Astrocytic aryl hydrocarbon receptor mediates chronic kidney disease-associated mental disorders involving GLT1 hypofunction and neuronal activity enhancement in the mouse brain.

Chronic kidney disease (CKD)-associated mental disorders have been attributed to the excessive accumulation of hemodialysis-resistant indoxyl-3-sulfate (I3S) in the brain. I3S not only induces oxidative stress but is also a potent endogenous agonist of the aryl hydrocarbon receptor (AhR). Here, we investigated the role of AhR in CKD-induced brain disorders using a 5/6 nephrectomy-induced CKD mouse model, which showed increased I3S concentration in both blood and brain, anxiety and impaired novelty recognition, and AhR activation in the anterior cortex. GFAP+ reactive astrocytes were increased accompanied with the reduction of glutamate transporter 1 (GLT1) on perineuronal astrocytic processes (PAPs) in the anterior cingulate cortex (ACC) in CKD mice, and these alterations were attenuated in both neural lineage-specific and astrocyte-specific Ahr conditional knockout mice (nAhrCKO and aAhrCKO). By using chronic I3S treatment in primary astrocytes and glia-neuron (GN) mix cultures to mimic the CKD brain microenvironment, we also found significant reduction of GLT1 expression and activity in an AhR-dependent manner. Chronic I3S treatment induced AhR-dependent pro-oxidant Nox1 and AhR-independent anti-oxidant HO-1 expressions. Notably, AhR mediates chronic I3S-induced neuronal activity enhancement and synaptotoxicity in GN mix, not neuron-enriched cortical culture. In CKD mice, neuronal activity enhancement was observed in ACC and hippocampal CA1, and these responses were abrogated by both nAhrCKO and aAhrCKO. Finally, intranasal AhR antagonist CH-223191 administration significantly ameliorated the GLT1/PAPs reduction, increase in c-Fos+ neurons, and memory impairment in the CKD mice. Thus, astrocytic AhR plays a crucial role in the CKD-induced disturbance of neuron-astrocyte interaction and mental disorders.

Curcumin Facilitates Aryl Hydrocarbon Receptor Activation to Ameliorate Inflammatory Astrogliosis.

Curcumin is an anti-inflammatory and neuroprotective compound in turmeric. It is a potential ligand of the aryl hydrocarbon receptor (AhR) that mediates anti-inflammatory signaling. However, the AhR-mediated anti-inflammatory effect of curcumin within the brain remains unclear. We investigated the role of AhR on the curcumin effect in inflammatory astrogliosis. Curcumin attenuated lipopolysaccharide (LPS)-induced proinflammatory IL-6 and TNF-α gene expression in primary cultured rat astrocytes. When AhR was knocked down, LPS-induced IL-6 and TNF-α were increased and curcumin-decreased activation of the inflammation mediator NF-κB p65 by LPS was abolished. Although LPS increased AhR and its target gene CYP1B1, curcumin further enhanced LPS-induced CYP1B1 and indoleamine 2,3-dioxygenase (IDO), which metabolizes tryptophan to AhR ligands kynurenine (KYN) and kynurenic acid (KYNA). Potential interactions between curcumin and human AhR analyzed by molecular modeling of ligand-receptor docking. We identified a new ligand binding site on AhR different from the classical 2,3,7,8-tetrachlorodibenzo-p-dioxin site. Curcumin docked onto the classical binding site, whereas KYN and KYNA occupied the novel one. Moreover, curcumin and KYNA collaboratively bound onto AhR during molecular docking, potentially resulting in synergistic effects influencing AhR activation. Curcumin may enhance the inflammation-induced IDO/KYN axis and allosterically regulate endogenous ligand binding to AhR, facilitating AhR activation to regulate inflammatory astrogliosis.

New paradigm of functional regulation by DNA mimic proteins: Recent updates.

For many, "DNA mimic protein" (DMP) remains an unfamiliar term. The key feature of these proteins is their DNA-like shape and charge distribution, and they affect the activity of DNA-binding proteins by occupying their DNA-binding domains. Functionally, DMPs regulate mechanisms such as gene expression, restriction, and DNA repair as well as the nucleosome package. Although a few DMPs, such as phage uracil DNA glycosylase inhibitor (UGI) and overcome classical restriction (Ocr), were reported about 20 years ago, only a small number of DMPs have been studied to date. In 2014, we reviewed the functional and structural features of 16 DMPs that were known at the time. Now, seven new DMPs, namely anti-CRISPR suppressors AcrF2, AcrF10 and AcrIIA4, human immunodeficiency virus essential factor VPR, multi-functional inhibitor anti-restriction nuclease (Arn), translational regulator AbbA, and putative Z-DNA mimic MBD3, have been reported. In addition, further study of two previously known DMPs, DMP19 and SAUGI, increased our knowledge of their importance and function. Here, we discuss these updated results and address how several characteristics of the structure/sequence of DMPs (e.g. the DNA-like charge distribution and structural D/E-rich repeats) might someday be used to identify new DMPs using bioinformatic approach. © 2018 IUBMB Life, 71(5):539-548, 2019.

