RESUMO
Extremophiles and their products have been a major focus of research interest for over 40 years. Through this period, studies of these organisms have contributed hugely to many aspects of the fundamental and applied sciences, and to wider and more philosophical issues such as the origins of life and astrobiology. Our understanding of the cellular adaptations to extreme conditions (such as acid, temperature, pressure and more), of the mechanisms underpinning the stability of macromolecules, and of the subtleties, complexities and limits of fundamental biochemical processes has been informed by research on extremophiles. Extremophiles have also contributed numerous products and processes to the many fields of biotechnology, from diagnostics to bioremediation. Yet, after 40 years of dedicated research, there remains much to be discovered in this field. Fortunately, extremophiles remain an active and vibrant area of research. In the third decade of the twenty-first century, with decreasing global resources and a steadily increasing human population, the world's attention has turned with increasing urgency to issues of sustainability. These global concerns were encapsulated and formalized by the United Nations with the adoption of the 2030 Agenda for Sustainable Development and the presentation of the seventeen Sustainable Development Goals (SDGs) in 2015. In the run-up to 2030, we consider the contributions that extremophiles have made, and will in the future make, to the SDGs.
Assuntos
Extremófilos , Extremófilos/metabolismo , Extremófilos/fisiologia , Desenvolvimento Sustentável , Adaptação Fisiológica , Ambientes Extremos , BiotecnologiaRESUMO
BACKGROUND: T1 colorectal cancer (CRC) without histological high-risk factors for lymph node metastasis (LNM) can potentially be cured by endoscopic resection, which is associated with significantly lower morbidity, mortality and costs compared to radical surgery. An important prerequisite for endoscopic resection as definite treatment is the histological confirmation of tumour-free resection margins. Incomplete resection with involved (R1) or indeterminate (Rx) margins is considered a strong risk factor for residual disease and local recurrence. Therefore, international guidelines recommend additional surgery in case of R1/Rx resection, even in absence of high-risk factors for LNM. Endoscopic full-thickness resection (eFTR) is a relatively new technique that allows transmural resection of colorectal lesions. Local scar excision after prior R1/Rx resection of low-risk T1 CRC could offer an attractive minimal invasive strategy to achieve confirmation about radicality of the previous resection or a second attempt for radical resection of residual luminal cancer. However, oncologic safety has not been established and long-term data are lacking. Besides, surveillance varies widely and requires standardization. METHODS/DESIGN: In this nationwide, multicenter, prospective cohort study we aim to assess feasibility and oncological safety of completion eFTR following incomplete resection of low-risk T1 CRC. The primary endpoint is to assess the 2 and 5 year luminal local tumor recurrence rate. Secondary study endpoints are to assess feasibility, percentage of curative eFTR-resections, presence of scar tissue and/or complete scar excision at histopathology, safety of eFTR compared to surgery, 2 and 5 year nodal and/or distant tumor recurrence rate and 5-year disease-specific and overall-survival rate. DISCUSSION: Since the implementation of CRC screening programs, the diagnostic rate of T1 CRC is steadily increasing. A significant proportion is not recognized as cancer before endoscopic resection and is therefore resected through conventional techniques primarily reserved for benign polyps. As such, precise histological assessment is often hampered due to cauterization and fragmentation and frequently leads to treatment dilemmas. This first prospective trial will potentially demonstrate the effectiveness and oncological safety of completion eFTR for patients who have undergone a previous incomplete T1 CRC resection. Hereby, substantial surgical overtreatment may be avoided, leading to treatment optimization and organ preservation. Trial registration Nederlands Trial Register, NL 7879, 16 July 2019 ( https://trialregister.nl/trial/7879 ).
