Cardiolipin and Its Different Properties in Mitophagy and Apoptosis

Cardiolipin (CL) is a unique dimeric phospholipid that exists almost exclusively in the inner mitochondrial membrane (IMM) in eukaryotic cells. Two chiral carbons and four fatty acyl chains in CL result in a flexible body allowing interactions with respiratory chain complexes and mitochondrial substrate carriers. Due to its high content of unsaturated fatty acids, CL is particularly prone to reactive oxygen species (ROS)-induced oxidative attacks. Under mild mitochondrial damage, CL is redistributed to the outer mitochondrial membrane (OMM) and serves as a recognition signal for dysfunctional mitochondria, which are rapidly sequestered by autophagosomes. However, peroxidation of CL is far greater in response to severe stress than under normal or mild-damage conditions. The accumulation of oxidized CL on the OMM results in recruitment of Bax and formation of the mitochondrial permeability transition pore (MPTP), which releases Cytochrome c (Cyt c) from mitochondria. Over the past decade, the significance of CL in the function of mitochondrial bioenergy has been explored. Moreover, approaches to analyzing CL have become more effective and accurate. In this review, we discuss the unique structural features of CL as well as the current understanding of CL-based molecular mechanisms of mitophagy and apoptosis.     

Coupling spatial segregation with synthetic circuits to control bacterial survival

Engineered bacteria have great potential for medical and environmental applications. Fulfilling this potential requires controllability over engineered behaviors and scalability of the engineered systems. Here, we present a platform technology, microbial swarmbot, which employs spatial arrangement to control the growth dynamics of engineered bacteria. As a proof of principle, we demonstrated a safeguard strategy to prevent unintended bacterial proliferation. In particular, we adopted several synthetic gene circuits to program collective survival in Escherichia coli: the engineered bacteria could only survive when present at sufficiently high population densities. When encapsulated by permeable membranes, these bacteria can sense the local environment and respond accordingly. The cells inside the microbial swarmbot capsules will survive due to their high densities. Those escaping from a capsule, however, will be killed due to a decrease in their densities. We demonstrate that this design concept is modular and readily generalizable. Our work lays the foundation for engineering integrated and programmable control of hybrid biological–material systems for diverse applications.     

Twist and Degrade—Impact of Molecular Structure on the Photostability of Nonfullerene Acceptors and Their Photovoltaic Blends

Nonfullerene acceptors (NFAs) dominate organic photovoltaic (OPV) research due to their promising efficiencies and stabilities. However, there is very little investigation into the molecular processes of degradation, which is critical to guiding design of novel NFAs for long‐lived, commercially viable OPVs. Here, the important role of molecular structure and conformation in NFA photostability in air is investigated by comparing structurally similar but conformationally different promising NFAs: planar O‐IDTBR and nonplanar O‐IDFBR. A three‐phase degradation process is identified: i) initial photoinduced conformational change (i.e., torsion about the core–benzothiadiazole dihedral), induced by noncovalent interactions with environmental molecules, ii) followed by photo‐oxidation and fragmentation, leading to chromophore bleaching and degradation product formation, and iii) finally complete chromophore bleaching. Initial conformational change is a critical prerequisite for further degradation, providing fundamental understanding of the relative stability of IDTBR and IDFBR, where the already twisted IDFBR is more prone to degradation. When blended with the donor polymer poly(3‐hexylthiophene), both NFAs exhibit improved photostability while the photostability of the polymer itself is significantly reduced by the more miscible twisted NFA. The findings elucidate the important role of NFA molecular structure in photostability of OPV systems, and provide vital insights into molecular design rules for intrinsically photostable NFAs.     

