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1.  Sumoylation in Aspergillus nidulans: sumO inactivation, overexpression and live-cell imaging 
Sumoylation, the reversible covalent attachment of small ubiquitin-like modifier (SUMO) peptides has emerged as an important regulator of target protein function. In Saccharomyces cerevisiae, but not in Schizosaccharyomes pombe, deletion of the gene encoding SUMO peptides is lethal. We have characterized the SUMO-encoding gene, sumO, in the filamentous fungus Aspergillus nidulans. The sumO gene was deleted in a diploid and sumO• haploids were recovered. The mutant was viable but exhibited impaired growth, reduced conidiation and self-sterility. Overexpression of epitope-tagged SumO peptides revealed multiple sumoylation targets in A. nidulans and SumO overexpression resulted in greatly increased levels of protein sumoylation without obvious phenotypic consequences. Using five-piece fusion PCR, we generated a gfp-sumO fusion gene expressed from the sumO promoter for live cell imaging of GFP-SumO and GFP-SumO-conjugated proteins. Localisation of GFP-SumO is dynamic, accumulating in punctate spots within the nucleus during interphase, lost at the onset of mitosis and re-accumulating during telophase.
doi:10.1016/j.fgb.2007.12.009
PMCID: PMC4220683  PMID: 18262811
SUMO; conidiation; sexual development; fusion PCR; cell cycle
2.  An Efficient System for Heterologous Expression of Secondary Metabolite Genes in Aspergillus nidulans 
Fungal secondary metabolites (SMs) are an important source of medically valuable compounds. Genome projects have revealed that fungi have many SM biosynthetic gene clusters that are not normally expressed. To access these potentially valuable, cryptic clusters, we have developed a heterologous expression system in Aspergillus nidulans. We have developed an efficient system for amplifying genes from a target fungus, placing them under control of a regulatable promoter, transferring them into A. nidulans and expressing them. We have validated this system by expressing non-reducing polyketide synthases of Aspergillus terreus and additional genes required for compound production and release. We have obtained compound production and release from six of these NR-PKSs and have identified the products. To demonstrate that the procedure allows transfer and expression of entire secondary metabolite biosynthetic pathways, we have expressed all the genes of a silent A. terreus cluster and demonstrate that it produces asperfuranone. Further, by expressing the genes of this pathway in various combinations, we have clarified the asperfuranone biosynthetic pathway. We have also developed procedures for deleting entire A. nidulans SM clusters. This allows us to remove clusters that might interfere with analyses of heterologously expressed genes and to eliminate unwanted toxins.
doi:10.1021/ja401945a
PMCID: PMC3697937  PMID: 23621425
3.  Molecular genetic analysis of the orsellinic acid/F9775 gene cluster of Aspergillus nidulans†‡ 
Molecular bioSystems  2009;6(3):587-593.
F-9775A and F-9775B are cathepsin K inhibitors that arise from a chromatin remodelling deletant strain of Aspergillus nidulans. A polyketide synthase gene has been determined to be responsible for their formation and for the simpler, archetypical polyketide orsellinic acid. We have discovered simple culture conditions that result in the production of the three compounds, and this facilitates analysis of the genes responsible for their synthesis. We have now analysed the F9775/orsellinic acid gene cluster using a set of targeted deletions. We find that the polyketide synthase alone is required for orsellinic acid biosynthesis and only two additional genes in the cluster are required for F9775 A and B synthesis. Our deletions also yielded the bioactive metabolites gerfelin and diorcinol.
doi:10.1039/b904541d
PMCID: PMC2903553  PMID: 20174687
4.  Identification and Characterization of the Asperthecin Gene Cluster of Aspergillus nidulans▿ † 
Applied and Environmental Microbiology  2008;74(24):7607-7612.
The sequencing of Aspergillus genomes has revealed that the products of a large number of secondary metabolism pathways have not yet been identified. This is probably because many secondary metabolite gene clusters are not expressed under normal laboratory culture conditions. It is, therefore, important to discover conditions or regulatory factors that can induce the expression of these genes. We report that the deletion of sumO, the gene that encodes the small ubiquitin-like protein SUMO in A. nidulans, caused a dramatic increase in the production of the secondary metabolite asperthecin and a decrease in the synthesis of austinol/dehydroaustinol and sterigmatocystin. The overproduction of asperthecin in the sumO deletion mutant has allowed us, through a series of targeted deletions, to identify the genes required for asperthecin synthesis. The asperthecin biosynthesis genes are clustered and include genes encoding an iterative type I polyketide synthase, a hydrolase, and a monooxygenase. The identification of these genes allows us to propose a biosynthetic pathway for asperthecin.
doi:10.1128/AEM.01743-08
PMCID: PMC2607171  PMID: 18978088
5.  Cytoplasmic Dynein's Mitotic Spindle Pole Localization Requires a Functional Anaphase-promoting Complex, γ-Tubulin, and NUDF/LIS1 in Aspergillus nidulans 
Molecular Biology of the Cell  2005;16(8):3591-3605.