Assuntos
Neoplasias Colorretais , Recidiva Local de Neoplasia , Humanos , Cicatriz/complicações , Cicatriz/patologia , Neoplasias Colorretais/patologia , Metástase Linfática , Estudos Multicêntricos como Assunto , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Neoplasia Residual/patologia , Estudos Prospectivos , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Failures of mine tailings storage facilities (TSF) can have profound and long-lasting effects on the downstream receiving environment. Virtually all spills to date have been into river systems without large lakes that may buffer downstream impacts. In August 2014, the failure of the Mount Polley copper (Cu)-gold mine TSF in British Columbia, Canada, released ~25 × 106 m3 of water and solids; globally, this is the second largest TSF spill in history. Over 18 × 106 m3 was delivered to Quesnel Lake, which is ~9 km from the TSF and is the third deepest lake in North America, and a crucial habitat for Pacific salmon and trout populations. We determined the sediment-associated Cu concentrations and fluxes in Quesnel River, downstream of the lake, from August 2014 to February 2021 based on the analysis of >400 samples of sediment, mainly collected using a continuous-flow centrifuge. During each winter since the spill, Cu concentrations in the fluvial sediment in the upper reaches of the river (~35 km from the TSF) were elevated relative to regional background concentrations and samples collected before the spill. Maximum Cu concentrations were ~410 mg kg-1 which exceeds Canadian sediment quality guidelines for the protection of aquatic organisms (197 mg kg-1). Monitoring of Quesnel Lake since the spill shows that these annual pulses in the winter are due to resuspension of unconsolidated tailings and sediments at the bottom of Quesnel Lake, during autumnal lake turnover, which become mixed throughout the water column and subsequently flow into Quesnel River. Results show that while large lakes may buffer downstream aquatic systems from contaminated sediment, they may prolong the environmental impact. These findings are crucial in understanding how lake processes may modify the effects of TSF spills on downstream aquatic systems.
Assuntos
Lagos , Poluentes Químicos da Água , Colúmbia Britânica , Cobre/análise , Monitoramento Ambiental , Sedimentos Geológicos/análise , Água/análise , Poluentes Químicos da Água/análiseRESUMO
Protein phosphorylation is known to occur in Archaea. However, knowledge of phosphorylation in the third domain of life is rather scarce. Homology-based searches of archaeal genome sequences reveals the absence of two-component systems in crenarchaeal genomes but the presence of eukaryotic-like protein kinases and protein phosphatases. Here, the influence of the offered carbon source (glucose versus tryptone) on the phospho-proteome of Sulfolobus solfataricus P2 was studied by precursor acquisition independent from ion count (PAcIFIC). In comparison to previous phospho-proteome studies, a high number of phosphorylation sites (1318) located on 690 phospho-peptides from 540 unique phospho-proteins were detected, thus increasing the number of currently known archaeal phospho-proteins from 80 to 621. Furthermore, a 25.8/20.6/53.6 Ser/Thr/Tyr percentage ratio with an unexpectedly high predominance of tyrosine phosphorylation was detected. Phospho-proteins in most functional classes (21 out of 26 arCOGs) were identified, suggesting an important regulatory role in S. solfataricus. Focusing on the central carbohydrate metabolism in response to the offered carbon source, significant changes were observed. The observed complex phosphorylation pattern hints at an important physiological function of protein phosphorylation in control of the central carbohydrate metabolism, which might particularly operate in channeling carbon flux into the respective metabolic pathways.
Assuntos
Proteínas Arqueais/metabolismo , Metabolismo dos Carboidratos , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Sulfolobus solfataricus/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Meios de Cultura , Glucose/metabolismo , Anotação de Sequência Molecular , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Peptonas/metabolismo , Fosfoproteínas/química , Fosforilação , Processamento de Proteína Pós-Traducional , Proteoma/química , Espectrometria de Massas em TandemRESUMO
Compared to Sulfolobus solfataricus P2, the S. solfataricus mutant PBL2025 misses 50 genes (SSO3004-3050), including genes coding for a multitude of enzymes possibly involved in sugar degradation or metabolism. We complemented PBL2025 with two of the missing proteins, the α-mannosidase (SSO3006, Ssα-man) and the ß-galactosidase LacS (SSO3019), and performed comparative fluorescence microscopy and confocal laser scanning microscopy to analyze the recombinant strains. We demonstrated that the Ssα-man complemented strain resembled the S. solfataricus P2 behavior with respect to attachment of cells to glass and growth of cells in static biofilms. During expression of the Ssα-man, but not LacS, glucose and mannose-containing extracellular polymeric substance (EPS) levels changed in the recombinant strain during surface attachment and biofilm formation. These results suggest that the Ssα-man might be involved in the modulation of the EPS composition and/or in the de-mannosylation of the glycan tree, which is attached to extracellular glycosylated proteins in S. solfataricus. On the other hand, LacS expression in PBL2025 reduced the carbohydrate content of the isolated total EPS implying a role in the modulation of the produced EPS during static biofilm formation. These are the first enzymes identified as playing a role in archaeal EPS formation.
