Optimizing SNP microarray probe design for high accuracy microbial genotyping

Microarrays to characterize single nucleotide polymorphisms (SNPs) provide a cost-effective and rapid method (under 24 h) to genotype microbes as an alternative to sequencing. We developed a pipeline for SNP discovery and microarray design that scales to 100's of microbial genomes. Here we tested various SNP probe design strategies against 8 sequenced isolates of Bacillus anthracis to compare sequence and microarray data. The best strategy allowed probe length to vary within 32–40 bp to equalize hybridization free energy. This strategy resulted in a call rate of 99.52% and concordance rate of 99.86% for finished genomes. Other probe design strategies averaged substantially lower call rates (94.65–96.41%) and slightly lower concordance rates (99.64–99.80%). These rates were lower for draft than finished genomes, consistent with higher incidence of sequencing errors and gaps. Highly accurate SNP calls were possible in complex soil and blood backgrounds down to 1000 copies, and moderately accurate SNP calls down to 100 spiked copies. The closest genome to the spiked strain was correctly identified at only 10 spiked copies. Discrepancies between sequence and array data did not alter the SNP-based phylogeny, regardless of the probe design strategy, indicating that SNP arrays can accurately place unsequenced isolates on a phylogeny.     

An engineered ultra‐high affinity Fab‐Protein G pair enables a modular antibody platform with multifunctional capability

Engineered recombinant antibody‐based reagents are rapidly supplanting traditionally derived antibodies in many cell biological applications. A particularly powerful aspect of these engineered reagents is that other modules having myriad functions can be attached to them either chemically or through molecular fusions. However, these processes can be cumbersome and do not lend themselves to high throughput applications. Consequently, we have endeavored to develop a platform that can introduce multiple functionalities into a class of Fab‐based affinity reagents in a “plug and play” fashion. This platform exploits the ultra‐tight binding interaction between affinity matured variants of a Fab scaffold (Fab^S) and a domain of an immunoglobulin binding protein, protein G (GA1). GA1 is easily genetically manipulatable facilitating the ability to link these modules together like beads on a string with adjustable spacing to produce multivalent and bi‐specific entities. GA1 can also be fused to other proteins or be chemically modified to engage other types of functional components. To demonstrate the utility for the Fab‐GA1 platform, we applied it to a detection proximity assay based on the β‐lactamase (BL) split enzyme system. We also show the bi‐specific capabilities of the module by using it in context of a Bi‐specific T‐cell engager (BiTE), which is a therapeutic assemblage that induces cell killing by crosslinking T‐cells to cancer cells. We show that GA1‐Fab modules are easily engineered into potent cell‐killing BiTE‐like assemblages and have the advantage of interchanging Fabs directed against different cell surface cancer‐related targets in a plug and play fashion.     

Comprehensive epitope mapping of the Epstein–Barr virus latent membrane protein-2 in normal, non tumor-bearing individuals

Latent membrane protein (LMP)-2 is one of the Epstein–Barr virus (EBV)-encoded proteins consistently expressed by nasopharyngeal carcinoma (NPC). EBV-transformed lymphoblastoid cell lines (LCL) have been used in patients with NPC to induce LMP-2-recognizing T cell lines which have been in turn utilized for protein-wide mapping of T cell epitopes. However, comprehensive mapping of naturally recognized LMP-2 epitopes in non tumor-bearing individuals has not been reported. Here, we applied a low sensitivity epitope-defining technique for the identification of LMP-2 CTL responses detectable ex vivo in EBV-experienced individuals. This screening tool has been previously validated by analyzing memory CTL responses to Flu, cytomegalovirus (CMV), and the melanoma associated antigen gp100/Mel17. Peripheral blood monocytes (PBMC) from ten Caucasian and ten Chinese individuals were stimulated ex vivo with pools of nonamer (9-mer) peptides overlapping in a stepwise fashion each single amino acid of the LMP-2 sequence. No obvious differences were observed between the immune response of the two ethnic groups save for those related to the divergence in the ethnic prevalence of HLA haplotypes. Several novel and known LMP-2 epitopes were identified. Reactivity toward at least one LMP-2 epitope was detected in 18 of the 20 donors but no prevalent human leukocyte antigen (HLA)/epitope combination was observed confirming that LMP-2 reactivity in the context of common HLA alleles is more pleiotropic than that of FLU and CMV. We believe that the usefulness of these epitopes occurring naturally in non-cancer bearing patients as reagents for the immunization of patients with early or advanced stage NPC deserves further evaluation. Maurizio Provenzano and Silvia Selleri equally contributed to this work.     

