Coli Extract Research Articles

One-Step Purification of Recombinant Cutinase from an E. coli Extract Using a Stabilizing Triazine-Scaffolded Synthetic Affinity Ligand.

Cutinase from Fusarium solani pisi is an enzyme that bridges functional properties between lipases and esterases, with applications in detergents, food processing, and the synthesis of fine chemicals. The purification procedure of recombinant cutinase from E. coil extracts is a well-established but time-consuming process, which involves a sequence of two anionic exchange chromatography steps followed by dialysis. Affinity chromatography is the most efficient method for protein purification, the major limitation of its use being often the availability of a ligand selective for a given target protein. Synthetic affinity ligands that specifically recognize certain sites on the surface of proteins are highly desirable for affinity processes due to their cost-effectiveness, durability, and reusability across multiple cycles. Additionally, these ligands establish moderate affinity interactions with the target protein, making it possible to purify proteins under gentle conditions while maintaining high levels of activity recovery. This study aimed to develop a new method for purifying cutinase, utilizing triazine-scaffolded biomimetic affinity ligands. These ligands were previously screened from a biased-combinatorial library to ensure their binding ability to cutinase without compromising its biological function. A lead ligand, designated as 11/3', [4-({4-chloro-6-[(2-methylbutyl)amino]-1,3,5-triazin-2-yl}amino)benzoic acid], was chosen and directly synthesized onto agarose. Experiments conducted at different scales demonstrated that this ligand (with an affinity constant Ka ≈ 104 M-1) exhibited selectivity towards cutinase, enabling the purification of the enzyme from an E. coli crude production medium in a single step. Under optimized conditions, the protein and activity yields reached 25% and 90%, respectively, with a resulting cutinase purity of 85%.

Biosynthesis and characterization of silver nanoparticles using Bacillus subtilis, Escherichia coli, and leaf extracts of Jatropha and Ocimum species

Biosynthesis and characterization of silver nanoparticles using Bacillus subtilis, Escherichia coli, and leaf extracts of Jatropha and Ocimum species

Prevalence and characterisation of antimicrobial resistance genes and class 1 and 2 integrons in multiresistant Escherichia coli isolated from poultry production

A global increase in the populations of drug resistant bacteria exerts negative effects on animal production and human health. Our study has been focused on the assessment of resistance determinants in relation to phenotypic resistance of the 74 commensal E. coli isolates present in different ecological environments. The samples were collected from poultry litter, feces, and neck skin. Among the microorganisms isolated from the poultry litter (group A), the highest resistance was noted against AMP and DOX (100%). In the E. coli extracts from the cloacal swabs (group B), the highest resistance was observed against AMP (100%) and CIP (92%). The meat samples (group C) were characterized by resistance to AMP (100%) and STX (94.7%). Genes encoding resistance to β-lactams (blaTEM, blaCTX-M), fluoroquinolones (qnrA, qnrB, qnrS), aminoglycosides (strA-strB, aphA1, aac(3)-II), sulfonamides (sul1, sul2, sul3), trimethoprim (dfr1, dfr5, dfr7/17) and tetracyclines (tetA, tetB) were detected in the studied bacterial isolates. The presence of class 1 and 2 integrons was confirmed in 75% of the MDR E. coli isolates (plasmid DNA), of which 60% contained class 1 integrons, 15% contained class 2 integrons, and 11.7% carried integrons of both classes. Thus, it may be concluded that integrons are the common mediators of antimicrobial resistance among commensal multidrug resistant Escherichia coli at important stages of poultry production.

Selection of Ovalbumin-specific Binding Peptides through Instant Translation in Ribosome Display Using E. coli Extract.

