Effect of an additionally introduced degQ gene on di-d-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) production by recombinant Bacillus subtilis in a single culture production system

Di-d-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) was produced directly from sucrose using a single culture of recombinant Bacillus subtilis 168 carrying the levan fructotransferase (lft) gene. In this study, three plasmids carrying the degQ36 gene, which is a degQ allele of B. subtilis (degQ36) with a degQ36 mutation on its promoter, were constructed to overproduce intact DegQ in B. subtilis 168. The transformant B. subtilis/pHT-D36 (with the degQ36 gene) consumed sucrose and produced levan at a higher rate than B. subtilis/pHT43 (without the degQ36 gene). The transformant B. subtilis/pLFT-GD36, carrying the lft and degQ36 genes, also consumed sucrose at a higher rate and produced more DFA IV than B. subtilis/pLFT-G, carrying the lft but without the degQ36 gene. B. subtilis/pLFT-GD36 produced 43.5 g/l of DFA IV and consumed 240 g/l of sucrose (96% of added sucrose) by 72 h of cultivation, whereas B. subtilis/pLFT-G produced 23.4 g/l of DFA IV with 76.9 g/l of sucrose still remaining in the system. Sucrose-inducible expression vectors were also constructed, which made it possible to produce DFA IV without IPTG induction. Using these vectors, sucrose consumption rates were enhanced and DFA IV production was increased upon introduction of the degQ36 gene. From these results, it can be concluded that the additionally introduced regulatory gene, degQ, was able to stimulate sucrose conversion to levan, and therefore increased DFA IV production in this system.     

Positive and Negative Regulators of Sclerostin Expression

Sclerostin is secreted from osteocytes, binds to the Wnt co-receptor Lrp5/6, and affects the interaction between Wnt ligands and Lrp5/6, which inhibits Wnt/β-catenin signals and suppresses bone formation. Sclerostin plays an important role in the preservation of bone mass by functioning as a negative regulator of bone formation. A sclerostin deficiency causes sclerosteosis, which is characterized by an excess bone mass with enhanced bone formation in humans and mice. The expression of sclerostin is positively and negatively regulated by many factors, which also govern bone metabolism. Positive and negative regulators of sclerostin expression and their effects are introduced and discussed herein based on recent and previous findings, including our research.     

Simultaneous measurement of temperature and chemical species concentrations with a holographic interferometer and infrared absorption

What is believed to be a new technique that allows for the simultaneous measurement of 2D temperature and chemical species concentration profiles with high spatial resolution and fast time response was developed and tested successfully by measuring a thin layer of fuel vapor created over a volatile fuel surface. Normal propanol was placed in an open-top rectangular container, and n-propanol fuel vapor was formed over the propanol surface in a quiescent laboratory environment. An IR beam with a wavelength of 8-13 mum emitted from a heated plate and a He-Ne laser beam with a wavelength of 632 nm were combined and passed through the n-propanol vapor layer, and both beams were absorbed by the vapor layer. The absorption of the IR beam was recorded by an IR camera, and the He-Ne laser was used to form a holographic interferogram. Two-dimensional temperature and propanol vapor concentration profiles were, respectively, determined by the IR absorption and the fringe pattern associated with the holographic interferogram. This new measurement technique is a significant improvement over the dual wavelength holographic interferometry that has been used previously to measure temperature and fuel concentration, and it is ready for application under different types of fire and flame conditions.     

Microstructure and fracture of 52100 steel

The fracture behavior of 52100 steel hardened and tempered to R_C62 has been investigated as a function of austenitizing over the temperature range from 800 to 1100°C. Specimens were homogenized at 1150°C and either furnace cooled or isothermally transformed at 580°C to produce a pearlitic microstructure prior to austenitizing for hardening. Furnace-cooled specimens developed a proeutectoid carbide network that did not dissolve during subsequent austenitizing below A_cm . The residual proeutectoid carbides and the carbide-free martensite-austenite structure between them controlled fracture and produced K_IC of 19 MPa \ m^1/2, the highest determined in this investigation. The specimens isothermally transformed prior to austenitizing below A_cm produced a microstructure of fine spherical carbides dispersed throughout a fine martensitic matrix and did not contain residual proeutectoid carbides. The transgranular fracture of the latter specimens by microvoid coalescence around the closely spaced spherical carbides resulted in the lowest values of fracture toughness, 14 to 16 MPa\ m^1/2, determined in these experiments. Austenitizing above A_cm caused solution of all carbides, a gradual coarsening of the austenitic grain size, a transition to plate martensite, and an increase in retained austenite. Fracture toughness increased slightly with increasing austenitizing temperature above A_cm despite the fact that fracture propagated primarily along the austenitic grain boundaries. The improved fracture toughness, verified by scanning electron microscopy of the fatigue crack-overload fracture interface, is believed to be caused in part by transgranular crack propagation during the first stages of crack extension that are most important in determining K_1C.     

An intraocular lens power calculation for high myopia

The accuracy of intraocular lens power calculation formulas for the axial high myopia were examined, especially regarding the point of the predictive refraction. We examined 170 eyes with axial lengths of 27 mm or longer, with postoperative visual acuity of 0.5 or more, and postoperative astigmatism of less than +/- 2D. Five formulas were tested for accuracy in predicting postoperative refraction. They were the L-SRK, SRK, SRK II, SRK/T, and Binkhorst formulas. The SRK formula tended to predict less myopia than the actual postoperative refraction. The SRK II and Binkhorst formulas predicted more myopia than the actual postoperative refraction. The best results were produced by the L-SRK and SRK/T formulas. The accuracy of the L-SRK formula predictions were measured for each of the four myopic levels. The same was done for the SRK/T formula. For both formulas, there was no statistically significant difference in accuracy of prediction for the four myopic categories. The two formulas are considered to be useful for high myopic cases.     

