Conformational Analysis of the Mannosidase Inhibitor Kifunensine: A Quantum Mechanical and Structural Approach

The varied yet family‐specific conformational pathways used by individual glycoside hydrolases (GHs) offer a tantalising prospect for the design of tightly binding and specific enzyme inhibitors. A cardinal example of a GH‐family‐specific inhibitor, and one that finds widespread practical use, is the natural product kifunensine, which is a low‐nanomolar inhibitor that is selective for GH family 47 inverting α‐mannosidases. Here we show, through quantum‐mechanical approaches, that kifunensine is restrained to a “ring‐flipped” ¹C₄ conformation with another accessible, but higher‐energy, region around the ^1,4B conformation. The conformations of kifunensine in complex with a range of GH47 enzymes—including an atomic‐level resolution (1 Å) structure of kifunensine with Caulobacter sp. CkGH47 reported herein and with GH family 38 and 92 α‐mannosidases—were mapped onto the kifunensine free‐energy landscape. These studies revealed that kifunensine has the ability to mimic the product state of GH47 enzymes but cannot mimic any conformational states relevant to the reaction coordinate of mannosidases from other families.     

Membrane technology for advanced wastewater reclamation for sustainable agriculture production

Treated domestic secondary effluent is a valuable water source that can be reused for diverse purposes. However, in order to minimize health and environmental risks and to maintain adequate levels of sustainable agriculture production on a long range time scale, advanced treatment is required. Advanced effluent quality maintaining minimal risks can be primarily attained by implementing the membrane technology. Field experiments are in progress for secondary wastewater polishing for unrestricted reuse for sustainable agricultural production. The two stage membrane treatment system for the secondary effluent polishing consists of combining two main stages: ultrafiltration (UF) and reverse osmosis (RO) membrane treatment. The UF stage is efficient in the removal of the organic matter and the pathogens while the RO provides the dissolved solids (salinity) removal. Effluent of various qualities is applied for irrigation along with continuous monitoring of the membrane components performance. The experimental data was obtained in the ongoing pilot studies carried out near the City of Arad (Israel) wastewater treatment system (the pilot plant performs in a feed and bleed operation mode). The results indicate the importance of maintaining high quality effluent for sustainable agriculture production. The management modeling gives an idea of the importance in maintaining adequate UF flushing policy in order to minimize expenses due to fouling.     

Structural Basis for Phospholyase Activity of a Class III Transaminase Homologue

Pyridoxal‐phosphate (PLP)‐dependent enzymes catalyse a remarkable diversity of chemical reactions in nature. A1RDF1 from Arthrobacter aurescens TC1 is a fold type I, PLP‐dependent enzyme in the class III transaminase (TA) subgroup. Despite sharing 28 % sequence identity with its closest structural homologues, including β‐alanine:pyruvate and γ‐aminobutyrate:α‐ketoglutarate TAs, A1RDF1 displayed no TA activity. Activity screening revealed that the enzyme possesses phospholyase (E.C. 4.2.3.2) activity towards O‐phosphoethanolamine (PEtN), an activity described previously for vertebrate enzymes such as human AGXT2L1, enzymes for which no structure has yet been reported. In order to shed light on the distinctive features of PLP‐dependent phospholyases, structures of A1RDF1 in complex with PLP (internal aldimine) and PLP ? PEtN (external aldimine) were determined, revealing the basis of substrate binding and the structural factors that distinguish the enzyme from class III homologues that display TA activity.     

气泡城市

Spacebuster是个动态的充气结构，它是便携式、可拓展的场馆。设计旨在将各种公共空间转变为可以进行社交集会的场所。事实上，Spacebuster是Raumlabo健筑设计事务所对上一个项目Kuechenmonumenl的迭代创新，它使得曼哈顿和布鲁克林的春天大放异彩，承办了很多各种社交和公众活动。     

Comparing the Catalytic and Structural Characteristics of a ‘Short’ Unspecific Peroxygenase (UPO) Expressed in Pichia pastoris and Escherichia coli

