A multicenter, dose-finding, phase 1b study of imatinib in combination with alpelisib as third-line treatment in patients with advanced gastrointestinal stromal tumor.

BACKGROUND: Acquired resistance to approved tyrosine kinase inhibitors limits their clinical use in patients with gastrointestinal stromal tumor (GIST). This study investigated the safety, tolerability and efficacy of alpelisib, a phosphatidylinositol 3-kinase inhibitor, used in combination with imatinib in patients with advanced GIST who had failed prior therapy with both imatinib and sunitinib. METHODS: This phase 1b, multicenter, open-label study consisted of 2 phases: dose escalation and dose expansion. Dose escalation involved 200 mg once daily (QD) alpelisib, initially, followed by 250 and 350 mg. These were combined with 400 mg QD imatinib until maximum tolerated dose (MTD) and/or a recommended phase 2 dose (RP2D) of alpelisib in combination with imatinib was determined. This MTD/RP2D dose was tested to evaluate the clinical activity of this combination in dose expansion. RESULTS: Fifty-six patients were enrolled, 21 and 35 in the dose escalation and expansion phases, respectively. The MTD of alpelisib given with imatinib was determined as 350 mg QD. Combination treatment showed partial response in 1 (2.9%) and stable disease in 15 (42.9%) patients. Median progression-free survival was 2 months (95% CI 1.8-4.6). Overall, 92.9% patients had adverse events (AEs) while 46.4% had grade 3/4 AEs, hyperglycemia being the most common (23.2%). CONCLUSIONS: The MTD of alpelisib was estimated as 350 mg QD when used in combination with imatinib 400 mg QD after oral administration in patients with advanced GIST. The safety and tolerability profile of this combination was acceptable; however, the combination did not demonstrate sufficient clinical activity to justify additional clinical testing. TRIAL REGISTRATION: ClinicalTrials.gov NCT01735968 (date of initial registration 28/11/2012).

A dialogue-like cell communication mechanism is conserved in filamentous ascomycete fungi and mediates interspecies interactions.

In many filamentous fungi, germinating spores cooperate by fusing into supracellular structures, which develop into the mycelial colony. In the model fungus Neurospora crassa, this social behavior is mediated by an intriguing mode of communication, in which two fusing cells take turns in signal sending and receiving. Here we show that this dialogue-like cell communication mechanism is highly conserved in distantly related fungal species and mediates interspecies interactions. In mixed populations, cells of N. crassa and the phytopathogenic gray mold Botrytis cinerea coordinate their behavior over a spatial distance and establish physical contact. Subsequent cell­cell fusion is, however, restricted to germlings of the same species, indicating that species specificity of germling fusion has evolved not on the level of the signal/receptor but at subsequent levels of the fusion process. In B. cinerea, fusion and infectious growth are mutually exclusive cellular programs. Remarkably, the presence of N. crassa can reprogram this behavior and induce fusion of the gray mold on plant surfaces, potentially weakening its pathogenic potential. In a third fungal species, the nematode-trapping fungus Arthrobotrys flagrans, the conserved signaling mechanism mediates vegetative fusion within mycelial colonies but has also been repurposed for the formation of nematode-catching traps. In summary, this study identified the cell dialogue mechanism as a conserved complex trait and revealed that even distantly related fungi possess a common molecular language, which promotes cellular contact formation across species borders.

Plasma Membrane Fusion Is Specifically Impacted by the Molecular Structure of Membrane Sterols During Vegetative Development of Neurospora crassa.

Cell-to-cell fusion is crucial for the development and propagation of most eukaryotic organisms. Despite this importance, the molecular mechanisms mediating this process are only poorly understood in biological systems. In particular, the step of plasma membrane merger and the contributing proteins and physicochemical factors remain mostly unknown. Earlier studies provided the first evidence of a role of membrane sterols in cell-to-cell fusion. By characterizing different ergosterol biosynthesis mutants of the fungus Neurospora crassa, which accumulate different ergosterol precursors, we show that the structure of the sterol ring system specifically affects plasma membrane merger during the fusion of vegetative spore germlings. Genetic analyses pinpoint this defect to an event prior to engagement of the fusion machinery. Strikingly, this effect is not observed during sexual fusion, suggesting that the specific sterol precursors do not generally block membrane merger, but rather impair subcellular processes exclusively mediating fusion of vegetative cells. At a colony-wide level, the altered structure of the sterol ring system affects a subset of differentiation processes, including vegetative sporulation and steps before and after fertilization during sexual propagation. Together, these observations corroborate the notion that the accumulation of particular sterol precursors has very specific effects on defined cellular processes rather than nonspecifically disturbing membrane functioning. Given the phenotypic similarities of the ergosterol biosynthesis mutants of N. crassa during vegetative fusion and of Saccharomyces cerevisiae cells undergoing mating, our data support the idea that yeast mating is evolutionarily and mechanistically more closely related to vegetative than sexual fusion of filamentous fungi.