Prenatal diagnosis--principles of diagnostic procedures and genetic counseling

The frequency of inherited malformations as well as genetic disorders in newborns account for around 3-5%. These frequency is much higher in early stages of pregnancy, because serious malformations and genetic disorders usually lead to spontaneous abortion. Prenatal diagnosis allowed identification of malformations and/or some genetic syndromes in fetuses during the first trimester of pregnancy. Thereafter, taking into account the severity of the disorders the decision should be taken in regard of subsequent course of the pregnancy taking into account a possibilities of treatment, parent's acceptation of a handicapped child but also, in some cases the possibility of termination of the pregnancy. In prenatal testing, both screening and diagnostic procedures are included. Screening procedures such as first and second trimester biochemical and/or ultrasound screening, first trimester combined ultrasound/biochemical screening and integrated screening should be widely offered to pregnant women. However, interpretation of screening results requires awareness of both sensitivity and predictive value of these procedures. In prenatal diagnosis ultrasound/MRI searching as well as genetic procedures are offered to pregnant women. A variety of approaches for genetic prenatal analyses are now available, including preimplantation diagnosis, chorion villi sampling, amniocentesis, fetal blood sampling as well as promising experimental procedures (e.g. fetal cell and DNA isolation from maternal blood). An incredible progress in genetic methods opened new possibilities for valuable genetic diagnosis. Although karyotyping is widely accepted as golden standard, the discussion is ongoing throughout Europe concerning shifting to new genetic techniques which allow obtaining rapid results in prenatal diagnosis of aneuploidy (e.g. RAPID-FISH, MLPA, quantitative PCR).     

A Novel p.E311K Mutation of Thyroid Receptor Beta Gene in Resistance to Thyroid Hormone Syndrome, Inherited in Autosomal Recessive Trait

Resistance to thyroid hormone (RTH) syndrome is caused by mutations in THRB gene and is inherited mainly as an autosomal dominant trait with dominant negative effect. Most of up-to-now described RTH cases were heterozygous. We studied a 19-year-old woman presenting severe mental impairment, hyperkinetic behavior, learning disability, hearing loss, tachycardia, goiter, strabismus, nystagmus, and normal stature. The laboratory findings revealed elevated TSH, T3, and T4 serum levels. Her parents were healthy with normal serum level of TSH, fT3, and fT4. Sequence based prediction of a substitution was analyzed by SDM, PolPhen, and SNAP software whereas structural visualizations were performed in UCSF Chimera. We found a novel mutation in THRB gene in position 1216 (G to A transition, codon 311) resulting in novel Glu-311-Lys (p.E311K) substitution, homozygous in proband presenting with severe symptoms of RTH and heterozygous in both of her healthy parents, thus suggesting autosomal recessive mode of inheritance. p.E311K substitution was not found in 50 healthy, unrelated individuals. p.E311K was shown to be deleterious by SDM, PolPhen, and SNAP software. Structural visualizations of mutated protein performed by UCSF Chimera software disclosed a loss of hydrogen bonds between E311, R383, and R429 along with abnormal residue-residue contact between K311 and L377. This is a very rare case of a homozygous mutation in a patient with severe symptoms of RTH and lack of symptoms in both heterozygous parents. Although, computational analyses have provided the evidence that p.E311K substitution may affect THRB function, lack of dominant negative effect typical for THRB mutations could not be explained by structure-based modeling. Further in vitro analysis is required to assess the functional consequences of this substitution.     

River macrophyte indices: not the Holy Grail!