Unspecific peroxygenases (UPOs) have emerged as valuable tools for the oxygenation of non-activated carbon atoms, as they exhibit high turnovers, good stability and depend only on hydrogen peroxide as the external oxidant for activity. However, the isolation of UPOs from their natural fungal sources remains a barrier to wider application. We have cloned the gene encoding an ‘artificial’ peroxygenase (artUPO), close in sequence to the ‘short’ UPO from Marasmius rotula (MroUPO), and expressed it in both the yeast Pichia pastoris and E. coli to compare the catalytic and structural characteristics of the enzymes produced in each system. Catalytic efficiency for the UPO substrate 5-nitro-1,3-benzodioxole (NBD) was largely the same for both enzymes, and the structures also revealed few differences apart from the expected glycosylation of the yeast enzyme. However, the glycosylated enzyme displayed greater stability, as determined by nano differential scanning fluorimetry (nano-DSF) measurements. Interestingly, while artUPO hydroxylated ethylbenzene derivatives to give the (R)-alcohols, also given by a variant of the ‘long’ UPO from Agrocybe aegerita (AaeUPO), it gave the opposite (S)-series of sulfoxide products from a range of sulfide substrates, broadening the scope for application of the enzymes. The structures of artUPO reveal substantial differences to that of AaeUPO, and provide a platform for investigating the distinctive activity of this and related'short’ UPOs.     

Detection of Alternaria fungal contamination in cereal grains by a polymerase chain reaction-based assay

Alternaria sp. are important fungal contaminants of grain products; they secrete four structural classes of compounds that are toxic or carcinogenic to plants and animals and cause considerable economic losses to growers and the food-processing industry. Alternaria toxins have been detected by high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay, and other techniques. Here, we report the development of a polymerase chain reaction (PCR)-based method for the detection of Alternaria DNA. PCR primers were designed to anneal to the ITS1 and ITS2 regions of the 5.8S rDNA gene of Alternaria alternata or Alternaria solani but not to other microbial or plant DNA. We compared the sensitivity of PCR in detecting Alternaria DNA, that of the HPLC method in detecting Alternaria alternariol and alternariol methyl ether toxins, and that of the morphological examination of mycelia and conidia in experimentally infested corn samples. The sensitivity of toxin detection for HPLC was above the level of contamination in a set of commercially obtained grain samples, resulting in negative scores for all samples, while the PCR-based method and mold growth plating followed by morphological identification of Alternaria gave parallel, positive results for 8 of 10 samples. The PCR assay required just 8 h, enabling the rapid and simultaneous testing of many samples at a low cost. PCR-based evidence for the presence of Alternaria DNA followed by positive assay results for Alternaria toxins would support the rejection of a shipment of grain.     

22-nm Half-pitch extreme ultraviolet node development at the SEMATECH Berkeley microfield exposure tool

Microfield exposure tools continue to play a dominant role in the development of extreme ultraviolet (EUV) resists. Here we present an update on the SEMATECH Berkeley 0.3-NA microfield exposure tool and summarize the latest test results from high-resolution line-space printing. Printing down to 20-nm is presented with large process latitude at 22-nm half-pitch lines. Also presented are line-edge roughness results along with a discussion of the importance of mask contributors to line-edge roughness measured in resist. Finally we briefly describe an upgrade to the tool that will enable EUV resist development at the 16-nm half-pitch node and beyond. (This paper was presented in MNE 2008 conference, <http://www.mne08.org>, <http://www.mne-conf.org>).     

Sparsity-based single-shot subwavelength coherent diffractive imaging

Coherent Diffractive Imaging (CDI) is an algorithmic imaging technique where intricate features are reconstructed from measurements of the freely diffracting intensity pattern. An important goal of such lensless imaging methods is to study the structure of molecules that cannot be crystallized. Ideally, one would want to perform CDI at the highest achievable spatial resolution and in a single-shot measurement such that it could be applied to imaging of ultrafast events. However, the resolution of current CDI techniques is limited by the diffraction limit, hence they cannot resolve features smaller than one half the wavelength of the illuminating light. Here, we present sparsity-based single-shot subwavelength resolution CDI: algorithmic reconstruction of subwavelength features from far-field intensity patterns, at a resolution several times better than the diffraction limit. This work paves the way for subwavelength CDI at ultrafast rates, and it can considerably improve the CDI resolution with X-ray free-electron lasers and high harmonics.