Cell-free Extracts Research Articles

Biosynthesis of selenium nanoparticles using cell-free extract of Xenorhabdus cabanillasii GU480990 and their potential mosquito larvicidal properties against yellow fever mosquito Aedes aegypti

Nanomaterials are successful due to their numerous applications in various domains such as cancer treatment, environmental applications, drug and gene delivery. Selenium is a metalloid element with broad biological activities and low toxicity especially at the nanoscale. Several studies have shown that nanoparticles synthesized from microbial and plant extracts are effective against important pests and pathogens. This study describes the bio fabrication of selenium nanoparticles using cell free extract of Xenorhabdus cabanillasii (XC-SeNPs) and assessed their mosquito larvicidal properties. Crystallographic structure and size of XC-SeNPs were determined with UV-a spectrophotometer, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD), Energy-dispersive X-ray spectroscopy (EDAX), Zeta potential and Transmission electron microscopy (TEM). The significant surface plasmon resonance at 275 nm indicated the synthesis of XC-SeNPs from the pure cell-free extract of X. cabanillasii. The XRD result exhibits the crystalline nature of XC-SeNPs. The Zeta potential analysis confirmed that the surface charge of XC-SeNPs was −24.17 mV. TEM analysis revealed that synthesized XC-SeNPs were monodispersed, spherically shaped, and sized about 80–200 nm range. In addition, the larvicidal potentials of the bio-fabricated XC-SeNPs were assessed against the 4th-instar Ae. aegypti. XC-SeNPs displayed a dose-dependent larvicidal effect; the larval mortality was 13.3 % at the minimum evaluated concentration and increased to 72 % at higher dose treatments. The LC50 and LC90 concentration of XC-SeNPs against mosquito larvae were 79.4 and 722.4 ppm, respectively.

Designing of an extract production protocol for industrial application of cell-free protein synthesis technology: Building from a current best practice to a quality by design approach.

Cell-Free Protein Synthesis (CFPS) has, over the past decade, seen a substantial increase in interest from both academia and industry. Applications range from fundamental research, through high-throughput screening to niche manufacture of therapeutic products. This review/perspective focuses on Quality Control in CFPS. The importance and difficulty of measuring the Raw Material Attributes (RMAs) of whole cell extract, such as constituent protein and metabolite concentrations, and of understanding and controlling these complicated enzymatic reactions is explored, for both centralised and distributed industrial production of biotherapeutics. It is suggested that a robust cell-free extract production process should produce cell extract of consistent quality; however, demonstrating this is challenging without a full understanding of the RMAs and their interaction with reaction conditions and product. Lack of technology transfer and knowledge sharing is identified as a key limiting factor in the development of CFPS. The article draws upon the experiences of industrial process specialists, discussions within the Future Targeted Healthcare Manufacturing Hub Specialist Working Groups and evidence drawn from various sources to identify sources of process variation and to propose an initial guide towards systematisation of CFPS process development and reporting. These proposals include the development of small scale screening tools, consistent reporting of selected process parameters and analytics and application of industrial thinking and manufacturability to protocol development.

Comparative oil extraction from mutt (Myliobatis goodei) liver by enzymatic hydrolysis: free versus immobilized biocatalyst.

The development and fine-tuning of biotechnological processes for fish oil extraction constitute a very important focus to contribute to the development of a food industry based on fish consumption. This work lies in a comparative analysis of the oil extraction yield of Myliobatis goodei livers using free and immobilized enzymes. An immobilized biocatalyst was designed from the cell-free extract of a Bacillus sp. Mcn4. A complete factorial design was used to study the components of the bacterial culture medium and select the condition with the highest titers of extracellular enzymatic activities. Wheat bran had a significant effect on the culture medium composition for enzymatic production. The immobilized biocatalyst was designed by covalent binding of the proteins present in the cocktail retaining a percentage of different types of enzymatic activities (Mult.Enz@MgFe2 O4 ). Among the biocatalyst used, Alcalase® 2.4 L and Purazyme® AS 60 L (free commercial proteases) showed extraction yields of 87.39% and 84.25%, respectively, while Mult.Enz@MgFe2 O4 achieved a better one of 89.97%. The oils obtained did not show significant differences in their physical-chemical properties while regarding the fatty acid content, the oil extracted with Purazyme® AS 60 L showed a comparatively lower proportion of polyunsaturated fatty acids. Our results suggest that the use of by-products of M. goodei is a valid alternative and encourages the use of immobilized multienzyme biocatalysts for the treatment of complex substrates in the fishing industry. © 2023 Society of Chemical Industry.

