Balancing redox cofactor generation and ATP synthesis: Key microaerobic responses in thermophilic fermentations

Geobacillus thermoglucosidasius is a Gram‐positive, thermophilic bacterium capable of ethanologenic fermentation of both C5 and C6 sugars and may have possible use for commercial bioethanol production [Tang et al., 2009; Taylor et al. (2009) Trends Biotechnol 27(7): 398–405]. Little is known about the physiological changes that accompany a switch from aerobic (high redox) to microaerobic/fermentative (low redox) conditions in thermophilic organisms. The changes in the central metabolic pathways in response to a switch in redox potential were analyzed using quantitative real‐time PCR and proteomics. During low redox (fermentative) states, results indicated that glycolysis was uniformly up‐regulated, the Krebs (tricarboxylic acid or TCA) cycle non‐uniformly down‐regulated and that there was little to no change in the pentose phosphate pathway. Acetate accumulation was accounted for by strong down‐regulation of the acetate CoA ligase gene (acs) in addition to up‐regulation of the pta and ackA genes (involved in acetate production), thus conserving ATP while reducing flux through the TCA cycle. Substitution of an NADH dehydrogenase (down‐regulated) by an up‐regulated NADH:FAD oxidoreductase and up‐regulation of an ATP synthase subunit, alongside the observed shifts in the TCA cycle, suggested that an oxygen‐scavenging electron transport chain likely remained active during low redox conditions. Together with the observed up‐regulation of a glyoxalase and down‐regulation of superoxide dismutase, thought to provide protection against the accumulation of toxic phosphorylated glycolytic intermediates and reactive oxygen species, respectively, the changes observed in G. thermoglucosidasius NCIMB 11955 under conditions of aerobic‐to‐microaerobic switching were consistent with responses to low pO₂ stress. Biotechnol. Bioeng. 2013; 110: 1057–1065. © 2012 Wiley Periodicals, Inc.     

Early‐Life Exposure to Pulsed LTE Radiofrequency Fields Causes Persistent Changes in Activity and Behavior in C57BL/6 J Mice

Despite much research, gaps remain in knowledge about the potential health effects of exposure to radiofrequency (RF) fields. This study investigated the effects of early‐life exposure to pulsed long term evolution (LTE) 1,846 MHz downlink signals on innate mouse behavior. Animals were exposed for 30 min/day, 5 days/week at a whole‐body average specific energy absorption rate (SAR) of 0.5 or 1 W/kg from late pregnancy (gestation day 13.5) to weaning (postnatal day 21). A behavioral tracking system measured locomotor, drinking, and feeding behavior in the home cage from 12 to 28 weeks of age. The exposure caused significant effects on both appetitive behaviors and activity of offspring that depended on the SAR. Compared with sham‐exposed controls, exposure at 0.5 W/kg significantly decreased drinking frequency (P ≤ 0.000) and significantly decreased distance moved (P ≤ 0.001). In contrast, exposure at 1 W/kg significantly increased drinking frequency (P ≤ 0.001) and significantly increased moving duration (P ≤ 0.005). In the absence of other plausible explanations, it is concluded that repeated exposure to low‐level RF fields in early life may have a persistent and long‐term effect on adult behavior. Bioelectromagnetics. 2019;40:498–511. © 2019 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.     

The spatial segregation of pericentric cohesin and condensin in the mitotic spindle

In mitosis, the pericentromere is organized into a spring composed of cohesin, condensin, and a rosette of intramolecular chromatin loops. Cohesin and condensin are enriched in the pericentromere, with spatially distinct patterns of localization. Using model convolution of computer simulations, we deduce the mechanistic consequences of their spatial segregation. Condensin lies proximal to the spindle axis, whereas cohesin is radially displaced from condensin and the interpolar microtubules. The histone deacetylase Sir2 is responsible for the axial position of condensin, while the radial displacement of chromatin loops dictates the position of cohesin. The heterogeneity in distribution of condensin is most accurately modeled by clusters along the spindle axis. In contrast, cohesin is evenly distributed (barrel of 500-nm width × 550-nm length). Models of cohesin gradients that decay from the centromere or sister cohesin axis, as previously suggested, do not match experimental images. The fine structures of cohesin and condensin deduced with subpixel localization accuracy reveal critical features of how these complexes mold pericentric chromatin into a functional spring.     

Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons

Non-viral vectors are promising vehicles for gene therapy but delivery of plasmid DNA to post-mitotic cells is challenging as nuclear entry is particularly inefficient. We have developed and evaluated a hybrid mRNA/DNA system designed to bypass the nuclear barrier to transfection and facilitate cytoplasmic gene expression. This system, based on co-delivery of mRNA(A64) encoding for T7 RNA polymerase (T7 RNAP) with a T7-driven plasmid, produced between 10- and 2200-fold higher gene expression in primary dorsal root ganglion neuronal (DRGN) cultures isolated from Sprague–Dawley rats compared to a cytomegalovirus (CMV)-driven plasmid, and 30-fold greater expression than the enhanced T7-based autogene plasmid pR011. Cell-free assays and in vitro transfections highlighted the versatility of this system with small quantities of T7 RNAP mRNA required to mediate expression at levels that were significantly greater than with the T7-driven plasmid alone or supplemented with T7 RNAP protein. We have also characterized a number of parameters, such as mRNA structure, intracellular stability and persistence of each nucleic acid component that represent important factors in determining the transfection efficiency of this hybrid expression system. The results from this study demonstrate that co-delivery of mRNA is a promising strategy to yield increased expression with plasmid DNA, and represents an important step towards improving the capability of non-viral vectors to mediate efficient gene transfer in cell types, such as in DRGN, where the nuclear membrane is a significant barrier to transfection.     

AGAP1, an endosome-associated,phosphoinositide-dependent ADP-ribosylation factor GTPase-activating protein that affects actin cytoskeleton

We have identified three members of the AGAP subfamily of ASAP family ADP-ribosylation factor GTPase-activating proteins (Arf GAPs). In addition to the Arf GAP domain, these proteins contain GTP-binding protein-like, ankyrin repeat and pleckstrin homology domains. Here, we have characterized the ubiquitously expressed AGAP1/KIAA1099. AGAP1 had Arf GAP activity toward Arf1>Arf5>Arf6. Phosphatidylinositol 4,5-bisphosphate and phosphatidic acid synergistically stimulated GAP activity. As found for other ASAP family Arf GAPs, the pleckstrin homology domain was necessary for activity. Deletion of the GTP-binding protein-like domain affected lipid dependence of Arf GAP activity. In vivo effects of AGAP1 were distinct from other ASAP family Arf GAPs. Overexpressed AGAP1 induced the formation of and was associated with punctate structures containing the endocytic markers transferrin and Rab4. AP1 was redistributed from the trans-Golgi to the punctate structures. Like other ASAP family members, AGAP1 overexpression inhibited the formation of PDGF-induced ruffles. However, distinct from other ASAP family members, AGAP1 also induced the loss of actin stress fibers. Thus, AGAP1 is a phosphoinositide-dependent Arf GAP that impacts both the endocytic compartment and actin.     

Androgen-induced mineralization by MC3T3-E1 osteoblastic cells reveals a critical window of hormone responsiveness

Despite their clinical importance for skeletal growth and homeostasis, the actions of androgens on osteoblastic cells are not well understood. MC3T3-E1 cells, a nontransformed murine preosteoblastic cell line, that traverse the stages of osteoblastic differentiation within 30 days in vitro, were exposed to mibolerone (an androgen receptor (AR) agonist) or 5alpha-dihydroxytestosterone (DHT) from days 3 to 30 post-plating. Cells exposed to this hormonal regimen exhibited a significant increase in mineralization (calcium deposition) compared to vehicle-treated cells. Delaying treatment for 4-11 days (treatment still completed on day 30 post-plating) enhanced mineralization further. Within 2 days post-plating, AR protein increased 7.2-fold in androgen-treated cells and 2.5-fold in vehicle-treated cells. MC3T3-E1 cells transfected with an androgen- and glucocorticoid-responsive reporter construct on day 1 post-plating followed by a 2 day exposure to DHT, mibolerone, or dexamethasone (dex; a glucocorticoid receptor agonist) exhibited reporter gene activation only with dex treatment. In contrast, delaying transfection and treatment for at least 1 day resulted in comparable androgen- and dex-mediated reporter gene transactivation. Therefore, the ability of MC3T3-E1 cells to respond to androgens is dependent on the timing of androgen administration.     

