TFG binds LC3C to regulate ULK1 localization and autophagosome formation.

The early secretory pathway and autophagy are two essential and evolutionarily conserved endomembrane processes that are finely interlinked. Although growing evidence suggests that intracellular trafficking is important for autophagosome biogenesis, the molecular regulatory network involved is still not fully defined. In this study, we demonstrate a crucial effect of the COPII vesicle-related protein TFG (Trk-fused gene) on ULK1 puncta number and localization during autophagy induction. This, in turn, affects formation of the isolation membrane, as well as the correct dynamics of association between LC3B and early ATG proteins, leading to the proper formation of both omegasomes and autophagosomes. Consistently, fibroblasts derived from a hereditary spastic paraparesis (HSP) patient carrying mutated TFG (R106C) show defects in both autophagy and ULK1 puncta accumulation. In addition, we demonstrate that TFG activity in autophagy depends on its interaction with the ATG8 protein LC3C through a canonical LIR motif, thereby favouring LC3C-ULK1 binding. Altogether, our results uncover a link between TFG and autophagy and identify TFG as a molecular scaffold linking the early secretion pathway to autophagy.

The Early Secretory Pathway Is Crucial for Multiple Aspects of the Hepatitis C Virus Life Cycle.

Although most of the early events of the hepatitis C virus (HCV) life cycle are well characterized, our understanding of HCV egress is still unclear. Some reports implicate the conventional endoplasmic reticulum (ER)-Golgi route, while some propose noncanonical secretory routes. Initially, the envelopment of HCV nucleocapsid occurs by budding into the ER lumen. Subsequently, the HCV particle exit from the ER is assumed to be mediated by coat protein complex II (COPII) vesicles. COPII vesicle biogenesis also involves the recruitment of cargo to the site of vesicle biogenesis via interaction with COPII inner coat proteins. We investigated the modulation and the specific role of the individual components of the early secretory pathway in HCV egress. We observed that HCV inhibits cellular protein secretion and triggers the reorganization of the ER exit sites and ER-Golgi intermediate compartments (ERGIC). Gene-specific knockdown of the components of this pathway such as SEC16A, TFG, ERGIC-53, and COPII coat proteins demonstrated the functional significance of these components and the distinct role played by these proteins in various aspects of the HCV life cycle. SEC16A is essential for multiple steps in the HCV life cycle, whereas TFG is specifically involved in HCV egress and ERGIC-53 is crucial for HCV entry. Overall, our study establishes that the components of the early secretory pathway are essential for HCV propagation and emphasize the importance of the ER-Golgi secretory route in this process. Surprisingly, these components are also required for the early stages of the HCV life cycle due to their role in overall intracellular trafficking and homeostasis of the cellular endomembrane system. IMPORTANCE The virus life cycle involves entry into the host, replication of the genome, assembly of infectious progeny, and their subsequent release. Different aspects of the HCV life cycle, including entry, genome replication, and assembly, are well characterized; however, our understanding of the HCV release is still not clear and subject to debate due to varied findings. Here, we attempted to address this controversy and enhance our understanding of HCV egress by evaluating the role of the different components of the early secretory pathway in the HCV life cycle. To our surprise, we found that the components of the early secretory pathway are not only essential for HCV release but also contribute to many other earlier events of the HCV life cycle. This study emphasizes the importance of the early secretory pathway for the establishment of productive HCV infection in hepatocytes.

TFG::MET-rearranged soft tissue tumor: A rare infantile neoplasm with a distinct low-grade triphasic morphology.

