H2A.Z-Dependent Regulation of Cohesin Dynamics on Chromosome Arms

Structural maintenance of chromosomes (SMC) complexes and DNA topoisomerases are major determinants of chromosome structure and dynamics. The cohesin complex embraces sister chromatids throughout interphase, but during mitosis most cohesin is stripped from chromosome arms by early prophase, while the remaining cohesin at kinetochores is cleaved at anaphase. This two-step removal of cohesin is required for sister chromatids to separate. The cohesin-related Smc5/6 complex has been studied mostly as a determinant of DNA repair via homologous recombination. However, chromosome segregation fails in Smc5/6 null mutants or cells treated with small interfering RNAs. This also occurs in Smc5/6 hypomorphs in the fission yeast Schizosaccharomyces pombe following genotoxic and replication stress, or topoisomerase II dysfunction, and these mitotic defects are due to the postanaphase retention of cohesin on chromosome arms. Here we show that mitotic and repair roles for Smc5/6 are genetically separable in S. pombe. Further, we identified the histone variant H2A.Z as a critical factor to modulate cohesin dynamics, and cells lacking H2A.Z suppress the mitotic defects conferred by Smc5/6 dysfunction. Together, H2A.Z and the SMC complexes ensure genome integrity through accurate chromosome segregation.     

A multifunctional serine protease primes the malaria parasite for red blood cell invasion

The malaria parasite Plasmodium falciparum replicates within an intraerythrocytic parasitophorous vacuole (PV). Rupture of the host cell allows release (egress) of daughter merozoites, which invade fresh erythrocytes. We previously showed that a subtilisin-like protease called PfSUB1 regulates egress by being discharged into the PV in the final stages of merozoite development to proteolytically modify the SERA family of papain-like proteins. Here, we report that PfSUB1 has a further role in ‘priming' the merozoite prior to invasion. The major protein complex on the merozoite surface comprises three proteins called merozoite surface protein 1 (MSP1), MSP6 and MSP7. We show that just before egress, all undergo proteolytic maturation by PfSUB1. Inhibition of PfSUB1 activity results in the accumulation of unprocessed MSPs on the merozoite surface, and erythrocyte invasion is significantly reduced. We propose that PfSUB1 is a multifunctional processing protease with an essential role in both egress of the malaria merozoite and remodelling of its surface in preparation for erythrocyte invasion.     

Evaluation of the "Cellscreen" system for proliferation studies on liver progenitor cells

Proliferation studies on mammalian cells have been disadvantaged by the limited availability of non-invasive assays as the majority of approaches are based on chemical treatment, sampling or staining of cells removed from culture. In this study, we utilised the Cellscreen system (Innovatis AG, Bielefeld, Germany), a non-invasive automated technique for measuring proliferation of adherent and suspension cells over time. We have evaluated the ability of the Cellscreen system to monitor and quantify growth of adherent liver progenitor cells over time and tested several applications, (i) serum reduction or (ii) treatment with a cytokine. Our results demonstrate that the Cellscreen system reproducibly documents pro- and anti-proliferative effects of cytokines and growth factors and quantifies changes by providing cell-doubling times for control and test cultures. However, we found that for the conversion of cell density values into absolute cell numbers different conversion factors, which better suit the individual growth phases, need to be established. Collectively, these findings reveal that the Cellscreen system is applicable for the determination of cell proliferation of adherent and suspension cells in response to a variety of (growth) factors. It minimises operator participation and thus enables more rapid and larger screens and, being non-invasive, permits multiple assays on the same culture of cells. Hence, this technique proves superior to the common proliferation assays opening up new dimensions of proliferation studies in cell biology.     

Enhanced neurally evoked responses and inhibition of norepinephrine reuptake in rat mesenteric arteries after spinal transection

In patients with high thoracic spinal lesions that remove most of the central drive to splanchnic preganglionic neurons, visceral or nociceptive stimuli below the lesion can provoke large increases in blood pressure (autonomic dysreflexia). We have examined the effects of T4 spinal transection on isometric contractions of mesenteric arteries isolated from spinalized rats. Nerve-evoked contractions involved synergistic roles for norepinephrine and ATP. At 7 wk after spinal transection, responses to perivascular stimulation at 1-5 Hz were enhanced fivefold, whereas the alpha1-adrenoceptor antagonist prazosin (10 nM) produced a twofold larger reduction in contraction (to 20 pulses at 10 Hz) than in unoperated controls. In contrast, the reduction in nerve-evoked contractions by the P2-purinoceptor antagonist suramin (0.1 mM) and the responses to the P2-purinoceptor agonist alpha,beta-methylene ATP or to high K+ concentration did not greatly differ between groups, indicating that arteries from spinalized rats were not generally hyperreactive. Sensitivity to the alpha1-adrenoceptor agonist phenylephrine was enhanced in arteries from spinalized rats, and the difference from controls was abolished by the norepinephrine uptake blocker desmethylimipramine. Sensitivity to the alpha1-adrenoceptor agonist methoxamine, which is not a substrate for the neuronal norepinephrine transporter, was similar among the groups. Thus the increased neurally evoked response after spinal transection appeared to be due to a reduction in neuronal uptake of released norepinephrine, a mechanism that did not explain the enhanced response of tail arteries after spinal transection that we previously reported. The findings provide further support for potentiated neurovascular responses contributing to the genesis of autonomic dysreflexia.     

