Resistance to pydiflumetofen in Botrytis cinerea: risk assessment and detection of point mutations in sdh genes that confer resistance.

BACKGROUND: Gray mold caused by Botrytis cinerea Pers. is one of the most significant airborne diseases. It can infest a wide range of crops, causing significant losses in yield and quality worldwide. Pydiflumetofen, a new generation succinate dehydrogenase inhibitor (SDHI), is currently being registered in China to control gray mold in a variety of crops. The baseline sensitivity, resistance risk, and resistance mechanism of Botrytis cinerea to pydiflumetofen were assessed in this study. RESULTS: A total of 138 strains of B. cinerea from 10 different regions were tested for their sensitivity to pydiflumetofen, and the mean EC50 value was 0.0056 µg mL-1 . Eight mutants were obtained by fungicide adaption from five sensitive parental isolates, and the resistance factor (RF) ranged from 51 to 135. The mutants exhibited strong adaptive traits in conidial production, conidial germination, and pathogenicity. Positive cross-resistance was only observed between other SDHIs (i.e. boscalid, fluopyram, and isopyrazam). Two different types of pydiflumetofen-resistant mutants were identified: point mutation P225L in sdhB and double mutation G85A and I93V in sdhC. The in vivo control efficacy of pydiflumetofen on the resistant mutants carrying P225L in sdhB as well as G85A and I93V in sdhC was significantly decreased to 52.62% and 32.27%, respectively. CONCLUSION: The fitness was significantly higher for all pydiflumetofen-resistant mutants than the corresponding parental. Two types of point mutations, sdhB-P225L and sdhC-G85A and I93V, might confer resistance to pydiflumetofen in B. cinerea. A precautionary resistance management strategy should be implemented. © 2021 Society of Chemical Industry.

Sensitivity assessment and SDHC-I86F mutation frequency of Phakopsora pachyrhizi populations to benzovindiflupyr and fluxapyroxad fungicides from 2015 to 2019 in Brazil.

BACKGROUND: Fungicides of the succinate dehydrogenase inhibitors (SDHIs) group have been used in soybean to control Asian soybean rust (ASR) caused by Phakopsora pachyrhizi. Fungal populations with less sensitivity to SDHI fungicides have been reported since 2015. RESULTS: In this study, fungal sensitivity to benzovindiflupyr (BZV) and fluxapyroxad (FXD) was assessed using a total of 770 P. pachyrhizi populations sampled over four soybean growing seasons. Cross-resistance, intrinsic activity, and frequency of SDHC-I86F mutation were also analyzed. The average effective concentration to inhibit 50% (EC50 ) and SDHC-I86F frequency increased over the 2015/2016, 2016/2017, 2017/2018 and 2018/2019 soybean-seasons. Fourteen P. pachyrhizi populations had the EC50 value above 10 mg L-1 for both carboxamides. No difference was found in intrinsic active to BZV and FXD fungicides for sensitive P. pachyrhizi populations. For P. pachyrhizi classified as less sensitive BZV showed the highest fungitoxicity effect. High frequency of the C-I86F mutation was observed in samples collected in volunteer soybean plants. The maximum frequency of SDHC-I86F mutation in the population was 50% and resulting in ASR populations with low sensitivity to SDHIs. A low correlation between bioassay and SDHC-I86F mutation was observed possible due to the dikaryotic nature of rust fungi or other mutations in the other succinate dehydrogenase subunits. CONCLUSION: The present work provides an overview of a large sampling size of P. pachyrhizi populations and their performance over the four crop seasons. The high frequency of SDHC-I86F mutation and low sensitivity to SDHIs are widely distributed in the main soybean growing regions in Brazil and present in volunteer plants in the soybean-free period. Further detailed studies are needed to identify novel point mutations affecting the effectiveness of SDHIs. © 2021 Society of Chemical Industry.

Field Assessment of Six Point-Mutations in SDH Subunit Genes Conferring Varying Resistance Levels to SDHIs in Clarireedia spp.