Assuntos
Biofilmes , Sulfolobus solfataricus/metabolismo , alfa-Manosidase/metabolismo , Sequência de Bases , Primers do DNA , Microscopia Confocal , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Sulfolobus solfataricus/enzimologia , Propriedades de SuperfícieRESUMO
We review and update the work on genetic elements, e.g., viruses and plasmids (exluding IS elements and transposons) in the kingdom Crenarchaeota (Thermoproteales and Sulfolobales) and the orders Thermococcales and Thermoplasmales in the kingdom Euryarchaeota of the archael domain, including unpublished data from our laboratory. The viruses of Crenarchaeota represent four novel virus families. The Fuselloviridae represented by SSVI of S. shibatae and relatives in other Sulfolobus strains have the form of a tailed spindle. The envelope is highly hydrophobic. The DNA is double-stranded and circular. Members of this group have also been found in Methanococcus and Haloarcula. The Lipothrivciridae (e.g., T TV1 to 3) have the form of flexible filaments. They have a core containing linear double-stranded DNA and DNA-binding proteins which is wrapped into a lipid membrane. The "Bacilloviridae" (e.g., TTV4 and SIRV) are stiff rods lacking this membrane, but also featuring linear double-stranded DNA and DNA-binding proteins. Both virus types carry on both ends structures involved in the attachment to receptors. Both types are represented in Thermoproteus and Sulfolobus. The droplet-formed novel Sulfolobus virus SNDV represents the "Guttaviridae" containing circular double-stranded DNA. Though head and tail viruses distantly resembling T phages or lambdoid phages were seen electronmicroscopically in solfataric water samples, no such virus has so far been isolated. SSV1 is temperate, TTV1 causes lysis after induction, the other viruses found so far exist in carrier states. The hosts of all but TTV1 survive virus production. We discuss the implications of the nature of these viruses for understanding virus evolution. The plasmids found so far range in size from 4.5 kb to about 40 kb. Most of them occur in high copy number, probably due to the way of their detection. Most are cryptic, pNOB8 is conjugative, the widespread pDL10 alleviates in an unknown way autotrophic growth of its host Desulfurolobus by sulfur reduction. The plasmid pTIK4 appears to encode a killer function. pNOB8 has been used as a vector for the transfer of the lac S (beta-galactosidase) gene into a mutant of S. solfataricus.
Assuntos
Archaea/genética , Archaea/virologia , Plasmídeos , Vetores Genéticos , Filogenia , Ensaio de Placa ViralRESUMO
The impairment of nitric oxide (NO)-mediated vasodilation in diabetes has been attributed to increased vascular oxidative stress. Lipoic acid has been shown to have substantial antioxidative properties. The aim of this study was to assess the effect of lipoic acid on NO-mediated vasodilation in diabetic patients in comparison with the well-recognized effect of ascorbic acid. Using venous occlusion plethysmography, we examined the effects of lipoic acid (0.2 mM) and ascorbic acid (1 and 10 mM) on forearm blood flow responses to acetylcholine, sodium nitroprusside and concomitant infusion of the NO-inhibitor, N(G)-monomethyl-L-arginine, in 39 diabetic patients and 11 control subjects. Plasma levels of antioxidants and parameters of lipid peroxidation were measured and correlated to endothelial function tests. Lipoic acid improved NO-mediated vasodilation in diabetic patients, but not in controls. NO-mediated vasodilation was improved by ascorbic acid at 10 mM, but not 1 mM. Improvements of endothelial function by ascorbic acid and lipoic acid were closely related. The beneficial effects of lipoic acid were positively related to plasma levels of malondialdehyde and inversely related to levels of ubiquinol-10. These findings support the concept that oxidative stress contributes to endothelial dysfunction and suggest a therapeutic potential of lipoic acid particularly in patients with imbalance between increased oxidative stress and depleted antioxidant defense.