The association of cellulitis incidence and meteorological factors in Taiwan.

Cellulitis is a common infection of the skin and soft tissue. Susceptibility to cellulitis is related to microorganism virulence, the host immunity status and environmental factors. This retrospective study from 2001 to 2013 investigated relationships between the monthly incidence rate of cellulitis and meteorological factors using data from the Taiwanese Health Insurance Dataset and the Taiwanese Central Weather Bureau. Meteorological data included temperature, hours of sunshine, relative humidity, total rainfall and total number of rainy days. In otal, 195 841 patients were diagnosed with cellulitis and the incidence rate was strongly correlated with temperature (γS = 0.84, P < 0.001), total sunshine hours (γS = 0.65, P < 0.001) and total rainfall (γS = 0.53, P < 0.001). The incidence rate of cellulitis increased by 3.47/100 000 cases for every 1° elevation in environmental temperature. Our results may assist clinicians in educating the public of the increased risk of cellulitis during warm seasons and possible predisposing environmental factors for infection.

Multiplexed Detection of Tumor Markers with Multicolor Polymer Dot-Based Immunochromatography Test Strip.

There have been ongoing efforts to develop more sensitive and fast quantitative screening of cancer markers by use of fluorometric immunochromatographic test strips (ICTS) since the remarkable advances in fluorescent nanomaterials. Semiconducting polymer dots (Pdots) have recently emerged as a new type of biocompatible fluorescent probe with extraordinary brightness which is suitable for biological and clinical use. Here, we developed Pdot-based ICTS for quantitative rapid screening of prostate-specific antigen (PSA), α-fetoprotein (AFP), and carcinoembryonic antigen (CEA) in 10 min. Through use of the ultrahigh fluorescence brightness of Pdots, this immunosensor enabled much better detection sensitivity (2.05, 3.30, and 4.92 pg/mL for PSA, AFP, and CEA, respectively), in which the detection limit is at least 2 orders of magnitude lower than that of conventional fluorometric ICTS. Furthermore, we performed proof-of-concept experiments for simultaneous determination of multiple tumor markers in a single test strip. These results demonstrated that this Pdot-based ICTS platform is a promising candidate for developing new generations of point-of-care diagnostics. To the best of our knowledge, this is the first example of Pdot-based ICTS with multiplexing capability.

Using structural-based protein engineering to modulate the differential inhibition effects of SAUGI on human and HSV uracil DNA glycosylase.

Uracil-DNA glycosylases (UDGs) are highly conserved proteins that can be found in a wide range of organisms, and are involved in the DNA repair and host defense systems. UDG activity is controlled by various cellular factors, including the uracil-DNA glycosylase inhibitors, which are DNA mimic proteins that prevent the DNA binding sites of UDGs from interacting with their DNA substrate. To date, only three uracil-DNA glycosylase inhibitors, phage UGI, p56, and Staphylococcus aureus SAUGI, have been determined. We show here that SAUGI has differential inhibitory effects on UDGs from human, bacteria, Herpes simplex virus (HSV; human herpesvirus 1) and Epstein-Barr virus (EBV; human herpesvirus 4). Newly determined crystal structures of SAUGI/human UDG and a SAUGI/HSVUDG complex were used to explain the differential binding activities of SAUGI on these two UDGs. Structural-based protein engineering was further used to modulate the inhibitory ability of SAUGI on human UDG and HSVUDG. The results of this work extend our understanding of DNA mimics as well as potentially opening the way for novel therapeutic applications for this kind of protein.

Expression, purification and enzymatic characterization of undecaprenyl pyrophosphate phosphatase from Vibrio vulnificus.

