Carotid free-floating thrombus in patients with acute ischaemic stroke and active cancer.

In patients with ischaemic stroke, a carotid free-floating thrombus (CFFT) raises diagnostic and therapeutic challenges. We describe two women, each taking tamoxifen for invasive non-metastatic breast cancer, who developed large-vessel occlusion ischaemic strokes. The first had a CFFT 24 hours after receiving intravenous thrombolysis and mechanical thrombectomy; the thrombus completely resolved after 1 week of therapeutic anticoagulation. The second had a tandem occlusion with a CFFT at admission; her neurological deficits rapidly improved after intravenous thrombolysis without needing a mechanical thrombectomy. However, subsequently, under therapeutic anticoagulation, distal migration of the CFFT caused a recurrent large vessel occlusion ischaemic stroke, requiring mechanical thrombectomy. The CTFF in both cases appeared to relate to a cancer-related prothrombotic state. Both received long-term oral anticoagulation and their tamoxifen was switched to anastrozole. At 3 months, both were functionally independent without recurrent vascular events.

Are we missing an opportunity? Prehospital delay in patients with acute ischemic stroke and known atrial fibrillation.

INTRODUCTION AND OBJECTIVES: The follow-up of patients with atrial fibrillation (AF) presents an opportunity to alert patients and their families on how to recognize and act in the event of stroke. Our aim was to compare stroke recognition-to-door time and prehospital stroke code activation in patients with known AF (KAF) and AF detected after stroke (AFDAS). METHODS: We performed a retrospective cohort study of consecutive patients receiving acute recanalization treatment for acute ischemic stroke between January 2016 and August 2022, with AF as a potential stroke cause. Patients were divided into KAF and AFDAS, and stroke recognition-to-door time and prehospital stroke code activation were compared. In the KAF subgroup, we assessed whether the use of preadmission anticoagulation was associated with the studied prehospital parameters. RESULTS: We included 438 patients, 290 female (66.2%), mean age 79.3±9.4 years. In total, 238 patients had KAF (54.3%) and 200 (45.7%) had AFDAS. Of those with KAF, 114 (48.1%) were pretreated with anticoagulation. Patients with KAF and AFDAS had no differences in stroke recognition-to-door time (74.0 [55.0-101.0] vs. 78.0 [60.0-112.0] min; p=0.097) or prehospital stroke code activation [148 (64.6%) vs. 128 (65.3%); p=0.965]. In the KAF subgroup, preadmission anticoagulation did not influence stroke recognition-to-door time or mode of hospital admission. CONCLUSION: Stroke recognition-to-door time and prehospital stroke code activation were similar between patients with known or newly diagnosed AF. Preadmission anticoagulation treatment also did not affect the studied parameters. Our findings highlight a missed opportunity to promote stroke knowledge in patients followed due to AF.

The role of neurohospitalism in a specialized cardiovascular hospital.

INTRODUCTION: Cardiology and cardiothoracic surgery are among the specialties that most commonly require neurology inpatient consultations. We aimed to study the neurology referrals by the cardiovascular-specialized hospital included in our tertiary hospital center. METHODS: Retrospective study of consecutive patients referred for neurology inpatient consultation between 01/01/2020 and 31/12/2022. We analyzed referrals, patients' characteristics, and the approach taken. A detailed subanalysis was performed for patients diagnosed with acute ischemic stroke (AIS). RESULTS: 143 patients were observed [mean age 67.3 years, 46 (32.2%) females]. Most frequent referral reasons were suspected AIS deficits (39.2%), altered mental status (19.6%), suspected seizures (13.3%), and neuroprognostication (11.9%). Mean referral-to-consult time was 2.7 days, and 117 (81.8%) consults were in-person. Additional investigation, treatment changes, and outpatient clinic referral were proposed, respectively in 79.7%, 60.1%, and 19.6% of patients. Most common diagnoses were AIS (45.5%), hypoxic-ischemic encephalopathy (18.9%) and delirium (7.0%). Regarding patients with AIS (n=62), most common stroke causes were post-cardiac procedure (44.6%), infective endocarditis (18.5%), aortic dissection (10.8%), acute myocardial infarction (10.8%) and anticoagulant withdrawal in patients with atrial fibrillation (6.2%). 34 AIS patients were diagnosed less than 24-hours since last seen well, of which four (6.2%) were treated (three with thrombolysis and one with mechanical thrombectomy). CONCLUSION: AIS is the most common reason for referral in our cardiovascular hospital. Our results highlight the importance of the availability of a neurologist/neurohospitalist with stroke expertise for consultation of inpatients admitted in a specialized cardiovascular hospital.

