7-Dehydrocholesterol: A sterol shield against an iron sword.

Li et al. and Freitas et al. recently identified 7-dehydrocholesterol (7-DHC), a sterol produced through the cholesterol biosynthetic pathway, as a lipid-soluble antioxidant that protects cells from ferroptosis, a cell death pathway triggered by iron-catalyzed phospholipid peroxidation.1,2.

Identification of hyperoxidized PRDX3 as a ferroptosis marker reveals ferroptotic damage in chronic liver diseases.

Ferroptosis, a regulated cell death pathway driven by accumulation of phospholipid peroxides, has been challenging to identify in physiological conditions owing to the lack of a specific marker. Here, we identify hyperoxidized peroxiredoxin 3 (PRDX3) as a marker for ferroptosis both in vitro and in vivo. During ferroptosis, mitochondrial lipid peroxides trigger PRDX3 hyperoxidation, a posttranslational modification that converts a Cys thiol to sulfinic or sulfonic acid. Once hyperoxidized, PRDX3 translocates from mitochondria to plasma membranes, where it inhibits cystine uptake, thereby causing ferroptosis. Applying hyperoxidized PRDX3 as a marker, we determined that ferroptosis is responsible for death of hepatocytes in mouse models of both alcoholic and nonalcoholic fatty liver diseases, the most prevalent chronic liver disorders. Our study highlights the importance of ferroptosis in pathophysiological conditions and opens the possibility to treat these liver diseases with drugs that inhibit ferroptosis.

A lactate-induced response to hypoxia.

Organisms must be able to respond to low oxygen in a number of homeostatic and pathological contexts. Regulation of hypoxic responses via the hypoxia-inducible factor (HIF) is well established, but evidence indicates that other, HIF-independent mechanisms are also involved. Here, we report a hypoxic response that depends on the accumulation of lactate, a metabolite whose production increases in hypoxic conditions. We find that the NDRG3 protein is degraded in a PHD2/VHL-dependent manner in normoxia but is protected from destruction by binding to lactate that accumulates under hypoxia. The stabilized NDRG3 protein binds c-Raf to mediate hypoxia-induced activation of Raf-ERK pathway, promoting angiogenesis and cell growth. Inhibiting cellular lactate production abolishes the NDRG3-mediated hypoxia responses. Our study, therefore, elucidates the molecular basis for lactate-induced hypoxia signaling, which can be exploited for the development of therapies targeting hypoxia-induced diseases.

Structural insights into vesicular monoamine storage and drug interactions.

Biogenic monoamines-vital transmitters orchestrating neurological, endocrinal and immunological functions1-5-are stored in secretory vesicles by vesicular monoamine transporters (VMATs) for controlled quantal release6,7. Harnessing proton antiport, VMATs enrich monoamines around 10,000-fold and sequester neurotoxicants to protect neurons8-10. VMATs are targeted by an arsenal of therapeutic drugs and imaging agents to treat and monitor neurodegenerative disorders, hypertension and drug addiction1,8,11-16. However, the structural mechanisms underlying these actions remain unclear. Here we report eight cryo-electron microscopy structures of human VMAT1 in unbound form and in complex with four monoamines (dopamine, noradrenaline, serotonin and histamine), the Parkinsonism-inducing MPP+, the psychostimulant amphetamine and the antihypertensive drug reserpine. Reserpine binding captures a cytoplasmic-open conformation, whereas the other structures show a lumenal-open conformation stabilized by extensive gating interactions. The favoured transition to this lumenal-open state contributes to monoamine accumulation, while protonation facilitates the cytoplasmic-open transition and concurrently prevents monoamine binding to avoid unintended depletion. Monoamines and neurotoxicants share a binding pocket that possesses polar sites for specificity and a wrist-and-fist shape for versatility. Variations in this pocket explain substrate preferences across the SLC18 family. Overall, these structural insights and supporting functional studies elucidate the mechanism of vesicular monoamine transport and provide the basis to develop therapeutics for neurodegenerative diseases and substance abuse.

An oncogenic JMJD6-DGAT1 axis tunes the epigenetic regulation of lipid droplet formation in clear cell renal cell carcinoma.