This article presents 2 cases of TFG::MET-rearranged mesenchymal tumor, an extremely rare molecular subset among an emerging group of mesenchymal neoplasms with kinase gene (NTRK, BRAF, RET and others) alterations. Both tumors were congenital, occurred in female patients and presented as huge masses on the trunk and thigh, measuring 18 and 20 cm in the largest dimension. Both cases showed identical areas with a distinctive triphasic morphology resembling fibrous hamartoma of infancy (FHI), consisting of haphazardly arranged ovoid to spindled cells traversed by variably cellular and hyalinized fascicles admixed with (most likely non-neoplastic) adipose tissue. In other areas, a high-grade infantile fibrosarcoma/malignant peripheral nerve sheath tumor-like (IFS/MPNST-like) morphology was present in both cases. While the first case co-expressed CD34 and S100 protein, the other case did not. When combined with the three previously reported MET-rearranged cases (of which two harbored TFG::MET fusion), 3/5 and 3/4 of MET-rearranged and TFG::MET fusion-associated tumors, respectively exhibited similar triphasic FHI-like low-grade morphology. This points toward the existence of a relatively distinct morphological subset among kinase-fusion-associated tumors which seems to be strongly associated with MET fusions. It seems some of these low-grade cases may transform into a high-grade variant with IFS/MPNST-like morphology as has been observed in other tumors with kinase gene fusions. While most cases seem to follow an indolent clinical course, the recognition of these tumors is clinically relevant as MET tyrosine kinase inhibitors might represent an effective treatment option for clinically aggressive or unresectable cases.

Mutation Screening of TFG in α-Synucleinopathy and Amyotrophic Lateral Sclerosis.

BACKGROUND: Recently, p.R383H in TFG was identified as the disease cause in a family with α-synucleinopathy and amyotrophic lateral sclerosis (ALS). However, no further replication has been conducted in larger cohorts. OBJECTIVE: The aim was to explore the genetic role of TFG in α-synucleinopathy and ALS. METHODS: We analyzed the rare protein-coding variants in patients with Parkinson's disease (PD), ALS, multiple system atrophy (MSA), spastic paraplegia (N = 2709), and 7536 controls with whole-exome sequencing. RESULTS: Nine rare variants were identified in PD and two in MSA. One PD patient carried the same variant p.R383H. Similarly, this patient developed early-onset PD with bradykinesia and rigidity on the left side as the initial symptoms. However, at the gene level, rare variants of TFG were not enriched in patients. CONCLUSIONS: Rare variants of TFG were not enriched in α-synucleinopathy and ALS. However, we could not deny the potential pathogenicity of specific variants such as p.R383H. Further exploration is still necessary. © 2022 International Parkinson and Movement Disorder Society.

A Novel TFG Mutation in a Korean Family with α-Synucleinopathy and Amyotrophic Lateral Sclerosis.

BACKGROUND: Tropomyosin-receptor kinase fused gene (TFG) functions as a regulator of intracellular protein packaging and trafficking at the endoplasmic reticulum exit sites. TFG has recently been proposed as a cause of multisystem proteinopathy. OBJECTIVES: Here, we describe a Korean family presenting with Parkinson's disease or amyotrophic lateral sclerosis caused by a novel variant of TFG (c.1148 G > A, p.Arg383His). METHODS: We collected clinical, genetic, dopamine transporter imaging, nerve conduction, and electromyography data from the seven subjects. To verify the pathogenicity of the R383H variant, we studied cell viability and the abnormal aggregation of α-synuclein and TAR DNA-binding protein 43 (TDP-43) in HeLa cells expressing R383H-TFG. RESULTS: The clinical phenotypes of the R383H-TFG mutation varied; of the five family members, one had Parkinson's disease, three had subclinical parkinsonism, and one (the proband) had amyotrophic lateral sclerosis. The individual with multiple system atrophy was the proband's paternal cousin, but the TFG genotype was not confirmed due to unavailability of samples. Our in vitro studies showed that R383H-TFG overexpression impaired cell viability. In cells co-expressing R383H-TFG and α-synuclein, insoluble α-synuclein aggregates increased in concentration and were secreted from the cells and co-localized with R383H-TFG. The levels of cytoplasmic insoluble aggregates of TDP-43 increased in HeLa cells expressing R383H-TFG and co-localized with R383H-TFG. CONCLUSIONS: Clinical and in vitro studies have supported the pathogenic role of the novel TFG mutation in α-synucleinopathy and TDP-43 proteinopathy. These findings expand the phenotypic spectrum of TFG and suggest a pivotal role of endoplasmic reticulum dysfunction during neurodegeneration. © 2021 International Parkinson and Movement Disorder Society.

