Discovery of a Unique Set of Dog-Seroreactive Coccidioides Proteins Using Nucleic Acid Programmable Protein Array.

Valley Fever (VF), caused by fungi in the genus Coccidioides, is a prevalent disease in southwestern and western parts of the United States that affects both humans and animals, such as dogs. Although the immune responses to infection with Coccidioides spp. are not fully characterized, antibody-detection assays are used in conjunction with clinical presentation and radiologic findings to aid in the diagnosis of VF. These assays often use Complement Fixation (CF) and Tube Precipitin (TP) antigens as the main targets of IgG and IgM reactivity, respectively. Our group previously reported evidence of over 800 genes expressed at the protein level in C. posadasii. However, antibody reactivity to the majority of these proteins has never been explored. Using a new, high-throughput screening technology, the Nucleic Acid Programmable Protein Array (NAPPA), we screened serum specimens from dogs against 708 of these previously identified proteins for IgG reactivity. Serum from three separate groups of dogs was analyzed and revealed a small panel of proteins to be further characterized for immuno-reactivity. In addition to CF/CTS1 antigen, sera from most infected dogs showed antibody reactivity to endo-1,3-betaglucanase, peroxisomal matrix protein, and another novel reactive protein, CPSG_05795. These antigens may provide additional targets to aid in antibody-based diagnostics.

Development of a rapid lateral flow assay for detection of anti-coccidioidal antibodies.

Coccidioides spp. are dimorphic fungi that are capable of infecting human and non-human mammals and can cause diverse manifestations of coccidioidomycosis or Valley fever (VF). In combination with clinical symptoms and radiographic findings, antibody-based diagnostic tests are often used to diagnose and monitor patients with VF. Chitinase 1 (CTS1) has previously been identified as the seroreactive antigen used in these diagnostic assays to detect anticoccidial IgG. Here, an indirect enzyme-linked immunosorbent assay to detect IgG to CTS1 demonstrated 165 of 178 (92.7%) patients with a positive result by immunodiffusion (ID) and/or complement fixation (CF) had antibodies to the single antigen CTS1. We then developed a rapid antibody lateral flow assay (LFA) to detect anti-CTS1 antibodies. Out of 143 samples tested, the LFA showed 92.9% positive percent agreement [95% confidence interval (CI), 84.3%-96.9%] and 97.7% negative percent agreement (95% CI, 87.9%-99.6%) with ID and CF assays. Serum or plasma from canines, macaques, and dolphins was also tested by the CTS1 LFA. Test line densities of the CTS1 LFA correlated in a linear manner with the reported CF and ID titers for human and non-human samples, respectively. This 10-min point-of-care test for the rapid detection of anti-coccidioidal antibodies could help to inform healthcare providers in real-time, potentially improving the efficiency of healthcare delivery.

Serological survey to estimate SARS-CoV-2 infection and antibody seroprevalence at a large public university: A cross-sectional study.

OBJECTIVE: This study investigated the seroprevalence of SARS-CoV-2 antibodies among adults over 18 years. DESIGN: Prospective cohort study. SETTINGS: A large public university. PARTICIPANTS: This study took volunteers over 5 days and recruited 1064 adult participants. PRIMARY OUTCOME MEASURES: Seroprevalence of SARS-CoV-2-specific antibodies due to previous exposure to SARS-CoV-2 and/or vaccination. RESULTS: The seroprevalence of the antireceptor binding domain (RBD) antibody was 90% by a lateral flow assay and 88% by a semiquantitative chemiluminescent immunoassay. The seroprevalence for antinucleocapsid was 20%. In addition, individuals with previous natural COVID-19 infection plus vaccination had higher anti-RBD antibody levels compared with those who had vaccination only or infection only. Individuals who had a breakthrough infection had the highest anti-RBD antibody levels. CONCLUSION: Accurate estimates of the cumulative incidence of SARS-CoV-2 infection can inform the development of university risk mitigation protocols such as encouraging booster shots, extending mask mandates or reverting to online classes. It could help us to have clear guidance to act at the first sign of the next surge as well, especially since there is a surge of COVID-19 subvariant infections.