Characterized by intracellular lipid droplet accumulation, clear cell renal cell carcinoma (ccRCC) is resistant to cytotoxic chemotherapy and is a lethal disease. Through an unbiased siRNA screen of 2-oxoglutarate (2-OG)-dependent enzymes, which play a critical role in tumorigenesis, we identified Jumonji domain-containing 6 (JMJD6) as an essential gene for ccRCC tumor development. The downregulation of JMJD6 abolished ccRCC colony formation in vitro and inhibited orthotopic tumor growth in vivo. Integrated ChIP-seq and RNA-seq analyses uncovered diacylglycerol O-acyltransferase 1 (DGAT1) as a critical JMJD6 effector. Mechanistically, JMJD6 interacted with RBM39 and co-occupied DGAT1 gene promoter with H3K4me3 to induce DGAT1 expression. JMJD6 silencing reduced DGAT1, leading to decreased lipid droplet formation and tumorigenesis. The pharmacological inhibition (or depletion) of DGAT1 inhibited lipid droplet formation in vitro and ccRCC tumorigenesis in vivo. Thus, the JMJD6-DGAT1 axis represents a potential new therapeutic target for ccRCC.

A lysine-cysteine redox switch with an NOS bridge regulates enzyme function.

Disulfide bonds between cysteine residues are important post-translational modifications in proteins that have critical roles for protein structure and stability, as redox-active catalytic groups in enzymes or allosteric redox switches that govern protein function1-4. In addition to forming disulfide bridges, cysteine residues are susceptible to oxidation by reactive oxygen species, and are thus central not only to the scavenging of these but also to cellular signalling and communication in biological as well as pathological contexts5,6. Oxidized cysteine species are highly reactive and may form covalent conjugates with, for example, tyrosines in the active sites of some redox enzymes7,8. However, to our knowledge, regulatory switches with covalent crosslinks other than disulfides have not previously been demonstrated. Here we report the discovery of a covalent crosslink between a cysteine and a lysine residue with a NOS bridge that serves as an allosteric redox switch in the transaldolase enzyme of Neisseria gonorrhoeae, the pathogen that causes gonorrhoea. X-ray structure analysis of the protein in the oxidized and reduced state reveals a loaded-spring mechanism that involves a structural relaxation upon redox activation, which is propagated from the allosteric redox switch at the protein surface to the active site in the protein interior. This relaxation leads to a reconfiguration of key catalytic residues and elicits an increase in enzymatic activity of several orders of magnitude. The redox switch is highly conserved in related transaldolases from other members of the Neisseriaceae; for example, it is present in the transaldolase of Neisseria meningitides (a pathogen that is the primary cause of meningitis and septicaemia in children). We surveyed the Protein Data Bank and found that the NOS bridge exists in diverse protein families across all domains of life (including Homo sapiens) and that it is often located at catalytic or regulatory hotspots. Our findings will inform strategies for the design of proteins and peptides, as well as the development of new classes of drugs and antibodies that target the lysine-cysteine redox switch9,10.

The ergodicity question when imaging DNA conformation using liquid cell electron microscopy.

Assessing the ergodicity of graphene liquid cell electron microscope measurements, we report that loop states of circular DNA interconvert reversibly and that loop numbers follow the Boltzmann distribution expected for this molecule in bulk solution, provided that the electron dose is low (80-keV electron energy and electron dose rate 1-20 e- Å-2 s-1). This imaging technique appears to act as a "slow motion" camera that reveals equilibrated distributions by imaging the time average of a few molecules without the need to image a spatial ensemble.

JLONMFSC: Clustering scRNA-seq data based on joint learning of non-negative matrix factorization and subspace clustering.

The development of single cell RNA sequencing (scRNA-seq) has provided new perspectives to study biological problems at the single cell level. One of the key issues in scRNA-seq data analysis is to divide cells into several clusters for discovering the heterogeneity and diversity of cells. However, the existing scRNA-seq data are high-dimensional, sparse, and noisy, which challenges the existing single-cell clustering methods. In this study, we propose a joint learning framework (JLONMFSC) for clustering scRNA-seq data. In our method, the dimension of the original data is reduced to minimize the effect of noise. In addition, the graph regularized matrix factorization is used to learn the local features. Further, the Low-Rank Representation (LRR) subspace clustering is utilized to learn the global features. Finally, the joint learning of local features and global features is performed to obtain the results of clustering. We compare the proposed algorithm with eight state-of-the-art algorithms for clustering performance on six datasets, and the experimental results demonstrate that the JLONMFSC achieves better performance in all datasets. The code is avalable at https://github.com/lanbiolab/JLONMFSC.