COPII-mediated trafficking at the ER/ERGIC interface.

Coat proteins play multiple roles in the life cycle of a membrane-bound transport intermediate, functioning in lipid bilayer remodeling, cargo selection and targeting to an acceptor compartment. The Coat Protein complex II (COPII) coat is known to act in each of these capacities, but recent work highlights the necessity for numerous accessory factors at all stages of transport carrier existence. Here, we review recent findings that highlight the roles of COPII and its regulators in the biogenesis of tubular COPII-coated carriers in mammalian cells that enable cargo transport between the endoplasmic reticulum and ER-Golgi intermediate compartments, the first step in a series of trafficking events that ultimately allows for the distribution of biosynthetic secretory cargoes throughout the entire endomembrane system.

Tyrosinase and laccase-producing Bacillus aryabhattai TFG5 and its role in the polymerization of phenols.

BACKGROUND: Tyrosinases and laccases are oxidoreductase enzymes that are used widely in the food, feed, textile, and biofuel industries. The rapidly growing industrial demand for bacterial oxido-reductases has encouraged research on this enzyme worldwide. These enzymes also play a key role in the formation of humic substances (HS) that are involved in controlling the biogeochemical carbon cycle, providing nutrients and bio-stimulants for plant growth, and interacting with inorganic and organic pollutants besides increasing carbon sequestration and mitigating greenhouse gas emission in the environment. The present study aimed to screen and characterize extracellular tyrosinase and laccase-producing soil bacteria that could be utilized in the polymerization of phenols. RESULTS: Twenty isolates from different soil samples collected from forest ecosystems were characterized through ARDRA using restriction digestion with AluI, HpaII, and HaeIII restriction enzymes. The results of Hierarchical Cluster Analysis (HCA) revealed a 60 % similarity coefficient among 13 out of 20 isolates, of which, the isolate TFG5 exhibited only 10 % similarity when compared to all the other isolates. The isolate TFG5 exhibited both tyrosinase (1.34 U.mL- 1) and laccase (2.01 U.mL- 1) activity and was identified as Bacillus aryabhattai. The increased polymerization activity was observed when B. aryabhattai TFG5 was treated with phenols. The monomers such as catechol, p-Hydroxy benzoic acid, ferulic acid, and salicylic acid were polymerized efficiently, as evidenced by their FT-IR spectra depicting increased functional groups compared to the standard mushroom tyrosinase. CONCLUSIONS: The polymerization ability of B. aryabhattai TFG5 could be applied to phenol-rich wastewater treatment for efficient precipitation of phenols. Furthermore, tyrosinases can be used for enhancing the synthesis of HS in soil.

Bacillus aryabhattai TFG5-mediated synthesis of humic substances from coir pith wastes.

BACKGROUND: Humic substances (HS) form the largest proportion among all the constituents of soil organic matter and are a key component of the terrestrial ecosystem. HS plays a multifunctional role in the environment by controlling the biogeochemical carbon cycle, providing nutrients and bio-stimulants for plant growth, and interacting with inorganic and organic pollutants. The rate of formation of HS in soils determines its productivity and carbon sequestration capacity. Enhancement of HS synthesis in the soil through the microbial route not only increases CO2 sequestration but also mitigates the greenhouse gas emissions in the environment. RESULT: In this study, we attempted to understand the mechanism of formation and enhancement of HS from coir pith wastes using the tyrosinase produced by Bacillus aryabhattai TFG5. The bacterium TFG5 isolated from the termite garden produced the tyrosinase (1.34 U mL-1) and laccase (2.1 U mL-1) at 48 h and 60 h of fermentation, respectively. The extracellular tyrosinase from B. aryabhattai TFG5 was designated as TyrB. Homology modeling of TyrB revealed a structure with a predicted molecular mass of 35.23 kDa and two copper ions in the active center with its conserved residues required for the tyrosinase activity. TyrB efficiently transformed and polymerized standard phenols, such as p-cresol, p-hydroxyl benzoic acid, Levo DOPA, and 2,6 DMP, besides transforming free phenols in coir pith wash water (CWW). Additionally, UV-Vis and FT-IR spectra of the degradation products of the coir pith treated with TyrB revealed the formation of HS within 3 days of incubation. Furthermore, the E472/664 ratio of the degradation products revealed a higher degree of condensation of the aromatic carbons and the presence of more aliphatic structures in the HS. CONCLUSION: The results confirmed the influence of TyrB for the effective synthesis of HS from coir pith wastes. The results of the present study also confirm the recently accepted theory of humification proposed by the International Humic Substances Society.

