A method for analysis and design of metabolism using metabolomics data and kinetic models: Application on lipidomics using a novel kinetic model of sphingolipid metabolism.

We present a model-based method, designated Inverse Metabolic Control Analysis (IMCA), which can be used in conjunction with classical Metabolic Control Analysis for the analysis and design of cellular metabolism. We demonstrate the capabilities of the method by first developing a comprehensively curated kinetic model of sphingolipid biosynthesis in the yeast Saccharomyces cerevisiae. Next we apply IMCA using the model and integrating lipidomics data. The combinatorial complexity of the synthesis of sphingolipid molecules, along with the operational complexity of the participating enzymes of the pathway, presents an excellent case study for testing the capabilities of the IMCA. The exceptional agreement of the predictions of the method with genome-wide data highlights the importance and value of a comprehensive and consistent engineering approach for the development of such methods and models. Based on the analysis, we identified the class of enzymes regulating the distribution of sphingolipids among species and hydroxylation states, with the D-phospholipase SPO14 being one of the most prominent. The method and the applications presented here can be used for a broader, model-based inverse metabolic engineering approach.

Prolonged starvation drives reversible sequestration of lipid biosynthetic enzymes and organelle reorganization in Saccharomyces cerevisiae.

Cells adapt to changing nutrient availability by modulating a variety of processes, including the spatial sequestration of enzymes, the physiological significance of which remains controversial. These enzyme deposits are claimed to represent aggregates of misfolded proteins, protein storage, or complexes with superior enzymatic activity. We monitored spatial distribution of lipid biosynthetic enzymes upon glucose depletion in Saccharomyces cerevisiae. Several different cytosolic-, endoplasmic reticulum-, and mitochondria-localized lipid biosynthetic enzymes sequester into distinct foci. Using the key enzyme fatty acid synthetase (FAS) as a model, we show that FAS foci represent active enzyme assemblies. Upon starvation, phospholipid synthesis remains active, although with some alterations, implying that other foci-forming lipid biosynthetic enzymes might retain activity as well. Thus sequestration may restrict enzymes' access to one another and their substrates, modulating metabolic flux. Enzyme sequestrations coincide with reversible drastic mitochondrial reorganization and concomitant loss of endoplasmic reticulum-mitochondria encounter structures and vacuole and mitochondria patch organelle contact sites that are reflected in qualitative and quantitative changes in phospholipid profiles. This highlights a novel mechanism that regulates lipid homeostasis without profoundly affecting the activity status of involved enzymes such that, upon entry into favorable growth conditions, cells can quickly alter lipid flux by relocalizing their enzymes.

Autophagy competes for a common phosphatidylethanolamine pool with major cellular PE-consuming pathways in Saccharomyces cerevisiae.

Autophagy is a highly regulated pathway that selectively degrades cellular constituents such as protein aggregates and excessive or damaged organelles. This transport route is characterized by engulfment of the targeted cargo by autophagosomes. The formation of these double-membrane vesicles requires the covalent conjugation of the ubiquitin-like protein Atg8 to phosphatidylethanolamine (PE). However, the origin of PE and the regulation of lipid flux required for autophagy remain poorly understood. Using a genetic screen, we found that the temperature-sensitive growth and intracellular membrane organization defects of mcd4-174 and mcd4-P301L mutants are suppressed by deletion of essential autophagy genes such as ATG1 or ATG7. MCD4 encodes an ethanolamine phosphate transferase that uses PE as a precursor for an essential step in the synthesis of the glycosylphosphatidylinositol (GPI) anchor used to link a subset of plasma membrane proteins to lipid bilayers. Similar to the deletion of CHO2, a gene encoding the enzyme converting PE to phosphatidylcholine (PC), deletion of ATG7 was able to restore lipidation and plasma membrane localization of the GPI-anchored protein Gas1 and normal organization of intracellular membranes. Conversely, overexpression of Cho2 was lethal in mcd4-174 cells grown at restrictive temperature. Quantitative lipid analysis revealed that PE levels are substantially reduced in the mcd4-174 mutant but can be restored by deletion of ATG7 or CHO2. Taken together, these data suggest that autophagy competes for a common PE pool with major cellular PE-consuming pathways such as the GPI anchor and PC synthesis, highlighting the possible interplay between these pathways and the existence of signals that may coordinate PE flux.

Systematic lipidomic analysis of yeast protein kinase and phosphatase mutants reveals novel insights into regulation of lipid homeostasis.

