Engineered Mutants of a Marine Photosynthetic Purple Nonsulfur Bacterium with Increased Volumetric Productivity of Polyhydroxyalkanoate Bioplastics.

Polyhydroxyalkanoates (PHAs) are green and sustainable bioplastics that could replace petrochemical synthetic plastics without posing environmental threats to living organisms. In addition, sustainable PHA production could be achieved using marine photosynthetic purple nonsulfur bacteria (PNSBs) that utilize natural seawater, sunlight, carbon dioxide gas, and nitrogen gas for growth. However, PHA production using marine photosynthetic PNSBs has not been economically feasible yet due to its high cost and low productivity. In this work, strain improvement, using genome-wide mutagenesis coupled with high-throughput screening via fluorescence-activated cell sorting, we were able to create Rhodovulum sulfidophilum mutants with enhanced volumetric PHA productivity, with an up to 1.7-fold increase. The best selected mutants (E6 and E6M4) reached the stationary growth phase 1 day faster and accumulated the maximum PHA content 2 days faster than the wild type. Maximizing volumetric PHA productivity before the stationary growth phase is indeed an additional advantage for R. sulfidophilum as a growth-associated PHA producer.

Visualizing developmentally programmed endoreplication in mammals using ubiquitin oscillators.

The majority of mammalian somatic cells maintain a diploid genome. However, some mammalian cell types undergo multiple rounds of genome replication (endoreplication) as part of normal development and differentiation. For example, trophoblast giant cells (TGCs) in the placenta become polyploid through endoreduplication (bypassed mitosis), and megakaryocytes (MKCs) in the bone marrow become polyploid through endomitosis (abortive mitosis). During the normal mitotic cell cycle, geminin and Cdt1 are involved in 'licensing' of replication origins, which ensures that replication occurs only once in a cell cycle. Their protein accumulation is directly regulated by two E3 ubiquitin ligase activities, APC(Cdh1) and SCF(Skp2), which oscillate reciprocally during the cell cycle. Although proteolysis-mediated, oscillatory accumulation of proteins has been documented in endoreplicating Drosophila cells, it is not known whether the ubiquitin oscillators that control normal cell cycle transitions also function during mammalian endoreplication. In this study, we used transgenic mice expressing Fucci fluorescent cell-cycle probes that report the activity of APC(Cdh1) and SCF(Skp2). By performing long-term, high temporal-resolution Fucci imaging, we were able to visualize reciprocal activation of APC(Cdh1) and SCF(Skp2) in differentiating TGCs and MKCs grown in our custom-designed culture wells. We found that TGCs and MKCs both skip cytokinesis, but in different ways, and that the reciprocal activation of the ubiquitin oscillators in MKCs varies with the polyploidy level. We also obtained three-dimensional reconstructions of highly polyploid TGCs in whole, fixed mouse placentas. Thus, the Fucci technique is able to reveal the spatiotemporal regulation of the endoreplicative cell cycle during differentiation.

Contrasting quiescent G0 phase with mitotic cell cycling in the mouse immune system.

A transgenic mouse line expressing Fucci (fluorescent ubiquitination-based cell-cycle indicator) probes allows us to monitor the cell cycle in the hematopoietic system. Two populations with high and low intensities of Fucci signals for Cdt1(30/120) accumulation were identified by FACS analysis, and these correspond to quiescent G0 and cycling G1 cells, respectively. We observed the transition of immune cells between quiescent and proliferative phases in lymphoid organs during differentiation and immune responses.

Proteomic analysis of lymphoblastoid cells derived from monozygotic twins discordant for bipolar disorder: a preliminary study.

Bipolar disorder is a severe mental illness characterized by recurrent manic and depressive episodes. In bipolar disorder, family and twin studies suggest contributions from genetic and environmental factors; however, the detailed molecular pathogenesis is yet unknown. Thus, identification of biomarkers may contribute to the clinical diagnosis of bipolar disorder. Monozygotic twins discordant for bipolar disorder are relatively rare but have been reported. Here we performed a comparative proteomic analysis of whole cell lysate derived from lymphoblastoid cells of monozygotic twins discordant for bipolar disorder by using two-dimensional differential in-gel electrophoresis (2D-DIGE). We found approximately 200 protein spots to be significantly differentially expressed between the patient and the co-twin (t test, p<0.05). Some of the proteins were subsequently identified by liquid chromatography tandem mass spectrometry and included proteins involved in cell death and glycolysis. To examine whether these proteins could serve as biomarkers of bipolar disorder, we performed Western blot analysis using case-control samples. Expression of phosphoglycerate mutase 1 (PGAM1), which is involved in glycolysis, was significantly up-regulated in patients with bipolar disorder (t test, p<0.05). Although PGAM1 cannot be regarded as a qualified biomarker of bipolar disorder from this preliminary finding, it could be one of the candidates for further study to identify biomarkers of bipolar disorder.