Imatinib in combination with phosphoinositol kinase inhibitor buparlisib in patients with gastrointestinal stromal tumour who failed prior therapy with imatinib and sunitinib: a Phase 1b, multicentre study.

BACKGROUND: The majority of patients with advanced gastrointestinal stromal tumours (GISTs) develop resistance to imatinib and sunitinib, the standard of care for these patients. This study evaluated the combination of buparlisib, an oral phosphoinositide 3-kinase (PI3K) inhibitor, with imatinib in patients with advanced GIST, who have failed prior therapy with imatinib and sunitinib. METHODS: This Phase 1b, multicentre, open-label study aimed to determine the maximum tolerated dose (MTD) and/or a recommended Phase 2 dose of buparlisib in combination with 400 mg of imatinib through a dose-escalation part and a dose-expansion part, and also evaluated the clinical profile of the combination. RESULTS: Sixty patients were enrolled, including 25 in the dose-escalation part and 35 in the dose-expansion part. In the combination, MTD of buparlisib was established as 80 mg. No partial or complete responses were observed. The estimated median progression-free survival was 3.5 months in the expansion phase. Overall, 98.3% of patients had treatment-related adverse events (AEs), including 45% with grade 3 or 4 AEs. CONCLUSIONS: Buparlisib in combination with imatinib provided no additional benefit compared with currently available therapies. Due to the lack of objective responses, further development of this combination was not pursued for third-line/fourth-line advanced/metastatic GIST. TRIAL REGISTRATION NUMBER: NCT01468688.

Phosphatidic acid produced by phospholipase D is required for hyphal cell-cell fusion and fungal-plant symbiosis.

Although lipid signaling has been shown to serve crucial roles in mammals and plants, little is known about this process in filamentous fungi. Here we analyze the contribution of phospholipase D (PLD) and its product phosphatidic acid (PA) in hyphal morphogenesis and growth of Epichloë festucae and Neurospora crassa, and in the establishment of a symbiotic interaction between E. festucae and Lolium perenne. Growth of E. festucae and N. crassa PLD deletion strains in axenic culture, and for E. festucae in association with L. perenne, were analyzed by light-, confocal- and electron microscopy. Changes in PA distribution were analyzed in E. festucae using a PA biosensor and the impact of these changes on the endocytic recycling and superoxide production investigated. We found that E. festucae PldB, and the N. crassa ortholog, PLA-7, are required for polarized growth and cell fusion and contribute to ascospore development, whereas PldA/PLA-8 are dispensable for these functions. Exogenous addition of PA rescues the cell-fusion phenotype in E. festucae. PldB is also crucial for E. festucae to establish a symbiotic association with L. perenne. This study identifies a new component of the cell-cell communication and cell fusion signaling network for hyphal morphogenesis and growth of filamentous fungi.

Plasma Membrane Integrity During Cell-Cell Fusion and in Response to Pore-Forming Drugs Is Promoted by the Penta-EF-Hand Protein PEF1 in Neurospora crassa.

Plasma membrane damage commonly occurs during cellular growth and development. To counteract these potentially lethal injuries, membrane repair mechanisms have evolved, which promote the integrity of the lipid bilayer. Although the membrane of fungi is the target of important clinical drugs and agricultural fungicides, the molecular mechanisms which mediate membrane repair in these organisms remain elusive. Here we identify the penta-EF-hand protein PEF1 of the genetic model fungus Neurospora crassa as part of a cellular response mechanism against different types of membrane injury. Deletion of the pef1 gene in the wild type and different lysis-prone gene knockout mutants revealed a function of the protein in maintaining cell integrity during cell-cell fusion and in the presence of pore-forming drugs, such as the plant defense compound tomatine. By fluorescence and live-cell imaging we show that green fluorescent protein (GFP)-tagged PEF1 accumulates at the sites of membrane injury in a Ca2+-dependent manner. Site-directed mutagenesis identified Ca2+-binding domains essential for the spatial dynamics and function of the protein. In addition, the subcellular localization of PEF1 revealed that the syncytial fungal colony undergoes compartmentation in response to antifungal treatment. We propose that plasma membrane repair in fungi constitutes an additional line of defense against membrane-disturbing drugs, thereby expanding the current model of fungal drug resistance mechanisms.

Functional analysis of active amino acid residues of the mercaptosuccinate dioxygenase of Variovorax paradoxus B4.

Thiol dioxygenases are non-heme mononuclear-iron proteins and belong to the cupin superfamily. In 2014, mercaptosuccinate dioxygenase (Msdo) of Variovorax paradoxus B4 was identified as another bacterial cysteine dioxygenase (Cdo) homolog catalyzing the conversion of mercaptosuccinate (MS) into succinate and sulfite. To gain further insights into potentially important amino acid residues for enzyme activity, seven enzyme variants were generated and analyzed. (i) Three variants comprised the substitution of one conserved histidine residue each by leucine, either supposed to be mandatory for coordination of the Fe(II) cofactor (H93 and H95) or to be important for substrate positioning within the active site (H163). The corresponding enzyme variants were completely inactive confirming their essential roles for enzyme activity. (ii) Mutation C100S resulted as well in an inactive enzyme demonstrating its importance for either stability or activity of the protein. (iii) For eukaryotic Cdo, a hydrogen bond network for substrate positioning was postulated, and the corresponding amino acids are basically present in Msdo. Albeit the MsdoQ64A mutation exhibited an increased Km of 0.29 mM when compared to the wildtype with 0.06 mM, it did not significantly affect the specific activity. (iv) The variant MsdoR66A showed only very low activity even when high amounts of enzyme were applied indicating that this residue might be important for catalysis. (v) No strong effect had the mutation Y165F for which a specific enzyme activity of 10.22 µmol min-1 mg-1 protein and a Km value of 0.06 mM with high similarity to those of the wildtype enzyme were obtained. This residue corresponds to Y157 of human Cdo, which is part of the catalytic triad and is supposed to be involved in substrate positioning. Apparently, another residue could fulfill this role in Msdo, since the loss of Y165 did not have a strong effect.

Spatio-temporal MAPK dynamics mediate cell behavior coordination during fungal somatic cell fusion.

Mitogen-activated protein kinases (MAPKs) are conserved regulators of proliferation, differentiation and adaptation in eukaryotic cells. Their activity often involves changes in their subcellular localization, indicating an important role for these spatio-temporal dynamics in signal transmission. A striking model illustrating these dynamics is somatic cell fusion in Neurospora crassa Germinating spores of this fungus rapidly alternate between signal sending and receiving, thereby establishing a cell-cell dialog, which involves the alternating membrane recruitment of the MAPK MAK-2 in both fusion partners. Here, we show that the dynamic translocation of MAK-2 is essential for coordinating the behavior of the fusion partners before physical contact. The activation and function of the kinase strongly correlate with its subcellular localization, indicating a crucial contribution of the MAPK dynamics in establishing regulatory feedback loops, which establish the oscillatory signaling mode. In addition, we provide evidence that MAK-2 not only contributes to cell-cell communication, but also mediates cell-cell fusion. The MAK-2 dynamics significantly differ between these two processes, suggesting a role for the MAPK in switching of the cellular program between communication and fusion.

[Migration of an axillary plexus catheter fragment : A severed catheter tip unnoticed on removal wandered into the central nervous system]. / Migration eines Plexus-axillaris-Katheter-Fragments : Eine bei der Entfernung unbemerkt abgetrennte Katheterspitze wandert ins Zentralnervensystem.

MIGRATION OF AN AXILLARY PLEXUS CATHETER FRAGMENT INTO THE INTRACEREBRAL COMPARTMENT: During removal of an axillary plexus catheter used for pain therapy, the catheter was probably inadvertently and unnoticed severed together with the suture fixation. The error went unnoticed and an approximately 14 cm long catheter fragment remained in the patient. The patient complained of neck pain, nausea and vomiting 2.5 years later. A computed tomography scan of the cranium and neck region revealed a tubular foreign body with a diameter of ca. 1 mm and a length of ca. 14 cm. The foreign body was identified to be the sheared catheter fragment. In the meantime, the fragment had obviously migrated from the axilla into the intracranial compartment. The tip of the catheter was found at the ventral surface of the pons and surgical extraction was not possible. Following a futile intervention by the hospital's liability insurance and despite evidence from an expert opinion for a gross treatment error, the patient took civil legal action against the hospital. A settlement was reached and the accused hospital committed itself to pay a compensation of 200,000 € plus any additional costs.

Establishment of Neurospora crassa as a host for heterologous protein production using a human antibody fragment as a model product.

BACKGROUND: Filamentous fungi are commonly used as production hosts for bulk enzymes in biotechnological applications. Their robust and quick growth combined with their ability to secrete large amounts of protein directly into the culture medium makes fungi appealing organisms for the generation of novel production systems. The red bread mold Neurospora crassa has long been established as a model system in basic research. It can be very easily genetically manipulated and a wealth of molecular tools and mutants are available. In addition, N. crassa is very fast growing and non-toxic. All of these features point to a high but so far untapped potential of this fungus for biotechnological applications. In this study, we used genetic engineering and bioprocess development in a design-build-test-cycle process to establish N. crassa as a production host for heterologous proteins. RESULTS: The human antibody fragment HT186-D11 was fused to a truncated version of the endogenous enzyme glucoamylase (GLA-1), which served as a carrier protein to achieve secretion into the culture medium. A modular expression cassette was constructed and tested under the control of different promoters. Protease activity was identified as a major limitation of the production strain, and the effects of different mutations causing protease deficiencies were compared. Furthermore, a parallel bioreactor system (1 L) was employed to develop and optimize a production process, including the comparison of different culture media and cultivation parameters. After successful optimization of the production strain and the cultivation conditions an exemplary scale up to a 10 L stirred tank reactor was performed. CONCLUSIONS: The data of this study indicate that N. crassa is suited for the production and secretion of heterologous proteins. Controlling expression by the optimized promoter Pccg1nr in a fourfold protease deletion strain resulted in the successful secretion of the heterologous product with estimated yields of 3 mg/L of the fusion protein. The fungus could easily be cultivated in bioreactors and a first scale-up was successful. The system holds therefore much potential, warranting further efforts in optimization.

Proteomic analysis of organic sulfur compound utilisation in Advenella mimigardefordensis strain DPN7T.

2-Mercaptosuccinate (MS) and 3,3´-ditiodipropionate (DTDP) were discussed as precursor substance for production of polythioesters (PTE). Therefore, degradation of MS and DTDP was investigated in Advenella mimigardefordensis strain DPN7T, applying differential proteomic analysis, gene deletion and enzyme assays. Protein extracts of cells cultivated with MS, DTDP or 3-sulfinopropionic acid (SP) were compared with those cultivated with propionate (P) and/or succinate (S). The chaperone DnaK (ratio DTDP/P 9.2, 3SP/P 4.0, MS/S 6.1, DTDP/S 6.2) and a Do-like serine protease (DegP) were increased during utilization of all organic sulfur compounds. Furthermore, a putative bacterioferritin (locus tag MIM_c12960) showed high abundance (ratio DTDP/P 5.3, 3SP/P 3.2, MS/S 4.8, DTDP/S 3.9) and is probably involved in a thiol-specific stress response. The deletion of two genes encoding transcriptional regulators (LysR (MIM_c31370) and Xre (MIM_c31360)) in the close proximity of the relevant genes of DTDP catabolism (acdA, mdo and the genes encoding the enzymes of the methylcitric acid cycle; prpC,acnD, prpF and prpB) showed that these two regulators are essential for growth of A. mimigardefordensis strain DPN7T with DTDP and that they most probably regulate transcription of genes mandatory for this catabolic pathway. Furthermore, proteome analysis revealed a high abundance (ratio MS/S 10.9) of a hypothetical cupin-2-domain containing protein (MIM_c37420). This protein shows an amino acid sequence similarity of 60% to a newly identified MS dioxygenase from Variovorax paradoxus strain B4. Deletion of the gene and the adjacently located transcriptional regulator LysR, as well as heterologous expression of MIM_c37420, the putative mercaptosuccinate dioxygenase (Msdo) from A. mimigardefordensis, showed that this protein is the key enzyme of MS degradation in A. mimigardefordensis strain DPN7T (KM 0.2 mM, specific activity 17.1 µmol mg-1 min-1) and is controlled by LysR (MIM_c37410).

Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell-cell recognition and fusion.

Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell-cell communication and fusion in the fungus Neurospora crassa Genetically identical germinating spores of this fungus undergo cell-cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell-cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion.

Everolimus for the Treatment of Advanced Pancreatic Neuroendocrine Tumors: Overall Survival and Circulating Biomarkers From the Randomized, Phase III RADIANT-3 Study.

Purpose Everolimus improved median progression-free survival by 6.4 months in patients with advanced pancreatic neuroendocrine tumors (NET) compared with placebo in the RADIANT-3 study. Here, we present the final overall survival (OS) data and data on the impact of biomarkers on OS from the RADIANT-3 study. Methods Patients with advanced, progressive, low- or intermediate-grade pancreatic NET were randomly assigned to everolimus 10 mg/day (n = 207) or placebo (n = 203). Crossover from placebo to open-label everolimus was allowed on disease progression. Ongoing patients were unblinded after final progression-free survival analysis and could transition to open-label everolimus at the investigator's discretion (extension phase). OS analysis was performed using a stratified log-rank test in the intent-to-treat population. The baseline levels of chromogranin A, neuron-specific enolase, and multiple soluble angiogenic biomarkers were determined and their impact on OS was explored. Results Of 410 patients who were enrolled between July 2007 and March 2014, 225 received open-label everolimus, including 172 patients (85%) randomly assigned initially to the placebo arm. Median OS was 44.0 months (95% CI, 35.6 to 51.8 months) for those randomly assigned to everolimus and 37.7 months (95% CI, 29.1 to 45.8 months) for those randomly assigned to placebo (hazard ratio, 0.94; 95% CI, 0.73 to 1.20; P = .30). Elevated baseline chromogranin A, neuron-specific enolase, placental growth factor, and soluble vascular endothelial growth factor receptor 1 levels were poor prognostic factors for OS. The most common adverse events included stomatitis, rash, and diarrhea. Conclusion Everolimus was associated with a median OS of 44 months in patients with advanced, progressive pancreatic NET, the longest OS reported in a phase III study for this population. Everolimus was associated with a survival benefit of 6.3 months, although this finding was not statistically significant. Crossover of patients likely confounded the OS results.

Effects of omeprazole and aluminum hydroxide/magnesium hydroxide on riociguat absorption.

Riociguat, a soluble guanylate cyclase stimulator, is a novel therapy for the treatment of pulmonary hypertension. Riociguat bioavailability is reduced in neutral versus acidic conditions and therefore may be affected by concomitant use of medications that increase gastric pH. The effect of coadministration of the proton pump inhibitor omeprazole or the antacid AlOH/MgOH on the pharmacokinetics, safety, and tolerability of riociguat 2.5 mg was characterized in two open-label, randomized, crossover studies in healthy males. In study 1, subjects pretreated for 4 days with omeprazole 40 mg received cotreatment with omeprazole plus riociguat or riociguat alone (no pretreatment) on day 5 (n = 12). In study 2, subjects received cotreatment with 10 mL AlOH/MgOH plus riociguat or riociguat alone (n = 12). Pre- and cotreatment with omeprazole decreased riociguat bioavailability (mean decreases in area under the plasma concentration-time curve [AUC] and maximum concentration in plasma [C max] were 26% and 35%, respectively). Cotreatment with AlOH/MgOH resulted in greater decreases in riociguat bioavailability (mean decreases in AUC and C max were 34% and 56%, respectively). In both studies, most adverse events (AEs) were of mild intensity, and no serious AEs were reported. No additional safety signals were identified. Treatment with riociguat, with or without omeprazole or AlOH/MgOH, was well tolerated, with a good safety profile. Owing to the resulting increase of gastric pH, riociguat bioavailability is reduced by coadministration with AlOH/MgOH and, to a lesser extent, by coadministration with omeprazole. Thus, antacids should not be administered within an hour of receiving riociguat, but no dose adjustment is required for coadministration of proton pump inhibitors.

