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Abstract In this work, the cooperation problem between two populations in a periodically varying environment is discussed. In particular, the two‐population prisoner's dilemma game with periodically oscillating payoffs is discussed, such that the time‐average of these oscillations over the period of environmental variations vanishes. The possible overlaps of these oscillations generate completely new dynamical effects that drastically change the phase space structure of the two‐population evolutionary dynamics. Due to these effects, the emergence of some level of cooperators in both populations is possible under certain conditions on the environmental variations. In the domain of stable coexistence the dynamics of cooperators in each population form stable cycles. Thus, the cooperators in each population promote the existence of cooperators in the other population. However, the survival of cooperators in both populations is not guaranteed by a large initial fraction of them.

Abstract It has been shown that the tumor population growth dynamics in a periodically varying environment can drastically differ from the one in a fixed environment. Thus, the environment of a tumor can potentially be manipulated to suppress cancer progression. Diverse evolutionary processes play vital roles in cancer progression and accordingly, understanding the interplay between these processes is essential in optimizing the treatment strategy. Somatic evolution and genetic instability result in intra‐tumor cell heterogeneity. Various models have been developed to analyze the interactions between different types of tumor cells. Here, models of density‐dependent interaction between different types of tumor cells under fast periodical environmental changes are examined. It is illustrated that tumor population densities, which vary on a slow time scale, are affected by fast environmental variations. Finally, the intriguing density‐dependent interactions in metastatic castration‐resistant prostate cancer (mCRPC) in which the different types of tumor cells are defined with respect to the production of and dependence on testosterone are discussed.

Abstract Many predators produce dormant offspring to escape harsh environmental conditions, but the evolutionary stability of this adaptation has not been fully explored. Like seed banks in plants, dormancy provides a stable competitive advantage when seasonal variations occur, because the persistence of dormant forms under harsh conditions compensates for the increased cost of producing dormant offspring. However, dormancy also exists in environments with minimal abiotic variation—an observation not accounted for by existing theory. Here it is demonstrated that dormancy can out‐compete perennial activity under conditions of extensive prey density fluctuation caused by overpredation. It is shown that at a critical level of prey density fluctuations, dormancy becomes an evolutionarily stable strategy. This is interpreted as a manifestation of Parrondo's paradox: although neither the active nor dormant forms of a dormancy‐capable predator can individually out‐compete a perennially active predator, alternating between these two losing strategies can paradoxically result in a winning strategy. Parrondo's paradox may thus explain the widespread success of quiescent behavioral strategies such as dormancy, suggesting that dormancy emerges as a natural evolutionary response to the self‐destructive tendencies of overpredation and related biological phenomena.

Background Prior studies have demonstrated the association of air pollution with cardiovascular deaths. Singapore experiences seasonal transboundary haze. We investigated the association between air pollution and acute myocardial infarction (AMI) incidence in Singapore. Methods and Results We performed a time‐stratified case‐crossover study on all AMI cases in the Singapore Myocardial Infarction Registry (2010–2015). Exposure on days where AMI occurred (case days) were compared with the exposure on days where AMI did not occur (control days). Control days were chosen on the same day of the week earlier and later in the same month and year. We fitted conditional Poisson regression models to daily AMI incidence to include confounders such as ambient temperature, rainfall, wind‐speed, and Pollutant Standards Index. We assessed relationships between AMI incidence and Pollutant Standards Index in the entire cohort and subgroups of individual‐level characteristics. There were 53 948 cases. Each 30‐unit increase in Pollutant Standards Index was association with AMI incidence (incidence risk ratio [IRR] 1.04, 95% CI 1.03–1.06). In the subgroup of ST‐segment–elevation myocardial infarction the IRR was 1.00, 95% CI 0.98 to 1.03, while for non–ST‐segment–elevation myocardial infarction, the IRR was 1.08, 95% CI 1.05 to 1.10. Subgroup analyses showed generally significant. Moderate/unhealthy Pollutant Standards Index showed association with AMI occurrence with IRR 1.08, 95% CI 1.05 to 1.11 and IRR 1.09, 95% CI 1.01 to 1.18, respectively. Excess risk remained elevated through the day of exposure and for >2 years after. Conclusions We found an effect of short‐term air pollution on AMI incidence, especially non–ST‐segment–elevation myocardial infarction and inpatient AMI. These findings have public health implications for primary prevention and emergency health services during haze.