Sex Differences in Outcomes of Late-Window Endovascular Stroke Therapy.

BACKGROUND: The association between sex and outcome after endovascular thrombectomy of acute ischemic stroke is unclear. The aim of this study was to compare the clinical and safety outcomes between men and women treated with endovascular thrombectomy in the late 6-to-24-hour window period. METHODS: This multicenter, retrospective observational cohort study included consecutive patients who underwent endovascular thrombectomy of anterior circulation stroke in the late window from 66 clinical sites in 10 countries from January 2014 to May 2022. The primary outcome was the 90-day ordinal modified Rankin Scale score. Secondary outcomes included 90-day functional independence (FI), return of Rankin (RoR) to prestroke baseline, FI or RoR, symptomatic intracranial hemorrhage, and mortality. Multivariable and inverse probability of treatment weighting methods were used. We explored the interaction of sex with baseline characteristics on the outcomes ordinal modified Rankin Scale and FI or RoR. RESULTS: Of 1932 patients, 1055 were women and 877 were men. Women were older (77 versus 69 years), had higher rates of atrial fibrillation, hypertension, and greater prestroke disability, but there was no difference in baseline National Institutes of Health Stroke Scale score. Inverse probability of treatment weighting analysis showed no difference between women and men in ordinal modified Rankin Scale (odds ratio, 0.98 [95% CI, 0.79-1.21]), FI or RoR (odds ratio, 0.98 [95% CI, 0.78-1.22]), severe disability or mortality (odds ratio, 0.99 [95% CI, 0.80-1.23]). The multivariable analysis of the above end points was concordant. There were no interactions between baseline characteristics and sex on the outcomes of ordinal modified Rankin Scale and FI or RoR. CONCLUSIONS: In late presenting patients with anterior circulation stroke treated with endovascular thrombectomy in the 6 to 24-hour window, there was no difference in clinical or safety outcomes between men and women.

Epileptogenic focal lesions in Dravet syndrome: A warning to investigators.

OBJECTIVE: Most patients with Dravet syndrome (DS) have unremarkable neuroimaging studies. However, a small number of patients exhibit focal abnormalities that may modify the epilepsy phenotype. We report a case series of DS patients carrying SCN1A variants concurrent with additional focal brain lesions, aiming to provide details regarding their clinical course, electrographic findings, and imaging features. METHODS: We reviewed the electronic medical records of patients with developmental and epileptic encephalopathies in our center, from January 2000 to December 2022, identifying 90 patients with DS resulting from SCN1A variants. Of these, patients displaying focal brain lesions were eligible. RESULTS: Five patients (4 males and 1 female), with median age of 26 years, were included. All exhibited clinical and electroencephalographic features consistent with the DS spectrum. Sequencing analysis of the SCN1A gene identified pathogenic variants. Magnetic resonance imaging (MRI) revealed focal cortical dysplasia (FCD) in two patients, while the remaining three had cystic lesions. Three patients had previously undergone resective epilepsy surgery in other centers, with no improvement in seizure frequency. Neuropathology studies revealed the presence of FCD type IIA, intracranial teratomas, and dysembryoplastic neuroepithelial tumor (DNET). SIGNIFICANCE: When an individual with an established diagnosis of genetic epilepsy and a focal lesion on MRI is undergoing preoperative evaluation, it is crucial to conduct a comprehensive analysis to understand the relevance of the focal finding for the patient's phenotype and thus anticipate potential surgical outcomes. In instances where epilepsy in DS patients is influenced by a specific focal structural lesion, resective surgery should be carefully considered after precise pharmacological treatment, acknowledging the persistent influence of an SCN1A variant on expected outcomes.