Assuntos
Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Diabetes Mellitus Tipo 2/fisiopatologia , Endotélio Vascular/efeitos dos fármacos , Óxido Nítrico/metabolismo , Estresse Oxidativo , Ácido Tióctico/farmacologia , Vasodilatação/efeitos dos fármacos , Acetilcolina/farmacologia , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nitroprussiato/farmacologia , PletismografiaRESUMO
In extreme environments varying from hot to cold, acidic to alkaline, and highly saline, mainly Archaea are found. Thermophilic and extremely acidophilic Archaea have a membrane that contains membrane spanning tetraether lipids. These tetra-ether membranes have a limited permeability for protons even at the high temperatures of growth and this property makes it possible for thermophilic archaea to maintain a viable proton motive force under the extreme conditions. -Ether lipids cannot be degraded easily and are highly stable which is also a requirement for life under extreme conditions. Psychrophilic and mesophilic Bacteria, and all Archaea adjust the lipid composition of their membranes so that the proton permeability of their membranes remains within a narrow range. This phenomenon is termed 'homeoproton permeability adaptation'. Thermophilic Bacteria are the only prokaryotes that are unable to control the proton permeability of their membranes. These organisms have to rely on the less permeable sodium ions in energy transducing processes in their membrane.
Assuntos
Adaptação Fisiológica , Archaea/fisiologia , Resposta ao Choque Térmico , Fenômenos Fisiológicos Bacterianos , Transporte Biológico , Membrana Celular/fisiologia , Lipídeos de Membrana/fisiologia , Proteínas de Membrana/fisiologiaRESUMO
In this review, new knowledge about the potential use of glutamine containing dipeptides as substrates in the frame of parenteral nutrition is presented. Using chemical and biotechnological methods, the stable and highly soluble peptide L-alanyl-L-glutamine (Ala-Gln) can be synthesized in high yields. Studies in experimental rats and dogs demonstrate the effective utilization of intravenously supplied Ala-Gln and the rapid provision of free glutamine for maintenance of the intracellular muscle-free glutamine pool in catabolic situations. Subsequent studies in healthy volunteers provide firm evidence that the infused Ala-Gln is rapidly eliminated from plasma (t1/2:3.8 minutes), associated by a prompt equimolar increase in the concentrations of free alanine and glutamine. Bolus injection and continuous infusion of the peptide was not accompanied by any side effects, and no complaints by the subjects were noted. These results may indicate a safe and efficient use of Ala-Gln as source of free glutamine in parenteral nutrition.
Assuntos
Dipeptídeos/farmacocinética , Glutamina/metabolismo , Animais , Disponibilidade Biológica , Dipeptídeos/administração & dosagem , Dipeptídeos/síntese química , Meia-Vida , Humanos , Infusões Intravenosas , CinéticaRESUMO
Of the total pool of muscle free intracellular amino acids, glutamine represents about 60%. During catabolic stress, a marked reduction (50%) of this pool occurs; the depletion is not reversible by nutrition or other therapeutical endeavors. Since free glutamine is unstable in solutions, the question is whether maintenance of this pool and improvement of the nitrogen economy is feasible by intravenous provision of synthetic, stable glutamine-containing dipeptides. In vivo studies in man and animals provide firm evidence that a synthetic glutamine containing dipeptide, L-alanyl-L-glutamine (Ala-Gln), is readily hydrolyzed following intravenous administration. The results also indicate a safe and efficient use of Ala-Gln as a source of free glutamine for parenteral nutrition. In clinical studies, nitrogen balance was more positive in catabolic patients receiving a peptide supplemented solution as compared with control patients given isonitrogenous, isoenergetic TPN. Preoperative muscle glutamine concentrations were essentially maintained in the peptide group and markedly decreased in the control group. It is inferred that the increased intestinal requirement of metabolic fuel during catabolic stress is matched by an enhanced demand on muscle glutamine, resulting in intracellular glutamine depletion. Thus, the delivery of adequate amounts of glutamine is essential to maintain the integrity of intestinal mucosa, to preserve the muscle glutamine pool, and to improve overall nitrogen economy during conditions of stress.
