Combined dissolved oxygen tension and online viscosity measurements in shake flask cultivations via infrared fluorescent oxygen-sensitive nanoparticles

Oxygen supply is one of the most critical process parameters in aerobic cultivations. To assure sufficient oxygen supply, shake flasks are usually used in combination with orbital shaking machines. In this study, a measurement technique for the dissolved oxygen tension (DOT) in shake flask cultures with viscosity changes is presented. The movement of the shaker table is monitored by means of a Hall effect sensor. For DOT measurements, infrared fluorescent oxygen-sensitive nanoparticles are added to the culture broth. The position of the rotating bulk liquid needs to be determined to assure measurements inside the liquid. The leading edge of the bulk liquid is detected based on the fluorescence signal intensity of the oxygen-sensitive nanoparticles. Furthermore, online information about the viscosity of the culture broth is acquired due to the detection of the position of the leading edge of the bulk liquid relative to the direction of the centrifugal force, as described by Sieben et al. (2019. Sci. Rep., 9, 8335). The DOT measurement is combined with a respiration activity monitoring system which allows for the determination of the oxygen transfer rate (OTR) in eight parallel shake flasks. Based on DOT and OTR, the volumetric oxygen transfer coefficient (k_La) is calculated during cultivation. The new system was successfully applied in cultivations of Escherichia coli, Bacillus licheniformis, and Xanthomonas campestris.     

6-Shogaol,an active constituent of ginger,attenuates neuroinflammation and cognitive deficits in animal models of dementia

Recently, increased attention has been directed towards medicinal extracts as potential new drug candidates for dementia. Ginger has long been used as an important ingredient in cooking and traditional herbal medicine. In particular, ginger has been known to have disease-modifying effects in Alzheimer's disease (AD). However, there is no evidence of which constituents of ginger exhibit therapeutic effects against AD. A growing number of experimental studies suggest that 6-shogaol, a bioactive component of ginger, may play an important role as a memory-enhancing and anti-oxidant agent against neurological diseases. 6-Shogaol has also recently been shown to have anti-neuroinflammatory effects in lipopolysaccharide (LPS)-treated astrocytes and animal models of Parkinson’s disease, LPS-induced inflammation and transient global ischemia. However, it is still unknown whether 6-shogaol has anti-inflammatory effects against oligomeric forms of the Aβ (AβO) in animal brains. Furthermore, the effects of 6-shogaol against memory impairment in dementia models are also yet to be investigated. In this study, we found that administration of 6-shogaol significantly reduced microgliosis and astrogliosis in intrahippocampal AβO-injected mice, ameliorated AβO and scopolamine-induced memory impairment, and elevated NGF levels and pre- and post-synaptic marker in the hippocampus. All these results suggest that 6-shogaol may play a role in inhibiting glial cell activation and reducing memory impairment in animal models of dementia.     

Fibrin specific peptides derived by phage display: characterization of peptides and conjugates for imaging

Peptides that bind to fibrin but not to fibrinogen or serum albumin were selected from phage display libraries as targeting moieties for thrombus molecular imaging probes. Three classes of cyclic peptides (cyclized via disulfide bond between two Cys) were identified with consensus sequences XArXCPY(G/D)LCArIX (Ar = aromatic, Tn6), X(2)CXYYGTCLX (Tn7), and NHGCYNSYGVPYCDYS (Tn10). These peptides bound to fibrin at ～2 sites with K(d) = 4.1 μM, 4.0 μM, and 8.7 μM, respectively, whereas binding to fibrinogen was at least 100-fold weaker. The peptides also bind to the fibrin degradation product DD(E) with similar affinity to that measured for fibrin. The Tn7 and Tn10 peptides bind to the same site on fibrin, while the Tn6 peptides bind to a unique site. Alanine scanning identified the N- and C-terminal ends of the Tn6 and Tn7 peptides as most tolerant to modification. Peptide conjugates with either fluorescein or diethylenetriaminepentaaceto gadolinium(III) (GdDTPA) at the N-terminus were prepared for potential imaging applications, and these retained fibrin binding affinity and specificity in plasma. Relaxivity and binding studies on the GdDTPA derivatives revealed that an N-terminal glycyl linker had a modest effect on fibrin affinity but resulted in lower fibrin-bound relaxivity.     

Oviposition preference and larval performance of North American monarch butterflies on four Asclepias species

Monarch butterflies, Danaus plexippus L. (Lepidoptera: Nymphalidae), occur world‐wide and are specialist herbivores of plants in the milkweed family (Asclepiadaceae). In North America, two monarch populations breed east and west of the continental divide in areas populated by different host plant species. To examine the population variation in monarch responses to different Asclepias species, we measured oviposition preference and larval performance among captive progeny reared from adult butterflies collected in eastern and western North America. Host plant use was evaluated using two milkweed species widely distributed in eastern North America (A. incarnata and A. syriaca), and two species common to western North America (A. fascicularis and A. speciosa). We predicted that exposure to different host plant species in their respective breeding ranges could select for divergent host use traits, so that monarchs should preferentially lay more eggs on, and larvae should perform better on, milkweed species common to their native habitats. Results showed that across all adult female butterflies, oviposition preferences were highest for A. incarnata and lowest for A. fascicularis, but mean preferences did not differ significantly between eastern and western monarch populations. Larvae from both populations experienced the highest survival and growth rates on A. incarnata and A. fascicularis, and we again found no significant interactions between monarch source population and milkweed species. Moreover, the average rank order of larval performance did not correspond directly to mean female oviposition preferences, suggesting that additional factors beyond larval performance influence monarch oviposition behavior. Finally, significant family level variation was observed for both preference and performance responses within populations, suggesting an underlying genetic variation or maternal effects governing these traits.     