Proteomics to Identify New Blood Biomarkers for Diagnosing Patients With Acute Stroke.

Background Blood biomarkers are a potential tool for early stroke diagnosis. We aimed to perform a pilot and exploratory study on untargeted blood biomarkers in patients with suspected stroke by using mass spectrometry analysis. Methods and Results This was a prospective observational study of consecutive patients with suspected stroke admitted within 6 hours of last being seen well. Blood samples were collected at admission. Patients were divided into 3 groups: ischemic stroke (IS), intracerebral hemorrhage (ICH), and stroke mimics. Quantitative analysis from mass spectrometry data was performed using a supervised approach. Biomarker-based prediction models were developed to differentiate IS from ICH and ICH+stroke mimics. Models were built aiming to minimize misidentification of patients with ICH as having IS. We included 90 patients, one-third within each subgroup. The median age was 71 years (interquartile range, 57-81 years), and 49 participants (54.4%) were women. In quantitative analysis, C3 (complement component 3), ICAM-2 (intercellular adhesion molecule 2), PLGLA (plasminogen like A), STXBP5 (syntaxin-binding protein 5), and IGHV3-64 (immunoglobulin heavy variable 3-64) were the 5 most significantly dysregulated proteins for both comparisons. Biomarker-based models showed 88% sensitivity and 89% negative predictive value for differentiating IS from ICH, and 75% sensitivity and 95% negative predictive value for differentiating IS from ICH+stroke mimics. ICAM-2, STXBP5, PLGLA, C3, and IGHV3-64 displayed the highest importance score in our models, being the most informative for identifying patients with stroke. Conclusions In this proof-of-concept and exploratory study, our biomarker-based prediction models, including ICAM-2, STXBP5, PLGLA, C3, and IGHV3-64, showed 75% to 88% sensitivity for identifying patients with IS, while aiming to minimize misclassification of ICH. Although our methodology provided an internal validation, these results still need validation in other cohorts and with different measurement techniques.

Parkinson's disease patients' perspective on telephone visits during the COVID-19 pandemic.

Telemedicine is a promising modality to provide specialized care in periods when attendance is challenged, as recently observed during the Coronavirus disease 2019 (COVID-19) pandemic. We aimed to evaluate the satisfaction with telemedicine visits in a group of Parkinson´s disease (PD) patients during the COVID-19 pandemic, as well as to identify demographic or clinical factors associated with higher telemedicine satisfaction. Retrospective cohort study of PD patients who attended telemedicine visits at Centro Hospitalar de Lisboa Ocidental, from March 1, 2020 until March 31, 2021. An eleven-question telephone survey was used to assess satisfaction with telemedicine visits. Patients' answers were dichotomized into "satisfied" or "not satisfied" to study the factors associated with satisfaction with telemedicine. Mann-Whitney U for continuous variables and chi-square tests for categorical variables were performed to compare data between the two groups. Linear regression was used to study the factors associated with being satisfied with telemedicine. A total of 111 patients (87%) accepted to participate in this survey. The majority (n = 74.67%) reported being satisfied with telemedicine visits. Patients preferred a combination of in-person and telemedicine visits (n = 43.39%). Male gender (p < 0.001) and employed patients (p < 0.001) were associated with higher satisfaction. In the linear regression, therapeutic changes deemed clear (p < 0.014) and considering the absence of neurological examination non-detrimental (p < 0.001) were associated with a higher degree of satisfaction with telemedicine. Most patients are satisfied with telemedicine visits, even in urgent implementation due to the COVID-19 pandemic. Moreover, telemedicine does not pose a hurdle to a paradigm shift away from conventional in-person appointments.

