Protein structure, amino acid composition and sequence determine proteome vulnerability to oxidation-induced damage.

Oxidative stress alters cell viability, from microorganism irradiation sensitivity to human aging and neurodegeneration. Deleterious effects of protein carbonylation by reactive oxygen species (ROS) make understanding molecular properties determining ROS susceptibility essential. The radiation-resistant bacterium Deinococcus radiodurans accumulates less carbonylation than sensitive organisms, making it a key model for deciphering properties governing oxidative stress resistance. We integrated shotgun redox proteomics, structural systems biology, and machine learning to resolve properties determining protein damage by γ-irradiation in Escherichia coli and D. radiodurans at multiple scales. Local accessibility, charge, and lysine enrichment accurately predict ROS susceptibility. Lysine, methionine, and cysteine usage also contribute to ROS resistance of the D. radiodurans proteome. Our model predicts proteome maintenance machinery, and proteins protecting against ROS are more resistant in D. radiodurans. Our findings substantiate that protein-intrinsic protection impacts oxidative stress resistance, identifying causal molecular properties.

FATCAT 2.0: towards a better understanding of the structural diversity of proteins.

FATCAT 2.0 server (http://fatcat.godziklab.org/), provides access to a flexible protein structure alignment algorithm developed in our group. In such an alignment, rotations and translations between elements in the structure are allowed to minimize the overall root mean square deviation (RMSD) between the compared structures. This allows to effectively compare protein structures even if they underwent structural rearrangements in different functional forms, different crystallization conditions or as a result of mutations. The major update for the server introduces a new graphical interface, much faster database searches and several new options for visualization of the structural differences between proteins.

Cancer3D 2.0: interactive analysis of 3D patterns of cancer mutations in cancer subsets.

Our knowledge of cancer genomics exploded in last several years, providing us with detailed knowledge of genetic alterations in almost all cancer types. Analysis of this data gave us new insights into molecular aspects of cancer, most important being the amazing diversity of molecular abnormalities in individual cancers. The most important question in cancer research today is how to classify this diversity to identify subtypes that are most relevant for treatment and outcome prediction for individual patients. The Cancer3D database at http://www.cancer3d.org gives an open and user-friendly way to analyze cancer missense mutations in the context of structures of proteins they are found in and in relation to patients' clinical data. This approach allows users to find novel candidate driver regions for specific subgroups, that often cannot be found when similar analyses are done on the whole gene level and for large, diverse cohorts. Interactive interface allows user to visualize the distribution of mutations in subgroups defined by cancer type and stage, gender and age brackets, patient's ethnicity or vice versa find dominant cancer type, gender or age groups for specific three-dimensional mutation patterns.

Spectrum of PALB2 germline mutations and characteristics of PALB2-related breast cancer: Screening of 16,501 unselected patients with breast cancer and 5890 controls by next-generation sequencing.

BACKGROUND: Partner and localizer BRCA2 (PALB2) is a breast cancer predisposition gene, but the clinical relevance of PALB2 germline mutations in Chinese patients with breast cancer remains unknown. This study attempted to investigate the full prevalence and spectrum of PALB2 germline mutations in China and the associations between PALB2 germline mutations and breast cancer risk. METHODS: A total of 21,216 unselected patients with breast cancer were enrolled from 10 provinces in China, and 5890 Chinese women without cancer were enrolled as healthy controls. PALB2 screening was based on next-generation sequencing. RESULTS: A total of 16,501 BRCA1/2-negative patients with breast cancer were analyzed. Deleterious PALB2 mutation carriers accounted for 0.97% (n = 160) in the breast cancer cohort and for 0.19% (n = 11) in the healthy control cohort. Forty-one novel PALB2 germline mutations were identified. A high frequency of PALB2 c.751C>T was detected, and it accounted for 10.63% of the PALB2 germline mutations detected (17 of 160). PALB2 mutations were significantly associated with increased breast cancer risk (odds ratio [OR], 5.23; 95% confidence interval [CI], 2.84-9.65; P < .0001), especially among women 30 years old or younger (OR, 10.09; 95% CI, 3.95-25.79; P < .0001). Clinical characteristics, including a family history, bigger tumor size, triple-negative breast cancer, positive lymph nodes, and bilateral breast cancer, were closely related to PALB2 mutations. CONCLUSIONS: This study revealed a comprehensive spectrum of PALB2 germline mutations and characteristics of PALB2-related breast cancer in China. PALB2 germline mutations confer a moderately increased risk for breast cancer but profoundly increase breast cancer risk for those 30 years old or younger in the Chinese population.