In vitro selection has been widely used to generate molecular-recognition elements in analytical sciences. Although reconstituted types of in vitro transcription and translation (IVTT) system, such as PURE system, are nowadays widely used for ribosome display and mRNA/cDNA display, use of E. coli extract is often avoided, presumably because it contains unfavorable contaminants, such as ribonuclease. Nevertheless, the initial speed of protein translation in E. coli extract is markedly faster than that of PURE system. We thus hypothesized that E. coli extract is more appropriate for instant translation in ribosome display than PURE system. Here, we first revisit the potency of E. coli extract for ribosome display by shortening the translation time, and then applied the optimized condition for selecting peptide aptamers for ovalbumin (OVA). The OVA-binding peptides selected using E. coli extract exhibited specific binding to OVA, even in the presence of 50% serum. We conclude that instant translation in ribosome display using E. coli extract has the potential to generate easy-to-use and economical molecular-recognition elements in analytical sciences.

Antimicrobial, Antioxidative, Elastase and Tyrosinase Inhibitory Effect of Supercritical and Hydrothermal Halopteris scoparia Extract

To find out a good candidate of cosmetic source and screened for antimicrobial, antioxidant and whitening activities. In the antioxidant experiment, supercritical Halopteris scoparia extraction (SHE) and hydrothermal Halopteris scoparia extraction (HHE) were slightly different depending on the experimental method (Total polyphenol content measurement, DPPH radical scavenging ability, ABTS radical scavenging ability, and SOD-like activity) and in the enzyme experiment (Elastase, Tyrosinase inhibition activity), the supercritical extract was more effective. In supercritical fluid, Halopteris scoparia extracts showed good antimicrobial activity against Propionibacterium acnes, Staphylococcus aureus, Bacillus subtilis and Escherichia coli but hydrothermal extract had no effect about the rest of the strains. Therefore, it can be expected that the supercritical Halopteris scoparia extract with excellent antibacterial activity can be used as a cosmetic material.

Evaluating the Efficacy of Garlic and Cranberry Extracts against E. coli causing Urinary Tract Infections

Background: Escherichia coli (E. coli) is the major cause of community-based Urinary tract infections (UTI). Products from nature have been used in traditional medicine practice although with limited scientific validity. In this study garlic and cranberry extracts was assessed for its efficacy to inhibit E. coli growth. Materials and Methods: Crude garlic and cranberry extracts were obtained and E. coli was isolated from an infected urine sample. Two-fold serial dilution was carried out on the E. coli and crude extract inoculations. Incubation and plating was carried out for 24 hr at 30°C. Colony count was done using a digital Quebec Colony Counter. Results: A general trend of reducing number of average CFU with increasing concentration of both extracts was observed. Garlic was observed to be more potent than the cranberry extract even at the lowest concentration. Conclusion: This study establishes the merit of using garlic and cranberry extract as a household remedy for UTI caused by E. coli.

The Impact of Membrane Composition and Co‐Drug Synergistic Effects on Vancomycin Association with Model Membranes from Electrochemical Impedance Spectroscopy

AbstractThe interaction of the antibacterial drug, vancomycin with microcavity suspended lipid bilayers (MSLB) was investigated using non‐Faradaic electrochemical impedance spectroscopy (EIS). Five MSLB compositions were prepared with increasing complexity at gold substrates: DOPC, DOPC:Chol, DOPC:SM:Chol, mammalian plasma membrane mimetic (MPM), and E. coli polar lipid extract. The latter two, are intended to mimic eukaryotic and bacterial inner membrane compositions respectively. The extent of vancomycin association and the impact drug association has on the electrochemical properties of the membrane depended on biomembrane composition. Trends were similar across all membranes but the E. coli membrane composition. Vancomycin increased membrane resistance and decreased capacitance in a saturable, concentration dependent manner, but for E. coli membrane resistance change was negligible and capacitance increased. Membrane resistance data was fit to the Langmuir‐Freundlich model to give quantitative insight into the relative extent of association of drug with membrane as a function of membrane composition. Overall, electrochemical data indicates that vancomycin associates at the interface of lipid membranes rather than penetrates the layer and this is promoted by the presence of anionic phospholipid. Interestingly, co‐incubation of the E. coli extract bilayer with both rifampicin and vancomycin, known to act synergistically, significantly promotes vancomycin association to E. coli membrane.