Rho-Kinase/ROCK Phosphorylates PSD-93 Downstream of NMDARs to Orchestrate Synaptic Plasticity

The N-methyl-D-aspartate receptor (NMDAR)-mediated structural plasticity of dendritic spines plays an important role in synaptic transmission in the brain during learning and memory formation. The Rho family of small GTPase RhoA and its downstream effector Rho-kinase/ROCK are considered as one of the major regulators of synaptic plasticity and dendritic spine formation, including long-term potentiation (LTP). However, the mechanism by which Rho-kinase regulates synaptic plasticity is not yet fully understood. Here, we found that Rho-kinase directly phosphorylated discs large MAGUK scaffold protein 2 (DLG2/PSD-93), a major postsynaptic scaffold protein that connects postsynaptic proteins with NMDARs; an ionotropic glutamate receptor, which plays a critical role in synaptic plasticity. Stimulation of striatal slices with an NMDAR agonist induced Rho-kinase-mediated phosphorylation of PSD-93 at Thr612. We also identified PSD-93-interacting proteins, including DLG4 (PSD-95), NMDARs, synaptic Ras GTPase-activating protein 1 (SynGAP1), ADAM metallopeptidase domain 22 (ADAM22), and leucine-rich glioma-inactivated 1 (LGI1), by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among them, Rho-kinase increased the binding of PSD-93 to PSD-95 and NMDARs. Furthermore, we found that chemical-LTP induced by glycine, which activates NMDARs, increased PSD-93 phosphorylation at Thr612, spine size, and PSD-93 colocalization with PSD-95, while these events were blocked by pretreatment with a Rho-kinase inhibitor. These results indicate that Rho-kinase phosphorylates PSD-93 downstream of NMDARs, and suggest that Rho-kinase mediated phosphorylation of PSD-93 increases the association with PSD-95 and NMDARs to regulate structural synaptic plasticity.     

A cell-transverse display algorithm for regular and rectilinear 3D grid data

This paper proposes an efficient direct imaging algorithm for constructing iso-surfaces from regular and rectilinear 3D grid data in scientific and engineering visualization. The basic idea is to generate and draw polygons simultaneously by processing the cells spanned by grids in decreasing order of distance from the current viewpoint. Iso-surfaces are generated in five or six tetrahedrons into which the cells are subdivided, and are sent to a graphics device or drawn into a frame buffer on the fly. The execution order of each of the tetrahedrons is identical and is determined by the current viewpoint. Since the algorithm does not need to store intermediate polyhedral data and does not require a depth buffer memory for hidden surface removal, it is applicable to a large quantity of data on a 3D grid, such as computed tomography (CT) data. It is also particularly powerful for semi-transparent display, because transparency calculation can be reduced to image compositing operations if the polygons are drawn in order of their z-depth from the current viewpoint.     

Front Cover: Development and Characterization of Selective FAK Inhibitors and PROTACs with In Vivo Activity (ChemBioChem 19/2023)

Focal adhesion kinase (FAK) is an attractive drug target due to its overexpression in cancer. FAK functions as a non-receptor tyrosine kinase and scaffolding protein, coordinating several downstream signaling effectors and cellular processes. While drug discovery efforts have largely focused on targeting FAK kinase activity, FAK inhibitors have failed to show efficacy as single agents in clinical trials. Here, using structure-guided design, we report the development of a selective FAK inhibitor (BSJ-04-175) and degrader (BSJ-04-146) to evaluate the consequences and advantages of abolishing all FAK activity in cancer models. BSJ-04-146 achieves rapid and potent FAK degradation with high proteome-wide specificity in cancer cells and induces durable degradation in mice. Compared to kinase inhibition, targeted degradation of FAK exhibits pronounced improved activity on downstream signaling and cancer cell viability and migration. Together, BSJ-04-175 and BSJ-04-146 are valuable chemical tools to dissect the specific consequences of targeting FAK through small-molecule inhibition or degradation.     

New Sweet Potato Line having low Gelatinization Temperature and altered Starch Structure

A new sweet potato breeding line, Kanto 116, was developed, featuring low gelatinization temperature and an altered starch fine structure. Starch granules from Kanto 116 showed an abnormal morphology characterized by cracking into granules. Starch content, amylose content and tuberous root appearance of Kanto 116 were similar to those of the control and the parents. Pasting temperatures of Kanto 116 starch determined by the Rapid Visco Analyser were 51.4 — 52.6 °C, approximately 20 °C lower than those of the control and parents starches. Onset, peak, and conclusion temperature of gelatinization, and gelatinization enthalpy of Kanto 116 starch determined by differential scanning calorimetry were 39.0 °C, 46.9 °C, 64.8 °C, and 8.8 J/g, respectively, and much lower than those of the control and parents starches. The chain‐length distribution of the amylopectin molecules, determined by high‐performance anion‐exchange chromatography, showed that Kanto 116 starch had a higher proportion of short chains (DP 6 — 11) and a lower proportion of chains between DP 12 — 28 than control and parent starches. The debranched β‐limit dextrin of Kanto 116 starch also showed that the proportion of both short and long B1 chains was different from those of the control and parents starches.