Propofol Inhibits Proliferation, Migration, Invasion and Promotes Apoptosis Through Down-Regulating miR-374a in Hepatocarcinoma Cell Lines.

BACKGROUND: Propofol is a commonly used anaesthetic with controversial effects on cancer cells. We aimed to explore the functional roles of propofol in hepatocellular carcinoma (HCC) cells as well as the underlying mechanisms. METHODS: HepG2 and SMMC-7721 cells were used in this study. Firstly, the effects of propofol on cell viability, migration, invasion, apoptosis, and involved proteins were assessed by Cell Counting Kit-8 assay, Transwell assay, flow cytometry assay and Western blot analysis, respectively. Subsequently, alteration of miR-374a after stimulation of propofol was analyzed by qRT-PCR. miR-374a was overexpressed and the alteration of proteins in the Wnt/ß-catenin and PI3K/AKT pathways was detected by Western blot analysis. The downstream factor of miR-374a was finally studied. RESULTS: Propofol inhibited cell viability, migration and invasion but promoted apoptosis of HepG2 and SMMC-7721 cells. Meanwhile, cyclinD1, matrix metalloproteinase (MMP)-2 and MMP-9 were down-regulated while Bax/Bcl-2, cleaved caspase-3 and cleaved caspase-9 were up-regulated by propofol. Then, miR-374a level was reduced by propofol. Expression of Wnt3a, ß-catenin, p-PI3K and p-AKT was decreased by propofol, whereas these decreases were reversed by miR-374a overexpression. Finally, TP53 was proven to be target of miR-374a in HepG2 cells. CONCLUSION: Propofol inhibited cell proliferation, migration and invasion while promoted cell apoptosis of HepG2 and SMMC-7721 cells through inhibiting the Wnt/ß-catenin and PI3K/ AKT pathways via down-regulation of miR-374a. Besides, miR-374a affected propofol-treated HepG2 cells by targeting TP53.

PDBFlex: exploring flexibility in protein structures.

The PDBFlex database, available freely and with no login requirements at http://pdbflex.org, provides information on flexibility of protein structures as revealed by the analysis of variations between depositions of different structural models of the same protein in the Protein Data Bank (PDB). PDBFlex collects information on all instances of such depositions, identifying them by a 95% sequence identity threshold, performs analysis of their structural differences and clusters them according to their structural similarities for easy analysis. The PDBFlex contains tools and viewers enabling in-depth examination of structural variability including: 2D-scaling visualization of RMSD distances between structures of the same protein, graphs of average local RMSD in the aligned structures of protein chains, graphical presentation of differences in secondary structure and observed structural disorder (unresolved residues), difference distance maps between all sets of coordinates and 3D views of individual structures and simulated transitions between different conformations, the latter displayed using JSMol visualization software.

Serum Human Epidermal Growth Factor 2 is a Novel Biomarker for Recurrence and Metastasis in Triple Negative Breast Cancer.

BACKGROUND: The aim of this study was to evaluate the predictive value of serum human epidermal growth factor 2 (HER2) for recurrence and metastasis in triple negative breast cancer (TNBC). METHODS: A total of 200 patients with benign breast tumors and 300 patients with breast cancer treated in the Department of Breast Surgery, Women and Children's Hospital of Ningbo City (China) between December 2006 and December 2013 were enrolled. Another 500 age- and gender-matched healthy individuals served as controls. The serum level of HER2 was determined using suspension array technology. Patients with breast cancer were further divided into ER-/PR-/HER2- and ER-/PR-/HER2+ groups and followed up for 5 years to analyze the serum concentration of HER2. RESULTS: The serum HER2 concentration was significantly higher in patients with breast cancer than in healthy controls or patients with benign tumors (both p < 0.01). The serum HER2 concentration also was significantly higher in patients with TNBC than in healthy controls (p < 0.01). The serum concentration of HER2 was significantly higher in TNBC patients who experienced recurrence and metastasis than in TNBC patients who did not experience recurrence and metastasis (both p < 0.01). Notably, the serum HER2 concentration in TNBC patients who experienced recurrence and metastasis was increased to a level statistically similar to that in patients with HER2+ breast cancer (p > 0.05). CONCLUSIONS: Patients with TNBC still have an increased serum HER2 concentration, and serum HER2 may be a valuable, novel biomarker for recurrence and metastasis in TNBC.

