Screening and characterization of butanol‐tolerant micro‐organisms

Aims: Poor butanol tolerance of solventogenic stains directly limits their butanol production during industrial‐scale fermentation process. This study was performed to search for micro‐organisms possessing elevated tolerance to butanol. Methods and Results: Two strains, which displayed higher butanol tolerance compared to commonly used solventogenic Clostridium acetobutylicum, were isolated by evolution and screening strategies. Both strains were identified as lactic acid bacteria (LAB). On this basis, a LAB culture collection was tested for butanol tolerance, and 60% of the strains could grow at a butanol concentration of 2·5% (v/v). In addition, an isolated strain with superior butanol tolerance was transformed using a certain plasmid. Conclusions: The results indicate that many strains of LAB possessed inherent tolerance of butanol. Significance and Impact of the Study: This study suggests that LAB strains may be capable of producing butanol to elevated levels following suitable genetic manipulation.     

Front‐Loading Natural‐Product‐Screening Libraries for log P: Background,Development, and Implementation

In the period from January 1981 to December 2010, 1068 small‐molecule new chemical entities (NCEs) were introduced, of which ca. 34% are either a natural product or a close analogue. While this metric reflects the impact natural products have played in delivering new chemical starting points (leads) for the pharmaceutical industry, it does not capture the decline this approach has suffered over the last 20 years as the high‐throughput screening (HTS) of pure compound libraries has become more popular. An impediment to natural‐product drug discovery in the HTS paradigm is the lack of a clear strategy that enables front‐loading of an extract or fraction's chemical constituents so that they are compliant with lead‐ and drug‐like chemical space. To address this imbalance, an approach based on lipophilicity, as measured by clog P has been developed that, together with advances being made in isolation and structural elucidation, can afford natural product leads in timelines compatible with pure compound screening.     

Cystine‐knot peptides targeting cancer‐relevant human cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4)

Cystine‐knot peptides sharing a common fold but displaying a notably large diversity within the primary structure of flanking loops have shown great potential as scaffolds for the development of therapeutic and diagnostic agents. In this study, we demonstrated that the cystine‐knot peptide MCoTI‐II, a trypsin inhibitor from Momordica cochinchinensis, can be engineered to bind to cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4), an inhibitory receptor expressed by T lymphocytes, that has emerged as a target for the treatment of metastatic melanoma. Directed evolution was used to convert a cystine‐knot trypsin inhibitor into a CTLA‐4 binder by screening a library of variants using yeast surface display. A set of cystine‐knot peptides possessing dissociation constants in the micromolar range was obtained; the most potent variant was synthesized chemically. Successive conjugation with neutravidin, fusion to antibody Fc domain or the oligomerization domain of C4b binding protein resulted in oligovalent variants that possessed enhanced (up to 400‐fold) dissociation constants in the nanomolar range. Our data indicate that display of multiple knottin peptides on an oligomeric scaffold protein is a valid strategy to improve their functional affinity with ramifications for applications in diagnostics and therapy. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular recognition of HER‐1 in whole‐blood samples

Multimode sensing was proposed for molecular screening and recognition of HER‐1 in whole blood. The tools used for molecular recognition were platforms based on nanostructured materials such as the complex of Mn(III) with meso‐tetra (4‐carboxyphenyl) porphyrin, and maltodextrin (dextrose equivalence between 4 and 7), immobilized in diamond paste, graphite paste or C₆₀ fullerene paste. The identification of HER‐1 in whole‐blood samples, at molecular level, is performed using stochastic mode and is followed by the quantification of it using stochastic and differential pulse voltammetry modes. HER‐1 can be identified in the concentration range between 280 fg/ml and 4.86 ng/ml using stochastic mode, this making possible the early detection of cancers such as gastrointestinal, pancreatic and lung cancers. The recovery tests performed using whole‐blood samples proved that the platforms can be used for identification and quantification of HER‐1 with high sensitivity and reliability in such samples, these making them good molecular screening tools. Copyright © 2014 John Wiley & Sons, Ltd.     