Purification and characterization of formaldehyde dismutases of Methylobacterium sp. FD1

In the present study, we purified and characterized three formaldehyde dismutases (Fdms) (EC 1.2.98.1) (Fdm1, Fdm2, and Fdm3) of Methylobacterium sp. FD1. These Fdms (with His-tag) were produced in the recombinant E. coli and purified by immobilized metal affinity chromatography from the E. coli extracts. In each of the three Fdms, the enzyme-bound coenzyme was nicotinamide adenine dinucleotide (NAD(H)) and the enzyme-bound metal was zinc. The quaternary structures of these Fdms were estimated as homotetrameric. The optimal pHs and temperatures of Fdm1, Fdm2, and Fdm3 were approximately 6.5, 6.0, and 6.0, and 35°C, 25°C, and 30°C, respectively. The Km values of Fdm1, Fdm2, and Fdm3 were 621, 865, and 414mM, respectively. These results were similar to the properties of already-known Fdms. However, each of the Fdms of FD1 had methanol:p-nitroso-N,N-dimethylaniline oxidoreductase activity that is not found in already-known Fdms.

In vitro evaluation of novel titania-containing borate bioactive glass scaffolds.

Titanium-containing borate bioactive glass scaffolds (0, 5, 15, and 20 mol %, identified as BRT0, BRT1, BRT3, and BRT4) with a microstructure similar to that of human trabecular bone were prepared and evaluated in vitro for potential bone loss applications in revision total knee arthroplasty (rTKA). Methyl thiazolyl tetrazolium (MTT) cell viability assays of scaffold ion release extracts revealed that BRT0 scaffolds (0 mol % titanium) inhibited cell proliferation and activity at day 14. At day 30, all scaffold extracts decreased cell proliferation and activity significantly. However, live/dead cell assay results demonstrated that degradation products from all the scaffolds had no inhibitory effect on cell viability. Significant bactericidal efficacies of BRT3 extracts against Escherishia coli (Gram-negative) and BRT1 extracts against Staphylococcus aureus and Staphylococcus epidermidis (both Gram-positive bacteria) were demonstrated. Finally, evaluation of the cell/bioactive glass surface interactions showed well-spread cells on the surface of the BRT3 glass discs and BRT1 and BRT3 scaffolds, when compared to BRT0 and BRT4 scaffolds. The results indicate that by changing the Ti4+ :B3+ ratio, the ion release and consequently cell proliferation could be improved. in vitro results in this study demonstrate that BRT3 scaffolds could be a promising candidate for addressing bone loss in rTKAs; however, in vivo studies would be required to evaluate the effect of a dynamic environment on the cell and tissue response to the fabricated scaffolds.

Identification of additive formulations for enhanced shelf‐life and productivity of E. coli cell‐free protein synthesis extracts

Cell‐Free Protein Synthesis (CFPS) is a platform biotechnology that enables a breadth of applications, from expressing traditionally intractable proteins to metabolic engineering and point‐of‐care diagnostics. However, field applications have remained limited due to the poor shelf stability of aqueous E. coli cell‐free extracts, which contain a complex mixture of cellular machinery. Lyophilization of the E. coli extract improves shelf‐life, but remains insufficient for maintaining extract productivity over extended storage periods at room temperature. For many pure protein‐based biologics, a variety of additives have been identified that help to stabilize the protein for lyophilization, transportation, and storage. However, the advances made for pure proteins cannot be directly applied to the complex cell extract, and current investigations into improving cell extract stability via additives has been limited. Thus, we have addressed the cold chain limitation in cell‐free by mapping the chemical space of ten low‐cost additives with four distinct mechanisms of action in a combinatorial manner to elucidate their capacity to further stabilize lyophilized cell extract. We report three key findings: 1) unique additive formulations that maintain full productivity of the cell extract at 4°C and 23°C; 2) additive formulations that enhance extract productivity up to 195%; 3) a machine learning algorithm that provides predictive capacity for the stabilizing effects of additive formulations that were not tested experimentally. The identified additive formulations provide a simple and low‐cost advance toward making CFPS field‐ready.Support or Funding InformationBill and Linda Frost Fund, Center for Applications in Biotechnology’s Chevron Biotechnology Applied Research Endowment Grant, Cal Poly Research, Scholarly, and Creative Activities Grant Program (RSCA 2017), and the National Science Foundation (NSF‐1708919)Comparison of traditional cellfree extract production and reaction setup with our modified workflow for improved stability and productivity to enable field applications.Figure 1

