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NAD + synthetase is an essential enzyme of de novo and recycling pathways of NAD + biosynthesis in Mycobacterium tuberculosis but not in humans. This bifunctional enzyme couples the NAD + synthetase and glutaminase activities through an ammonia tunnel but free ammonia is also a substrate. Here we show that the Homo sapiens NAD + synthetase (hsNadE) lacks substrate specificity for glutamine over ammonia and displays a modest activation of the glutaminase domain compared to tbNadE. We report the crystal structures of hsNadE and NAD + synthetase from M. tuberculosis (tbNadE) with synthetase intermediate analogues. Based on the observed exclusive arrangements of the domains and of the intra- or inter-subunit tunnels we propose a model for the inter-domain communication mechanism for the regulation of glutamine-dependent activity and NH 3 transport. The structural and mechanistic comparison herein reported between hsNadE and tbNadE provides also a starting point for future efforts in the development of anti-TB drugs. M. tuberculosis NAD + synthetase (tbNadE) is of interest as a drug target. Here the authors present the actively trapped Homo sapiens NAD + synthetase (hsNadE) and tbNadE structures and show key differences in the synthetase active site and in structural elements possibly involved in the allosteric regulation of catalysis to be leveraged for the development of M. tuberculosis selective inhibitors.

Gram-negative bacteria have a large variety of channel-forming proteins in their outer membrane, generally referred to as porins. Some display weak voltage dependence. A similar trimeric channel former, named Triplin, displays very steep voltage dependence, rivaling that responsible for the electrical excitability of mammals, and high inter-subunit cooperativity. We report detailed insights into the molecular basis for these very unusual properties explored at the single-molecule level. By using chemical modification to reduce the charge on the voltage sensors, they were shown to be positively charged structures. Trypsin cleavage of the sensor eliminates voltage gating by cleaving the sensor. From asymmetrical addition of these reagents, the positively charged voltage sensors translocate across the membrane and are, thus, responsible energetically for the steep voltage dependence. A mechanism underlying the cooperativity was also identified. Theoretical calculations indicate that the charge on the voltage sensor can explain the rectification of the current flowing through the open pores if it is located near the pore mouth in the open state. All results support the hypothesis that one of the three subunits is oriented in a direction opposite to that of the other two. These properties make Triplin perhaps the most complex pore-forming molecular machine described to date.

Smart material-based actuators have attracted much interest because of their envisioned applications in the fields of soft robotics, sensors, and artificial muscles. In this study, we fabricated liquid crystal (LC) actuators via polydopamine (PDA) assistance based on controlling the order parameters. The irregular aggregation of PDA particles coated on the LC film surface was evaluated. To synthesize LC actuators, a predesigned glass sample cell filled with tilt-arranged LCs was fabricated. The LC mixtures were polymerized by 254 nm UV irradiation in glass sample cells. The polymerized LC films showed reversible bending and helical motions depending on predesigned molecular arrangement of the films. This phenomenon was attributed to the phase transition from monodomain to isotropic LC films triggered by temperature and near-infrared light exposure. Adding of chiral dopant shows much higher curling efficiency on thermal stimulation. A hand-shaped actuator with gripping ability was prepared from the synthesized LC networks. The near-infrared light sensitivity of the LC networks was further enhanced using PDA coated on the surface. The observed results suggested that the synthesized LC actuators effectively converted thermal energy and photo-energy to mechanical power. These predesigned LC actuators hold potential for applications as artificial muscles and in micro robotics.A series of thermally tunable liquid crystal elastomeric films was synthesized. Reversible shape variation controlled by temperature or light was achieved based on the order parameter control via the assistance of polydopamine.

This paper reports on the discovery of a novel three-membrane channel unit exhibiting very steep voltage dependence and strong cooperative behavior. It was reconstituted into planar phospholipid membranes formed by the monolayer method and studied under voltage-clamp conditions. The behavior of the novel channel-former, isolated from Escherichia coli, is consistent with a linearly organized three-channel unit displaying steep voltage-gating (a minimum of 14 charges in the voltage sensor) that rivals that of channels in mammalian excitable membranes. The channels also display strong cooperativity in that closure of the first channel permits the second to close and closure of the second channel permits closure of the third. All three have virtually the same conductance and selectivity, and yet the first and third close at positive potentials whereas the second closes at negative potentials. Thus, is it likely that the second channel-former is oriented in the membrane in a direction opposite to that of the other two. This novel structure is named “triplin.” The extraordinary behavior of triplin indicates that it must have important and as yet undefined physiological roles.

