A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit

ПЕРВЫЙ КЛИНИЧЕСКИЙ ОПЫТ ПРИМЕНЕНИЯ КОЛОНОК ДЛЯ ЛПС-АДСОРБЦИИ «ТОКСИПАК» В ЛЕЧЕНИИ ПАЦИЕНТОВ С СЕПСИСОМ

Endotoxicosis is one of the main factors of the pathogenesis of multiple organic dysfunctions in gram-negative sepsis, induced by the accumulation of lipopolysaccharides - wall components of gram-negative bacteria. In vitro removal of them (LPS-adsorption) promotes better treatment outcomes in sepsis patients. Development and introduction of the Russian tools for LPS-adsorption becomes more crucial. Objective: to evaluate the efficiency and safety of the column of Toxipak for endotoxin removal, manufactured by POCARD Ltd. Materials and methods: 7 adult sepsis patients who had LPS-adsorption were included into the study. The changes of the following parameters were monitored in the patients: temperature, pulse, arterial pressure, respiration rate, central venous pressure, oxygen saturation, hourly rate of diuresis, oxygenation index was calculated, hematologic and biochemical blood parameters were tested as well as coagulation system rates, endotoxin level, C-reactive protein (CRP), procalcitonin, and interleukins (IL) 1, 6, 8. Criteria of the system inflammatory response and SOFA score were used for the comprehensive evaluation of clinical status. The adverse events, duration of stay in the intensive care ward and 18- and 24-day mortality were followed up during manipulations. Results: the studied column provided no negative effect on the cellular and biochemical composition, main parameters of clotting system and acid-base balance of blood. Its use resulted in the reduction of endotoxin (LPS) (by 64%), CRP (by 14%), IL-1 (by 38%) in blood, increase of oxygenation index, and renal function improvement. LPS adsorption promoted the reduction of clinical and laboratory signs of system inflammatory response, and severity of organ dysfunction (from 6.0 to 3.0 as per SOFA score) in patients with sepsis and septic shock. Conclusion: Toxipak column is safe and efficient for removal of endotoxins from blood. Одним из основных факторов патогенеза множественной органной дисфункции при грамотрицательном сепсисе является эндотоксикоз, обусловленный накоплением липополисахаридов – компонентов стенки грамотрицательных бактерий. Их экстракорпоральное удаление (ЛПС-адсорбция) способствует улучшению результатов лечения пациентов с сепсисом. Разработка и внедрение в практику отечественных устройств для ЛПС-адсорбции является актуальной задачей. Цель: провести оценку эффективности и безопасности колонок «Токсипак» для удаления эндотоксинов производства ЗАО НПФ «ПОКАРД». Материал и методы: в исследование включено 7 взрослых пациентов с сепсисом, которым выполнено 10 ЛПС-адсорбций. У пациентов в динамике измеряли температуру тела, пульс, артериальное давление, частоту дыхания, центральное венозное давление, сатурацию кислорода, почасовой темп диуреза, рассчитывали индекс оксигенации, в крови определяли гематологические и биохимические показатели, показатели системы свертывания, уровень эндотоксина, С-реактивного белка (СРБ), прокальцитонина, интерлейкинов (ИЛ) 1, 6, 8. Для интегральной оценки клинического статуса использовали критерии системной воспалительной реакции и шкалу SOFA. Оценивали нежелательные явления в ходе проведения процедур, длительность пребывания в отделении реанимации и интенсивной терапии, 14- и 28-дневную летальность. Результаты: изученные колонки не оказывали отрицательного влияния на клеточный и биохимический состав крови, основные показатели системы свертывания и кислотно-основное состояние крови. В результате их использования в крови достоверно уменьшилась концентрация эндотоксинов (ЛПС) (на 64%), СРБ (на 14%) и ИЛ-1 (на 38%), повысился индекс оксигенации, улучшилась функция почек. ЛПС-адсорбция способствовала ослаблению клинических и лабораторных проявлений системной воспалительной реакции, снижению выраженности органной дисфункции (с 6,0 до 3,0 баллов по шкале SOFA) у пациентов с сепсисом и септическим шоком. Вывод: колонки «Токсипак» эффективно удаляют из крови эндотоксины и являются безопасными.