Harnessing selenium nanoparticles (SeNPs) for enhancing growth and germination, and mitigating oxidative stress in Pisum sativum L.

Selenium, an essential micronutrient for plants and animals, can cause selenium toxicity as an oxyanion or at elevated doses. However, the toxic selenite (SeO32−) oxyanion, can be converted into less harmful elemental nano-selenium (Se0), with various practical applications. This research aimed to investigate two methods for reducing SeO32−: abiotic reduction using cell-free extract from Enterococcus spp. (abiotic-SeNPs) and chemical reduction involving L-ascorbic acid (chemical-SeNPs). Analysis with XPS confirmed the presence of Se0, while FTIR analysis identified surface functional groups on all SeNPs. The study evaluated the effects of SeO32−, abiotic-SeNPs, and chemical-SeNPs at different concentrations on the growth and germination of Pisum sativum L. seeds. SeO32− demonstrated detrimental effects on germination at concentrations of 1 ppm (germination index (GI) = 0.3). Conversely, both abiotic- and chemical-SeNPs had positive impacts on germination, with GI > 120 at 10 ppm. Through the DPPH assay, it was discovered that SeNPs exhibited superior antioxidant capabilities at 80 ppm, achieving over 70% inhibition, compared to SeO32− (less than 20% inhibition), therefore evidencing significant antioxidant properties. This demonstrates that SeNPs have the potential to be utilized as an agricultural fertilizer additive, benefiting seedling germination and development, while also protecting against oxidative stress.

An Enzymatic Cofactor Regeneration System for the in-Vitro Reduction of Isolated C=C Bonds by Geranylgeranyl Reductases.

Cofactor regeneration systems are of major importance for the applicability of oxidoreductases in biocatalysis. Previously, geranylgeranyl reductases have been investigated for the enzymatic reduction of isolated C=C bonds. However, an enzymatic cofactor-regeneration system for in vitro use is lacking. In this work, we report a ferredoxin from the archaea Archaeoglobus fulgidus that regenerates the flavin of the corresponding geranylgeranyl reductase. The proteins were heterologously produced, and the regeneration was coupled to a ferredoxin reductase from Escherichia coli and a glucose dehydrogenase from Bacillus subtilis, thereby enabling the reduction of isolated C=C bonds by purified enzymes. The system was applied in crude, cell-free extracts and gave conversions comparable to those of a previous method using sodium dithionite for cofactor regeneration. Hence, an enzymatic approach to the reduction of isolated C=C bonds can be coupled with common systems for the regeneration of nicotinamide cofactors, thereby opening new perspectives for the application of geranylgeranyl reductases in biocatalysis.

Efficient synthesis of malonyl-CoA by an acyl-CoA synthetase from Streptomyces sp.

Malonyl-CoA is a precursor of fatty acids, polyketides, and bio-based chemicals with potential applications in medicine, antibiotics, and fuels. However, its low intracellular concentration and high cost have led to difficulties in research and production. To develop an efficient method for producing malonyl-CoA, we screened the acyl-CoA synthetase (ACS) gene from Streptomyces sp. using sequence-structure alignment. This protein contains conserved sequences and active sites for malonyl-CoA synthetases. The purified recombinant enzyme ACS was heterologously expressed in Escherichia coli BL21 and characterised. The results showed that it converted the substrates malonate and CoA into malonyl-CoA. Under the optimal conditions, the specific activity of the purified ACS was 32.3 U·mg−1 and the conversion rate reached 98.8%. In addition, when the cell-free extracts were used as catalysts, the highest yield of malonyl-CoA was obtained after 4 h, yielding 24.2 g·L−1 with a conversion rate of 90.3%. After the product was purified and vacuum freeze-dried, a solid powder of malonyl-CoA was obtained. This study characterised and identified a new ACS and optimised the reaction conditions to efficiently synthesise pure malonyl-CoA in vitro in high yield using enzyme-mediated methods.