In Aspergillus nidulans, cytoplasmic dynein and NUDF/LIS1 are found at the spindle poles during mitosis, but they seem to be targeted to this location via different mechanisms. The spindle pole localization of cytoplasmic dynein requires the function of the anaphase-promoting complex (APC), whereas that of NUDF does not. Moreover, although NUDF's localization to the spindle poles does not require a fully functional dynein motor, the function of NUDF is important for cytoplasmic dynein's targeting to the spindle poles. Interestingly, a γ-tubulin mutation, mipAR63, nearly eliminates the localization of cytoplasmic dynein to the spindle poles, but it has no apparent effect on NUDF's spindle pole localization. Live cell analysis of the mipAR63 mutant revealed a defect in chromosome separation accompanied by unscheduled spindle elongation before the completion of anaphase A, suggesting that γ-tubulin may recruit regulatory proteins to the spindle poles for mitotic progression. In A. nidulans, dynein is not apparently required for mitotic progression. In the presence of a low amount of benomyl, a microtubule-depolymerizing agent, however, a dynein mutant diploid strain exhibits a more pronounced chromosome loss phenotype than the control, indicating that cytoplasmic dynein plays a role in chromosome segregation.
doi:10.1091/mbc.E04-12-1071
PMCID: PMC1182300  PMID: 15930134
6.  γ-Tubulin Plays an Essential Role in the Coordination of Mitotic EventsV⃞ 
Molecular Biology of the Cell  2004;15(3):1374-1386.
Recent data from multiple organisms indicate that γ-tubulin has essential, but incompletely defined, functions in addition to nucleating microtubule assembly. To investigate these functions, we examined the phenotype of mipAD159, a cold-sensitive allele of the γ-tubulin gene of Aspergillus nidulans. Immunofluorescence microscopy of synchronized material revealed that at a restrictive temperature mipAD159 does not inhibit mitotic spindle formation. Anaphase A was inhibited in many nuclei, however, and after a slight delay in mitosis (∼6% of the cell cycle period), most nuclei reentered interphase without dividing. In vivo observations of chromosomes at a restrictive temperature revealed that mipAD159 caused a failure of the coordination of late mitotic events (anaphase A, anaphase B, and chromosomal disjunction) and nuclei reentered interphase quickly even though mitosis was not completed successfully. Time-lapse microscopy also revealed that transient mitotic spindle abnormalities, in particular bent spindles, were more prevalent in mipAD159 strains than in controls. In experiments in which microtubules were depolymerized with benomyl, mipAD159 nuclei exited mitosis significantly more quickly (as judged by chromosomal condensation) than nuclei in a control strain. These data reveal that γ-tubulin has an essential role in the coordination of late mitotic events, and a microtubule-independent function in mitotic checkpoint control.
doi:10.1091/mbc.E03-06-0405
PMCID: PMC363147  PMID: 14668489
7.  TINA Interacts with the NIMA Kinase in Aspergillus nidulans and Negatively Regulates Astral Microtubules during Metaphase ArrestV⃞ 
Molecular Biology of the Cell  2003;14(8):3169-3179.
The tinA gene of Aspergillus nidulans encodes a protein that interacts with the NIMA mitotic protein kinase in a cell cycle-specific manner. Highly similar proteins are encoded in Neurospora crassa and Aspergillus fumigatus. TINA and NIMA preferentially interact in interphase and larger forms of TINA are generated during mitosis. Localization studies indicate that TINA is specifically localized to the spindle pole bodies only during mitosis in a microtubule-dependent manner. Deletion of tinA alone is not lethal but displays synthetic lethality in combination with the anaphase-promoting complex/cyclosome mutation bimE7. At the bimE7 metaphase arrest point, lack of TINA enhanced the nucleation of bundles of cytoplasmic microtubules from the spindle pole bodies. These microtubules interacted to form spindles joined in series via astral microtubules as revealed by live cell imaging. Because TINA is modified and localizes to the spindle pole bodies at mitosis, and lack of TINA causes enhanced production of cytoplasmic microtubules at metaphase arrest, we suggest TINA is involved in negative regulation of the astral microtubule organizing capacity of the spindle pole bodies during metaphase.
doi:10.1091/mbc.E02-11-0715
PMCID: PMC181558  PMID: 12925754
8.  Spindle Formation in Aspergillus Is Coupled to Tubulin Movement into the NucleusV⃞ 
Molecular Biology of the Cell  2003;14(5):2192-2200.