A protein-lipid complex that detoxifies free fatty acids.

Fatty acids (FAs) are well known to serve as substrates for reactions that provide cells with membranes and energy. In contrast to these metabolic reactions, the physiological importance of FAs themselves known as free FAs (FFAs) in cells remains obscure. Since accumulation of FFAs in cells is toxic, cells must develop mechanisms to detoxify FFAs. One such mechanism is to sequester free polyunsaturated FAs (PUFAs) into a droplet-like structure assembled by Fas-Associated Factor 1 (FAF1), a cytosolic protein. This sequestration limits access of PUFAs to Fe2+ , thereby preventing Fe2+ -catalyzed PUFA peroxidation. Consequently, assembly of the FAF1-FFA complex is critical to protect cells from ferroptosis, a cell death pathway triggered by PUFA peroxidation. The observations that free PUFAs in cytosol are not randomly diffused but rather sequestered into a membraneless complex should open new directions to explore signaling pathways by which FFAs regulate cellular physiology.

FAF1 blocks ferroptosis by inhibiting peroxidation of polyunsaturated fatty acids.

Iron-dependent peroxidation of polyunsaturated fatty acids (PUFAs) leads to ferroptosis. While detoxification reactions removing lipid peroxides in phospholipids such as that catalyzed by glutathione peroxidase 4 (GPX4) protect cells from ferroptosis, the mechanism through which cells prevent PUFA peroxidation was not completely understood. We previously identified Fas-associated factor 1 (FAF1) as a protein directly interacting with free PUFAs through its UAS domain. Here we report that this interaction is crucial to protect cells from ferroptosis. In the absence of FAF1, cultured cells became sensitive to ferroptosis upon exposure to physiological levels of PUFAs, and mice developed hepatic injury upon consuming a diet enriched in PUFA. Mechanistically, we demonstrate that FAF1 assembles a globular structure that sequesters free PUFAs into a hydrophobic core, a reaction that prevents PUFA peroxidation by limiting its access to iron. Our study suggests that peroxidation of free PUFAs contributes to ferroptosis, and FAF1 acts upstream of GPX4 to prevents initiation of ferroptosis by limiting peroxidation of free PUFAs.

Diverging Maternal and Cord Antibody Functions From SARS-CoV-2 Infection and Vaccination in Pregnancy.

Maternal immunity impacts the infant, but how is unclear. To understand the implications of the immune exposures of vaccination and infection in pregnancy for neonatal immunity, we evaluated antibody functions in paired peripheral maternal and cord blood. We compared those who in pregnancy received mRNA coronavirus disease 2019 (COVID-19) vaccine, were infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the combination. We found that vaccination enriched a subset of neutralizing activities and Fc effector functions that was driven by IgG1 and was minimally impacted by antibody glycosylation in maternal blood. In paired cord blood, maternal vaccination also enhanced IgG1. However, Fc effector functions compared to neutralizing activities were preferentially transferred. Moreover, changes in IgG posttranslational glycosylation contributed more to cord than peripheral maternal blood antibody functional potency. These differences were enhanced with the combination of vaccination and infection as compared to either alone. Thus, Fc effector functions and antibody glycosylation highlight underexplored maternal opportunities to safeguard newborns.

Kahweol Inhibits Pro-Inflammatory Cytokines and Chemokines in Tumor Necrosis Factor-α/Interferon-γ-Stimulated Human Keratinocyte HaCaT Cells.

