A description of the upgraded beamline X25 at the NSLS, operated by the PXRR and the Photon Sciences Directorate serving the Macromolecular Crystallography community, is presented.
Beamline X25 at the NSLS is one of the five beamlines dedicated to macromolecular crystallography operated by the Brookhaven National Laboratory Macromolecular Crystallography Research Resource group. This mini-gap insertion-device beamline has seen constant upgrades for the last seven years in order to achieve mini-beam capability down to 20 µm × 20 µm. All major components beginning with the radiation source, and continuing along the beamline and its experimental hutch, have changed to produce a state-of-the-art facility for the scientific community.
beamline; mini-κ; Pilatus 6M; PXRR; macromolecular crystallography; wBPM
Traditional single-marker and multimarker molecular profiling approaches in bladder cancer do not account for major risk factors and their influence on clinical outcome. This study examined the prognostic value of molecular alterations across all disease stages after accounting for clinicopathological factors and smoking, the most common risk factor for bladder cancer in the developed world, in a population-based cohort.
Primary bladder tumors from 212 cancer registry patients (median follow-up, 13.2 years) were immunohistochemically profiled for Bax, caspase-3, Apaf-1, Bcl-2, p53, p21, cyclooxygenase-2, vascular endothelial growth factor, and E-cadherin alterations. “Smoking intensity” quantified the impact of duration and daily frequency of smoking.
Age, pathological stage, surgical modality, and adjuvant therapy administration were significantly associated with survival. Increasing smoking intensity was independently associated with worse outcome (P<0.001). Apaf-1, E-cadherin and p53 were prognostic for outcome (P=0.005, 0.014 and 0.032, respectively); E-cadherin remained prognostic following multivariable analysis (P=0.040). Combined alterations in all nine biomarkers were prognostic by univariable (P<0.001) and multivariable (P=0.006) analysis. A multivariable model that included all nine biomarkers and smoking intensity had greater accuracy in predicting prognosis than models comprising of standard clinicopathological covariates without or with smoking intensity (P<0.001 and P=0.018, respectively).
Apaf-1, E-cadherin and p53 alterations individually predicted survival in bladder cancer patients. Increasing number of biomarker alterations was significantly associated with worsening survival, although markers comprising the panel were not necessarily prognostic individually. Predictive value of the nine-biomarker panel with smoking intensity was significantly higher than that of routine clinicopathological parameters alone.
Exposure of a single crystal of the nitrite adduct of ferric myoglobin (Mb) at 100 K to high-intensity synchrotron X-ray radiation resulted in changes in the UV-vis spectrum that can be attributed to reduction of the ferric compound to the ferrous derivative. We employed correlated single-crystal spectroscopy with crystallography to further characterize this photoproduct. The 1.55 Å resolution crystal structure of the photoproduct reveals retention of the O-binding mode of nitrite to the iron center. The data are consistent with the cryogenic generation and trapping, at 100 K, of a ferrous d6 MbII(ONO)* complex by photoreduction of the ferric precursor crystals using high-intensity X-ray radiation.
The instrumentation and methods available for collecting almost simultaneous single-crystal electronic absorption correlated with X-ray diffraction data at NSLS beamline X26-C are reviewed, as well as a very brief outline of its Raman spectroscopy capability.
The research philosophy and new capabilities installed at NSLS beamline X26-C to support electronic absorption and Raman spectroscopies coupled with X-ray diffraction are reviewed. This beamline is dedicated full time to multidisciplinary studies with goals that include revealing the relationship between the electronic and atomic structures in macromolecules. The beamline instrumentation has been fully integrated such that optical absorption spectra and X-ray diffraction images are interlaced. Therefore, optical changes induced by X-ray exposure can be correlated with X-ray diffraction data collection. The installation of Raman spectroscopy into the beamline is also briefly reviewed. Data are now routinely generated almost simultaneously from three complementary types of experiments from the same sample. The beamline is available now to the NSLS general user population.
metalloenzymes; cofactors; electronic absorption spectroscopy; Raman spectroscopy
Microdeletions within chromosome 15q13.3 are associated both with a recently recognised syndrome of mental retardation, seizures, and dysmorphic features, and with schizophrenia.
