Evaluation and validation of a novel Arxula adeninivorans estrogen screen (nAES) assay and its application in analysis of wastewater, seawater, brackish water and urine

A novel Arxula adeninivorans yeast estrogen screen (nAES) assay has been developed for detection of estrogenic activity in various liquid samples such as wastewater, seawater, brackish water and swine urine. Two bio-components were engineered to co-express the human estrogen receptor α (hERα) and an inducible reporter gene; either the non-conventional phytase gene (phyK, derived from Klebsiella sp. ASR1) or the non-conventional tannase gene (ATAN1, derived from Arxula). Both reporters were put under the control of an Arxula derived glucoamylase (GAA) promoter, which was modified by the insertion of two estrogen-responsive elements (EREs). The Arxula transformation/expression platform Xplor® 2, which lacks resistance markers and E. coli elements, was used to select stable mitotic transformants. They were then analyzed for robustness and suitability as the bio-component for the nAES assay. Two types of the nAES assay based on the reporter proteins phytase and tannase (nAES-P, nAES-T) were used in this work. The nAES-P type is more suitable for the analysis of seawater, brackish water and urine whereas the nAES-T type exhibited higher robustness to NaCl. Both assay types have similar characteristics for the determination of estrogen in sewage and urine samples e.g. 6-25 h assay period with detection and determination limits and EC₅₀ values for 17β-estradiol of 2.8 ng L^− 1, 5.9 ng L^− 1, 33.2 ng L^− 1 (nAES-P) and 3.1 ng L^− 1, 6.7 ng L^− 1 and 39.4 ng L^− 1 (nAES-T). Substrate specificity and analytical measurement range (AMR) for both assay types are also similar. These characteristics show that the nAES assay based on non-conventional salt tolerant yeast is applicable for a high throughput estrogen analysis in the environmental and regulatory control sectors.     

Characterization of water in bacterial cellulose using dielectric spectroscopy and electron microscopy

It is shown that only 10% of the 99 wt% water present in bacterial cellulose (BC) gels, produced by Acetobacter xylinum, behave like free bulk water; the majority of the water molecules in the gels is more or less tightly bound to the cellulose. The magnitude of the diffusion coefficients of ions transported in the water phase of the BC gels as well as the information contained in freeze fracture transmission electron microscopic images of the gel structures indicates that the bulk-like water is confined in “lakes” rather than forming a continuous phase throughout the gel. Water desorption isotherms suggest that these “lakes” decrease in size with increasing oxygen concentration used during the biosynthesis process of the gels.     

Electrospinning of polymer melts: Phenomenological observations

Melt electrospinning is an alternative to solution electrospinning, however, melt electrospinning has typically resulted in fibers with diameters of tens of microns. In this paper we demonstrate that polypropylene fibers can be reduced from 35 ± 8 μm in diameter, to 840 ± 190 nm with a viscosity-reducing additive. Melt electrospun blends of poly(ethylene glycol)-block-poly(?-caprolactone) (PEG₄₇-b-PCL₉₅) and poly(?-caprolactone) (PCL) produced fibers with micron-scale diameters (2.0 ± 0.3 μm); this was lowered to 270 ± 100 nm by using the gap method of alignment for collection. The collected melt electrospun fibers often fused together where they touched, allowing the stabilization of relatively thick non-woven felts. The melt electrospun collection also included coiled circles and looped patterns of fibers approximately 150-250 μm in diameter. The polymer jet was visible between the collector and spinneret for particularly significant lengths, and underwent coiling and buckling instabilities close to the collector. The focused deposition of melt electrospun fibers was maintained when multiple jets were observed, with the collections from multiple jets separated by 3.8 ± 0.5 mm for a 5 cm collector gap. The frequent fusion points between melt electrospun fibers, and a reduction in diameter for the gap method of alignment, indicated that the melt electrospun fibers are still slightly molten at collection.     

Ferrite‐Based Exchange‐Coupled Hard–Soft Magnets Fabricated by Spark Plasma Sintering

Ferrite‐based, hard‐soft magnetic nanocomposites with the composition (100%?x)SrFe₁₂O₁₉–xCoFe₂O₄, where x = 5, 10, and 15 wt%, were prepared by mixing the constituent powders, followed by spark plasma sintering. In order to control the particle size of the constituent materials, the SrFe₁₂O₁₉ and CoFe₂O₄ powders were synthesized using the hydrothermal method, mixed and then consolidated with spark plasma sintering. The conditions during the spark plasma sintering process (sintering temperature, time, and applied pressure) were varied in order to prepare composites with a high density and exchange‐coupled hard and soft magnetic phases, leading to an increase in the maximum energy product, when compared with pure SrFe₁₂O₁₉. The microstructural analysis revealed that the relative density of the sintered composite exceeded 90% of the theoretical value and that the CoFe₂O₄ was uniformly distributed in the SrFe₁₂O₁₉ matrix. Magnetic measurements of the sintered composites showed a single‐phase magnetic behavior. When compared with the single‐phase SrFe₁₂O₁₉ used in this study, the SPS composites exhibited a 22% increase in the maximum energy product (26.1 kJ/m³).     