In the present study, we functionally analysed two yapsin genes of the thermotolerant methylotrophic yeast Hansenula polymorpha, HpYPS1 and HpYPS7, for their roles in maintaining cell wall integrity and proteolytic processing. Both HpYPS1 and HpYPS7 proteins were shown to largely localize on the cell wall via glycosylphosphatidylinositol anchor. Heterologous expression of HpYPS1 completely restored all of the growth defects of the Saccharomyces cerevisiae yps1-deletion strains, while HpYPS7 expression exhibited a limited complementation effect on the S. cerevisiae yps7-deletion strain. However, different from S. cerevisiae, deletion of the HpYPS genes generated only minor influence on the sensitivity to cell wall stress. Likewise, HpYPS1 expression was significantly induced only by a subset of stressor agents, such as sodium dodecyl sulphate and tunicamycin. HpYps1p was shown to consist of two subunits, whereas HpYps7p comprises a single long polypeptide chain. Biochemical analysis revealed that HpYps1p has much stronger proteolytic cleavage activity at basic amino acids, compared to HpYps7p. Consistent with the much higher proteolytic activity and expression level of HpYps1p compared to HpYps7p, the sole disruption of HpYPS1 was sufficient in eliminating the aberrant proteolytic cleavage of recombinant proteins secreted by H. polymorpha. The results indicate that, although their roles in the maintenance of cell wall integrity are not critical, HpYps1p and HpYps7p are functional aspartic proteases at the cell surface of H. polymorpha. Furthermore, our data present the high biotechnological potential of H. polymorpha yps1-mutant strains as hosts useful for the production of secretory recombinant proteins., (Copyright © 2011 John Wiley & Sons, Ltd.)

Background and Purpose: The objective of this study was to investigate the combined influence of genetic polymorphisms in ABCB1 and CYP2D6 genes on risperidone pharmacokinetics., Experimental Approach: Seventy-two healthy Korean volunteers receiving a single oral dose of 2 mg risperidone were included in this study., Key Results: Significant differences were observed between the ABCB1 3435C>T genotypes for the pharmacokinetic parameters (peak serum concentration) of risperidone and the active moiety (risperidone and its main metabolite, 9-hydroxyrisperidone). There were no significant differences in the area under the serum concentration-time curves of risperidone and the active moiety among the ABCB1 2677G>T/A and 3435C>T genotypes. However, the peak serum concentration and area under the serum concentration-time curves were significantly different among the ABCB1 3435C>T genotypes in CYP2D6*10/*10., Conclusions and Implications: These findings indicate that polymorphisms of ABCB1 3435C>T in individuals with CYP2D6*10/*10, which has low metabolic activity, could play an important role in the potential adverse effects or toxicity of risperidone., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Since elevated ethanol is a major stress during ethanol fermentation, yeast strains tolerant to ethanol are highly desirable for the industrial scale ethanol production. A technology called global transcriptional machinery engineering (gTME), which exploits a mutant library of SPT15 encoding the TATA-binding protein of Saccharomyces cerevisiae (Alper et al., 2006; Science 314: 1565-1568), seems to a powerful tool for creating ethanol-tolerant strains. However, the ability of created strains to tolerate high ethanol on rich media remains unproven. In this study, a similar strategy was used to obtain five strains with enhanced ethanol tolerance (ETS1-5) of S. cerevisiae. Comparing global transcriptional profiles of two selected strains ETS2 and ETS3 with that of the control identified 42 genes that were commonly regulated with twofold change. Out of 34 deletion mutants available from a gene knockout library, 18 were ethanol sensitive, suggesting that these genes were closely associated with ethanol tolerance. Eight of them were novel with most being functionally unknown. To establish a basis for future industrial applications, strains iETS2 and iETS3 were created by integrating the SPT15 mutant alleles of ETS2 and ETS3 into the chromosomes, which also exhibited enhanced ethanol tolerance and survival upon ethanol shock on a rich medium. Fermentation with 20% glucose for 24 h in a bioreactor revealed that iETS2 and iETS3 grew better and produced approximately 25% more ethanol than a control strain. The ethanol yield and productivity were also substantially enhanced: 0.31 g/g and 2.6 g/L/h, respectively, for control and 0.39 g/g and 3.2 g/L/h, respectively, for iETS2 and iETS3. Thus, our study demonstrates the utility of gTME in generating strains with enhanced ethanol tolerance that resulted in increase of ethanol production. Strains with enhanced tolerance to other stresses such as heat, fermentation inhibitors, osmotic pressure, and so on, may be further created by using gTME., (Copyright © 2011 Wiley Periodicals, Inc.)