IKK/NF-kappaB regulates skeletal myogenesis via a signaling switch to inhibit differentiation and promote mitochondrial biogenesis

Nuclear factor kappaB (NF-kappaB) is involved in multiple skeletal muscle disorders, but how it functions in differentiation remains elusive given that both anti- and promyogenic activities have been described. In this study, we resolve this by showing that myogenesis is controlled by opposing NF-kappaB signaling pathways. We find that myogenesis is enhanced in MyoD-expressing fibroblasts deficient in classical pathway components RelA/p65, inhibitor of kappaB kinase beta (IKKbeta), or IKKgamma. Similar increases occur in myoblasts lacking RelA/p65 or IKKbeta, and muscles from RelA/p65 or IKKbeta mutant mice also contain higher fiber numbers. Moreover, we show that during differentiation, classical NF-kappaB signaling decreases, whereas the induction of alternative members IKKalpha, RelB, and p52 occurs late in myogenesis. Myotube formation does not require alternative signaling, but it is important for myotube maintenance in response to metabolic stress. Furthermore, overexpression or knockdown of IKKalpha regulates mitochondrial content and function, suggesting that alternative signaling stimulates mitochondrial biogenesis. Together, these data reveal a unique IKK/NF-kappaB signaling switch that functions to both inhibit differentiation and promote myotube homeostasis.     

Engineering antibody heavy chain CDR3 to create a phage display Fab library rich in antibodies that bind charged carbohydrates

A number of small charged carbohydrate moieties have been associated with inflammation and cancer. However, the development of therapeutic Abs targeting these moieties has been hampered by their low immunogenicity and their structural relationship to self-Ag. We report the design of an Ab repertoire enriched in Abs binding to small charged carbohydrates and the construction of a human Fab phagemid library, "FAB-CCHO." This library combines L chain Ig sequences from human donors and H chain synthetic diversity constructed in key Ag contact sites in CDRs 1, 2, and 3 of the human framework V(H)3-23. The H chain CDR3 has been engineered to enrich the library in Abs that bind charged carbohydrates by the introduction of basic residues at specific amino acid locations. These residues were selected on the basis of anti-carbohydrate Ab sequence alignment. The success of this design is demonstrated by the isolation of phage Abs against charged carbohydrate therapeutic target Ags such as sulfated sialyl-Lewis X glycan and heparan sulfate.     

A comparative biochemical and structural analysis of the intracellular chorismate mutase (Rv0948c) from Mycobacterium tuberculosis H(37)R(v) and the secreted chorismate mutase (y2828) from Yersinia pestis

The Rv0948c gene from Mycobacterium tuberculosis H(37)R(v) encodes a 90 amino acid protein as the natural gene product with chorismate mutase (CM) activity. The protein, 90-MtCM, exhibits Michaelis-Menten kinetics with a k(cat) of 5.5+/-0.2s(-1) and a K(m) of 1500+/-100microm at 37 degrees C and pH7.5. The 2.0A X-ray structure shows that 90-MtCM is an all alpha-helical homodimer (Protein Data Bank ID: 2QBV) with the topology of Escherichia coli CM (EcCM), and that both protomers contribute to each catalytic site. Superimposition onto the structure of EcCM and the sequence alignment shows that the C-terminus helix3 is shortened. The absence of two residues in the active site of 90-MtCM corresponding to Ser84 and Gln88 of EcCM appears to be one reason for the low k(cat). Hence, 90-MtCM belongs to a subfamily of alpha-helical AroQ CMs termed AroQ(delta.) The CM gene (y2828) from Yersinia pestis encodes a 186 amino acid protein with an N-terminal signal peptide that directs the protein to the periplasm. The mature protein, *YpCM, exhibits Michaelis-Menten kinetics with a k(cat) of 70+/-5s(-1) and K(m) of 500+/-50microm at 37 degrees C and pH7.5. The 2.1A X-ray structure shows that *YpCM is an all alpha-helical protein, and functions as a homodimer, and that each protomer has an independent catalytic unit (Protein Data Bank ID: 2GBB). *YpCM belongs to the AroQ(gamma) class of CMs, and is similar to the secreted CM (Rv1885c, *MtCM) from M.tuberculosis.     

Identification of a twin-arginine leader-binding protein

The transport and targeting of a number of periplasmic proteins is carried out by the Sec-independent Mtt (also referred to as Tat) protein translocase. Proteins using this translocase have a distinct twin-arginine-containing leader. We hypothesized that specific leader-binding proteins exist to escort proteins to the translocase complex. A fusion was constructed with the twin-arginine leader from dimethyl sulphoxide (DMSO) reductase, subunit DmsA, to the N-terminus of glutathione-S-transferase. This leader fusion was bound to a glutathione affinity column through which an Escherichia coli anaerobic cell-free extract was passed. Proteins that bound to the leader were then separated and identified by N-terminal sequencing, which identified DnaK and a protein originating from the uncharacterized reading frame ynfI. This gene has been designated dmsD based on the findings presented in this paper. DmsD was purified as a His6 fusion and was shown to interact with preprotein forms of DmsA and TorA (trimethyl amine N-oxide reductase). A strain carrying a dmsD knock-out mutation showed a loss of anaerobic growth on glycerol-DMSO medium and reduced growth on glycerol-fumarate medium. This work suggests that DmsD is a twin-arginine leader-binding protein.