Comparative Analysis of Antimicrobial Antibodies between Mild and Severe COVID-19.

Patients with 2019 coronavirus disease (COVID-19) exhibit a broad spectrum of clinical presentations. A person's antimicrobial antibody profile, as partially shaped by past infection or vaccination, can reflect the immune system health that is critical to control and resolve the infection. We performed an explorative immunoproteomics study using microbial protein arrays displaying 318 full-length antigens from 77 viruses and 3 bacteria. We compared antimicrobial antibody profiles between 135 patients with mild COVID-19 disease and 215 patients with severe disease in 3 independent cohorts from Mexico and Italy. Severe disease patients were older with higher prevalence of comorbidities. We confirmed that severe disease patients elicited a stronger anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) response. We showed that antibodies against HCoV-229E and HcoV-NL63 but not against HcoV-HKU1 and HcoV-OC43 were also higher in those who had severe disease. We revealed that for a set of IgG and IgA antibodies targeting coronaviruses, herpesviruses, and other respiratory viruses, a subgroup of patients with the highest reactivity levels had a greater incidence of severe disease compared to those with mild disease across all three cohorts. On the contrary, fewer antibodies showed consistent greater prevalence in mild disease in all 3 cohorts. IMPORTANCE The clinical presentations of COVID-19 range from asymptomatic to critical illness that may lead to intensive care or even death. The health of the immune system, as partially shaped by past infections or vaccinations, is critical to control and resolve the infection. Using an innovative protein array platform, we surveyed antibodies against hundreds of full-length microbial antigens from 80 different viruses and bacteria in COVID-19 patients from different geographic regions with mild or severe disease. We not only confirmed the association of severe COVID-19 disease with higher reactivity of antibody responses to SARS-CoV-2 but also uncovered known and novel associations with antibody responses against herpesviruses and other respiratory viruses. Our study represents a significant step forward in understanding the factors contributing to COVID-19 disease severity. We also demonstrate the power of comprehensive antimicrobial antibody profiling in deciphering risk factors for severe COVID-19. We anticipate that our approach will have broad applications in infectious diseases.

Antibody responses to the host microbiome in chronic rhinosinusitis.

BACKGROUND: The role of microbes in chronic rhinosinusitis (CRS) is poorly understood. We hypothesize that analyzing prior microbial exposures via assessing microbial protein serological reactivity in CRS versus controls may offer insights for CRS etiopathogenesis. METHODS: We profiled IgG and IgA antibodies to individual microbial proteins in serum samples of CRS patients and controls using a novel high-throughput microarray protein technology, Nucleic Acid Programmable Protein Array (NAPPA). The study was conducted on 118 subjects (39 CRS, 79 controls). A CRS-focused NAPPA array, with 1557 potentially sero-reactive microbial proteins elected from a pre-screening of 6500 genes of interest was constructed. It included membrane-associated proteins from 47 bacterial species and all proteins from 43 viral strains. Differences between CRS and controls were compared across individual antimicrobial antibodies and the species. RESULTS: Chronic rhinosinusitis patients had significantly elevated antimicrobial antibodies compared with controls. One bacterium (Staphylococcus aureus) and three viral strains (human metapneumovirus, human herpesvirus 5, and human herpesvirus 4) were identified as sources of the proteins that showed significantly elevated sero-reactivity in CRS patients. Within CRS, patients with polyps had elevated antibodies against S. aureus, influenza A virus (H1N1, H3N2), and rhinovirus B14. CRS patients without polyps showed more antibodies against human herpesvirus 1 and vaccinia virus WR. CONCLUSIONS: Compared with healthy controls, CRS patients' serum samples showed significantly increased sero-reactivity to both bacterial and viral proteins, reflecting recent or current infection or active colonization. Significantly higher antibodies against S. aureus, human metapneumovirus, human herpesvirus 5, and human herpesvirus 4 in CRS need further study.

Identification of anti-Helicobacter pylori antibody signatures in gastric intestinal metaplasia.