In many important organisms, including many algae and most fungi, the nuclear envelope does not disassemble during mitosis. This fact raises the possibility that mitotic onset and/or exit might be regulated, in part, by movement of important mitotic proteins into and out of the nucleoplasm. We have used two methods to determine whether tubulin levels in the nucleoplasm are regulated in the fungus Aspergillus nidulans. First, we have used benomyl to disassemble microtubules and create a pool of free tubulin that can be readily observed by immunofluorescence. We find that tubulin is substantially excluded from interphase nuclei, but is present in mitotic nuclei. Second, we have observed a green fluorescent protein/α-tubulin fusion in living cells by time-lapse spinning-disk confocal microscopy. We find that tubulin is excluded from interphase nuclei, enters the nucleus seconds before the mitotic spindle begins to form, and is removed from the nucleoplasm during the M-to-G1 transition. Our data indicate that regulation of intranuclear tubulin levels plays an important, perhaps essential, role in the control of mitotic spindle formation in A. nidulans. They suggest that regulation of protein movement into the nucleoplasm may be important for regulating mitotic onset in organisms with intranuclear mitosis.
doi:10.1091/mbc.E02-10-0641
PMCID: PMC165107  PMID: 12802085
9.  Isolation of mip (microtubule-interacting protein) mutations of Aspergillus nidulans. 
Molecular and Cellular Biology  1986;6(8):2963-2968.
We identified four mutations in two previously undescribed loci involved in microtubule function in Aspergillus nidulans as extragenic suppressors of benA33, a heat-sensitive beta-tubulin mutation. Three of the four mutations map to a locus closely linked to riboB on linkage group VIII; we designated this locus mipA (for microtubule-interacting protein). We were not able to map the remaining suppressor because of chromosomal rearrangements. However, since it recombines with riboB at a significantly higher frequency than the mipA alleles, it is unlikely to be in mipA; thus, we designated it mipB1. The mip mutations are not allelic to the previously identified loci that encode alpha- and beta-tubulin, and it is likely that mipA and mipB encode previously unidentified nontubulin proteins involved in microtubule function. Each of the mip mutations suppresses the heat sensitivity conferred by benA33 and suppresses the blockage of nuclear division and movement conferred by this mutation at high temperatures. Interactions between mipA and benA are allele specific. All of the mipA mutations are cryptic in a wild-type benA background but cause cold sensitivity in combination with benA33. These mutations also confer cold sensitivity in combination with benA31 and benA32 and reduce the resistance conferred by these mutations to the antimicrotubule agent benomyl but do not suppress the heat sensitivity conferred by these alleles. Finally, the mipA alleles suppress the heat sensitivity conferred by benA11, benA17, and benA21 but do not confer cold sensitivity in combination with these alleles.
Images
PMCID: PMC367866  PMID: 3537728
10.  Management of pregnancy in patients with hypertrophic cardiomyopathy. 
British Medical Journal  1979;1(6180):1749-1750.
The outcome of 54 pregnancies in 23 patients with hypertrophic cardiomyopathy was analysed. No mother or infant died in the perinatal period. Six patients developed dyspnoea requiring treatment with diuretics. Beta-adrenergic blocking drugs were given in 18 pregnancies and three of the infants in this were small for dates and in two fetal bradycardia occurred. The results comfirmed that pregnancy is safe in patients with hypertrophic cardiomyopathy. A flexible approach should be adopted towards administering beta-adrenergic blocking drugs to pregnant women with hypertrophic cardiomyopathy. Many such patients do well without these drugs and can thus avoid the potential hazards--namely, small-for-dates babies and fetal bradycardia--that are associated with them.
PMCID: PMC1599373  PMID: 572730
11.  γ-Tubulin and the C-Terminal Motor Domain Kinesin-like Protein, KLPA, Function in the Establishment of Spindle Bipolarity in Aspergillus nidulans 
Molecular Biology of the Cell  2001;12(10):3161-3174.
Previous research has found that a γ-tubulin mutation in Schizosaccharomyces pombe is synthetically lethal with a deletion of the C-terminal motor domain kinesin-like protein gene pkl1, but the lethality of the double mutant prevents a phenotypic analysis of the synthetic interaction. We have investigated interactions between klpA1, a deletion of an Aspergillus nidulans homolog of pkl1, and mutations in the mipA, γ-tubulin gene. We find that klpA1 dramatically increases the cold sensitivity and slightly reduces the growth rate at all temperatures, of three mipA alleles. In synchronized cells we find that klpA1 causes a substantial but transient inhibition of the establishment of spindle bipolarity. At a restrictive temperature, mipAD123 causes a slight, transient inhibition of spindle bipolarity and a more significant inhibition of anaphase A. In the mipAD123/klpA1 strain, formation of bipolar spindles is more strongly inhibited than in the klpA1 single mutant and many spindles apparently never become bipolar. These results indicate, surprisingly, that γ-tubulin and the klpA kinesin have overlapping roles in the establishment of spindle bipolarity. We propose a model to account for these data.
PMCID: PMC60164  PMID: 11598200
12.  Alanine-scanning Mutagenesis of Aspergillus γ-Tubulin Yields Diverse and Novel PhenotypesV⃞ 
Molecular Biology of the Cell  2001;12(7):2119-2136.
