Incubation Time Research Articles

Dispersal of microbes from grassland fire smoke to soils.

Wildland fire is increasingly recognized as a driver of bioaerosol emissions, but the effects that smoke-emitted microbes have on the diversity and community assembly patterns of the habitats where they are deposited remain unknown. In this study, we examined whether microbes aerosolized by biomass burning smoke detectably impact the composition and function of soil sinks using lab-based mesocosm experiments. Soils either containing the native microbial community or presterilized by γ-irradiation were inundated with various doses of smoke from native tallgrass prairie grasses. Smoke-inundated, γ-irradiated soils exhibited significantly higher respiration rates than both smoke-inundated, native soils and γ-irradiated soils exposed to ambient air only. Microbial communities in γ-irradiated soils were significantly different between smoke-treated and control soils, which supports the hypothesis that wildland fire smoke can act as a dispersal agent. Community compositions differed based on smoke dose, incubation time, and soil type. Concentrations of phosphate and microbial biomass carbon and nitrogen together with pH were significant predictors of community composition. Source tracking analysis attributed smoke as contributing nearly 30% of the taxa found in smoke-inundated, γ-irradiated soils, suggesting smoke may play a role in the recovery of microbial communities in similar damaged soils. Our findings demonstrate that short-distance microbial dispersal by biomass burning smoke can influence the assembly processes of microbial communities in soils and has implications for a broad range of fields including agriculture, restoration, plant disease, and biodiversity.

Effect of Ultrasonic Waves on Aspire Biodiesel and Comparison of Its Properties with Petroleum Diesel

This study aimed to determine the effect of ultrasonic sound waves on modifying the chemical structure of biodiesel to bring its physical properties closer to petroleum diesel. In this direction, safflower oil was selected because its fatty acid composition is similar to fatty acid esters of petroleum diesel and is a sustainable source. Refined safflower oil, the free fatty acid content of which was determined, was reacted with methanol under NaOH catalyst to perform the transesterification reaction. After biodiesel production, samples were incubated in an ultrasonic bath for 60, 120, and 180 minutes. FTIR, density, free fatty acid content, flash point, viscosity, and cloud point analyses investigated the effect of incubation times on biodiesel's chemical structure and properties. FTIR spectra showed that ultrasonic sound waves partially decomposed fatty acid methyl esters and increased the number of volatile components in biodiesel. The flash point of biodiesel has been associated with a decrease of 89°C, and the low flash point is expected to increase fuel efficiency. Kinematic viscosity values were measured in the 3.4583-3.5115 mm²/s range, and density values were measured in the 0.8820-0.8872 g/ml range. These values show that biodiesel complies with national and international standards. As a result, the ultrasonic bath process applied to biodiesel showed a similar result to chemical modification methods by affecting the structure of fatty acid chains. Thus, it brought the physical properties of biodiesel closer to petroleum diesel. It is seen that this method is a more efficient alternative for biodiesel production because it does not use additional chemicals, and the process is faster. In conclusion, by increasing the production of the drought-resistant safflower plant, sustainable energy resources will be contributed, while its waste can be evaluated as animal feed. Ultrasonicated safflower biodiesel can also be used as an efficient, environmentally and mechanically friendly alternative fuel source.

Microstructure and Precipitation of a Cu–Ni Alloyed Medium‐Mn Duplex Low‐Density Steel Prepared by Near‐Rapid Solidification

A Cu–Ni‐alloyed medium‐Mn duplex low‐density steel with a composition of Fe–9Mn–5.5Al–0.3C–3Ni–1.5Cu (wt%) is prepared by near‐rapid solidification. The microstructure features of the alloy under as‐cast, cold‐rolling, and heat treatments are studied. The effect of Cu‐ and Ni‐alloying elements on the microstructure and precipitates is discussed. In the results, it is shown that martensite and nanoscale precipitates are found in the microstructure of near‐rapid solidification. The enrichment of Cu is found at the phase boundaries of austenite and ferrite. A large number of annealing twins are present in the microstructure of cold‐rolled and annealed alloy. In the first principles and thermodynamic calculations, it is shown that Ni element is capable to shorten the nucleation incubation time of Cu precipitates. In addition, the interface segregation of Ni and Al elements reduces the adhesion work of Cu particles with the matrix. Both Cu and Ni elements increase the stacking fault energy of the alloy, which is increased from 37.3 to 44.2 mJ m−2. The contributions of precipitation strengthening and twinning strengthening to the strength of the alloy are 72 and 86 MPa, respectively, while the dominant contribution of dislocation hardening to the strength is 326 MPa.