BACKGROUND: Chronic Helicobacter pylori infection may induce gastric intestinal metaplasia (IM). We compared anti-H. pylori antibody profiles between IM cases and non-atrophic gastritis (NAG) controls. METHODS: We evaluated humoral responses to 1528 H. pylori proteins among a discovery set of 50 IM and 50 NAG using H. pylori protein arrays. Antibodies with ≥ 20% sensitivity at 90% specificity for either group were selected and further validated in an independent set of 100 IM and 100 NAG using odds ratios (OR). A validated multi-signature was evaluated using the area under the receiver operating characteristics curve (AUC) and net reclassification improvement (NRI). RESULTS: Sixty-two immunoglobulin (Ig) G and 11 IgA antibodies were detected in > 10%. Among them, 22 IgG and 6 IgA antibodies were different between IM and NAG in the discovery set. Validated antibodies included 11 IgG (anti-HP1177/Omp27/HopQ [OR = 8.1, p < 0.001], anti-HP0547/CagA [4.6, p < 0.001], anti-HP0596/Tipα [4.0, p = 0.002], anti-HP0103/TlpB [3.8, p = 0.001], anti-HP1125/PalA/Omp18 [3.1, p = 0.001], anti-HP0153/RecA [0.48, p = 0.03], anti-HP0385 [0.41, p = 0.006], anti-HP0243/TlpB [0.39, p = 0.016], anti-HP0371/FabE [0.37, p = 0.017], anti-HP0900/HypB/AccB [0.35, p = 0.048], and anti-HP0709 [0.30, p = 0.003]), and 2 IgA (anti-HP1125/PalA/Omp18 [2.7, p = 0.03] and anti-HP0596/Tipα [2.5, p = 0.027]). A model including all 11 IgG antibodies (AUC = 0.81) had better discriminated IM and NAG compared with an anti-CagA only (AUC = 0.77) model (NRI = 0.44; p = 0.001). CONCLUSIONS: Our study represents the most comprehensive assessment of anti-H. pylori antibody profiles in IM. The target antigens for these novel antibodies may act together with CagA in the progression to IM. Along with other biomarkers, specific H. pylori antibodies may identify IM patients, who would benefit from surveillance.

Oxidized LDL is stable in human serum under extended thawed-state conditions ranging from -20 °C to room temperature.

Introduction: Oxidized LDL (oxLDL) is formed by the spontaneous reaction between aldehyde byproducts of lipid peroxidation and lysine residues of apolipoprotein B within LDL. Clinically, oxLDL is used as a marker of coronary artery disease and predictor of metabolic syndrome risk. Despite its popularity as a clinical marker, no systematic studies of oxLDL stability, in which serum or plasma has been pre-analytically exposed to an array of different time and temperature conditions, have been carried out. Objective: To systematically evaluate the stability of oxLDL in human serum samples exposed to thawed conditions (> -30 °C) for varying periods of time while monitoring a second protein/small molecule redox system as a positive control for non-enzymatic biomolecular activity. Methods: OxLDL was measured in serum samples, from 24 different humans, that had been pre-exposed to three different time courses at 23 °C, 4 °C and -20 °C using ELISA kits from Mercodia that employ the 4E6 mouse monoclonal antibody. A liquid chromatography/mass spectrometry-based marker of serum exposure to thawed conditions known as ΔS-Cys-Albumin was employed as a positive control. Results: OxLDL was stable in serum exposed to 23 °C for up to 48 h, 4 °C for 21 days, or -20 °C for 65 days. ΔS-Cys-Albumin changed dramatically during these time courses (p < 0.001). Conclusions: OxLDL is remarkably stable ex vivo in human serum samples exposed to thawed conditions.

Comparative Microbiomics Analysis of Antimicrobial Antibody Response between Patients with Lung Cancer and Control Subjects with Benign Pulmonary Nodules.