Mechanisms of Inflammatory Caspase Activation in Sickle Cell Disease

Background: Sickle cell disease (SCD) is an inherited red blood cell disorder that impacts millions of people worldwide. Patients with SCD experience chronic hemolysis and have elevated serum levels of free heme. Free heme activates a variety of inflammatory pathways that contribute to the serious clinical complications of SCD. One of the inflammatory pathways activated by heme is the inflammatory caspase pathway. Our lab previously showed that heme exposure in macrophages leads to the activation of inflammatory caspase proteins caspase-1 and caspase-4. Activation of these proteins by heme leads to IL-1β release and pyroptosis, a form of inflammatory cell death. Patients with SCD have increased circulating IL-1β levels, indicating this pathway is active in patients. In mouse models, inflammatory caspases are required for hemolysis-induced neutrophil recruitment and lethality, indicating these proteins are central to hemolysis-induced inflammation. Despite the key role these proteins play following hemolysis, it remains unknown how heme activates inflammatory caspases. Interestingly, both caspase-1 and caspase-4 are required for heme-induced IL-1β release while caspase-4 alone is required for heme-induced pyroptosis. In fact, pyroptosis likely is required for IL-1β release from the cell. Based on this data, we hypothesized that caspase-4 is the apical step in this pathway and a key druggable target for controlling unchecked inflammation in SCD. Results: We first sought to determine if caspase-4 activation is upstream of caspase-1 activation. To assess this, we used an imaging-based caspase activation reporter assay. Using this system, we found that siRNA knock-down of caspase-4 in primary human macrophages significantly inhibited heme-induced caspase-1 activation (63.8% vs 26.3%, n =4, p = 0.015). This result shows that caspase-4 is upstream of heme-induced caspase-1 activation and confirmed caspase-4 activation is the initial step in this pathway. Next, we sought to determine how caspase-4 activation occurs. Other known activators of caspase-4 induce activation by directly binding to caspase-4 and inducing self-oligomerization. To determine if heme acts in a similar manner, we used a published heme-binding prediction algorithm called HeMoQuest. We found multiple predicted heme-binding sites in caspase-4. The dissociation constants for these binding sites are less than 1 µM, suggesting strong binding affinity. Importantly, there are no predicted heme binding sites in caspase-1, indicating caspase-4 is the only direct heme receptor in this pathway. To validate these binding predictions, we performed molecular docking experiments and confirmed there are multiple heme-binding sites in caspase-4. Specifically, we found two heme-binding regions in the p10 catalytic subunit of caspase-4 comprising 2-4 essential residues. We next confirmed that heme directly interacts with caspase-4 in vitro. Using a pull-down assay with heme conjugated to agarose beads, we observed heme bound to purified caspase-4 and to casapase-4 in macrophage cell-free extracts. Heme did not interact with proteins structurally related to caspase-4 including caspase-1, caspase-7, and caspase-9 and caspase-4 was not pulled down by control agarose. This confirms the specificity of the heme-caspase-4 interaction. To determine if heme binding to caspase-4 results in activation, we performed native PAGE analysis of purified caspase-4 after exposure to heme. Heme induced formation of a caspase-4 oligomer around 200 kDa in size, four-fold larger than the inactive caspase-4 monomer. Other compounds known to induce IL-1β release from cells, like microbial toxin nigericin, did not induce caspase-4 oligomerization, demonstrating the specificity of heme's effect on caspase-4. Conclusions: We have determined that caspase-4 is a direct intracellular heme receptor and that heme exposure leads to caspase-4 activation, which in turn facilitates caspase-1 activation, IL-1β release and cell death. Our future work will elucidate the functional effects of heme binding to caspase-4 in mouse models of sickle cell disease. Caspase-4 is an attractive therapeutic target as it is required for both functional outputs of inflammatory caspase activation, IL-1β release and pyroptosis. Inhibiting heme-caspase-4 interactions would provide a specific way to block the harmful effects of this pathway in patients.