[Evaluation of the Knowledge of Women of Childbearing Age with Epilepsy about the Impact of Their Disease in Contraception, Pregnancy and Breastfeeding: A Multicenter Cross-Sectional Study]. / Avaliação do Conhecimento das Mulheres com Epilepsia em Idade Fértil sobre o Impacto da sua Doença na Contraceção, Gravidez e Amamentação: Um Estudo Transversal Multicêntrico.

INTRODUCTION: The interaction of antiseizure medication with contraceptives, its potential teratogenicity and implications in pregnancy and breastfeeding are aspects to consider in the neurological care of women with epilepsy of childbearing age. To ensure the commitment in therapeutic decisions and the appropriate planning of maternity, it is essential that women are informed about the implications of their disease in these domains. The main aim of this study was to assess the knowledge of women of childbearing age with epilepsy concerning the impact of epilepsy in contraception, pregnancy and breastfeeding. As secondary aims we defined (1) the demographic, clinical and therapeutic characterization of this group of patients, (2) the identification of variables that correlated with the level of knowledge of women with epilepsy, and (3) the identification of preferential methods to acquire new knowledge about epilepsy. MATERIAL AND METHODS: The study was observational, cross-sectional and multicentric, and was carried out in five hospitals of the Lisbon metropolitan area. After identifying all women of childbearing age with epilepsy followed in the epilepsy clinic of each center, we applied an electronic questionnaire based on a non-systematic review of the literature. RESULTS: One hundred and fourteen participants were validated, with a median age of 33 years. Half of the participants were on monotherapy, and the majority had no seizures in the last six months. We identified important gaps in the participants' knowledge. Sections about complications and administration of antiseizure medication during pregnancy were the ones with the worst results. None of the clinical and demographic variables correlated with the final questionnaire score. Having had a previous pregnancy and the desire to breastfeed in a future pregnancy were positively correlated with the performance in breastfeeding section. Face-to-face discussion during medical outpatient visits was selected as the preferential method to learn about epilepsy, and the internet and social media were the least preferred ones. CONCLUSION: The knowledge of women of childbearing age with epilepsy in the Lisbon metropolitan area concerning the impact of epilepsy in contraception, pregnancy and breastfeeding seems to have significant gaps. Medical teams should consider engaging in patient education particularly during outpatient clinics.

Introdução: A interação dos fármacos anticrise epilética com os métodos contracetivos, a sua potencial teratogenicidade e as implicações na gravidez e amamentação são aspetos a considerar no acompanhamento de mulheres com epilepsia em idade fértil. Para o seu desejado envolvimento nas decisões terapêuticas e o adequado planeamento da maternidade, é essencial que as mulheres estejam corretamente informadas acerca das implicações da sua doença. O objetivo principal do presente estudo foi avaliar o conhecimento das mulheres com epilepsia em idade fértil sobre o impacto da epilepsia na contraceção, gravidez e amamentação. Como objetivos secundários definiram-se (1) a caracterização demográfica, clínica e terapêutica deste grupo de doentes, (2) a identificação de variáveis correlacionadas com o nível de conhecimento das mulheres com epilepsia, e (3) a identificação de meios e suportes preferenciais para aquisição de novos conhecimentos sobre epilepsia. Material e Métodos: O estudo foi observacional, transversal e multicêntrico, tendo decorrido em cinco centros hospitalares da região metropolitana de Lisboa. Após identificação das mulheres com epilepsia em idade fértil seguidas na Consulta de Epilepsia de cada centro, aplicou-se um questionário eletrónico construído após revisão não sistemática da literatura. Resultados: Foram validadas 114 participantes, com uma idade mediana de 33 anos. Metade das participantes apresentavam-se sob monoterapia, tendo a maioria a epilepsia controlada há pelo menos seis meses. Identificaram-se importantes lacunas no conhecimento das participantes. Conceitos sobre complicações dos fármacos anticrise epilética e a sua administração durante a gravidez motivaram piores resultados. Não houve correlação entre variáveis clinico-demográficas e o resultado no questionário. A ocorrência de gravidez prévia e o desejo de amamentar numa gravidez futura correlacionaram-se com o desempenho na secção sobre amamentação. A discussão oral na consulta foi a forma preferencial para aquisição de novos conhecimentos sobre epilepsia, tendo a internet e as redes sociais sido os meios menos escolhidos. Conclusão: O conhecimento das mulheres com epilepsia em idade fértil na área metropolitana de Lisboa sobre o impacto da sua doença na contraceção, gravidez e amamentação parece apresentar lacunas importantes. A educação para a saúde deste grupo deverá constituir uma preocupação por parte das equipas médicas, devendo privilegiar-se a consulta como local de ensino.