Large‐scale molecular genetic analysis in plant‐pathogenic fungi: a decade of genome‐wide functional analysis

Plant‐pathogenic fungi cause diseases to all major crop plants world‐wide and threaten global food security. Underpinning fungal diseases are virulence genes facilitating plant host colonization that often marks pathogenesis and crop failures, as well as an increase in staple food prices. Fungal molecular genetics is therefore the cornerstone to the sustainable prevention of disease outbreaks. Pathogenicity studies using mutant collections provide immense function‐based information regarding virulence genes of economically relevant fungi. These collections are rich in potential targets for existing and new biological control agents. They contribute to host resistance breeding against fungal pathogens and are instrumental in searching for novel resistance genes through the identification of fungal effectors. Therefore, functional analyses of mutant collections propel gene discovery and characterization, and may be incorporated into disease management strategies. In the light of these attributes, mutant collections enhance the development of practical solutions to confront modern agricultural constraints. Here, a critical review of mutant collections constructed by various laboratories during the past decade is provided. We used Magnaporthe oryzae and Fusarium graminearum studies to show how mutant screens contribute to bridge existing knowledge gaps in pathogenicity and fungal–host interactions.     

Review of established and innovative detection methods for carbapenemase‐producing Gram‐negative bacteria

The minimal antibiotic options for carbapenemase‐producing Gram‐negative bacteria necessitate their rapid detection. A literature review of a variety of phenotypic and genotypic methods is presented. Advances in culture methods and screening media are still subject to long incubation hours. Biochemical methods have shorter turnaround times and higher sensitivities and specificities, but cannot differentiate between various types and variants. Spectrophotometric methods are cheap and efficient, but are uncommon in many clinical settings, while the MALDI‐TOF MS is promising for species identification, typing and resistance gene determination. Although next generation sequencing (NGS) technologies provide a better platform to detect, type and characterize carbapenem‐resistant bacteria, the different NGS platforms, the large computer memories and space needed to process and store genomic data and the nonuniformity in data analysis platforms are still a challenge. The sensitivities, specificities and turnaround times recorded in the various studies reviewed favours the use of the biochemical tests (Carba NP or Rapid Carb screen tests) for the detection of putative carbapenemase‐producing isolates. MALDI‐TOF MS and/or molecular methods like microarray, loop‐mediated isothermal amplification and real‐time multiplex PCR assays could be used for further characterization in a reference laboratory. NGS may be used for advanced epidemiological and molecular studies.     

A computationally inspired investigation of the solid forms of (R)‐1‐phenylethylammonium‐(S)‐2‐phenylbutyrate

Following the computation of a lattice energy landscape which predicted that there should be more stable, denser forms of (R)‐1‐phenylethylammonium‐(S)‐2‐phenylbutyrate, crystallizations from a range of solvents were performed to search for other polymorphs and investigate the possibility that the known P4₁ structure could be a hydrate. Extensive crystallization experiments from a wide range of solvents gave fine needles or microcrystalline samples. A redetermination of the P4₁ structure by powder X‐ray diffraction located all protons, and in conjunction with other experimental and computational evidence showed that the structure was anhydrous. Evidence for two additional forms was found as mixtures with form I. These include an orthorhombic form, possibly a Z′ = 3 polymorph, and another as yet unidentified form obtained as a minor component from dichloromethane solution. However, both these forms appear to be metastable with respect to form I (P4₁), which is therefore probably the most thermodynamically stable form that can be crystallized from solution under ambient conditions. This determination of the solid state behavior of the less readily crystallized member of the diastereomeric salt system (R)‐1‐phenylethylammonium‐(R/S)‐2‐phenylbutyrate provides a challenge to the theoretical modeling to explain its ideal resolution behavior. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Culture storage age and fungal re‐isolation from host‐tissue influence Colletotrichum spp. virulence to pepper fruits