Atopic dermatitis (AD), marked by intense itching and eczema-like lesions, is a globally increasing chronic skin inflammation. Kahweol, a diterpene that naturally occurs in coffee beans, boasts anti-inflammatory, antioxidative, and anti-cancer properties. This research explores the anti-inflammatory action of kahweol on HaCaT human keratinocytes stimulated by tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), focusing on key signal transduction pathways. Our results demonstrate that kahweol markedly reduces the production of IL-1ß, IL-6, C-X-C motif chemokine ligand 8, and macrophage-derived chemokine in TNF-α/IFN-γ-activated HaCaT cells. Furthermore, it curtails the phosphorylation of key proteins in the mitogen-activated protein kinase (MAPK) pathways, including c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38. Additionally, kahweol impedes the phosphorylation and nuclear translocation of the NF-κB p65 subunit and constrains its DNA-binding capability. It also hampers the phosphorylation, nuclear translocation, and DNA-binding activities of signal transducer and activator of transcription 1 (STAT1) and STAT3. Collectively, these findings suggest that kahweol hinders the generation of cytokines and chemokines in inflamed keratinocytes by inhibiting the MAPK, NF-κB, and STAT cascades. These insights position kahweol as a promising agent for dermatological interventions, especially in managing inflammatory skin conditions such as AD.

FAM Tag Size Separation-Based Capture-Systematic Evolution of Ligands by Exponential Enrichment for Sterigmatocystin-Binding Aptamers with High Specificity.

Sterigmatocystin (ST) is a known toxin whose aptamer has rarely been reported because ST is a water-insoluble small-molecule target with few active sites, leading to difficulty in obtaining its aptamer using traditional target fixation screening methods. To obtain aptamer for ST, we incorporated FAM tag size separation into the capture-systematic evolution of ligands by exponential enrichment and combined it with molecular activation for aptamer screening. The screening process was monitored using a quantitative polymerase chain reaction fluorescence amplification curve and recovery of negative-, counter-, and positive-selected ssDNA. The affinity and specificity of the aptamer were verified by constructing an aptamer-affinity column, and the binding sites were predicted using molecular docking simulations. The results showed that the Kd value of the H Seq02 aptamer was 25.3 nM. The aptamer-affinity column based on 2.3 nmol of H Seq02 exhibited a capacity of about 80 ng, demonstrating better specificity than commercially available antibody affinity columns. Molecular simulation docking predicted the binding sites for H Seq02 and ST, further explaining the improved specificity. In addition, circular dichroism and isothermal titration calorimetry were used to verify the interaction between the aptamer and target ST. This study lays the foundation for the development of a new ST detection method.

Characterization of T cell receptor repertoire in penile cancer.

Tumor-infiltrating lymphocytes (TILs) play a key role in regulating the host immune response and shaping tumor microenvironment. It has been previously shown that T cell infiltration in penile tumors was associated with clinical outcomes. However, few studies have reported the T cell receptor (TCR) repertoire in patients with penile cancer. In the present study, we evaluated the TCR repertoires in tumor and adjacent normal tissues from 22 patients with penile squamous cell carcinoma (PSCC). Analysis of the T cell receptor beta-variable (TRBV) and joining (TRBJ) genes usage and analysis of complementarity determining region 3 (CDR3) length distribution did not show significant differences between tumor and matched normal tissues. Moreover, analysis of the median Jaccard index indicated a limited overlap of TCR repertoire between these groups. Compared with normal tissues, a significantly lower diversity and higher clonality of TCR repertoire was observed in tumor samples, which was associated with clinical characteristics. Further analysis of transcriptional profiles demonstrated that tumor samples with high clonality showed increased expression of genes associated with CD8 + T cells. In addition, we analyzed the TCR repertoire of CD4 + T cells and CD8 + T cells isolated from tumor tissues. We identified that expanded clonotypes were predominantly in the CD8 + T cell compartment, which presented with an exhausted phenotype. Overall, we comprehensively compared TCR repertoire between penile tumor and normal tissues and demonstrated the presence of distinct T cell immune microenvironments in patients with PSCC.

Surface Pinning of Mn by Oxidation State Control for the Synthesis of Cobalt-Free, Ni-Rich, Core/Shell Structured Cathode Materials.

Motivated by the increasing cost, environmental concerns, and limited availability of Co, researchers are actively seeking alternative cathode materials for lithium-ion batteries. A promising strategy involves structure-modified materials, such as a NiMn core/shell system. This design leverages the high energy density of a Ni-rich core while employing an Mn-rich shell to enhance interfacial stability by suppressing unwanted reactions with the electrolyte. This approach offers improved cycling stability and reduced reliance on Co. However, the interdiffusion of Mn ions between the core and shell remains a significant challenge during synthesis. This work presents a facile approach to address the issue of Mn interdiffusion in core/shell cathode materials. The study demonstrates that partial oxidation of the precursor during the drying stage effectively enhances the Mn oxidation state. This strategy successfully suppresses Mn interdiffusion during subsequent calcination, leading to the preservation of the core/shell architecture in the final cathode material. This optimized structure mitigates interfacial reactions, enhances chemomechanical properties, and reduces crosstalk, a major contributor to rollover failure. This work presents a novel approach for synthesizing high-performance core/shell cathode materials for next-generation lithium-ion batteries.