The Response of Lactococcus lactis to Membrane Protein Production

Background: The biogenesis of membrane proteins is more complex than that of water-soluble proteins, and recombinant expression of membrane proteins in functional form and in amounts high enough for structural and functional studies is often problematic. To better engineer cells towards efficient protein production, we set out to understand and compare the cellular consequences of the overproduction of both classes of proteins in Lactococcus lactis, employing a combined proteomics and transcriptomics approach. Methodology and Findings: Highly overproduced and poorly expressed membrane proteins both resulted in severe growth defects, whereas amplified levels of a soluble substrate receptor had no effect. In addition, membrane protein overproduction evoked a general stress response (upregulation of various chaperones and proteases), which is probably due to accumulation of misfolded protein. Notably, upon the expression of membrane proteins a cell envelope stress response, controlled by the two-component regulatory CesSR system, was observed. Conclusions: The physiological response of L. lactis to the overproduction of several membrane proteins was determined and compared to that of a soluble protein, thus offering better understanding of the bottlenecks related to membrane protein production and valuable knowledge for subsequent strain engineering.

Cashew nut allergy: clinical relevance and allergen characterisation

Cashew plant (Anacardium occidentale L.) is the most relevant species of the Anacardium genus. It presents high economic value since it is widely used in human nutrition and in several industrial applications. Cashew nut is a well-appreciated food (belongs to the tree nut group), being widely consumed as snacks and in processed foods by the majority of world's population. However, cashew nut is also classified as a potent allergenic food known to be responsible for triggering severe and systemic immune reactions (e.g. anaphylaxis) in sensitised/allergic individuals that often demand epinephrine treatment and hospitalisation. So far, three groups of allergenic proteins have been identified and characterised in cashew nut: Ana o 1 and Ana o 2 (cupin superfamily) and Ana o 3 (prolamin superfamily), which are all classified as major allergens. The prevalence of cashew nut allergy seems to be rising in industrialised countries with the increasing consumption of this nut. There is still no cure for cashew nut allergy, as well as for other food allergies; thus, the allergic patients are advised to eliminate it from their diets. Accordingly, when carefully choosing processed foods that are commercially available, the allergic consumers have to rely on proper food labelling. In this sense, the control of labelling compliance is much needed, which has prompted the development of proficient analytical methods for allergen analysis. In the recent years, significant research advances in cashew nut allergy have been accomplished, which are highlighted and discussed in this review.This work was supported by FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 with grant no. UID/QUI/50006/2013–POCI/01/ 0145/FEDER/007265. Joana Costa is grateful to FCT post-doctoral grant (SFRH/BPD/102404/2014) financed by POPH-QREN (subsidised by FSE and MCTES).info:eu-repo/semantics/publishedVersio

FIRST CLINICAL EXPERIENCE OF USING COLUMN FOR LPS-ADSORPTION OF TOXIPAK IN TREATMENT OF SEPSIS PATIENTS

Endotoxicosis is one of the main factors of the pathogenesis of multiple organic dysfunctions in gram-negative sepsis, induced by the accumulation of lipopolysaccharides - wall components of gram-negative bacteria. In vitro removal of them (LPS-adsorption) promotes better treatment outcomes in sepsis patients. Development and introduction of the Russian tools for LPS-adsorption becomes more crucial. Objective: to evaluate the efficiency and safety of the column of Toxipak for endotoxin removal, manufactured by POCARD Ltd. Materials and methods: 7 adult sepsis patients who had LPS-adsorption were included into the study. The changes of the following parameters were monitored in the patients: temperature, pulse, arterial pressure, respiration rate, central venous pressure, oxygen saturation, hourly rate of diuresis, oxygenation index was calculated, hematologic and biochemical blood parameters were tested as well as coagulation system rates, endotoxin level, C-reactive protein (CRP), procalcitonin, and interleukins (IL) 1, 6, 8. Criteria of the system inflammatory response and SOFA score were used for the comprehensive evaluation of clinical status. The adverse events, duration of stay in the intensive care ward and 18- and 24-day mortality were followed up during manipulations. Results: the studied column provided no negative effect on the cellular and biochemical composition, main parameters of clotting system and acid-base balance of blood. Its use resulted in the reduction of endotoxin (LPS) (by 64%), CRP (by 14%), IL-1 (by 38%) in blood, increase of oxygenation index, and renal function improvement. LPS adsorption promoted the reduction of clinical and laboratory signs of system inflammatory response, and severity of organ dysfunction (from 6.0 to 3.0 as per SOFA score) in patients with sepsis and septic shock. Conclusion: Toxipak column is safe and efficient for removal of endotoxins from blood