Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli

<p>Abstract</p> <p>Background</p> <p>Biotin is an essential enzyme cofactor that acts as a CO₂carrier in carboxylation and decarboxylation reactions. The <it>E. coli </it>genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for ³⁵S labeling of biotin.</p> <p>Results</p> <p>In this study, we produced [³⁵S]-biotin from Na³⁵SO₄and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (<it>Pf</it>BCCP-79) from the <it>Plasmodium falciparum </it>acetyl-CoA carboxylase (ACC) was expressed in <it>E. coli </it>as a biotinylation substrate. We found that overexpression of the <it>E. coli </it>biotin synthase, BioB, and biotin ligase, BirA, increased <it>Pf</it>BCCP-79 biotinylation 160-fold over basal levels. Biotinylated <it>Pf</it>BCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [<it>D</it>]-biotin that specifically labels biotinylated proteins through reuptake in <it>E. coli</it>.</p> <p>Conclusions</p> <p>The strategy described in our report provides a simple and effective method for the production of [³⁵S]-biotin in <it>E. coli </it>based on affinity chromatography.</p

Genetic analysis for sooty mold resistance and heart of palm yield in Archontophoenix.

Palmeiras do gênero Archontophoenix, utilizadas tanto como ornamentais quanto produtoras de palmito de qualidade, são susceptíveis à fumagina, doença associada à infestação por pulgões, que afeta a fotossíntese, o crescimento e a aparência das plantas. Foram avaliados a campo a resistência à fumagina conjuntamente com três caracteres associados ao crescimento em 24 famílias de meios-irmãos, 28 meses após o plantio, a fim de identificar a variabilidade genética para os caracteres resistência à fumagina, altura, diâmetro e número de folhas; estimar as correlações genotípicas e fenotípicas envolvendo esses quatro caracteres; e aplicar a estratégia de seleção usando o índice multiefeitos. Houve diferenças entre as famílias para os caracteres avaliados, sugerindo a possibilidade de seleção. O baixo coeficiente de variação observado para resistência à fumagina (9,48%) indica que o método de avaliação adotado, baseado em escala de notas após observação visual, foi eficiente e prático para comparar níveis de infestação do complexo fungo+pulgão em palmeiras do gênero Archontophoenix. As estimativas da herdabilidade no sentido restrito foram baixas a médias para os caracteres relacionados ao crescimento (0,10, 0,26 e 0,26 para número de folhas, diâmetro e altura da planta, respectivamente) e muito altas (0,91) para resistência à fumagina. Correlação genética positiva foi observada entre resistência à fumagina e altura da planta, indicando que a eliminação de plantas muito susceptíveis pode ser feita sem interferência na seleção indireta para produção de palmito. A estratégia de seleção pelo índice multiefeitos (com ganhos genéticos esperados variando de 6,23 a 11,83%) mostrou-se adequada para melhorar simultaneamente caracteres relacionados ao crescimento e à produção de palmito

Light dependent redox catalysis by Photosystem I complexes encapsulated in organic nanoparticles

Photosystem I (PSI) is a pigment binding multi-subunit protein complexes involved in photosynthesis. PSI is localized in the thylakoid membranes and catalyze the electron transfer reaction from plastocyanin to ferredoxin, as one of the main steps involved in conversion of light energy into chemical energy. PSI is highly efficiency with a photochemical efficiency close to one. Several attempts have doing in the past in order to exploit the high efficiency and high stability of PSI in an extra-cellular context in order to catalyze electron transfer reactions: in this work we present an innovative solution for exploiting the photochemical properties of PSI, by encapsulation of PSI complexes in organic nanoparticles. Nanoparticles offer a protected environment to the encapsulated molecule, giving it the possibility of preserving its functional properties and studying how they change over time. In this work the complete characterization, both morphological and functional, of nanostructures obtained by encapsulation of PSI complexes purified from higher plants with PLGA (poly lactic-co-glycolic acid) polymer is presented. The results obtained by transient absorption and time-resolved fluorescence demonstrate that encapsulated PSI were characterized by an higher photochemcial activity compared to PSI complexes in detergent solution. Moreover, encapsulated PSI maintained the high efficiency observed for several weeks even if exposed to very strong light, being more stable compared to PSI in detergent solution. Finally, the nanostructures obtained by encapsulated PSI were able to catalyze light dependent redox reactions with electron acceptors and donors outside the nanostructures Potential application of these PLGA encapsulated PSI in different fields are thus presented and discussed