Antimicrobial potential of exotic fruits residues

The antibacterial potential of extracts from the residues of fruits dekopon, kiwifruit, blueberry, sapodilla, tamarillo (peel), and noni (peel, seed and mixture of peel and seed) was evaluated against Gram-positive bacteria viz. Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, and Staphylococcus aureus and the Gram-negative bacteria, Escherichia coli, Pseudomonas aeruginosa, Salmonella enteritidis, and Serratia marcescens. It was found that extracts of mixture of noni seed and peel residues in acetone 40–80%, ethanol 40–80% and methanol 50–80% were effective against 4, 3 and 2 bacteria, respectively, being B. cereus and S. marcescens not inhibited by any extract. Highlight to the extract of mix noni seed and peel in 80% ethanol which was most effective against E. coli and S. aureus, with diameters of the inhibition zone (DIZ) around 14.0 mm. The extracts of dekopon peel in methanol 50, 70 and 80% and extract of kiwifruit peel in ethanol 40% were the only ones that showed antimicrobial potential, inhibiting S. enteritidis and S. aureus, respectively (DIZ of 9.0 mm). The extracts of blueberry peel were most effective against the Gram-negative bacteria, being E. coli and S. marcescens more sensitive to extracts of this residue in 80% and 70% ethanol (DIZs of 12–13 mm), respectively. The extracts of sapodilla peel in 70% and 80% acetone were more effective against Gram-positive bacteria, with DIZs between 10.0 and 12.0 mm. Finally, the extracts of tamarillo peel only inhibited E. faecalis (extracts in 60 and 70% ethanol), E. coli (extracts in 50–80% ethanol) and S. enteritidis (extract in methanol 80%). The minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC) were determined for the bacteria more sensitive to extracts of fruits residues. The selected extracts inhibited the bacteria with the lowest MIC of 0.39 mg/mL and eliminate with MBC values >200 mg/mL. However, the 70% ethanol blueberry peel extract was the highlight, since that inhibited and eliminated E. coli with the lowest concentration (0.39 mg/mL). In this study, the residues of these exotic fruits, which are commonly discarded, showed a great potential for use as natural antibacterial therapies.

Proteome Analysis of Phase-Separated Condensed Proteins with Ionic Surfactants Revealed Versatile Formation of Artificial Biomolecular Condensates.

The formation of highly condensed, native proteins is important for the development of protein-based drugs and materials. In the cell, various types of liquid droplets with broad functions are formed by the spontaneous condensation of protein, as a physiological response. These droplets lack a surrounding membrane but are phase-separated from the water medium. These types of phase-separated states of proteins have potential applications in biotechnology. Recently, we have developed an artificial phase-separated liquid of condensed native proteins, termed a protein condensate (PC), formed by electrostatic complexation with ionic surfactants. Here we report the applicability of PC formation, studied using an E. coli extract as the protein source. The addition of anionic and cationic surfactants at a specific ratio to the E. coli extract resulted in PC formation. A proteome analysis showed that the PC thus formed contained about 600 kinds of proteins, representing 65% of the uniquely detected proteins and confirming the high versatility of PC formation. A statistical analysis revealed that a variety of types of proteins with a wide range of molecular weights and isoelectric points could form PCs.