Locating an antagonist in the 5-HT3 receptor binding site using modeling and radioligand binding

We have used a homology model of the extracellular domain of the 5-HT(3) receptor to dock granisetron, a 5-HT(3) receptor antagonist, into the binding site using AUTODOCK. This yielded 13 alternative energetically favorable models. The models fell into 3 groups. In model type A the aromatic rings of granisetron were between Trp-90 and Phe-226 and its azabicyclic ring was between Trp-183 and Tyr-234, in model type B this orientation was reversed, and in model type C the aromatic rings were between Asp-229 and Ser-200 and the azabicyclic ring was between Phe-226 and Asn-128. Residues located no more than 5 A from the docked granisetron were identified for each model; of 26 residues identified, 8 were found to be common to all models, with 18 others being represented in only a subset of the models. To identify which of the docking models best represents the ligand-receptor complex, we substituted each of these 26 residues with alanine and a residue with similar chemical properties. The mutant receptors were expressed in human embryonic kidney (HEK)293 cells and the affinity of granisetron determined using radioligand binding. Mutation of 2 residues (Trp-183 and Glu-129) ablated binding, whereas mutation of 14 other residues caused changes in the [(3)H]granisetron binding affinity in one or both mutant receptors. The data showed that residues both in and close to the binding pocket can affect antagonist binding and overall were found to best support model B.     

Nuclear congression is driven by cytoplasmic microtubule plus end interactions in S. cerevisiae

Nuclear movement before karyogamy in eukaryotes is known as pronuclear migration or as nuclear congression in Saccharomyces cerevisiae. In this study, S. cerevisiae is used as a model system to study microtubule (MT)-dependent nuclear movements during mating. We find that nuclear congression occurs through the interaction of MT plus ends rather than sliding and extensive MT overlap. Furthermore, the orientation and attachment of MTs to the shmoo tip before cell wall breakdown is not required for nuclear congression. The MT plus end-binding proteins Kar3p, a class 14 COOH-terminal kinesin, and Bik1p, the CLIP-170 orthologue, localize to plus ends in the shmoo tip and initiate MT interactions and depolymerization after cell wall breakdown. These data support a model in which nuclear congression in budding yeast occurs by plus end MT capture and depolymerization, generating forces sufficient to move nuclei through the cytoplasm. This is the first evidence that MT plus end interactions from oppositely oriented organizing centers can provide the force for organelle transport in vivo.     

An In Silico Agent-Based Model Demonstrates Reelin Function in Directing Lamination of Neurons during Cortical Development

The characteristic six-layered appearance of the neocortex arises from the correct positioning of pyramidal neurons during development and alterations in this process can cause intellectual disabilities and developmental delay. Malformations in cortical development arise when neurons either fail to migrate properly from the germinal zones or fail to cease migration in the correct laminar position within the cortical plate. The Reelin signalling pathway is vital for correct neuronal positioning as loss of Reelin leads to a partially inverted cortex. The precise biological function of Reelin remains controversial and debate surrounds its role as a chemoattractant or stop signal for migrating neurons. To investigate this further we developed an in silico agent-based model of cortical layer formation. Using this model we tested four biologically plausible hypotheses for neuron motility and four biologically plausible hypotheses for the loss of neuron motility (conversion from migration). A matrix of 16 combinations of motility and conversion rules was applied against the known structure of mouse cortical layers in the wild-type cortex, the Reelin-null mutant, the Dab1-null mutant and a conditional Dab1 mutant. Using this approach, many combinations of motility and conversion mechanisms can be rejected. For example, the model does not support Reelin acting as a repelling or as a stopping signal. In contrast, the study lends very strong support to the notion that the glycoprotein Reelin acts as a chemoattractant for neurons. Furthermore, the most viable proposition for the conversion mechanism is one in which conversion is affected by a motile neuron sensing in the near vicinity neurons that have already converted. Therefore, this model helps elucidate the function of Reelin during neuronal migration and cortical development.