Association of pretreatment hippocampal volume with neurocognitive function in patients treated with hippocampal avoidance whole brain radiation therapy for brain metastases: Secondary analysis of NRG Oncology/RTOG 0933

PURPOSE: Hippocampal volume (HV) is an established predicting factor for neurocognitive function (NCF) in neurodegenerative disease. Whether the same phenomenon exists with hippocampal-avoidant whole brain radiation therapy is not known; therefore, we assessed the association of baseline HV with NCF among patients enrolled on RTOG 0933. METHODS AND MATERIALS: Hippocampal volume and total brain volume were calculated from the radiation therapy plan. Hippocampal volume was correlated with baseline and 4-month NCF scores (Hopkins Verbal Learning Test-Revised [HVLT-R] Total Recall [TR], Immediate Recognition, and Delayed Recall [DR]) using Pearson correlation. Deterioration in NCF was defined per the primary endpoint of RTOG 0933(mean 4-month relative decline in HVLT-R DR). Comparisons between patients with deteriorated and nondeteriorated NCF were made using the Wilcoxon test. RESULTS: Forty-two patients were evaluable. The median age was 56.5 years (range, 28-83 years), and 81% had a class II recursive partitioning analysis. The median total, right, and left HVs were 5.4 cm CONCLUSIONS: Larger HV was positively associated with improved performance on baseline and 4-month HVLT-R TR and DR scores in patients with brain metastases undergoing hippocampal-avoidant whole brain radiation therapy but was not associated with a change in NCF

Effect of Remote Ischaemic Conditioning on the Inflammatory Cytokine Cascade of COVID-19 (RIC in COVID-19): a Randomized Controlled Trial

Purpose: Patients hospitalized with COVID-19 may develop a hyperinflammatory, dysregulated cytokine “storm” that rapidly progresses to acute respiratory distress syndrome, multiple organ dysfunction, and even death. Remote ischaemic conditioning (RIC) has elicited anti-inflammatory and cytoprotective benefits by reducing cytokines following sepsis in animal studies. Therefore, we investigated whether RIC would mitigate the inflammatory cytokine cascade induced by COVID-19. Methods: We conducted a prospective, multicentre, randomized, sham-controlled, single-blind trial in Brazil and South Africa. Non-critically ill adult patients with COVID-19 pneumonia were randomly allocated (1:1) to receive either RIC (intermittent ischaemia/reperfusion applied through four 5-min cycles of inflation (20 mmHg above systolic blood pressure) and deflation of an automated blood-pressure cuff) or sham for approximately 15 days. Serum was collected following RIC/sham administration and analyzed for inflammatory cytokines using flow cytometry. The endpoint was the change in serum cytokine concentrations. Participants were followed for 30 days. Results: Eighty randomized participants (40 RIC and 40 sham) completed the trial. Baseline characteristics according to trial intervention were overall balanced. Despite downward trajectories of all cytokines across hospitalization, we observed no substantial changes in cytokine concentrations after successive days of RIC. Time to clinical improvement was similar in both groups (HR 1.66; 95% CI, 0.938–2.948, p 0.08). Overall RIC did not demonstrate a significant impact on the composite outcome of all-cause death or clinical deterioration (HR 1.19; 95% CI, 0.616–2.295, p = 0.61). Conclusion: RIC did not reduce the hypercytokinaemia induced by COVID-19 or prevent clinical deterioration to critical care. Trial Registration: ClinicalTrials.gov Identifier: NCT04699227

Bio-nanotechnology application in wastewater treatment

The nanoparticles have received high interest in the field of medicine and water purification, however, the nanomaterials produced by chemical and physical methods are considered hazardous, expensive, and leave behind harmful substances to the environment. This chapter aimed to focus on green-synthesized nanoparticles and their medical applications. Moreover, the chapter highlighted the applicability of the metallic nanoparticles (MNPs) in the inactivation of microbial cells due to their high surface and small particle size. Modifying nanomaterials produced by green-methods is safe, inexpensive, and easy. Therefore, the control and modification of nanoparticles and their properties were also discussed