The tetraspanin TSP3 of Neurospora crassa is a vacuolar membrane protein and shares characteristics with IDI proteins.

The fungal vacuole is an organelle, which adopts pleiotropic morphologies and functions. In aging and starving hyphae it is the compartment of degradation and recycling of cellular constituents. Here we identified TSP3, one of three tetraspanins present in the filamentous ascomycete fungus Neurospora crassa, as a vacuolar membrane protein. The protein is detected only in aging and starving cultures and under other conditions, which induce autophagy, such as vegetative incompatibility or the presence of the macrolide antibiotic rapamycin. Mutant analysis revealed that TSP3 is dispensable for growth and development of the fungus under laboratory conditions. Together these findings indicate that tsp3 shares characteristics with idi (induced during incompatibility) genes and might promote vacuolar functions related to autophagy.

A jack-of-all-trades: 2-mercaptosuccinic acid.

2-Mercaptosuccinic acid (MS) is an important and versatile substance for diverse fields of applications of which the most significant are surveyed in this article. Biological, chemical, and physical properties of MS as well as the knowledge of its synthesis and microbial degradation are illustrated. In addition, exemplary structural analogs of the organic sulfur compound are commented. The key application of MS in nanotechnology is discussed in detail with particular emphasis on quantum dots (nanocrystals) and self-assembled monolayers in combination with gold or silver. Furthermore, some medical and pharmaceutical applications are given, inter alia in bioimaging, as a nanocarrier, and with regard to the antimicrobial activity of MS-silver and MS-gold nanoparticles. Moreover, biological and chemical applications of MS are exemplified: the thiol compound can serve as an inhibitor for glutathione peroxidase, or the toxicity of substances can be increased due to the presence of MS in the respective cells or tissues. In the field of cosmetics, MS is widely utilized as a reducing agent for numerous products as explained in this article. Additionally, the microbial utilization of MS as a carbon and energy source for growth is elucidated in-depth, providing insight into different catabolic mechanisms.

The AngFus3 Mitogen-Activated Protein Kinase Controls Hyphal Differentiation and Secondary Metabolism in Aspergillus niger.

Adaptation to a changing environment is essential for the survival and propagation of sessile organisms, such as plants or fungi. Filamentous fungi commonly respond to a worsening of their growth conditions by differentiation of asexually or sexually produced spores. The formation of these specialized cell types is, however, also triggered as part of the general life cycle by hyphal age or density. Spores typically serve for dispersal and, therefore, translocation but can also act as resting states to endure times of scarcity. Eukaryotic differentiation in response to environmental and self-derived signals is commonly mediated by three-tiered mitogen-activated protein (MAP) kinase signaling cascades. Here, we report that the MAP kinase Fus3 of the black mold Aspergillus niger (AngFus3) and its upstream kinase AngSte7 control vegetative spore formation and secondary metabolism. Mutants lacking these kinases are defective in conidium induction in response to hyphal density but are fully competent in starvation-induced sporulation, indicating that conidiation in A. niger is triggered by various independent signals. In addition, the mutants exhibit an altered profile of volatile metabolites and secrete dark pigments into the growth medium, suggesting a dysregulation of the secondary metabolism. By assigning the AngFus3 MAP kinase pathway to the transduction of a potentially self-derived trigger, this work contributes to the unraveling of the intricate signaling networks controlling fungal differentiation. Moreover, our data further support earlier observations that differentiation and secondary metabolism are tightly linked in filamentous fungi.

Phase I study of everolimus, cetuximab and irinotecan as second-line therapy in metastatic colorectal cancer.