BACKGROUND: CT screening can detect lung cancer early but suffers a high false-positive rate. There is a need for molecular biomarkers that can distinguish malignant and benign indeterminate pulmonary nodules (IPN) detected by CT scan. METHODS: We profiled antibodies against 901 individual microbial antigens from 27 bacteria and 29 viruses in sera from 127 lung adenocarcinoma (ADC), 123 smoker controls (SMC), 170 benign nodule controls (BNC) individuals using protein microarrays to identify ADC and BNC specific antimicrobial antibodies. RESULTS: Analyzing fourth quartile ORs, we found more antibodies with higher prevalence in the three BNC subgroups than in ADC or SMC. We demonstrated that significantly more anti-Helicobacter pylori antibodies showed higher prevalence in ADC relative to SMC. We performed subgroup analysis and found that more antibodies with higher prevalence in light smokers (≤20 pack-years) compared with heavy smokers (>20 pack-years), in BNC with nodule size >1 cm than in those with ≤1 cm nodules, and in stage I ADC than in stage II and III ADC. We performed multivariate analysis and constructed antibody panels that can distinguish ADC versus SMC and ADC versus BNC with area under the ROC curve (AUC) of 0.88 and 0.80, respectively. CONCLUSIONS: Antimicrobial antibodies have the potential to reduce the false positive rate of CT screening and provide interesting insight in lung cancer development. IMPACT: Microbial infection plays an important role in lung cancer development and the formation of benign pulmonary nodules.

Tracking the Stability of Clinically Relevant Blood Plasma Proteins with Delta-S-Cys-Albumin-A Dilute-and-Shoot LC/MS-Based Marker of Specimen Exposure to Thawed Conditions.

Biomolecular integrity can be compromised when blood plasma/serum (P/S) specimens are improperly handled. Compromised analytes can subsequently produce erroneous results-without any indication of having done so. We recently introduced an LC/MS-based marker of P/S exposure to thawed conditions called ΔS-Cys-Albumin which, aided by an established rate law, quantitatively tracks exposure of P/S to temperatures greater than their freezing point of -30 °C. The purposes of this study were to (1) evaluate ΔS-Cys-Albumin baseline values in gastrointestinal cancer patients and cancer-free control donors, (2) empirically assess the kinetic profiles of ΔS-Cys-Albumin at 23 °C, 4 °C, and -20 °C, and (3) empirically link ΔS-Cys-Albumin to the stability of clinically relevant proteins. ΔS-Cys-Albumin was measured at ≥ 9 different time points per exposure temperature in serum and K2EDTA plasma samples from 24 separate donors in aliquots kept separately at 23 °C, 4 °C, and -20 °C. Twenty-one clinically relevant plasma proteins were measured at four time points per temperature via a multiplexed immunoassay on the Luminex platform. Protein stability was assessed by mixed effects models. Coordinated shifts in stability between ΔS-Cys-Albumin and the unstable proteins were documented by repeated measures and Pearson correlations. Plasma ΔS-Cys-Albumin dropped from approximately 20% to under 5% within 96 h at 23 °C, 28 days at 4 °C, and 65 days at -20 °C. On average, 22% of the 21 proteins significantly changed in apparent concentration at each exposure temperature (p < 0.0008 with >10% shift). A linear inverse relationship was found between the percentage of proteins destabilized and ΔS-Cys-Albumin (r = -0.61; p < 0.0001)-regardless of the specific time/temperature of exposure. ΔS-Cys-Albumin tracks cumulative thawed-state exposure. These results now enable ΔS-Cys-Albumin to approximate the percentage of clinically relevant proteins that have been compromised by incidental plasma exposure to thawed-state conditions.

Serological profiling of Crohn's disease and ulcerative colitis patients reveals anti-microbial antibody signatures.

