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1.
J Orthod ; : 14653125241255139, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38845172

RESUMO

OBJECTIVE: To evaluate the effectiveness of bone anchored maxillary protraction (BAMP) in the management of class III skeletal malocclusion in children aged 11-14 years compared with an untreated control group in terms of perceived need for orthognathic surgery, skeletal and dental change, and psychological impact. DESIGN: A multicentre two-armed parallel randomised controlled trial. SETTING: Six UK hospital orthodontic units. METHODS: A total of 57 patients were randomly allocated into either the BAMP group (BAMPG) (n = 28) or a no treatment control group (CG) (n = 29). OUTCOMES: Data collection occurred at registration (DC1),18 months (DC2) and 3 years (DC3), where skeletal and dental changes were measured from lateral cephalograms and study models. Oral Aesthetic Subjective Impact Score (OASIS) and Oral Quality of Life (OHQOL) questionnaires were used to assess the psychological impact of treatment. RESULTS: The mean age was 12.9 ± 0.7 years and 12.6 ± 0.9 years in the BAMPG and CG, respectively. At DC2, the BAMPG achieved a class III ANB improvement of +0.6° compared with -0.7° in the CG (P = 0.004). The overjet improvement was +1.4 mm for the BAMPG and -0.2 mm for the CG (P = 0.002). There was no evidence of any other group differences for the other skeletal or dental cephalometric outcomes (P > 0.05) or the questionnaire data (OASIS P = 0.10, OHQOL P = 0.75). At DC2, the 18-month follow-up, 22% of the BAMPG achieved a positive overjet. At the 3-year follow-up (DC3), fewer patients in the BAMPG were perceived to need orthognathic surgery (48%) compared with 75% of patients in the CG (P = 0.04), with an odds ratio of 0.31 (95% confidence interval = 0.10-0.95). CONCLUSION: The BAMP technique did not show any social or psychological benefits; however, the skeletal class III improvement in ANB and the overjet change were sufficient to reduce the perceived need for orthognathic surgery by 27% compared with the CG.

2.
Front Oral Health ; 3: 892615, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35795156

RESUMO

Medication-related osteonecrosis of the jaw (MRONJ) is an often-severe complication found in patients receiving bisphosphonates in the management of Paget's, osteoporosis and metastatic bone cancer. Mucosal breakdown with bone exposure is a primary clinical presentation of MRONJ linked to the inhibitory effect of nitrogen-containing bisphosphonates (N-BP) on the mevalonate pathway. Geranylgeraniol (GGOH) has demonstrated a rescue effect on N-BP-treated osteoclasts but the biological effects on oral soft tissues and cells remain unclear. This study aimed to determine whether GGOH could prevent bisphosphonate induced toxicity to oral mucosa cells in vitro. Primary oral fibroblasts and keratinocytes were exposed to different GGOH concentrations or GGOH in combination with two nitrogen-containing bisphosphonates, zoledronic acid (ZA) or pamidronic acid (PA), for 72 h. The metabolic activity of each cell type was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. GGOH without bisphosphonates significantly reduced the metabolic activity of oral mucosa cells. Fibroblasts treated with GGOH and ZA in combination showed a slight increase in metabolic status compared to fibroblasts treated with ZA alone, however this positive effect was not observed in keratinocytes. In the presence of PA, GGOH was unable to increase the metabolic activity of either cell type. These findings demonstrate that GGOH is toxic to oral mucosa cells and that GGOH was not able to prevent bisphosphonate induced toxicity. These data show that GGOH does not have therapeutic potential for bisphosphonate-induced soft tissue toxicity in MRONJ and the use of GGOH as an MRONJ treatment should be strongly reconsidered.

3.
J Orthod ; 46(4): 374-377, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31595809

RESUMO

Inferior alveolar nerve (IAN) damage is a rare but recognised complication of dental procedures including third molar surgery, implant surgery, endodontic treatment and local anaesthetic injections. However, it is rarely caused by orthodontic tooth movement. This report highlights a case of temporary IAN anaesthesia to the right mental region, which was likely to have occurred secondary to the orthodontic uprighting of a lingually tilted molar using a high strength arch wire. Immediate deactivation of the appliance and an acute reducing dose of systemic steroids resulted in complete resolution of symptoms. To the best of the author's knowledge, there have been seven previously described cases of IAN paraesthesia but no cases reporting IAN anaesthesia secondary to orthodontic fixed-appliance treatment. This case highlights the importance of dentists practising orthodontics to have an awareness of the clinical and radiographic signs that may indicate a high-risk case requiring appropriate referral for cone beam imaging and careful orthodontic planning. Furthermore, this case emphasises the need to warn high-risk patients of the symptoms of this rare complication and how it may be managed. This will ultimately help to minimise the risk of litigation and optimise patient experience and care.