Widespread occurrence of covalent lysine-cysteine redox switches in proteins.

We recently reported the discovery of a lysine-cysteine redox switch in proteins with a covalent nitrogen-oxygen-sulfur (NOS) bridge. Here, a systematic survey of the whole protein structure database discloses that NOS bridges are ubiquitous redox switches in proteins of all domains of life and are found in diverse structural motifs and chemical variants. In several instances, lysines are observed in simultaneous linkage with two cysteines, forming a sulfur-oxygen-nitrogen-oxygen-sulfur (SONOS) bridge with a trivalent nitrogen, which constitutes an unusual native branching cross-link. In many proteins, the NOS switch contains a functionally essential lysine with direct roles in enzyme catalysis or binding of substrates, DNA or effectors, linking lysine chemistry and redox biology as a regulatory principle. NOS/SONOS switches are frequently found in proteins from human and plant pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and also in many human proteins with established roles in gene expression, redox signaling and homeostasis in physiological and pathophysiological conditions.

Enzymatic Synthesis of α-Glucan Microparticles Using Amylosucrases from Bifidobacterium Species and Its Physicochemical Properties.

α-Glucan microparticles (GMPs) have significant potential as high-value biomaterials in various industries. This study proposes a bottom-up approach for producing GMPs using four amylosucrases from Bifidobacterium sp. (BASs). The physicochemical characteristics of these GMPs were analyzed, and the results showed that the properties of the GMPs varied depending on the type of enzymes used in their synthesis. As common properties, all GMPs exhibited typical B-type crystal patterns and poor colloidal dispersion stability. Interestingly, differences in the physicochemical properties of GMPs were generated depending on the synthesis rate of linear α-glucan by the enzymes and the degree of polymerization (DP) distribution. Consequently, we found differences in the properties of GMPs depending on the DP distribution of linear glucans prepared with four BASs. Furthermore, we suggest that precise control of the type and characteristics of the enzymes provides the possibility of producing GMPs with tailored physicochemical properties for various industrial applications.

Hepatobiliary phase imaging in cirrhotic patients using compressed sensing and controlled aliasing in parallel imaging results in higher acceleration.

OBJECTIVE: We aimed to compare the image quality and focal lesion detection ability of hepatobiliary phase (HBP) images obtained using compressed sensing (CS) and controlled aliasing in parallel imaging results in higher acceleration (CAIPIRINHA) in patients with liver cirrhosis. MATERIALS AND METHODS: We retrospectively included 244 gadoxetic acid-enhanced liver MRI from 244 patients with cirrhosis obtained by two HBP images using CS and CAIPIRINHA from July 2020 to December 2020. The optimized resolution and scan time for CS-HBP and CAIPIRINHA-HBP were 0.9 × 0.9 × 1.5 mm3 and 15 s and 1.3 × 1.3 × 3 mm3 and 16 s, respectively. We compared the image quality between the two sets of images in 244 patients and focal lesion (n = 294) analyses for 112 patients. RESULTS: CS-HBP showed comparable overall image quality (3.7 ± 0.9 vs. 3.6 ± 0.8, p = 0.680), superior liver edge sharpness (3.9 ± 0.6 vs. 3.6 ± 0.5, p < 0.001), and fewer respiratory motion artifacts (4.0 ± 0.7 vs. 3.8 ± 0.5, p < 0.001), but higher non-respiratory artifacts (3.4 ± 0.7 vs. 3.6 ± 0.6, p < 0.001) and subjective image noise (3.5 ± 0.8 vs. 3.6 ± 0.7, p = 0.014) than CAIPIRINHA-HBP. CS-HBP showed a higher signal-to-noise ratio in the liver than CAIPIRINHA-HBP (20.9 ± 9.0 vs. 18.9 ± 7.1, p = 0.008). The pooled sensitivity, specificity, and AUC were 90.0%, 77.5%, and 0.84 for CS-HBP and 73.5%, 82.4%, and 0.78 for CAIPIRINHA-HBP, respectively. CONCLUSIONS: CS-HBP showed better focal lesion detection ability, comparable overall image quality, and fewer respiratory motion artifacts, but higher non-respiratory artifacts and noise compared to CAIPIRINHA-HBP. Thus, CS-HBP could be recommended for liver MRI in patients with cirrhosis to improve diagnostic performance. CLINICAL RELEVANCE STATEMENT: Thin-slice CS-HBP may be useful for detecting sub-centimeter hepatocellular carcinoma in cirrhotic patients with Child-Pugh classification A while maintaining comparable subjective image quality. KEY POINTS: • Compared with controlled aliasing in parallel imaging results in higher acceleration, compressed sensing hepatobiliary phase yielded thinner slices and shorter scan time at a higher accelerating factor. • Compressed sensing hepatobiliary phase showed comparable overall image quality, superior liver edge sharpness, and fewer respiratory motion artifacts, but higher non-respiratory artifacts and subjective image noise than controlled aliasing in parallel imaging results in higher acceleration-hepatobiliary phase. • Compressed sensing hepatobiliary phase can detect sub-centimeter hepatocellular carcinoma in cirrhotic patients with Child-Pugh classification A.

