Cell-free Extracts Research Articles

Pro-vasculogenic Fibers by PDA-Mediated Surface Functionalization Using Cell-Free Fat Extract (CEFFE).

Formation of adequate vascular network within engineered three-dimensional (3D) tissue substitutes postimplantation remains a major challenge for the success of biomaterials-based tissue regeneration. To better mimic the in vivo angiogenic and vasculogenic processes, nowadays increasing attention is given to the strategy of functionalizing biomaterial scaffolds with multiple bioactive agents. Aimed at engineering electrospun biomimicking fibers with pro-vasculogenic capability, this study was proposed to functionalize electrospun fibers of polycaprolactone/gelatin (PCL/GT) by cell-free fat extract (CEFFE or FE), a newly emerging natural "cocktail" of cytokines and growth factors extracted from human adipose tissue. This was achieved by having the electrospun PCL/GT fiber surface coated with polydopamine (PDA) followed by PDA-mediated immobilization of FE to generate the pro-vasculogenic fibers of FE-PDA@PCL/GT. It was found that the PDA-coated fibrous mat of PCL/GT exhibited a high FE-loading efficiency (∼90%) and enabled the FE to be released in a highly sustained manner. The engineered FE-PDA@PCL/GT fibers possess improved cytocompatibility, as evidenced by the enhanced cellular proliferation, migration, and RNA and protein expressions (e.g., CD31, vWF, VE-cadherin) in the human umbilical vein endothelial cells (huvECs) used. Most importantly, the FE-PDA@PCL/GT fibrous scaffolds were found to enormously stimulate tube formation in vitro, microvascular development in the in ovo chick chorioallantoic membrane (CAM) assay, and vascularization of 3D construct in a rat subcutaneous embedding model. This study highlights the potential of currently engineered pro-vasculogenic fibers as a versatile platform for engineering vascularized biomaterial constructs for functional tissue regeneration.

Molecular Targets of the 5-Amido-Carboxamide Bumped Kinase Inhibitor BKI-1748 in Cryptosporidium parvum and HCT-8 Host Cells.

Cryptosporidium parvum is an apicomplexan parasite causing persistent diarrhea in humans and animals. Issuing from target-based drug development, calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs), with excellent efficacies in vitro and in vivo have been generated. Some BKIs including BKI-1748 share a core structure with similarities to the first-generation antiprotozoal drug quinine, which is known to exert notorious side effects. Unlike quinine, BKI-1748 rapidly interfered with C. parvum proliferation in the human colon tumor (HCT) cell line HCT-8 cells and caused dramatic effects on the parasite ultrastructure. To identify putative BKI targets in C. parvum and in host cells, we performed differential affinity chromatography with cell-free extracts from non-infected and infected HCT-8 cells using BKI-1748 and quinine epoxy-activated sepharose columns followed by mass spectrometry. C. parvum proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from both BKI-1748 and quinine columns. However, no C. parvum proteins could be identified binding exclusively to BKI-1748. In contrast, 25 BKI-1748-specific binding proteins originating from HCT-8 cells were detected. Moreover, 29 C. parvum and 224 host cell proteins were identified in both BKI-1748 as well as in quinine eluates. In both C. parvum and host cells, the largest subset of binding proteins was involved in RNA binding and modification, with a focus on ribosomal proteins and proteins involved in RNA splicing. These findings extend previous results, showing that BKI-1748 interacts with putative targets involved in common, essential pathways such as translation and RNA processing.

A new and promiscuous α/β hydrolase from Acinetobacter tandoii DSM 14970 T inactivates the mycotoxin ochratoxin A

The presence of ochratoxin A (OTA) in food and feed represents a serious concern since it raises severe health implications. Bacterial strains of the Acinetobacter genus hydrolyse the amide bond of OTA yielding non-toxic OTα and L-β-phenylalanine; in particular, the carboxypeptidase PJ15_1540 from Acinetobacter sp. neg1 has been identified as an OTA-degrading enzyme. Here, we describe the ability to transform OTA of cell-free protein extracts from Acinetobacter tandoii DSM 14970 T, a strain isolated from sludge plants, and also report on the finding of a new and promiscuous α/β hydrolase (ABH), with close homologs highly distributed within the Acinetobacter genus. ABH from A. tandoii (AtABH) exhibited amidase activity against OTA and OTB mycotoxins, as well as against several carboxypeptidase substrates. The predicted structure of AtABH reveals an α/β hydrolase core composed of a parallel, six-stranded β-sheet, with a large cap domain similar to the marine esterase EprEst. Further biochemical analyses of AtABH reveal that it is an efficient esterase with a similar specificity profile as EprEst. Molecular docking studies rendered a consistent OTA-binding mode. We proposed a potential procedure for preparing new OTA-degrading enzymes starting from promiscuous α/β hydrolases based on our results.Key points• AtABH is a promiscuous αβ hydrolase with both esterase and amidohydrolase activities• AtABH hydrolyses the amide bond of ochratoxin A rendering nontoxic OTα• Promiscuous αβ hydrolases are a possible source of new OTA-degrading enzymes