We have created 41 clustered charged-to-alanine scanning mutations of the mipA, γ-tubulin, gene of Aspergillus nidulans and have created strains carrying these mutations by two-step gene replacement and by a new procedure, heterokaryon gene replacement. Most mutant alleles confer a wild-type phenotype, but others are lethal or conditionally lethal. The conditionally lethal alleles exhibit a variety of phenotypes under restrictive conditions. Most have robust but highly abnormal mitotic spindles and some have abnormal cytoplasmic microtubule arrays. Two alleles appear to have reduced amounts of γ-tubulin at the spindle pole bodies and nucleation of spindle microtubule assembly may be partially inhibited. One allele inhibits germ tube formation. The cold sensitivity of two alleles is strongly suppressed by the antimicrotubule agents benomyl and nocodazole and a third allele is essentially dependent on these compounds for growth. Together our data indicate that γ-tubulin probably carries out functions essential to mitosis and organization of cytoplasmic microtubules in addition to its well-documented role in microtubule nucleation. We have also placed our mutations on a model of the structure of γ-tubulin and these data give a good initial indication of the functionally important regions of the molecule.
PMCID: PMC55667  PMID: 11452008
13.  The Crystal Structure of the Hinge Domain of the Escherichia coli Structural Maintenance of Chromosomes Protein MukB 
Journal of molecular biology  2009;395(1):11-19.
MukB, a divergent structural maintenance of chromosomes (SMC) protein, is important for chromosomal segregation and condensation in γ-proteobacteria. MukB and canonical SMC proteins share a characteristic five-domain structure. Globular N- and C-terminal domains interact to form an ABC-like ATPase or “head” domain, which is connected to a smaller dimerization or “hinge” domain by a long, antiparallel coiled coil. In addition to mediating dimerization, this hinge region has been implicated in both conformational flexibility and dynamic protein-DNA interactions. We report here the first crystallographic model of the MukB hinge domain. This model also contains approximately 20% of the coiled coil domain, including an unusual coiled coil deviation. These results will facilitate studies to clarify the roles of both the hinge and coiled coil domains in MukB function.
doi:10.1016/j.jmb.2009.10.040
PMCID: PMC4335309  PMID: 19853611
MukB; Structural Maintenance of Chromosomes (SMC); hinge; coiled coil; condensin
14.  DNA Damage Induced Hyperphosphorylation of Replication Protein A. 1. Identification of Novel Sites of Phosphorylation in Response to DNA Damage† 
Biochemistry  2005;44(23):8428-8437.
Replication protein A (RPA) is the predominant eukaryotic single-stranded DNA binding protein composed of 70, 34, and 14 kDa subunits. RPA plays central roles in the processes of DNA replication, repair, and recombination, and the p34 subunit of RPA is phosphorylated in a cell-cycle-dependent fashion and is hyperphosphorylated in response to DNA damage. We have developed an in vitro procedure for the preparation of hyperphosphorylated RPA and characterized a series of novel sites of phosphorylation using a combination of in gel tryptic digestion, SDS–PAGE and HPLC, MALDI-TOF MS analysis, 2D gel electrophoresis, and phosphospecific antibodies. We have mapped five phosphorylation sites on the RPA p34 subunit and five sites of phosphorylation on the RPA p70 subunit. No modification of the 14 kDa subunit was observed. Using the procedures developed with in vitro phosphorylated RPA, we confirmed a series of phosphorylation events on RPA from HeLa cells that was hyperphosphorylated in vivo in response to the DNA damaging agents, aphidicolin and hydroxyurea.
doi:10.1021/bi0480584
PMCID: PMC4331072  PMID: 15938632
15.  DNA Damage Induced Hyperphosphorylation of Replication Protein A. 2. Characterization of DNA Binding Activity, Protein Interactions, and Activity in DNA Replication and Repair† 
Biochemistry  2005;44(23):8438-8448.
Replication protein A (RPA) is a heterotrimeric protein consisting of 70-, 34-, and 14- kDa subunits that is required for many DNA metabolic processes including DNA replication and DNA repair. Using a purified hyperphosphorylated form of RPA protein prepared in vitro, we have addressed the effects of hyperphosphorylation on steady-state and pre-steady-state DNA binding activity, the ability to support DNA repair and replication reactions, and the effect on the interaction with partner proteins. Equilibrium DNA binding activity measured by fluorescence polarization reveals no difference in ssDNA binding to pyrimidine-rich DNA sequences. However, RPA hyperphosphorylation results in a decreased affinity for purine-rich ssDNA and duplex DNA substrates. Pre-steady-state kinetic analysis is consistent with the equilibrium DNA binding and demonstrates a contribution from both the kon and koff to achieve these differences. The hyperphosphorylated form of RPA retains damage-specific DNA binding, and, importantly, the affinity of hyperphosphorylated RPA for damaged duplex DNA is 3-fold greater than the affinity of unmodified RPA for undamaged duplex DNA. The ability of hyperphosphorylated RPA to support DNA repair showed minor differences in the ability to support nucleotide excision repair (NER). Interestingly, under reaction conditions in which RPA is maintained in a hyperphosphorylated form, we also observed inhibition of in vitro DNA replication. Analyses of protein–protein interactions bear out the effects of hyperphosphorylated RPA on DNA metabolic pathways. Specifically, phosphorylation of RPA disrupts the interaction with DNA polymerase α but has no significant effect on the interaction with XPA. These results demonstrate that the effects of DNA damage induced hyperphosphorylation of RPA on DNA replication and DNA repair are mediated through alterations in DNA binding activity and protein–protein interactions.
