based on these results, we propose that breast cancer cells display a disrupted iron homeostasis due to iron release delays caused by alterations in the pH or ionic milieu of the early endosomes. Currently, we are testing a putative correlation between pH gradient alterations and iron release delays in different breast cancer cells. To further investigate cellular iron regulation, Ft cell-specific Raman signature was determined via evaluation of cells incubated in the presence or absence of iron-loaded Ft. Since cells may internalize Ft through an endocytic pathway, this approach allowed for identification of Ft Raman signature. However, the subcellular distribution of internalized iron-loaded Ft may not be reflective of normal cytoplasmic Ft in cells. Internalization of iron-loaded Ft results in multiple significantly different Raman spectra peaks at several wavelengths. Importantly, we have used multivariate analysis to show a significant separation of cell Raman spectra from that of cells incubated with Ft. Further we can map a previously known heme protein peak at 1372 cm-1, which is specifically associated with the ferric iron heme. In the future, following further validation of Ft peaks the mapping of Tf, Ft and heme can be evaluated across multiple cell lines and tissue samples. The application of this research has far reaching potential in understanding how dysregulation of endocytic pH may lead to alterations in intracellular iron transport and storage in breast cancer cells.

Anti-thrombotic activity of phenolic acids obtained from Salvia miltiorrhiza f. alba in TNF-α-stimulated endothelial cells via the NF-κB/JNK/p38 MAPK signaling pathway.

Over the past 100years, Salvia miltiorrhiza f. alba (Lamiaceae) (RSMA) roots have been used to cure thromboangiitis obliterans (TAO) in local clinics. This study aimed to confirm the anti-thrombotic efficacy of 12 phenolic acids obtained from RSMA and to clarify the possible underlying mechanisms. The results of quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that most of the phenolic acids markedly inhibited PAI-1 protein and mRNA levels but increased t-PA protein and mRNA levels in TNF-α-induced EA.hy926 cells (P < 0.05 or 0.001), with lithospermic acid displaying the strongest effect. In vitro anticoagulation and antiplatelet aggregation assays showed that lithospermic acid and salvianolic acid B significantly prolonged prothrombin time (PT), activated partial thromboplastin time (APTT), decreased fibrinogen concentration (FIB), and inhibited platelet aggregation induced by adenosine diphosphate (ADP) in rat blood. Both lithospermic acid and salvianolic acid B markedly down-regulated the expression of factor Xa and factor IIa on the external surface of EA.hy926 cells and demonstrated significant anti-factor IIa and anti-factor Xa activity using chromogenic substrates in vitro. Western blot results revealed that both lithospermic acid and salvianolic acid B also significantly inhibited the expression of TF, p-p65, p-p38, and pJNK proteins induced by TNF-α. These results indicated that all of the phenolic acids appeared to have some anti-thrombotic activity, with salvianolic acid B and lithospermic acid markedly decreasing the chance of thrombosis by regulating the NF-κB/JNK/p38 MAPK signaling pathway in response to TNF-α.

CSIG-16. SEXUAL DIMPORHISM IN IRON ACQUISITION IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most aggressive brain cancer. Sex differences in incidence and clinical outcomes have been reported, however, our knowledge of contributing mechanisms is limited. Iron acquisition is key to robust tumor growth. Upregulation of Transferrin (Tf, iron transport protein)/Transferrin receptor (TfR) is found in multiple different cancers. We have identified H-ferritin (FTH1) as involved in iron transport and explore its uptake in GBM in this study. We interrogated iron uptake in a syngeneic orthotopic mouse model (GL261 cells) using male and female mice. After the tumors were established, radioactive 125I labeled Tf and FTH1 proteins were injected retro-orbitally in the mice. After 24 hours, tumors were removed, homogenized and analyzed for Tf and FTH1 uptake. There was a significant difference in Tf uptake into the tumor versus matched non-tumor tissue in both males and females and the uptake in the tumors was 1.5-fold higher in males than females. There was no significant difference in FTH1 uptake between male and female tumors nor between tumor and matched non-tumor brain tissue. Binding analyses were performed on homogenized samples of human male and female GBM tissue samples using 125I labeled Tf and FTH1. Tumors from males had increased binding of both proteins compared to tumors from females. We next queried the TCGA database and found in females, high TfR expression was associated with poor survival but not in males. TCGA database revealed a robust expression of Tim1, a putative receptor for FTH1, but its expression did not relate to survival. In summary, this study demonstrates FTH1 binding to GBMs and sexual dimorphism in iron acquisition via Tf and survival.

MAPK Enzymes: a ROS Activated Signaling Sensors Involved in Modulating Heat Stress Response, Tolerance and Grain Stability of Wheat under Heat Stress.