Sargassum, Gracilaria and Ulva Exhibit Positive Antimicrobial Activity against Human Pathogens

Bacterial resistance to pharmaceutical drugs is on rise, which emphasizes the need for screening of new drugs from natural resources. Seaweeds from the marine ecosystem are important source of bioactive compounds making them one of the major subjects for screening of various pharmaceutical drugs. So here, we assessed the bacterial growth inhibitory functions of four seaweeds Sargassum wightii, Gracillaria edulis, G. corticata and Ulva lactuca of Andaman Sea and Bay of Bengal, India respectively against three pathogens Pseudomonas aeruginosa, Eischeira coli and Staphylococcus aureus. Solvent extraction of four seaweeds was performed using 70% methanol, ethanol and ethyl acetate. Agar well diffusion method was used to test the bioactivity of seaweeds against pathogens. S. wightii,G. edulis and U. lactuca were observed with better solvent extracts compared to G. corticata. Methanol extract of S. wightii was observed with the highest (29.0 ± 1.22) zone of inhibition (ZOI) and ethyl acetate extract of U. lactuca was observed with the lowest ZOI (5.0 ± 0.0) against S. aureus. Butanol extract of S. wightii was observed with the highest ZOI (14.0 ± 0.83) against P. aeruginosa, whereas G. edulis methanol extract and U. lactuca ethyl-acetate extract were observed with the lowest ZOI (6.0 ± 0.0). For E. coli, butanol and methanol extracts of G. edulis and U. lactuca showed the highest (12.0 ± 0.54) and the lowest (6.0 ± 0.0). Our preliminary results suggest bioactivity of S. wightii, G. edulis and U. lactuca showed positive results. Further biochemical characterization of S. wightiishould be carried out for potential bioactive compounds against human pathogens. Our results suggest bioactive compounds from seaweeds can be used as pharmaceutical drugs.

Challenges in Application of Langmuir Monolayer Studies To Determine the Mechanisms of Bactericidal Activity of Ruthenium Complexes.

The effects induced by antibiotics on the bacterial membrane may be correlated with their bactericidal activity, and such molecular-level interactions can be probed with Langmuir monolayers representing the cell membrane. In this study, we investigated the interaction between [Ru(mcbtz)2(PPh3)2] (RuBTZ, mcbtz = 2-mercaptobenzothiazoline) and [Ru(mctz)2(PPh3)2] (RuCTZ, mctz = 2-mercaptothiazoline) with Langmuir monolayers of a lipid extract of Escherichia coli, an extract of lipopolysaccharides (LPSs), and a zwitterionic phospholipid, dioleoylphosphatidyl choline (DOPC). RuBTZ and RuCTZ had little effects on DOPC, which is consistent with their negligible toxicity toward mammalian cells that may be approximated by a zwitterionic monolayer. Also little were their effects on LPSs. In contrast, RuBTZ and RuCTZ induced expansion in the surface pressure isotherms and decreased the compressional modulus of the E. coli lipid extract. While the more hydrophobic RuBTZ seemed to affect the hydrophobic tails of the E. coli extract monolayer to a larger extent, according to polarization modulation infrared reflection absorption spectroscopy results, evidence of a stronger RuBTZ interaction could not be confirmed unequivocally. Therefore, the interaction with the E. coli cell membrane cannot be directly correlated with the observed higher bactericidal activity of RuBTZ, in comparison to that of RuCTZ. This appears to be a case in which Langmuir monolayer studies do not suffice to determine the mechanisms responsible for the bactericidal activity.

New Heterofunctional Supports Based on Glutaraldehyde-Activation: A Tool for Enzyme Immobilization at Neutral pH.

Immobilization is an exciting alternative to improve the stability of enzymatic processes. However, part of the applied covalent strategies for immobilization uses specific conditions, generally alkaline pH, where some enzymes are not stable. Here, a new generation of heterofunctional supports with application at neutral pH conditions was proposed. New supports were developed with different bifunctional groups (i.e., hydrophobic or carboxylic/metal) capable of adsorbing biocatalysts at different regions (hydrophobic or histidine richest place), together with a glutaraldehyde group that promotes an irreversible immobilization at neutral conditions. To verify these supports, a multi-protein model system (E. coli extract) and four enzymes (Candida rugosa lipase, metagenomic lipase, β-galactosidase and β-glucosidase) were used. The immobilization mechanism was tested and indicated that moderate ionic strength should be applied to avoid possible unspecific adsorption. The use of different supports allowed the immobilization of most of the proteins contained in a crude protein extract. In addition, different supports yielded catalysts of the tested enzymes with different catalytic properties. At neutral pH, the new supports were able to adsorb and covalently immobilize the four enzymes tested with different recovered activity values. Notably, the use of these supports proved to be an efficient alternative tool for enzyme immobilization at neutral pH.