Mitochondria-Targeted Gold Biometallization for Photoacoustically Visualized Photothermal Cancer Therapy.

Subcellular biomineralization systems with cellular intervention functions have shown great potential in cancer theranostic applications. However, the lack of subcellular specificity, high ion concentrations, and long incubation time required for biomineralization still limit its in vivo therapeutic efficacy. Herein, we report a mitochondria-targeted polymer-gold complex (TPPM-Au) to realize mitochondrial biometallization, which involves analogous mechanisms during biomineralization, for cancer treatment in vivo. The TPP-containing TPPM-Au delivered more Au3+ selectively into the mitochondria of cancer cells than normal cells, rapidly mineralizing to gold nanoparticles (GNPs) and consuming a large amount of the antioxidant glutathione (GSH). The formed GNPs can further continue consuming GSH with the atomic economy by forming Au-S with GSH, which further results in the accumulation of reactive oxygen species (ROS), thereby impairing mitochondrial function and inducing cell apoptosis. More importantly, TPPM-Au is capable of having superior tumor-penetrating, excellent photothermal and photoacoustic properties, endowing it with the ability to inhibit tumor growth through spatiotemporally monitorable mitochondria-targeted biometallization and photothermal therapy. The mitochondria-targeted gold biometallization theranostic platform provides insight into the application of subcellularly targeted biometallization or biomineralization in cancer therapy.

Production of Sugars and Ethanol from Acid–Alkaline-Pretreated Agave sisalana Residue

Drylands in Brazil have been exploring sisal (Agave sisalana) as an essential source of income. However, the solid residues generated because of this activity still need suitable destinations; therefore, research has been carried out to transform them into added-value products. Therefore, the present study evaluated the potential of sisal or agave solid residue as a precursor feedstock for second-generation ethanol production. Acid and acid–alkaline pretreatments were carried out on sisal residues to enrich the biomass with cellulose and maximize enzymatic digestibility. Second-generation ethanol production was carried out using Semi-simultaneous saccharification and fermentation (SSSF). Regardless of catalyst dosage and incubation time, oxalic acid pretreatments generated samples with a similar chemical composition to those pretreated with sulfuric acid. However, samples pretreated with oxalic acid showed lower enzymatic digestibility. Samples pretreated with oxalic acid and sodium hydroxide obtained 14.28 g/L of glucose and cellulose conversion of 79.1% (at 5% solids), while 21.49 g/L glucose and 91.2% of cellulose conversion were obtained in the hydrolysis of pretreated samples with sulfuric acid and sodium hydroxide combined pretreatments. The pretreatment sequence efficiently reduced cellulase dosage from 20 to 10 FPU/g without compromising sugar release. SSSF achieved maximum production of 40 g/L ethanol and 43% ethanol conversion using 30% solids and gradually adding biomass and cellulases.

Fast and on-site detection of fenthion in rice using core-shell Au@Ag nanoparticles and a portable Raman spectrometer

The residue of pesticide fenthion is a common issue in food safety, and how to conveniently detect it is a challenge in the food industry. Therefore, combined with a portable Raman spectrometer, a surface enhanced Raman spectroscopy (SERS) method based on a high-performance substrate Au@AgNPs had been designed for the rapid and sensitive detection of fenthion. Au@AgNPs can be induced to generate more hot spots to improve SERS signals because of their core-shell structure. The quantitative detection of fenthion was achieved under the optimal conditions: pH 7.0, 10−3 M CaCl2, 70 % Au@AgNPs concentration, and 4 min of incubation time. The results showed that the method had a wider detection range of 0.1–100 mg/L, a low detection limit (LOD) of 0.036 mg/L, excellent anti-interference ability, and reproducibility. In addition, the method was successfully validated with the actual sample rice. Therefore, the method had the advantages of being fast, on-site, and easy to use, which is useful for promoting its application.