doi:10.1021/bi048057b
PMCID: PMC4328999  PMID: 15938633
16.  Recent advances in genome mining of secondary metabolite biosynthetic gene clusters and the development of heterologous expression systems in Aspergillus nidulans 
Fungi are prolific producers of secondary metabolites (SMs) that show a variety of biological activities. Recent advances in genome sequencing have shown that fungal genomes harbor far more SM gene clusters than are expressed under conventional laboratory conditions. Activation of these “silent” gene clusters is a major challenge, and many approaches have been taken to attempt to activate them and, thus, unlock the vast treasure chest of fungal SMs. This review will cover recent advances in genome mining of SMs in Aspergillus nidulans. We will also discuss current updates in gene annotation of A. nidulans and recent developments in A. nidulans as a molecular genetic system, both of which are essential for rapid and efficient experimental verification of SM gene clusters on a genome-wide scale. Finally, we will describe advances in the use of A. nidulans as a heterologous expression system to aid in the analysis of SM gene clusters from other fungal species that do not have an established molecular genetic system.
doi:10.1007/s10295-013-1386-z
PMCID: PMC4324552  PMID: 24342965
Aspergillus; secondary metabolite; polyketide synthase; nonribosomal peptide synthetase; gene cluster
17.  Eye-specification genes in the bacterial light organ of the bobtail squid Euprymna scolopes, and their expression in response to symbiont cues 
Mechanisms of development  2013;131:111-126.
The squid Euprymna scolopes has evolved independent sets of tissues capable of light detection, including a complex eye and a photophore or ‘light organ’, which houses the luminous bacterial symbiont Vibrio fischeri. As the eye and light organ originate from different embryonic tissues, we examined whether the eye-specification genes, pax6, eya, six, and dac, are shared by these two organs, and if so, whether they are regulated in the light organ by symbiosis. We obtained sequences of the four genes with PCR, confirmed orthology with phylogenetic analysis, and determined that each was expressed in the eye and light organ. With in situ hybridization (ISH), we localized the gene transcripts in developing embryos, comparing the patterns of expression in the two organs. The four transcripts localized to similar tissues, including those associated with the visual system ~1/4 into embryogenesis (Naef stage 18) and the light organ ~3/4 into embryogenesis (Naef stage 26). We used ISH and quantitative real-time PCR to examine transcript expression and differential regulation in postembryonic light organs in response to the following colonization conditions: wild-type, luminescent V. fischeri; a mutant strain defective in light production; and as a control, no symbiont. In ISH experiments light organs showed down regulation of the pax6, eya, and six transcripts in response to wild-type V. fischeri. Mutant strains also induced down regulation of the pax6 and eya transcripts, but not of the six transcript. Thus, luminescence was required for down regulation of the six transcript. We discuss these results in the context of symbiont-induced light-organ development. Our study indicates that the eye-specification genes are expressed in light-interacting tissues independent of their embryonic origin and are capable of responding to bacterial cues. These results offer evidence for evolutionary tinkering or the recruitment of eye development genes for use in a light-sensing photophore.
doi:10.1016/j.mod.2013.09.004
PMCID: PMC4000693  PMID: 24157521
immunity; microbe; photoreceptor; rhabdomere; retina; vision
18.  Comparative Analysis of the Intestinal Bacterial and RNA Viral Communities from Sentinel Birds Placed on Selected Broiler Chicken Farms 
PLoS ONE  2015;10(1):e0117210.
There is a great deal of interest in characterizing the complex microbial communities in the poultry gut, and in understanding the effects of these dynamic communities on poultry performance, disease status, animal welfare, and microbes with human health significance. Investigations characterizing the poultry enteric virome have identified novel poultry viruses, but the roles these viruses play in disease and performance problems have yet to be fully characterized. The complex bacterial community present in the poultry gut influences gut development, immune status, and animal health, each of which can be an indicator of overall performance. The present metagenomic investigation was undertaken to provide insight into the colonization of specific pathogen free chickens by enteric microorganisms under field conditions and to compare the pre-contact intestinal microbiome with the altered microbiome following contact with poultry raised in the field. Analysis of the intestinal virome from contact birds (“sentinels”) placed on farms revealed colonization by members of the Picornaviridae, Picobirnaviridae, Reoviridae, and Astroviridae that were not present in pre-contact birds or present in proportionally lower numbers. Analysis of the sentinel gut bacterial community revealed an altered community in the post-contact birds, notably by members of the Lachnospiracea/Clostridium and Lactobacillus families and genera. Members of the avian enteric Reoviridae and Astroviridae have been well-characterized and have historically been implicated in poultry enteric disease; members of the Picobirnaviridae and Picornaviridae have only relatively recently been described in the poultry and avian gut, and their roles in the recognized disease syndromes and in poultry performance in general have not been determined. This metagenomic analysis has provided insight into the colonization of the poultry gut by enteric microbes circulating in commercial broiler flocks, and has identified enteric viruses and virus communities that warrant further study in order to understand their role(s) in avian gut health and disease.
doi:10.1371/journal.pone.0117210
PMCID: PMC4311960  PMID: 25635690
19.  A New Finite Element Approach for Near Real-Time Simulation of Light Propagation in Locally Advanced Head and Neck Tumors 
Lasers in surgery and medicine  2015;47(1):60-67.
