Application of β-glucuronidase-immobilised silica gel formulation to microfluidic platform for biotransformation of β-glucuronides

Objective

To improve the efficiency of reactions of β-glucuronidase (GUS)-assisted glucuronic acid (GluA) removal within a microfluidic system.

Results

β-glucuronidase from Helix pomatia was immobilised and characterised in silica-based sol–gel monoliths. Efficiency of the GUS-doped silica monoliths was tested for hydrolysis of p-Nitrophenyl-β-d-glucuronide (pNP–GluA) in both ml-scaled medium via batch reactions and microfluidic environment via continuous-flow reactions. In the microfluidic platform, within a duration of 150 min of continuous operation (flow rate: 1 µL/min), the obtained highest pNP yield was almost 50% higher than that of the corresponding batchwise reaction. However, increased flow rates (3, 5, and 10 µL/min) resulted in lower conversion yields compared to 1 µL/min. The microfluidic platform demonstrated continuous hydrolytic activity for 7 days with considerable reaction yields while using a small amount of the enzyme.

Conclusion

These results revealed that usage of the microreactors has considerable potential to efficiently obtain bioactive GluA-free aglycons from various plant-derived β-glucuronides for pharmaceutical applications.

Graphical Abstract

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Onychomycoses in a Military Population in Brazil

Purpose of Review

This study aimed to isolate and characterize filamentous fungi onychomycosis agents in a military population assisted at a hospital outpatient clinic.

Recent Findings

In onychomycosis, the fungi colonize the subungual region causing thickening, discoloration, or cracking of the nail bed. Samples were collected from patients with clinical sights of onychomycosis.

Summary

Among 80 samples collected, 50 (62.5%) had positive culture. Isolated dermatophytes (86%) were Trichophyton rubrum (21; 42%), T. mentagrophytes var. interdigitale (19; 38%), and Microsporum gypseum (3; 6%) and non-dermatophyte molds were Fusarium spp. (1; 2%), Scytalidium spp. (1; 2%), and Chaetomium globosum (5; 10%). Minimal inhibitory concentrations (mg/L) of terbinafine, itraconazole, and fluconazole necessary to inhibit 50/90% of the isolates were respectively 0.015/0.06, 0.06/0.12, and 32/32. Etiological agents of onychomycosis in a military hospital are similar as reported in studies for the general population. High prevalence of non-dermatophytic agents was observed, especially for Chaetomium globosum.

     

Degradation of carbazole,dibenzothiophene and polyaromatic hydrocarbons by recombinant <Emphasis Type="Italic">Rhodococcus</Emphasis> sp.

Objectives

With the view of designing a single biocatalyst for biorefining, carbazole dioxygenase was cloned from Pseudomonas sp. and expressed in Rhodococcus sp.

Results

The recombinant, IGTS8, degraded both carbazole and dibenzothiophene at 400 mg/l in 24 h. Maximum carbazole degradation was in 1:1 (v/v) hexadecane/aqueous phase. Anthracene, phenanthrene, pyrene, fluoranthene and fluorine were also degraded without affecting the aliphatic component.

Conclusions

Recombinant Rhodococcus sp. IGTS8 can function as a single biocatalyst for removing major contaminants of fossil fuels viz. dibenzothiophene, carbazole and polyaromatic compounds.

     

Corneal Collagen Cross-Linking for the Management of Mycotic Keratitis

Purpose

To evaluate the efficiency of corneal collagen cross-linking (CXL) in addition to topical voriconazole in cases with mycotic keratitis.

Design

Retrospective case series in a tertiary university hospital.

Participants

CXL was performed on 13 patients with mycotic keratitis who presented poor or no response to topical voriconazole treatment.

Methods

The clinical features, symptoms, treatment results and complications were recorded retrospectively. The corneal infection was graded according to the depth of infection into the stroma (from grade 1 to grade 3). The visual analogue scale was used to calculate the pain score before and 2 days after surgery.

Main Outcome Measures

Grade of the corneal infection.

Results

Mean age of 13 patients (6 female and 7 male) was 42.4 ± 17.7 years (20–74 years). Fungus was demonstrated in culture (eight patients) or cytological examination (five patients). Seven of the 13 patients (54%) were healed with topical voriconazole and CXL adjuvant treatment in 26 ± 10 days (15–40 days). The remaining six patients did not respond to CXL treatment; they initially presented with higher grade ulcers. Pre- and post-operative pain score values were 8 ± 0.8 and 3.5 ± 1, respectively (p < 0.05).

Conclusions

The current study suggests that adjunctive CXL treatment is effective in patients with small and superficial mycotic ulcers. These observations require further research by large randomized clinical trials.