Immobilization of aldoxime dehydratases on metal affinity resins and use of the immobilized catalysts for the synthesis of nitriles important in fragrance industry

Nitriles have a wide range of uses as building blocks, solvents, and alternative fuels, but also as intermediates and components of flavors and fragrances. The enzymatic synthesis of nitriles by aldoxime dehydratase (Oxd) is an emerging process with significant advantages over conventional approaches. Here we focus on the immobilization of His-tagged Oxds on metal affinity resins, an approach that has not been used previously for these enzymes. The potential of the immobilized Oxd was demonstrated for the synthesis of phenylacetonitrile (PAN) and E-cinnamonitrile, compounds applicable in the fragrance industry. A comparison of Talon and Ni-NTA resins showed that Ni-NTA with its higher binding capacity was more suitable for the immobilization of Oxd. Immobilized Oxds were prepared from purified enzymes (OxdFv from Fusarium vanettenii and OxdBr1 from Bradyrhizobium sp.) or the corresponding cell-free extracts. The immobilization of cell-free extracts reduced time and cost of the catalyst production. The immobilized OxdBr1 was superior in terms of recyclability (22 cycles) in the synthesis of PAN from 15 mM E/Z-phenylacetaldoxime at pH 7.0 and 30 °C (100% conversion, 61% isolated yield after product purification). The volumetric and catalyst productivity was 10.5 g/L/h and 48.3 g/g of immobilized protein, respectively.

Biosynthesis of Silver Nanoparticles From Marine Actinobacterium Micromonospora sp. and Their Bioactive Potential.

Background The biosynthesis of nanoparticles represents a rapid, environmentally friendly, cost-effective, and straightforward technology. This approach allows for the production of nanoparticles with a wide range of chemical compositions, sizes, shapes, high uniformity, and scalability. One of the principal advantages of biogenic nanoparticles is their water solubility and compatibility with biological systems. Biologically synthesized nanoparticles have demonstrated superior efficiency compared to conventionally synthesized particles. Among biosynthesis, microbial-mediated biosynthesis is a promising one that has a selectively reducing ability on specific metal ions through electron transfer. Objectives Evaluation of antimicrobial and antioxidant activity of silver nanoparticle synthesized by actinobacteria Micromonospora sp. which is isolated from marine environment. Materials and methods In this study, actinobacteria were isolated from the marine sediment using the spread plate method. The isolates were identified based on morphological observation, cell wall amino acids, sugar analysis, and micromorphological analysis. The silver nanoparticle synthesis from microbes and their inhibition against clinical pathogens have been evaluated by the disc diffusion method.Antioxidant efficiency was evaluated in termsof total antioxidant activity through ammonium molybdenum assay. Results A total of five isolates were isolated from the sediment sample. The cell-free extract of MBIT-MSA4 can synthesize silver nanoparticles that have potential antimicrobial activity against the clinical pathogens Streptococcus mutans at a zone of inhibition 6 mm, 10 mm inhibition zone of Klebsiella pneumonia,and 8 mm zone of inhibition of Staphylococcus aureus. Also, it has significant antioxidant activity up to 73% of free radical inhibition. Conclusion Marine microbial-mediated biosynthesized silver nanoparticles have potential antimicrobial activity against S.mutansand methicillin-resistantStaphylococcus aureus(MRSA) and inhibit the oxidation process through antioxidant activity. Thisenhanced efficient biosynthesisednanoparticle has significantly reduced the concentration of free radicals caused by pathogens.

Antiproliferative and Antitelomerase Effects of Silymarin on Human Colorectal and Hepatocellular Carcinoma Cells.