Monitoring of Gas Emboli During Hysteroscopic Surgery: A Prospective Study.

OBJECTIVES: Previous studies have demonstrated a high frequency of gas emboli during hysteroscopy, but guidelines for the prevention, early detection, and intervention of gas embolism during hysteroscopic procedures are still lacking. This study aimed to gain a clearer understanding of risk factors and specific signs and symptoms associated with gas emboli. METHODS: This prospective study enrolled 120 women scheduled for hysteroscopy using 5% glucose as distension medium. The gas bubbles were monitored sequentially in internal iliac vein, common iliac vein, inferior vena cava, superior vena cava, heart, and pulmonary artery under the gray-scale imaging of Doppler ultrasound. The frequency, extent, and the hemodynamic and respiratory effects of gas emboli were evaluated. The interventions and outcomes were recorded. The risk factors associated with gas emboli, and their relationship with the frequency and extent of gas emboli, were assessed. RESULTS: In our study, evidence of gas emboli under Doppler ultrasound monitoring was observed in 44 (36.7%) patients. The operation was continued and finished as soon as possible for patients presenting with stable vital signs or transient hemodynamic and respiratory changes, which resolved spontaneously without intervention. The operation was paused for patients presenting with significant hemodynamic changes or loss of consciousness, and the operation was resumed shortly after resumption of stable vital signs following symptomatic treatment. All patients in our study finished the operation and recovered without developing serious complications. Data analysis showed prolonged procedure duration and increased bleeding volume were both positively correlated with the frequency and extent of gas emboli. CONCLUSION: Our study demonstrated a high frequency of gas emboli during hysteroscopy. Doppler ultrasonic monitoring combined with a clearer understanding of specific signs, symptoms, and risk factors will facilitate early detection and intervention of gas emboli during hysteroscopy.

AIDA: ab initio domain assembly for automated multi-domain protein structure prediction and domain-domain interaction prediction.

MOTIVATION: Most proteins consist of multiple domains, independent structural and evolutionary units that are often reshuffled in genomic rearrangements to form new protein architectures. Template-based modeling methods can often detect homologous templates for individual domains, but templates that could be used to model the entire query protein are often not available. RESULTS: We have developed a fast docking algorithm ab initio domain assembly (AIDA) for assembling multi-domain protein structures, guided by the ab initio folding potential. This approach can be extended to discontinuous domains (i.e. domains with 'inserted' domains). When tested on experimentally solved structures of multi-domain proteins, the relative domain positions were accurately found among top 5000 models in 86% of cases. AIDA server can use domain assignments provided by the user or predict them from the provided sequence. The latter approach is particularly useful for automated protein structure prediction servers. The blind test consisting of 95 CASP10 targets shows that domain boundaries could be successfully determined for 97% of targets. AVAILABILITY AND IMPLEMENTATION: The AIDA package as well as the benchmark sets used here are available for download at http://ffas.burnham.org/AIDA/. CONTACT: adam@sanfordburnham.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

AIDA: ab initio domain assembly server.

AIDA: ab initio domain assembly server, available at http://ffas.burnham.org/AIDA/ is a tool that can identify domains in multi-domain proteins and then predict their 3D structures and relative spatial arrangements. The server is free and open to all users, and there is an option for a user to provide an e-mail to get the link to result page. Domains are evolutionary conserved and often functionally independent units in proteins. Most proteins, especially eukaryotic ones, consist of multiple domains while at the same time, most experimentally determined protein structures contain only one or two domains. As a result, often structures of individual domains in multi-domain proteins can be accurately predicted, but the mutual arrangement of different domains remains unknown. To address this issue we have developed AIDA program, which combines steps of identifying individual domains, predicting (separately) their structures and assembling them into multiple domain complexes using an ab initio folding potential to describe domain-domain interactions. AIDA server not only supports the assembly of a large number of continuous domains, but also allows the assembly of domains inserted into other domains. Users can also provide distance restraints to guide the AIDA energy minimization.

POSA: a user-driven, interactive multiple protein structure alignment server.