Spitz nevus with a novel TFG-NTRK2 fusion: The first case report of NTRK2-rearranged Spitz/Reed nevus.

Fusions of ALK, ROS1, NTRK1, NTRK3, RET, MET, MERTK, FGFR1, ERBB4, LCK, BRAF, MAP3K8, MAP3K3, and PRKDC and mutation of HRAS have so far been discovered as the genetic alterations associated with the pathogenesis of Spitz neoplasms. This report presents the first case of NTRK2-rearranged Spitz/Reed nevus. The patient was a 39-year-old male with a pigmented macule rapidly growing on his shoulder. Histopathologically, the lesion was a junctional melanocytic nevus composed of large nests of spindled melanocytes with abundant eosinophilic cytoplasm associated with a hyperplastic epidermis. These findings fulfilled the diagnostic criteria of a pigmented spindle cell nevus of Reed (variant of Spitz nevus). Immunohistochemistry for pan-Trk revealed diffuse cytoplasmic positivity in the tumor cells, but immunoexpression of ALK, ROS1, and BRAF V600E was not seen. A novel, in-frame, TFG-NTRK2 fusion was identified by RNA sequencing. This case report expands the list of genetic alterations in Spitz neoplasms and the spectrum of NTRK2-rearranged tumors.

Preeclampsia: Cardiotonic Steroids, Fibrosis, Fli1 and Hint to Carcinogenesis.

Despite prophylaxis and attempts to select a therapy, the frequency of preeclampsia does not decrease and it still takes the leading position in the structure of maternal mortality and morbidity worldwide. In this review, we present a new theory of the etiology and pathogenesis of preeclampsia that is based on the interaction of Na/K-ATPase and its endogenous ligands including marinobufagenin. The signaling pathway of marinobufagenin involves an inhibition of transcriptional factor Fli1, a negative regulator of collagen synthesis, followed by the deposition of collagen in the vascular tissues and altered vascular functions. Moreover, in vitro and in vivo neutralization of marinobufagenin is associated with the restoration of Fli1. The inverse relationship between marinobufagenin and Fli1 opens new possibilities in the treatment of cancer; as Fli1 is a proto-oncogene, a hypothesis on the suppression of Fli1 by cardiotonic steroids as a potential anti-tumor therapeutic strategy is discussed as well. We propose a novel therapy of preeclampsia that is based on immunoneutralization of the marinobufagenin by monoclonal antibodies, which is capable of impairing marinobufagenin-Na/K-ATPase interactions.

Continuum of phenotypes in hereditary motor and sensory neuropathy with proximal predominance and Charcot-Marie-Tooth patients with TFG mutation.

Charcot-Marie-Tooth (CMT) is a common neuropathy, and hereditary motor and sensory neuropathy with proximal predominance (HMSN-P) is a recently described rare neuromuscular disease. Although many genes have been implicated for CMT, TFG is the only known HMSN-P-causing gene. Within the framework of diagnostic criteria, clinical variation is evident among CMT-diagnosed and also HMSN-P-diagnosed individuals. Mutations that cause p.(Pro285Leu) and p.(Gly269Val) in TFG were earlier reported as cause of HMSN-P in two Iranian pedigrees. Here, we report the identification of p.(Gly269Val) in TFG as cause of CMT in a large Iranian pedigree. The clinical features of patients of the three pedigrees are presented and critically compared. Similarities between the two HMSN-P-diagnosed pedigrees with different TFG mutations, and differences between the two differentially diagnosed pedigrees with the same p.(Gly269Val) mutation were evident. The clinical features of the HMSN-P pedigree with the p.(Pro285Leu) and the CMT pedigree with the p.(Gly269Val) mutation were clearly congruent with the respective diagnoses, whereas the features of the HMSN-P-diagnosed pedigree with the p.(Gly269Val) were intermediate between the other two pedigrees. It is therefore suggested that the clinical features of the three Iranian pedigrees with TFG mutations and diagnosed with HMSN-P or CMT represent a continuum.