Development of Novel Biocomposites with Antimicrobial-Activity-Based Magnesium-Doped Hydroxyapatite with Amoxicillin.

Background/Objectives: A biocomposite based on magnesium-doped hydroxyapatite and enriched with amoxicillin (MgHApOx) was synthesized using the coprecipitation method and is presented here for the first time. Methods: The stability of MgHAp and MgHApOx suspensions was evaluated by ultrasound measurements. The structure of the synthesized MgHAp and MgHApOx was examined with X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The crystalline structure was determined by X-ray diffraction. The FTIR data were collected in the range of 4000-400 cm-1. The morphology of the nanoparticles was evaluated by scanning electron microscopy (SEM). Furthermore, the biocompatible properties of MgHAp, MgHApOx and amoxicillin (Ox) suspensions were assessed using human fetal osteoblastic cells (hFOB 1.19 cell line). The antimicrobial properties of the MgHAp, MgHApOx and Ox suspension nanoparticles were assessed using the standard reference microbial strains Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Candida albicans ATCC 10231. Results: X-ray studies have shown that the biocomposite retains the characteristics of HAp and amoxicillin. The SEM assessment exhibited that the apatite contains particles at nanometric scale with acicular flakes morphology. The XRD and SEM results exhibited crystalline nanoparticles. The average crystallite size calculated from XRD analysis increased from 15.31 nm for MgHAp to 17.79 nm in the case of the MgHApOx sample. The energy-dispersive X-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS) analysis highlighted the presence of the constituent elements of MgHAp and amoxicillin. Moreover, XPS confirmed the substitution of Ca2+ ions with Mg2+ and the presence of amoxicillin constituents in the MgHAp lattice. The results of the in vitro antimicrobial assay demonstrated that MgHAp, MgHApOx and Ox suspensions exhibited good antimicrobial activity against the tested microbial strains. The results showed that the antimicrobial activity of the samples was influenced by the presence of the antibiotic and also by the incubation time. Conclusions: The findings from the biological assays indicate that MgHAp and MgHApOx are promising candidates for the development of new biocompatible and antimicrobial agents for biomedical applications.

Enhanced Removal of Chlorpyrifos, Cu(II), Pb(II), and Iodine from Aqueous Solutions Using Ficus Nitida and Date Palm Biochars

This study explores the adsorption efficiency of biochar derived from palm trees and Ficus nitida for the removal of various contaminants, including Cu(II), Pb(II), iodine, and chlorpyrifos from aqueous solutions. Biochar was prepared using a two-step pyrolysis process for date palm biochar and single-step pyrolysis for Ficus nitida biochar. Characterization techniques such as SEM, EDX, and FTIR revealed a significant surface area and a variety of functional groups in both types of biochar, essential for effective adsorption. The date palm biochar exhibited superior adsorption capacities for Cu(II) and Pb(II) ions, achieving efficiencies up to 99.9% and 100%, respectively, due to its high content of oxygen-containing functional groups that facilitated strong complexation and ion exchange mechanisms. Conversely, Ficus nitida biochar demonstrated a higher adsorption capacity for iodine, reaching 68% adsorption compared to 39.7% for date palm biochar, owing to its greater surface area and microporosity. In the case of chlorpyrifos, Ficus nitida biochar again outperformed date palm biochar, achieving a maximum adsorption efficiency of 87% after 24 h of incubation, compared to 50.8% for date palm biochar. The study also examines the effect of incubation time on adsorption efficiency, showing that the adsorption of chlorpyrifos by date palm biochar increased significantly with time, reaching a maximum of 62.9% after 48 h, with no further improvement beyond 12 h. These results highlight the importance of biochar characteristics, such as surface area, pore structure, and functional groups, in determining adsorption efficiency. The findings suggest that optimizing pyrolysis conditions and surface modifications could further enhance the performance of biochar as a cost-effective and sustainable solution for water purification and environmental remediation.