AIM: To evaluate feasible doses of weekly everolimus and irinotecan given with cetuximab for previously treated metastatic colorectal cancer (mCRC). PATIENTS AND METHODS: Adults with mCRC that progressed after 5-fluorouracil or capecitabine-plus-oxaliplatin were treated using a sequential dose escalation scheme. Dosing decisions were based on the probability of experiencing a dose-limiting toxicity (DLT) during the first two 21-day treatment cycles. RESULTS: Patients received everolimus 30 mg/week plus irinotecan 350 mg/m2 q3w (n=5; dose A1) or everolimus 30 mg/week plus irinotecan 250 mg/m2 q3w (n=14; dose B1). Among patients evaluable for the maximum tolerated dose, two out of four in A1 and one out of eight in B1 experienced four DLTs. The trial was terminated early based on changes in clinical practice and emerging data on everolimus dosing. CONCLUSION: The feasible doses of everolimus and irinotecan administered with cetuximab as second-line therapy in mCRC were 30 mg/week and 250 mg/m2, respectively.

Mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, from Variovorax paradoxus strain B4 is the key enzyme of mercaptosuccinate degradation.

The versatile thiol mercaptosuccinate has a wide range of applications, e.g. in quantum dot research or in bioimaging. Its metabolism is investigated in Variovorax paradoxus strain B4, which can utilize this compound as the sole source of carbon and sulfur. Proteomic studies of strain B4 resulted in the identification of a putative mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, possibly representing the key enzyme in the degradation of mercaptosuccinate. Therefore, the putative mercaptosuccinate dioxygenase was heterologously expressed, purified, and characterized in this study. The results clearly demonstrated that the enzyme utilizes mercaptosuccinate with concomitant consumption of oxygen. Thus, the enzyme is designated as mercaptosuccinate dioxygenase. Succinate and sulfite were verified as the final reaction products. The enzyme showed an apparent Km of 0.4 mM, and a specific activity (Vmax) of 20.0 µmol min(-1) mg(-1) corresponding to a kcat of 7.7 s(-1). Furthermore, the enzyme was highly specific for mercaptosuccinate, no activity was observed with cysteine, dithiothreitol, 2-mercaptoethanol, and 3-mercaptopropionate. These structurally related thiols did not have an inhibitory effect either. Fe(II) could clearly be identified as metal cofactor of the mercaptosuccinate dioxygenase with a content of 0.6 mol of Fe(II)/mol of enzyme. The recently proposed hypothesis for the degradation pathway of mercaptosuccinate based on proteome analyses could be strengthened in the present study. (i) Mercaptosuccinate is first converted to sulfinosuccinate by this mercaptosuccinate dioxygenase; (ii) sulfinosuccinate is spontaneously desulfinated to succinate and sulfite; and (iii) whereas succinate enters the central metabolism, sulfite is detoxified by the previously identified putative molybdopterin oxidoreductase.

Mercaptosuccinate metabolism in Variovorax paradoxus strain B4--a proteomic approach.

Variovorax paradoxus B4 was isolated due to its ability to degrade the organic thiol compound mercaptosuccinate, which could be a promising precursor for novel polythioesters. The analysis of the proteome of this Gram-negative bacterium revealed several proteins with significantly increased expression during growth of cells with mercaptosuccinate as carbon source when compared to cells grown with gluconate or succinate. Among those, a large number of proteins involved in amino acid metabolism were identified, e.g., adenosylhomocysteinase and glutamate-ammonia ligase. Additionally, detection of superoxide dismutase strengthened the assumption of enhanced stress levels in mercaptosuccinate-grown cells. Several isoforms of a rhodanese domain-containing protein exhibited particularly increased expression during growth with mercaptosuccinate in comparison to gluconate (factor 14.2, stationary phase) or to succinate (factor 15.4, stationary phase). Besides this, augmented expression of the hypothetical protein VAPA_1c41240 raised attention. VAPA_1c41240 exhibited up to 13.3-fold (mercaptosuccinate vs gluconate) or 9.5-fold (mercaptosuccinate vs succinate) increased expression levels, and in silico searches revealed that this protein might be a thiol dioxygenase. Based on these results, a novel degradation pathway is proposed for mercaptosuccinate. The newly identified putative mercaptosuccinate dioxygenase could convert mercaptosuccinate to sulfinosuccinate by the introduction of two molecules of oxygen. Subsequently, sulfinosuccinate would be cleaved into succinate and sulfite either by a yet unknown enzyme, by spontaneous hydrolysis, or by the putative mercaptosuccinate dioxygenase itself. Succinate could then enter the central metabolism, while detoxification of sulfite could be achieved by the previously identified putative molybdopterin oxidoreductase. Biochemical studies will be done in the future to confirm this pathway.