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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.
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.
PMCID: PMC3697937  PMID: 23621425
3.  CSF-1R inhibition alters macrophage polarization and blocks glioma progression 
Nature medicine  2013;19(10):10.1038/nm.3337.
Glioblastoma multiforme (GBM) comprises several molecular subtypes including proneural GBM. Most therapeutic approaches targeting glioma cells have failed. An alternative strategy is to target cells in the glioma microenvironment, such as tumor-associated macrophages and microglia (TAMs). Macrophages depend upon colony stimulating factor (CSF)-1 for differentiation and survival. A CSF-1R inhibitor was used to target TAMs in a mouse proneural GBM model, which dramatically increased survival, and regressed established tumors. CSF-1R blockade additionally slowed intracranial growth of patient-derived glioma xenografts. Surprisingly, TAMs were not depleted in treated mice. Instead, glioma-secreted factors including GM-CSF and IFN-γ facilitated TAM survival in the context of CSF-1R inhibition. Alternatively activated/ M2 macrophage markers decreased in surviving TAMs, consistent with impaired tumor-promoting functions. These gene signatures were associated with enhanced survival in proneural GBM patients. Our results identify TAMs as a promising therapeutic target for proneural gliomas, and establish the translational potential of CSF-1R inhibition for GBM.
PMCID: PMC3840724  PMID: 24056773
4.  Microenvironment-mediated resistance to anticancer therapies 
Cell Research  2012;23(2):179-181.
PMCID: PMC3567818  PMID: 22945358
5.  A prospective study of chemotherapy-induced febrile neutropenia in the South West London Cancer Network. Interpretation of study results in light of NCAG/NCEPOD findings 
British Journal of Cancer  2010;104(3):407-412.
Chemotherapy-induced febrile neutropenia is a medical emergency complicating the treatment of many cancer patients. It is associated with considerable morbidity and mortality, as well as impacting on healthcare resources.
A prospective study of all cases of chemotherapy-induced febrile neutropenia in the South West London Cancer Network was conducted over a 4-month period. Factors including demographics, treatment history, management of febrile neutropenia and outcome were recorded.
Results and conclusi:
Our results reflect those of the recent National Chemotherapy Advisory Group (NCEPOD, 2008)/National Confidential Enquiry into Patient Outcomes and Death reports (NCAG, 2009) and highlight the need for network-wide clinical care pathways to improve outcomes in this area.
PMCID: PMC3049562  PMID: 21179036
neutropenic sepsis; chemotherapy; infection; febrile neutropenia
6.  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.
PMCID: PMC2903553  PMID: 20174687
7.  Safe practice and nursing care of patients receiving oral anti-cancer medicines: a position statement from UKONS 
ecancermedicalscience  2010;4:177.
This position statement has been formulated by oncology nurses from the UK Oncology Nursing Society (UKONS) to provide guidance for nurses on safe practice with oral anti-cancer medicines. The ultimate aim is to ensure that patients are not only well informed and capable of managing their oral anti-cancer medicines, but also supported safely and effectively while they are receiving these treatments.
PMCID: PMC3234027  PMID: 22276031
8.  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.
PMCID: PMC2607171  PMID: 18978088
9.  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.
PMCID: PMC1182300  PMID: 15930134
10.  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.
PMCID: PMC363147  PMID: 14668489
11.  Endocarditis: problems—patients being treated for endocarditis and not doing well 
Heart  2001;85(4):470-474.
PMCID: PMC1729693  PMID: 11250980
12.  Myocarditis, pericarditis and other pericardial diseases 
Heart  2000;84(4):449-454.
PMCID: PMC1729455  PMID: 10995424
13.  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.
PMCID: PMC181558  PMID: 12925754
14.  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.
PMCID: PMC165107  PMID: 12802085
15.  Marked variation in the cardiomyopathy associated with Friedreich's ataxia 
Heart  1999;81(2):141-147.
Objective—To document the cardiac phenotype associated with Friedreich's ataxia, a recessively inherited disorder characterised by spinocerebellar degeneration.
Setting—Individuals with Friedreich's ataxia who accepted the invitation to participate in the study.
Hypothesis—The cardiomyopathy associated with Friedreich's ataxia may offer a human model for the study of factors modulating cardiac hypertrophy.
Methods—55 patients (mean (SD) age 30 (9) years) with a clinical diagnosis of Friedreich's ataxia were studied by clinical examination, electrocardiography, cross sectional and Doppler echocardiography, and analysis of the GAA repeat in the first intron of the frataxin gene.
Results—A wide variety of cardiac morphology was documented. Subjects with normal frataxin alleles had no evidence of cardiomyopathy. In homozygous subjects, a relation was found between the thickness of the interventricular septum (r = 0.53, p < 0.005), left ventricular mass (r = 0.48, p < 0.01), and the number of GAA repeats on the smaller allele of the frataxin gene. No relation was shown between the presence of electrocardiographic abnormalities (mainly repolarisation changes) and either the pattern of ventricular hypertrophy (if present) and degree of neurological disability or the length of time since diagnosis. No tendency to ventricular thinning or dilatation with age was found. Although ventricular systolic function appeared impaired in some cases, Doppler studies of ventricular filling were within the normal range for age.
Conclusions—The cardiomyopathy associated with Friedreich's ataxia shows a variable phenotype which is not concordant with the presence of ECG abnormalities or the neurological features of the condition. As the genetic basis for Friedreich's ataxia has been established, further studies will help to clarify the molecular mechanisms of the cardiac hypertrophy.

 Keywords: cardiomyopathy; Friedreich's ataxia; genetics
PMCID: PMC1728941  PMID: 9922348
16.  Pregnancy and congenital heart disease. 
Heart  1997;78(1):12-14.
PMCID: PMC484856  PMID: 9290394
17.  Myocardial beta adrenoceptors and left ventricular function in hypertrophic cardiomyopathy. 
Heart  1996;75(1):50-54.
OBJECTIVE--To assess the relation between left ventricular function and myocardial beta adrenoceptor density. METHODS--17 patients with hypertrophic cardiomyopathy, six with and 11 without heart failure, were studied. Left ventricular function was assessed by echocardiography, and myocardial beta adrenoceptors by positron emission tomography. Patient data were compared with those obtained in normal controls. RESULTS--Myocardial beta adrenoceptor density in the 17 patients was 7.00 (SD 1.90) pmol/g v 11.50 (2.18) pmol/g in normal controls (P < 0.01). beta Adrenoceptor density in the six patients with left ventricular failure was 5.61 (0.88) pmol/g v 7.71 (1.86) pmol/g in the 11 patients with normal ventricular function (P < 0.05), and there was a significant correlation (r = 0.52; P < 0.05) between left ventricular fractional shortening and myocardial beta adrenoceptor density. A positive correlation (r = 0.51; P < 0.05) was also found between myocardial beta adrenoceptor density and the E/A transmitral flow ratio, an index of left ventricular diastolic function. CONCLUSIONS--There is myocardial beta adrenoceptor downregulation in patients with hypertrophic cardiomyopathy with or without signs of heart failure.
PMCID: PMC484222  PMID: 8624872
24.  Anticoagulants in pregnancy. 
British Heart Journal  1995;74(2):107-111.
PMCID: PMC483982  PMID: 7546985

Results 1-25 (162)