Undecaprenyl pyrophosphate phosphatase (UppP), a cell membrane integral enzyme, catalyzes the dephosphorylation of undecaprenyl pyrophosphate to undecaprenyl phosphate, which is an essential carrier lipid in bacterial cell wall synthesis. We previously purified E. coli UppP and concluded that its catalytic site is likely located in the periplasm. To search for additional natural UppP homologs to elucidate what constitutes a common catalytic mechanism and to gain a better chance of obtaining high-resolution crystal structural information, we expressed and purified recombinant Vibrio vulnificus UppP using E. coli as a host. Mutagenesis analysis demonstrates that the proposed catalytic residues Gln-13, Glu-17, His-26 and Arg-166 are directly involved in enzyme catalysis. Additionally, mutations of most of the conserved serine and glycine residues within the proposed catalytic site (S22A, G163A and S165A) lead to complete inactivity, very low activity (<1.3% of the wild type) or no protein expression at all (G163R and G168A), whereas S23A and S167A retain enzyme activity (65% and 34%). Kinetic analysis indicates that S23A and S167A result in 1.4- and 5-fold decreases in kcat, whereas the substrate Km value exhibits only minor changes compared with wild-type UppP, implying that they are involved in enzyme catalysis. The structural modeling and molecular dynamics simulation analyses also provide a plausible structure of the catalytic core, centered on a conserved histidine (His-26) that initiates the hydrolysis of phosphate esters, rationalizing the mutagenesis data. This conclusion can be applied generally to all bacterial UppP enzymes.

Proposed carrier lipid-binding site of undecaprenyl pyrophosphate phosphatase from Escherichia coli.

Undecaprenyl pyrophosphate phosphatase (UppP), an integral membrane protein, catalyzes the dephosphorylation of undecaprenyl pyrophosphate to undecaprenyl phosphate, which is an essential carrier lipid in the bacterial cell wall synthesis. Sequence alignment reveals two consensus regions, containing glutamate-rich (E/Q)XXXE plus PGXSRSXXT motifs and a histidine residue, specific to the bacterial UppP enzymes. The predicted topological model suggests that both of these regions are localized near the aqueous interface of UppP and face the periplasm, implicating that its enzymatic function is on the outer side of the plasma membrane. The mutagenesis analysis demonstrates that most of the mutations (E17A/E21A, H30A, S173A, R174A, and T178A) within the consensus regions are completely inactive, indicating that the catalytic site of UppP is constituted by these two regions. Enzymatic analysis also shows an absolute requirement of magnesium or calcium ions in enzyme activity. The three-dimensional structural model and molecular dynamics simulation studies have shown a plausible structure of the catalytic site of UppP and thus provides insights into the molecular basis of the enzyme-substrate interaction in membrane bilayers.

Crowning proteins: modulating the protein surface properties using crown ethers.

Crown ethers are small, cyclic polyethers that have found wide-spread use in phase-transfer catalysis and, to a certain degree, in protein chemistry. Crown ethers readily bind metallic and organic cations, including positively charged amino acid side chains. We elucidated the crystal structures of several protein-crown ether co-crystals grown in the presence of 18-crown-6. We then employed biophysical methods and molecular dynamics simulations to compare these complexes with the corresponding apoproteins and with similar complexes with ring-shaped low-molecular-weight polyethylene glycols. Our studies show that crown ethers can modify protein surface behavior dramatically by stabilizing either intra- or intermolecular interactions. Consequently, we propose that crown ethers can be used to modulate a wide variety of protein surface behaviors, such as oligomerization, domain-domain interactions, stabilization in organic solvents, and crystallization.

Interception of teicoplanin oxidation intermediates yields new antimicrobial scaffolds.

In the search for new efficacious antibiotics, biosynthetic engineering offers attractive opportunities to introduce minor alterations to antibiotic structures that may overcome resistance. Dbv29, a flavin-containing oxidase, catalyzes the four-electron oxidation of a vancomycin-like glycopeptide to yield A40926. Structural and biochemical examination of Dbv29 now provides insights into residues that govern flavinylation and activity, protein conformation and reaction mechanism. In particular, the serendipitous discovery of a reaction intermediate in the crystal structure led us to identify an unexpected opportunity to intercept the normal enzyme mechanism at two different points to create new teicoplanin analogs. Using this method, we synthesized families of antibiotic analogs with amidated and aminated lipid chains, some of which showed marked potency and efficacy against multidrug resistant pathogens. This method offers a new strategy for the development of chemical diversity to combat antibacterial resistance.

14-3-3 proteins regulate cullin 7-mediated Eag1 degradation.