Dollar spot, caused by Clarireedia spp. (formerly Sclerotinia homoeocarpa F.T. Bennett), is the most economically important turfgrass disease causing considerable damage on golf courses. While cultural practices are available for reducing dollar spot infection, chemical fungicide use is often necessary for maintaining optimal turf quality. Since the release of boscalid in 2003, the succinate dehydrogenase inhibitor (SDHI) class has become an invaluable tool for managing dollar spot. However, resistance to this class has recently been reported in Clarireedia spp. and many other plant pathogenic fungi. After SDHI field failure on four golf courses and one university research plot, a total of six unique SDH mutations conferring differential in vitro sensitivities to SDHIs have been identified in Clarireedia spp. In 2018 and 2019, turf research plots were inoculated with sensitive, non-mutated isolates of Clarireedia spp., as well as resistant isolates harboring each unique identified mutation. Fungicide efficacy trials were conducted on inoculated plots to assess differential sensitivity to five SDHI active ingredients (boscalid, fluxapyroxad, isofetamid, fluopyram, and pydiflumetofen) across mutations under field conditions. Results indicate unique mutations are associated with distinct SDHI field efficacy profiles as shown in in-vitro sensitivity assays. Isolate populations with B subunit mutations (H267Y/R) were more sensitive to fluopyram, whereas isolate populations with C subunit mutations (C-G91R, C-G150R) showed resistance to all SDHIs tested. Mutation-associated differential sensitivity observed under field conditions indicates a need for a nation-wide survey and frequent monitoring of SDHI sensitivity of dollar spot populations on golf courses in the USA. Further, the information gained from this study will be useful in providing sustainable management recommendations for controlling site-specific resistant populations of Clarireedia spp.

Site-directed mutagenesis of the succinate dehydrogenase subunits B and D from Corynespora cassiicola reveals different fitness costs and sensitivities to succinate dehydrogenase inhibitors.

Carboxamide fungicides target succinate dehydrogenase (SDH). Recently published monitoring studies have shown that Corynespora cassiicola isolates are resistant to one or several SDH inhibitors (SDHIs) with amino acid substitutions in the SDH B and D subunits. We confirmed, by site-directed mutagenesis of the sdhB and sdhD genes, that each of the mutations identified in the field strains of C. cassiicola conferred resistance to boscalid and, in some cases, cross-resistance to other SDHIs (fluopyram, carboxin and penthiopyrad). Analyses of the enzyme activity and sdhB and sdhD gene expression show that modifications (SdhB_H278Y and SdhD_H105R) that result in a decline in SDH enzyme activity may be complemented by gene overexpression. The SdhB_H278Y, SdhB_I280V and SdhD_H105R mutants suffered large fitness penalties based on their biological properties, including conidia production and germination, mycelial growth, pathogenicity or survival abilities under environment stress. However, fitness cost was not found in the SdhB_H278R, SdhD_D95E and SdhD_G109V mutants. In the evaluation of resistance to boscalid in 2018 and 2019, the frequency of the SdhD_D95E and SdhD_G109V genotypes in the Liaoning and Shandong provinces changed dramatically compared with 2005-2017, from low resistance frequency (0.53% for D95E and 2.53% for G109V) to dominant resistance frequency (17.28% for D95E and 15.38% for G109V). Considering both the fitness and increased frequency of these genotypes, we may infer that the SdhD_D95E and SdhD_G109V mutants will be the dominant resistance mutants in field.

Resistance risk assessment for a novel succinate dehydrogenase inhibitor pydiflumetofen in Fusarium asiaticum.

BACKGROUND: Fusarium asiaticum is one of predominant pathogens of Fusarium head blight (FHB) in China. Pydiflumetofen (Pyd) is a novel succinate dehydrogenase inhibitor (SDHI) which has been commercialized in China for the controlling of wheat FHB since 2019. In the current study, a risk assessment of the pydiflumetofen-resistance selected in Fusarium asiaticum was investigated. RESULTS: One PydMR mutant [resistance factor (RF) < 80] and four PydHR mutants (RF > 3000) were generated by fungicide-taming from 1000 mycelial discs of the wild-type strain 2021. Nucleotide sequences alignment results of FaSdh from the wild-type strain and resistant mutants showed that all the mutations were categorized into three genotypes, i.e. FaSdhBH248Y from PydMR mutant, both FaSdhC1 A64V and FaSdhC1 R67K from PydHR mutants. All the resistant mutants possessed no fitness penalty based on the data of mycelial linear growth, conidiation and virulence. In addition, the FaSdhC1 A64V mutants showed positive cross-resistance between pydiflumetofen and boscalid or thifluzamide, but no cross-resistance between pydiflumetofen and Y13149 or Y12196, while the FaSdhC1 R67K mutants exhibited positive cross-resistance between pydiflumetofen and boscalid, thifluzamide or Y12196, and no cross-resistance between pydiflumetofen and Y13149. Furthermore, positive cross-resistance between the five tested SDHIs was detected in the FaSdhBH248Y mutants. CONCLUSION: The results suggest a moderate to high resistance risk of F. asiaticum to pydiflumetofen, and provide essential data for monitoring the emergence of resistance and resistance management strategies for pydiflumetofen, which will be useful for scientific application of this fungicide in China.