Establishing a versatile toolkit of flux enhanced strains and cell extracts for pathway prototyping

Building and optimizing biosynthetic pathways in engineered cells holds promise to address societal needs in energy, materials, and medicine, but it is often time-consuming. Cell-free synthetic biology has emerged as a powerful tool to accelerate design-build-test-learn cycles for pathway engineering with increased tolerance to toxic compounds. However, most cell-free pathway prototyping to date has been performed in extracts from wildtype cells which often do not have sufficient flux towards the pathways of interest, which can be enhanced by engineering. Here, to address this gap, we create a set of engineered Escherichia coli and Saccharomyces cerevisiae strains rewired via CRISPR-dCas9 to achieve high-flux toward key metabolic precursors; namely, acetyl-CoA, shikimate, triose-phosphate, oxaloacetate, α-ketoglutarate, and glucose-6-phosphate. Cell-free extracts generated from these strains are used for targeted enzyme screening in vitro. As model systems, we assess in vivo and in vitro production of triacetic acid lactone from acetyl-CoA and muconic acid from the shikimate pathway. The need for these platforms is exemplified by the fact that muconic acid cannot be detected in wildtype extracts provided with the same biosynthetic enzymes. We also perform metabolomic comparison to understand biochemical differences between the cellular and cell-free muconic acid synthesis systems (E. coli and S. cerevisiae cells and cell extracts with and without metabolic rewiring). While any given pathway has different interfaces with metabolism, we anticipate that this set of pre-optimized, flux enhanced cell extracts will enable prototyping efforts for new biosynthetic pathways and the discovery of biochemical functions of enzymes.

Effect of Yogurt Addition on the Stability of Anthocyanin during Cold Storage of Strawberry, Raspberry, and Blueberry Smoothies.

The addition of yogurt to fruit smoothies enhances their nutritional value by introducing components not naturally found in fruit products. However, the addition of fermented products can affect the stability of fruit bioactive components in fruits, such as anthocyanins. This study aimed to evaluate the effect of varying yogurt additions (0, 10, 20, and 30%) on the stability of anthocyanins during a 4-week refrigerated storage period. The smoothies were obtained from purees of strawberry, raspberry, and blueberry, combined with apple juice and apple puree. In addition, to elucidate the causes of the observed changes in the smoothies, model studies were conducted using purified anthocyanin extracts obtained from the analyzed fruits. We assessed the effects of pH, hydrogen peroxide concentration, and the addition of cell-free extracts from Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus on changes in anthocyanin content during storage. We found that adding yogurt led to a decrease in anthocyanin stability during the 4-week cold storage period. Specifically, a 30% yogurt addition decreased anthocyanin stability in all tested beverages, while a 20% yogurt addition impacted the strawberry and raspberry smoothies. The degree to which yogurt affected anthocyanin stability was dependent on the source of the raw material. The most notable impact was observed in strawberry smoothies and the least in blueberry smoothies. The variability could be attributed to differences in anthocyanin profiles among the fruits, the chemical composition of the beverages, and the observed difference in the survival rates of lactic acid bacteria. Model studies showed that during the storage of anthocyanin extracts, the addition of hydrogen peroxide and cell-free extract had a significant effect, whereas pH within the examined range (3.0-4.5) did not affect anthocyanin stability.

Biocontrol of Fusarium wilt disease by endophytic Bacillus xiamenensis BS10 isolated from the root nodules of Brassica nigra (mustard plant) and analysis of defence molecular biomarkers in the greenhouse and field trial

Endophytic bacteria possess positive effects on their host plants, including, secondary metabolite induction, disease-resistance improvement, and growth promotion. Bacillus spp. are considered to be an eco-friendly and bio-safe alternative to traditional chemical fungicides. In our study, a collection of 12 endophytic bacteria were isolated from Brassica nigra root nodules and analyzed for plant growth-promoting activities. The endophytic strain BS10 significantly solubilise phosphate (19 ± 1 mm halo zone), siderophore (17 ± 1 mm halo zone), HCN (18 ± 1 halo zone), and indole acetic acid (205.1 ± 2.8 μg/mL) significantly (p < 0.05) than other isolates. The antifungal activity of isolated endophytes was analyzed using a dual-culture screening method. The antifungal activity against Fusarium oxysporum revealed that the strain BS10 exhibited maximum activity against F. oxysporum and the inhibition rate ranged from 20 ± 1 to 70.2 ± 3.5 % after 4 days of treatment and increased after 8 days (36.9 ± 3.5–75.8 ± 3.2 %) among other endophytes. The cell-free extract decreased fungal growth and the inhibition rate varied between 12 % and 47 % (p < 0.05). The results revealed that the fungal infection of the mustard plants with wilt disease causing F. oxysporum significantly reduced the growth of the mustard plant, and root, shoot length, and dry weight compared with the control (p < 0.05). The infected plants treated with endophyte showed an improvement in the growth parameters (p < 0.05). The endophytes-treated plants showed fewer symptoms of rot disease in mustard plants. The chlorophyll content (2.27-fold), soluble leaf protein (1.22-fold), proline (26 ± 2.8 nM/g fresh weight) were improved in the plant leaves treated with endophyte. The biosynthesis of ascorbate peroxidase, phenylalanine ammonia-lyase, and catalase was improved in the endophyte-treated seeds compared with control seeds (p > 0.05). The isolated B. xiamenensis is an important resource to biocontrol Fusarium wilt diseases in the mustard plant.