The assessment of the phototoxic potential of drugs forming complexes with melanin - Screening in vitro studies using normal skin cells with varying pigmentation irradiated by a sunlight simulator

Phototoxic reactions are among the most common skin-related adverse effects induced by drugs. It is believed that the binding of chemicals to melanin biopolymers is a significant factor influencing skin toxicity. The formation of drug-melanin complexes can lead to the accumulation of drugs or their photodegradation products in pigmented cells, potentially affecting phototoxic reactions. Current procedures for assessing the phototoxic potential of drugs are based on tests using immortalized mouse fibroblasts.This study aimed to assess the phototoxic potential of selected drugs that form complexes with melanin (chloroquine, chlorpromazine, doxycycline) using human melanocytes with varying degrees of pigmentation. Parallel research was conducted on human dermal fibroblasts. To induce phototoxicity, cell cultures were irradiated using a sunlight simulator (5 J/cm2 for UVA spectrum). To account for the process of drug accumulation, two experimental models with different incubation times of cells with drugs before irradiation were used. The photo-irritation factor (PIF) was calculated based on NRU and WST-1 screening tests. Additionally, cell viability was examined cytometrically, and analyses of the cell cycle and reduced glutathione levels were conducted.The results indicated that drugs binding with melanin exhibited different levels of cytotoxicity and phototoxicity towards fibroblasts and melanocytes. These observed differences impact the values of PIF, potentially complicating the interpretation of the studies. Additional analyses, such as examining cell subpopulations in the sub-G1 phase and determining the level of reduced glutathione, can enhance the assessment of the phototoxicity of drugs on pigmented cells.

Label-free and sensitive detection of Citrus Bark Cracking Viroid in hop using Ti3C2Tx MXene-modified genosensor

Since the discovery of Citrus bark cracking viroid (CBCVd) in hops in 2007, affected hop-growing countries such as Slovenia and Germany have been actively pursuing efficient and easily accessible diagnostic tools that could contribute to the early detection of CBCVd in the field. In the early stages of CBCVd infection, typical symptoms or subtle signs of spread may not be evident. Detection becomes feasible only when the plant begins to display symptoms. Unfortunately, there is currently no treatment available, which requires the removal of infected plants as the only viable solution. However, this approach leads to significant economic losses on a large scale. This work demonstrates the development and study of a sensitive, selective, and label-free impedimetric genosensor for the detection of CBCVd in total RNA hop samples. The genosensor is based on a supporting glassy carbon electrode modified with streptavidin-agarose beads, which serve as an effective immobilization layer for a biotinylated single-stranded DNA capture probe. The integration of a 2D-layered Ti3C2Tx MXene into the sensing architecture resulted in a significantly improved electroanalytical performance of the genosensor. Several fabrication and operational parameters were optimized, such as the streptavidin-agarose beads deposition time, capture probe immobilization time and concentration, and sample incubation time. The optimized genosensor exhibited a limit of detection of only 0.5 fg μL⁻¹ (5.5 fmol L−1) in combination with a one-hour incubation with the denatured total RNA hop extract, thus eliminating the need for an additional and laborious amplification step.

NUTRIENT CONTENT, IN VITRO GAS PRODUCTION AND POST INCUBATION PARAMETERS OF INDIGENOUS BROWSE FODDERS IN DIFFERENT AGRO-ECOLOGICAL ZONES OF NORTHWEST ETHIOPIA