Mitogen-activated protein kinase (MAPK) signaling cascade is highly conserved across the species triggering the self-adjustment of the cells by transmitting the external signals to the nucleus. The cascade consists of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs) and MAPKs. These kinases are functionally interrelated through activation by sequential phosphorylation. MAPK cascade is involved in modulating the tolerance and regulating the growth and developmental processes in plants through transcriptional programming. The cascade has been well characterized in Arabidopsis, Tobacco and rice, but limited information is available in wheat due to complexity of genome. MAPK-based sensors have been reported to be highly specific for the external or intracellular stimuli activating specific TF, stress-associated genes (SAGs) and stress-associated proteins (SAPs) linked with heat-stress tolerance and other biological functions especially size, number and quality of grains. Even, MAPKs have been reported to influence the activity of ATP-binding cassette (ABC) transporter superfamily involved in stabilizing the quality of the grains under adverse conditions. Wheat has also diverse network of MAPKs involved in transcriptional reprogramming upon sensing the terminal HS and in turn protect the plants. Current review mainly focuses on the role of MAPKs as signaling sensor and modulator of defense mechanism for mitigating the effect of heat on plants with focus on wheat. It also indirectly protects the nutrient depletion from the grains under heat stress. MAPKs, lying at pivotal positions, can be utilized for manipulating the heat-stress response (HSR) of wheat to develop plant for future (P4F).

Programmed Self-Assembly of Protein-Coated AIE-Featured Nanoparticles with Dual Imaging and Targeted Therapy to Cancer Cells.

Modifying different functional moieties into one platform is a conventional strategy for constructing theranostic systems. However, this strategy usually suffers from the unsatisfied efficiency of each individual function. Herein, a programmed self-assembly strategy is presented to fabricate theranostic nanoparticles, which significantly exhibit a dual-modality imaging function involving fluorescence imaging and magnetic resource imaging (MRI), and an efficient targeted therapy to cancer cells. Fluorescent vesicles are first self-assembled by aggregation-induced emission (AIE)-active molecules. Gd3+, serving as an MRI agent, is subsequently bound to the vesicles to provide highly positive charges, which have been realized to be anticancer active. Thereafter, transferrin (Tf) protein is introduced onto the surface of Gd3+ coordinated vesicles, shielding the positive charges and making the nanoparticles nontoxic to cells. With the assistance of Tf protein, the constructed nanoparticles are specifically targeted to cancer cells. Moreover, Tf proteins further peel off from nanoparticles in lysosomes due to their charge reversion, resulting in highly positive charges and heavy toxicity of nanoparticles to kill cancer cells. In the nanoparticles, each of the functional components acts as double-sided adhesive tape to glue the next layer, so that the abilities of functional components are not compromised. This strategy holds great potential for theranostic nanomedicine.

Evolutionary conservation of transferrin genomic organization and expression characterization in seven freshwater turtles

Serum transferrin (tf), encoding an iron-binding glycoprotein, has been revealed to play important roles in iron transportation and immune response, and it also has been demonstrated to be valuable for phylogenetic analysis in vertebrates. However, the evolutionary conservation, expression profiles and positive selection of transferrin genes among freshwater turtle species remain largely unclear. Here, the genomic DNA and coding sequences of transferrin genes were cloned and characterized in seven freshwater turtles including Mauremys mutica, Mauremys sinensis, Cyclemys dentate, Mauremyssi reevesi, Heosemys grandis, Trachemys scripta and Chrysemys picta. The isolated coding sequences of turtles' tf genes were 2118 bp or 2121 bp, encoding 706 or 707 amino acids. The predicted Tf proteins of turtles share high identities with M. mutica Tf, up to 91%–98% and the M. mutica Tf has the highest identity (91%) in amino acid with the Chelomia mydas Tf, the moderate with other reptiles' Tfs (65%–59%), chicken (58%), and Human Tf (∼55%), and the lowest with zebrafish Tf (41%). Additionally, tf genes were consistently composed of 17 exons and 16 introns with the same splicing sites in introns in all the turtles examined. Moreover, 12 positive selected sites were detected in these turtles’ Tf and mainly distributed on the surface of transferrin protein. Importantly, it was found that transferrin genes in all turtles examined were predominantly expressed in adult liver via real-time quantitative PCR. The molecular characterizations and expression profiles of transferrin would shed new insights into understanding the conversations and divergences of transferrin genes in turtles, even in vertebrates.