The regulatory pathways required to maintain eukaryotic lipid homeostasis are largely unknown. We developed a systematic approach to uncover new players in the regulation of lipid homeostasis. Through an unbiased mass spectrometry-based lipidomic screening, we quantified hundreds of lipid species, including glycerophospholipids, sphingolipids, and sterols, from a collection of 129 mutants in protein kinase and phosphatase genes of Saccharomyces cerevisiae. Our approach successfully identified known kinases involved in lipid homeostasis and uncovered new ones. By clustering analysis, we found connections between nutrient-sensing pathways and regulation of glycerophospholipids. Deletion of members of glucose- and nitrogen-sensing pathways showed reciprocal changes in glycerophospholipid acyl chain lengths. We also found several new candidates for the regulation of sphingolipid homeostasis, including a connection between inositol pyrophosphate metabolism and complex sphingolipid homeostasis through transcriptional regulation of AUR1 and SUR1. This robust, systematic lipidomic approach constitutes a rich, new source of biological information and can be used to identify novel gene associations and function.

Violência contra a mulher e a criança: condutas médicas à paciente agredida sexualmente / Violence against the woman and the child: medical management of the sexually assaulted patient

A assistência a pacientes vítimas de abuso sexual deve ser intersetorial e interdisciplinar, incluindo interface com questões policiais e judiciais. Nesse contexto, o médico tem dever ético e legal de prestar atendimento prioritário a essas pacientes. É necessário que o serviço de saúde realize exame físico, exame ginecológico, coleta de amostras para diagnóstico de infecções genitais e coleta de material para identificação do agressor. As evidências mostram que a violência contra a mulher tem impacto sobre a saúde física e mental desta. A equipe médica, portanto, deve estar preparada para oferecer apoio psicossocial. Outra preocupação médica é diminuir ao máximo as consequências do ato de violência, seja protegendo a mulher de doenças sexualmente transmissíveis ou de gravidez traumática. A paciente vítima de violência sexual, ocasional e sem uso de preservativo, deve receber no serviço de urgência profilaxia contra HIV, hepatite B, sífilis, gonorreia, clamidiose, cancro mole, tricomoníase e tétano. Além de ter direito a receber anticoncepção de emergência, o aborto nessas pacientes pode ser feito legalmente. Para isso, basta a autorização da paciente e/ou do responsável, dependendo da idade da paciente, sendo incorreta e ilegal a exigência de Boletim de Ocorrência Policial ou laudo do Instituto Médico Legal.

The assistance to patients who are victims of sexual abuse should be intersectoral and interdisciplinary, including interface with police and justice affairs. In this context the physician has an ethical and legal obligation to give priority attention to these patients. It is necessary for the health service to perform physical examination, gynecological examination, collection of samples for diagnosis of genital infections and collection of material for the aggressor’s identification. Evidences shows that violence against women has an impact on both their mental and physical health. The medical team, therefore, must be prepared to offer psychosocial support. Another medical concern is to minimize as much as possible the consequences of the act of violence, either by protecting the woman from Sexually transmitted Diseases or from traumatic pregnancy. The patient who was suffered occasional sexual violence without the use of a condom must receive in the emergency service prophylaxis against HIV, type B hepatitis, syphilis, gonorrhea, chlamydia, chancroid, trichomoniasis and tetanus. Besides having the right to receive emergency contraception, abortion in these patients can be legally done. It is only required the written consent of the patient and/or the person legally responsible for her, depending on the patient’s age, and the requirement of the Police or Forensic Medicine reports is considered both incorrect and illegal.

Abuso sexual em serviços de emergência: relato de caso / Sexual abuse in emergency centers: case report

Este artigo trata de paciente com 54 anos, atendida no Hospital Júlia Kubitschek, com relato de ter sido vítima de violência sexual. O agressor armado invadiu sua casa e a obrigou, mediante ameaça de morte, a manter com ele sexo vaginal, anal e oral, sem uso de preservativo. Apresentava-se ansiosa e chorosa. Havia lesão contusa em região escapular esquerda e leve hiperemia de grande lábio direito. O exame especular e a inspeção da região anal não apresentavam alterações. Foram realizadas avaliação laboratorial e profilaxia para doenças sexualmente transmissíveis (DSTs), incluindo administração de antirretrovirais. Foi encaminhada ao ambulatório de vítimas de violência sexual.

This article is about a 54-year-old female patient, assisted at Júlia Kubitschek Hospital, who reported having been victim of sexual violence. According to her, the armed aggressor broke into her house and forced her by threat of death to have vaginal, anal and oral sex with him without using condom. The patient was anxious and tearful. There were blunt injury in the left scapular region (shoulder blade area) and mild hyperemia of the right labium. Speculum examination and inspection of the anal region showed no alterations. The treatment included laboratory tests and prophylaxis for Sexually Transmitted Diseases (STDs), including administration of antiretroviral medication. The patient was referred to an outpatient center for victims of sexual violence.