Assuntos
Carcinoma/metabolismo , Neoplasias do Colo/metabolismo , Glutamina/metabolismo , Necessidades Nutricionais , Ferimentos e Lesões/metabolismo , Animais , Carcinoma/terapia , Neoplasias do Colo/terapia , Dipeptídeos/metabolismo , Dipeptídeos/farmacocinética , Dipeptídeos/uso terapêutico , Glutamina/uso terapêutico , Humanos , Infusões Intravenosas , Músculos/metabolismo , Nutrição Parenteral , Valores de Referência , Estresse Fisiológico/metabolismo , Ferimentos e Lesões/terapiaRESUMO
Of the total pool of muscle free intracellular amino acids, glutamine represents about 60%. During catabolic stress, a marked reduction (50%) of this pool occurs; the depletion is not reversible by therapeutic efforts or conventional nutritional means. If maintenance of the intracellular glutamine pool promotes conservation of muscle protein, there is a theoretical case for use of glutamine supplements in the parenteral nutrition of patients with injury and infection. Glutamine is too unstable and poorly soluble for addition to existing preparations in its native form, but this drawback can be overcome by the use of synthetic stable and highly soluble glutamine-containing dipeptides. In vivo studies in humans and animals provide firm evidence that a synthetic glutamine-containing dipeptide, L-alanyl-L-glutamine (Ala-Gln), is readily hydrolyzed following its intravenous administration. The results also indicate a safe and efficient use of Ala-Gln as a source of free glutamine in parenteral nutrition. In clinical studies, nitrogen balance was more positive in catabolic patients receiving a peptide-supplemented solution than in control patients given isonitrogenous, isoenergetic total parenteral nutrition. Muscle glutamine concentrations were markedly decreased in the control groups. The intracellular concentrations were not influenced following severe injury, but were maintained in postoperative trauma. It is inferred that the increased intestinal requirement and cellular demand for metabolic fuel during catabolic stress is matched by an enhanced demand on muscle glutamine, resulting in intracellular glutamine depletion. Thus, the delivery of adequate amounts of glutamine is essential to maintain the integrity of intestinal mucosa and rapidly proliferating cells, to preserve the muscle glutamine pool, and to improve overall nitrogen economy during conditions of stress.
Assuntos
Dipeptídeos/metabolismo , Glutamina/metabolismo , Nutrição Parenteral , Animais , Dipeptídeos/administração & dosagem , Cães , Glutamina/administração & dosagem , Humanos , Hidrólise , Mucosa Intestinal/metabolismo , Taxa de Depuração Metabólica , Ratos , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/terapiaRESUMO
A retrospective study was performed to determine the frequency of new symptoms and diseases after silicone breast implantation. Questionnaires were mailed to 826 women who made up a breast implant group (n = 516) and a control group who had undergone blepharoplasty (n = 124), liposuction (n = 111), or rhinoplasty (n = 75). Responses were obtained from 370 women (45 percent); however, 68 of these patients (18 percent) were considered ineligible. The overall response rate was 59 percent for the breast implant group and 46 percent for controls. The 302 eligible women included patients with silicone breast implants (n = 222) and controls (n = 80). Women with implants were significantly younger than controls, the median age of women with breast implants being 37 years compared with 46.5 years for controls (p < 0.0001). We compared the incidence of 23 symptoms and 4 connective-tissue diseases after cosmetic surgery in the two groups. The symptoms of swollen glands under arms (p < 0.05) and tender glands under arms (p < 0.01) were statistically more frequent in the breast implant group. The symptom change in skin color was more common in the controls (p < 0.001). The Bonferroni correction for multiple (27) endpoints adjusts the 5 and 1 percent significance cutoff points to 0.00185 and 0.00037, respectively, leaving only change of skin color significant at the 5 percent level on the adjusted data. No cases of scleroderma or lupus were found, and the incidence of arthritis was not significantly different between the implant and control groups.