Secreted Flavin Cofactors for Anaerobic Respiration of Fumarate and Urocanate by Shewanella oneidensis: Cost and Role.

Shewanella oneidensis strain MR-1, a facultative anaerobe and model organism for dissimilatory metal reduction, uses a periplasmic flavocytochrome, FccA, both as a terminal fumarate reductase and as a periplasmic electron transfer hub for extracellular respiration of a variety of substrates. It is currently unclear how maturation of FccA and other periplasmic flavoproteins is achieved, specifically in the context of flavin cofactor loading, and the fitness cost of flavin secretion has not been quantified. We demonstrate that deletion of the inner membrane flavin adenine dinucleotide (FAD) exporter Bfe results in a 23% slower growth rate than that of the wild type during fumarate respiration and an 80 to 90% loss in fumarate reductase activity. Exogenous flavin supplementation does not restore FccA activity in a Δbfe mutant unless the gene encoding the periplasmic FAD hydrolase UshA is also deleted. We demonstrate that the small Bfe-independent pool of FccA is sufficient for anaerobic growth with fumarate. Strains lacking Bfe were unable to grow using urocanate as the sole electron acceptor, which relies on the periplasmic flavoprotein UrdA. We show that periplasmic flavoprotein maturation occurs in careful balance with periplasmic FAD hydrolysis, and that the current model for periplasmic flavin cofactor loading must account for a Bfe-independent mechanism for flavin transport. Finally, we determine that the metabolic burden of flavin secretion is not significant during growth with flavin-independent anaerobic electron acceptors. Our work helps frame the physiological motivations that drove evolution of flavin secretion by ShewanellaIMPORTANCEShewanella species are prevalent in marine and aquatic environments, throughout stratified water columns, in mineral-rich sediments, and in association with multicellular marine and aquatic organisms. The diversity of niches shewanellae can occupy are due largely to their respiratory versatility. Shewanella oneidensis is a model organism for dissimilatory metal reduction and can respire a diverse array of organic and inorganic compounds, including dissolved and solid metal oxides. The fumarate reductase FccA is a highly abundant multifunctional periplasmic protein that acts to bridge the periplasm and temporarily store electrons in a variety of respiratory nodes, including metal, nitrate, and dimethyl sulfoxide respiration. However, maturation of this central protein, particularly flavin cofactor acquisition, is poorly understood. Here, we quantify the fitness cost of flavin secretion and describe how free flavins are acquired by FccA and a homologous periplasmic flavoprotein, UrdA.

Risk assessment of maternally inherited SDHD paraganglioma and phaeochromocytoma.

BACKGROUND: Germline mutations in the SDHD tumour suppressor gene (11q23.1) predispose to phaeochromocytomas and paragangliomas (PPGL) mainly on a paternal transmission. However, PPGL have been recently reported in three carriers of a maternally inherited SDHD mutation. OBJECTIVE: To assess the risk of PPGL occurrence on maternal transmission of SDHD mutation. METHODS: Pedigrees of 80 SDHD-related families have been reviewed. 35 asymptomatic subjects carrying a maternally transmitted SDHD mutation were identified. 20 of them accepted to benefit from a PPGL imaging screening. RESULTS: A unique histologically proven biochemically negative phaeochromocytoma has been diagnosed in a 35-year-old woman. Molecular investigations carried out on tumour tissue revealed that the loss of heterozygosity encompassed the paternally derived q arm and the maternally derived p arm of chromosome 11. CONCLUSIONS: This study demonstrates that the risk of developing PPGL for a subject carrying a germline SDHD mutation on the maternal allele remains a rare scenario but does exist. Our data suggest an adjustment of current genetic counselling and clinical care recommendations for at-risk subjects. A targeted familial genetic test should be proposed from the age of 18 years to every subject having a mother carrying a germline SDHD mutation and a first medical workup, including imaging, should be recommended to SDHD-positive mutation carriers.

Fitness measurement reveals contrasting costs in homologous recombinant mutants of Botrytis cinerea resistant to succinate dehydrogenase inhibitors.

Botrytis cinerea is a pathogenic ascomycete fungus that causes gray mold on many crops. Chemical control remains the principal method for curbing this disease. However, fungicide efficacy may be compromised by the selection of resistant strains. Assessments of the fitness of resistant strains is important, to evaluate the risk of their establishment in populations. Strains resistant to boscalid, the first succinate dehydrogenase inhibitor (SDHI) registered for the treatment of gray mold on grapevine in France, have recently been detected in the field. Most of these strains harbor mutations of the sdhB gene, encoding subunit B of SDH. In this study, we used sdhB recombinant mutants to investigate the impact of mutations conferring SDHI resistance on the fitness of B. cinerea. We have shown that sdhB mutations (except for the sdhB(H272Y) mutation) affect SDH activity and respiration rate. Our results suggest that different sdhB mutations have different effects on fitness. In particular, mutants displaying an inhibition of SDH activity do not suffer the same effects on fitness. We discuss the results in the context of mutant frequencies in field populations and the possible occurrence of compensatory mechanisms that modulate fitness losses.

Usefulness of Succinate dehydrogenase B (SDHB) immunohistochemistry in guiding mutational screening among patients with pheochromocytoma-paraganglioma syndromes.

Genetic testing of pheochromocytomas (PCC) and paragangliomas (PGL), although expensive, is gradually becoming a part of the routine laboratory investigation for patients with PCC-PGL syndrome. Recently, Succinate dehydrogenase B (SDHB) immunochemistry has been shown to be an excellent indicator of germline mutations in the SDH genes and could help significantly reduce cost. This study assesses the utility of SDHB immunohistochemical analysis when used to guide genetic analysis, with emphasis on cost benefits it could provide in a resource-limited setting. Forty-four cases of PCC/PGL characterized by genetic analysis were included to determine their SDHB expression pattern by immunohistochemistry. SDHB antibody expression was negative among three cases each, with SDHB and SDHD mutations. Immunohistochemistry results were positive for all three cases of RET, a single case of neurofibromatosis and for two cases with Von Hippel-Lindau (VHL) mutations while the remaining two cases with VHL mutations showed a diffuse 'cytoplasmic blush'. Thirty of the remaining 31 samples demonstrated positive staining and were negative for mutations, while a lone sample that was negative for staining and mutation was not included in the final analysis as the internal control for the sample was not adequately stained. Cost analysis in our settings showed that triaging with SDHB immunohistochemistry could potentially reduce costs by USD 320-500 per patient. SDHB immunohistochemistry, when used as a guide to genetic testing, can significantly reduce the effort, time and costs of testing among patients with PCC-PGL, a huge benefit in resource limited settings.

Usefulness of negative and weak-diffuse pattern of SDHB immunostaining in assessment of SDH mutations in paragangliomas and pheochromocytomas.

This is a confirmatory study about usefulness of SDHB and SDHA immunostaining in assessment of SDH mutations in paragangliomas and pheochromocytomas. Paraganglioma/pheochromocytoma syndrome (PGL/PCC syndrome) consists of different entities, associated with germline mutations in five different genes: SDHD, SDHAF2, SDHC, SDHA and SDHB. It has been suggested that negative immunostaining of SDHB can be taken as an indicator of the presence of a mutation in one of the five SDH genes. We have performed SDHB and SDHA immunohistochemical staining in a series of paragangliomas and pheochromocytomas from 64 patients. The patients had been previously checked for mutations in SDHD, SDHC and SDHB, but also for mutation in RET and VHL. All 14 patients with SDH mutations (9 with SDHB and 5 with SDHD mutations) exhibited negative or weak-diffuse SDHB staining pattern in tumour tissue, whereas cells of the 23 RET mutated and 8 VHL mutated tumours showed a positive SDHB immunostaining. Sixteen of the patients that did not exhibit a mutation in any gene showed positive SDHB immunostaining in tumour tissue, while only three of the patients without mutation exhibited negative staining. All patients exhibited positive pattern of SDHA immunostaining. The results confirm the value of SDHB immunohistochemical status in assessment of germline mutations in PGL/PCC syndrome.