<p><strong>Background: </strong>In Ethiopia indigenous browse species have the potential to serve as a sustainable supplement for poor quality feeds and enhance ruminant livestock production. <strong>Objective:</strong> To analyze the nutrient content, in vitro gas production, and post incubation parameters of indigenous browse species in northwestern Ethiopia. <strong>Methodology: </strong>Eight indigenous browse fodder trees were collected from each agroecological zone for this experiment. Leaf and pod samples of the fodder trees were collected, dried and ground for laboratory analysis. Analysis of variance was carried out for nutrient content, in vitro gas, and methane production of the samples within agroecological zones using standard analytical procedures. The statistical design used to analyze the data was Completely Randomized Design (CRD). <strong> Results:</strong> The highest CP content was recorded from <em>Vernonia</em><em> amygdalina </em>(223.4 g/kg DM)<em>,</em> <em>Dodonaea viscosa </em>(207.7 g/kg DM), and <em>Acacia abyssinica </em>(216.0 g/kg DM) in the highland, midland and lowland agro-ecology respectively. In lowland area, NDF, ADF and ADL contents were highest for <em>Albezia amara</em> leaves and lowest for <em>Acacia brevispica</em> respectively. <em>V. amygdalina</em>, <em>Stereospermum kunthianum</em> and <em>Ficus vasta</em> had the highest gas volume at 24-hour incubation time in high, mid and lowland, respectively. The estimated organic matter digestibility, metabolizable energy and short chain fatty acid at 24 hour of incubation was the highest for <em>V. amygdalina</em>, <em>S kunthianum</em> and <em>F. vasta</em> in high, mid and lowland, respectively. <em>Ficus sycomorus</em> and <em>Myrica salicifolia</em> had the lowest methane production among the species in the low, mid and highland study areas, respectively. <strong>Implications: </strong>The present study provides a valuable resource for selecting suitable feed options for livestock in different agroecological zones. High CP and digestible browse species like <em>V. amygdalina,</em> <em>S. kunthianum</em>, and <em>F. vasta</em> could be prioritized for livestock feed. Low methane-producing species like <em>F. sycomorus</em> and <em>M. salicifolia</em> could be incorporated into diets to reduce enteric methane emissions. <strong>Conclusion</strong>: Based on their nutrient composition and in vitro gas production potential, these feed resources are of better quality to supplement grazing livestock during the seasons of critical feed shortage as the main feed resources are either limited in availability or lower in nutrient composition and digestibility in the study areas.</p>

X-ray crystal structure of proliferating cell nuclear antigen 1 from Aeropyrum pernix.

Proliferating cell nuclear antigen (PCNA) plays a critical role in DNA replication by enhancing the activity of various proteins involved in replication. Inthis study, the crystal structure of ApePCNA1, one of three PCNAs from thethermophilic archaeon Aeropyrum pernix, was elucidated. ApePCNA1 was cloned and expressed in Escherichia coli and the protein was purified and crystallized. The resulting crystal structure determined at 2.00 Å resolution revealed that ApePCNA1 does not form a trimeric ring, unlike PCNAs from other domains of life. It has unique structural features, including a long interdomain-connecting loop and a PIP-box-like sequence at the N-terminus, indicating potential interactions with other proteins. These findings provide insights into the functional mechanisms of PCNAs in archaea and their evolutionary conservation across different domains of life. A modified medium and protocol were used to express recombinant protein containing the lac operon. The expression of the target protein increased and the total incubation time decreased when using this system compared with those of previous expression protocols.

Thermodynamic–kinetic calculations and dilatometric verification of the martensitic and bainitic transformations in 4% medium-Mn steel

AbstractThe present study investigated the theoretical and experimental phase transitions phenomena during continuous cooling and isothermal holding above and below Ms temperature in 4 mass. % Mn medium-Mn steel. The thermodynamic–kinetic calculations were performed using JMatPro software, and phase transformations were recorded using a BÄHR high-resolution DIL805A/D dilatometer. The research covered continuous cooling rates from 60 to 0.05 °C s–1 and isothermal holding temperatures in a range between 420 °C and 230 °C. The issues related to both modelling and dilatometric methodology were discussed. The CCT and TTT diagrams were prepared on the basis of dilatometry and compared with the results of light microscopy and hardness tests. The alloy containing about 4 mass.% Mn and 0.22% Mo exhibited very high hardenability as only continuous cooling with rates lower than 1 °C s–1 allowed the bainitic transformation to be initiated. The bainitic transformation is accelerated after passing the Ms temperature. Both the incubation time and the time needed to complete the transformation were significantly reduced (the incubation time from 100 s to below 1 s and the completion time from over 4000 s to below 1000 s). The obtained microstructures were homogeneous and refined.