Using resistant cultivars is the most sustainable and practical approach against plant diseases. Plant germplasm and breeding lines are selected and assayed against, usually, the most aggressive or virulent strains of a pathogen (e.g., fungus) that causes the disease. However, prolong storage of the pathogen in culture media could affect virulence that, consequently, also influence the outcome of the resistance assay. This study demonstrates that long‐term storage (at least a year) of Colletotrichum truncatum and C. scovillei, causal agents of pepper anthracnose, in potato dextrose agar (PDA) medium decreased the aggressiveness and virulence of the fungus in host‐pepper fruits. However, reintroduction of the pathogen to the host and isolation of the pathogen as the new inoculum, prior to inoculation assays, increased the virulence of the fungi. These findings suggest that re‐inoculation and re‐isolation of Colletotichum truncatum and C. scovillei that have been stored for at least 1 year in PDA medium are necessary when using fungal cultures in pathogenicity and plant resistance assays to achieve desirable, comparable and reliable results.     

A multi‐wavelength,laser‐based optical spectroscopy device for breast density and breast cancer risk pre‐screening

Optical Breast Spectroscopy (OBS) has been shown to predict mammographic breast density, a strong breast cancer risk factor. OBS is a low‐cost technique applicable at any age. OBS information may be useful for personalizing breast cancer screening programs based on risk to improve consensus on and adherence to screening guidelines. To facilitate the use of OBS in population‐wide studies, a research prototype OBS device was modified to make it portable and cheaper and to require less operator interaction. Two major changes were made: (1) the broadband light source was replaced with a laser module with 13 individual wavelengths turned on sequentially, enabling the use of photodiode detectors instead of a spectrometer, and (2) the light sources and detectors were placed in fixed positions within 4 sizes of cup, eliminating the need for placement by the operator. Wavelengths were selected using data from two previous studies. The reduction in spectral content did not significantly reduce the ability to distinguish between different risk groups. Positions for the light sources and detectors were chosen based on Monte Carlo simulations to match the optically interrogated volumes of the original device. Two light sources and six detectors per cup were used in the final design.

     

Quantification of length‐bias in screening trials with covariate‐dependent test sensitivity

Length‐biased sampling exists in screening programs where longer duration disease is detected during the preclinical stage because a longer sojourn time (preclinical duration) has a higher probability of being screen detected. By modeling the course of disease, we quantify the effect of length‐biased sampling on clinical duration when cases are subject to periodic screening with variable test sensitivity. We use the highly flexible bivariate lognormal density to jointly model preclinical and clinical durations, and we model screening test sensitivity as a function of the sojourn time and number of previous false negative screens. We show that the mean clinical duration among screen‐detected cases can be up to 40% higher, with shrinking standard deviation, than those among nonscreen‐detected cases, due to biased sampling alone, irrespective of any possible benefit (increased survival time arising from earlier detection or reduction in mortality). These findings will aid in the design and interpretation of screening trials.     

Fast Screening of Whole Blood Samples and Pharmaceutical Compounds for Enantiorecognition of Free L‐T3, L‐T4, and D‐T4

A fast screening method of whole blood was proposed for enantiorecognition of free L‐T₃ , L‐T₄, and D‐T₄. Stochastic microsensors based on four inulins (IN, IQ, TEX, and HD) immobilized on diamond paste (DP) were used for recognition of free L‐T₃ , L‐T₄, and D‐T₄. For the enantiorecognition of free L‐T₄ and D‐T₄ in whole blood and pharmaceutical samples, the best microsensor was the one based on TEX/DP (wide linear concentration ranges, and low limits of quantification). The best limit of detection for the assay of free L‐T₃ (400 fmol/L) was recorded using the microsensors based on HD/DP, while for the assay of free L‐T4, and D‐T4 the best limit of determination (1 pmol/L) was recorded using the TX/DP‐based microsensor. For the enantiorecognition of free L‐T₃ in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification – of pmol/L magnitude order). For the enantiorecognition of free L‐T₃ in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification – of pmol/L magnitude order). Free L‐T₃ , L‐T₄, and D‐T₄ were recovered with high reliabilities in whole blood samples (recoveries higher than 99.00%, with RSD values lower than 1.00%) and pharmaceutical samples (recoveries higher than 95.00% with RSD values lower than 1.00%). Chirality 27:973–978, 2015. © 2015 Wiley Periodicals, Inc.