In vitro DNA repair genomics using XR-seq with Escherichia coli and mammalian cell-free extracts

The XR-seq (eXcision Repair-sequencing) method has been extensively used to map nucleotide excision repair genome-wide in organisms ranging from Escherichia coli to yeast, Drosophila, Arabidopsis, mice, and humans. The basic feature of the method is to capture the excised oligomers carrying DNA damage, sequence them, and align their sequences to the genome. We wished to perform XR-seq in vitro with cell-free extract supplemented with a damaged DNA substrate so as to have greater flexibility in investigating factors that affect nucleotide excision repair in the cellular context [M. J. Smerdon, J. J. Wyrick, S. Delaney, J. Biol. Chem. 299, 105118 (2023)]. We report here the successful use of ultraviolet light-irradiated plasmids as substrates for repair in vitro and in vivo by E. coli and E. coli cell-free extracts and by mammalian cell-free extract. XR-seq analyses demonstrated common excision product length and sequence characteristics in vitro and in vivo for both the bacterial and mammalian systems. This approach is expected to help understand the effects of epigenetics and other cellular factors and conditions on DNA repair.

Nitrification activity in the presence of 2-chlorophenol using whole nitrifying cells and cell-free extracts: batch and SBR assays.

The online version contains supplementary material available at 10.1007/s13205-023-03764-z.

Cell free extract-mediated biogenic synthesis of ZnONPs and their application with kanamycin as a bactericidal combination.

Antimicrobial resistance (AMR) is a main public health issue and a challenge for the scientific community all over the globe. Hence, there is a burning need to build new bactericides that resist the AMR. The ZnONPs were produced by cell free extract of mint (Mentha piperita L.) leaves. Antibiotics that are ineffective against resistant bacteria like Escherichia coli and Staphylococcus aureus were treated. The antibiotics were first screened, and then antibacterial activity was checked by disk diffusion, and MIC of Mp-ZnONPs individually and using Kanamycin (KAN) were determined against these pathogens by broth microdilution method. The synergism between Mp-ZnONPs and KAN was confirmed by checkerboard assay. The MIC showed robust antibacterial activity against the tested pathogens. The combination of KAN and Mp-ZnONPs reduces the MIC of KAN as it efficiently inhibits E. coli's growth, and KAN significantly enhances the antibacterial activity of Mp-ZnONPs. Taken together, Mp-ZnONPs have strong antimicrobial activity, and KAN significantly improves it against the tested pathogens, which would offer an effective, novel, and benign therapeutic methodology to regulate the incidence. The combination of Mp-ZnONPs and KAN would lead to the development of novel bactericides, that could be used in the formulation of pharmaceutical products.

β-Glucosidase activity and antimicrobial properties of potentially probiotic autochthonous lactic cultures.

The demand for lactic acid bacteria products, especially probiotics, has increased. Bacteria that increase polyphenol bioavailability and act as bio preservatives are sought after. This study aims to identify autochthonous lactic acid cultures from EMBRAPA that demonstrate β-glucosidase activity and inhibitory effect on microbial sanitary indicators. Cell-free extracts were obtained by sonicating every 5 s for 40 min. The extracts were mixed with cellobiose and incubated at 50 °C. The reaction was stopped by immersing the tubes in boiling water. The GOD-POD reagent was added for spectrophotometer readings. Antimicrobial activity was tested against reference strains using the agar well diffusion method. Lactic cultures in MRS broth were added to 0.9 cm wells and incubated. The diameter of the inhibition zones was measured to determine the extension of inhibition. Only L. rhamnosus EM1107 displayed extracellular β-glucosidase activity, while all autochthonous strains except L. plantarum CNPC020 demonstrated intracellular activity for this enzyme. L. plantarum CNPC003 had the highest values. On the other hand, L. plantarum CNPC020, similarly to L. mucosae CNPC007, exhibited notable inhibition against sanitary indicators. These two strains significantly differed from the other five autochthonous cultures regarding S. enterica serovar Typhimurium ATCC 14028 inhibition (P < 0.05). However, they did not differ from at least one positive control in terms of inhibition against S. aureus ATCC 25923 and E. coli ATCC 25922 (P > 0.05). Therefore, it is advisable to consider these cultures separately for different technological purposes, such as phenolics metabolism or bio preservative activity. This will facilitate appropriate selection based on each specific property required for the intended product development.