POSA (Partial Order Structure Alignment), available at http://posa.godziklab.org, is a server for multiple protein structure alignment introduced in 2005 (Ye,Y. and Godzik,A. (2005) Multiple flexible structure alignment using partial order graphs. Bioinformatics, 21, 2362-2369). It is free and open to all users, and there is no login requirement, albeit there is an option to register and store results in individual, password-protected directories. In the updated POSA server described here, we introduce two significant improvements. First is an interface allowing the user to provide additional information by defining segments that anchor the alignment in one or more input structures. This interface allows users to take advantage of their intuition and biological insights to improve the alignment and guide it toward a biologically relevant solution. The second improvement is an interactive visualization with options that allow the user to view all superposed structures in one window (a typical solution for visualizing results of multiple structure alignments) or view them individually in a series of synchronized windows with extensive, user-controlled visualization options. The user can rotate structure(s) in any of the windows and study similarities or differences between structures clearly visible in individual windows.

bNAber: database of broadly neutralizing HIV antibodies.

The discovery of broadly neutralizing antibodies (bNAbs) has provided an enormous impetus to the HIV vaccine research and to entire immunology. The bNAber database at http://bNAber.org provides open, user-friendly access to detailed data on the rapidly growing list of HIV bNAbs, including neutralization profiles, sequences and three-dimensional structures (when available). It also provides an extensive list of visualization and analysis tools, such as heatmaps to analyse neutralization data as well as structure and sequence viewers to correlate bNAbs properties with structural and sequence features of individual antibodies. The goal of the bNAber database is to enable researchers in this field to easily compare and analyse available information on bNAbs thereby supporting efforts to design an effective vaccine for HIV/AIDS. The bNAber database not only provides easy access to data that currently is scattered in the Supplementary Materials sections of individual papers, but also contributes to the development of general standards of data that have to be presented with the discovery of new bNAbs and a universal mechanism of how such data can be shared.

FFAS-3D: improving fold recognition by including optimized structural features and template re-ranking.

MOTIVATION: Homology detection enables grouping proteins into families and prediction of their structure and function. The range of application of homology-based predictions can be significantly extended by using sequence profiles and incorporation of local structural features. However, incorporation of the latter terms varies a lot between existing methods, and together with many examples of distant relations not recognized even by the best methods, suggests that further improvements are still possible. RESULTS: Here we describe recent improvements to the fold and function assignment system (FFAS) method, including adding optimized structural features (experimental or predicted), 'symmetrical' Z-score calculation and re-ranking the templates with a neural network. The alignment accuracy in the new FFAS-3D is now 11% higher than the original and comparable with the most accurate template-based structure prediction algorithms. At the same time, FFAS-3D has high success rate at the Structural Classification of Proteins (SCOP) family, superfamily and fold levels. Importantly, FFAS-3D results are not highly correlated with other programs suggesting that it may significantly improve meta-predictions. FFAS-3D does not require 3D structures of the templates, as using predicted features instead of structure-derived does not lead to the decrease of accuracy. Because of that, FFAS-3D can be used for databases other than Protein Data Bank (PDB) such as Protein families database or Clusters of orthologous groups thus extending its applications to functional annotations of genomes and protein families. AVAILABILITY AND IMPLEMENTATION: FFAS-3D is available at http://ffas.godziklab.org.

Structural genomics analysis of uncharacterized protein families overrepresented in human gut bacteria identifies a novel glycoside hydrolase.

BACKGROUND: Bacteroides spp. form a significant part of our gut microbiome and are well known for optimized metabolism of diverse polysaccharides. Initial analysis of the archetypal Bacteroides thetaiotaomicron genome identified 172 glycosyl hydrolases and a large number of uncharacterized proteins associated with polysaccharide metabolism. RESULTS: BT_1012 from Bacteroides thetaiotaomicron VPI-5482 is a protein of unknown function and a member of a large protein family consisting entirely of uncharacterized proteins. Initial sequence analysis predicted that this protein has two domains, one on the N- and one on the C-terminal. A PSI-BLAST search found over 150 full length and over 90 half size homologs consisting only of the N-terminal domain. The experimentally determined three-dimensional structure of the BT_1012 protein confirms its two-domain architecture and structural analysis of both domains suggests their specific functions. The N-terminal domain is a putative catalytic domain with significant similarity to known glycoside hydrolases, the C-terminal domain has a beta-sandwich fold typically found in C-terminal domains of other glycosyl hydrolases, however these domains are typically involved in substrate binding. We describe the structure of the BT_1012 protein and discuss its sequence-structure relationship and their possible functional implications. CONCLUSIONS: Structural and sequence analyses of the BT_1012 protein identifies it as a glycosyl hydrolase, expanding an already impressive catalog of enzymes involved in polysaccharide metabolism in Bacteroides spp. Based on this we have renamed the Pfam families representing the two domains found in the BT_1012 protein, PF13204 and PF12904, as putative glycoside hydrolase and glycoside hydrolase-associated C-terminal domain respectively.