     

Catesbeianin-1, a novel antimicrobial peptide isolated from the skin of <Emphasis Type="Italic">Lithobates catesbeianus</Emphasis> (American bullfrog)

Objectives

To identify and characterize a novel antimicrobial peptide, catesbeianin-1.

Results

Catesbeianin-1 is 25 amino acids long and is α-helical, cationic and amphipathic. It had antimicrobial activity against Gram-positive and Gram-negative bacteria. It was resistant against trypsin and pepsin. Catesbeianin-1 exhibited moderate hemolytic activity (approx 8%) at 100 μg/ml, and its HC₅₀ (50% hemolytic concentration) was 300 μg/ml. Its cytotoxicity was approx 10–20% at 100 μg/ml, and its CC₅₀ (50% cytotoxic concentration) was >100 μg/ml. The LD₅₀ of catesbeianin-1 in mice was 80 mg/kg. At 3.1 µg/ml, catesbeianin-1 significantly inhibited the growth of methicillin-resistant Staphylococcus aureus.

Conclusions

A new antimicrobial peptide from the skin of Lithobates catesbeianus (American bullfrog) may represent a template for the development of novel antimicrobial agents.

     

A novel ethanol-tolerant laccase,Tvlac, from <Emphasis Type="Italic">Trametes versicolor</Emphasis>

Objectives

To produce and characterize novel laccases with ethanol tolerance from Trametes versicolor using agriculture by-products as energy source.

Results

Trametes versicolor 1017 produces two laccase isoenzymes with a total activity of 10 U ml^?1 within 8 days when using wheat bran and peanut powder as energy sources in liquid culture medium. A novel isoenzyme, named Tvlac, was identified, purified and characterized. Its optimum pH and temperature were from 4.5 to 5 and 55 to 60 °C, respectively. Its activity was stimulated by ethanol at 10 % (v/v) which increased the V ₀.

Conclusions

The biochemical properties of Tvlac substantiate the potential of this enzyme for applications under an aqueous ethanol mixture environment.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

Selective oxidation of UDP-glucose to UDP-glucuronic acid using permeabilized <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> expressing human UDP-glucose 6-dehydrogenase

Objectives

To use permeabilized cells of the fission yeast, Schizosaccharomyces pombe, that expresses human UDP-glucose 6-dehydrogenase (UGDH, EC 1.1.1.22), for the production of UDP-glucuronic acid from UDP-glucose.

Results

In cell extracts no activity was detected. Therefore, cells were permeabilized with 0.3 % (v/v) Triton X-100. After washing away all low molecular weight metabolites, the permeabilized cells were directly used as whole cell biocatalyst. Substrates were 5 mM UDP-glucose and 10 mM NAD⁺. Divalent cations were not added to the reaction medium as they promoted UDP-glucose hydrolysis. With this reaction system 5 mM UDP-glucose were converted into 5 mM UDP-glucuronic acid within 3 h.

Conclusions

Recombinant permeabilized cells of S. pombe can be used to synthesize UDP-glucuronic acid with 100 % yield and selectivity.

     

Pranlukast,a novel binding ligand of human Raf1 kinase inhibitory protein

Objectives

To study the binding of pranlukast to hRKIP and its regulatory role in the Raf1/MEK/ERK signal pathway.

Results

NMR and fluorescence experiments demonstrated hRKIP could bind pranlukast with a binding constant of 1016 mM^?1. Residues (Y81, S109 and Y181) on the conserved ligand-binding pocket of hRKIP played a crucial role in binding pranlukast, and their mutations reduced the binding affinity more than 85 %. Furthermore, 25 μM pranlukast could up-regulate the ERK phosphorylation by about 17 %.

Conclusion

Pranlukast may be used as a potential drug precursor for treating hRKIP involved diseases.

     

Biodegradation of saline phenolic wastewater in a biological contact oxidation reactor with immobilized cells of <Emphasis Type="Italic">Oceanimonas</Emphasis> sp.

Objective

To develop a method to treat saline phenolic wastewater in a biological contact oxidation reactor (BCOR) with immobilized cells of a marine microorganism, Oceanimonas sp., isolated from seawater.

Results

Cells were immobilized on fibre carriers in the BCOR. Saline wastewater with phenol at 1.5 g/l and NaCl at 6 % (w/v) was treated. In continuous assays, 99 % removal of phenol was achieved and a kinetic model for the phenol degradation is presented based on Monod’s equation.

Conclusion

The BOCR system using immobilized cells of Oceanimonas efficiently treats saline phenolic wastewaters without having decrease the salinity of the wastewater.

     

Minimal variation of the plasma lipidome after delayed processing of neonatal cord blood

Background

Cord blood lipids are potential disease biomarkers. We aimed to determine if their concentrations were affected by delayed blood processing.

Method

Refrigerated cord blood from six healthy newborns was centrifuged every 12 h for 4 days. Plasma lipids were analysed by liquid chromatography/mass spectroscopy.