Assuntos
Anestesia , Traumatismos do Nervo Trigêmeo , Humanos , Nervo Mandibular , Dente Serotino , Extração Dentária , Técnicas de Movimentação Dentária
6.
BMJ ; 364: l477, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30709797
7.
J Anat ; 219(5): 638-45, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21812777

RESUMO

Microsurgical repair of transected peripheral nerves is compromised by the formation of scar tissue and the development of a neuroma, thereby limiting the success of regeneration. The aim of this study was to quantify histomorphometrically the structural changes in neural tissue that result from repair, and determine the effect of mannose-6-phosphate (M6P), a scar-reducing agent previously shown to enhance regeneration. In anaesthetised C57-black-6 mice, the left sciatic nerve was sectioned and repaired using four epineurial sutures. Either 100 µL of 600 mm M6P (five animals) or 100 µL of phosphate-buffered saline (placebo controls, five animals) was injected into and around the nerve repair site. A further group acted as sham-operated controls. After recovery for 6 weeks, the nerve was harvested for analysis using light and electron microscopy. Analysis revealed that when compared with sham controls, myelinated axons had smaller diameters both proximal and distal to the repair. Myelinated axon counts, axonal density and size all decreased across the repair site. There were normal numbers and densities of non-myelinated axons both proximal and distal to the repair. However, there were more Remak bundles distal to the repair site, and fewer non-myelinated axons per Remak bundle. Application of M6P did not affect any of these parameters.


Assuntos
Manosefosfatos/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Nervo Isquiático/lesões , Nervo Isquiático/patologia , Nervo Isquiático/fisiologia
8.
Brain Res ; 1394: 40-8, 2011 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-21550591

RESUMO

We have determined the effect of applying Mannose-6-Phosphate (M6P), a scar reducing agent, to a site of sciatic nerve repair. In anaesthetised C57-Black-6 mice, the left sciatic nerve was sectioned and repaired using 4 epineurial sutures. Either 100 µl of 600 mM Mannose-6-Phosphate (29 animals), or 100 µl of phosphate buffered saline as a placebo control (29 animals), was injected into and around the nerve repair site. A further group acted as sham-operated controls. After 6 or 12 weeks of recovery the extent of regeneration was assessed electrophysiologically and the percentage area of collagen staining at the repair site was analysed using picrosirius red and image analysis. Gait analysis was undertaken pre-operatively and at 1, 3, 6, 9 and 12 weeks postoperatively, to assess functional recovery. At 6 weeks the compound action potentials recorded from the regenerated nerves in the M6P group were significantly larger than in the placebo controls (P=0.015), and the conduction velocities were significantly faster (P=0.005), but there were no significant differences between these groups at 12 weeks. Gait analysis suggested better early functional recovery in the M6P group. In both repair groups there was a significant reduction in collagen staining between 6 and 12 weeks, suggestive of scar remodelling. We conclude that the normal scar remodelling process aids long term recovery in repaired nerves. Administration of 600 mM M6P to the nerve repair site enhances nerve regeneration and functional recovery in the early stages, and may lead to improved outcomes.


Assuntos
Cicatriz/prevenção & controle , Manosefosfatos/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Axotomia , Colágeno/análise , Eletrofisiologia , Camundongos , Camundongos Endogâmicos C57BL , Nervo Isquiático/lesões , Nervo Isquiático/patologia , Nervo Isquiático/fisiologia
9.
Pest Manag Sci ; 67(9): 1082-92, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21495152