Clinical pattern and response to treatment of primary stabbing headache: Retrospective case series study from a Portuguese tertiary hospital.

OBJECTIVES: This retrospective case series study aimed to investigate the demographic and clinical patterns of primary stabbing headache (PSH). In addition, we tried to identify subgroups of treatment responses in a neurology outpatient consultation at a Portuguese tertiary hospital. METHODS: Clinical records were retrospectively reviewed and patients meeting the International Classification of Headache Disorders, 3rd edition, criteria for PSH were identified from January 2014 to December 2020. We collected data regarding demographic characteristics, clinical features of the headache, primary headache comorbidities, and information about treatment-related do PSH. RESULTS: Of 1857 patients, 32 (1.7%; mean [SD] age of onset 56 [3.5] years) had the final diagnosis of PSH. Regarding headache characteristics, 20 patients (62.5%) reported episodes of stabbing in fixed locations and 12 (37.5%) in multiple areas; the duration of each attack was between ≤5 s (seven [21.9%]), 5-60 s (20 [62.5%]), and ≥60 s (five [15.6%]). In all, 18 patients (56.3%) had an episodic course (vs. six of 32 [18.8%] an acute course and eight of 32 [25%] a chronic course). In all, 17 patients started medical treatment (53.1%), with total or partial improvement in 10 (58.8%) of them. It was found that patients with pain in fixed locations had a better response to treatment when compared to patients with multiple locations, in a statistically significant way (eight of 11 vs. two of six, p = 0.023). CONCLUSION: In our sample, the mean age of onset of PSH was >50 years and there was a wide range of PSH duration. The duration of each attack (>5 s), the pain in fixed locations, non-daily episodes of the pain in each attack, and the intermittent course of headache were the most prevalent clinical features. Finally, patients with stabbing in localized areas had a better response to treatment.

REM sleep absence in patients referred to polysomnography for REM sleep behavior disorder.

Detection of REM sleep behavior disorder (RBD) with polysomnography (PSG) is relevant for the diagnosis of α-synucleinopathies. However, some patients referred for suspicion of RBD do not present REM sleep at PSG (NoREMSusRBD), rendering the study inconclusive. Our objective was to investigate disorders possibility associated with REM sleep absence in patients referred to PSG for investigation of RBD, in particular α-synucleinopathies. A sleep-lab database was revised to select NoREMSusRBD (n = 15) and patients: with no REM sleep referred for suspicion of other sleep disorder (NoREMSusOther, n = 28); referred for RBD suspicion with negative PSG (NegativeRBD, n = 24); α-synucleinopathies with no REM sleep (NoREMα, n = 23) and idiopathic RBD (iRBD, n = 26). NoREMSusRBD patients were compared with the other groups regarding PSG data and the emergence of prodromal features or established criteria for α-synucleinopathy. Severe Obstructive Sleep Apnea (OSA) was significantly more frequent in the NoREMsusRBD compared to the NoREMα and iRBD groups. No patient in the NoREMSusRBD developed a α-synucleinopathy (2 cases on the iRBD group). The prevalence of prodromal features in NoREMSusRBD (n = 7, 46.7%) was similar to that of iRBD (n = 18, 69.2%) and significantly higher than in the other groups. Apnea-Hypopnea Indices (AHI) were significantly higher in the NoREMSusRBD compared with iRBD and NoREMα. Our study suggests that the absence of REM sleep in NoREMSusRBD could be caused by OSA but does not exclude the possibility of underlying α- synucleinopathy, suggested by an increased prevalence of prodromal features. These data support the need for excluding OSA in patients suspected for RBD and recommends follow-up of NoREMSusRBD patients to uncover a possible α- synucleinopathy.