Screening and Optimization of Pathogenic Strains of Pseudomonas sp. for Cholinesterase Enzyme Activity

The identification and optimization of pathogenic strains of Pseudomonas sp. with significant Cholinesterase enzyme activity has promising implications for environmental and medical applications. This study aimed to screen various Pseudomonas strains to identify those exhibiting high cholinesterase activity and to optimize the conditions for maximal enzyme production. Multiple Pseudomonas strains were isolated and characterized from diverse clinical samples, employing selective media and biochemical assays to determine cholinesterase activity. The strains with maximum enzyme activity were further subjected to optimization experiments, including variations in growth conditions such as Temperature, pH and Nutrient availability. The findings revealed sample no.39 among the collected 50 samples had significant enzyme activity of 0.19151 IU is obtained when choline chloride was 0.3999%, Ammonium Chloride was 0.01M, pH was 7.0, Temperature was 27°C and Time of incubation was 24hrs. These results not only advance our understanding of Pseudomonas sp. enzyme capabilities but also offer potential applications in bioremediation, biosensor development and pharmaceutical industries. Future research will focus on the molecular characterization of the cholinesterase enzyme and its applications in various industrial processes.

Extraction and evaluation of chitosan as an insecticide against saw-toothed grain beetle, <i>Oryzaephilus surinamensis</i> L. (Coleoptera: Silvanidae)

Rice samples infested with saw-toothed grain beetles have been collected from a local market in the city of Kirkuk/Iraq. The study was conducted in Kirkuk during the period 15 December 2023 to 25 April 2024, at College of Pure Sciences at the University of Kirkuk, Iraq. Chitosan was utilised in the study to examine its toxic effects at three different concentrations (0.5, 1.0, 1.5 ppm) on the life cycle of the saw-toothed grain beetle, focusing on oviposition rate, duration of larval and pupal stages, and mortality rate of its adults. The chemical demonstrated a substantial effect in lowering the number of eggs laid as the concentration increased, with the control treatment registering the highest oviposition rate of 314.14 eggs compared to 209.31, 117.03, and 61.12 eggs for chitosan concentrations of 0.5, 1.0, and 1.5 ppm, respectively. The shortest egg incubation period recorded was 8.17 days, Incubation times for chitosan treatments increased significantly to 9.66, 11.69, and 14.00 days for concentrations of 0.5, 1.0, and 1.5 ppm, respectively, demonstrating an inverse association with concentration levels. Furthermore, the emergence rate of beetles decreased as concentration increased. The emergence counts for the chitosan treatments at concentrations of 0.5, 1.0, and 1.5 ppm dropped to 133.11, 55.69, and 30.12 beetles, respectively, with the control treatment having the highest average emergence of 289.43 beetles. The larval stage duration showed a direct proportionality with chitosan concentrations, with the 1.5 ppm concentration marking the longest larval duration at 22.00 days, significantly surpassing all other concentrations. The control treatment recorded the shortest duration at 13.56 days, whereas 0.5 and 1.0 ppm concentrations resulted in duration of 17.19 and 19.66 days, respectively. For the pupal stage, significant differences were observed with increasing chitosan concentration; the control treatment displayed the shortest pupal duration at 4.33 days. A direct relationship was found between the concentrations and pupal stage duration, reducing the period to 6.00, 7.59, and 9.07 days for concentrations of 0.5, 1.0, and 1.5 ppm, respectively. Chitosan exhibited significant differences from the second day of the experiment, as the mortality rate increased with concentration and over time. The concentration of 1.5 ppm showed the highest mortality rate at 98.62% after 16 days, whereas the control treatment recorded the lowest rate at 33.17%. Based on the results of the current study, chitosan can be utilized as an effective pesticide for controlling stored-product pests, particularly the saw-toothed grain beetle.