In the yeast Saccharomyces cerevisiae, the unfolded protein response (UPR) involves the unconventional splicing of HAC1 mRNA, which is mediated by the activated Ire1p transmembrane kinase/endonuclease. In this study, we isolated and characterized a Yarrowia lipolytica HAC1 (YlHAC1) encoding a basic-leucine zipper transcription factor. The null mutant strain of YlHAC1 (DeltaYlhac1) displayed a significantly increased sensitivity to dithiothreitol (DTT) and tunicamycin (TM), along with a defect in hyphal growth, suggesting the essential function of YlHAC1 in UPR. The unconventional splicing of YlHAC1 mRNA occurred under the UPR conditions induced by DTT or TM treatment. Unlike S. cerevisiae HAC1 mRNA with an intron of 252 nt, YlHAC1 mRNA was shown to harbour a short intron of length 29 nt. The YlHAC1 mRNA harboured the nucleotides CAG, conserved at the intron borders in the filamentous fungi hac1/hacA and mammalian XBP1, as well as a conserved bipartite element within the 3' untranslated region. The expression of the spliced form of YlHAC1 mRNA in the wild-type andDeltaYlhac1 strains resulted in an increased resistance to DTT, thereby indicating that the spliced form is translated into a functional YlHac1p.

Interest has been increasing in the thermotolerant methylotrophic yeast Hansenula polymorpha as a useful system for fundamental research and applied purposes. Only a few genetic marker genes and auxotrophic hosts are yet available for this yeast. Here we isolated and developed H. polymorpha TRP1, MET2 and ADE2 genes as selectable markers for multiple genetic manipulations. The H. polymorpha TRP1 (HpTRP1), MET2 (HpMET2) and ADE2 (HpADE2) genes were sequentially disrupted, using an HpURA3 pop-out cassette in H. polymorpha to generate a series of new multiple auxotrophic strains, including up to a quintuple auxotrophic strain. Unexpectedly, the HpTRP1 deletion mutants required additional tryptophan supplementation for their full growth, even on complex media such as YPD. Despite the clearly increased resistance to 5-fluoroanthranilic acid of the HpTRP1 deletion mutants, the HpTRP1 blaster cassette does not appear to be usable as a counter-selection marker in H. polymorpha. Expression vectors carrying HpADE2, HpTRP1 or HpMET2 with their own promoters and terminators as selectable markers were constructed and used to co-transform the quintuple auxotrophic strain for the targeted expression of a heterologous gene, Aspergillus saitoi MsdS, at the ER, the Golgi and the cell surface, respectively.

Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1 Delta mutant strain was constructed and its induction kinetics investigated. As expected, the gal1 Delta strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05-0.1 g/L). However, the gal1 Delta strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1 Delta mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1 Delta mutant strain, generating gal1 Delta mig1 Delta and gal1 Delta hxk2 Delta double strains. The gal1 Delta hxk2 Delta strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1 Delta strain, whereas the gal1 Delta mig1 Delta strain showed similar patterns to the gal1 Delta strain. Furthermore, the gal1 Delta hxk2 Delta strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1 Delta hxk2 Delta strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae.

The TRP1 gene encoding N-(5'-phosphoribosyl)-anthranilate isomerase was isolated from the yeast Yarrowia lipolytica, in which only a few genetic marker genes are available. The Y. lipolytica TRP1 gene (YlTRP1) cloned by complementation of Y. lipolytica trp1 mutation was found to be a functional homologue of Saccharomyces cerevisiae TRP1. Since YlTRP1 could be used for counterselection in medium containing 5-fluoroanthranilic acid (5-FAA), we constructed TRP blasters that contained YlTRP1 flanked by a direct repeat of a sequence and allowed the recycling of the YlTRP1 marker. Using the TRP blasters the sequential disruption of target genes could be carried out within the same strain of Y. lipolytica. The nucleotide sequence of the YlTRP1 gene has been deposited at GenBank under Accession No. AF420590., (Copyright 2003 John Wiley & Sons, Ltd.)