Methods and results
Based on routine diagnostic testing of ~8200 samples using array comparative genomic hybridisation, we identified 20 individuals (14 children and six parents in 12 families) with microdeletions of 15q13.3. Phenotypes in the children included developmental delay, mental retardation, or borderline IQ in most and autistic spectrum disorder (6/14), speech delay, aggressiveness, attention deficit hyperactivity disorder, and other behavioural problems. Both parents were available in seven families, and the deletion was de novo in one, inherited from an apparently normal parent in four, and inherited from a parent with learning disability and bipolar disorder in two families. Of the 14 children, six in five families were adopted, and DNA was available for only one of these 10 biological parents; the deletion was very likely inherited for one of these families with two affected children. Among the unavailable parents, two mothers were described as having mental retardation, another mother as having “mental illness”, and one father as having schizophrenia. We hypothesise that some of the unavailable parents have the deletion.
The occurrence of increased adoption, frequent autism, bipolar disorder, and lack of penetrance are noteworthy findings in individuals with deletion 15q13.3. A high rate of adoption may be related to the presence of the deletion in biological parents.
Unconfirmed histories of antisocial behaviours in unavailable biological parents raise the concern that future research may show that deletion 15q13.3 is associated with such behaviours.
Ovules are the female reproductive structures that develop into seeds. Angiosperm ovules include one, or more commonly two, integuments that cover the nucellus and female gametophyte. Mutations in Arabidopsis KANADI (KAN) and YABBY polarity genes result in amorphous or arrested integument growth, suggesting that polarity determinants play key roles in ovule development. We show that the Class III Homeodomain Leucine-Zipper (HD-ZIPIII) genes CORONA (CNA), PHABULOSA (PHB), and PHAVOLUTA (PHV) are expressed adaxially in the inner integument during ovule development, independent of ABERRANT TESTA SHAPE (ATS, also known as KANADI4) activity. Loss of function of these genes leads to aberrant integument growth. Additionally, over-expression of PHB or PHV in ovules is not sufficient to repress ATS expression, and can produce phenotypes similar to those of the HD-ZIPIII loss of function lines. The absence of evidence of mutual negative regulation by KAN and HD-ZIPIII transcription factors is in contrast to known mechanisms in leaves. Loss of HD-ZIPIII activity can partially compensate for loss of ATS activity in the ats cna phb phv quadruple mutant, demonstrating that CNA/PHB/PHV act in concert with ATS to control integument morphogenesis. In a parallel pathway, ATS acts with REVOLUTA (REV) to restrict INNER NO OUTER (INO) expression and outer integument growth. Based on these expression and genetic studies we propose a model in which a balance between the relative levels of adaxial/abaxial activities, rather than the maintenance of boundaries of expression domains, is necessary to support laminar growth of the two integuments.
integument; adaxial-abaxial polarity; HD-ZIPIII; KANADI; YABBY; Arabidopsis
Light chain (LC) amyloidosis (AL) is a fatal disease in which immunoglobulin LC deposit as fibrils. Although the LC amyloid-forming propensity is attributed primarily to the variable region, fibrils also contain full-length LC comprised of variable-joining (VL) and constant (CL) regions. To assess the role of CL in fibrillogenesis, we compared the thermal stability of full-length LC and corresponding VL and CL fragments. Protein unfolding and aggregation were monitored by circular dichroism and light scattering. A full-length λ6 LC purified from urine of a patient with AL amyloidosis showed irreversible unfolding coupled to aggregation. The transition temperature decreased at slower heating rates, indicating kinetic effects. Next, we studied five recombinant λ6 proteins: full-length amyloidogenic LC, its VL, germline LC, germline VL, and CL. Amyloidogenic and germline proteins showed similar rank order of stability, VL < LC < CL; hence, in the full-length LC, VL destabilizes CL. Amyloidogenic proteins were less stable than their germline counterparts, suggesting that reduction in VL stability destabilizes the full-length LC. Thermal unfolding of the full-length amyloidogenic and germline LC required high activation energy and involved irreversible aggregation, yet the unfolding of the isolated VL and CL fragments was partially reversible. Therefore, compared to their fragments, full-length LCs are more likely to initiate aggregation during unfolding and provide a template for the VL deposition. The kinetic barrier for this aggregation is regulated by the stability of the VL region. This represents a paradigm shift in AL fibrillogenesis and suggests CL region as a potential therapeutic target.
We show in human immunodeficiency virus–positive persons that the coronary artery disease effect of an unfavorable genetic background is comparable to previous studies in the general population, and comparable in size to traditional risk factors and antiretroviral regimens known to increase cardiovascular risk.
Background Persons infected with human immunodeficiency virus (HIV) have increased rates of coronary artery disease (CAD). The relative contribution of genetic background, HIV-related factors, antiretroviral medications, and traditional risk factors to CAD has not been fully evaluated in the setting of HIV infection.
Methods In the general population, 23 common single-nucleotide polymorphisms (SNPs) were shown to be associated with CAD through genome-wide association analysis. Using the Metabochip, we genotyped 1875 HIV-positive, white individuals enrolled in 24 HIV observational studies, including 571 participants with a first CAD event during the 9-year study period and 1304 controls matched on sex and cohort.
Results A genetic risk score built from 23 CAD-associated SNPs contributed significantly to CAD (P = 2.9×10−4). In the final multivariable model, participants with an unfavorable genetic background (top genetic score quartile) had a CAD odds ratio (OR) of 1.47 (95% confidence interval [CI], 1.05–2.04). This effect was similar to hypertension (OR = 1.36; 95% CI, 1.06–1.73), hypercholesterolemia (OR = 1.51; 95% CI, 1.16–1.96), diabetes (OR = 1.66; 95% CI, 1.10–2.49), ≥1 year lopinavir exposure (OR = 1.36; 95% CI, 1.06–1.73), and current abacavir treatment (OR = 1.56; 95% CI, 1.17–2.07). The effect of the genetic risk score was additive to the effect of nongenetic CAD risk factors, and did not change after adjustment for family history of CAD.
Conclusions In the setting of HIV infection, the effect of an unfavorable genetic background was similar to traditional CAD risk factors and certain adverse antiretroviral exposures. Genetic testing may provide prognostic information complementary to family history of CAD.
HIV infection; coronary artery disease; genetics; traditional risk factors; antiretroviral therapy
Genome-wide association studies (GWASs) have identified multiple common genetic variants associated with an increased risk of testicular germ cell tumors (TGCTs). A previous GWAS reported a possible TGCT susceptibility locus on chromosome 1q23 in the UCK2 gene, but failed to reach genome-wide significance following replication. We interrogated this region by conducting a meta-analysis of two independent GWASs including a total of 940 TGCT cases and 1559 controls for 122 single-nucleotide polymorphisms (SNPs) on chromosome 1q23 and followed up the most significant SNPs in an additional 2202 TGCT cases and 2386 controls from four case–control studies. We observed genome-wide significant associations for several UCK2 markers, the most significant of which was for rs3790665 (PCombined = 6.0 × 10−9). Additional support is provided from an independent familial study of TGCT where a significant over-transmission for rs3790665 with TGCT risk was observed (PFBAT = 2.3 × 10−3). Here, we provide substantial evidence for the association between UCK2 genetic variation and TGCT risk.
The Mothers After Gestational Diabetes in Australia Diabetes Prevention Program (MAGDA-DPP) is a randomized controlled trial (RCT) that aims to assess the effectiveness of a structured diabetes prevention intervention for women who had gestational diabetes.
The original protocol was published in Trials (http://www.trialsjournal.com/content/14/1/339). This update reports on an additional exclusion criterion and change in first eligibility screening to provide greater clarity. The new exclusion criterion “surgical or medical intervention to treat obesity” has been added to the original protocol. The risks of developing diabetes will be affected by any medical or surgical intervention as its impact on obesity will alter the outcomes being assessed by MAGDA-DPP. The screening procedures have also been updated to reflect the current recruitment operation. The first eligibility screening is now taking place either during or after pregnancy, depending on recruitment strategy.
Australian New Zealand Clinical Trials Registry ANZCTRN 12610000338066.
