Evolution of Organic Solvent-Resistant DNA Polymerases.

The introduction of thermostable polymerases revolutionized the polymerase chain reaction (PCR) and biotechnology. However, many GC-rich genes cannot be PCR-amplified with high efficiency in water, irrespective of temperature. Although polar organic cosolvents can enhance nucleic acid polymerization and amplification by destabilizing duplex DNA and secondary structures, nature has not selected for the evolution of solvent-tolerant polymerase enzymes. Here, we used ultrahigh-throughput droplet-based selection and deep sequencing along with computational free-energy and binding affinity calculations to evolve Taq polymerase to generate enzymes that are both stable and highly active in the presence of organic cosolvents, resulting in up to 10% solvent resistance and over 100-fold increase in stability at 97.5 °C in the presence of 1,4-butanediol, as well as tolerance to up to 10 times higher concentrations of the potent cosolvents sulfolane and 2-pyrrolidone. Using these polymerases, we successfully amplified a broad spectrum of GC-rich templates containing regions with over 90% GC content, including templates recalcitrant to amplification with existing polymerases, even in the presence of cosolvents. We also demonstrated dramatically reduced GC bias in the amplification of genes with widely varying GC content in quantitative polymerase chain reaction (qPCR). By expanding the scope of solvent systems compatible with nucleic acid polymerization, these organic solvent-resistant polymerases enable a dramatic reduction of sequence bias not achievable through thermal resistance alone, with significant implications for a wide range of applications including sequencing and synthetic biology in mixed aqueous-organic media.

Identification of a novel peptide ligand for the cancer-specific receptor mutation EGFRvIII using high-throughput sequencing of phage-selected peptides.

We report here the selection and characterization of a novel peptide ligand using phage display targeted against the cancer-specific epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII). This receptor is expressed in several kinds of cancer: ovarian cancer, breast cancer and glioblastoma, but not in normal tissues. A 12-mer random peptide library was screened against EGFRvIII. Phage-selected peptides were sequenced in high-throughput by next generation sequencing (NGS), and their diversity was studied to identify highly abundant clones expected to bind with the highest affinities to EGFRvIII. The enriched peptides were characterized and their binding capacity towards stable cell lines expressing EGFRvIII, EGFR wild type (EGFR WT), or a low endogenous level of EGFR WT was confirmed by flow cytometry analysis. The best peptide candidate, VLGREEWSTSYW, was synthesized, and its binding specificity towards EGFRvIII was validated in vitro. Additionally, computational docking analysis suggested that the identified peptide binds selectively to EGFRvIII. The novel VLGREEWSTSYW peptide is thus a promising EGFRvIII-targeting agent for future applications in cancer diagnosis and therapy.

MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot.

Antibody (Ab) validation is the procedure in which an Ab is thoroughly assayed for sensitivity and specificity in a given application. Validation of Abs against post-translationally modified (PTM) targets is particularly challenging because it requires specifically prepared antigen. Here we describe a novel validation method using surrogate proteins in a Western blot. The surrogate protein, which we termed 'MILKSHAKE,' is a modified maltose binding protein enzymatically conjugated to a peptide from the chosen target that is either modified or nonmodified at the residue of interest. The certainty of the residue's modification status can be used to confirm Ab specificity. This method also allows for Ab validation even in the absence or limited availability of treated cell lysates.

Interleukin-36-receptor antagonist deficiency and generalized pustular psoriasis.

BACKGROUND: Generalized pustular psoriasis is a life-threatening disease of unknown cause. It is characterized by sudden, repeated episodes of high-grade fever, generalized rash, and disseminated pustules, with hyperleukocytosis and elevated serum levels of C-reactive protein, which may be associated with plaque-type psoriasis. METHODS: We performed homozygosity mapping and direct sequencing in nine Tunisian multiplex families with autosomal recessive generalized pustular psoriasis. We assessed the effect of mutations on protein expression and conformation, stability, and function. RESULTS: We identified significant linkage to an interval of 1.2 megabases on chromosome 2q13-q14.1 and a homozygous missense mutation in IL36RN, encoding an interleukin-36-receptor antagonist (interleukin-36Ra), an antiinflammatory cytokine. This mutation predicts the substitution of a proline residue for leucine at amino acid position 27 (L27P). Homology-based structural modeling of human interleukin-36Ra suggests that the proline at position 27 affects both the stability of interleukin-36Ra and its interaction with its receptor, interleukin-1 receptor-like 2 (interleukin-1 receptor-related protein 2). Biochemical analyses showed that the L27P variant was poorly expressed and less potent than the nonvariant interleukin-36Ra in inhibiting a cytokine-induced response in an interleukin-8 reporter assay, leading to enhanced production of inflammatory cytokines (interleukin-8 in particular) by keratinocytes from the patients. CONCLUSIONS: Aberrant interleukin-36Ra structure and function lead to unregulated secretion of inflammatory cytokines and generalized pustular psoriasis. (Funded by Agence Nationale de la Recherche and Société Française de Dermatologie.).

Only four genes (EDA1, EDAR, EDARADD, and WNT10A) account for 90% of hypohidrotic/anhidrotic ectodermal dysplasia cases.

Hypohidrotic and anhidrotic ectodermal dysplasia (HED/EDA) is a rare genodermatosis characterized by abnormal development of sweat glands, teeth, and hair. Three disease-causing genes have been hitherto identified, namely, (1) EDA1 accounting for X-linked forms, (2) EDAR, and (3) EDARADD, causing both autosomal dominant and recessive forms. Recently, WNT10A gene was identified as responsible for various autosomal recessive forms of ectodermal dysplasias, including onycho-odonto-dermal dysplasia (OODD) and Schöpf-Schulz-Passarge syndrome. We systematically studied EDA1, EDAR, EDARADD, and WNT10A genes in a large cohort of 65 unrelated patients, of which 61 presented with HED/EDA. A total of 50 mutations (including 32 novel mutations) accounted for 60/65 cases in our series. These four genes accounted for 92% (56/61 patients) of HED/EDA cases: (1) the EDA1 gene was the most common disease-causing gene (58% of cases), (2)WNT10A and EDAR were each responsible for 16% of cases. Moreover, a novel disease locus for dominant HED/EDA mapped to chromosome 14q12-q13.1. Although no clinical differences between patients carrying EDA1, EDAR, or EDARADD mutations could be identified, patients harboring WNT10A mutations displayed distinctive clinical features (marked dental phenotype, no facial dysmorphism), helping to decide which gene should be first investigated in HED/EDA.