Reduced plasma APOA1 level is associated with Gastric Tumor Growth in MKN45 mouse xenograft model

There is no suitable diagnostic and prognostic biomarker for gastric cancer. The biggest hurdles in biomarker discovery are (i) the low abundance of cancer cell-specific proteins that limits their detection and (ii) complex inter-patient variations that complicate the discovery process. To circumvent these issues, we conducted proteomics on the plasma of gastric cancer mouse xenograft and attempted to identify proteins released by cancer cells. MKN45 gastric cancer cells were subcutaneously implanted into immune-incompetent nude mice. Plasma samples collected from mice with different tumor sizes (low, mid and high tumor loads) were subjected to iTRAQ and mass spectrometric analyses. Detection of human APOA1 in mouse plasma was verified and its expression level was shown to be lower in mice with large tumors compared to those with small tumors. Studies on a panel of about 14 gastric cancer cell lines supported the notion that APOA1 in mouse plasma was of human gastric cancer cell origin. While the clinical utility of APOA1 remains to be ascertained with a larger scale study, the current work supported the feasibility of using mouse xenograft model for gastric cancer biomarker discovery.     

A model‐driven approach towards rational microbial bioprocess optimization

Due to sustainability concerns, bio‐based production capitalizing on microbes as cell factories is in demand to synthesize valuable products. Nevertheless, the nonhomogenous variations of the extracellular environment in bioprocesses often challenge the biomass growth and the bioproduction yield. To enable a more rational bioprocess optimization, we have established a model‐driven approach that systematically integrates experiments with modeling, executed from flask to bioreactor scale, and using ferulic acid to vanillin bioconversion as a case study. The impacts of mass transfer and aeration on the biomass growth and bioproduction performances were examined using minimal small‐scale experiments. An integrated model coupling the cell factory kinetics with the three‐dimensional computational hydrodynamics of bioreactor was developed to better capture the spatiotemporal distributions of bioproduction. Full‐factorial predictions were then performed to identify the desired operating conditions. A bioconversion yield of 94% was achieved, which is one of the highest for recombinant Escherichia coli using ferulic acid as the precursor.     

Developing a method for customized induction of flowering

Background  

The ability to induce flowering on demand is of significant biotechnological interest. FT protein has been recently identified as an important component of the mobile flowering hormone, florigen, whose function is conserved across the plant kingdom. We therefore focused on manipulation of both endogenous and heterologous FT genes to develop a floral induction system where flowering would be inhibited until it was induced on demand. The concept was tested in the model plant Arabidopsis thaliana (Arabidopsis).     

Potential Distribution of the Australian Native Chloris truncata Based on Modelling Both the Successful and Failed Global Introductions

Our aim was to model the current and future potential global distribution of Chloris truncata (windmill grass) based on the plant's biology, soil requirements and colonisation success. The growth response of C. truncata to constant temperatures and soil moisture levels were measured and estimated respectively, to develop parameters for a CLIMEX bioclimatic model of potential distribution. The native distribution in eastern Australia and naturalised distribution in Western Australia was also used to inform the model. Associations with soil types were assessed within the suitable bioclimatic region in Australia. The global projection of the model was tested against the distribution of soil types and the known successful and failed global introductions. The verified model was then projected to future conditions due to climate change. Optimal temperature for plant development was 28°C and the plant required 970 degree-days above a threshold of 10°C. Early collection records indicate that the species is native to Queensland, New South Wales and Victoria. The plant has been introduced elsewhere in Australia and throughout the world as a wool contaminant and as a potential pasture species, but some of the recorded establishments have failed to persist. The CLIMEX model projected to the world reflected effectively both the successful and failed distributions. The inclusion of soil associations improved the explanation of the observed distribution in Australia, but did not improve the ability to determine the potential distribution elsewhere, due to lack of similarity of soil types between continents. The addition of a climate change projection showed decreased suitability for this species in Australia, but increased suitability for other parts of the world, including regions where the plant previously failed to establish.