Organic solutes in the deepest phylogenetic branches of the Bacteria: identification of α(1-6)glucosyl-α(1-2)glucosylglycerate in Persephonella marina.

The accumulation of organic solutes was investigated in the thermophilic bacteria Persephonella marina and Marinitoga piezophila, two representatives of the deepest lineages in the domain Bacteria. These organisms grow optimally at around 70 °C in medium containing 3 % NaCl. A new disaccharide, accumulating in Persephonella marina, was identified as α(1-6)glucosyl-α(1-2)glucosylglycerate (GGG), by nuclear magnetic resonance. This identification was validated by comparison with the spectra of the compound obtained by chemical synthesis. Besides GGG, the solute pool of Persephonella marina comprised ß-glutamate, di-myo-inositol-1,3'-phosphate and 2-O-α-glucosylglycerate. In contrast, amino acids such as α-glutamate, proline and alanine were the dominant components of the solute pool of Marinitoga piezophila and sugar derivatives were absent. The ability of GGG to protect protein structure against heat denaturation was assessed using model proteins. A genomic search for the biosynthetic pathways of known ionic solutes in Aquificales and Thermotogales shows the inability of this analysis to predict the nature of compatible solutes and underlines the need for efficient cultivation techniques.

Processing the interspecies quorum-sensing signal autoinducer-2 (AI-2): characterization of phospho-(S)-4,5-dihydroxy-2,3-pentanedione isomerization by LsrG protein.

The molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD) is produced by many different species of bacteria and is the precursor of the signal molecule autoinducer-2 (AI-2). AI-2 mediates interspecies communication and facilitates regulation of bacterial behaviors such as biofilm formation and virulence. A variety of bacterial species have the ability to sequester and process the AI-2 present in their environment, thereby interfering with the cell-cell communication of other bacteria. This process involves the AI-2-regulated lsr operon, comprised of the Lsr transport system that facilitates uptake of the signal, a kinase that phosphorylates the signal to phospho-DPD (P-DPD), and enzymes (like LsrG) that are responsible for processing the phosphorylated signal. Because P-DPD is the intracellular inducer of the lsr operon, enzymes involved in P-DPD processing impact the levels of Lsr expression. Here we show that LsrG catalyzes isomerization of P-DPD into 3,4,4-trihydroxy-2-pentanone-5-phosphate. We present the crystal structure of LsrG, identify potential catalytic residues, and determine which of these residues affects P-DPD processing in vivo and in vitro. We also show that an lsrG deletion mutant accumulates at least 10 times more P-DPD than wild type cells. Consistent with this result, we find that the lsrG mutant has increased expression of the lsr operon and an altered profile of AI-2 accumulation and removal. Understanding of the biochemical mechanisms employed by bacteria to quench signaling of other species can be of great utility in the development of therapies to control bacterial behavior.

Assessment of the efficacy of solutes from extremophiles on protein aggregation in cell models of Huntington's and Parkinson's diseases.

Protein misfolding and deposition in the brain are implicated in the etiology of numerous neurodegenerative disorders. Here, organic solutes characteristic of microorganisms adapted to hot environments, were tested on experimental cell models of Huntington's and Parkinson's diseases. Diglycerol phosphate, di-myo-inositol phosphate, mannosylglycerate, and mannosylglyceramide were not toxic to the cells, at 10 mM concentration, but caused a decrease in cell density, which suggested an effect on proliferation. In contrast, mannosyl-lactate, an artificial analogue of mannosylglycerate, had a negative impact on cell viability. Concerning protein aggregation, inclusions of mutant huntingtin were reduced in the presence of diglycerol phosphate and di-myo-inositol phosphate, increased with mannosylglycerate, while mannosyl-lactate and mannosylglyceramide had no significant effect. α-Synuclein aggregation was not affected by the solutes tested, except for di-myo-inositol phosphate that led to a slight increased percentage of cells displaying visible aggregates. These solutes might be useful in the development of therapies for protein misfolding diseases.

