A “plug‐n‐play” modular metabolic system for the production of apocarotenoids

Apocarotenoids, such as α‐, β‐ionone, and retinol, have high commercial values in the food and cosmetic industries. The demand for natural ingredients has been increasing dramatically in recent years. However, attempts to overproduce β‐ionone in microorganisms have been limited by the complexity of the biosynthetic pathway. Here, an Escherichia coli‐based modular system was developed to produce various apocarotenoids. Incorporation of enzyme engineering approaches (N‐terminal truncation and protein fusion) into modular metabolic engineering strategy significantly improved α‐ionone production from 0.5 mg/L to 30 mg/L in flasks, producing 480 mg/L of α‐ionone in fed‐batch fermentation. By modifying apocarotenoid genetic module, this platform strain was successfully re‐engineered to produce 32 mg/L and 500 mg/L of β‐ionone in flask and bioreactor, respectively (>80‐fold higher than previously reported). Similarly, 33 mg/L of retinoids was produced in flask by reconstructing apocarotenoid module, demonstrating the versatility of the “plug‐n‐play” modular system. Collectively, this study highlights the importance of the strategy of simultaneous modular pathway optimization and enzyme engineering to overproduce valuable chemicals in microbes.     

Hydrology of winter–spring “red tides” in Bahía de Mazatlán, Sinaloa, México

“Red tide” events are frequent and periodical in Bahía de Mazatlán, Sinaloa, México. Yet, the ones observed from 4 February to 4 June 2000, showed some distinctive features: First, the dinoflagellates Prorocentrum balticum (85%), P. mexicanum (5%), and Ceratium furca (5%), dominated the composition of the blooms; Second, the average cell abundance by date was 1.3×10⁶ cells l⁻¹, with a range of 3.5×10³ to 24,500 × 10³ cells l⁻¹, well above previous records; Third, the temperature registered at 10–20 m deep was unusually cold (19 °C), below the normal average of 21.5 °C observed over the last 10 years. Salinity was high (35.9 psu) and showed very little influence on the water density gradient. A mean thermal stratification index (TSI), of 3.4, with a maximum of 7 °C, was observed throughout the period, in spite of a weak upwelling activity and intermittent strong NW winds which were unable to break up water column stratification. Temperature fluctuations at the surface and at the bottom layers showed a 30-day periodicity, suggesting some association with the lunar cycle. To explain the characteristics of the “red tides” registered in Bahía de Mazatlán during the winter–spring period of year 2000, it is proposed that the temperature and density stratification, stabilized further by internal waves that compensated for the weak upwelling activity and provided the necessary nutrients to the surface layer, favored the persistence and intensity of the harmful algal bloom events then observed.     

Effects of filtration through Sephadex columns improve overall quality parameters and “in vivo” fertility of subfertile refrigerated boar-semen

This study was performed to test the effects of filtration through several chromatographic resins on the semen quality parameters (percentages of viability, altered acrosomes and morphological abnormalities, motion characteristicis and the response to the Osmotic Resistance Test) of boar ejaculates of poor quality. Our results indicate that filtration through a non-ionic Sephadex resin bed (Sephadex G-15), combined with a glasswool subjection bed, induced an overall improvement of semen quality parameters, especially seen in a significant (P < 0.05) decrease in the percentages of morphological abnormalities and an increase of several motility parameters related to velocity and linearity. Similar results, although less intense, were observed when the filtration through G-15 resin was accompanied by an ionically neutral polypropylene disk bed instead of glasswool. On the other hand, filtration through two separate ion-exchange Sephadex resins, cationic C-50 and anionic A-50, have less beneficial, and even detrimental, effects on boar-semen quality. In all cases, filtration was accompanied by a significant (P < 0.01) decrease in the final concentration of the samples. Ultrastructural and lectin studies showed that the interaction between sperm and chromatographic resins depends on the resin type utilized, and in the case of G-15 it seems that it works by trapping that sperm with not enough strength to overcome the physical resistance associated with chromatographic particles. When semen of poor quality was filtered through G-15 resin and then was utilized for “in vivo” fertility trials, a significant (P < 0.05) increase in the percentage of fertility was observed, when compared with the same, but unfiltered, samples. In summary, our results strongly indicate that filtration through ionically inert, Sephadex chromatographic resins could be a very useful and practical method to improve both boar-semen quality and fertilizing ability, especially from mediocre and/or subfertile samples.     