BACKGROUND: Mutations in the human gene encoding the neuron-specific Eag1 (KV10.1; KCNH1) potassium channel are linked to congenital neurodevelopmental diseases. Disease-causing mutant Eag1 channels manifest aberrant gating function and defective protein homeostasis. Both the E3 ubiquitin ligase cullin 7 (Cul7) and the small acid protein 14-3-3 serve as binding partners of Eag1. Cul7 mediates proteasomal and lysosomal degradation of Eag1 protein, whereas over-expression of 14-3-3 notably reduces Eag1 channel activity. It remains unclear whether 14-3-3 may also contribute to Eag1 protein homeostasis. RESULTS: In human cell line and native rat neurons, disruptions of endogenous 14-3-3 function with the peptide inhibitor difopein or specific RNA interference up-regulated Eag1 protein level in a transcription-independent manner. Difopein hindered Eag1 protein ubiquitination at the endoplasmic reticulum and the plasma membrane, effectively promoting the stability of both immature and mature Eag1 proteins. Suppression of endogenous 14-3-3 function also reduced excitotoxicity-associated Eag1 degradation in neurons. Difopein diminished Cul7-mediated Eag1 degradation, and Cul7 knock-down abolished the effect of difopein on Eag1. Inhibition of endogenous 14-3-3 function substantially perturbed the interaction of Eag1 with Cul7. Further structural analyses suggested that the intracellular Per-Arnt-Sim (PAS) domain and cyclic nucleotide-binding homology domain (CNBHD) of Eag1 are essential for the regulatory effect of 14-3-3 proteins. Significantly, suppression of endogenous 14-3-3 function reduced Cul7-mediated degradation of disease-associated Eag1 mutant proteins. CONCLUSION: Overall these results highlight a chaperone-like role of endogenous 14-3-3 proteins in regulating Eag1 protein homeostasis, as well as a therapeutic potential of 14-3-3 modulators in correcting defective protein expression of disease-causing Eag1 mutants.

Identification of an emerging cucumber virus in Taiwan using Oxford nanopore sequencing technology.

BACKGROUND: In June 2020, severe symptoms of leaf mosaic and fruit malformation were observed on greenhouse-grown cucumber plants in Xizhou Township of Changhua County, Taiwan. An unknown virus, designated CX-2, was isolated from a diseased cucumber sample by single lesion isolation on Chenopodium quinoa leaves. Identification of CX-2 was performed. Moreover, the incidence of cucumber viruses in Taiwan was also investigated. METHODS: Transmission electron microscopy was performed to examine virion morphology. The portable MinION sequencer released by Oxford Nanopore Technologies was used to detect viral sequences in dsRNA of CX-2-infected leaf tissue. The whole genome sequence of CX-2 was completed by Sanger sequencing and analyzed. Reverse transcription-polymerase chain reaction (RT-PCR) with species-specific primers and indirect enzyme-linked immunosorbent assay (ELISA) with anti-coat protein antisera were developed for virus detection in the field [see Additional file 1]. RESULTS: Icosahedral particles about 30 nm in diameter were observed in the crud leaf sap of CX-2-infected C. quinoa plant. The complete genome sequence of CX-2 was determined as 4577 nt long and shared 97.0-97.2% of nucleotide identity with that of two cucumber Bulgarian latent virus (CBLV) isolates in Iran and Bulgaria. Therefore, CX-2 was renamed CBLV-TW. In 2020-2022 field surveys, melon yellow spot virus (MYSV) had the highest detection rate of 74.7%, followed by cucurbit chlorotic yellows virus (CCYV) (32.0%), papaya ringspot virus virus watermelon type (PRSV-W) (10.7%), squash leaf curl Philippines virus (SLCuPV) (9.3%), CBLV (8.0%) and watermelon silver mottle virus (WSMoV) (4.0%). Co-infection of CBLV and MYSV could be detected in field cucumbers. CONCLUSION: The emerging CBLV-TW was identified by nanopore sequencing. Whole genome sequence analysis revealed that CBLV-TW is closely related, but phylogenetically distinct, to two known CBLV isolates in Bulgaria and Iran. Detection methods including RT-PCR and indirect ELISA have been developed to detect CBLV and to investigate cucumber viruses in central Taiwan. The 2020-2022 field survey results showed that MYSV and CCYV were the main threats to cucumbers, with CBLV, SLCuPV and WSMoV were occasionally occurring.

In Vivo Validation of Diffuse Optical Imaging with a Dual-Direction Measuring Module of Parallel-Plate Architecture for Breast Tumor Detection.