BACKGROUND: The healthcare burden of inflammatory bowel disease (IBD) is rising globally and there are limited non-invasive biomarkers for accurate and early diagnosis. AIM: To understand the important role that intestinal microbiota play in IBD pathogenesis and identify anti-microbial antibody signatures that benefit clinical management of IBD patients. METHODS: We performed serological profiling of 100 Crohn's disease (CD) patients, 100 ulcerative colitis (UC) patients and 100 healthy controls against 1173 bacterial and 397 viral proteins from 50 bacteria and 33 viruses on protein microarrays. The study subjects were randomly divided into discovery (n = 150) and validation (n = 150) sets. Statistical analysis was performed using R packages. RESULTS: Anti-bacterial antibody responses showed greater differential prevalence among the three subject groups than anti-viral antibody responses. We identified novel antibodies against the antigens of Bacteroidetes vulgatus (BVU_0562) and Streptococcus pneumoniae (SP_1992) showing higher prevalence in CD patients relative to healthy controls. We also identified antibodies against the antigen of Streptococcus pyogenes (SPy_2009) showing higher prevalence in healthy controls relative to UC patients. Using these novel antibodies, we built biomarker panels with area under the curve (AUC) of 0.81, 0.87, and 0.82 distinguishing CD vs control, UC vs control, and CD vs UC, respectively. Subgroup analysis revealed that penetrating CD behavior, colonic CD location, CD patients with a history of surgery, and extensive UC exhibited highest antibody prevalence among all patients. We demonstrated that autoantibodies and anti-microbial antibodies in CD patients had minimal correlation. CONCLUSION: We have identified antibody signatures for CD and UC using a comprehensive analysis of anti-microbial antibody response in IBD. These antibodies and the source microorganisms of their target antigens improve our understanding of the role of specific microorganisms in IBD pathogenesis and, after future validation, should aid early and accurate diagnosis of IBD.

Exploration of autoantibody responses in canine diabetes using protein arrays.

Canine diabetes has been considered a potential model of human type 1 diabetes (T1D), however the detection of autoantibodies common in humans with T1D in affected dogs is inconsistent. The aim of this study was to compare autoantibody responses in diabetic and healthy control dogs using a novel nucleic acid programmable protein array (NAPPA) platform. We performed a cross-sectional study of autoantibody profiles of 30 diabetic and 30 healthy control dogs of various breeds. Seventeen hundred human proteins related to the pancreas or diabetes were displayed on NAPPA arrays and interrogated with canine sera. The median normalized intensity (MNI) for each protein was calculated, and results were compared between groups to identify candidate autoantibodies. At a specificity of 90%, six autoantibodies had sensitivity greater than 10% (range 13-20%) for distinguishing diabetic and control groups. A combination of three antibodies (anti-KANK2, anti-GLI1, anti-SUMO2) resulted in a sensitivity of 37% (95% confidence interval (CI) 0.17-0.67%) at 90% specificity and an area under the receiver operating characteristics curve of 0.66 (95% CI 0.52-0.80). While this study does not provide conclusive support for autoimmunity as an underlying cause of diabetes in dogs, future studies should consider the use of canine specific proteins in larger numbers of dogs of breeds at high risk for diabetes.

Identification of anti-Epstein-Barr virus (EBV) antibody signature in EBV-associated gastric carcinoma.

BACKGROUND: Around 10% of gastric carcinomas (GC) contain Epstein-Barr virus (EBV) DNA. We characterized the GC-specific antibody response to this common infection, which may provide a noninvasive method to detect EBV-positive GC and elucidate its contribution to carcinogenesis. METHODS: Plasma samples from EBV-positive (n = 28) and EBV-negative (n = 34) Latvian GC patients were immune-profiled against 85 EBV proteins on a multi-microbial Nucleic Acid Programmable Protein Array (EBV-NAPPA). Antibody responses were normalized for each sample as ratios to the median signal intensity (MNI) across all antigens, with seropositivity defined as MNI ≥ 2. Antibodies with ≥ 20% sensitivity at 95% specificity for tumor EBV status were verified by enzyme-linked immunosorbent assay (ELISA) and validated in independent samples from Korea and Poland (n = 24 EBV-positive, n = 65 EBV-negative). RESULTS: Forty anti-EBV IgG and eight IgA antibodies were detected by EBV-NAPPA in ≥ 10% of EBV-positive or EBV-negative GC patients, of which nine IgG antibodies were discriminative for tumor EBV status. Eight of these nine were verified and seven were validated by ELISA: anti-LF2 (odds ratio = 110.0), anti-BORF2 (54.2), anti-BALF2 (44.1), anti-BaRF1 (26.7), anti-BXLF1 (12.8), anti-BRLF1 (8.3), and anti-BLLF3 (5.4). The top three had areas under receiver operating characteristics curves of 0.81-0.85 for distinguishing tumor EBV status. CONCLUSIONS: The EBV-associated GC-specific humoral response was exclusively directed against lytic cycle immediate-early and early antigens, unlike other EBV-associated malignancies such as nasopharyngeal carcinoma and lymphoma where humoral response is primarily directed against late lytic antigens. Specific anti-EBV antibodies could have utility for clinical diagnosis, epidemiologic studies, and immune-based precision treatment of EBV-positive GC.

