Structural basis for the minimal bifunctional alginate epimerase AlgE3 from Azotobacter chroococcum.

Among the epimerases specific to alginate, some of them in Azotobacter genera convert ß-d-mannuronic acid to α-l-guluronic acid but also have lyase activity to degrade alginate. The remarkable characteristics of these epimerases make it a promising enzyme for tailoring alginates to meet specific demands. Here, we determined the structure of the bifunctional mannuronan C-5 epimerase AlgE3 from Azotobacter chroococcum (AcAlgE3) in complex with several mannuronic acid oligomers as well as in apo form, which allowed us to elucidate the binding manner of each mannuronic acid oligomer, and the structural plasticity, which is dependent on calcium ions. Moreover, a comprehensive analysis of the lyase activity profiles of AcAlgE3 combined with structural characteristics explained the preference for different chain length oligomers.

Lonicerin, an anti-algE flavonoid against Pseudomonas aeruginosa virulence screened from Shuanghuanglian formula by molecule docking based strategy.

ETHNOPHARMACOLOGICAL RELEVANCE: The Shuanghuanglian formula (SF) is a famous antimicrobial and antiviral traditional Chinese medicine that is made of Lonicera japonica Thunb., Scutellaria baicalensis Georgi, and Forsythia suspensa (Thunb.) Vahl. According to the Chinese Pharmacopoeia, the SF is commonly administered in the forms of oral liquid, tablets, and injection. It has long been used to treat acute respiratory tract infections, especially lung infection. AIM OF THE STUDY: In the light of the increasing incidence of multidrug resistance to conventional antibiotics, the aim of this study was to screen potential anti-virulence agents against Pseudomonas aeruginosa from the extract of the SF. MATERIALS AND METHODS: The SF was used for effective compounds screening via the combination of the molecule docking approach and ultra-high-performance liquid chromatography-quadrupole/time of flight mass spectrometry. Fifty-one anti-virulence-related proteins were docked, 26 identified compounds were from SF. Subsequently, the top-scoring screened compound was assessed via bioactive-related assays, including the quantification of alginate biosynthesis, anti-biofilm assays, and the A549 human lung cells infection. RESULT: A flavonoid Lonicerin was found to be bonded with the active site of the alginate secretion protein (AlgE) with the highest score in molecule docking. Furthermore, we validated that Lonicerin could significantly reduce alginate secretion (25 µg/mL) and biofilm formation (12.5 µg/mL) at a sub-MIC concentration without inhibiting the proliferation of P. aeruginosa or influencing the expression of AlgE, which suggested that Lonicerin may directly inhibit AlgE. In addition, Lonicerin was proven to inhibit the infection of P. aeruginosa in the A549 cells. CONCLUSION: This work reported on the first potential AlgE antagonist that was derived from herbal resources. Lonicerin was proven to be an effective inhibitor in-vitro of P. aeruginosa infection.

Wafer-Level Vacuum Packaging of Smart Sensors.

The reach and impact of the Internet of Things will depend on the availability of low-cost, smart sensors-"low cost" for ubiquitous presence, and "smart" for connectivity and autonomy. By using wafer-level processes not only for the smart sensor fabrication and integration, but also for packaging, we can further greatly reduce the cost of sensor components and systems as well as further decrease their size and weight. This paper reviews the state-of-the-art in the wafer-level vacuum packaging technology of smart sensors. We describe the processes needed to create the wafer-scale vacuum microchambers, focusing on approaches that involve metal seals and that are compatible with the thermal budget of complementary metal-oxide semiconductor (CMOS) integrated circuits. We review choices of seal materials and structures that are available to a device designer, and present techniques used for the fabrication of metal seals on device and window wafers. We also analyze the deposition and activation of thin film getters needed to maintain vacuum in the ultra-small chambers, and the wafer-to-wafer bonding processes that form the hermetic seal. We discuss inherent trade-offs and challenges of each seal material set and the corresponding bonding processes. Finally, we identify areas for further research that could help broaden implementations of the wafer-level vacuum packaging technology.

In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures.

Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ diffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ serial crystallography (IMISX) method that enables crystallographic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and ß-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins.

In meso in situ serial X-ray crystallography of soluble and membrane proteins.

The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method. Among the listings are some of the highest profile membrane proteins, including the ß2-adrenoreceptor-Gs protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing in meso crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development.