Accurate geometries for “Mountain pass” regions of the Ramachandran plot using quantum chemical calculations

Unusual local arrangements of protein in Ramachandran space are not well represented by standard geometry tools used in either protein structure refinement using simple harmonic geometry restraints or in protein simulations using molecular mechanics force fields. In contrast, quantum chemical computations using small poly‐peptide molecular models can predict accurate geometries for any well‐defined backbone Ramachandran orientation. For conformations along transition regions—ϕ from −60 to 60°—a very good agreement with representative high‐resolution experimental X‐ray (≤1.5 Å) protein structures is obtained for both backbone C⁻¹‐N‐C_α angle and the nonbonded O⁻¹…C distance, while “standard geometry” leads to the “clashing” of O…C atoms and Amber FF99SB predicts distances too large by about 0.15 Å. These results confirm that quantum chemistry computations add valuable support for detailed analysis of local structural arrangements in proteins, providing improved or missing data for less understood high‐energy or unusual regions.     

Comparative study of the molecularly imprinted polymers prepared by reversible addition–fragmentation chain transfer “bulk” polymerization and traditional radical “bulk” polymerization

Bisphenol A (BPA) and propranolol‐imprinted polymers have been prepared via both reversible addition–fragmentation chain transfer “bulk” polymerization (RAFTBP) and traditional radical “bulk” polymerization (TRBP) under similar reaction conditions, and their equilibrium binding properties were compared in detail for the first time. The chemical compositions, specific surface areas, equilibrium bindings, and selectivity of the obtained molecularly imprinted polymers (MIPs) were systematically characterized. The experimental results showed that the MIPs with molecular imprinting effects and quite fast binding kinetics could be readily prepared via RAFTBP, but they did not show improved template binding properties in comparison with those prepared via TRBP, which is in sharp contrast to many previous reports. This could be attributed to the heavily interrupted equilibrium between the dormant species and active radicals in the RAFT mechanism because of the occurrence of fast gelation during RAFTBP. The findings presented here strongly demonstrates that the application of controlled radical polymerizations (CRPs) in molecular imprinting does not always benefit the binding properties of the resultant MIPs, which is of significant importance for the rational use of CRPs in generating MIPs with improved properties. Copyright © 2013 John Wiley & Sons, Ltd.     

Role of oxygen supply in α, ω‐dodecanedioic acid biosynthesis from n‐dodecane by Candida viswanathii ipe‐1: Effect of stirring speed and aeration

α, ω‐Dodecanedioic acid (DC₁₂) usually serves as a monomer of polyamides or some special nylons. During the biosynthesis, oxygenation cascaded in conversion of hydrophobic n‐dodecane to DC₁₂, while the oxidation of n‐dodecane took place in the intracellular space. Therefore, it was important to investigate the role of oxygen supply on the cell growth and DC₁₂ biosynthesis. It was found that stirring speed and aeration influenced the dissolved oxygen (DO) concentration which in turn affected cell growth as well as DC₁₂ biosynthesis. However, the effect of culture redox potential (Orp) level on DC₁₂ biosynthesis was more significant than that of DO level. For DC₁₂ biosynthesis, the first step was to form the emulsion droplets through the interaction of n‐dodecane and the cell. When the stirring speed was enhanced, slits in the surface layer of the emulsion droplets would be increased. Thus, the substances transportation by water through the slits would be intensified, leading to an enhanced DC₁₂ production. Compared with the batch culture at a lower stirring speed (400 rpm) without culture redox potential (Orp) control, the DC₁₂ concentration was increased by 5 times up to 201.3 g/L with Orp controlled above 0 mV at a higher stirring speed (800 rpm).     