Ubiquitin-specific peptidase USP22 negatively regulates the STAT signaling pathway by deubiquitinating SIRT1.

BACKGROUND/AIMS: The ubiquitin-specific peptidase USP22 mediates various cellular and organismal processes, such as cell growth, apoptosis, and tumor malignancy. However, the molecular mechanisms that regulate USP22 activity remain poorly understood. Here we identify STAT3 as a new USP22 interactor. METHODS: · We used western blotting and RT-PCR to measure key protein, acetylated STAT3, and mRNA levels in HEK293 and colorectal cancer cell lines transfected with expression plasmids or specific siRNAs. Co-immunoprecipitation was used to demonstrate protein-protein interaction and protein complex composition. RESULTS: USP22 overexpression down-regulated STAT3 acetylation by deubiquitinating SIRT1. The three proteins were found to be present in a single protein complex. SiRNA-mediated depletion of endogenous USP22 resulted in SIRT1 destabilization and elevated STAT3 acetylation. Consistent with this finding, USP22 also down-regulated the expression of two known STAT3 target genes, MMP9 and TWIST. CONCLUSION: We show that USP22 is a new regulator of the SIRT1-STAT3 signaling pathway and report a new mechanistic explanation for cross talk between USP22 and the SIRT1-STAT pathways.

CD133 affects the invasive ability of HCT116 cells by regulating TIMP-2.

CD133 is widely expressed in colorectal cancer (CRC) tissues and cell lines. This protein has been used as a marker of CRC cancer stem cells, although the function and mechanism of CD133 in CRC invasion and metastasis remain unclear. In our study, we examined the role of CD133 in CRC invasion in vitro and investigated the mechanism involved in CD133-related invasion. CD133(high) and CD133(low) HCT116 cells were isolated, and the proliferation and invasive ability of these two subpopulations were tested. CD133(high) HCT116 cells exhibited greater proliferation and invasion compared with CD133(low) HCT116 cells. CD133 knockdown (using CD133 small-interfering [si]RNA) inhibited the invasive activity of CD133si-HCT116 cells. For the first time, we found that the expression of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) was down-regulated in CD133si-HCT116 cells. In addition, for the TIMP-2si-HCT116 cells (transfected with TIMP-2 siRNA), in vitro invasion was significantly decreased, whereas the expression of CD133 remained unchanged. Finally, the metalloproteinase 2 and MEK/ERK signaling pathways were examined, and no significant change was observed after the knockdown of CD133 or TIMP-2 in HCT116 cells. In conclusion, we demonstrated that CD133 plays an important role in HCT116 cell invasion, and for the first time, we found that CD133 knockdown significantly down-regulated TIMP-2 expression, which suggests that CD133 affects the invasive ability of HCT116 cells by regulating TIMP-2.

[Enhanced growth inhibition by combined two pathway inhibitors on K-ras mutated non-small cell lung cancer cells].

OBJECTIVE: To evaluate the effect of combined targeting of MEK and PI3K signaling pathways on K-ras mutated non-small cell lung cancer cell line A549 cells and the relevant mechanisms. METHODS: A549 cells were treated with different concentrations of two inhibitors. Growth inhibition was determined by MTT assay. According to the results of MTT test, the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941,0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244+0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244+5.0 µmol/L GDC-0941). The cell cycle and apoptosis were analyzed by flow cytometry. The expression of proteins related to apoptosis was tested with Western blot. RESULTS: Both GDC-0941 and AZD6244 inhibited the cell proliferation. The combination group II led to a stronger growth inhibition. The combination group I showed an antagonistic effect and combination group II showed an additive or synergistic effect. Compared with the control group, the combination group I led to reduced apoptotic rate [(20.70 ± 0.99)% vs. (18.65 ± 0.92 )%, P > 0.05]; Combination group II exhibited enhanced apoptotic rate [(37.85 ± 3.18)% vs. (52.27 ± 4.36)%, P < 0.01]. In addition, in the combination group II, more A549 cells were arrested in G0/G1 phase and decreased S phase (P < 0.01), due to the reduced expressions of CyclinD1 and Cyclin B1, the increased cleaved PARP and the diminished ratio of Bcl-2/Bax. CONCLUSIONS: For single K-ras mutated NSCLC cell line A549 cells, combination of RAS/MEK/ERK and PI3K/AKT/mTOR inhibition showed synergistic effects depending on the drug doses. Double pathways targeted therapy may be beneficial for these patients.