Construction and characterization of two recombinant bacteria that grow on ortho- and para-substituted chlorobiphenyls

Cloning and expression of the aromatic ring dehalogenation genes in biphenyl-growing, polychlorinated biphenyl (PCB)-cometabolizing Comamonas testosteroni VP44 resulted in recombinant pathways allowing growth on ortho- and para-chlorobiphenyls (CBs) as a sole carbon source. The recombinant variants were constructed by transformation of strain VP44 with plasmids carrying specific genes for dehalogenation of chlorobenzoates (CBAs). Plasmid pE43 carries the Pseudomonas aeruginosa 142 ohb genes coding for the terminal oxygenase (ISPOHB) of the ortho-halobenzoate 1,2-dioxygenase, whereas plasmid pPC3 contains the Arthrobacter globiformis KZT1 fcb genes, which catalyze the hydrolytic para-dechlorination of 4-CBA. The parental strain, VP44, grew only on low concentrations of 2- and 4-CB by using the products from the fission of the nonchlorinated ring of the CBs (pentadiene) and accumulated stoichiometric amounts of the corresponding CBAs. The recombinant strains VP44(pPC3) and VP44(pE43) grew on, and completely dechlorinated high concentrations (up to 10 mM), of 4-CBA and 4-CB and 2-CBA and 2-CB, respectively. Cell protein yield corresponded to complete oxidation of both biphenyl rings, thus confirming mineralization of the CBs. Hence, the use of CBA dehalogenase genes appears to be an effective strategy for construction of organisms that will grow on at least some congeners important for remediation of PCBs.     

Surface plasmon resonance study of the molecular recognition between polymerase and DNA containing various mismatches and conformational changes of DNA-protein complexes

Although surface plasmon resonance (SPR) biosensor technique has been used to study protein-protein interactions and to detect conformational changes of proteins, it has not been shown whether the SPR biosensor can be used to study a complex kinetic system such as the protein-DNA binding, which sometimes involves several binding steps as well as dynamic conformational changes of the complexes. In this study, we have used SPR biosensor and T7 polymerase as the model system to study the interactions of the polymerase with a series of DNA template-primer duplexes containing different number of mismatches and GC contents at various positions near the primer 3'-end. In general, the binding constants measured by the SPR are several magnitudes smaller than those determined in solution, indicating the limitation of the surface-based technique for measuring solution-based interactions. However, the distinct polymerase binding profiles obtained for DNA duplexes differed by as low as a single mismatch suggest that the SPR data can be used for relative comparison purpose among a set of experiments carried out under identical conditions. The successful fitting of the binding profiles using the established translocation model also demonstrated that SPR can be used to monitor conformational changes, as well as to derive relative kinetic values, within a complicated DNA-protein interaction system. The results also demonstrated that SPR biosensor may be used as a sensitive technique for studying molecular recognition events, such as single-base discrimination involved in protein-DNA interactions.     

Assessing Mitochondrial DNA Variation and Copy Number in Lymphocytes of ~2,000 Sardinians Using Tailored Sequencing Analysis Tools

DNA sequencing identifies common and rare genetic variants for association studies, but studies typically focus on variants in nuclear DNA and ignore the mitochondrial genome. In fact, analyzing variants in mitochondrial DNA (mtDNA) sequences presents special problems, which we resolve here with a general solution for the analysis of mtDNA in next-generation sequencing studies. The new program package comprises 1) an algorithm designed to identify mtDNA variants (i.e., homoplasmies and heteroplasmies), incorporating sequencing error rates at each base in a likelihood calculation and allowing allele fractions at a variant site to differ across individuals; and 2) an estimation of mtDNA copy number in a cell directly from whole-genome sequencing data. We also apply the methods to DNA sequence from lymphocytes of ~2,000 SardiNIA Project participants. As expected, mothers and offspring share all homoplasmies but a lesser proportion of heteroplasmies. Both homoplasmies and heteroplasmies show 5-fold higher transition/transversion ratios than variants in nuclear DNA. Also, heteroplasmy increases with age, though on average only ~1 heteroplasmy reaches the 4% level between ages 20 and 90. In addition, we find that mtDNA copy number averages ~110 copies/lymphocyte and is ~54% heritable, implying substantial genetic regulation of the level of mtDNA. Copy numbers also decrease modestly but significantly with age, and females on average have significantly more copies than males. The mtDNA copy numbers are significantly associated with waist circumference (p-value = 0.0031) and waist-hip ratio (p-value = 2.4×10^-5), but not with body mass index, indicating an association with central fat distribution. To our knowledge, this is the largest population analysis to date of mtDNA dynamics, revealing the age-imposed increase in heteroplasmy, the relatively high heritability of copy number, and the association of copy number with metabolic traits.     