Background and Objectives:
Several clinical studies suggest that interstitial photodynamic therapy (I-PDT) may benefit patients with locally advanced head and neck cancer (LAHNC). For I-PDT, the therapeutic light is delivered through optical fibers inserted into the target tumor. The complex anatomy of the head and neck requires careful planning of fiber insertions. Often the fibers’ location and tumor optical properties may vary from the original plan therefore pretreatment planning needs near real-time updating to account for any changes. The purpose of this work was to develop a finite element analysis (FEA) approach for near real-time simulation of light propagation in LAHNC.
Methods:
Our previously developed FEA for modeling light propagation in skin tissue was modified to simulate light propagation from interstitial optical fibers. The modified model was validated by comparing the calculations with measurements in a phantom mimicking tumor optical properties. We investigated the impact of mesh element size and growth rate on the computation time, and defined optimal settings for the FEA. We demonstrated how the optimized FEA can be used for simulating light propagation in two cases of LAHNC amenable to I-PDT, as proof-of-concept.
Results:
The modified FEA was in agreement with the measurements (P=0.0271). The optimal maximum mesh size and growth rate were 0.005-0.02 m and 2-2.5 m/m, respectively. Using these settings the computation time for simulating light propagation in LAHNC was reduced from 25.9 to 3.7 min in one case, and 10.1 to 4 minutes in another case. There were minor differences (1.62%, 1.13%) between the radiant exposures calculated with either mesh in both cases.
Conclusions:
Our FEA approach can be used to model light propagation from diffused optical fibers in complex heterogeneous geometries representing LAHNC. There is a range of maximum element size (MES) and maximum element growth rate (MEGR) that can be used to minimize the computation time of the FEA to 4 minutes.
doi:10.1002/lsm.22313
PMCID: PMC4304874  PMID: 25559426
20.  Bayesian History Matching of Complex Infectious Disease Models Using Emulation: A Tutorial and a Case Study on HIV in Uganda 
PLoS Computational Biology  2015;11(1):e1003968.
Advances in scientific computing have allowed the development of complex models that are being routinely applied to problems in disease epidemiology, public health and decision making. The utility of these models depends in part on how well they can reproduce empirical data. However, fitting such models to real world data is greatly hindered both by large numbers of input and output parameters, and by long run times, such that many modelling studies lack a formal calibration methodology. We present a novel method that has the potential to improve the calibration of complex infectious disease models (hereafter called simulators). We present this in the form of a tutorial and a case study where we history match a dynamic, event-driven, individual-based stochastic HIV simulator, using extensive demographic, behavioural and epidemiological data available from Uganda. The tutorial describes history matching and emulation. History matching is an iterative procedure that reduces the simulator's input space by identifying and discarding areas that are unlikely to provide a good match to the empirical data. History matching relies on the computational efficiency of a Bayesian representation of the simulator, known as an emulator. Emulators mimic the simulator's behaviour, but are often several orders of magnitude faster to evaluate. In the case study, we use a 22 input simulator, fitting its 18 outputs simultaneously. After 9 iterations of history matching, a non-implausible region of the simulator input space was identified that was times smaller than the original input space. Simulator evaluations made within this region were found to have a 65% probability of fitting all 18 outputs. History matching and emulation are useful additions to the toolbox of infectious disease modellers. Further research is required to explicitly address the stochastic nature of the simulator as well as to account for correlations between outputs.
Author Summary
An increasing number of scientific disciplines, and biology in particular, rely on complex computational models. The utility of these models depends on how well they are fitted to empirical data. Fitting is achieved by searching for suitable values for the models' input parameters, in a process known as calibration. Modern computer models typically have a large number of input and output parameters, and long running times, a consequence of their increasing computational complexity. The above two things hinder the calibration process. In this work, we propose a method that can help the calibration of models with long running times and several inputs and outputs. We apply this method on an individual based, dynamic and stochastic HIV model, using HIV data from Uganda. The final system has a 65% probability of selecting an input parameter set that fits all 18 model outputs.
doi:10.1371/journal.pcbi.1003968
PMCID: PMC4288726  PMID: 25569850
21.  A surgeon led smoking cessation intervention in a head and neck cancer centre 
Background
The government has recognised the role of healthcare professionals in smoking cessation interventions with integrated care pathways for identification and referral of at-risk patients who smoke. Referral for suspected cancers has been suggested as a ‘teachable moment’, whereby individuals are motivated and more likely to adopt risk-reducing behaviours. A head and neck cancer referral clinic could therefore provide opportunities for smoking cessation intervention.