A 6.0 kb genomic DNA segment was isolated by its ability to rescue the temperature-sensitive growth defect and the hypersensitivity to sodium deoxycholate of a spontaneous vanadate-resistant mutant derived from Hansenula polymorpha DL-1. The genomic fragment contains four open reading frames homologous to the Saccharomyces cerevisiae genes YPT1 (which codes for a GTP-binding protein; 75% amino acid identity), PMI40 (encoding phosphomannose isomerase; 61% identity), YLR065c (30% identity) and CST13 (28% identity). The H. polymorpha YPT1 homologue (HpYPT1) was found to be responsible for the complementation of the temperature-sensitive phenotype and the sodium deoxycholate sensitivity of the mutant strain. Disruption of the H. polymorpha PMI40 homologue (HpPMI40) resulted in the auxotrophic requirement for D-mannose. The heterologous expressions of HpYPT1 and HpPMI40 were able to complement the temperature-sensitive phenotype of S. cerevisiae ypt1-1 mutant and the mannose auxotrophy of S. cerevisiae pmi40 null mutant, respectively, indicating that the H. polymorpha genes encode the functional homologues of S. cerevisiae YPT1 and PMI40 proteins. The nucleotide sequence has been submitted to GenBank under Accession No. AF454544., (Copyright 2002 John Wiley & Sons, Ltd.)

To optimize the secretory expression of recombinant human serum albumin (HSA) under the control of methanol oxidase (MOX) promoter in the methylotrophic yeast Hansenula polymorpha DL-1, we analyzed several parameters affecting the expression of HSA from the MOX promoter. Removal of the 5'-untranslated region derived from HSA cDNA in the expression cassette led to at least a fivefold improvement of HSA expression efficiency at the translational level. With the optimized expression cassette, the gene dosage effect on HSA expression was abolished and thus, a single copy of the expression vector integrated into the MOX locus became sufficient for the maximal expression of HSA. Northern blot analysis revealed that the levels of HSA transcript did not increase any further upon increasing copy number. The mox-disrupted (mox Delta) transformant was constructed, in which the genomic MOX gene was transplaced with the HSA expression cassette, to examine the effect of the methanol oxidase-deficient phenotype of the host on HSA expression. The mox Delta transformant showed higher levels of HSA production in shake-flask cultures than the MOX wild-type transformant, especially at low concentrations of methanol and a twofold higher specific HSA production rate in fed-batch fermentation with an abrupt induction mode. The native prepro signal sequence of HSA secreted in H. polymorpha was correctly processed and the mature recombinant protein had a pI value identical to that of the authentic HSA. Our results suggest that the H. polymorpha expression systems developed in this study are suitable for large-scale production of recombinant albumin., (Copyright John Wiley & Sons, Inc.)

A gene homologous to Saccharomyces cerevisiae MNN9 has been cloned and characterized in the methylotrophic yeast Hansenula polymorpha. This gene was cloned from a H. polymorpha genomic DNA library using the S. cerevisiae MNN9 gene as a probe. The H. polymorpha MNN9 homologue (HpMNN9) contained a 1062 bp open reading frame encoding a predicted protein of 354 amino acids. The deduced amino acid sequence showed 58% and 51% identity, respectively, with the S. cerevisiae and Candida albicans Mnn9 proteins. Disruption of HpMNN9 leads to phenotypic effects suggestive of cell wall defects, including detergent sensitivity and hygromycin B sensitivity. The hygromycin B sensitivity of S. cerevisiae mnn9 null mutant was complemented in the presence of the HpMNN9 gene. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession No. AF264786., (Copyright 2001 John Wiley & Sons, Ltd.)

A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha. The partial DNA fragment of the H. polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids. The putative gene product contains all ten of the conserved regions observed in P-type ATPase. The cloned gene product exhibits 60.3% amino acid identity to the S. cerevisiae PMR1 gene product and complemented the growth defect of a S. cerevisiae pmr1 null mutant in the EGTA-containing medium. The results demonstrate that the H. polymorpha gene encodes the functional homologue of the S. cerevesiae PMR1 gene product, a P-type Ca(2+)-ATPase.

Human alpha 1-antitrypsin (alpha 1-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human alpha 1-AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant alpha 1-AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant alpha 1-AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted alpha 1-AT. The human alpha 1-AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of alpha 1-AT in the methylotrophic yeasts appeared to be relatively shorter than those of alpha 1-AT in S. cerevisiae. The alpha 1-AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of alpha 1-AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein.