Succinate Dehydrogenase Subunit B Research Articles

Sensitivity of Botrytis cinerea from vineyards to Boscalid, Isofetamid and Pydiflumetofen in Shandong province, China

Succinate dehydrogenase inhibitor (SDHI) fungicides are the most commonly and effectively used class of fungicides for controlling gray mold. Among them, only boscalid has been registered in China for controlling grape gray mold, while isofetamid and pydiflumetofen are two new SDHI fungicides that have demonstrated high efficacy against various fungal diseases. However, the sensitivity to these three fungicides against B. cinerea isolates from vineyards in China are currently unknown. In this study, the sensitivity of 55 B. cinerea isolates from vineyards to boscalid, isofetamid and pydiflumetofen were determined, with EC50 values ranging from 1.10 to 393, 0.0300 to 42.0 and 0.0990 to 25.5 μg mL-1, respectively. The resistance frequencies for boscalid, isofetamid and pydiflumetofen were 60.0%, 7.2% and 12.8%, respectively. Three mutations (H272R, H272Y, P225F) were detected in the SdhB subunit, with H272R being the most prevalent (75.7%), followed by H272Y (16.2%) and P225F (8.1%). All three mutations are associated with resistance to boscalid, of which H272R mutants exhibited high resistance. Only P225F and H272Y mutants exhibited resistance to isofetamid and pydiflumetofen, respectively. A weakly positive cross-resistance relationship was observed between boscalid and pydiflumetofen (r = 0.38, P < 0.05). Additionally, the H272R mutants showed no significant fitness costs, whereas the remaining mutants exhibited reduced mycelial growth (P225F) and sporulation (H272Y and P225F). These results suggest that isofetamid and pydiflumetofen are effective fungicides against B. cinerea in vineyards, but appropriate rotation strategies must be implemented to reduce the selection of existing SDHI-resistant isolates.

Resistance of the Ginseng Gray Mold Pathogen, Botrytis cinerea, to Boscalid and Pyraclostrobin Fungicides in China.

Gray mold caused by Botrytis cinerea severely threatens the yield of ginseng (Panax ginseng). Various categories of fungicides have been utilized to control gray mold on this crop. In this study, the resistance of 102 isolates of B. cinerea from 11 commercial ginseng-growing regions in China to fungicides was examined. A total of 32.4% were resistant to boscalid, with EC50 values that ranged from 12.26 to 235.87 μg/ml, and 94.1% were resistant to pyraclostrobin, with EC50 values that ranged from 5.88 to 487.72 μg/ml. Except for sdhA and sdhD, the amino acid substitutions of P225F, P225L, N230I, H272Y, and H272R in the sdhB subunit from 24 (4 sensitive [S] and 20 resistant [R]), 5 (1 S and 4 R), 1 (S), 1 (R), and 8 (4 S and 4 R) strains, respectively, and the concurrent amino acid substitutions of G85A + I93V + M158V + V168I in the sdhC subunit from 5 (4 S and 1 R) strains were identified. A G143A substitution in cytochrome b was identified in 96 isolates that were resistant to pyraclostrobin and three that were sensitive to it. The Bcbi-143/144 intron was identified in the other three isolates sensitive to pyraclostrobin, but it was absent in the isolates that harbored the G143A mutation. The results showed that the populations of B. cinerea on ginseng have developed strong resistance to pyraclostrobin. Therefore, it is not recommended to continue using this fungicide to control gray mold on P. ginseng. Boscalid is still effective against most isolates. However, to prevent fungicide resistance, it is recommended to use a mixture of boscalid with other categories of fungicides.

Responses to systemic therapy in metastatic pheochromocytoma/paraganglioma: a retrospective multicenter cohort study.

The therapeutic options for metastatic pheochromocytomas/paragangliomas (mPPGLs) include chemotherapy with cyclophosphamide/vincristine/dacarbazine (CVD), temozolomide monotherapy, radionuclide therapies, and tyrosine kinase inhibitors such as sunitinib. The objective of this multicenter retrospective study was to evaluate and compare the responses of mPPGLs including those with pathogenic variants in succinate dehydrogenase subunit B (SDHB), to different systemic treatments. This is a retrospective analysis of treatment responses of mPPGL patients (n = 74) to systemic therapies. Patients with mPPGLs treated at 6 specialized national centers were selected based on participation in the ENSAT registry. Survival until detected progression (SDP) and disease-control rates (DCRs) at 3 months were evaluated based on imaging reports. For the group of patients with progressive disease at baseline (83.8% of 74 patients), the DCR with first-line CVD chemotherapy was 75.0% (n = 4, SDP 11 months; SDHB [n = 1]: DCR 100%, SDP 30 months), with somatostatin peptide receptor-based radionuclide therapy (PPRT) 85.7% (n = 21, SDP 17 months; SDHB [n = 10]: DCR 100%, SDP 14 months), with 131I-meta-iodobenzylguanidine (131I-MIBG) 82.6% (n = 23, SDP 43 months; SDHB [n = 4]: DCR 100%, SDP 24 months), with sunitinib 100% (n = 7, SDP 18 months; SDHB [n = 3]: DCR 100%, SDP 18 months), and with somatostatin analogs 100% (n = 4, SDP not reached). The DCR with temozolomide as second-line therapy was 60.0% (n = 5, SDP 10 months; SDHB [n = 4]: DCR 75%, SDP 10 months). We demonstrate in a real-life clinical setting that all current therapies show reasonable efficacy in preventing disease progression, and this is equally true for patients with germline SDHB mutations.

SAT305 Clinical Characteristics And Outcomes Of Patient With Metastatic Pheochromocytoma/Paraganglioma (PPGL) And Extreme Elevated Biochemistry

Abstract Disclosure: T. Prodanov: None. A. Jha: None. S. Talvacchio: None. M. Nazari, MD: None. K. Pacak, MD, PhD: None. Background: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrinetumors arising from chromaffin cells in adrenal or extra-adrenal locations,respectively. PPGLs are characterized by an excessive production ofcatecholamines and/or metanephrines. Most of the symptoms of PPGLs, likehypertension, headaches, palpitation, sweating, and anxiety are an effect ofoverproduction of catecholamines and/or metanephrines. Metastatic PPGLs mayoccur in as many as 35% of patients, with higher metastatic potential observed in succinatedehydrogenase subunit B (SDHB)-related tumors (up to 90%). ClinicalCase: A retrospective analysis of 422patients with metastatic PPGL diagnosed and treated from 2000 to 2023 at theNational Institutes of Health, in Bethesda, Maryland, was conducted. We found42 patients with extremely elevated free plasma normetanephrine levels.Extremely elevated level was defined if the value was 100 times or more higherthan the upper reference limit. Free plasma normetanephrine and metanephrinewere measured. Therelated descriptive analyses of this cohort are as follows: 18 (42.8%) werefemales, 24 (57.2%) were males. The average age of patients at the time of theprimary tumor diagnosis was 39.3 ± 44 years. At theirinitial presentation, there were 29 (69.1%) patients with solitary tumors; and13 (30.9%) with metastatic tumors. In 20 (47.6%) patients, primary tumor waslocated in the adrenal gland, 19 (45.3%) had extra-adrenal PGL and head andneck PGL was seen in 3 (7.1%) patients. The average time interval to metastaticdisease was 5.7 ± 20 years. Thirteen(30.9%) patients had metastatic disease at the initial presentation. Theelevated plasma normetanephrine values ranged from 11407 to 69697 pg/mL (normalrange 18-112 pg/mL). The highest level was 622 times above upper referencelimit. None of the patients were found with elevated metanephrine 100 timesthat of upper reference limit.Genetictesting showed pathogenic variants in SDHB in 22 (52.4%), SDHA in2 (4.7%), NF1 in 2 (4.7%), VHL in 1 (2.3%), IDH1 in 1 (2.3%),and apparently sporadic disease in 12 (28.6%) patients, respectively. Overallsurvival, defined as time from diagnosis until last follow up/death was 11.47 ± 22.5 years.Twentypatients received CVD therapy, 13 patients received 131I-MIBGtherapy, 4 patients received 177Lu-DOTATATE therapy, 9 receivedlocalized radiation therapy. Thirty-nine out of 42 (92.8%) patients died due tometastatic PPGL most likely related to cardiovascular complications. Conclusion:Patients presented with extremely elevatednormetanephrine levels were found to have poorer prognosis as 92.8% (39/42) ofpatients died due to complications associated with their disease. Patients whopresent with extremely elevated plasma metanephrines should be carefullymonitored and managed. Presentation: Saturday, June 17, 2023

The aggressiveness of succinate dehydrogenase subunit B-deficient chromaffin cells is reduced when their bioelectrical properties are restored by glibenclamide.

Pheochromocytomas/paragangliomas (PPGLs) are neuroendocrine tumours, mostly resulting from mutations in predisposing genes. Mutations of succinate dehydrogenase (SDH) subunit B (SDHB) are associated with high probability of metastatic disease. Since bioelectrical properties and signalling in cancer are an emerging field, we investigated the metabolic, functional and electrophysiological characteristics in human succinate dehydrogenase subunit B (SDHB)-deficient pheochromocytoma cells. These cells exhibited reduced SDH function with elevated succinate-to-fumarate ratio and reduced intracellular ATP levels. The analysis of membrane passive properties revealed a more hyperpolarized membrane potential and a lower cell capacitance of SDHB-deficient cells compared to the parental ones. These bioelectrical changes were associated with reduced proliferation and adhesion capacity of SDHB-deficient cells. Only in SDHB-deficient cells, we also observed an increased amplitude of potassium currents suggesting an activation of ATP-sensitive potassium channels (KATP). Indeed, exposure of the SDHB-deficient cells to glibenclamide, a specific KATP inhibitor, or to ATP caused normalization of potassium current features and altered proliferation and adhesion. In this work, we show for the first time that reduced intracellular ATP levels in SDHB-deficient chromaffin cells impaired cell bioelectrical properties, which, in turn, are associated with an increased cell aggressiveness. Moreover, we first ever demonstrated that glibenclamide not only reduced the outward potassium currents in SDHB-deficient cells but increased their growth capacity, reduced their ability to migrate and shifted their phenotype towards one more similar to that of parental one.

Abdominal nonfunctional paraganglioma in which succinate dehydrogenase subunit B (SDHB) immunostaining was performed: a case report

BackgroundAbdominal nonfunctional paraganglioma is rare. Malignant potential of paraganglioma is assessed by Grading of Adrenal Pheochromocytoma and Paraganglioma score and genetic testing, but genetic testing is not common. We present a case of abdominal nonfunctional paraganglioma whose malignant potential was assessed by grading of adrenal pheochromocytoma and paraganglioma score and succinate dehydrogenase subunit B staining alternative to genetic testing.Case presentationA 39-year-old Japanese man had a right retroperitoneal tumor without symptoms. Uptake in the tumor was shown by 123I-meta-iodobenzylguanidine scintigraphy. There were no metastases. The results of biochemical workups including blood hormones and urinary metanephrines were normal. We performed retroperitoneoscopic surgery. The tumor was positive for chromogranin A staining but negative for tyrosine hydroxylase. On the basis of the preoperative biochemical workups and pathology results, we diagnosed the tumor as nonfunctional paraganglioma. The Grading of Adrenal Pheochromocytoma and Paraganglioma score classified the tumor as moderately differentiated. Furthermore, negative succinate dehydrogenase subunit B staining suggested the patient has the SDHx (SDHA, SDHB, SDHC and SDHD) mutation.ConclusionAbdominal nonfunctional PGLs are associated with SDHB mutation, and SDHB staining should be performed as a screening.

Succinate dehydrogenase variants in paraganglioma: why are B subunit variants 'bad'?

Mutations that predispose to familial pheochromocytoma and paraganglioma include inherited variants in the four genes (SDHA, SDHB, SDHC and SDHD) encoding subunits of succinate dehydrogenase (SDH), an enzyme of the mitochondrial tricarboxylic acid cycle and complex II of the electron transport chain. In heterozygous variant carriers, somatic loss of heterozygosity is thought to result in tumorigenic accumulation of succinate and reactive oxygen species. Inexplicably, variants affecting the SDHB subunit predict worse clinical outcomes. Why? Here we consider two hypotheses. First, relative to SDH A, C and D subunits, the small SDHB subunit might be more intrinsically 'fragile' to missense mutations because of its relatively large fraction of amino acids contacting prosthetic groups and other SDH subunits. We show evidence that supports this hypothesis. Second, the natural pool of human SDHB variants might, by chance, be biased toward severe truncating variants and missense variants causing more disruptive amino acid substitutions. We tested this hypothesis by creating a database of known SDH variants and predicting their biochemical severities. Our data suggest that natural SDHB variants are more pathogenic. It is unclear if this bias is sufficient to explain clinical data. Other explanations include the possibility that SDH subcomplexes remaining after SDHB loss have unique tumorigenic gain-of-function characteristics, and/or that SDHB may have additional unknown tumor-suppressor functions.

Metabolic Characterization and Glyceraldehyde-3-Phosphate Dehydrogenase-Dependent Regulation of Epithelial Sodium Channels in hPheo1 Wild-type and SDHB Knockdown Cells.

Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited curative treatment options outside of surgical resection. Patients with mutations in succinate dehydrogenase subunit B (SDHB) are at an increased risk of malignant and aggressive disease. As cation channels are associated with tumorigenesis, we studied the expression and activity of cation channels from the Degenerin superfamily in a progenitor cell line derived from a human PCC. hPheo1 wild-type (WT) and SDHB knockdown (KD) cells were studied to investigate whether epithelial sodium channels (ENaC) and acid-sensing ion channels (ASIC) are regulated by the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). First, we performed targeted metabolomic studies and quantified changes in glycolysis pathway intermediates and citric acid cycle intermediates using hPheo1 WT cells and SDHB KD cells. Next, we performed protein biochemistry and electrophysiology studies to characterize the protein expression and activity, respectively, of these ion channels. Our western blot experiments show both ENaC alpha and ASIC1/2 are expressed in both hPheo1 WT and SDHB KD cells, with lower levels of a cleaved 60 kDa form of ENaC in SDHB KD cells. Single-channel patch clamp studies corroborate these results and further indicate channel activity is decreased in SDHB KD cells. Additional experiments showed a more significant decreased membrane potential in SDHB KD cells, which were sensitive to amiloride compared to WT cells. We provide evidence for the differential expression and activity of ENaC and ASIC hybrid channels in hPheo1 WT and SDHB KD cells, providing an important area of investigation in understanding SDHB-related disease.

SNORA14A inhibits hepatoblastoma cell proliferation by regulating SDHB-mediated succinate metabolism

Hepatoblastoma (HB) is the most common paediatric liver malignancy. Dysregulation of small nucleolar RNAs (snoRNAs) is a critical inducer of tumour initiation and progression. However, the association between snoRNAs and HB remains unknown. Here, we conducted snoRNA expression profiling in HB by snoRNA sequencing and identified a decreased level of SNORA14A, a box H/ACA snoRNA, in HB tissues. Low expression of SNORA14A was correlated with PRETEXT stage and metastasis in patients. Functionally, overexpression of SNORA14A suppressed HB cell proliferation and triggered cell apoptosis and G2/M phase arrest. Mechanistically, SNORA14A overexpression promoted the processing and maturation of the 18 S ribosomal RNA (rRNA) precursor to increase succinate dehydrogenase subunit B (SDHB) protein levels. In accordance with SNORA14A downregulation, SDHB protein expression was significantly reduced in HB tissues and cells, accompanied by abnormal accumulation of succinate. Overexpression of SDHB showed antiproliferative and proapoptotic effects and the capacity to induce G2/M phase arrest, while succinate dose-dependently stimulated HB cell growth. Furthermore, the inhibition of SNORA14A in HB malignant phenotypes was mediated by SDHB upregulation-induced reduction of cellular succinate levels. Therefore, the SNORA14A/18 S rRNA/SDHB axis suppresses HB progression by preventing cellular accumulation of the oncometabolite succinate and provides promising prognostic biomarkers and novel therapeutic targets for HB.

ONC201 Suppresses Neuroblastoma Growth by Interrupting Mitochondrial Function and Reactivating Nuclear ATRX Expression While Decreasing MYCN.

Neuroblastoma (NB) is characterized by several malignant phenotypes that are difficult to treat effectively without combination therapy. The therapeutic implication of mitochondrial ClpXP protease ClpP and ClpX has been verified in several malignancies, but is unknown in NB. Firstly, we observed a significant increase in ClpP and ClpX expression in immature and mature ganglion cells as compared to more malignant neuroblasts and less malignant Schwannian-stroma-dominant cell types in human neuroblastoma tissues. We used ONC201 targeting ClpXP to treat NB cells, and found a significant suppression of mitochondrial protease, i.e., ClpP and ClpX, expression and downregulation of mitochondrial respiratory chain subunits SDHB and NDUFS1. The latter was associated with a state of energy depletion, increased reactive oxygen species, and decreased mitochondrial membrane potential, consequently promoting apoptosis and suppressing cell growth of NB. Treatment of NB cells with ONC201 as well as the genetic attenuation of ClpP and ClpX through specific short interfering RNA (siRNA) resulted in the significant upregulation of the tumor suppressor alpha thalassemia/mental retardation X-linked (ATRX) and promotion of neurite outgrowth, implicating mitochondrial ClpXP proteases in MYCN-amplified NB cell differentiation. Furthermore, ONC201 treatment significantly decreased MYCN protein expression and suppressed tumor formation with the reactivation of ATRX expression in MYCN-amplified NB-cell-derived xenograft tumors. Taken together, ONC201 could be the potential agent to provide diversified therapeutic application in NB, particularly in NB with MYCN amplification.

PSUN27 Largest Cohort of Cervical Spine Metastases of Pheochromocytoma/Paraganglioma in the Setting of SDHB Mutation

Abstract Background Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors arising from chromaffin cells in adrenal or extra-adrenal locations, respectively. Overall, 10 to 20% of PPGLs are metastatic, with higher metastatic potential observed in succinate dehydrogenase subunit B (SDHB)-related tumors (up to 90%). Metastatic PPGLs are diagnosed based on the presence of tumor cells of chromaffin origin in a location outside the site of normal development (adrenal medulla and the sympathetic chain) in the bones and lymphatic nodes per 2017 WHO guidelines. Studies evaluating both metastatic pheochromocytomas and extra-adrenal PGLs have reported bone to be the most common site of metastasis. Clinical Case We reviewed the metastatic patients in our PPGL cohort and found 40 (29.85%) out of 134 patients with SDHB-related metastatic PPGLs developing metastatic lesions in the cervical spine. The related descriptive analyses were as follows: 25 (62.5%) were males, 15 (37.5%) females. Fourteen patients (35%) have died due to metastatic PPGL complications. The average age of patients at the time of the primary tumor diagnosis was 30.3 ± 35 years and the age at the initial metastatic tumor diagnosis was 34 ± 33 years. The average time interval to metastatic disease was 7.4 ± 38 years. At their initial presentation, there were 25 (62.5%) patients with solitary tumors; 1 (2.5%) with multiple primary tumors; and 14 (35%) with metastatic tumors. All the patients either developed or had metastatic lesions in the cervical vertebrae. Nine (22.5%) patients required surgical fusions of the vertebrae to manage their pathological fractures and another 9 (22.5%) patients received radiation therapy. Conclusion Genetic testing is recommended for all patients with PPGLs. Genetic analysis, especially finding a SDHB mutation may be helpful in determining metastatic potential. Most patients with spinal metastatic lesions are asymptomatic, but when clinical symptoms arise, the most common clinical symptom involves pain and neurological deficit. Metastasis to the spine is an important complication that can lead to significant pain and disability and requires surgical interventions or radiotherapy. Awareness for such spinal tumors is essential for offering the correct treatment to the patients. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

P.80 Respiratory chain dysfunction in dermatomyositis is associated with mitochondrial DNA depletion

Patients with dermatomyositis suffer from reduced aerobic metabolism contributing to impaired muscle function, which has been linked to cytochrome c oxidase (COX) deficiency in muscle tissue. This mitochondrial respiratory chain dysfunction is typically seen in perifascicular regions, which also show the most intense inflammatory reaction along with capillary loss and muscle fiber atrophy. The objective of this study was to investigate the pathobiology of the oxidative phosphorylation deficiency in dermatomyositis. Muscle biopsy specimens with perifascicular COX deficiency from five juveniles and seven adults with early untreated dermatomyositis were investigated. Immunohistochemical analyses of subunits in the respiratory chain were performed including complex I (subunit NDUFB8), complex II (succinate dehydrogenase, subunit SDHB) and complex IV (COX, subunit MTCO1). The results showed a profound deficiency of complex I and IV in the perifascicular regions with enzyme histochemical COX deficiency, whereas succinate dehydrogenase activity and complex II were preserved. In situ hybridization of mitochondrial RNA showed depletion of mitochondrial DNA (mtDNA) transcripts in the perifascicular regions. Analysis of mtDNA by next generation deep sequencing and quantitative PCR in affected muscle regions showed an overall reduction of mtDNA copy number particularly in the perifascicular regions. In conclusion, the respiratory chain dysfunction in dermatomyositis muscle is associated with mtDNA depletion causing deficiency of complex I and IV which are partially encoded by mtDNA, whereas complex II which is entirely nuclear gene encoded is preserved. The depletion of mtDNA indicates a perturbed replication of mtDNA partially explaining the muscle pathology and the disturbed aerobic metabolism.

EP-630 Pheochromocytoma Associated with SDHB Mutation: A case report

Abstract Introduction Pheochromocytomas and paragangliomas are rare neuroendocrine tumors arising from the chromaffin cells of the adrenal medulla or extra-adrenal tissues. 10% to 20% of tumors are detected before age 18 years. Approximately 10%–15% of pheochromocytoma and 20%–50% of paraganglioma are malignant. Up to 24% of the cases are familial where they may be associated with genetic syndromes like MEN 2, succinate dehydrogenase subunit B (SDHB) gene mutations and VHL syndrome. Germline mutations in SDHB gene increase the risk of PGLs/ PHEOs and RCC with 38%-83% risk of malignancy. We represent a rare case of pheochromocytoma associated with SDHB mutation in a child. Case presentation A 17-year old patient presented with repeated vomiting and hypertension. BMI was 14.4 kg/m2, BP was 140/112 mmgh, PR was127 peats/min. The rest of the clinical examination was unremarkable. Plasma and 24-hour urinary metanephrines levels were highly elevated together with Chromogranin A level. Abdominal CT and MRI scans revealed an enlarged right adrenal gland measuring approximately 45 mm, indenting but not invading the posterior aspect of the IVC. Management Treatment was initiated with α-blockers; (Doxazosin then phenoxybenzamine); followed by β-blockers. The patient underwent laparoscopic right adrenalectomy. The tumor was 50×30×30 mm and weighting 58g. The tumor was positive with chromogranin, and S100. The PASS score was 2. Genetic mutation was detected at SDGB (c.72+1G>A). Conclusion We recommend genetic testing for all suspecting cases of PHEO/PGLs to establish the appropriate follow-up plan.

Respiratory chain dysfunction in perifascicular muscle fibres in patients with dermatomyositis is associated with mitochondrial DNA depletion.

Patients with dermatomyositis (DM) suffer from reduced aerobic metabolism contributing to impaired muscle function, which has been linked to cytochrome c oxidase (COX) deficiency in muscle tissue. This mitochondrial respiratory chain dysfunction is typically seen in perifascicular regions, which also show the most intense inflammatory reaction along with capillary loss and muscle fibre atrophy. The objective of this study was to investigate the pathobiology of the oxidative phosphorylation deficiency in DM. Muscle biopsy specimens with perifascicular COX deficiency from five juveniles and seven adults with DM were investigated. We combined immunohistochemical analyses of subunits in the respiratory chain including complex I (subunit NDUFB8), complex II (succinate dehydrogenase, subunit SDHB) and complex IV (COX, subunit MTCO1) with in situ hybridisation, next generation deep sequencing and quantitative polymerase chain reaction (PCR). There was a profound deficiency of complexes I and IV in the perifascicular regions with enzyme histochemical COX deficiency, whereas succinate dehydrogenase activity and complex II were preserved. In situ hybridisation of mitochondrial RNA showed depletion of mitochondrial DNA (mtDNA) transcripts in the perifascicular regions. Analysis of mtDNA by next generation deep sequencing and quantitative PCR in affected muscle regions showed an overall reduction of mtDNA copy number particularly in the perifascicular regions. The respiratory chain dysfunction in DM muscle is associated with mtDNA depletion causing deficiency of complexes I and IV, which are partially encoded by mtDNA, whereas complex II, which is entirely encoded by nuclear DNA, is preserved. The depletion of mtDNA indicates a perturbed replication of mtDNA explaining the muscle pathology and the disturbed aerobic metabolism.

Identification of Putative SDHI Target Site Mutations in the SDHB, SDHC, and SDHD Subunits of the Grape Powdery Mildew Pathogen Erysiphe necator.

Succinate dehydrogenase inhibitors (SDHIs) are fungicides used in control of numerous fungal plant pathogens, including Erysiphe necator, the causal agent of grapevine powdery mildew (GPM). Here, the sdhb, sdhc, and sdhd genes of E. necator were screened for mutations that may be associated with SDHI resistance. GPM samples were collected from 2017 to 2020 from the U.S. states of California, Oregon, Washington, and Michigan, and the Canadian province of British Columbia. Forty-five polymorphisms were identified in the three sdh genes, 17 of which caused missense mutations. Of these, the SDHC-p.I244V substitution was shown in this study to reduce sensitivity of E. necator to boscalid and fluopyram, whereas the SDHC-p.G25R substitution did not affect SDHI sensitivity. Of the other 15 missense mutations, the SDHC-p.H242R substitution was shown in previous studies to reduce sensitivity of E. necator toward boscalid, whereas the equivalents of the SDHB-p.H242L, SDHC-p.A83V, and SDHD-p.I71F substitutions were shown to reduce sensitivity to SDHIs in other fungi. Generally, only a single amino acid substitution was present in the SDHB, SDHC, or SDHD subunit of E. necator isolates, but missense mutations putatively associated with SDHI resistance were widely distributed in the sampled areas and increased in frequency over time. Finally, isolates that had decreased sensitivity to boscalid or fluopyram were identified but with no or only the SDHC-p.G25R amino acid substitution present in SDHB, SDHC, and SDHD subunits. This suggests that target site mutations probably are not the only mechanism conferring resistance to SDHIs in E. necator.

Baseline Sensitivity and Resistance of Botrytis cinerea to Penthiopyrad in Hebei Province, China

Tomato gray mold (TGM), caused by Botrytis cinerea, is a destructive disease of greenhouse tomatoes. Its control mainly depends on the application of chemical fungicides. In China, penthiopyrad has been registered for the management of TGM for about 3 years. In this study, the baseline sensitivity of B. cinerea to penthiopyrad was measured based on mycelial growth and spore germination, using 131 wild isolates collected from Hebei Province. The mean EC50 values 1.054 ± 0.633 mg/L (on mycelial growth) and 0.101 ± 0.037 mg/L (on conidia germination) were used as the baseline sensitivity. Isolates (187) of B. cinerea, sampled from eight regions in Hebei province during 2021 and 2022, were used for resistance studies by mycelial growth method. Based on the resistance factor, these isolates were considered: sensitive (=65 isolates), low (=39 isolates), moderate (=38 isolates), and high (=45 isolates) resistance. All 15 isolates from Raoyang were sensitive to penthiopyrad. Four mutation types in the sdhB subunit, namely H272R, N230I, P225F, and P225L, were associated with the resistance to penthiopyrad. A positive cross-resistance was also recorded between penthiopyrad and fluopyram, boscalid, or pydiflumetofen. These results provide useful information on the chemical control of TGM.

Metformin Treatment Induces Different Response in Pheochromocytoma/Paraganglioma Tumour Cells and in Primary Fibroblasts.

Simple SummaryPheochromocytoma/paragangliomas (PPGLs) are neuroendocrine tumours and are often non-metastatic. However, no effective treatment is available for their metastatic form. Recent studies have shown that metformin exhibits antiproliferative activity in many human cancers, including PPGLs. Nevertheless, no data are available concerning whether metformin is also able to inhibit PPGL metastatic spread. A tumour is a very complex system, comprising not only cancer cells, but also other cells that all together form the so-called tumour microenvironment. Cancer-associated fibroblasts are residential or recruited fibroblasts, transformed by cancer cells, to promote tumour growth and spread. Therefore, the interplay between tumour cells and cancer-associated fibroblasts has become an interesting target for cancer therapy. Here, we demonstrate that metformin has different effects on cancer cells and fibroblasts, providing evidence that metformin may hold promise for altering tumour microenvironment homeostasis. Improving our knowledge on malignant tumour microenvironment properties could lead to develop complementary strategies to target tumour spread and progression.Pheochromocytoma/paragangliomas (PPGLs) are neuroendocrine tumours, often non-metastatic, but without available effective treatment for their metastatic form. Recent studies have shown that metformin exhibits antiproliferative activity in many human cancers, including PPGLs. Nevertheless, no data are available on the role of metformin on PPGL cells (two-dimension, 2D) and spheroids (three-dimension, 3D) migration/invasion. In this study, we observed that metformin exerts an antiproliferative effect on 2D and 3D cultures of pheochromocytoma mouse tumour tissue (MTT), either silenced or not for the SDHB subunit. However, metformin did not affect MTT migration. On the other hand, metformin did not have a short-term effect on the proliferation of mouse primary fibroblasts, but significantly decreased their ability to migrate. Although the metabolic changes induced by metformin were similar between MTT and fibroblasts (i.e., an overall decrease of ATP production and an increase in intracellular lactate concentration) the activated signalling pathways were different. Indeed, after metformin administration, MTT showed a reduced phosphorylation of Akt and Erk1/2, while fibroblasts exhibited a downregulation of N-Cadherin and an upregulation of E-Cadherin. Herein, we demonstrated that metformin has different effects on cell growth and spread depending on the cell type nature, underlining the importance of the tumour microenvironment in dictating the drug response.