Gestational diabetes; Lifestyle intervention; Post-natal; Type 2 diabetes prevention
Protein function is regulated by diverse posttranslational modifications. The mitochondrial sirtuin SIRT5 removes malonyl and succinyl moieties from target lysines. The spectrum of protein substrates subject to these modifications is unknown. We report systematic profiling of the mammalian succinylome, identifying 2,565 succinylation sites on 779 proteins. Most of these do not overlap with acetylation sites, suggesting differential regulation of succinylation and acetylation. Our analysis reveals potential impacts of lysine succinylation on enzymes involved in mitochondrial metabolism; e.g., amino acid degradation, the tricarboxylic acid cycle (TCA) cycle, and fatty acid metabolism. Lysine succinylation is also present on cytosolic and nuclear proteins; indeed, we show that a substantial fraction of SIRT5 is extra-mitochondrial. SIRT5 represses biochemical activity of, and cellular respiration through, two protein complexes identified in our analysis, pyruvate dehydrogenase complex and succinate dehydrogenase. Our data reveal widespread roles for lysine succinylation in regulating metabolism and potentially other cellular functions.
A variety of environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of disease and phenotypic variation. The process involves exposure of a gestating female and the developing fetus to environmental factors that promote permanent alterations in the epigenetic programming of the germline. The molecular aspects of the phenomenon involve epigenetic modifications (epimutations) in the germline (e.g. sperm) that are transmitted to subsequent generations. The current study integrates previously described experimental epigenomic transgenerational data and web-based bioinformatic analyses to identify genomic features associated with these transgenerationally transmitted epimutations. A previously identified genomic feature associated with these epimutations is a low CpG density (<12/100bp). The current observations suggest the transgenerational differential DNA methylation regions (DMR) in sperm contain unique consensus DNA sequence motifs, zinc finger motifs and G-quadruplex sequences. Interaction of molecular factors with these sequences could alter chromatin structure and accessibility of proteins with DNA methyltransferases to alter de novo DNA methylation patterns. G-quadruplex regions can promote the opening of the chromatin that may influence the action of DNA methyltransferases, or factors interacting with them, for the establishment of epigenetic marks. Zinc finger binding factors can also promote this chromatin remodeling and influence the expression of non-coding RNA. The current study identified genomic features associated with sperm epimutations that may explain in part how these sites become susceptible for transgenerational programming.
Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify pathways supporting the Myc oncogenic program, we employed a genome-wide RNAi screen for Myc-synthetic-lethal genes and uncovered a role for the SUMO-activating-enzyme (SAE1/2). Loss of SAE1/2 enzymatic activity drives synthetic lethality with Myc. Inactivation of SAE2 leads to mitotic catastrophe and cell death selectively upon Myc hyper-activation. Mechanistically, SAE2 inhibition switches a transcriptional subprogram of Myc from activated to repressed. A subset of these SUMOylation-dependent-Myc-switchers (SMS genes) is required for mitotic spindle function and to support the Myc oncogenic program. SAE2 is required for Myc-dependent tumor growth, and patient survival significantly correlates with SAE1/SAE2 levels in Myc-high tumors. These studies reveal a mitotic vulnerability of Myc-driven cancers, demonstrate that inhibiting sumoylation impairs Myc-dependent tumorigenesis, and suggest inhibiting SUMOylation may have therapeutic benefits for patients with Myc-driven cancer.
Shiga toxin-producing E. coli (STEC) are a group of common and potentially deadly intestinal pathogens expressing Shiga toxin (Stx) as a primary virulence factor. Of the two types of Stx, Stx2 is responsible for more severe symptoms during infection, while Stx1 is almost identical to the Shiga toxin from Shigella dysenteriae, a ubiquitous pathogen in developing countries. Although antibodies against Stx1 have been reported, few have reached the affinity needed for assembling highly sensitive immunoassays. Sensitive and affordable immunoassays for Stx1 and Stx2 could help improve detection of STEC in livestock, food, the environment, and in clinical samples resulting in improved food safety and human health.
Method and Findings
Three new monoclonal antibodies (mAbs) against the B subunit of Stx1 were generated using recombinant toxoid Stx1E167Q and hybridoma technology. These new mAbs recognize all subtypes of Stx1, but do not cross-react with any subtype of Stx2. In addition, they exhibited the ability to neutralize Stx1 toxicity in Vero cell assays. An optimized sandwich ELISA using of a pair of these mAbs had a limit of detection of 8.7 pg/mL, which is superior to any existing assay of this kind. Using one of these Stx1 mAbs in concert with Stx2 mAbs, the presence of hybrid Stx1/Stx2 toxin in the culture media of STEC strains that express both Stx1 and Stx2 was demonstrated.