Helicobacter pylori Immunoproteomic Profiles in Gastric Cancer.

Chronic Helicobacter pylori infection is the major risk factor for gastric cancer (GC). However, only some infected individuals develop this neoplasia. Previous H. pylori serology studies have been limited by investigating small numbers of candidate antigens. Therefore, we evaluated humoral responses to a nearly complete H. pylori immunoproteome (1527 proteins) among 50 GC cases and 50 controls using Nucleic Acid Programmable Protein Array (NAPPA). Seropositivity was defined as median normalized intensity ≥2 on NAPPA, and 53 anti-H. pylori antibodies had >10% seroprevalence. Anti-GroEL exhibited the greatest seroprevalence (77% overall), which agreed well with ELISA using whole-cell lysates of H. pylori cells. After an initial screen by H. pylori-NAPPA, we discovered and verified that 12 antibodies by ELISA in controls had ≥15% of samples with an optical reading value exceeding the 95th percentile of the GC group. ELISA-verified antibodies were validated blindly in an independent set of 100 case-control pairs. As expected, anti-CagA seropositivity was positively associated with GC (odds ratio, OR = 5.5; p < 0.05). After validation, six anti-H. pylori antibodies showed lower seropositivity in GC, with ORs ranging from 0.44 to 0.12 (p < 0.05): anti-HP1118/Ggt, anti-HP0516/HsIU, anti-HP0243/NapA, anti-HP1293/RpoA, anti-HP0371/FabE, and anti-HP0875/KatA. Among all combinations, a model with anti-Ggt, anti-HslU, anti-NapA, and anti-CagA had an area under the curve of 0.73 for discriminating GC vs. controls. This study represents the first comprehensive assessment of anti-H. pylori humoral profiles in GC. Decreased responses to multiple proteins in GC may reflect mucosal damage and decreased bacterial burden. The higher prevalence of specific anti-H. pylori antibodies in controls may suggest immune protection against GC development.

Burnout and self-reported quality of care in community mental health.

Staff burnout is widely believed to be problematic in mental healthcare, but few studies have linked burnout directly with quality of care. The purpose of this study was to examine the relationship between burnout and a newly developed scale for quality of care in a sample of community mental health workers (N=113). The Self-Reported Quality of Care scale had three distinct factors (Client-Centered Care, General Work Conscientiousness, and Low Errors), with good internal consistency. Burnout, particularly personal accomplishment, and to a lesser extent depersonalization, were predictive of overall self-rated Quality of Care, over and above background variables.

The Fanconi anemia proteins FANCD2 and FANCJ interact and regulate each other's chromatin localization.

Fanconi anemia is a genetic disease resulting in bone marrow failure, birth defects, and cancer that is thought to encompass a defect in maintenance of genomic stability. Mutations in 16 genes (FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P, and Q) have been identified in patients, with the Fanconi anemia subtype J (FA-J) resulting from homozygous mutations in the FANCJ gene. Here, we describe the direct interaction of FANCD2 with FANCJ. We demonstrate the interaction of FANCD2 and FANCJ in vivo and in vitro by immunoprecipitation in crude cell lysates and from fractions after gel filtration and with baculovirally expressed proteins. Mutation of the monoubiquitination site of FANCD2 (K561R) preserves interaction with FANCJ constitutively in a manner that impedes proper chromatin localization of FANCJ. FANCJ is necessary for FANCD2 chromatin loading and focus formation in response to mitomycin C treatment. Our results suggest not only that FANCD2 regulates FANCJ chromatin localization but also that FANCJ is necessary for efficient loading of FANCD2 onto chromatin following DNA damage caused by mitomycin C treatment.