Design of new enzyme stabilizers inspired by glycosides of hyperthermophilic microorganisms.

In response to stressful conditions like supra-optimal salinity in the growth medium or temperature, many microorganisms accumulate low-molecular-mass organic compounds known as compatible solutes. In contrast with mesophiles that accumulate neutral or zwitterionic compounds, the solutes of hyperthermophiles are typically negatively charged. (2R)-2-(alpha-D-Mannopyranosyl)glycerate (herein abbreviated as mannosylglycerate) is one of the most widespread solutes among thermophilic and hyperthermophilic prokaryotes. In this work, several molecules chemically related to mannosylglycerate were synthesized, namely (2S)-2-(1-O-alpha-D-mannopyranosyl)propionate, 2-(1-O-alpha-D-mannopyranosyl)acetate, (2R)-2-(1-O-alpha-D-glucopyranosyl)glycerate and 1-O-(2-glyceryl)-alpha-D-mannopyranoside. The effectiveness of the newly synthesized compounds for the protection of model enzymes against heat-induced denaturation, aggregation and inactivation was evaluated, using differential scanning calorimetry, light scattering and measurements of residual activity. For comparison, the protection induced by natural compatible solutes, either neutral (e.g., trehalose, glycerol, ectoine) or negatively charged (di-myo-inositol-1,3'-phosphate and diglycerol phosphate), was assessed. Phosphate, sulfate, acetate and KCl were also included in the assays to rank the solutes and new compounds in the Hofmeister series. The data demonstrate the superiority of charged organic solutes as thermo-stabilizers of enzymes and strongly support the view that the extent of protein stabilization rendered by those solutes depends clearly on the specific solute/enzyme examined. The relevance of these findings to our knowledge on the mode of action of charged solutes is discussed.

Bifunctional CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase, the key enzyme for di-myo-inositol-phosphate synthesis in several (hyper)thermophiles.

The pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts of Archaeoglobus fulgidus and structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Gonçalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol. 188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression in Escherichia coli of the putative genes for CTP:l-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3'-phosphate-1'-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus, Pyrococcus furiosus, Thermococcus kodakaraensis, Aquifex aeolicus, and Rubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP and l-myo-inositol-1-phosphate into CDP-l-myo-inositol, as well as the synthesis of DIPP from CDP-l-myo-inositol and l-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP and l-myo-inositol-1-P; the DIPP synthase domain used only l-myo-inositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-l-myo-inositol and CDP-d-myo-inositol, were recognized as alcohol donors. Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (l-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted in Aeropyrum pernix, Thermotoga maritima, and Hyperthermus butylicus. Additionally, using l-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3'-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.

Biosynthetic pathways of inositol and glycerol phosphodiesters used by the hyperthermophile Archaeoglobus fulgidus in stress adaptation.

Archaeoglobus fulgidus accumulates di-myo-inositol phosphate (DIP) and diglycerol phosphate (DGP) in response to heat and osmotic stresses, respectively, and the level of glycero-phospho-myo-inositol (GPI) increases primarily when the two stresses are combined. In this work, the pathways for the biosynthesis of these three compatible solutes were established based on the detection of the relevant enzymatic activities and characterization of the intermediate metabolites by nuclear magnetic resonance analysis. The synthesis of DIP proceeds from glucose-6-phosphate via four steps: (i) glucose-6-phosphate was converted into l-myo-inositol 1-phosphate by l-myo-inositol 1-phosphate synthase; (ii) l-myo-inositol 1-phosphate was activated to CDP-inositol at the expense of CTP; this is the first demonstration of CDP-inositol synthesis in a biological system; (iii) CDP-inositol was coupled with l-myo-inositol 1-phosphate to yield a phosphorylated intermediate, 1,1'-di-myo-inosityl phosphate 3-phosphate (DIPP); (iv) finally, DIPP was dephosphorylated into DIP by the action of a phosphatase. The synthesis of the two other polyol-phosphodiesters, DGP and GPI, proceeds via the condensation of CDP-glycerol with the respective phosphorylated polyol, glycerol 3-phosphate for DGP and l-myo-inositol 1-phosphate for GPI, yielding the respective phosphorylated intermediates, 1X,1'X-diglyceryl phosphate 3-phosphate (DGPP) and 1-(1X-glyceryl) myo-inosityl phosphate 3-phosphate (GPIP), which are subsequently dephosphorylated to form the final products. The results disclosed here represent an important step toward the elucidation of the regulatory mechanisms underlying the differential accumulation of these compounds in response to heat and osmotic stresses.