These new mAbs provide a mix of availability, utility, versatility, and most importantly, increased sensitivity for detection of Stx1. There are numerous potential applications for these mAbs, including low-cost detection assays and therapeutic use. Analysis of hybrid Stx1/2 could provide new insights on the structure, activity, and cellular targets of Shiga toxins.
Fracture of a modern ceramic head component in total hip replacement is an uncommon but catastrophic complication. Hence, the occurrence of a second ceramic head fracture in the same hip replacement of an individual represents a perishingly rare event. We present the case as a means of highlighting potential risk factors for ceramic head fracture and suggest possible management strategies in such cases.
Low back pain (LBP) is the leading cause of disability worldwide. Of those patients who present to primary care with acute LBP, 40% continue to report symptoms 3 months later and develop chronic LBP. Although it is possible to identify these patients early, effective interventions to improve their outcomes are not available. This double-blind (participant/outcome assessor) randomised controlled trial will investigate the efficacy of a brief educational approach to prevent chronic LBP in ‘at-risk’ individuals.
Participants will be recruited from primary care practices in the Sydney metropolitan area. To be eligible for inclusion participants will be aged 18–75 years, with acute LBP (<4 weeks’ duration) preceded by at least a 1 month pain-free period and at-risk of developing chronic LBP. Potential participants with chronic spinal pain and those with suspected serious spinal pathology will be excluded. Eligible participants who agree to take part will be randomly allocated to receive 2×1 h sessions of pain biology education or 2×1 h sessions of sham education from a specially trained study physiotherapist. The study requires 101 participants per group to detect a 1-point difference in pain intensity 3 months after pain onset. Secondary outcomes include the incidence of chronic LBP, disability, pain intensity, depression, healthcare utilisation, pain attitudes and beliefs, global recovery and recurrence and are measured at 1 week post-intervention, and at 3, 6 and 12 months post LBP onset.
Ethical approval was obtained from the University of New South Wales Human Ethics Committee in June 2013 (ref number HC12664). Outcomes will be disseminated through publication in peer-reviewed journals and presentations at international conference meetings.
Trial registration number
Pain Management; Preventive Medicine; Primary Care
Neurodegenerative disorders with high iron in the basal ganglia encompass an expanding collection of single gene disorders collectively known as neurodegeneration with brain iron accumulation. These disorders can largely be distinguished from one another by their associated clinical and neuroimaging features. The aim of this study was to define the phenotype that is associated with mutations in WDR45, a new causative gene for neurodegeneration with brain iron accumulation located on the X chromosome. The study subjects consisted of WDR45 mutation-positive individuals identified after screening a large international cohort of patients with idiopathic neurodegeneration with brain iron accumulation. Their records were reviewed, including longitudinal clinical, laboratory and imaging data. Twenty-three mutation-positive subjects were identified (20 females). The natural history of their disease was remarkably uniform: global developmental delay in childhood and further regression in early adulthood with progressive dystonia, parkinsonism and dementia. Common early comorbidities included seizures, spasticity and disordered sleep. The symptoms of parkinsonism improved with l-DOPA; however, nearly all patients experienced early motor fluctuations that quickly progressed to disabling dyskinesias, warranting discontinuation of l-DOPA. Brain magnetic resonance imaging showed iron in the substantia nigra and globus pallidus, with a ‘halo’ of T1 hyperintense signal in the substantia nigra. All patients harboured de novo mutations in WDR45, encoding a beta-propeller protein postulated to play a role in autophagy. Beta-propeller protein-associated neurodegeneration, the only X-linked disorder of neurodegeneration with brain iron accumulation, is associated with de novo mutations in WDR45 and is recognizable by a unique combination of clinical, natural history and neuroimaging features.
iron; NBIA; autophagy; basal ganglia; Rett syndrome
To characterize and identify determinants of HIV disease progression among a predominantly injection drug use (IDU) HIV population in the highly active antiretroviral therapy era.
The present retrospective study was based on 343 HIV patients diagnosed from 2005 to 2010 from two clinics in Saskatoon, Saskatchewan. Disease progression was defined as the time from diagnosis to immunological AIDS (CD4 count <200 cells/μL) and death. Uni- and multivariable Cox proportional hazards models were used.