RESUMO

BACKGROUND: The U.K. government has published plans to reduce U.K. agriculture's greenhouse gas (GHG) emissions. At the same time, the goal of global food security requires an increase in arable crop yields. Foliar disease control measures such as fungicides have an important role in meeting both objectives. RESULTS: It is estimated that U.K. winter barley production is associated with GHG emissions of 2770 kg CO2 eq. ha(-1) of crop and 355 kg CO2 eq. t(-1) of grain. Foliar disease control by fungicides is associated with decreases in GHG emissions of 42-60 kg CO2 eq. t(-1) in U.K. winter barley and 29-39 kg CO2 eq. t(-1) in U.K. spring barley. The sensitivity of these results to the impact of disease control on yield and to variant GHG emissions assumptions is presented. Fungicide treatment of the major U.K. arable crops is estimated to have directly decreased U.K. GHG emissions by over 1.5 Mt CO2 eq. in 2009. CONCLUSION: Crop disease control measures such as fungicide treatment reduce the GHG emissions associated with producing a tonne of grain. As national demand for food increases, greater yields as a result of disease control also decrease the need to convert land from non-arable to arable use, which further mitigates GHG emissions.


Assuntos
Dióxido de Carbono/metabolismo , Produtos Agrícolas/metabolismo , Fungicidas Industriais/farmacologia , Efeito Estufa , Hordeum/metabolismo , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Ciclo do Carbono , Dióxido de Carbono/análise , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/microbiologia , Abastecimento de Alimentos , Fungos/efeitos dos fármacos , Fungos/fisiologia , Hordeum/crescimento & desenvolvimento , Hordeum/microbiologia , Estações do Ano , Reino Unido
10.
J Invertebr Pathol ; 107(2): 155-8, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21414321

RESUMO

Interactions between the entomopathogenic fungi Zoophthora radicans and Pandora blunckii infecting larvae of Plutella xylostella were investigated. This is the first report to quantify within-host growth of one fungus in the presence of another competing for the same host resource using quantitative PCR (qPCR) at regular time points during the infection process. In larvae inoculated only with Z. radicans, there was a cumulative increase in the quantity of Z. radicans DNA throughout the time course of infection. However, in dual-inoculated larvae, there was an initial accelerated rate of growth of Z. radicans compared to when it was inoculated alone, but by the time of host death it had been effectively excluded by P. blunckii. The implications of these results for co-existence of these fungal pathogens in the field are discussed.


Assuntos
Entomophthorales/genética , Interações Hospedeiro-Patógeno/genética , Mariposas/microbiologia , Controle Biológico de Vetores , Animais , DNA Fúngico/análise , Entomophthorales/crescimento & desenvolvimento , Larva/microbiologia
11.
Trends Microbiol ; 18(8): 365-73, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20598545

RESUMO

Microbe-host interactions can be categorised as pathogenic, parasitic or mutualistic, but in practice few examples exactly fit these descriptions. New molecular methods are providing insights into the dynamics of microbe-host interactions, with most microbes changing their relationship with their host at different life-cycle stages or in response to changing environmental conditions. Microbes can transition between the trophic states of pathogenesis and symbiosis and/or between mutualism and parasitism. In plant-based systems, an understanding of the true ecological niche of organisms and the dynamic state of their trophic interactions with their hosts has important implications for agriculture, including crop rotation, disease control and risk management.


Assuntos
Bactérias/patogenicidade , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , Plantas/microbiologia , Simbiose , Interações Microbianas , Fenômenos Fisiológicos Vegetais
12.
Mycol Res ; 113(Pt 8): 867-76, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19422912

RESUMO

For the first time, the specific activities of chitinases, esterases, lipases and a serine protease (VCP1) produced by different isolates of the nematophagous fungus Pochonia chlamydosporia were quantified and compared. The isolates were grown for different time periods in a minimal liquid medium or media supplemented with 1 % chitin, 0.2 % gelatin or 2 % olive oil. Enzyme-specific activities were quantified in filtered culture supernatants using chromogenic p-nitrophenyl substrates (for chitinases, lipases and esterases) and a p-nitroanilide substrate (to measure the activity of the proteinase VCP1). Additionally, information on parasitic growth (nematode egg parasitism) and saprotrophic growth (plant rhizosphere colonisation) was collected. Results showed that the production of extracellular enzymes was influenced by the type of medium (p<0.05) in which P. chlamydosporia was grown. Enzyme activity differed with time (p<0.05), and significant differences were found between isolates (p<0.001) and the amounts of enzymes produced (p<0.001). However, no significant relationships were found between enzyme activities and parasitic or saprotrophic growth using Kendall's coefficient of concordance or Spearman rank correlation coefficient. The results provided new information about enzyme production in P. chlamydosporia and suggested that the mechanisms which regulate the trophic switch in this fungus are complex and dependent on several factors.


Assuntos
Espaço Extracelular/enzimologia , Proteínas Fúngicas/metabolismo , Hidrolases/metabolismo , Hypocreales/enzimologia , Hypocreales/isolamento & purificação , Nematoides/microbiologia , Animais , Meios de Cultura/metabolismo
13.
Mycol Res ; 112(Pt 10): 1227-40, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18693001

RESUMO

Species-specific primers for Zoophthora radicans and Pandora bluckii were developed. To achieve this, partial sequences of DNA that encode for rRNA, more specifically, the ITS region (rDNA-ITS) were obtained from different isolates and analysed. Seven Z. radicans isolates (four from P. xylostella, and three from other lepidopteran hosts) and one P. blunckii isolate (from P. xylostella) were used. These isolates were selected based on PCR-RFLP patterns obtained from 22 isolates of P. blunckii and 39 isolates of Z. radicans. All P. blunckii isolates were from the same host (P. xylostella); 20 isolates were from Mexico, one from the Philippines, and one from Germany. The Z. radicans isolates were more diverse in geographical origin (Mexico, Kenya, Japan, New Zealand, Australia, Taiwan, Philippines, Malaysia, Uruguay, France, USA, Poland, Indonesia, Switzerland, Israel, China, and Denmark) and host origin (Lepidoptera, Hemiptera, Hymentoptera, and Diptera). Using conventional PCR, each pair of species-specific primers successfully detected each species of fungus from DNA extracted from infected host larvae either single- or dual-inoculated with both fungal species. The PCR-RFLP analysis also showed that Z. radicans was genetically more diverse than P. blunckii, although only a limited number of P. blunckii isolates from one country were considered. There was no direct relationship between genetic diversity and host or geographical origin. The relationship between genetic variation within both fungal species and host specificity or ecological adaptation is discussed.


Assuntos
Primers do DNA/genética , Entomophthorales/isolamento & purificação , Mariposas/microbiologia , Reação em Cadeia da Polimerase/métodos , Animais , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Entomophthorales/classificação , Entomophthorales/genética , Variação Genética , Larva/microbiologia , México , Dados de Sequência Molecular , Filogenia , Polimorfismo de Fragmento de Restrição , Especificidade da Espécie
14.
Trends Microbiol ; 16(8): 380-7, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18595713

RESUMO

Plant, animal and human diseases spread by microscopic airborne particles have had major economic and social impacts during history. Special air-sampling devices have been used to collect such particles since the 19th century but it has often been impossible to identify them accurately. Exciting new opportunities to combine air sampling with quantitative PCR to identify and count these particles are reviewed, using crop pathogen examples. These methods can be used to predict the risk of unexpected outbreaks of airborne diseases by identifying increases in pathogen inoculum or genetic changes in pathogen populations that render control ineffective. The predictions can provide guidance to policymakers, health professionals or the agricultural industry for the development of strategies to minimise the risk of severe pandemics.


Assuntos
Microbiologia do Ar , Fungos/isolamento & purificação , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase/métodos , Fungos/classificação , Plantas
15.
J Peripher Nerv Syst ; 12(4): 269-76, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18042137

RESUMO

Axonal regeneration at a site of peripheral nerve repair can be impeded by the formation of scar tissue, which creates a mechanical barrier and initiates the development of multiple branched axonal sprouts that form a neuroma. We have investigated the hypothesis that the application of a scar-reducing agent to the nerve repair site would permit better axonal regeneration. In anaesthetised C57 Black-6 mice, the left sciatic nerve was sectioned and immediately re-approximated using four epineurial sutures. In five groups of eight mice, we injected transforming growth factor-beta3 (50 or 500 ng), interleukin-10 (IL-10) (125 or 500 ng), or saline into and around the repair site, both before and after the nerve section. Another group of eight animals acted as sham-operated controls. After 6 weeks, the outcome was assessed by recording compound action potentials (CAPs), measuring collagen levels using picrosirius red staining, and counting the number of myelinated axons proximal and distal to the repair. CAPs evoked by electrical stimulation distal to the repair were significantly smaller in all repair groups except for the low-dose IL-10 group, where they were not significantly different from that in controls. The area of staining for collagen had significantly increased in all repair groups except for the low-dose IL-10 group, which was not significantly different from that in controls. The myelinated fibre counts were always higher distal to the repair site, but there were no significant differences between groups. We conclude that administration of a low-dose of IL-10 to a site of sciatic nerve repair reduces scar formation and permits better regeneration of the damaged axons.


