Crystal Structure of a Bioactive Pactamycin Analog Bound to the 30S Ribosomal Subunit

Biosynthetically and chemically derived analogs of the antibiotic pactamycin and de-6-methylsalicylyl (MSA)-pactamycin have attracted recent interest as potential antiprotozoal and antitumor drugs. Here, we report a 3.1-Å crystal structure of de-6-MSA-pactamycin bound to its target site on the Thermus thermophilus 30S ribosomal subunit. Although de-6-MSA-pactamycin lacks the MSA moiety, it shares the same binding site as pactamycin and induces a displacement of nucleic acid template bound at the E-site of the 30S. The structure highlights unique interactions between this pactamycin analog and the ribosome, which paves the way for therapeutic development of related compounds.     

Nod1-mediated lipolysis promotes diacylglycerol accumulation and successive inflammation via PKCδ-IRAK axis in adipocytes

Chronic inflammation contributes to obesity mediated metabolic disturbances, including insulin resistance. Obesity is associated with altered microbial load in metabolic tissues that can contribute to metabolic inflammation. Different bacterial components such as, LPS, peptidoglycans have been shown to underpin metabolic disturbances through interaction with host innate immune receptors. Activation of Nucleotide-binding oligomerization domain-containing protein 1 (Nod1) with specific peptidoglycan moieties promotes insulin resistance, inflammation and lipolysis in adipocytes. However, it was not clear how Nod1-mediated lipolysis and inflammation is linked. Here, we tested if Nod1-mediated lipolysis caused accumulation of lipid intermediates and promoted cell autonomous inflammation in adipocytes. We showed that Nod1-mediated lipolysis caused accumulation of diacylglycerol (DAG) and activation of PKCδ in 3T3-L1 adipocytes, which was prevented with a Nod1 inhibitor. Nod1-activated PKCδ caused downstream stimulation of IRAK1/4 and was associated with increased expression of proinflammatory cytokines such as, IL-1β, IL-18, IL-6, TNFα and MCP-1. Pharmacological inhibition or siRNA mediated knockdown of IRAK1/4 attenuated Nod1-mediated activation of NF-κB, JNK, and the expression of proinflammatory cytokines. These results reveal that Nod1-mediated lipolysis promoted accumulation of DAG, which engaged PKCδ and IRAK1/4 to augment inflammation in 3T3-L1 adipocytes.     

Preparation and characterization of flurbiprofen beads by melt solidification technique

Amelt solidification technique has been developed to obtain sustained-release waxy beads of flurbiprofen. Low glass transition temperature (t _g) and shear-induced crystallization of flurbiprofen made it a suitable candidate for melt solidification technique. The process involved emulsification and solidification of flurbiprofen-cetyl alcohol melt at significantly low temperature (5°C). The effect of variables, namely, the amount of cetyl alcohol and the speed of agitation, was studied using 3² factorial design. The technique and the beads were evaluated on the basis of process and desired yield, surface topography, Fourier-transform infrared (FT-IR), differential scanning calorimetry (DSC), particle size distribution, crushing strength, and drug release. Average values for process and desired yields were 97% wt/wt and 26% wt/wt, respectively. No interaction was observed between drug and excipient. Multiple regression analysis was carried out, and response surfaces were obtained. A curvilinear relationship was observed between percentage of desired yield and the amount of cetyl alcohol. Linear decrease in crushing strength was observed with increase in the amount of cetyl alcohol. Drug released from the beads followed zero order kinetics. Burst release was shown to a greater extent in beads containing a lower amount of cetyl alcohol. Response surfaces of time required for certain percentage of drug (t _D%) showed that after critical concentration of about 20% of cetyl alcohol (400 mg/batch), no significant release retardant effect was observed.     

Resilience to Freezing in the Vegetative Cells of the Microalga Lobosphaera incisa (Trebouxiophyceae,Chlorophyta)

The chlorophyte microalga Lobosphaera incisa was isolated from the snowy slopes of Mt. Tateyama in Japan. This microalga stores exceptionally high amounts of the omega-6 LC-PUFA arachidonic acid in triacylglycerols, and therefore represents a potent photosynthetic source for this essential LC-PUFA. Assuming that freezing tolerance may play a role in adaptation of L. incisa to specific ecological niches, we examined the capability of L. incisa to tolerate extreme sub-zero temperatures. We report here, that the vegetative cells of L. incisa survived freezing at −20°C and −80°C (over 1 month), without cryoprotective agents or prior treatments. Cells successfully recovered upon thawing and proliferated under optimal growth conditions (25°C). However, cells frozen at −80°C showed better recovery and lower cellular ROS generation upon thawing, compared to those preserved at −20°C. Photosynthetic yield of PSII, estimated by F_v/F_m, temporarily decreased at day 1 post freezing and resumed to the original level at day 3. Interestingly, the thawed algal cultures produced a higher level of chlorophylls, exceeding the control culture. The polar metabolome of the vegetative cells comprised a range of compatible solutes, dominated by glutamate, sucrose, and proline. We posit that the presence of endogenous cryoprotectants, a rigid multilayer cell wall, the high LC-PUFA content in membrane lipids, and putative cold-responsive proteins may contribute to the retention of functionality upon recovery from the frozen state, and therefore for the survival under cryospheric conditions. From the applied perspective, this beneficial property holds promise for the cryopreservation of starter cultures for research and commercial purposes.     