Complete Genome Sequence of Staphylococcus lugdunensis Strain HKU09-01

Staphylococcus lugdunensis is a member of the coagulase-negative staphylococci and commonly found as part of the human skin flora. It is a significant cause of catheter-related bacteremia and also causes serious infections like native valve endocarditis in previously healthy individuals. We report the complete genome sequence of this medically important bacterium.Staphylococcus lugdunensis is a member of the coagulase-negative staphylococci (CoNS) commonly colonizing the human skin and mucosal membranes. While the genus Staphylococcus contains 48 named species currently, only a few species, notably S. aureus, are coagulase positive. Thus, the phenotypic characteristic is routinely tested in the medical microbiological laboratory for rapid differentiation of the highly pathogenic S. aureus from the other staphylococci. Among the CoNS, only a few species are known to cause human disease, usually in the form of opportunistic infections only (6). However, S. lugdunensis is an important exception (3). Besides causing catheter-related bacteremia similar to other CoNS, it causes a variety of severe nosocomial and community-acquired infections, including native valve endocarditis, a devastating and potentially fatal disease that can affect previously healthy individuals. Another unusual feature are the susceptibilities of S. lugdunensis isolates to multiple antimicrobial agents even when the incidence of multiple-drug-resistant CoNS and S. aureus occurrences are increasing in both hospital and community settings (4, 5).The genome sequence of S. lugdunensis strain HKU09-01 was determined by high-throughput sequencing performed on a GS FLX system (Roche Diagnostics, Basel, Switzerland), with approximately 45-fold coverage of the genome. This clinical strain was previously isolated from the culture of pus from a skin swab. Genome assembly was performed using the Newbler assembler, resulting in 30 large contigs (>500 bp in size). The contigs were then ordered and oriented into one scaffold using OSLay (11). The genome-finishing strategy for S. lugdunensis was similar to that employed for our previously sequenced Laribacter hongkongensis genome (12). Briefly, gap closures were performed by genomic PCR followed by DNA sequencing of amplification products on an ABI 3130xl sequencer (Applied Biosystems, CA). The finished sequence was validated by genome macrorestriction analysis using multiple rare-cutting enzymes and visualization by pulsed-field gel electrophoresis. Protein coding regions were predicted with Glimmer3 (2), and automatic genome annotation was performed on the RAST server (1). Additionally, annotation of tRNA and transfer-messenger RNA (tmRNA) genes was performed using tRNAScan-SE (10) and ARAGORN (9). Identification of rRNA genes was performed using RNAmmer (8).The genome of S. lugdunensis strain HKU09-01 consists of a circular 2,658,366-bp chromosome with G+C content of 33.87%, similar to those of other staphylococci. No plasmids are present in the sequenced strain. The genome contains 61 tRNA genes for all amino acids and 2,489 predicted protein-coding genes. Eight putative genomic islands were identified, and one actually consists of a pair of duplicated 32-kb genomic regions. Similar to Staphylococcus saprophyticus (7), but different from the other staphylococci, the genome contains 6 rRNA operons, one of them having the unusual organization 5S-16S-23S-5S.With the availability of the present genome sequence, S. lugdunensis now joins other staphylococcal species with human pathogenic potential, like S. aureus, S. epidermidis, S. haemolyticus, and S. saprophyticus, to have at least one reference genome available. Further in-depth analysis will be necessary to fully elucidate the genomic differences that may explain the variation in virulence of the staphylococcal species.