Visualizing the “sandwich” structure of osteocytes in their native environment deep in bone in vivo

Osteocytes are the most abundant cells in bone and always the focus of bone research. They are embedded in the highly scattering mineralized bone matrix. Consequently, visualizing osteocytes deep in bone with subcellular resolution poses a major challenge for in vivo bone research. Here we overcome this challenge by demonstrating 3‐photon imaging of osteocytes through the intact mouse skull in vivo. Through broadband transmittance characterization, we establish that the excitation at the 1700‐nm window enables the highest optical transmittance through the skull. Using label‐free third‐harmonic generation (THG) imaging excited at this window, we visualize osteocytes through the whole 140‐μm mouse skull and 155 μm into the brain in vivo. By developing selective labeling technique for the interstitial space, we visualize the “sandwich” structure of osteocytes in their native environment. Our work provides novel imaging methodology for bone research in vivo.      

Low fluences of ultraviolet irradiation stimulate hela cell surface aminopeptidase and candidate “TGFαase” activity

Several forms of perturbation result in the release of bioactive molecules into the microenvironment of injured cells to mediate the inflammatory or reparative reactions which restore normal tissue structure and function. Amongst other products, ultraviolet irradiation (UV) causes the release of the growth factor TGFα from a variety of epithelial cell sources, apparently by a post-translational mechanism. Here we have explored the hypothesis that UV results in the activation of cell surface proteases which may then be capable of excising mature TGFα from its plasma membrane-bound precursor. Using a recently described, sensitive assay of peptidase activity tailored to the substrate requirements for cleavage of the scissile bonds in proTGFα, we have found that nonlethal fluences of UV ( < 12 Jm^?2) to HeLa cell cultures are followed by large increases in cell surface proteolytic activities. Amongst these, endopeptidase activity produces a similar product profile from the nonapeptide substrate to that of human leukocyte elastase, an enzyme previously shown to be capable of releasing a bioactive, mature form of TGFα from its cell-bound precursor. However, in addition to this candidate “TGFase” activity, cell surface aminopeptidase activity was also very significantly increased. The increase in the two classes of peptidase function differed in the timing of their responses. Aminopeptidase activation occurred immediately following UV, peaking after some 15–20 h, whereas the increase in endopeptidase activity lagged 6 h behind, cresting after 20–24 h. No evidence for a role for aminopeptidase in the activation of the endopeptidase could be found. Also, there was no increase in the total proteolytic activity demonstratable in cell extracts following UV. Attempts to interrupt the UV peptidase activation by inhibiting protein synthesis with cycloheximide were unsuccessful; rather, the inhibitor itself caused an increase in both classes of peptidase activity during the first 20 h. Unlike the UV response, both the aminopeptidase and endopeptidase ectoactivities increased simultaneously within a few hours of introducing cycloheximide into the medium of unirradiated cultures. The cycloheximide induced activity peaked after 20 h. Interestingly, cycloheximide alone has previously been shown to potentiate TGFα release from a cell line producing its precursor constitutively. These data suggest that both UV and cycloheximide can initiate reactions in HeLa cells which result in ectopeptidase activation of a global nature. Since both agents result in rapid interruption of DNA synthesis, it is possible that this cell surface proteolytic response may be analogous to, or part of, the “mammalian genetic stress response.” The mechanism of the activation of the cell surface proteases appears to be post-translational, perhaps part of a proteolytic cascade originating from perturbed macromolecular synthesis. © 1993 Wiley-Liss, Inc.     

Probing the interaction of distamycin A with S100β: the “unexpected” ability of S100β to bind to DNA‐binding ligands

DNA‐minor‐groove‐binding ligands are potent antineoplastic molecules. The antibiotic distamycin A is the prototype of one class of these DNA‐interfering molecules that have been largely used in vitro. The affinity of distamycin A for DNA is well known, and the structural details of the complexes with some B‐DNA and G‐quadruplex‐forming DNA sequences have been already elucidated. Here, we show that distamycin A binds S100β, a protein involved in the regulation of several cellular processes. The reported affinity of distamycin A for the calcium(II)‐loaded S100β reinforces the idea that some biological activities of the DNA‐minor‐groove‐binding ligands arise from the binding to cellular proteins. Copyright © 2015 John Wiley & Sons, Ltd.     