Drug-induced cell cycle modulation leading to cell-cycle arrest, nuclear mis-segregation, or endoreplication.

BACKGROUND: Cancer cell responses to chemotherapeutic agents vary, and this may reflect different defects in DNA repair, cell-cycle checkpoints, and apoptosis control. Cytometry analysis only quantifies dye-incorporation to examine DNA content and does not reflect the biological complexity of the cell cycle in drug discovery screens. RESULTS: Using population and time-lapse imaging analyses of cultured immortalized cells expressing a new version of the fluorescent cell-cycle indicator, Fucci (Fluorescent Ubiquitination-based Cell Cycle Indicator), we found great diversity in the cell-cycle alterations induced by two anticancer drugs. When treated with etoposide, an inhibitor of DNA topoisomerase II, HeLa and NMuMG cells halted at the G2/M checkpoint. HeLa cells remained there, but NMuMG cells then overrode the checkpoint and underwent nuclear mis-segregation or avoided the checkpoint and entered the endoreplication cycle in a drug concentration dependent manner. In contrast, an inhibitor of Cdk4 led to G1 arrest or endoreplication in NMuMG cells depending upon the initial cell-cycle phase of drug exposure. CONCLUSIONS: Drug-induced cell cycle modulation varied not only between different cell types or following treatment with different drugs, but also between cells treated with different concentrations of the same drug or following drug addition during different phases of the cell cycle. By combining cytometry analysis with the Fucci probe, we have developed a novel assay that fully integrates the complexity of cell cycle regulation into drug discovery screens. This assay system will represent a powerful drug-discovery tool for the development of the next generation of anti-cancer therapies.

Tracing the silhouette of individual cells in S/G2/M phases with fluorescence.

The APC(Cdh1) E3 ligase is active in the late M and G(1) phases. Geminin is a direct substrate of the APC(Cdh1) complex, and accumulates during the S, G(2), and M phases. By fusing the amino-terminal region of Geminin to fluorescent proteins, we have developed cell cycle markers that accumulate in the S/G(2)/M phases in both the nucleus and the cytoplasm. These markers reveal the morphology of individual cells that have undergone DNA replication, allowing us to monitor cell growth relative to differentiation of various cell types. After electroporating the developing mouse embryos, we highlighted neuroepithelial progenitors in the S/G(2)/M phases, which possessed an elongated morphology with an apical and/or a basal attachment. We also show that nuclear localization of the ubiquitin ligase for Geminin is essential for full performance of the markers.

Purification of polyglutamine aggregates and identification of elongation factor-1alpha and heat shock protein 84 as aggregate-interacting proteins.

Aggregates of green fluorescent protein (GFP)-fused truncated N-terminal huntingtin containing abnormally long polyglutamine tracts (150 repeats of glutamine residue) were purified from an ecdysone-inducible mutant neuro2A cell line (HD150Q-28) by using a fluorescence-activated cell sorter. To analyze the aggregate-interacting proteins, we subjected the purified aggregates to SDS-PAGE; prominent protein bands in the gel were digested with Achromobactor lysyl endopeptidase, followed by a HPLC-mass spectrometry (MS) analysis. The resulting data of tandem MS analysis revealed that, in addition to ubiquitin and widely reported chaperone proteins such as heat shock cognate 70 (HSC70), human DNA J-1 (HDJ-1), and HDJ-2, the translational elongation factor-1alpha (EF-1alpha) and heat shock protein 84 (HSP84) also were recognized as aggregate-interacting proteins. Sequestration of these proteins to aggregates was confirmed further by several immunochemical methods. We confirmed that, in addition to the other known proteins, EF-1alpha and HSP84 also colocalized with the intracellular aggregates. An assay of the transient expression of EF-1alpha and HSP84 in HD150Q-28 cells revealed that both proteins improved cell viability. Moreover, the rate of aggregate formation decreased in both transfectants. Our study suggests that both EF-1alpha and HSP84 are involved in the neurodegenerative process of polyglutamine diseases.