Muscle pathology of hereditary motor and sensory neuropathy with proximal dominant involvement with TFG mutation.

BACKGROUND: Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) is characterized by adult onset, a slowly progressive course and autosomal dominant inheritance. It remains unclear whether myopathic changes occur histopathologically. METHODS: We encountered 2 patients in a family with a heterozygous p.P285L mutation in TRK-fused gene (TFG), which is known to cause HMSN-P. The affected individuals developed proximal-dominant muscle weakness in their 40s, which slowly progressed to a motor neuron disease-like phenotype. RESULTS: Muscle biopsy showed myopathic pathology including fiber size variability, increased internal nuclei, fiber splitting, and core-like structures, associated with neurogenic changes: large groups of atrophic fibers and fiber type-grouping. Immunohistochemistry revealed sarcoplasmic aggregates of TFG, TDP-43, and p62 without congophilic material. CONCLUSIONS: The present study demonstrates myopathic changes in HMSN-P. Although the mechanisms underlying the skeletal muscle involvement remain to be elucidated, immunohistochemistry suggests that abnormal protein aggregation may be involved in the myopathic pathology.

Adrenomedullin mediates pro-angiogenic and pro-inflammatory cytokines in asthma and COPD.

PURPOSE: Adrenomedullin (AM) is a pluripotent peptide hormone with contradictory effects in human health and disease. In chronic inflammatory lung diseases, such as asthma and COPD, AM has been shown to inhibit inflammation and cell proliferation. In the present study, we aimed to investigate the effect of AM on pro-angiogenic and pro-inflammatory cytokines in asthma and COPD. PATIENTS AND METHODS: Serum levels of pro-AM were measured in patients with asthma, COPD and matched controls. The effect of AM on intracellular signaling proteins and cytokine secretion was assessed in primary cultures of epithelial cells (EC) and airway smooth muscle cells (ASMC) established from endo-bronchial biopsies of patients with asthma, COPD and controls. RESULTS: Serum pro-AM was higher in patients with asthma and COPD, compared to controls. AM stimulated cAMP in ASMC but not in EC. In EC, AM decreased Erk1/2 MAPK expression and activation but in ASMC, AM activated Erk1/2. This effect was similar in asthma, COPD and controls. AM stimulated the secretion of pro-angiogenic CXCL1 by EC of controls and CXCL5 by EC of asthma patients. AM did not affect the secretion of IL-6 or IL-8 by EC but stimulated the secretion of IL-6 by ASMC. In EC, AM inhibited the stimulatory effect of TGF-ß and IL-4 on the secretion of IL-6 and IL-8 but had an additive stimulatory effect with TGF-ß in ASMC. CONCLUSIONS: These data suggest that AM mediates the secretion of pro-angiogenic and pro-inflammatory cytokines in a cell-type and/or a disease-specific way, explaining its association with clinical outcomes in COPD.

Sural nerve pathology in TFG-associated motor neuron disease with sensory neuropathy.

The tropomyosin-receptor kinase fused gene (TFG) functions in vesicles formation and egress at the endoplasmic reticulum (ER). A heterozygous missense mutation c.854C > T (p.Pro285Leu) within TFG has been reported as causative for hereditary motor and sensory neuropathy with proximal predominance. Here, we describe two unrelated Chinese pedigrees with 13 affected members harboring the same variant. The clinical, electrophysiological and pathological findings are consistent with motor neuron disease with sensory neuropathy. The main symptoms were painful muscle cramps, slowly progressive proximal predominant weakness, muscle atrophy, fasciculation and distal sensory disturbance. Electromyography revealed widespread denervation and reinnervation. Sural nerve biopsy revealed severe loss of myelinated fibers. Electron microscopy revealed aggregation of ER with enlarged lumen and small vesicles in the remaining myelinated and unmyelinated axons. The mitochondria are smaller in Schwann cells and axons. Some unmyelinated axons showed disappearance of neurofilament and microtubular structures. This is the first report of c.854C > T mutation within TFG in Chinese population. Our findings not only extend the geographical and phenotypic spectrum of TFG-related neurological disorders, but also confirm the abnormalities of ER and mitochondria in sural nerves.