Results

Of 262 lipids identified, only eight varied significantly over time. These comprised three dihexosylceramides, two phosphatidylserines and two phosphatidylethanolamines whose relative concentrations increased and one sphingomyelin that decreased.

Conclusion

Delay in separation of plasma from refrigerated cord blood has minimal effect overall on the plasma lipidome.

     

Accumulation of Fascin<Superscript>+</Superscript> cells during experimental autoimmune neuritis

Background

Experimental autoimmune neuritis (EAN) is a well-known animal model of human demyelinating polyneuropathies and is characterized by inflammation and demyelination in the peripheral nervous system. Fascin is an evolutionarily highly conserved cytoskeletal protein of 55 kDa containing two actin binding domains that cross-link filamentous actin to hexagonal bundles.

Methods

Here we have studied by immunohistochemistry the spatiotemporal accumulation of Fascin?+?cells in sciatic nerves of EAN rats.

Results

A robust accumulation of Fascin?+?cell was observed in the peripheral nervous system of EAN which was correlated with the severity of neurological signs in EAN.

Conclusion

Our results suggest a pathological role of Fascin in EAN.

Virtual slides

The virtual slides for this article can be found here: http://www.diagnosticphatology.diagnomx.eu/vs/6734593451114811

     

PEGylation of cytochrome <Emphasis Type="Italic">c</Emphasis> at the level of lysine residues mediated by a microbial <Emphasis Type="Italic">trans</Emphasis>glutaminase

Objectives

To establish a method for microbial transglutaminase (mTG)-mediated PEGylation of proteins at the level of lysine (Lys) residues.

Results

Carboxybenzyl-glutaminyl–glycinyl-methoxypolyethylene glycol (CBZ-QG-mPEG) was prepared by introducing carboxybenzyl-glutaminyl-glycine (CBZ-QG) to mPEG amine. The analysis by Fourier transform infrared spectroscopy and SDS-PAGE showed that CBZ-QG-mPEG was successfully synthesized and can be recognized by mTG as an acyl donor to modify therapeutic protein, cytochrome c (cyt c). Finally, under an optimized condition (cyt c 0.5 mg/ml, CBZ-QG-mPEG 11.25 mg/ml, mTG 0.5 mg/ml, 37 °C, 2 h), the PEGylation yield reached 76.5 %.

Conclusions

This is the first study regarding the PEGylation of protein at the level of Lys residues catalyzed by mTG. The novel method could be employed to immobilize active proteins and modify therapeutic proteins.

     

Fusion expression of cecropin B-like antibacterial peptide in <Emphasis Type="Italic">Pichia</Emphasis> GS115 and its antibacterial mechanism

Objectives

To establish an efficient expression system for a fusion protein of glutathione S-transferase and cecropin B (GST-CB) and to clarify the antibacterial mechanism of CB.

Results

The optimal incubation time and methanol concentration for induced expression of CB were 36 h and 1 % w/v, respectively. The yield of GST-CB was 2.2 g/l. The minimum inhibitory concentrations of GST-CB towards Staphylococcus aureus subsp. saprophyticus (ATCC 15305) and Escherichia coli strain CFT073 were 250 and 125 μg/ml, respectively. Notably, mutations of proline 24 (P24) in CB produced a polypeptide without antimicrobial activity.

Conclusion

The fusion protein GST-CB, which has a broad spectrum antimicrobial activity, can be abundantly expressed in Pichia pastoris GS115, and P24 may be an important amino acid for the antimicrobial activity of GST-CB.

     

Reengineering substrate specificity of <Emphasis Type="Italic">E. coli</Emphasis> glutamate dehydrogenase using a position-based prediction method

Objective

To re-engineer the active site of proteins for non-natural substrates using a position-based prediction method (PBPM).

Results

The approach has been applied to re-engineer the E. coli glutamate dehydrogenase to alter its substrate from glutamate to homoserine for a de novo 1,3-propanediol biosynthetic pathway. After identification of key residues that determine the substrate specificity, residue K92 was selected as a candidate site for mutation. Among the three mutations (K92V, K92C, and K92M) suggested by PBPM, the specific activity of the best mutant (K92 V) was increased from 171 ± 35 to 1328 ± 71 μU mg^?1.

Conclusion

The PBPM approach has a high efficiency for re-engineering the substrate specificity of natural enzymes for new substrates.

     

Cadmium reduces zinc uptake but enhances its translocation in the cadmium-accumulator, <Emphasis Type="Italic">Carpobrotus rossii</Emphasis>, without affecting speciation

Background and aims

Interactions between Cd and Zn occur in soils and plants but are inconsistent. This study examined how Cd/Zn interactions influence the growth of Carpobrotus rossii (Haw.) and the accumulation of Cd and Zn in plants.