Assuntos
Cicatriz/patologia , Cicatriz/prevenção & controle , Interleucina-10/uso terapêutico , Regeneração Nervosa/efeitos dos fármacos , Neuropatia Ciática/patologia , Neuropatia Ciática/prevenção & controle , Animais , Interleucina-10/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Regeneração Nervosa/fisiologia , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/crescimento & desenvolvimento , Nervo Isquiático/patologia
16.
J Peripher Nerv Syst ; 11(4): 286-93, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17117936

RESUMO

Scar formation at a site of nerve injury can cause a mechanical barrier to axonal regeneration and lead to the development of multiple axonal sprouts to form a neuroma. We have investigated the hypothesis that the application of a scar-preventing agent to a nerve repair site would enhance regeneration of the nerve and reduce neuroma formation. The left sciatic nerve was exposed under general anaesthesia in 18 adult Sprague-Dawley rats. In 12 animals, the nerve was sectioned and immediately re-approximated using four epineurial sutures, and in 6 of these animals neutralising antibodies to transforming growth factor (TGF)-beta1 and TGF-beta2 were injected into and around the repair site. The six other animals acted as controls. After 7 weeks, the outcome was assessed by recording compound action potential (CAP) ratios, measuring collagen levels using picrosirius red staining, and counting the number of myelinated axons proximal and distal to the repair. After repair alone, the mean percentage of area of staining (PAS) for collagen within the nerve had significantly increased. However, after repair with the administration of antibodies, the PAS was not significantly different from that in the sham controls. After administration of antibodies, the CAP ratios were significantly smaller than in controls but not after repair alone. In both nerve injury groups, the myelinated fibre counts were significantly increased distal to the injury site, but there was no difference between these two groups. We conclude that administration of antibodies to TGF-beta1 and TGF-beta2 reduced scar formation at the repair site but did not enhance regeneration of the nerve or reduce the development of multiple axonal sprouts.


Assuntos
Anticorpos/uso terapêutico , Cicatriz/prevenção & controle , Regeneração Nervosa/efeitos dos fármacos , Nervo Isquiático/lesões , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta2/antagonistas & inibidores , Potenciais de Ação/efeitos dos fármacos , Animais , Axotomia , Masculino , Neuroma/prevenção & controle , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/imunologia , Fator de Crescimento Transformador beta1/imunologia , Fator de Crescimento Transformador beta2/imunologia
17.
Neuroreport ; 17(12): 1245-9, 2006 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-16951563

RESUMO

We have investigated the effect of scarring at a site of peripheral nerve repair by comparing regeneration of the sciatic nerve in normal mice and two transgenic strains with an increased or decreased propensity for scarring. The outcome was assessed by quantifying collagen at the repair site, recording compound action potentials and counting myelinated nerve fibres on each side of the repair. We found that higher levels of collagen scar formation were associated with smaller compound action potentials, slower conduction velocities and a reduction in fibre numbers across the repair site. We conclude that scarring impedes regeneration at sites of nerve repair and suggest that this could be amenable to therapeutic manipulation.


Assuntos
Cicatriz/fisiopatologia , Regeneração Nervosa/fisiologia , Neuropatia Ciática/fisiopatologia , Cicatrização/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Cicatriz/metabolismo , Colágeno/metabolismo , Estimulação Elétrica/métodos , Fator de Crescimento Insulin-Like II/deficiência , Interleucina-10/deficiência , Interleucina-4/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Nervosas/patologia , Fibras Nervosas/fisiologia , Condução Nervosa/genética , Condução Nervosa/efeitos da radiação , Receptor IGF Tipo 2/efeitos dos fármacos , Neuropatia Ciática/genética , Neuropatia Ciática/metabolismo , Neuropatia Ciática/patologia , Cicatrização/genética
18.
FEMS Microbiol Ecol ; 51(2): 257-64, 2005 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16329874