6651 A Traceable Clinical Free Thyroxine Assay Based on Equilibrium Dialysis ID-LC/MS/MS

Abstract Disclosure: D. Spector: None. L. Zhang: None. A. Ribera: None. A. Doty: None. C. Tse: None. O. Sugahara: None. N. Vazquez: None. U. Danilenko: None. H.W. Vesper: None. Accurate diagnosis and effective treatment of thyroid diseases rely on reliable thyroid function tests. Serum free thyroxine (FT4) immunoassays (IAs) are most commonly used for FT4 measurements in clinical practice. However, FT4 IAs have not been standardized with large variations between different assay platforms. A reference measurement procedure (RMP) for FT4 based on well-defined equilibrium dialysis (ED) of serum has been established for CDC standardization program to support the standardization of clinical assays for patient care. In the current study, we aim to develop a routine clinical FT4 assay based on ED-LC/MS/MS that is traceable to the RMP. ED-LC/MS/MS analysis was performed using a commercially available micro-ED plate. FT4 in dialysate was extracted with a 96-well C18 SPE plate. FT4 in the samples was quantitated by using LC/MS/MS in positive ion mode. The assay was calibrated with the certified primary reference material. By using RMP as a reference, we compared the analysis of 40 single donor sera between the ED-LC/MS/MS assay and FT4 IA on Roche e401platform. ED-LC/MS/MS assay could resolve T4, T3, reverse triiodothyronine (rT3), and other interferences in the samples with C18 chromatography in 4 min. The linear range of the routine FT4 assay was from 0.5-100 pg/mL covering clinically relevant FT4 concentrations including hypo-, hyperthyroid patient samples. The intra and inter-run CV% of the assay in 20 days was 3.72 and 5.25%, respectively. The effect of minor changes to the method conditions was studied. It was determined that serum/buffer ratios from 1:1 to 1:2 in the ED inserts, speeds of shaker for ED, and ED incubation time, did not influence the FT4 measurement. However, elevated ED temperature at 38°C could result in a more than 10% increase in FT4 values. A microplate format for the procedures of ED and SPE is amiable to the application of an automation system which significantly improves the throughput of the procedure. Method comparison showed good agreement between ED-LC/MS/MS assay and RMP in Deming regression analysis with a slope close to 1, while there was significant bias of IA from RMP. ED-LC/MS/MS assay was also applied for pregnancy serum samples with minimal difference on the FT4 measurement from RMP. We have developed a routine clinical FT4 assay that is traceable to RMP. The accuracy, precision, and sensitivity of the assay are suitable for high-throughput FT4 measurements in clinical laboratories and large epidemiologic studies. We demonstrate that the developed method can provide accurate FT4 measurements in patients including pregnant women. Presentation: 6/3/2024

Microbially Induced Calcium Carbonate Precipitation UsingLysinibacillussp.: A Ureolytic Bacteriumfrom Uttarakhand for Soil Stabilization.

Microbially induced calcium carbonate precipitation (MICP) is a soil remediation method that has emerged as a viable and long-term solution for enhancing soil mechanical qualities. The technique of MICP that has been extensively researched is urea hydrolysis, which occurs naturally in the environment by urease-producing bacteria as part of their fundamental metabolic processes. The objectives of the current study include screening and identifying native ureolytic bacteria from soil in Uttarakhand, optimizing growth factors for increased urease activity, and calcite precipitation by the bacteria using response surface methodology. Additionally, it was assessed how well the isolated bacteria in the medium biomineralized when using synthetic media and cheaper alternatives such as cow urine and eggshell as sources of urea and Ca2+, respectively. The isolated strain identified as Lysinibacillus sp. was found to be the very active strain after soil samples were screened for ureolytic bacteria. It was discovered that optimization studies with values of pH 8, urea concentration (0.8M), inoculum concentration (3%), and incubation time (48h) yielded a higher activity of 33.7 U/mL (threefold increase), and a higher calcium carbonate precipitation (enzyme activity: 10.96 U/mL, pH: 8.92, soluble Ca2⁺: 25.53mM and insoluble Ca2⁺: 0.856g). The calcite precipitation in broth media supplemented with ready-made substrates and alternative sources demonstrated a similar result of increased pH and ammonia release. Thus, the current study successfully paves the way for several possibilities to stabilize the slopy soils prone to landslides and erosion in Uttarakhand and pinpoint an economic approach through biomineralization.