Methods

Plants were grown in nutrient solutions containing 5–100 μM Zn and 0, 5 or 15 μM Cd. Plant growth and tissue concentrations were measured, and the speciation of Zn within the plant tissues determined using synchrotron-based X-ray absorption spectroscopy.

Results

There was an additive negative interaction between Cd and Zn on root growth. Only the highest level of Zn (100 μM) decreased Cd concentrations in root and shoot tissues (by 40–64%), whilst 100 μM Zn enhanced Cd translocation at 5 μM Cd but decreased it at 15 μM Cd. In contrast, both 5 and 15 μM Cd decreased Zn concentrations in root and shoot tissues but increased Zn translocation by 30–90%. This interaction was not associated with changes in Zn speciation within the plants, with most Zn associated with oxalate (48–87%).

Conclusions

The presence of Zn and Cd resulted in an additive negative effect on root growth, but an antagonistic pattern in their accumulation in shoots of C. rossii.

     

Expression of a high sweetness and heat-resistant mutant of sweet-tasting protein,monellin, in <Emphasis Type="Italic">Pichia pastoris</Emphasis> with a constitutive GAPDH promoter and modified <Emphasis Type="Italic">N</Emphasis>-terminus

Objectives

To improve the stability and sweetness of the sweet-tasting protein, monellin, by using site-directed mutagenesis and a Pichia pastoris expression system with a GAPDH constitutive promoter.

Results

Both wild-type and E2 N mutant of single-chain monellin gene were cloned into the PGAPZαA vector and expressed in Pichia pastoris. The majority of the secreted recombinant protein, at 0.15 g/l supernatant, was monellin. This was purified by Sephadex G50 chromatography. The sweetness threshold of wild-type and E2 N were 30 μg/ml and 20 μg/ml, respectively. Compared with the proteins expressed in Escherichia coli, the thermostability of both proteins was improved. The N-terminal sequence is determinative for the sweetness of the proteins expressed in yeast strains.

Conclusions

Site-directed mutagenesis, modification of the N-terminus of monellin, and without the need of methanol induction in P. pastoris expression system, indicate the possibility for large-scale production of this sweet-tasting protein.

     

A novel method to harvest <Emphasis Type="Italic">Chlorella</Emphasis> sp. by co-flocculation/air flotation

Objectives

To develop a more effective dissolved air flotation process for harvesting microalgae biomass, a co-flocculation/air flotation (CAF) system was developed that uses an ejector followed by a helix tube flocculation reactor (HTFR) as a co-flocculation device to harvest Chlorella sp. 64.01.

Results

The optimal size distribution of micro-bubbles and an air release efficiency of 96 % were obtained when the flow ratio of inlet fluid (raw water) to motive fluid (saturated water) of the ejector was 0.14. With a reaction time of 24 s in the HTFR, microalgae cells and micro-bubbles were well flocculated, and these aerated flocs caused a fast rising velocity (96 m/h) and high harvesting efficiency (94 %).

Conclusions

In a CAF process, micro-bubbles can be encapsulated into microalgae flocs, which makes aerated flocs more stable. CAF is an effective approach to harvesting microalgae.

     

Using a lipidomics approach for nutritional phenotyping in response to a test meal containing gamma-linolenic acid

Introduction

Plasma fatty acids are derived from preformed sources in the diet and de novo synthesis through the action of desaturase and elongase enzymes.

Objective

This study was designed to examine the elongation of gamma-linolenic acid (GLA, 18:3n6) into dihomo-gamma-linolenic acid (DGLA, 20:3n6) over an 8-h period using both targeted gas chromatography–flame ionization detection and untargeted liquid chromatography–mass spectrometry-based lipidomics utilizing the sequential window acquisition of all theoretical fragment-ion spectra (SWATH).

Methods

In a single blind, placebo-controlled, crossover design, seven healthy subjects consumed a test meal that consisted of GLA fat (borage oil) or a control fat (a mixture of corn, safflower, sunflower and extra-virgin light olive oils) on three separate test days for each test meal.

Results

Total plasma fatty acid concentrations and 366 unique lipid species were measured at 0, 2, 4, 6 and 8 h in response to the test meals. Mean plasma 18:3n6 was 7-fold higher to the GLA challenge compared with baseline and the control meal. By 8 h, mean plasma 20:3n6 was significantly higher in response to the GLA test meal than baseline and the control group. Five of the seven subjects were “responders” in converting GLA into DGLA, but two subjects did not show this conversion. The conversion was independent of physical activity level.

Conclusion

Using polyunsaturated fatty acid metabolism as an example, this study demonstrates inter-individual differences in enzymatic capacities to inform exact nutritional and metabolic phenotyping that could be used for precision medicine.

Graphical Abstract

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