Naltrexone reduces weight gain, alters “β-endorphin”, and reduces insulin output from pancreatic islets of genetically obese mice

Naltrexone, an opiate antagonist, was administered to young obese (ob/ob) and lean mice for five weeks. Animals had continuous access to food and received 10 mg/kg SC twice daily with equivalent volumes of saline given to controls. The effects on body weight, and pituitary and plasma levels of β-endorphin-like material were measured. Naltrexone-injected obese animals gained weight more slowly over the first three weeks while the weight gain of lean animals was not affected by naltrexone. Plasma levels of β-endorphin were shown to be significantly higher in untreated ob/ob mice and this difference increased with age (4–20 weeks). With naltrexone treatment, plasma levels in +/? mice rose and exceeded those in ob/ob. Saline treatment appeared to be a stress, and pituitary β-endorphins rose 4–6 fold in ob/ob compared with +/?. While naltrexone reduced the levels in ob/ob pituitary towards normal, no effect on β-endorphin levels in pituitary of lean mice was obtained. In vitro studies of effects of the opiate antagonists, naloxone, on insulin secretion by isolated islets provided additional evidence of resistance of lean mice to naloxone relative to ob/ob. (IRI secretion fell only in naloxone treated ob/ob islets.) These observations support the contention that this form of genetic obesity is characterized by elevated endogenous opiate levels and an increased sensitivity to opiate antagonists such as naltrexone or naloxone.     

Understanding the disrupting mechanism of the Tau aggregation motif “306VQIVYK311” by phenylthiazolyl‐hydrazides inhibitors

Alzheimer's disease is a progressive neurodegenerative disorder characterized by the abnormal processing of the Tau and the amyloid precursor proteins. The unusual aggregation of Tau is based on the formation of intermolecular β‐sheets through two motifs: ²⁷⁵VQIINK²⁸⁰ and ³⁰⁶VQIVYK³¹¹. Phenylthiazolyl‐hydrazides (PTHs) are capable of inhibiting/disassembling Tau aggregates. However, the disaggregation mechanism of Tau oligomers by PTHs is still unknown. In this work, we studied the disruption of the oligomeric form of the Tau motif ³⁰⁶VQIVYK³¹¹ by PTHs through molecular docking, molecular dynamics, and free energy calculations. We predicted hydrophobic interactions as the major driving forces for the stabilization of Tau oligomer, with V306 and I308 being the major contributors. Nonpolar component of the binding free energy is essential to stabilize Tau‐PTH complexes. PTHs disrupted mainly the van der Waals interactions between the monomers, leading to oligomer destabilization. Destabilization of full Tau filament by PTHs and emodin was not observed in the sampled 20 ns; however, in all cases, the nonpolar component of the binding free energy is essential for the formation of Tau filament‐PTH and Tau filament‐emodin. These results provide useful clues for the design of more effective Tau‐aggregation inhibitors.     

A de novo chromosome‐level genome assembly of Coregonus sp. “Balchen”: One representative of the Swiss Alpine whitefish radiation

Salmonids are of particular interest to evolutionary biologists due to their incredible diversity of life‐history strategies and the speed at which many salmonid species have diversified. In Switzerland alone, over 30 species of Alpine whitefish from the subfamily Coregoninae have evolved since the last glacial maximum, with species exhibiting a diverse range of morphological and behavioural phenotypes. This, combined with the whole genome duplication which occurred in the ancestor of all salmonids, makes the Alpine whitefish radiation a particularly interesting system in which to study the genetic basis of adaptation and speciation and the impacts of ploidy changes and subsequent rediploidization on genome evolution. Although well‐curated genome assemblies exist for many species within Salmonidae, genomic resources for the subfamily Coregoninae are lacking. To assemble a whitefish reference genome, we carried out PacBio sequencing from one wild‐caught Coregonus sp. “Balchen” from Lake Thun to ~90× coverage. PacBio reads were assembled independently using three different assemblers, falcon , canu and wtdbg2 and subsequently scaffolded with additional Hi‐C data. All three assemblies were highly contiguous, had strong synteny to a previously published Coregonus linkage map, and when mapping additional short‐read data to each of the assemblies, coverage was fairly even across most chromosome‐scale scaffolds. Here, we present the first de novo genome assembly for the Salmonid subfamily Coregoninae. The final 2.2‐Gb wtdbg2 assembly included 40 scaffolds, an N50 of 51.9 Mb and was 93.3% complete for BUSCOs. The assembly consisted of ~52% transposable elements and contained 44,525 genes.     

