Fabrication of elliptically constructed liquid crystalline elastomeric scaffolds for 3D artificial tissues.

Inspired by the orientation and the fibrous structure of human muscle tissues, we fabricated preconstructed porous liquid crystalline (LC) scaffolds through a two-step polymerization and salt leaching method. A novel strategy combining the aligning properties of LCs and the ease of processing of elastomers for the preparation of elliptical scaffolds for muscle cell culture was proposed in this research. Different from the other types of scaffolds, our biocompatible LC scaffold that can be implanted into the human body using a supporting unit to improve the mechanical properties compared with those of natural muscle. To evaluate the synthesized scaffolds, in vitro experiments using normal human dermal fibroblast (NHDF) cells and smooth muscle cells from rats were carried out, and the sample cells were cultured on each sample scaffold. Based on the results of long-term culture of NHDF cells on the LC scaffolds, it can be confirmed that all three kinds of LC scaffolds have good biocompatibility and provide enough space for cell growth. The addition of gelatin can significantly enhance the biocompatibility of the synthesized scaffolds. Evaluation of scaffold morphologies on cell growth indicates that the molecular arrangement on the scaffolds can induce the growth direction of smooth muscle cells to a certain extent, thereby increasing the formation of highly ordered arrangement tissues. The population doubling time of NHDF cells on the different scaffolds suggest that gelatin can improve the attachment and growth of cells. Investigation of cell viability on LC scaffolds shows that the original LC scaffolds already possess excellent biocompatibility. Additionally, the average cell viability of smooth muscle cells was above 90%, showing that the LC scaffolds in this research are suitable for application in muscle tissue engineering. Based on the results, the gelatin-coated scaffolds are more conducive to the growth of cells in this research and provide promising candidates for tissue engineering in biomedical fields and research fields.

Triplin: Functional Probing of Its Structure and the Dynamics of the Voltage-Gating Process.

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.

Aberrant cyclin C nuclear release induces mitochondrial fragmentation and dysfunction in MED13L syndrome fibroblasts.

MED13L syndrome is a haploinsufficiency developmental disorder characterized by intellectual disability, heart malformation, and hypotonia. MED13L controls transcription by tethering the cyclin C-Cdk8 kinase module (CKM) to the Mediator complex. In addition, cyclin C has CKM-independent roles in the cytoplasm directing stress-induced mitochondrial fragmentation and regulated cell death. Unstressed MED13L S1497 F/fs patient fibroblasts exhibited aberrant cytoplasmic cyclin C localization, mitochondrial fragmentation, and a 6-fold reduction in respiration. In addition, the fibroblasts exhibited reduced mtDNA copy number, reduction in mitochondrial membrane integrity, and hypersensitivity to oxidative stress. Finally, transcriptional analysis of MED13L mutant fibroblasts revealed reduced mRNA levels for several genes necessary for normal mitochondrial function. Pharmacological or genetic approaches preventing cyclin C-mitochondrial localization corrected the fragmented mitochondrial phenotype and partially restored organelle function. In conclusion, this study found that mitochondrial dysfunction is an underlying defect in cells harboring the MED13L S1497 F/fs allele and identified cyclin C mis-localization as the likely cause. These results provide a new avenue for understanding this disorder.

Different ways to transport ammonia in human and Mycobacterium tuberculosis NAD+ synthetases.

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 NH3 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.

Self-Healable Porous Polyampholyte Hydrogels with Higher Water Content as Cell Culture Scaffolds for Tissue Engineering Applications.

In this paper, we first demonstrate the control of the film pore size using neutral hydrophilic 2-hydroxyethyl methacrylate (HEMA) content. To improve the mechanical properties of a polyampholyte (PA), both HEMA and the cross-linker N,N'-methylenebisacrylamide (Bis-Am) were introduced into the PA chain. The predesigned copolymers showed great mechanical properties and optical behavior. The introduction of HEMA significantly increased the water content of the polymer, leading to the formation of porous structures in xerogels. The dynamic interaction between the positive and negative termini of the PA endowed the hydrogels with self-healing ability. The synthesized chemically cross-linked PA gels showed high stability in saline solution. The biocompatibility of the PA gels was confirmed using a cytotoxicity test of cells attached to the synthesized PA-X-2 and PA/HEMA-90/10-X-0.5. The results of this investigation indicate that the synthesized PA gels are applicable as a polymeric scaffold for cell culture.

Cooperativity and Steep Voltage Dependence in a Bacterial Channel.

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.

Mitochondrial translocation of cyclin C stimulates intrinsic apoptosis through Bax recruitment.

Intrinsic apoptosis requires mitochondrial outer membrane disruption triggered by recruitment, activation, and oligomerization of the Bcl-2 homology protein Bax. Following oxidative stress, we demonstrated that the transcriptional regulator cyclin C is released into the cytosol where it directs mitochondrial fragmentation and efficient apoptotic induction. This study reveals that cytoplasmic cyclin C is required for both normal Bax activation and its efficient mitochondrial localization. This activity appears direct as cyclin C co-immunoprecipitates with active Bax in stressed cells and binds recombinant Bax in vitro. In addition, stable cyclin C-Bax association requires the fission complex. Pharmacologically stimulating cyclin C nuclear release is sufficient for Bax association and their mitochondrial localization in the absence of any stress signals. However, these cells do not undergo cell death as Bax fails to oligomerize. These data support a model that cyclin C association defines an initial step in Bax mitochondrial recruitment and provides a physical connection between the fission and apoptotic factors. This strategy allows the cell to discriminate stress-induced fission able to recruit Bax from other types of mitochondrial divisions.

Cyclin C Regulated Oxidative Stress Responsive Transcriptome in Mus musculus Embryonic Fibroblasts.