Chemokine-Like Receptor 1 mRNA Weakly Correlates with Non-Alcoholic Steatohepatitis Score in Male but Not Female Individuals

The chemokine-like receptor 1 (CMKLR1) ligands resolvin E1 and chemerin are known to modulate inflammatory response. The progression of non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH) is associated with inflammation. Here it was analyzed whether hepatic CMKLR1 expression is related to histological features of NASH. Therefore, CMKLR1 mRNA was quantified in liver tissue of 33 patients without NAFLD, 47 patients with borderline NASH and 38 patients with NASH. Hepatic CMKLR1 mRNA was not associated with gender and body mass index (BMI) in the controls and the whole study group. CMKLR1 expression was similar in controls and in patients with borderline NASH and NASH. In male patients weak positive correlations with inflammation, fibrosis and NASH score were identified. In females CMKLR1 was not associated with features of NAFLD. Liver CMKLR1 mRNA tended to be higher in type 2 diabetes patients of both genders and in hypercholesterolemic women. In summary, this study shows that hepatic CMKLR1 mRNA is weakly associated with features of NASH in male patients only.     

Lipidomic Analysis of Serum from High Fat Diet Induced Obese Mice

Lipid metabolites regulate fatty acid and glucose homeostasis. The intention of the current study is to identify circulating lipid species, which are altered in rodent obesity and strongly correlate with the classically measured metabolites glucose, triglycerides, and cholesterol. Mice fed a high fat diet (HFD) for 14 weeks have increased body weight and fasting glucose. Serum triglycerides are not altered, while cholesterol tends to be increased. Accordingly, major cholesteryl ester (CE) species and free cholesterol are not significantly raised in obesity while minor metabolites, including CE 20:3 and CE 18:3, are increased or reduced, respectively. Distinct sphingomyelin (SM) species are elevated while ceramides are not raised. Phosphatidylinositol (PI) species, including PI 34:1, are raised while others are decreased. PI 34:1 strongly correlates with fasting glucose and proinsulin levels. Phosphatidylcholine (PC) 26:0, 40:2, and 40:5, which are induced in obesity, correlate with cholesterol. PC 38:4 and PC 40:6 are also raised in fat fed mice and positively correlate with fasting glucose. Lysophosphatidylcholine (LPC) species are also changed in obesity and the already shown reduction of LPC 16:1 has been confirmed. LPC 22:4, which is increased, correlates with serum cholesterol. The data indicate that circulating levels of various lipid species are changed in the obesity model studied and some of them are strongly associated with classically measured metabolites.     

Generic Structures of Cytotoxic Liprotides: Nano‐Sized Complexes with Oleic Acid Cores and Shells of Disordered Proteins

The cytotoxic complex formed between α‐lactalbumin and oleic acid (OA) has inspired many studies on protein–fatty acid complexes, but structural insight remains sparse. After having used small‐angle X‐ray scattering (SAXS) to obtain structural information, we present a new, generic structural model of cytotoxic protein–oleic acid complexes, which we have termed liprotides (lipids and partially denatured proteins). Twelve liprotides formed from seven structurally unrelated proteins and prepared by different procedures all displayed core–shell structures, each with a micellar OA core and a shell consisting of flexible, partially unfolded protein, which stabilizes the OA micelle. The common structure explains similar effects exerted on cells by different liprotides and is consistent with a cargo off‐loading of the OA into cell membranes.     

A Thioacetal Photocage Designed for Dual Release: Application in the Quantitation of Therapeutic Release by Synchronous Reporter Decaging

Despite the immense potential of existing photocaging technology, its application is limited by the paucity of advanced caging tools. Here, we report on the design of a novel thioacetal ortho‐nitrobenzaldehyde (TNB) dual arm photocage that enabled control of the simultaneous release of two payloads linked to a single TNB unit. By using this cage, which was prepared in a single step from commercial 6‐nitroverataldehyde, three drug–fluorophore conjugates were synthesized: Taxol‐TNB‐fluorescein, Taxol‐TNB‐coumarin, and doxorubicin‐TNB‐coumarin, and long‐wavelength UVA light‐triggered release experiments demonstrated that dual payload release occurred with rapid decay kinetics for each conjugate. In cell‐based assays performed in vitro, dual release could also be controlled by UV exposure, resulting in increased cellular fluorescence and cytotoxicity with potency equal to that of unmodified drug towards the KB carcinoma cell line. The extent of such dual release was quantifiable by reporter fluorescence measured in situ and was found to correlate with the extent of cytotoxicity. Thus, this novel dual arm cage strategy provides a valuable tool that enables both active control and real‐time monitoring of drug activation at the delivery site.