R106C TFG variant causes infantile neuroaxonal dystrophy "plus" syndrome.

TFG (tropomyosin-receptor kinase fused gene) encodes an essential protein in the regulation of vesicular trafficking between endoplasmic reticulum and Golgi apparatus. The homozygous variant c.316C > T within TFG has been previously associated with a complicated hereditary spastic paraplegia (HSP) phenotype in two unrelated Indian families. Here, we describe the first Italian family with two affected siblings harboring the same variant, who in childhood were classified as infantile neuroaxonal dystrophy (INAD) based on clinical and neuropathological findings. Twenty years after the first diagnosis, exome sequencing was instrumental to identify the genetic cause of this disorder and clinical follow-up of patients allowed us to reconstruct the natural history of this clinical entity. Investigations on patient's fibroblasts demonstrate the presence of altered mitochondrial network and inner membrane potential, associated with metabolic impairment. Our study highlights phenotypic heterogeneity characterizing individuals carrying the same pathogenic variant in TFG and provides an insight on tight connection linking mitochondrial efficiency and neuronal health to vesicular trafficking.

Vibration-Induced Errors in MEMS Tuning Fork Gyroscopes with Imbalance.

This paper discusses the vibration-induced error in non-ideal MEMS tuning fork gyroscopes (TFGs). Ideal TFGs which are thought to be immune to vibrations do not exist, and imbalance between two gyros of TFGs is an inevitable phenomenon. Three types of fabrication imperfections (i.e., stiffness imbalance, mass imbalance, and damping imbalance) are studied, considering different imbalance radios. We focus on the coupling types of two gyros of TFGs in both drive and sense directions, and the vibration sensitivities of four TFG designs with imbalance are simulated and compared. It is found that non-ideal TFGs with two gyros coupled both in drive and sense directions (type CC TFGs) are the most insensitive to vibrations with frequencies close to the TFG operating frequencies. However, sense-axis vibrations with in-phase resonant frequencies of a coupled gyros system result in severe error outputs to TFGs with two gyros coupled in the sense direction, which is mainly attributed to the sense capacitance nonlinearity. With increasing stiffness coupled ratio of the coupled gyros system, the sensitivity to vibrations with operating frequencies is cut down, yet sensitivity to vibrations with in-phase frequencies is amplified.

TFG associated hereditary spastic paraplegia: an addition to the phenotypic spectrum.

Hereditary spastic paraplegias (HSPs) constitute movement disorders with extreme lower limb spasticity caused by axonopathies of the upper motor neurons. We describe two siblings affected with a recessive form of movement disorder. Whole-exome sequencing revealed a homozygous missense mutation c.64 C>T (p.Arg22Trp) in TFG as cause of the disorder. Comparison of the phenotype of the patients of this study, with that reported previously, revealed differences in the severity of the disorder as well as new clinical findings. These include presence of clonus, undeveloped speech, and sleep disturbances. Our findings extend the phenotypic spectrum associated with the TFG mutations in HSP.

Novel Genetic, Clinical, and Pathomechanistic Insights into TFG-Associated Hereditary Spastic Paraplegia.

Hereditary spastic paraplegias (HSPs) are genetically and clinically heterogeneous axonopathies primarily affecting upper motor neurons and, in complex forms, additional neurons. Here, we report two families with distinct recessive mutations in TFG, previously suggested to cause HSP based on findings in a single small family with complex HSP. The first carried a homozygous c.317G>A (p.R106H) variant and presented with pure HSP. The second carried the same homozygous c.316C>T (p.R106C) variant previously reported and displayed a similarly complex phenotype including optic atrophy. Haplotyping and bisulfate sequencing revealed evidence for a c.316C>T founder allele, as well as for a c.316_317 mutation hotspot. Expression of mutant TFG proteins in cultured neurons revealed mitochondrial fragmentation, the extent of which correlated with clinical severity. Our findings confirm the causal nature of bi-allelic TFG mutations for HSP, broaden the clinical and mutational spectra, and suggest mitochondrial impairment to represent a pathomechanistic link to other neurodegenerative conditions.