Temporal evolution of financial-market correlations.

We investigate financial market correlations using random matrix theory and principal component analysis. We use random matrix theory to demonstrate that correlation matrices of asset price changes contain structure that is incompatible with uncorrelated random price changes. We then identify the principal components of these correlation matrices and demonstrate that a small number of components accounts for a large proportion of the variability of the markets that we consider. We characterize the time-evolving relationships between the different assets by investigating the correlations between the asset price time series and principal components. Using this approach, we uncover notable changes that occurred in financial markets and identify the assets that were significantly affected by these changes. We show in particular that there was an increase in the strength of the relationships between several different markets following the 2007-2008 credit and liquidity crisis.

Functional and physical interaction between the mismatch repair and FA-BRCA pathways.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure and an increased risk for leukemia and cancer. Fifteen proteins thought to function in the repair of DNA interstrand crosslinks (ICLs) comprise what is known as the FA-BRCA pathway. Activation of this pathway leads to the monoubiquitylation and chromatin localization of FANCD2 and FANCI. It has previously been shown that FANCJ interacts with the mismatch repair (MMR) complex MutLα. Here we show that FANCD2 interacts with the MMR proteins MSH2 and MLH1. FANCD2 monoubiquitylation, foci formation and chromatin loading are greatly diminished in MSH2-deficient cells. Human or mouse cells lacking MSH2 or MLH1 display increased sensitivity and radial formation in response to treatment with DNA crosslinking agents. Studies in human cell lines and Drosophila mutants suggest an epistatic relationship between FANCD2, MSH2 and MLH1 with regard to ICL repair. Surprisingly, the interaction between MSH2 and MLH1 is compromised in multiple FA cell lines, and FA cell lines exhibit deficient MMR. These results suggest a significant role for MMR proteins in the activation of the FA pathway and repair of ICLs. In addition, we provide the first evidence for a defect in MMR in FA cell lines.

The E3 ubiquitin ligase RAD18 regulates ubiquitylation and chromatin loading of FANCD2 and FANCI.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenital abnormalities, and an increased risk for cancer and leukemia. Components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand cross-links. Central to this pathway is the monoubiquitylation and chromatin localization of 2 FA proteins, FA complementation group D2 (FANCD2) and FANCI. In the present study, we show that RAD18 binds FANCD2 and is required for efficient monoubiquitylation and chromatin localization of both FANCD2 and FANCI. Human RAD18-knockout cells display increased sensitivity to mitomycin C and a delay in FANCD2 foci formation compared with their wild-type counterparts. In addition, RAD18-knockout cells display a unique lack of FANCD2 and FANCI localization to chromatin in exponentially growing cells. FANCD2 ubiquitylation is normal in cells containing a ubiquitylation-resistant form of proliferating cell nuclear antigen, and chromatin loading of FA core complex proteins appears normal in RAD18-knockout cells. Mutation of the RING domain of RAD18 ablates the interaction with and chromatin loading of FANCD2. These data suggest a key role for the E3 ligase activity of RAD18 in the recruitment of FANCD2 and FANCI to chromatin and the events leading to their ubiquitylation during S phase.

Dynamic communities in multichannel data: an application to the foreign exchange market during the 2007-2008 credit crisis.

We study the cluster dynamics of multichannel (multivariate) time series by representing their correlations as time-dependent networks and investigating the evolution of network communities. We employ a node-centric approach that allows us to track the effects of the community evolution on the functional roles of individual nodes without having to track entire communities. As an example, we consider a foreign exchange market network in which each node represents an exchange rate and each edge represents a time-dependent correlation between the rates. We study the period 2005-2008, which includes the recent credit and liquidity crisis. Using community detection, we find that exchange rates that are strongly attached to their community are persistently grouped with the same set of rates, whereas exchange rates that are important for the transfer of information tend to be positioned on the edges of communities. Our analysis successfully uncovers major trading changes that occurred in the market during the credit crisis.