Cell-free fat extract-loaded microneedles attenuate inflammation-induced apoptosis and mitochondrial damage in tendinopathy.

Existing clinical treatments for tendinopathy mainly focus on reducing pain, whereas inhibiting or reversing disease progression remains challenging. Local therapeutic drugs, such as glucocorticoids, cause adverse effects on the metabolism of tendon tissues and injection-related complications. Therefore, new administration modalities for tendinopathy need to be developed. In this study, we designed a hydrogel-based microneedle (MN) system for the long-term transdermal delivery of our novel biological cell-free fat extract (CEFFE) to treat tendinopathies. We found that CEFFE-loaded MNs (CEFFE-MNs) had good biosafety and inhibited lipopolysaccharide (LPS)-induced apoptosis and matrix degradation in Achilles tendon cells of rats. The Achilles tendons of rats returned to their maximum mechanical strength after applying CEFFE-MNs. The administration of CEFFE-MNs had better anti-apoptosis and tendon repair-promoting effects than CEFEF injections in vivo. Transcriptome sequencing indicated that the anti-apoptosis effect of CEFFE-MNs was highly related to tumor necrosis factor (TNF) signaling. CEFFE-MNs inhibited the expression of TNF, TNF receptor 1, and downstream nuclear factor-kappa B signaling. Additionally, CEFFE-MNs rescued LPS-induced mitochondrial dynamics in tendon cells via the TNF-Drp1 axis. Our study reports a novel CEFFE-MN system that exhibits long-term anti-inflammatory and anti-apoptotic effects, suggesting it as a new treatment route for tendinopathy with broad clinical translation prospects.

Specific antioxidant enzymes are involved in the freeze-thawing response of industrial baker's yeasts.

In this study, the biochemical basis of resistance to slow freezing and thawing (F-T) stress was explored in two baker yeast industrial strains of Saccharomyces cerevisiae that presented differential tolerance to freezing in order to be in the frozen bakery industry. Strain Y8, used commercially in sweet baking doughs, exhibited greater stress tolerance than Y9, a strain employed in regular doughs. Survival of Y8 was higher than that of Y9 (30%vs 12%) after F-T or other reactive oxygen species (ROS) inducing stresses compared to their non-stressed controls. The superior F-T tolerance of Y8 was related to its lower ROS accumulation capacity, determined by fluorometry in cell-free extracts and in vivo, by fluorescence microscopy upon F-T, being Y8 ROS accumulation 2-fold lower than that of Y9. That, in turn, could be positively associated with Y8's higher constitutive activities of cytosolic catalase (CAT) and superoxide dismutase by a significant activation (25%) of Y8 CAT after F-T. That would complement the protective effects of other protectant molecules like trehalose, present at high concentration in this strain.

CfOmics: a cell-free multi-Omics database for diseases.

Liquid biopsy has emerged as a promising non-invasive approach for detecting, monitoring diseases, and predicting their recurrence. However, the effective utilization of liquid biopsy data to identify reliable biomarkers for various cancers and other diseases requires further exploration. Here, we present cfOmics, a web-accessible database (https://cfomics.ncRNAlab.org/) that integrates comprehensive multi-omics liquid biopsy data, including cfDNA, cfRNA based on next-generation sequencing, and proteome, metabolome based on mass-spectrometry data. As the first multi-omics database in the field, cfOmics encompasses a total of 17 distinct data types and 13 specimen variations across 69 disease conditions, with a collection of 11345 samples. Moreover, cfOmics includes reported potential biomarkers for reference. To facilitate effective analysis and visualization of multi-omics data, cfOmics offers powerful functionalities to its users. These functionalities include browsing, profile visualization, the Integrative Genomic Viewer, and correlation analysis, all centered around genes, microbes, or end-motifs. The primary objective of cfOmics is to assist researchers in the field of liquid biopsy by providing comprehensive multi-omics data. This enables them to explore cell-free data and extract profound insights that can significantly impact disease diagnosis, treatment monitoring, and management.