A comprehensive next generation sequencing-based genetic testing strategy to improve diagnosis of inherited pheochromocytoma and paraganglioma.

CONTEXT: Pheochromocytomas and paragangliomas are notable for a high frequency of inherited cases, many of which present as apparently sporadic tumors. OBJECTIVE: The objective of this study was to establish a comprehensive next generation sequencing (NGS)-based strategy for the diagnosis of patients with pheochromocytoma and paraganglioma by testing simultaneously for mutations in MAX, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL. DESIGN: After the methodology for the assay was designed and established, it was validated on DNA samples with known genotype and then patients were studied prospectively. SETTING: The study was performed in a diagnostic genetics laboratory. PATIENTS: DNA samples from 205 individuals affected with adrenal or extraadrenal pheochromocytoma/head and neck paraganglioma (PPGL/HNPGL) were analyzed. A proof-of-principle study was performed using 85 samples known to contain a variant in 1 or more of the genes to be tested, followed by prospective analysis of an additional 120 samples. MAIN OUTCOME MEASURES: We assessed the ability to use an NGS-based method to perform comprehensive analysis of genes implicated in inherited PPGL/HNPGL. RESULTS: The proof-of-principle study showed that the NGS assay and analysis gave a sensitivity of 98.7%. A pathogenic mutation was identified in 16.6% of the prospective analysis cohort of 120 patients. CONCLUSIONS: A comprehensive NGS-based strategy for the analysis of genes associated with predisposition to PPGL and HNPGL was established, validated, and introduced into diagnostic service. The new assay provides simultaneous analysis of 9 genes and allows more rapid and cost-effective mutation detection than the previously used conventional Sanger sequencing-based methodology.

Evaluation of SDHB, SDHD and VHL gene susceptibility testing in the assessment of individuals with non-syndromic phaeochromocytoma, paraganglioma and head and neck paraganglioma.

OBJECTIVES: Research studies have reported that about a third of individuals with phaeochromocytoma/paraganglioma (PPGL) have an inherited predisposition, although the frequency of specific mutations can vary between populations. We evaluated VHL, SDHB and SDHD mutation testing in cohorts of patients with non-syndromic PPGL and head and neck paraganglioma (HNPGL). DESIGN: Prospective, observational evaluation of NHS practice. PATIENTS: Individuals with PPGL/HNPGL referred to a supraregional genetics testing service over a 10-year period. MEASUREMENTS: Clinical (age, tumour site, malignancy, etc.), mutation frequencies and characteristics. RESULTS: A total of 501 probands with PPGL (n = 413) or HNPGL (n = 88) were studied. Thirty-one percent of patients with PPGL presented had a pathogenic mutation in SDHB, SDHD or VHL. Mutation detection rates were highest in those with a positive family history (62%), malignancy (53%), multiple tumours (33%) or PGL (44%). Twenty-eight percent of individuals with a single sporadic phaeochromocytoma had a mutation. Overall, 63% of patients with HNPGL had a mutation (92% of those with a family history, 89% of those with multicentric tumours and 34% of those with a single sporadic HNPGL). Penetrance was calculated in 121 SDHB mutation-positive probands and 187 of their mutation-positive relatives. Most relatives were asymptomatic and lifetime penetrance in non-proband SDHB mutation carriers was <50%. CONCLUSIONS: Practice-based evaluations of genetic testing in PPGL reveal high mutation detection rates. Although clinical criteria can be used to prioritize mutation testing, mutations were detected in 'low risk groups' indicating a need for comprehensive and inexpensive genetic testing strategies for PPGL and HNPGL.

Somatic mutation analysis of the SDHB, SDHC, SDHD, and RET genes in the clinical assessment of sporadic and hereditary pheochromocytoma.