The transcriptional changes that occur in response to oxidative stress help direct the decision to maintain cell viability or enter a cell death pathway. Cyclin C-Cdk8 is a conserved kinase that associates with the RNA polymerase II Mediator complex that stimulates or represses transcription depending on the locus. In response to oxidative stress, cyclin C, but not Cdk8, displays partial translocation into the cytoplasm. These findings open the possibility that cyclin C relocalization is a regulatory mechanism governing oxidative stress-induced transcriptional changes. In the present study, the cyclin C-dependent transcriptome was determined and compared to transcriptional changes occurring in oxidatively stressed Mus musculus embryonic fibroblasts. We observed a similar number (∼2000) of genes up or downregulated in oxidatively stressed cells. Induced genes include cellular repair/survival factors while repressed loci were generally involved in proliferation or differentiation. Depleting cyclin C in unstressed cells produced an approximately equal number of genes (∼2400) that were repressed by, or whose transcription required, cyclin C. Consistent with the possibility that cyclin C nuclear release contributes to transcriptional remodeling in response to oxidative stress, we found that 37% cyclin C-dependent genes were downregulated following stress. Moreover, 20% of cyclin C- repressed genes were induced in response to stress. These findings are consistent with a model that cyclin C relocalization to the cytoplasm, and corresponding inactivation of Cdk8, represents a regulatory mechanism to repress and stimulate transcription of stress-responsive genes.

Terrestrial mud volcanism represents the prominent surface geological feature, where fluids and hydrocarbons are discharged along deeply rooted structures in tectonically active regimes. Terrestrial mud volcanoes (MVs) directly emit the major gas phase, methane, into the atmosphere, making them important sources of greenhouse gases over geological time. Quantification of methane emission would require detailed insights into the capacity and efficiency of microbial metabolisms either consuming or producing methane in the subsurface, and establishment of the linkage between these methane-related metabolisms and other microbial or abiotic processes. Here we conducted geochemical, microbiological and genetic analyses of sediments, gases, and pore and surface fluids to characterize fluid processes, community assemblages, functions and activities in a methane-emitting MV of southwestern Taiwan. Multiple lines of evidence suggest that aerobic/anaerobic methane oxidation, sulfate reduction and methanogenesis are active and compartmentalized into discrete, stratified niches, resembling those in marine settings. Surface evaporation and oxidation of sulfide minerals are required to account for the enhanced levels of sulfate that fuels subsurface sulfate reduction and anaerobic methanotrophy. Methane flux generated by in situ methanogenesis appears to alter the isotopic compositions and abundances of thermogenic methane migrating from deep sources, and to exceed the capacity of microbial consumption. This metabolic stratification is sustained by chemical disequilibria induced by the mixing between upward, anoxic, methane-rich fluids and downward, oxic, sulfate-rich fluids.

The class I cyclin family is a well-studied group of structurally conserved proteins that interact with their associated cyclin-dependent kinases (Cdks) to regulate different stages of cell cycle progression depending on their oscillating expression levels. However, the role of class II cyclins, which primarily act as transcription factors and whose expression remains constant throughout the cell cycle, is less well understood. As a classic example of a transcriptional cyclin, cyclin C forms a regulatory sub-complex with its partner kinase Cdk8 and two accessory subunits Med12 and Med13 called the Cdk8-dependent kinase module (CKM). The CKM reversibly associates with the multi-subunit transcriptional coactivator complex, the Mediator, to modulate RNA polymerase II-dependent transcription. Apart from its transcriptional regulatory function, recent research has revealed a novel signaling role for cyclin C at the mitochondria. Upon oxidative stress, cyclin C leaves the nucleus and directly activates the guanosine 5’-triphosphatase (GTPase) Drp1, or Dnm1 in yeast, to induce mitochondrial fragmentation. Importantly, cyclin C-induced mitochondrial fission was found to increase sensitivity of both mammalian and yeast cells to apoptosis. Here, we review and discuss the biology of cyclin C, focusing mainly on its transcriptional and non-transcriptional roles in tumor promotion or suppression.