Overcoming the “Light‐Soaking” Issue in Inverted Organic Solar Cells by the Use of Al:ZnO Electron Extraction Layers

A common phenomenon of organic solar cells (OSCs) incorporating metal‐oxide electron extraction layers is the requirement to expose the devices to UV light in order to improve device characteristics – known as the so‐called “light‐soaking” issue. This behaviour appears to be of general validity for various metal‐oxide layers, various organic donor/acceptor systems, and regardless if single junction devices or multi stacked cells are considered. The requirement of UV exposure of OSCs may impose severe problems if substrates with limited UV transmission, UV blocking filters or UV to VIS down‐conversion concepts are applied. In this paper, we will demonstrate that this issue can be overcome by the use of Al doped ZnO (AZO) as electron extraction interlayer. In contrast to devices based on TiO_x and ZnO, the AZO devices show well‐behaved solar cell characteristics with a high fill factor (FF) and power conversion efficiency (PCE) even without the UV spectral components of the AM1.5 solar spectrum. As opposed to previous claims, our results indicate that the origin of s‐shaped characteristics of the OSCs is the metal‐oxide/organic interface. The electronic structures of the TiO_x/fullerene and AZO/fullerene interfaces are studied by photoelectron spectroscopy, revealing an electron extraction barrier for the TiO_x/fullerene case and facilitated electron extraction for AZO/fullerene. These results are of general relevance for organic solar cells based on various donor acceptor active systems.     

Oxidation of Dimethylsulphoxide to Formaldehyde by Oxyhaemoglobin and Oxyleghaemoglobin in the Presence of Hydrogen Peroxide is Not Mediated by “Free” Hydroxyl Radicals

In the presence of excess hydrogen peroxide. human oxyhaemoglobin and oxyleghaemoglobin from soybean root nodules cause oxidation of dimethylsulphoxide to formaldehyde. This reaction is inhibited by thiourea but not by phenylalanine. HEPES. mannitol or arginine. It is concluded that dimethylsulphoxide oxidation is not mediated by “free” hydroxyl radicals. consistent with previous conclusions that intact haemoglobin, leghaemoglobin or myoglobin molecules do not react with H₂O₂ to form hydroxyl radicals detectable outside the protein.     

Phylogenetic signals in thermal traits remain stronger in the tropics if we can believe published physiological data. A reply to McKechnie et al., “Data quality problems undermine analyses of endotherm upper critical temperatures”

Due to concerns about data quality, McKechnie, Coe, Gerson, and Wolf ( 2016 ) questioned the conclusions of our study (Khaliq et al., 2015 ) published in this journal. Here, we argue that most of the questioned data points are in fact useful for macrophysiological analyses, mostly because the vast majority of data are explicitly reported in the peer‐reviewed physiological literature. Furthermore, we show that our conclusions remain largely robust irrespective of the data inclusion criterion. While we think that constructive debates about the adequate use of primary data in meta‐studies as well as more transparency in data inclusion criteria are indeed useful, we also emphasize that data suitability should be evaluated in the light of the scope and scale of the study in which they are used. We hope that this discussion will not discourage the exchange between disciplines such as biogeography and physiology, as this integration is needed to address some of the most urgent scientific challenges.     

The development of a “microtitre” fluctuation test for the detection of indirect mutagens, and its use in the evaluation of mixed enzyme induction of the liver

The “Microtitre” Fluctuation test recently introduced for the detection of direct mutagens has been adapted for the detection of indirect mutagens through the incorporation of an “S9-mix” metabolic system. It compares favourably with Greens' original method for the detection of a range of chemical mutagens.

The technique has been employed in the evaluation of mixed enzyme induction using phenobarbitone and β-naphthoflavone (benzoflavone). as a safe substitute for Aroclor-1254. The post-mitochondrial preparations from rats induced with the combined inducers had a similar “metabolic competence” to those derived from Aroclor induced animals. Such a combination would therefore provide a useful alternative to Aroclor-1254 for routine screening.

It was found that the level of “S9” present in the metabolic system greatly affected the quantitative mutagenic response. This varied considerably from chemical to chemical and underlined the need for such preliminary investigations in routine screening.