Systemic analysis of somatic mutations of other susceptibility genes in syndromic tumors as well as apparently sporadic tumors in well-characterized specimens is lacking. Its clinical relevance has not been studied. Our objective was to determine the frequency of second allele inactivation in syndromic tumors and determine the frequency and potential clinical impact of somatic mutations and loss of heterozygosity (LOH) of the known susceptibility genes in syndromic and sporadic tumors. Nine tumor specimens from clinically characterized VHL mutation, five from SDHB mutation, four from SDHD mutation, two from RET mutation carriers, and eight from apparently sporadic cases were analyzed. Tumor DNA mutation screening of the SDHx, VHL, and RET genes and LOH analyses of the SDHx and VHL genes were performed. The Yates-corrected chi-squared test was used for comparison of the clinical data and the molecular-genetic results. Second allele inactivation in tumors was identified in 83% of VHL, 80% of SDHB, and 50% of SDHD specimen. High prevalence of VHL (6/6, p=0.024) and SDHB (7/7, p=0.018) somatic mutations has been identified in the sporadic group compared to all others. In the group of the VHL tumors the SDHB somatic events were significantly lower (2/6; p=0.045). In 18/19 (95%) of cases, we were able to demonstrate the presence of at least two concomitant affected susceptibility genes. We conclude that LOH is the most prevalent second allele-inactivating event. SDHB and VHL somatic mutation might play a role in the sporadic forms of tumor development. There is no clinical impact of mutation screening or LOH analysis of tumor specimens.

Molecular characterization of boscalid- and penthiopyrad-resistant isolates of Didymella bryoniae and assessment of their sensitivity to fluopyram.

BACKGROUND: Didymella bryoniae has a history of developing resistance to single-site fungicides. A recent example is with the succinate-dehydrogenase-inhibiting fungicide (SDHI) boscalid. In laboratory assays, out of 103 isolates of this fungus, 82 and seven were found to be very highly resistant (B(VHR) ) and highly resistant (B(HR) ) to boscalid respectively. Cross-resistance studies with the new SDHI penthiopyrad showed that the B(VHR) isolates were only highly resistant to penthiopyrad (B(VHR) -P(HR) ), while the B(HR) isolates appeared sensitive to penthiopyrad (B(HR) -P(S) ). In this study, the molecular mechanism of resistance in these two phenotypes (B(VHR) -P(HR) and B(HR) -P(S) ) was elucidated, and their sensitivity to the new SDHI fluopyram was assessed. RESULTS: A 456 bp cDNA amplified fragment of the succinate dehydrogenase iron sulfur gene (DbSDHB) was initially cloned and sequenced from two sensitive (B(S) -P(S) ), two B(VHR) -P(HR) and one B(HR) -P(S) isolate of D. bryoniae. Comparative analysis of the DbSDHB protein revealed that a highly conserved histidine residue involved in the binding of SDHIs and present in wild-type isolates was replaced by tyrosine (H277Y) or arginine (H277R) in the B(VHR) -P(HR) and B(HR) -P(S) variants respectively. Further examination of the role and extent of these alterations showed that the H/Y and H/R substitutions were present in the remaining B(VHR) -P(HR) and B(HR) -P(S) variants respectively. Analysis of the sensitivity to fluopyram of representative isolates showed that both SDHB mutants were sensitive to this fungicide as the wild-type isolates. CONCLUSION: The genotype-specific cross-resistance relationships between the SDHIs boscalid and penthiopyrad and the lack of cross-resistance between these fungicides and fluopyram should be taken into account when selecting SDHIs for gummy stem blight management.

Analysis of the production process of optically pure D-lactic acid from raw glycerol using engineered Escherichia coli strains.

Glycerol has become an ideal feedstock for producing fuels and chemicals. Here, five technological schemes for optically pure D: -lactic acid production from raw glycerol were designed, simulated, and economically assessed based on five fermentative scenarios using engineered Escherichia coli strains. Fermentative scenarios considered different qualities of glycerol (pure, 98 wt.%, and crude, 85 wt.%) with concentrations ranging from 20 to 60 g/l in the fermentation media, and two fermentation stages were also analyzed. Raw glycerol (60 wt.%) was considered as the feedstock feeding the production process in all cases; then a purification process of raw glycerol up to the required quality was required. Simulation processes were carried out using Aspen Plus, while economic assessments were performed using Aspen Icarus Process Evaluator. D: -Lactic acid recovery and purification processes were based on reactive extraction with tri-n-octylamine using dichloromethane as active extractant agent. The use of raw glycerol represents only between 2.4% and 7.8% of the total production costs. Also, the total production costs obtained of D: -lactic acid in all cases were lower than its sale price indicating that these processes are potentially profitable. Thus, the best configuration process requires the use of crude glycerol diluted at 40 g/l with total glycerol consumption and with D: -lactic acid recovering by reactive extraction. The lowest obtained total production cost was 1.015 US$/kg with a sale price/production cost ratio of 1.53.