We demonstrate a working prototype of an optical breast imaging system involving parallel-plate architecture and a dual-direction scanning scheme designed in combination with a mammography machine; this system was validated in a pilot study to demonstrate its application in imaging healthy and malignant breasts in a clinical environment. The components and modules of the self-developed imaging system are demonstrated and explained, including its measuring architecture, scanning mechanism, and system calibration, and the reconstruction algorithm is presented. Additionally, the evaluation of feature indices that succinctly demonstrate the corresponding transmission measurements may provide insight into the existence of malignant tissue. Moreover, five cases are presented including one subject without disease (a control measure), one benign case, one suspected case, one invasive ductal carcinoma, and one positive case without follow-up treatment. A region-of-interest analysis demonstrated significant differences in absorption between healthy and malignant breasts, revealing the average contrast between the abnormalities and background tissue to exceed 1.4. Except for ringing artifacts, the average scattering property of the structure densities was 0.65-0.85 mm-1.

Alcohol-Soluble Zwitterionic 4-(Dimethyl(pyridin-2-yl)ammonio)butane-1-sulfonate Small Molecule as a Cathode Modifier for Nonfullerene Acceptor-Based Organic Solar Cells.

A new zwitterionic small molecule 4-(dimethyl(pyridin-2-yl)ammonio)butane-1-sulfonate (PAS), synthesized from 2-dimethylaminopyrindine (2-DMAP), was developed for the ITO cathode modifier. PAS and 2-DMAP dissolved in methanol can form a thin layer on ITO cathode by a simple spin-coating process. The heteroatom moieties in 2-DMAP (sp2 and sp3 nitrogen) and PAS (sp2 nitrogen and sulfonate ion) can coordinate to the ITO surface and decrease the ITO work function by the induced surface dipoles. The fullerene-based (PBDTT-FTTE:PC71BM) inverted OSCs using PAS and 2-DMAP interlayer can achieve PCEs of 8.95 and 8.26%, respectively, which are superior to the devices without a modifier (PCE = 3.25%) and comparable to the corresponding ZnO-based device (PCE = 8.57%). Nevertheless, 2-DMAP, like other nitrogen-containing polymer interlayer materials, turns out to be not applicable to inverted organic solar cells (I-OSCs) with IT-4F as the n-type electron acceptor because the amino group of 2-DMAP can act as a nucleophile to attack the end-group of IT-4F at the interface. The decomposition of IT-4F by 2-DMAP was carefully proved to be via retro-aldol condensation. As a result, the device (PBDBT-F:IT-4F) modified with 2-DMAP displayed a low PCE of 7.34%. The zwitterionic PAS with reduced nucleophilicity and basicity can modify the ITO surface without decomposing IT-4F. The PBDBT-F:IT-4F-based device modified with PAS maintained a high PCE of 11.41%. Most importantly, the PAS-based device using the well-known Y6 acceptor (PBDBT-F:Y6) can achieve a PCE of 13.82%. This new interfacial material can be universally applied to I-OSCs employing various A-D-A-type acceptors installed with the electrophilic 1,1-dicyanamethylene-5,6-difluoro-3-indanone (FIC) end-group.

Crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of human coronavirus OC43 nucleocapsid protein.

The N-terminal domain of nucleocapsid protein from human coronavirus OC43 (HCoV-OC43 N-NTD) mostly contains positively charged residues and has been identified as being responsible for RNA binding during ribonucleocapsid formation in the coronavirus. In this study, the crystallization and preliminary crystallographic analysis of HCoV-OC43 N-NTD (amino acids 58-195) with a molecular weight of 20 kDa are reported. HCoV-OC43 N-NTD was crystallized at 293 K using PEG 1500 as a precipitant and a 99.9% complete native data set was collected to 1.7 A resolution at 100 K with an overall R(merge) of 5.0%. The crystals belonged to the hexagonal space group P6(5), with unit-cell parameters a = 81.57, c = 42.87 A. Solvent-content calculations suggest that there is likely to be one subunit of N-NTD in the asymmetric unit.

Mismatched dNTP incorporation by DNA polymerase beta does not proceed via globally different conformational pathways.