Mitochondrial Transfusion Improves Mitochondrial Function Through Up-regulation of Mitochondrial Complex II Protein Subunit SDHB in the Hippocampus of Aged Mice

The mitochondrial theory of aging is characterized by mitochondrial electron transport chain dysfunction. As a hallmark of aging, an increasing number of investigations have attempted to improve mitochondrial function in both aging and age-related disease. Emerging from these attempts, methods involving mitochondrial isolation, transfusion, and transplantation have taken center stage. In particular, mitochondrial transfusion refers to the administration of mitochondria from healthy tissue into the bloodstream or into tissues affected by injury, disease, or aging. In this study, methods of mitochondrial isolation and transfusion were developed and utilized. First, we found a significant decrease (p < 0.05) in the expression of mitochondrial complex proteins (I-V) in aged (12 months old) mouse brain tissue (C57BL/6 mice) in comparison to healthy young brain tissue (1 month old). To investigate whether healthy young mitochondria taken from the liver could improve mitochondrial function in older animals, we intravenously injected mitochondria isolated from young C57BL/6 mice into aged mice from the same strain. This study, for the first time, demonstrates that mitochondrial transfusion significantly (p < 0.05) improves mitochondrial function via the up-regulation of the mitochondrial complex II protein subunit SDHB in the hippocampus of aged mice. This result has identified a role for mitochondrial complex II in the aging process. Therefore, mitochondrial complex II could serve as a putative target for therapeutic interventions against aging. However, more importantly, methods of mitochondrial transfusion should be further tested to treat a variety of human diseases or disorders and to slow down or reverse processes of aging.

Rapidly Increasing Boscalid Resistance in Corynespora cassiicola in China.

Corynespora leaf spot caused by Corynespora cassiicola is an important foliar disease in cucumber. Succinate dehydrogenase inhibitors are the main fungicides used to control this disease. With the application of succinate dehydrogenase inhibitors (SDHIs) in the field, boscalid-resistant isolates have been continuously detected in the field. Resistance monitoring programs were performed to investigate the frequency and genotypes of resistant isolates. In our resistance monitoring, the frequency of resistant isolates rapidly increased from 9.68 to 85.88% in 2005 to 2020. Nine genotypes conferring SDHI resistance were found in resistant isolates, with different levels of resistance to SDHIs: B-H278R, B-H278L, B-H278Y, B-I280V, C-N75S, C-S73P, D-D95E, D-H105R, and D-G109V. The first sdh mutation was detected in Hebei Province in China, conferring an amino acid substitution at codon 278 in the sdhB subunit from histidine to tyrosine (B-H278Y), and it was the dominant resistance genotype in 2014 to 2015. Subsequently, other genotypes were gradually detected in the field, and the dominant mutations varied across years and across regions. The newest genotype (B-H278L) conferring SDHI resistance was found in 2020. To the best of our knowledge, this is the first report of C. cassiicola in cucumber. To date, multiple resistance to SDHIs, quinone outside inhibitors, benzimidazole fungicides, and dicarboximide fungicides have been detected, accounting for 75.64% of SDHI-resistant isolates. Therefore, the above four fungicides must be strictly restricted, and further monitoring work in other provinces with more isolates should be performed in the future.

Molecular Mechanism of Sclerotinia sclerotiorum Resistance to Succinate Dehydrogenase Inhibitor Fungicides.

Succinate dehydrogenase inhibitor (SDHI) fungicides have a wide spectrum of fungicidal effects on a variety of fungi causing plant diseases, including Sclerotinia stem rot caused by Sclerotinia sclerotiorum. However, the consistent use of site-specific SDHI fungicides can result in the development of resistant isolates with mutations in the SDHB, SDHC, or SDHD subunit thereby leading to a rapid decline of fungicide performance. In this study, we found that SDHC was genetically evolved into two isotypes SDHC1 and SDHC2 in S. sclerotiorum but not involved in the sensitivity to SDHI fungicides. In addition, we demonstrated that the A11V substitution in SDHB was not involved in the resistance of S. sclerotiorum to boscalid, and this substitution widely emerged in the field populations. Meanwhile, the P226L substitution in SDHB was demonstrated to confer boscalid resistance in S. sclerotiorum. The result of cross-resistance showed that the SDHB-P226L substitution exhibited a positive cross-resistance between boscalid and carboxin, fluopyram, pydiflumetofen, flubeneteram, pyraziflumid, fluindapyr, or penthiopyrad. Taken together, our results indicated that the P226L substitution in SDHB resulted in the resistance of S. sclerotiorum to SDHI fungicides but suffered from fitness penalty, especially the homozygous mutants conferring the P226L substitution in SDHB.