Feasibility of calculating rocuronium dosage by skeletal muscle weight in patients with obesity.

This study aimed to investigate the dose-response relationship of rocuronium administered based on skeletal muscle weight and to assess the feasibility of calculating rocuronium dosage by skeletal muscle weight in short surgeries for patients with obesity. This single-center, randomized controlled clinical trial included 71 patients with obesity aged 28-70 years, with body fat percentages (PBF) >20% in men and > 28% in women, ASA status I-III, scheduled for tracheoscopy. Patients were randomly allocated into two groups: skeletal muscle group (SM group) received rocuronium based on the skeletal muscle content (1.0 mg/kg, n = 31), and the conventional administration group (conventional group) received rocuronium based on total body weight (0.45 mg/kg, n = 30). General anesthesia was administered using the same protocol. Parameters recorded included patients' general condition, muscle relaxant usage, onset time of muscle relaxants, non-response time, clinical effect time, 75% recovery time, and recovery index. Additionally, occurrences of body movement, choking, and incomplete muscle relaxation during surgery were recorded. Compared to the conventional group, the SM group required significantly less rocuronium dosage, resulting in significantly lower non-response time, clinical effect time, 75% recovery time, and recovery index (p < 0.05), and the onset time is slightly longer. Neither group experienced body movement, choking, or incomplete muscle relaxation (p > 0.05). Utilizing skeletal muscle weight to calculate rocuronium dosage in short surgeries for patients with obesity can reduce dosage, shorten recovery time, and prevent residual muscle relaxation while achieving satisfactory muscle relaxation to meet surgical requirements.

[Thinking of the treatment for lumbar spinal stenosis with high-dense silven needle therapy based on the theory of six-meridian differentiation].

The paper introduces the thinking of the diagnosis and treatment with high-dense silver needle therapy for lumbar spinal stenosis (LSS) based on the theory of six-meridian differentiation. According to the severity of LSS and the depth of illness location, LSS is differentiated as six syndromes/patterns, including taiyang disorder, yangming disorder, shaoyang disorder, shaoyin disorder, jueyin disorder and taiyin disorder. The high-dense silver needle therapy is used. The main points include the bilateral Jiaji points (EX-B 2) from L1 to L5 and the acupoints of the bladder meridian of foot-taiyang (1.5 cun lateral to each side of L1 to L5); and the supplementary points are selected from the affected meridians. According to the disorders of six meridians, the length of moxa stick is adjusted in warm acupuncture, targeting the tender sites of soft tissue damage. In order to obtain the satisfactory effects, the appropriate physical exercise is applicable rather than absolutely limiting the movement of affected vertebrae during the treatment.

FFAS server: novel features and applications.

The Fold and Function Assignment System (FFAS) server [Jaroszewski et al. (2005) FFAS03: a server for profile-profile sequence alignments. Nucleic Acids Research, 33, W284-W288] implements the algorithm for protein profile-profile alignment introduced originally in [Rychlewski et al. (2000) Comparison of sequence profiles. Strategies for structural predictions using sequence information. Protein Science: a Publication of the Protein Society, 9, 232-241]. Here, we present updates, changes and novel functionality added to the server since 2005 and discuss its new applications. The sequence database used to calculate sequence profiles was enriched by adding sets of publicly available metagenomic sequences. The profile of a user's protein can now be compared with ∼20 additional profile databases, including several complete proteomes, human proteins involved in genetic diseases and a database of microbial virulence factors. A newly developed interface uses a system of tabs, allowing the user to navigate multiple results pages, and also includes novel functionality, such as a dotplot graph viewer, modeling tools, an improved 3D alignment viewer and links to the database of structural similarities. The FFAS server was also optimized for speed: running times were reduced by an order of magnitude. The FFAS server, http://ffas.godziklab.org, has no log-in requirement, albeit there is an option to register and store results in individual, password-protected directories. Source code and Linux executables for the FFAS program are available for download from the FFAS server.