Understanding how DNA polymerases control fidelity requires elucidation of the mechanisms of matched and mismatched dNTP incorporations. Little is known about the latter because mismatched complexes do not crystallize readily. In this report, we employed small-angle X-ray scattering (SAXS) and structural modeling to probe the conformations of different intermediate states of mammalian DNA polymerase beta (Pol beta) in its wild-type and an error-prone variant, I260Q. Our structural results indicate that the mismatched ternary complex lies in-between the open and the closed forms, but more closely resembles the open form for WT and the closed form for I260Q. On the basis of molecular modeling, this over-stabilization of mismatched ternary complex of I260Q is likely caused by formation of a hydrogen bonding network between the side chains of Gln(260), Tyr(296), Glu(295) and Arg(258), freeing up Asp(192) to coordinate MgdNTP. These results argue against recent reports suggesting that mismatched dNTP incorporations follow a conformational path distinctly different from that of matched dNTP incorporation, or that its conformational closing is a major contributor to fidelity.

Tools for teaching biochemistry and molecular biology: A parallel session at the IUBMB/PSBMB 2019 "Harnessing Interdisciplinary Education in Biochemistry and Molecular Biology" conference.

Approaches to learning and teaching have been undergoing massive changes. Technology has enabled many innovations while other methods have embedded authentic research approaches or looked to other disciplines. The tools in education session of the conference looked at tools being used to teach biochemistry and molecular biology ranging from online platforms, authentic research experiences to the use of music.

Biochemical and molecular dynamics studies of archaeal polyisoprenyl pyrophosphate phosphatase from Saccharolobus solfataricus.

The undecaprenyl pyrophosphate phosphatase (UppP) is an integral membrane pyrophosphatase. In bacteria, UppP catalyzes the dephosphorylation of undecaprenyl pyrophosphate (C55-pp) to undecaprenyl phosphate (C55-P) in the periplasmic space, which is an essential step for the isoprenyl lipid carrier to reenter the peptidoglycan synthesis cycle. Besides bacteria, the UppP homologs are widely distributed in archaea genome. However, all archaea lack peptidoglycan structure in their cell wall components, and the major archaeal lipid carriers are dolichol phosphate (Dol-p) and dolichol pyrophosphate (Dol-pp), so the functions of the UppP homolog in archaea remain unclear. Here, we purified a recombinant polyisoprenyl pyrophosphatase of a thermoacidophilic archaeon, Saccharolobus solfataricus (SsUppP), and characterized its enzymatic properties. Two isoprenyl pyrophosphate, farnesyl pyrophosphate (Fpp) and geranylgeranyl pyrophosphate (Ggpp), were used as the surrogate substrates, simulating the bacterial and archaeal lipid carriers. SsUppP dephosphorylated Fpp and Ggpp at 37 °C, but retained the phosphatase activity at high temperatures. The optimal condition for the enzymatic activity was found to be at pH 7 and 70 °C. The thermostability of SsUppP was also supported by molecular dynamics simulation studies. Our results indicated that the archaeal SsUppP can dephosphorylate isoprenyl pyrophosphates at the natural environment of high temperature, and the possibility to catalyze the dephosphorylation of archaeal lipid carriers.

Diffuse expression of RNA-binding protein IMP3 predicts high-stage lymph node metastasis and poor prognosis in colorectal adenocarcinoma.

BACKGROUND: IMP3 (insulinlike growth factor II mRNA-binding protein 3) is a newly identified oncofetal RNA-binding protein that is involved in cell growth and cell migration during the early stages of embryogenesis. This study sought to elucidate its role in tumor progression and prognosis of colorectal adenocarcinoma (CRA). METHODS: IMP3 expression in 186 surgically resected unifocal primary CRAs was analyzed by immunohistochemistry. The proportions of tumor cells positive for IMP3 were scored into diffuse (> or =50%), focal or heterogeneous (10-50%), and trace (<10%), and the expression levels were correlated with clinicopathologic features and patient survival. RESULTS: Cytoplasmic immunoreactivity for IMP3 was diffuse in 66 (35%), focal or heterogeneous in 38 (21%), and trace in 34 (18%) samples. No staining was seen in the adjacent nontumorous tissue. Diffuse IMP3 expression correlated with large tumor (>3 cm, P = .0452), high-stage tumor (IIIa-IV, P = .0417), lymph node metastasis (P = .0232), high lymph node ratio (LNR > or = .7, P = .0016), and lower 5-year survival (P = .0012). Further analysis showed that patients with high-stage CRA and diffuse IMP3 expression had the worst survival rate (P < .0001)-far worse than those without diffuse IMP3 expression (P = .0038). Moreover, multivariant analysis showed diffuse IMP3 expression, serosal invasion, LNR, tumor stage, and adjuvant chemotherapy were independent prognostic factors in CRA. CONCLUSION: Diffuse IMP3 protein expression correlates with invasion and aggressiveness during cancer growth and metastasis, and it is an important prognostic factor of CRAs.