Recombinant DNA technology mediated by restriction enzymes and ligases allows in vitro manipulation of a DNA segment isolated from the genome. Short overhangs generated by restriction enzymes facilitate efficient pasting together a DNA sequence and a vector. We adopted this recombinant DNA strategy to develop an in vivo recombinant-genome genome editing approach. Using the programmable endonuclease Cas9 or Cas12a as a restriction enzyme, we devised an in situ cut-and-paste (iCAP) genome editing method that was tested in both mouse germline and human cell line platforms. Mouse gene loci Slc35f2 and Slc35f6 were each edited with in-frame insertion of a large APEX2-Cre cassette and concurrent FRT3 insertion at a second location providing proof of principle for the iCAP method. Further, a de nova single nucleotide mutation associated with MED13L syndrome was efficiently corrected in patient cells. Altogether, the iCAP method provides a single genome editing platform with flexibility and multiutility enabling versatile and precise sequence alterations, such as insertion, substitution, and deletion, at single or multiple locations within a genomic segment in mammalian genomes. Competing Interest Statement P.J., K.K. and S.C. are inventors on a patent application. Other authors declare no competing interests.

Ceramide forms a novel type of channel in the mitochondrial outer membrane and these channels are involved the release of intermembrane space proteins from mitochondria, a decision-making step in the apoptotic process. An antiapoptotic protein, Bcl-xL, regulates the apoptotic process and inhibits the formation of ceramide channels. However, there is no precedent to indicate how a protein regulates a lipid channel. We investigated the mechanism of this regulation and identified the hydrophobic groove of the Bcl-xL as the binding site by which Bcl-xL binds to the channel. This was demonstrated by using a combination of experimental and modeling methods, including site-directed mutagenesis, a fluorescence quenching assay, a mitochondrial outer membrane permeability assay, and molecular dynamic simulations. Interestingly, the hydrophobic groove serves to inhibit another channel former, Bax. We found that the binding sites for Bax and ceramide on Bcl-xL are distinct but overlapping. We used that fact to generate mutants that have differential abilities to inhibit one or the other of these channels. These are useful because although ceramide is important in apoptosis, it is still controversial that whether ceramide channels result in apoptosis in vivo. To probe the relative importance of these two channels in apoptosis, Bcl-xL mutant proteins were expressed in Bcl-xL deficient cells. Weakening the inhibitory potency of Bcl-xL on either Bax or ceramide channels resulted in cells being more sensitive to the induction of apoptosis. This is the first evidence for the role of ceramide channels in the apoptotic process in vivo. In a separate investigation, a novel voltage-gated channel unit was found in E. coli extracts. The unit is consistent with three channels forming the functional triplet. These channels are highly voltage gated and highly cooperative. Those results indicated that one of the channels is oriented in an antiparallel fashion compared to the rest. This arrangement is very rare in protein channels. Rectification of the current flowing through the channels was used to identify the orientation of the channels to provide evidence for or against the antiparallel hypothesis. The results favor the antiparallel hypothesis but also reveal an unexpected asymmetry in the transmembrane electrostatics.

Linear stability of two immisible liquids flowing through a long vertical annulus in the gravitational field is investigated. The inner and the outer walls of the annulus are kept at different fixed temperatures and the temperature dependence of viscosity is considered. For Newtonian fluids, interfacial instabilities induced by viscosity stratification and density stratification have been extensively studied in the literature. For non-isothermal flows, the interfacial instability due to thermal conductivity stratification is also identified. It is found in this dissertation that when the densities of the two fluids are matched at a reference temperature, there still exists an interfacial instability if the thermal expansion coefficients of the fluids are different. This mechanism is similar to the instability caused by a density jump at an interface for isothermal flow. When temperature dependency of the viscosity is considered, thermal conductivity stratification is found capable of inducing two types of interfacial instability. The first type, investigated previously, is from the continuous temperature condition at the interface; the second type, which has not been studied before, is from the continuous shear-stress condition at the interface. It is also found that a jump in the products of thermal conductivity and the second derivative of the basic state temperature at the interface can produce a new instability. This new instability exists, for example, when a heat source term needs to be included in the energy equation and there is a discontinuity of the heat source at the interface. Another possibility could come from the inclusion of viscous dissipation in the energy equation. The interfacial instability induced by viscous dissipation is analyzed here since lubricated pipelining, which is used to transport heavy crude oil, is of interest. The viscous-dissipation effect is considered because of the high viscosity of the heavy oil. An energy analysis based on the corresponding eigenfunctions is used to investigate the sources and the mechanisms of the instability.