RESUMO

The Paecilomyces lilacinus is the most widely tested fungus for the control of root-knot and cyst nematodes. The fungus has also been implicated in a number of human and animal infections, difficulties in diagnosis often result in misdiagnosis or delays in identification leading to a delay in treatment. Here, we report the development of species-specific primers for the identification of P. lilacinus based on sequence information from the ITS gene, and their use in identifying P. lilacinus isolates, including clinical isolates of the fungus. The primer set generated a single PCR fragment of 130 bp in length that was specific to P. lilacinus and was also used to detect the presence of P. lilacinus from soil, roots and nematode eggs. Real-time PCR primers and a TaqMan probe were also developed and provided quantitative data on the population size of the fungus in two field sites. PCR, bait and culture methods were combined to investigate the presence and abundance of the fungus from two field sites in the United Kingdom where potato cyst nematode populations were naturally declining, and results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.


Assuntos
Primers do DNA/genética , Paecilomyces/genética , Paecilomyces/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , Nematoides/microbiologia , Óvulo/microbiologia , Paecilomyces/classificação , Microbiologia do Solo , Especificidade da Espécie
19.
Mycol Res ; 108(Pt 6): 654-61, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15323248

RESUMO

The nematophagous fungus Pochonia chlamydosporia is a potential biocontrol agent against root knot and cyst nematodes. Genetic transformation of the fungus to introduce visual marker genes, novel traits, or changes in expression levels of endogenous genes, would greatly enhance understanding of its behaviour on nematode-infested roots and of its interactions with other soil and rhizosphere microorganisms. A transformation system for the introduction of novel genes into P. chlamydosporia has been developed. Methods to generate protoplasts, introduce DNA and regenerate transformed viable fungal mycelium have been optimised, using plasmids carrying the green fluorescent protein marker gene gfp and the hygromycin resistance gene hph. Cultures of P. chlamydosporia were resistant to high levels of a range of fungal inhibitors, including hygromycin, that are commonly used with dominant selectable marker genes in the transformation of other fungi. However, regenerating protoplasts transformed with hph could be selected by their ability to grow through an agar overlay containing 1 mg ml(-1) hygromycin. Green fluorescence was observed in protoplasts and regenerating mycelium after transformation with gfp, but the GFP phenotype was lost on subculture. Maintenance of introduced genes was not stable, and during subculture, PCR assays indicated that the transformants lost both hph and gfp. When these genes were introduced on the same plasmid, segregation of hph and gfp was observed prior to their loss. It was unclear whether the introduced plasmids were able to replicate autonomously in P. chlamydosporia, or if they integrated transiently into the fungal genome. Possible reasons for the instability of the transformants are discussed.


Assuntos
Marcadores Genéticos , Transformação Genética , Tylenchoidea/microbiologia , Verticillium/genética , Animais , Resistência Microbiana a Medicamentos/genética , Proteínas de Fluorescência Verde , Higromicina B/farmacologia , Proteínas Luminescentes , Plasmídeos , Protoplastos , Tylenchoidea/crescimento & desenvolvimento , Verticillium/classificação , Verticillium/isolamento & purificação
20.
J Appl Genet ; 45(1): 3-15, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-14960763

RESUMO

Conventional methods to identify fungi have often relied on identification of disease symptoms, isolation and culturing of environmental organisms, and laboratory identification by morphology and biochemical tests. Although these methods are still fundamental there is an increasing move towards molecular diagnostics of fungi in all fields. In this review, some of the molecular approaches to fungal diagnostics based on polymerase chain reaction (PCR) and DNA/RNA probe technology are discussed. This includes several technological advances in PCR-based methods for the detection, identification and quantification of fungi including real-time PCR which has been successfully used to provide rapid, quantitative data on fungal species from environmental samples. PCR and probe based methods have provided new tools for the enumeration of fungal species, but it is still necessary to combine the new technology with more conventional methods to gain a fuller understanding of interactions occurring in the environment. Since its introduction in the mid 1980's PCR has provided many molecular diagnostic tools, some of which are discussed within this review, and with the advances in micro-array technology and real-time PCR methods the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual fungal species but also on whole communities.


Assuntos
Fungos/genética , Fungos/isolamento & purificação , Doenças das Plantas/microbiologia , Sondas de DNA , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Fungos/patogenicidade , Micologia/métodos , Reação em Cadeia da Polimerase/métodos
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