Of the 343 patients, 79% had a history of IDU, 77% were hepatitis C virus (HCV) coinfected and 67% were of Aboriginal descent. The one-year and three-year immunological AIDS-free probabilities were 78% and 53%, respectively. The one-year and three-year survival probabilities were 97% and 88%, respectively. Multicollinearity among IDU, HCV and ethnicity was observed and, thus, separate models were built. HCV coinfection (HR 2.9 [95% CI 1.2 to 6.9]) was a significant predictor of progression to immunological AIDS when controlling for baseline CD4 counts, treatment, age at diagnosis and year of diagnosis. For survival, only treatment use was a significant predictor (HR 0.34 [95% CI 0.1 to 0.8]). HCV coinfection was marginally significant (P=0.067).
Baseline CD4 count, HCV coinfection, year of diagnosis and treatment use were significant predictors of disease progression. This highlights the importance of early treatment and the need for targeted interventions for these particularly vulnerable populations to slow disease progression.
Aboriginal ethnicity; Disease progression; Hepatitis C coinfection; HIV/AIDS; Injection drug use; Survival
A conventional electron capture dissociation (ECD) spectrum of a protein is uniquely characteristic of the first dimension of its linear structure. This sequence information is indicated by summing the primary cm+ and zm+• products of cleavage at each of its molecular ion's inter-residue bonds. For example, the ECD spectra of Ubiquitin (M + nH)n+ ions, n = 7-13, provide sequence characterization of 72 of its 75 cleavage sites from 1843 ions in seven c(1-7)+ and eight z(1-8)+• spectra and their respective complements. Now we find that each of these c/z spectra is itself composed of “charge site (CS)” spectra, the cm+ or zm+• products of electron capture at a specific protonated basic residue. This charge site has been H-bonded to multiple other residues, producing multiple precursor ion forms; ECD at these residues yields the multiple products of that CS spectrum. Closely similar CS spectra are often formed from a range of charge states of Ubiquitin and KIX ions; this indicates a common secondary conformation, but not the conventional α-helicity postulated previously. CS spectra should provide new capabilities for comparing regional conformations of gaseous protein ions and delineating ECD fragmentation pathways.
There is considerable regional variation in Medicare outpatient visit rates; such variations may be the consequence of patient health, race/ethnicity differences, patient preferences, or physician supply and beliefs about the efficacy of frequently scheduled visits.
To test associations between varying regional Medicare outpatient visit rates and beneficiaries’ health, race/ethnicity, preferences, and physician practice norms and supply.
We used Medicare claims from 2006 and 2007, and data from national surveys of three different groups in 2005 – Medicare beneficiaries, cardiologists, and primary care physicians. Regression analysis tested explanations for outpatient visit rates: patient health (self-reported and hierarchical condition category (HCC) score), self-reported race/ethnicity, preferences for care, and local physician practice norms and supply in beneficiaries’ Hospital Referral Regions (HRRs) of residence.
Beneficiaries in the highest quintile of HCC scores experienced 4.99 more visits than those in the lowest. Beneficiaries who were black experienced 2.14 fewer visits than others with similar health and preferences. Higher care-seeking preferences were marginally significantly associated with more visits, while education and poverty were insignificant. HRRs with high physician supply and high frequency practice norms were associated with 2.04 additional visits per year, while HRRs with high supply but low frequency norms were associated with 1.45 additional visits. Adjusting for all individual beneficiary covariates explained less than 20% of the original associations between visit rates and physician supply and practice norms.
Medicare beneficiaries’ health status, race, and preferences help explain individual office visit frequency; in particular, African-American patients appear to experience lower access to care. Yet, these factors explain a small fraction of the observed regional differences associated with physician supply and beliefs about the appropriate frequency of office visits.
healthcare utilization; physician visits; geographic variation; physician supply; patient preferences
Epithelioid hemangioendotheliomas are rare vascular tumors, often arising from medium to large veins in the extremities. Symptoms of these tumors vary depending upon location. Rarely, tumors may arise in chest and involve large vessels in the mediastinum. We present a case of a 17-year-old male presenting with compressive symptoms of the left upper extremity who was found to have a large epithelioid hemangioendothelioma encasing the left brachiocephalic vein.
Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies) and pooled horseradish peroxidase (HRP)-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB), the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1–2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic) and/or B-PER (a cell-disrupting, protein extraction reagent). Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef.
B-PER; colorimetry; detection; ELISA; high-throughput; microarray STEC; microtiter plate; precipitating; toxin typing