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.

Cyclin C: The Story of a Non-Cycling Cyclin.

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.

Cyclin C directly stimulates Drp1 GTP affinity to mediate stress-induced mitochondrial hyperfission.

Mitochondria exist in an equilibrium between fragmented and fused states that shifts heavily toward fission in response to cellular damage. Nuclear-to-cytoplasmic cyclin C relocalization is essential for dynamin-related protein 1 (Drp1)-dependent mitochondrial fission in response to oxidative stress. This study finds that cyclin C directly interacts with the Drp1 GTPase domain, increases its affinity to GTP, and stimulates GTPase activity in vitro. In addition, the cyclin C domain that binds Drp1 is contained within the non-Cdk binding second cyclin box domain common to all cyclin family members. This interaction is important, as this domain is sufficient to induce mitochondrial fission when expressed in mouse embryonic fibroblasts in the absence of additional stress signals. Using gel filtration chromatography and negative stain electron microscopy, we found that cyclin C interaction changes the geometry of Drp1 oligomers in vitro. High-molecular weight low-GTPase activity oligomers in the form of short filaments and rings were diminished, while dimers and elongated filaments were observed. Our results support a model in which cyclin C binding stimulates the reduction of low-GTPase activity Drp1 oligomers into dimers capable of producing high-GTPase activity filaments.

Ceramide channels: destabilization by Bcl-xL and role in apoptosis.

Ceramide is a bioactive sphingolipid involved in mitochondrial-mediated apoptosis. Our data suggest that ceramides directly regulate a key initiation step in apoptosis: mitochondrial outer membrane permeabilization (MOMP). MOMP allows release of intermembrane space proteins to the cytosol, inducing the execution of the cell. Ceramides form channels in planar phospholipid membranes and outer membranes of isolated mitochondria, channels large enough to facilitate passage of proteins released during MOMP. Bcl-xL inhibits MOMP in vivo and inhibits the formation of ceramide channels in vitro. However the significance of Bcl-xL's regulation of ceramide channel formation within cells was untested. We engineered Bcl-xL point mutations that specifically affect the interaction between ceramide and Bcl-xL to probe the mechanism of ceramide channel regulation and the role of ceramide channels in apoptosis. Using these mutants and fluorescently-labeled ceramide, we identified the hydrophobic groove on Bcl-xL as the critical ceramide binding site and regulator of ceramide channel formation. Bcl-xL mutants with weakened interaction with ceramide also have reduced ability to interfere with ceramide channel formation. Some mutants have similar altered ability to inhibit both ceramide and Bax channel formation, whereas others act differentially, suggesting distinct but overlapping binding sites. To probe the relative importance of these channels in apoptosis, Bcl-xL mutant proteins were stably expressed in Bcl-xL deficient cells. Weakening the inhibition of either Bax or ceramide channels decreased the ability of Bcl-xL to protect cells from apoptosis in a stimulus-dependent manner. These studies provide the first in vivo evidence for the role of ceramide channels in MOMP.

The dynamics of Bax channel formation: influence of ionic strength.

Mitochondrial outer membrane permeabilization (MOMP) is a complex multistep process. Studies of MOMP in vivo are limited by the stochastic variability of MOMP between cells and rapid completion of IMS protein release within single cells. In vitro models have provided useful insights into MOMP. We have investigated the dynamics of Bax-mediated MOMP in isolated mitochondria using ionic strength as a tool to control the rate of MOMP. We find that Bax can induce both transient permeabilization, detected by protein release, and more substantial long-lasting permeabilization, measured by the rate of oxidation of added cytochrome c. We found that higher ionic strength causes Bax to form small channels quickly but the expansion of these early channels is impeded. This inhibitory effect of ionic strength is independent of tBid. Channels formed under low ionic strength are not destabilized by raising the ionic strength. Increase in ionic strength also increases the ability of Bcl-xL to inhibit Bax-mediated MOMP. Ionic strength does not affect Bax insertion into mitochondria. Thus, ionic strength influences the assembly of Bax molecules already in membrane into channels. Ionic strength can be used as an effective biophysical tool to study Bax-mediated channel formation.

Metabolic stratification driven by surface and subsurface interactions in a terrestrial mud volcano.

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.

Mechanism of the maturation process of SARS-CoV 3CL protease.

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus. Viral maturation requires a main protease (3CL(pro)) to cleave the virus-encoded polyproteins. We report here that the 3CL(pro) containing additional N- and/or C-terminal segments of the polyprotein sequences undergoes autoprocessing and yields the mature protease in vitro. The dimeric three-dimensional structure of the C145A mutant protease shows that the active site of one protomer binds with the C-terminal six amino acids of the protomer from another asymmetric unit, mimicking the product-bound form and suggesting a possible mechanism for maturation. The P1 pocket of the active site binds the Gln side chain specifically, and the P2 and P4 sites are clustered together to accommodate large hydrophobic side chains. The tagged C145A mutant protein served as a substrate for the wild-type protease, and the N terminus was first digested (55-fold faster) at the Gln(-1)-Ser1 site followed by the C-terminal cleavage at the Gln306-Gly307 site. Analytical ultracentrifuge of the quaternary structures of the tagged and mature proteases reveals the remarkably tighter dimer formation for the mature enzyme (K(d) = 0.35 nm) than for the mutant (C145A) containing 10 extra N-terminal (K(d) = 17.2 nM) or C-terminal amino acids (K(d) = 5.6 nM). The data indicate that immature 3CL(pro) can form dimer enabling it to undergo autoprocessing to yield the mature enzyme, which further serves as a seed for facilitated maturation. Taken together, this study provides insights into the maturation process of the SARS 3CL(pro) from the polyprotein and design of new structure-based inhibitors.