Assuntos
Mamoplastia , Complicações Pós-Operatórias/etiologia , Próteses e Implantes , Silicones , Cirurgia Plástica , Saúde da Mulher , Adulto , Doenças do Tecido Conjuntivo/etiologia , Pálpebras/cirurgia , Feminino , Humanos , Lipectomia , Mamoplastia/efeitos adversos , Pessoa de Meia-Idade , Transtornos da Pigmentação/etiologia , Estudos Retrospectivos , Rinoplastia , Cirurgia Plástica/efeitos adversos , Inquéritos e QuestionáriosRESUMO
Skin changes associated with aging often manifest as cosmetic disabilities. As the population of elderly persons continues to rise, these aging skin changes and patients' dissatisfaction with them will increasingly command the attention of the primary care physician. The cosmetic aging changes and management strategies addressed here include wrinkles, hair changes, common benign neoplasms, dyspigmentation, and telangiectasias. While none of these conditions is a direct threat to the physical well being of the patient, their psychological impact, particularly with regard to self-perception, can be significant and even profound. They therefore merit a response from physicians caring for such patients.
Assuntos
Envelhecimento da Pele , Dermatopatias/terapia , Idoso , Alopecia/tratamento farmacológico , Alopecia/etiologia , Colágeno/administração & dosagem , Criocirurgia , Dermabrasão , Humanos , Minoxidil/uso terapêutico , Tretinoína/uso terapêuticoRESUMO
Kwashiorkor is part of the spectrum of protein-energy malnutrition. The condition results from a lack of nutritional protein coupled with carbohydrate excess. Protein malnutrition is much more common in the Third World; however, it is also the most common form of nutritional deficiency among hospitalized patients in the United States. Cutaneous clues to the diagnosis of protein deficiency include the "flag sign," hypochromotrichia, alopecia, "crazy pavement" dermatosis, pallor, and glossitis. Systemic manifestations include peripheral edema, liver disease, neurologic changes, diarrhea, weight loss, and hypoalbuminemia. We report here a case of profound kwashiorkor in a nonhospitalized patient who presented with multiple cutaneous and systemic findings indicative of protein malnutrition.
Assuntos
Kwashiorkor/patologia , Dermatopatias/patologia , Adulto , Feminino , Doenças do Cabelo/patologia , Humanos , Hipopigmentação/patologia , Doenças da Língua/patologiaRESUMO
Children with congenital heart disease need adequate diagnostic classification regarding their cardiovascular status (CVS). N-terminal brain natriuretic peptide (N-BNP) plasma concentration indicates dysfunction of the cardiovascular system and guides decisions concerning treatment and prognosis. Reference values are established for adults, with age-dependent increasing values and higher values in women. To avoid misclassification concerning the CVS, a large group of healthy children and adolescents can be used show the relationship between gender, age, and N-BNP and these can serve as reference values. N-BNP was measured in 434 healthy subjects (240 female and 194 male) with ages ranging from 0 to 32 years without any cardiovascular disease or renal or hepatic impairment. Measurements were performed with an electrochemiluminescence immunoassay from Roche Diagnostics. Mean N-BNP decreased from 12.6 fmol/ml (0-9 years; n = 79) to 9.41 fmol/ml (10-14 years; n = 154) and in adolescents from 6.1 (15-19 years; n = 99) to 4.8 fmol/ml (> 19 years; n = 102) in adults (p < 0.05). Mean N-BNP concerning gender did not differ in any age group younger than 19 years. In contrast, the adult female group had 78% higher N-BNP compared to the male group (p < 0.05). There was a significant peak in N-BNP at the age of 12-14 years. This study shows that reference values for N-BNP differed profoundly in children compared to adults and were up to 260% higher in children without any gender difference. Therefore, these reference values will help to avoid CVS misclassification in children for the biomarker N-BNP.
Assuntos
Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Adolescente , Adulto , Fatores Etários , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Valores de Referência , Fatores SexuaisRESUMO
Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80 degrees C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol. 48:1241-1252, 2003). Here we have refined this system for high-level gene expression in S. solfataricus with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from Acidianus ambivalens, an Fe-S protein of the ABC class from S. solfataricus, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon.
Assuntos
Proteínas Arqueais/metabolismo , Vetores Genéticos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Proteínas Recombinantes/metabolismo , Sulfolobus solfataricus/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas Arqueais/genética , Regulação da Expressão Gênica em Archaea , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Sulfolobus solfataricus/genéticaRESUMO
Qualitative analysis of spontaneous motility was performed in 22 preterm infants (gestational age 25-31 weeks) on the intensive care unit. The infants were videorecorded once a week in the late afternoon during 1 h until 36 weeks of gestation. Quality of movement was analyzed by 8 observers using visual 'Gestalt perception' and compared with the neurological outcome 1 year after term. A normal quality of movement consistently predicted a normal neurological outcome with a probability of 90-100%. An abnormal quality of movement predicted an abnormal outcome with a probability of only 56% in the first, but with a probability of 82% in the third postnatal week. The average interobserver agreement was 78%. The analysis of spontaneous motility for the early diagnosis of neurological dysfunctions can reliably be applied on very immature preterms under intensive care conditions from the 3rd postnatal week on.