Analysis of ribosomal inter-subunit sites as targets for complementary oligonucleotides.

The bacterial ribosome has many functional ribosomal RNA (rRNA) sites. We have computationally analyzed the rRNA regions involved in the interactions between the 30S and 50S subunits. Various properties of rRNA such as solvent accessibility, opening energy, hydrogen bonding pattern, van der Waals energy, thermodynamic stability were determined. Based on these properties we selected rRNA targets for hybridization with complementary 2'-O-methyl oligoribonucleotides (2'-OMe RNAs). Further, the inhibition efficiencies of the designed ribosome-interfering 2'-OMe RNAs were tested using a β-galactosidase assay in a translation system based on the E. coli extract. Several of the oligonucleotides displayed IC50 values below 1 μM, which were in a similar range as those determined for known ribosome inhibitors, tetracycline and pactamycin. The calculated opening and van der Waals stacking energies of the rRNA targets correlated best with the inhibitory efficiencies of 2'-OMe RNAs. Moreover, the binding affinities of several oligonucleotides to both 70S ribosomes and isolated 30S and 50S subunits were measured using a double-filter retention assay. Further, we applied heat-shock chemical transformation to introduce 2'-OMe RNAs to E. coli cells and verify inhibition of bacterial growth. We observed high correlation between IC50 in the cell-free extract and bacterial growth inhibition. Overall, the results suggest that the computational analysis of potential rRNA targets within the conformationally dynamic regions of inter-subunit bridges can help design efficient antisense oligomers to probe the ribosome function.

Antibacterial and Antibiotic-Potentiation Activities of Levisticum officinale L. Extracts on Pathogenic Bacteria

Background: Multidrug resistant (MDR) Gram-negative bacteria have resistance to many antimicrobial compounds by multiple mechanisms including reduced outer membrane permeability, and active efflux mechanisms by efflux pumps. Objectives: The current study was planned to search the antibacterial activities of the ethanol and chloroform extracts of Levisticum officinale L., and their synergistic effects with ciprofloxacin against some Gram-negative pathogenic bacteria; also to analyze the extracts if they contain inhibitors of efflux pumps of the examined bacteria. Materials and Methods: After grinding, the resulting powder of Levisticum officinale L. was extracted with 85% ethanol and chloroform by maceration method. The studied microorganisms included the reference strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Salmonella enteritidis. Broth micro-dilution methods were used to determine the minimum inhibitory concentrations (MICs) of the extracts alone or in association with ciprofloxacin and phenylalanine-arginine-β-naphtylamide (PAβN) as an efflux pump inhibitor (EPI). The presumptive efflux activity was detected by the ethidium bromide (EB) well diffusion method. Results: MIC determination indicated that the Levisticum officinale L. extracts inhibited the growth of all the studied bacteria within a concentration range of 3125 to 25000 μg/mL. The synergistic effects were noted between the Levisticum officinale L. extracts and ciprofloxacin on all tested bacteria. In S. enertidis and E. coli both extracts of L. officinale, but in P. aeruginosa and A. baumannii only ethanolic extract increased the amount of EB accumulation (i e, reduced efflux). Conclusion: The overall results of the current study provided information for the possible use of the Levisticum officinale L. extracts to control bacterial infections caused by the examined bacteria.