Canonical and single-cell Hi-C reveal distinct chromatin interaction sub-networks of mammalian transcription factors

BackgroundTranscription factor (TF) binding to regulatory DNA sites is a key determinant of cell identity within multi-cellular organisms and has been studied extensively in relation to site affinity and chromatin modifications. There has been a strong focus on the inference of TF-gene regulatory networks and TF-TF physical interaction networks. Here, we present a third type of TF network, the spatial network of co-localized TF binding sites within the three-dimensional genome.ResultsUsing published canonical Hi-C data and single-cell genome structures, we assess the spatial proximity of a genome-wide array of potential TF-TF co-localizations in human and mouse cell lines. For individual TFs, the abundance of occupied binding sites shows a positive correspondence with their clustering in three dimensions, and this is especially apparent for weak TF binding sites and at enhancer regions. An analysis between different TF proteins identifies significantly proximal pairs, which are enriched in reported physical interactions. Furthermore, clustering of different TFs based on proximity enrichment identifies two partially segregated co-localization sub-networks, involving different TFs in different cell types. Using data from both human lymphoblastoid cells and mouse embryonic stem cells, we find that these sub-networks are enriched within, but not exclusive to, different chromosome sub-compartments that have been identified previously in Hi-C data.ConclusionsThis suggests that the association of TFs within spatial networks is closely coupled to gene regulatory networks. This applies to both differentiated and undifferentiated cells and is a potential causal link between lineage-specific TF binding and chromosome sub-compartment segregation.

Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions.

Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

Mathematical modeling of mutant transferrin-CRM107 molecular conjugates for cancer therapy

The transferrin (Tf) trafficking pathway is a promising mechanism for use in targeted cancer therapy due to the overexpression of transferrin receptors (TfRs) on cancerous cells. We have previously developed a mathematical model of the Tf/TfR trafficking pathway to improve the efficiency of Tf as a drug carrier. By using diphtheria toxin (DT) as a model toxin, we found that mutating the Tf protein to change its iron release rate improves cellular association and efficacy of the drug. Though this is an improvement upon using wild-type Tf as the targeting ligand, conjugated toxins like DT are unfortunately still highly cytotoxic at off-target sites. In this work, we address this hurdle in cancer research by developing a mathematical model to predict the efficacy and selectivity of Tf conjugates that use an alternative toxin. For this purpose, we have chosen to study a mutant of DT, cross-reacting material 107 (CRM107). First, we developed a mathematical model of the Tf-DT trafficking pathway by extending our Tf/TfR model to include intracellular trafficking via DT and DT receptors. Using this mathematical model, we subsequently investigated the efficacy of several conjugates in cancer cells: DT and CRM107 conjugated to wild-type Tf, as well as to our engineered mutant Tf proteins (K206E/R632A Tf and K206E/R534A Tf). We also investigated the selectivity of mutant Tf-CRM107 against non-neoplastic cells. Through the use of our mathematical model, we predicted that (i) mutant Tf-CRM107 exhibits a greater cytotoxicity than wild-type Tf-CRM107 against cancerous cells, (ii) this improvement was more drastic with CRM107 conjugates than with DT conjugates, and (iii) mutant Tf-CRM107 conjugates were selective against non-neoplastic cells. These predictions were validated with in vitro cytotoxicity experiments, demonstrating that mutant Tf-CRM107 conjugates is indeed a more suitable therapeutic agent. Validation from in vitro experiments also confirmed that such whole-cell kinetic models can be useful in cancer therapeutic design.

A novel quantification strategy of transferrin and albumin in human serum by species-unspecific isotope dilution laser ablation inductively coupled plasma mass spectrometry (ICP-MS)

Species-specific (SS) isotope dilution analysis with gel electrophoresis (GE)-laser ablation (LA)-ICP-MS is a promising technique for the quantification of particular metal-binding proteins in biological samples. However, unavailable isotopically enriched spike and metal losses in GE separation are main limitations for SS-isotope dilution PAGE-LA-ICP-MS. In this study, we report for the first time the absolute quantification of transferrin (Tf) and albumin (Alb) in human serum by non-denaturing (native) GE combined with species-unspecific isotope dilution mass spectrometry (IDMS). In order to achieve a homogeneous distribution of both protein and isotope-enriched spike (simulated isotope equilibration), immersing the protein strips with 34S spike solution after gel electrophoresis was demonstrated to be an effective way of spike addition. Furthermore, effects of immersion time and 34S spike concentration were investigated to obtain optimal conditions of the post-electrophoresis isotope dilution method. The relative mass of spike and ablated sample (msp/msam) in IDMS equation was calculated by standard Tf and Alb proteins, which could be applied to the quantification of Tf and Alb in ERM-DA470k/IFCC for method confirmation. The results were in agreement with the certified value with good precision and small uncertainty (1.5–3%). In this method, species-specific spike protein is not necessary and the integrity of the heteroatom-protein could be maintained in sample preparation process. Moreover, the application of species-unspecific isotope dilution GE-LA-ICP-MS has the potential to offer reliable, direct and simultaneous quantification of proteins after conventional 1D and 2D gel electrophoretic separations.