Comparative height crown allometry and mechanical design in 22 tree species of Kuala Belalong rainforest, Brunei, Borneo

In rainforests, trunk size, strength, crown position, and geometry of a tree affect light interception and the likelihood of mechanical failure. Allometric relationships of tree diameter, wood density, and crown architecture vs. height are described for a diverse range of rainforest trees in Brunei, northern Borneo. The understory species follow a geometric model in their diameter-height relationship (slope, β = 1.08), while the stress-elasticity models prevail (β = 1.27-1.61) for the midcanopy and canopy/emergent species. These relationships changed with ontogeny, especially for the understory species. Within species, the tree stability safety factor (SSF) and relative crown width decreased exponentially with increasing tree height. These trends failed to emerge in across-species comparisons and were reversed at a common (low) height. Across species, the relative crown depth decreased with maximum potential height and was indistinguishable at a common (low) height. Crown architectural traits influence SSF more than structural property of wood density. These findings emphasize the importance of applying a common reference size in comparative studies and suggest that forest trees (especially the understory group) may adapt to low light by having deeper rather than wider crowns due to an efficient distribution and geometry of their foliage.     

Microresonator mass sensors for detection of Bacillus anthracis Sterne spores in air and water

Towards the goal of developing a real-time monitoring device for microorganisms, we demonstrate the use of microcantilevers as resonant mass sensors for detection of Bacillus anthracis Sterne spores in air and liquid. The detection scheme was based on measuring resonant frequency decrease driven by thermally induced oscillations, as a result of the added mass of the spores with the use of a laser Doppler vibrometer (LDV). Viscous effects were investigated by comparing measurements in air and deionized (DI) water along with theoretical values. Moreover, biological experiments were performed which involved suspending spores onto the cantilevers and performing mass detection in air and water. For detection of spores in water, the cantilevers were functionalized with antibodies in order to fix the spores onto the surface. We demonstrate that as few as 50 spores on the cantilever can be detected in water using the thermal noise as excitation source. Measurement sensitivity of 9.23 Hz/fg for air and 0.1 Hz/fg for water were obtained. These measurements were compared with theoretical values and sources of improvement in cantilever sensitivity in a viscous medium were also discussed. It is expected that by driving the cantilevers and using higher order modes, detection of a single spore in liquids should be achievable.     

Nuclear but not cytosolic phosphoinositide 3-kinase beta has an essential function in cell survival

Class I(A) phosphoinositide 3-kinases (PI3Ks) are heterodimeric enzymes composed of a p85 regulatory and a p110 catalytic subunit that induce the formation of 3-polyphosphoinositides, which mediate cell survival, division, and migration. There are two ubiquitous PI3K isoforms p110α and p110β that have nonredundant functions in embryonic development and cell division. However, whereas p110α concentrates in the cytoplasm, p110β localizes to the nucleus and modulates nuclear processes such as DNA replication and repair. At present, the structural features that determine p110β nuclear localization remain unknown. We describe here that association with the p85β regulatory subunit controls p110β nuclear localization. We identified a nuclear localization signal (NLS) in p110β C2 domain that mediates its nuclear entry, as well as a nuclear export sequence (NES) in p85β. Deletion of p110β induced apoptosis, and complementation with the cytoplasmic C2-NLS p110β mutant was unable to restore cell survival. These studies show that p110β NLS and p85β NES regulate p85β/p110β nuclear localization, supporting the idea that nuclear, but not cytoplasmic, p110β controls cell survival.     

Evaluating the roles of the heme a side chains in cytochrome c oxidase using designed heme proteins

Heme a is a redox cofactor unique to cytochrome c oxidases and vital to aerobic respiration. Heme a differs from the more common heme b by two chemical modifications, the C-8 formyl group and the C-2 hydroxyethylfarnesyl group. The effects of these porphyrin substituents on ferric and ferrous heme binding and electrochemistry were evaluated in a designed heme protein maquette. The maquette scaffold chosen, [Delta7-H3m](2), is a four-alpha-helix bundle that contains two bis(3-methyl-l-histidine) heme binding sites with known absolute ferric and ferrous heme b affinities. Hemes b, o, o+16, and heme a, those involved in the biosynthesis of heme a, were incorporated into the bis(3-methyl-l-histidine) heme binding sites in [Delta7-H3m](2). Spectroscopic analyses indicate that 2 equiv of each heme binds to [Delta7-H3m](2), as designed. Equilibrium binding studies of the hemes with the maquette demonstrate the tight affinity for hemes containing the C-2 hydroxyethylfarnesyl group in both the ferric and ferrous forms. Coupled with the measured equilibrium midpoint potentials, the data indicate that the hydroxyethylfarnesyl group stabilizes the binding of both ferrous and ferric heme by at least 6.3 kcal/mol via hydrophobic interactions. The data also demonstrate that the incorporation of the C-8 formyl substituent in heme a results in a 179 mV, or 4.1 kcal/mol, positive shift in the heme reduction potential relative to heme o due to the destabilization of ferric heme binding relative to ferrous heme binding. The two substituents appear to counterbalance each other to provide for tighter heme a affinity relative to heme b in both the ferrous and ferric forms by at least 6.3 and 2.1 kcal/mol, respectively. These results also provide a rationale for the reaction sequence observed in the biosynthesis of heme a.