Neoteric Biofilms Applied to Enhance the Safety Characteristics of Ras Cheese during Ripening.

The milk's natural flora, or the starter, can preserve cheesemaking and allow for microbial competition. This investigation aimed to improve cheese safety and assess its characteristics using probiotic cell pellets (LCP) or cell-free extracts (CFS). Cheese samples were collected from different areas to investigate the current contamination situation. Six CFSs of probiotics were assessed as antifungal against toxigenic fungi using liquid and solid media and their aflatoxin reduction impact. The most effective CFS was chosen for cheese coating in nanoemulsion. Coated cheese with CFS, LCP, and LCP-CFS was assessed against control for changes in chemical composition, ripening indications, rheological properties, and microbiology. Results showed significant contamination levels in the collected samples, and toxic fungi were present. Lactobacillus rhamnosus CFS has aflatoxins reducibility in liquid media. During cheese ripening, uncoated cheese showed higher fat, protein, salt content, soluble nitrogen, total volatile fatty acids, tyrosine, and tryptophan contents than coated samples, except for LCP-coating treatment. Cheese rheology indicated that coating treatments had the lowest hardness, cohesiveness, gumminess, chewiness, and springiness compared to uncoated cheese. Uncoated cheese had the highest yeast and mold counts compared to the treated ones. The LCP-CFS-coated cheese showed no Aspergillus cells for up to 40 days. Uncoated Ras cheese recorded slightly lower flavor, body, texture, and appearance scores than coated cheeses. In conclusion, coating cheese with L. rhamnosus nanoemulsion has antifungal and antiaflatoxigenic properties, even for LCP, CFS, and CFS-LCP, which could extend cheese shelf life.

In Search of Complementary Extraction Methods for Comprehensive Coverage of the Escherichia coli Metabolome.

Escherichia coli is an invaluable research tool for many fields of biology, in particular for the production of recombinant enzymes. However, the activity of many such recombinant enzymes cannot be determined using standard biochemical assays, as often, the relevant substrates are not known, or the products produced are not detectable. Today, the biochemical footprints of such unknown enzyme activities can be revealed via the analysis of the metabolomes of the recombinant E. coli clones in which they are expressed, using sensitive technologies such as mass spectrometry. However, before any metabolites can be identified, it is necessary to achieve as high a coverage of the potential metabolites present within E. coli as possible. We have therefore analyzed a wide range of different extraction methods against the cell free extracts of various recombinant E. coli clones. The results were analyzed to determine the minimum number of extractions that achieved high recovery and coverage of metabolites. Two methods were selected for further analysis due to their ability to produce not only high numbers of ions, but also wide mass coverage and a high degree of complementarity. One extraction method uses acetonitrile and water, in a 4:1 ratio, which is then dried down and reconstituted in the chromatography running buffer prior to injection onto the chromatography column, and the other extraction method uses a combination of methanol, water and chloroform, in a 3:1:1 ratio, which is injected directly onto the chromatography column. These two extraction methods were shown to be complementary to each other, as regards the respective metabolites extracted, and to cover a large range of metabolites.

An-ecofriendly synthesis of silver nanoparticles using microalga Desmodesmus protuberans and evaluation of their antimicrobial activity

Due to the numerous uses for nanoparticles, nanoparticle production is currently a particularly fascinating topic of research. Since no harmful chemicals are employed during the biological process, it is thought to be the safest and cleanest method. The strong ability of algae to absorb metals and decrease metal ions makes algal synthesis of Ag-NPs particularly intriguing. The current work concentrated on the green synthesis of silver nanoparticles (AgNPs) using the microalgae Desmodesmus protuberans. The preparation of nanoparticles was confirmed by the observation of the color change of the mixture of silver nitrate, after the addition of the cell free algal extract, from bright green to reddish yellow. The biosynthesis of AgNPs was further con􀂿rmed by scanning electron microscopic and energy dispersive X-ray analysis. Furthermore, silver nanoparticles demonstrated a signi􀂿cant antimicrobial activity against the pathogenic microorganisms.