Vacuna oral (OM-89) en la profilaxis de infección urinaria recurrente: una revisión sistemática realista con metaanálisis

ObjectiveTo evaluate the efficacy of Escherichia Coli extract (OM-89) in the prophylaxis of recurrent uncomplicated urinary tract infection (UTI) through a contemporary systematic review and meta-analysis. MethodsInclusion criteria were double-blind randomized trials using orally administrated OM-89, 6mg daily, during three months with a minimum of three months of monitoring. Outcomes were the frequency of bacteriuria in 3 and 6 months, dysuria in 6 months and UTI in 6 months. Databases: PubMed, MEDLINE, Cochrane Collaboration and their key references. After analysis by three independent reviewers, 15 double-blind randomized trials were identified, 10 papers excluded due to methods flaws and 5 used for data analysis due to double blinding and reporting drop-outs. ResultsAmong 5 selected studies the date of publication ranged from 1985 to 2005, totalizing 396 patients in the OM-89 group and 392 in the control group. Overall, there were 61 dropouts in the control group and 76 in the OM-89 group. As a major limitation there was no appropriate description of their methodologies and none of the studies described conflict of interest or commitment to the pharmaceutical industry. All studies were multi-centric, except for two, which showed no clarity on allocation concealment. All studies show benefit in favor of vaccine. ConclusionsCurrent literature on prospective randomized controlled trials evaluating the use of oral OM-89 vaccine in the recurrent UTI prophylaxis is of low quality, limited to the first six months only and with variable definition of bacteriuria and UTI. Although all studies show benefit in favor of vaccine, no robust trial was identified, resulting in a high heterogeneity in the data analyzed. Also, publication bias could not be excluded and future higher quality studies are warranted adding intermediate (>12 months) and long-term follow-up.

Warpgroup: increased precision of metabolomic data processing by consensus integration bound analysis

Current informatic techniques for processing raw chromatography/mass spectrometry data break down under several common, non-ideal conditions. Importantly, hydrophilic liquid interaction chromatography (a key separation technology for metabolomics) produces data which are especially challenging to process. We identify three critical points of failure in current informatic workflows: compound specific drift, integration region variance, and naive missing value imputation. We implement the Warpgroup algorithm to address these challenges. Warpgroup adds peak subregion detection, consensus integration bound detection, and intelligent missing value imputation steps to the conventional informatic workflow. When compared with the conventional workflow, Warpgroup made major improvements to the processed data. The coefficient of variation for peaks detected in replicate injections of a complex Escherichia Coli extract were halved (a reduction of 19%). Integration regions across samples were much more robust. Additionally, many signals lost by the conventional workflow were 'rescued' by the Warpgroup refinement, thereby resulting in greater analyte coverage in the processed data. I: MPLEMENTATION: Warpgroup is an open source R package available on GitHub at github.com/nathaniel-mahieu/warpgroup. The package includes example data and XCMS compatibility wrappers for ease of use. Supplementary data are available at Bioinformatics online. nathaniel.mahieu@wustl.edu or gjpattij@wustl.edu.

Protein-templated biomimetic silica nanoparticles.

Biomimetic silica particles can be synthesized as a nanosized material within minutes in a process mimicked from living organisms such as diatoms and sponges. In this work, we have studied the effect of bovine serum albumin (BSA) as a template to direct the synthesis of silica nanoparticles (NPs) with the potential to associate proteins on its surface. Our approach enables the formation of spheres with different physicochemical properties. Particles using BSA as a protein template were smaller (∼250-380 nm) and were more monodisperse than those lacking the proteic core (∼700-1000 nm) as seen by dynamic light scattering (DLS), scanning electron microscopy (SEM), and environmental scanning electron microscopy (ESEM) analysis. The absence of BSA during synthesis produced silica nanoparticles without any porosity that was detectable by nitrogen adsorption, whereas particles containing BSA developed porosity in the range of 4 to 5 nm which collapsed on the removal of BSA, thus producing smaller pores. These results were in accordance with the pore size calculated by high-resolution transmission electron microscopy (HTEM). The reproducibility of the BSA-templated nanoparticle properties was determined by analyzing four batches of independent synthesizing experiments that maintained their properties. The high positive superficial charge of the nanoparticles facilitated adsorption under mild conditions of a range of proteins from an E. coli extract and a commercial preparation of laccase from Trametes versicolor. All of the proteins were quantitatively desorbed. Experiments conducted showed the reusability of the particles as supports for the ionic adsorption of the biomolecules. The protein loading capacity of the BSA-based biomimetic particles was determined using laccase as 98.7 ± 6.6 mg·g(-1) of particles.