Familial paraganglioma syndrome: applying genetic screening in otolaryngology.

CONTEXT: sporadic head and neck paragangliomas often represent familial paraganglioma syndrome (FPS). FPS patients require close follow-up, and family members will benefit from screening. No clear guideline for following these patients exists in the otolaryngology literature. OBJECTIVE: to present a series of FPS patients, illustrating the importance of a cost-effective FPS genetic screening algorithm applicable in otolaryngology. DESIGN: case series, literature review, clinical guidelines. SETTING: tertiary care hospital. PATIENTS: adult patients with SDHx mutations were identified in the University of Alberta's head and neck mass database. Medical records were reviewed for presentation, diagnosis, findings, treatment, follow-up, and genetic testing. A literature review of FPS clinical features, treatment, and genetic screening methods was performed. INTERVENTION: genetic screening. MAIN OUTCOME MEASURE: cost-effectiveness of genetic testing for FPS screening. RESULTS: two patients with FPS were surgically treated by otolaryngologists. All patients presented with multifocal disease and carried SDHB or SDHD mutations. A screening and genetic testing protocol was implemented leading to early detection in a third patient, thus reducing morbidity. The literature review supports the contention that all patients with head and neck paragangliomas should undergo genetic testing. An algorithm to screen such patients is proposed. Cost analysis showed savings of over $2400 ($US 2200) every 6 years with this approach. CONCLUSION: owing to the potential morbidity associated with head and neck paragangliomas, it is prudent that FPS be ruled out. Those found to have SDHx mutations, and first-degree relatives, should be offered genetic testing with enrolment in a screening protocol. This provides a cost-effective, early detection approach to FPS.

Quantitative assessment of succinate dehydrogenase activity of rat lateral pterygoid muscle in undecalcified fresh-frozen section.

The quantitative assessment of enzyme activities in situ in single muscle fibres is essential for understanding the functions of skeketal muscles. The function of the lateral pterygoid muscle (LPM) is not fully understood because it is a deeply located masticatory muscle and cannot be dissected in an intact configuration. Here we report how to measure the activities of mitochondrial succinate dehydrogenase (SDH) in single muscle fibres in situ in the LPM in sections of rat heads. Unfixed head sections were incubated on gel films containing SDH substrate and nitroblue tetrazolium. During incubation, images of the section due to deposition of the final reaction products, formazans, were captured at intervals of 10 s using a real-time image analysis system for absorbance measurements. We found that the belly of the LPM consisted of four areas with different mean activities of SDH. The lateral and upper areas of the muscle showed similarly high SDH activities. Mean activity in the lower area was the lowest, about half of those of the lateral and upper areas. These results agree with the hypothesis that the superior head of the LPM participates in more continuous contraction and is more resistant to fatigue than the inferior head.

Antisense oligodeoxynucleotides: useful tool for search and assessment of new targets for anti-malarial drugs.

We investigated about targeting for new antimalarial drugs using antisense (AS) oligodeoxynucleotides (ODNs). Synthetic nuclease-resistant ODNs (phosphorothioate (PS) ODNs and ODNs containing 4'alpha-C-(2-aminoethyl)thymidines (4'-amino ODNs)) which target mitochondrial succinate dehydrogenase (SDH) iron-sulfur subunit (IP), had antimalarial activity (EC50; about 1.0 microM). Furthermore we showed that intra-parasitic SDH IP mRNA levels, which were detected using quantitative RT-PCR assay, were decreased 13% of control after the 24 h expose to SDH IP AS. From the results, we conclude that SDH has potential as the target for novel antimalarials, and AS ODNs is effective for search and assessment of targets for new antimalarial drugs.