Effect of pyruvate kinase overproduction on glucose metabolism of Lactococcus lactis.

Lactococcus lactis strain NZ9000(pNZpyk), which overproduces pyruvate kinase (PK), was constructed. The pNZpyk plasmid carries the P(nisA)-pyk transcriptional fusion, and the overexpression of its pyk gene was accomplished by using the nisin-inducible expression system of the NZ9000 strain. In vivo (13)C- and (31)P-NMR spectroscopy was used to evaluate the effect of this modification on the metabolism of glucose in non-growing cells. A detailed description of the kinetics of glucose, end products, glycolytic intermediates, NAD(+) and NADH was obtained. A 15-fold increase in the level of PK did not increase the overall glycolytic flux, which, on the contrary, was slightly reduced. Significant differences were observed in (i) the level of 3-phosphoglycerate (3-PGA) and phosphoenolpyruvate (PEP), metabolites associated with starvation; (ii) the rate of fructose 1,6-bisphosphate (FBP) depletion upon glucose exhaustion; and (iii) the NAD(+)/NADH ratio during glucose catabolism. In the mutant, the rate of FBP consumption after glucose depletion was notably accelerated under anaerobic conditions, whereas 3-PGA and PEP decreased to undetectable levels. Furthermore, the level of NAD(+) decreased steadily during the utilization of glucose, probably due to the unanticipated reduction in the lactate dehydrogenase activity in comparison with the control strain, NZ9000(pNZ8020). The results show that PK is an important bottleneck to carbon flux only when glucose becomes limiting; in the overproducer this constriction was no longer present, as evidenced by the faster FBP consumption and lack of accumulation of 3-PGA and PEP in anaerobic as well as aerobic conditions. Despite these clear changes, the PK-overproducing strain showed typical homolactic metabolism under anaerobic conditions, as did the strain harbouring the vector plasmid without the pyk insert. However, under an oxygen atmosphere, there was increased channelling of carbon to the production of acetate and acetoin, to the detriment of lactate production.

Protein stabilization by compatible solutes. Effect of diglycerol phosphate on the dynamics of Desulfovibrio gigas rubredoxin studied by NMR.

Heteronuclear NMR relaxation measurements and hydrogen exchange data have been used to characterize protein dynamics in the presence or absence of stabilizing solutes from hyperthermophiles. Rubredoxin from Desulfovibrio gigas was selected as a model protein and the effect of diglycerol phosphate on its dynamic behaviour was studied. The presence of 100 mM diglycerol phosphate induces a fourfold increase in the half-life for thermal denaturation of D. gigas rubredoxin. A model-free analysis of the protein backbone relaxation parameters shows an average increase of generalized order parameters of 0.015 reflecting a small overall reduction in mobility of fast-scale motions. Hydrogen exchange data acquired over a temperature span of 20 degrees C yielded thermodynamic parameters for the structural opening reactions that allow for the exchange. This shows that the closed form of the protein is stabilized by an additional 1.6 kJ x mol(-1) in the presence of the solute. The results seem to indicate that the stabilizing effect is due mainly to a reduction in mobility of the slower, larger-scale motions within the protein structure with an associated increase in the enthalpy of interactions.