This study aims to pilot a brief smoking cessation intervention during a consultation visit for patients referred with suspected head and neck cancer and evaluate its acceptability and impact.
Methods
A brief script for smoking cessation intervention which included a smoking cessation referral was designed to be delivered to patients attending a rapid access clinic. Patient outcome data was collected by the stop smoking team for patients who accepted the referral. A subset of these patients was also interviewed by telephone; these findings were combined with data provided by the stop smoking services to assess the acceptability and impact of pilot smoking cessation intervention on patients.
Results
In total, 473 new patients attended the clinic during the study period, of whom 102 (22%) were smokers. Of these, 80 (78%) accepted a referral to stop smoking services. A total of 75 (74%) patients were approached subsequently in a telephone survey. Of the 80 newly referred patients, 29 (36%) quit smoking at least temporarily. Another eight patients reduced their smoking or set a quit date (10%), so the experience of attending the clinic and the intervention impacted favourably on almost half of the patients (46%). The patient survey found the intervention to be acceptable for 94% (n = 50) of patients. Qualitative analysis of patient responses revealed five elements which support the acceptability of the intervention.
Conclusions
The findings of this pilot study suggest that discussion of smoking cessation with patients referred for suspected head and neck cancer may have an impact and facilitate the process towards quitting. A possible diagnosis of cancer appears to present a ‘teachable moment’ to encourage positive health behaviour change.
doi:10.1186/s12913-014-0636-8
PMCID: PMC4301942  PMID: 25527115
Smoking cessation; Stop smoking; Head and neck cancer; Oral cancer; Prevention
22.  Comparison of Speech Recognition and Localization Performance in Bilateral and Unilateral Cochlear Implant Users Matched on Duration of Deafness and Age at Implantation 
Ear and hearing  2008;29(3):352-359.
Objective
The purpose of this investigation was to compare speech recognition and localization performance of subjects who wear bilateral cochlear implants (CICI) with subjects who wear a unilateral cochlear implant (true CI-only).
Design
A total of 73 subjects participated in this study. Specifically, of the 73 subjects, 64 (32 CICI and 32 true CI-only) participated in the word recognition testing; 66 (33 CICI and 33 true CI-only) participated in the sentence recognition testing; and 24 (12 CICI and 12 true CI-only) participated in the localization testing. Because of time constraints not all subjects completed all testing. The average age at implantation for the CICI and true CI-only listeners who participated in the speech perception testing was 54 and 55 yrs, respectively, and the average duration of deafness was 8 yrs for both groups of listeners. The average age at implantation for the CICI and true CI-only listeners who participated in the localization testing was 54 and 53 yrs, respectively, and the average duration of deafness was 10 yrs for the CICI listeners and 11 yrs for the true CI-only listeners. All speech stimuli were presented from the front. The test setup for everyday-sound localization comprised an eight-speaker array spanning, an arc of approximately 108° in the frontal horizontal plane.
Results
Average group results were transformed to Rationalized Arcsine Unit scores. A comparison in performance between the CICI score and the true CI-only score in quiet revealed a significant difference between the two groups with the CICI group scoring 19% higher for sentences and 24% higher for words. In addition, when both cochlear implants were used together (CICI) rather than when either cochlear implant was used alone (right CI or left CI) for the CICI listeners, results indicated a significant binaural summation effect for sentences and words.
Conclusion
The average group results in this study showed significantly greater benefit on words and sentences in quiet and localization for listeners using two cochlear implants over those using only one cochlear implant. One explanation of this result might be that the same information from both sides are combined, which results in a better representation of the stimulus. A second explanation might be that CICI allow for the transfer of different neural information from two damaged peripheral auditory systems leading to different patterns of information summating centrally resulting in enhanced speech perception. A future study using similar methodology to the current one will have to be conducted to determine if listeners with two cochlear implants are able to perform better than listeners with one cochlear implant in noise.
doi:10.1097/AUD.0b013e318167b870
PMCID: PMC4266575  PMID: 18453885
23.  Ethnic variation in stillbirth risk and the role of maternal obesity: analysis of routine data from a London maternity unit 
Background
Approximately 5 in 1,000 deliveries in England and Wales result in stillbirth, with little improvement in figures over the last few decades. The aim of this study was to investigate the association between clinical and socio-demographic factors and stillbirth, with a particular focus on ethnicity and obesity.
Methods
Analysis of routine maternity data on 53,293 singleton births occurring in a large London teaching hospital between 2004 and 2012. Logistic regression was used to investigate risk factors for stillbirth and to explore potential effect modification.