Assuntos
Recém-Nascido Prematuro/fisiologia , Movimento/fisiologia , Doenças do Sistema Nervoso/fisiopatologia , Fatores Etários , Idade Gestacional , Humanos , Recém-Nascido , Terapia Intensiva Neonatal , Prognóstico , Gravação de VideoteipeRESUMO
Screening of newborn infants for genetic disease began over 35 years ago as a public health measure to prevent mental retardation in phenylketonuria (PKU). It was so successful that tests for several other genetic disorders were added. We review the current status of this screening, including discussions of the genetic disorders often covered and the results of newborn screening for them. We emphasize recent advances. These include expansion of coverage for genetic disorders with the new methodology of tandem mass spectrometry (MS-MS) and the introduction of molecular (DNA) testing to increase the specificity of testing for several disorders, thereby reducing false-positive rates. These and other advances have also produced issues of criteria for screening, missed cases, and appropriate use of stored newborn specimens.
Assuntos
Doenças Genéticas Inatas/diagnóstico , Testes Genéticos/métodos , Fatores Etários , DNA/genética , DNA/isolamento & purificação , Erros de Diagnóstico , Feminino , Doenças Genéticas Inatas/genética , Testes Genéticos/organização & administração , Humanos , Recém-Nascido , Masculino , Espectrometria de Massas/métodosRESUMO
Of the total pool of muscle free intracellular amino acids glutamine represents about 60%. A uniform reduction of approximately 50% of the intracellular free glutamine pool is the most typical feature in various catabolic conditions. Since nutritional or therapeutical efforts to beneficially influence cellular glutamine pool failed and because free glutamine cannot be infused owing to its instability, the question arose as to whether maintenance of this pool is feasible by intravenous provision of glutamine-containing peptides. Our basic research plan attempted to combine the synthesis and characterization of, among other peptides, L-alanyl-L-glutamine (Ala-Gln) with investigations aimed at examining in vivo uptake and subsequent utilization of this solute. The synthesis of Ala-Gln was performed by applying the N-carboxy anhydride method in the aqueous phase. The purity in the final product approached 100% and the structure could be fully confirmed by field-desorption mass spectrometry and proton magnetic resonance spectrometry. The synthetic peptide Ala-Gln is highly soluble (568 g/l H2O; 20 degrees C) and stable during heat sterilization at various pH. Thus, Ala-Gln complies with each criterion to be included in future parenteral solutions. Isotope studies with Ala[U14C]Gln in experimental rat and dog strongly indicate that the peptide is easily available and the constituent amino acids are rapidly used for protein synthesis, preferentially in muscle tissue. In catabolic rats, continuous TPN without inclusion of Ala-Gln resulted in a profound decrease in tissue free glutamine levels compared with normal rats. Inclusion of Ala-Gln to TPN was followed by an increase in tissue free glutamine pool, considerably in liver and markedly in muscle. These findings indicate a preferential capacity of muscle tissue to take up Ala-Gln and suggest subsequent utilization of the liberated free glutamine in this tissue. For the first time, in vivo utilization of Ala-Gln was evaluated in healthy humans and substantiated with kinetic studies and under conditions of continuous infusion of peptide-supplemented amino acid solution. The peptide elimination t1/2 was 3.1 +/- 0.16 min and that for the liberated free amino acids glutamine and alanine 8.2 +/- 0.82 and 6.8 +/- 0.34 min, respectively. During infusion of an amino acid solution supplemented with Ala-Gln and Gly-Tyr, the increments of plasma glutamine and tyrosine were 33% +/- 2.2 and 67% +/- 5.7 over the initial values. No peptide could be detected in the urine.(ABSTRACT TRUNCATED AT 400 WORDS)