The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data

Metabolic profiling is increasingly being used to investigate a diverse range of biological questions. Due to the rapid turnover of intracellular metabolites it is important to have reliable, reproducible techniques for sampling and sample treatment. Through the use of non-targeted analytical techniques such as NMR and GC-MS we have performed a comprehensive quantitative investigation of sampling techniques for Pichia pastoris. It was clear that quenching metabolism using solutions based on the standard cold methanol protocol caused some metabolite losses from P. pastoris cells. However, these were at a low level, with the NMR results indicating metabolite increases in the quenching solution below 5% of their intracellular level for 75% of metabolites identified; while the GC-MS results suggest a slightly higher level with increases below 15% of their intracellular values. There were subtle differences between the four quenching solutions investigated but broadly, they all gave similar results. Total culture extraction of cells + broth using high cell density cultures typical of P. pastoris fermentations, was an efficient sampling technique for NMR analysis and provided a gold standard of intracellular metabolite levels; however, salts in the media affected the GC-MS analysis. Furthermore, there was no benefit in including an additional washing step in the quenching process, as the results were essentially identical to those obtained just by a single centrifugation step. We have identified the major high-concentration metabolites found in both the extra- and intracellular locations of P. pastoris cultures by NMR spectroscopy and GC-MS. This has provided us with a baseline metabolome for P. pastoris for future studies. The P. pastoris metabolome is significantly different from that of Saccharomyces cerevisiae, with the most notable difference being the production of high concentrations of arabitol by P. pastoris

Intramedullary spinal cord paracoccidioidomycosis: report of two cases

Two cases of intramedullary paracoccidioidomycosis are reported. Paracoccidioidomycosis is a systemic disease that involves the buccopharyngeal mucosa, lungs, lymph nodes and viscera and infrequently the central nervous system. Localization in the spinal cord is rare. Case 1: a 55-year old male admitted with crural pararesis, tactile/painful hypesthesia and sphincter disturbances of 15 days duration. Cutaneous-pulmonary blastomycosis was diagnosed 17 years ago. Myelotomography showed a blockade of T3-T4 (intramedullary lesion). The lesion surgically removed was a Paracoccidioides brasiliensis granuloma. Treatment with sulfadiazine was started after the surgery. Follow-up of 15 month showed an improvement of the clinical signs. Case 2: a 57-year old male was admitted elsewhere 6 months ago and, with a radiologic diagnosis of pulmonary paracoccidioidomycosis, was treated with amphotericin B. He progressively developed paresthesia and tactile/ pain anaesthesia on the left side, sphincter disturbances and tetraparesis with bilateral extensor plantar response and clonus of the feet. Myelotomography showed a blockade of C4-C6 (intramedullary lesion). The lesion was not found during surgical exploration and the patient deteriorated and died. Post-mortem examination revealed an intramedullary tumor above the site of the mielotomy (Paracoccidioides brasiliensis granuloma). The preoperative diagnosis of intramedullary paracoccidioidomycotic granulomas is difficult because the clinical and radiologic manifestations are uncharacteristic. Clinical suspicion was possible in our cases based on the history of previous systemic disease. Contrary to intracranial localizations, paracoccidioidomycotic granulomas causing progressive spinal cord compression may require early surgery because response to clinical treatment is slow and the reversibility of neurological deficits depends on the promptness of the decompression.São relatados dois casos de granuloma blastomicótico intramedular. A paracoccidioidomicose é micose sistêmica que atinge predominantemente a mucosa bucofaríngea, pulmões, linfonodos e vísceras e infrequentemente o sistema nervoso. A localização medular é rara. Caso 1: paciente masculino, de 55 anos, admitido com parestesias, hipoestesia táctil/dolorosa, paraparesia crural e distúbios esfincterianos. Tinha diagnóstico de blastomicose cutâneo-pulmonar há 17 anos. A mielotomografia mostrou bloqueio em T3-T4 (lesão intramedular). A lesão removida cirurgicamente revelou-se um granuloma blastomiótico. Após a cirurgia foi tratado com sulfadiazina. Durante o seguimento (15 meses) apresentou melhora do quadro clínico. Caso 2: paciente masculino, de 57 anos, internado em outro hospital há 6 meses por blastomicose pulmonar e tratado com anfotericina B. Desenvolveu parestesias, hipoestesia táctil/dolorosa à esquerda, distúrbios esfincterianos e tetraparesia com sinal de Babinski bilateral e clonus nos pés. A mielotomografia mostrou bloqueio de C4-C6 (lesão intramedular). A lesão não foi encontrada durante a cirurgia e o paciente piorou e faleceu. A necrópsia revelou um granuloma blastomicótico intramedular, acima do local da mielotomia. O diagnóstico dos granulomasblastomicóticos intramedulares apresenta dificuldades porque as manisfestações clínicas e radiológicas são incaracterísticas. Nos casos relatados, a suspeita clínica foi baseada nos antecedentes de doença sistêmica prévia. Para os granulomas blastomicóticos raquídeos que causam compressão medular progressiva, ao contrário dos encefálicos, a cirurgia precoce deve ser considerada, pois a regressão com o tratamento clínico é lenta e a reversibilidade dos deficits neurológicos depende da rapidez da descompressão medular