Results
53,293 deliveries occurred during the time period, of which 329 resulted in a stillbirth (6.2 per 1,000 births). Compared to White women, non-White ethnicity was associated with a doubling of the odds of stillbirth (aOR for Black women 2.15, 95% CI 1.56-2.97; aOR for South Asian women 2.33, 95% CI 1.42-3.83). Obese women had a trend towards higher odds of stillbirth compared to women of recommended BMI (aOR 1.38, 95% CI 0.98-1.96), though this was not significant (p 0.07). Both higher parity (≥2 compared to para 1) and hypertension were associated with a higher odds of stillbirth (parity ≥2 aOR 1.65, 95% CI 1.13-2.39; hypertension aOR 1.84, 95% CI 1.22-2.78) but there was no evidence that area deprivation or maternal age were independently associated with stillbirth in this population. There was some evidence of effect modification between ethnicity and obesity (p value for interaction 0.06), with obesity a particularly strong risk factor for stillbirth in South Asian women (aOR 4.64, 95% CI 1.84-11.70).
Conclusions
There was a high prevalence of stillbirth in this multi-ethnic urban population. The increased risk of stillbirth observed in non-White women remains after adjusting for other factors. Our finding of possible effect modification between ethnicity and obesity suggests that further research should be conducted in order to improve understanding of the interplay between ethnicity, obesity and stillbirth.
Electronic supplementary material
The online version of this article (doi:10.1186/s12884-014-0404-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12884-014-0404-0
PMCID: PMC4272534  PMID: 25481783
Stillbirth; Obesity; Ethnicity
24.  Gene Pathways That Delay Caenorhabditis elegans Reproductive Senescence 
PLoS Genetics  2014;10(12):e1004752.
Reproductive senescence is a hallmark of aging. The molecular mechanisms regulating reproductive senescence and its association with the aging of somatic cells remain poorly understood. From a full genome RNA interference (RNAi) screen, we identified 32 Caenorhabditis elegans gene inactivations that delay reproductive senescence and extend reproductive lifespan. We found that many of these gene inactivations interact with insulin/IGF-1 and/or TGF-β endocrine signaling pathways to regulate reproductive senescence, except nhx-2 and sgk-1 that modulate sodium reabsorption. Of these 32 gene inactivations, we also found that 19 increase reproductive lifespan through their effects on oocyte activities, 8 of them coordinate oocyte and sperm functions to extend reproductive lifespan, and 5 of them can induce sperm humoral response to promote reproductive longevity. Furthermore, we examined the effects of these reproductive aging regulators on somatic aging. We found that 5 of these gene inactivations prolong organismal lifespan, and 20 of them increase healthy life expectancy of an organism without altering total life span. These studies provide a systemic view on the genetic regulation of reproductive senescence and its intersection with organism longevity. The majority of these newly identified genes are conserved, and may provide new insights into age-associated reproductive senescence during human aging.
Author Summary
Female reproductive senescence is one hallmark of human aging, and as the germline ages, there is increased incidence of chromosome non-dysjunction and DNA damage. Delayed childbearing is a general feature in modern society, resulting in high risk of infertility, miscarriage and birth defects. Thus, understanding the molecular mechanisms regulating reproductive senescence and its association with somatic senescence is increasingly relevant to human health, and will shed light on the prolongation of reproductive longevity and the improvement of post-reproductive health. Here we conducted a genomic screen in Caenorhabditis elegans, searching for genetic regulators of reproductive senescence. We identified 32 gene inactivations that extend reproductive lifespan. Functional characterization of these genes has revealed their interactions with insulin/IGF-1 and TGF-β signaling pathways, their effects in different genders on the regulation of reproductive longevity, and their implications in the control of healthy life expectancy. Many of these genes are conserved between worms and humans. Our studies thus provide new insights into the molecular control of reproductive aging and the mechanistic link between reproductive senescence and organism longevity.
doi:10.1371/journal.pgen.1004752
PMCID: PMC4256158  PMID: 25474471
25.  Ethical and scientific considerations for patient enrollment into concurrent clinical trials 
Trials  2014;15(1):470.
Researchers and institutional review boards often consider it inappropriate for patients to be asked to consent to more than one study despite there being no regulatory prohibition on co-enrollment in most countries. There are however ethical, safety, statistical, and practical considerations relevant to co-enrollment, particularly in surgery and perioperative medicine, but co-enrollment can be done if such concerns can be resolved. Preventing eligible patients from co-enrolling in studies which they would authentically value participating in, and whose material risks and benefits they understand, violates their autonomy - and thus contravenes a fundamental principle of research ethics. Statistical issues must be considered but can be addressed. In most cases each trial can be analyzed separately and validly using standard intention to treat principles; selection and other biases can be avoided if enrollment into the second trial is not dependent upon randomized treatment in the first trial; and valid interaction analyses can be performed for each trial by considering the patient’s status in the other trial at the time of randomization in the index trial. Clinical research with a potential to inform and improve clinical practice is valuable and should be supported. The ethical, safety, statistical, and practical aspects of co-enrollment can be managed, providing greater opportunity for research-led improvements in clinical practice.
doi:10.1186/1745-6215-15-470
PMCID: PMC4258295  PMID: 25433679
Autonomy; Co-enrollment; Drug interactions; Ethics; Perioperative medicine; Safety; Statistics; Surgery

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