The TFG-TEC oncoprotein induces transcriptional activation of the human ß-enolase gene via chromatin modification of the promoter region.

Recurrent chromosome translocations are the hallmark of many human cancers. A proportion of human extraskeletal myxoid chondrosarcomas (EMCs) are associated with the characteristic chromosomal translocation t(3;9)(q11-12;q22), which results in the formation of a chimeric protein in which the N-terminal domain of the TRK-fused gene (TFG) is fused to the translocated in extraskeletal chondrosarcoma (TEC; also called CHN, CSMF, MINOR, NOR1, and NR4A3) gene. The oncogenic effect of this translocation may be due to the higher transactivation ability of the TFG-TEC chimeric protein; however, downstream target genes of TFG-TEC have not yet been identified. The results presented here, demonstrate that TFG-TEC activates the human ß-enolase promoter. EMSAs, ChIP assays, and luciferase reporter assays revealed that TFG-TEC upregulates ß-enolase transcription by binding to two NGFI-B response element motifs located upstream of the putative transcription start site. In addition, northern blot, quantitative real-time PCR, and Western blot analyses showed that overexpression of TFG-TEC up-regulated ß-enolase mRNA and protein expression in cultured cell lines. Finally, ChIP analyses revealed that TFG-TEC controls the activity of the endogenous ß-enolase promoter by promoting histone H3 acetylation. Overall, the results presented here indicate that TFG-TEC triggers a regulatory gene hierarchy implicated in cancer cell metabolism. This finding may aid the development of new therapeutic strategies for the treatment of human EMCs. © 2015 Wiley Periodicals, Inc.

The c.*229C > T gene polymorphism in 3'UTR region of the topoisomerase IIß binding protein 1 gene and LOH in BRCA1/2 regions and their effect on the risk and progression of human laryngeal carcinoma.

Topoisomerase IIß binding protein 1 (TopBP1), a multiple-BRCT-domain, protein plays crucial roles in chromosome replication, DNA damage repair, apoptosis, and cell cycle checkpoint signalling. The aim of this study was to identify five SNPs at loci potentially located in the 3'UTR region of the TopBP1 gene (rs185903567, rs116645643, rs115160714, rs116195487, rs112843513), their relationship with the risk of squamous cell laryngeal cancer (SCLC), tumor invasiveness, and prognosis. Genotyping was performed in 323 genetically unrelated individuals with SCLC and 418 randomly selected healthy volunteers. Allele-specific TopBP1 mRNA and protein expressions were determined by using real-time PCR and Western blotting techniques, respectively. LOH in BRCA1/BRCA2 was determined by using microsatellite markers. Compared to homozygous common allele carriers, heterozygosity for the T variant was associated with increased risk of SCLC (adjusted odds ratio [OR] = 9.83, 95 % confidence interval [CI]: 3.12-22.16, p dominant < 0.0001). The presence of risk allele at rs115160714 TopBP1 determined a higher incidence of nodal metastases (OR = 7.98, 95 % CI: 3.94-16.00, p = 0.001) and higher tumor grade (OR = 6.48, 95 % CI: 0.86-48.01, p = 0.03). The heterozygotes displayed diffuse tumor growth with no distinct borderline (OR = 3.10, 95 % Cl: 0.92-10.62, p = 0.049) and higher depth of invasion (OR = 2.66, 95 % Cl: 0.78-9.03, p = 0.04). Relationships were also identified between TopBP1 mRNA/protein expression and overall survival (p < 0.0001). The incidence of LOH in BRCA1/BRCA2 was significantly related to higher tumor grade and TFG (p < 0.05). The results of this study suggest that rs115160714 TopBP1 may be a genetic marker of etiology and progression in laryngeal cancer.