The functional role of P2 purinergic receptors in the progression of gastric cancer.

Studies have confirmed that P2 purinergic receptors (P2X receptors and P2Y receptors) expressed in gastric cancer (GC) cells and GC tissues and correlates with their function. Endogenous nucleotides including ATP, ADP, UTP, and UDP, as P2 purinergic receptors activators, participate in P2 purinergic signal transduction pathway. These activated P2 purinergic receptors regulate the progression of GC mainly by mediating ion channels and intracellular signal cascades. It is worth noting that there is a difference in the expression of P2 purinergic receptors in GC, which may play different roles in the progression of GC as a tumor promoting factor or a tumor suppressor factor. Among them, P2 × 7, P2Y2 and P2Y6 receptors have certain clinical significance in patients with GC and may be used as biological molecular markers for the prediction of patients with GC. Therefore, in this paper, we discuss the functional role of nucleotide / P2 purinergic receptors signal axis in regulating the progression of GC and that these P2 purinergic receptors may be used as potential molecular targets for the prevention and treatment of GC.

The Prognostic Value of Tumor Cell Clusters in the Fallopian Tube Lumen in Stage I Endometrioid Carcinoma.

The aim of this study was to investigate the pathologic prognostic factors such as tumor cell clusters (TCCs) in the fallopian tube lumen, myometrial invasion patterns, and positive peritoneal cytology (PPC) in women with Stage I endometrial endometrioid carcinoma (EEC). From 2009 to 2020, consecutive patients with Stage I EEC who underwent hysterectomy and bilateral salpingectomy were included. The primary outcome was the recurrence-free survival (RFS) rate, and the clinicopathological factors affecting RFS were analyzed. A total of 765 patients were enrolled. Seventeen patients (2.2%) had TCC in the fallopian tube lumen, and 58 patients showed a microcystic elongated and fragmented pattern (7.6%). PPC was found in 19 patients (2.5%). The median follow-up period was 61.0 months (range: 2.0-149.7). The majority (88.6%) of patients had Stage IA EEC. The 5-year RFS and overall survival rates were 97.5% and 98.5%, respectively. In multivariate analysis for RFS, the significant prognostic factors were lymphovascular invasion (hazard ratio = 4.604; 95% CI: 1.387-15.288; P = 0.013) and grade (grade 2; hazard ratio = 4.949; 95% CI: 1.035-23.654; P = 0.045, and grade 3; hazard ratio = 5.469; 95% CI: 1.435-20.848; P = 0.013). Other pathologic factors including TCC in the fallopian tube lumen, myometrial invasion patterns, PPC, and hormonal status had no prognostic significance. TCC in the fallopian tube lumen, myometrial invasion pattern, PPC, and estrogen and progesterone receptor positivity were not significant prognostic factors in Stage I EEC. In contrast, lymphovascular invasion and grade were significant prognostic factors.