1. Recent studies have demonstrated that there is generally no unambiguous relationship between plant species composition and specific environmental conditions in rivers. Nevertheless, indices of environmental pressures based on macrophytes are flourishing, because of the requirements of the Water Framework Directive (WFD). 2. We first reviewed nine such indices against 13 criteria for bioindicators. Then, using data from France and England, we tested whether the IBMR (Macrophyte Biological Index for Rivers) and LEAFPACS (predictions and classification system for macrophytes) methods could reliably indicate nutrient and hydromorphological pressures. Finally, we used an improved bootstrapping method to estimate accuracy. 3. Currently, most indices lack ecological meaning for a variety of reasons, including partial sampling (backwaters are excluded); reliance on list of taxa (there are identification difficulties) rather than structure and functions; correlation rather than causation; application within a limited biogeographical area; reliance on ‘expert’ judgement; high precision but poor accuracy; poorly defined reference conditions; lack of independent tests; and an inability to discriminate reliably between the target pressures of interest from confounding background variables. 4. IBMR was a far better indicator of pH (or HCO₃‐pCO₂) than it was of soluble reactive phosphorus, SRP (or SRP‐NH₄). While there was a highly significant correlation between IBMR and SRP after removing the effect of pH, the relationship was weak (r² = 0.08, n = 215, P < 0.001). 5. LEAFPACS is a multi‐metric method summing up five individual indices, each compliant with the WFD. Its individual metrics were not better correlated with nutrient and hydromorphological pressures (with r² < 0.1, n = 62, P < 0.05) than was the IBMR. The meaning of the overall metric is questionable. 6. There are problems in determining the precision of the indices, owing to uncertainties in recording, but they are less than the uncertainties in determining accuracy (because species optima and tolerances are sometimes poorly known). 7. Reliable information is needed to improve the state of our rivers. Macrophyte indices are able to detect statistically significant pressures from a large population of sites but cannot be applied at specific sites, as required by the WFD, owing to large uncertainties and low explanatory power. Typically, more than 90% of the variability in macrophyte indices is attributed to factors other than human pressure. The WFD would be better served by a simpler, holistic approach based on our current mechanistic understanding of river processes. These findings are likely to apply also to other taxonomic groups (macroinvertebrates, diatoms, fish) used in the assessment of purported ecological quality and to palaeolimnological measures of reference status.     

Use of Glucose,Insulin, and C‐Reactive Protein to Determine Need for Glucose Tolerance Testing

Objective: Glucose intolerance has been shown to be a better predictor of morbidity and mortality than impaired fasting glucose. However, glucose tolerance tests are inconvenient and expensive. This study evaluated the relative frequencies of glucose intolerance and impaired fasting glucose and sought to determine if 2‐hour glucose could be predicted from simple demographic and laboratory data in an obese population. Research Methods and Procedures: Eighty‐nine obese subjects (median BMI 35 kg/m², range 30 to 40 kg/m²) underwent glucose tolerance testing. Using step‐wise linear and logistic regression analysis, fasting glucose, high‐sensitivity C‐reactive protein (hsCRP), fasting insulin, high‐density lipoprotein cholesterol, triglycerides, weight, height, BMI, waist circumference, hip circumference, waist‐to‐hip ratio, sex, and age were assessed as predictors of glucose intolerance. Results: Impaired glucose tolerance was more prevalent (27%) than impaired fasting glucose (5.6%). Only fasting glucose and hsCRP were significant (p < 0.05) independent predictors of impaired 2‐hour glucose (>140 mg/dL). A fasting glucose ≥ 100 mg/dL or an hsCRP > 0.32 mg/dL (upper quartile of the normal range) detected 81% (sensitivity) of obese subjects with impaired glucose tolerance; however, specificity was poor (46%). Fasting insulin ≥ 6 μU/mL had better sensitivity (92%) but poorer specificity (30%). Discussion: Impaired glucose tolerance is more common than impaired fasting glucose in an obese population. Possible strategies to avoid doing glucose tolerance tests in all obese patients would be to do glucose tolerance testing only in those whose fasting glucose is ≥ 100 mg/dL or whose hsCRP exceeds 0.32 mg/dL or those whose fasting insulin is ≥ 6 μU/mL.