How to isolate channel-forming membrane proteins using the E. coli expression system.

The recombinant expression, isolation and characterization of pore-forming proteins is one of the most commonly used strategies for understanding the permeability properties of the biological membrane into which they are embedded. This protocol describes how to quantify the expression of your protein of interest and use this information to optimize its production using the Escherichia coli strain BL21Gold(de3)ΔABCF. It explains with a step-by-step approach how to separate the bacterial compartments according to their solubility and how to extract your protein of interest in its native conformation using detergent solutions. Finally, it describes how to improve its purity via ion-exchange chromatography and insert the purified porins into outer membrane vesicles, from which they can be copurified. The protocol is simpler and less empirical than those described for most channel-forming membrane proteins and also provides a solid foundation for the isolation of soluble proteins. Several parameters can be optimized on a case-by-case basis: expression time and temperature, concentration of the inducer, nature and concentration of the detergent, incubation time and temperature, pH and ionic strength of the purification buffers. This protocol is effective with prokaryotic channel-forming membrane proteins and can be employed for the production of pore-forming proteins from chloroplasts, mitochondria or eukaryotes in general. With minor optimization, this protocol can be adapted for the isolation of receptors, carrier, pumps or any other membrane-active proteins.

Fast preparing bioelectrode with conductive bioink for nitrite detection in high sensitivity and stability

Electrochemically active biofilms (EABs) for nitrite detection have high specificity, rapid response, operational simplicity, and extended lifespan advantages. However, their scale production remains challenging due to time-consuming and uniform preparation. In this study, a novel approach was proposed to fast fabricate an EAB biosensor with a synthetic biofilm electrode for nitrite detection. The biofilm electrode was prepared by coating bioinks with varying conductive materials onto the surface of the graphite sheets, showing short incubation time and good reproducibility. Incorporating conductive materials into the bioinks remarkably enhanced the maximum voltage of the first cycle of bioelectrode incubation, with an increase of up to 633% for carbon nanofibers. The nitrite reduction current was amplified by a factor of 2.97, due to the enhancement of extracellular electron transfer (EET). The developed nitrite biosensor exhibited a detection range of 0.1–15 mg NO2--N L−1, with a high sensitivity of 610.8 μA mM−1 cm−2, and a stabilization operation time of at least 280 cycles. This study not only provided valuable insights into conductive materials for synthetic biofilms but also presented a practical approach for the rapid preparation, scale production, and optimization of highly sensitive and stable EAB sensors.

Electrochemical immunosensor based on gold nanoparticles for the detection of creatine kinase as a cardiac marker

This study presents the development of an electrochemical immunosensor utilizing gold nanoparticles for the detection of creatine kinase (CK), a crucial cardiac marker. Gold nanoparticles with an average diameter of 18.3 ± 2.1 nm were synthesized and characterized, exhibiting a characteristic surface plasmon resonance peak at 520 nm. The construction of the immunosensor was observed through cyclic voltammetry and electrochemical impedance spectroscopy, showing notable alterations in electron transfer rates during each step of the assembly process. Optimal performance was achieved at pH 7.4, with an antibody concentration of 100 μg/mL and an incubation time of 45 minutes. The sensor demonstrated a sensitivity of 152.6 Ω/(ng/mL) and maintained its response in the presence of common interfering substances, with maximum signal deviation of 7.2 %. Testing with spiked human serum samples yielded recovery rates between 96.5 % and 103.8 %. Comparison with ELISA using clinical samples further validated the sensor's accuracy. This AuNP-based immunosensor offers a promising platform for rapid and sensitive CK detection in cardiac health assessment.