Silymarin, a widely-used hepatoprotective agent, has shown antitumor properties in both in vitro and animal studies. Currently, there is limited knowledge regarding silymarin's antitelomerase effects on human colorectal cancer and hepatocyte carcinoma cells. In this study, we investigated the antiproliferative and antitelomerase effects of silymarin on four human colorectal cancer and HepG2 hepatocyte carcinoma cell lines. The cell viability and telomerase activity were assessed using MTT and the telomerase repeat amplification protocol assay, respectively. We also investigated the effects of silymarin on the expression of human telomerase reverse transcriptase and its promoter methylation in HepG2 cells by real-time RT-PCR and methylation-specific PCR, respectively. Silymarin treatment inhibited cell proliferation and telomerase activity in all cancer cells. After 24 h of treatment, silymarin exhibited IC50 values ranging from 19 - 56.3 µg/mL against these cancer cells. A 30-min treatment with silymarin at the IC50 concentration effectively inhibited telomerase activity in cell-free extracts of both colorectal cancer and hepatocyte carcinoma cells. Treatment of HepG2 cells with 10 and 30 µg/mL of silymarin for 48 h resulted in a decrease in human telomerase reverse transcriptase expression to 75 and 35% of the level observed in the untreated control (p < 0.01), respectively. Treatment with silymarin (10, 30, and 60 µg/mL) for 48 h did not affect human telomerase reverse transcriptase promoter methylation in HepG2 cells. In conclusion, our findings suggest that silymarin inhibits cancer cell growth by directly inhibiting telomerase activity and downregulating its human telomerase reverse transcriptase catalytic subunit. However, silymarin did not affect human telomerase reverse transcriptase promoter methylation at the concentrations of 10 - 60 µg/mL used in this study.

Production of geraniol and geranic acid: High value compounds of citral biosynthetic pathway from lemongrass oil using Aspergillus niger AUD-C2 as catalyst

Microbial biotransformation of lemongrass oil to geraniol and geranic acid, high value compounds of citral biosynthetic pathway has been reported. Biotransformation studies were carried out using a fungus Aspergillus niger strain AUD-C2 isolated from the rhizospheric soil of lemongrass, as catalyst. Three different methodologies using culture broth, spore suspension and cell-free extract, have been utilized for biotransformation and successfully demonstrated at litre scale in bioreactors. Overall 55% geraniol and 75% geranic acid were obtained by biotransformation of lemongrass oil in 48 h using spore suspension and cell free extract, respectively.

Cell-free supernatant crude extracts of mold-competing bacteria protect peanut crops against yellow mold disease caused by Aspergillus flavus AF1

Bacillus sp. 16060 (Bacillus subtilis group), Serratia marcescens SE1, and S. marcescens SE2, isolated from malted barley and peanut seedlings with yellow molds, inhibited the growth of Aspergillus flavus AF1 on various culture media and peanut seeds. Ethyl acetate extracts from these bacteria, cultured in Nutrient Broth (NB) and Peptone Glycerol Broth (PGB) with A. flavus AF1 biomass as an inducer, exhibited fungicidal properties. The Minimum Fungicidal Concentration was 100 μg/ml for strain 16060 and 250 μg/ml for strains SE1 and SE2. UHPLC-ESI-QTOF/MS and GNPS analysis revealed many unsaturated fatty acids in the crude extract of Bacillus sp. 16060, whereas polyketides were primarily identified as metabolites from two Serratia marcescens strains. All three crude extracts contained cyclic lipopeptide of the icosalide family. These extracts, with biosurfactant properties, effectively reduced yellow mold Disease incidence on peanut seeds from 93.3% to 65.0% after seed treatment. The Disease severity index for treated seeds ranged between 0.29 and 0.39, compared to 0.67 for untreated seeds. The Disease control efficacy of strain 16060's crude extract matched commercial fungicides, outperforming those of strains SE1 and SE2. All extracts effectively protected peanut seedlings from yellow mold disease, promoting vigorous growth. There was a 2.96–3.25 fold rise in Germination percentage, 4.61–4.80 fold in Vigor index, and 2.45 fold in biomass relative to the control. Hence, antifungal crude extracts derived from the mold-competing bacteria Bacillus sp. 16060, S. marcescens SE1, and S. marcescens SE2 may serve as multicomponent bacterial fungicides for peanut cultivation, providing protection against phytopathogenic mold.

Extracellular synthesis of silver nanoparticle using yeast extracts: antibacterial and seed priming applicationss.

The evolution and rapid spread of multidrug-resistant (MDR) bacterial pathogens have become a major concern for human health and demand the development of alternative antimicrobial agents to combat this emergent threat. Conventional intracellular methods for producing metal nanoparticles (NPs) using whole-cell microorganisms have limitations, including binding of NPs to cellular components, potential product loss, and environmental contamination. In contrast, this study introduces a green, extracellular, and sustainable methodology for the bio-materialization of silver NPs (AgNPs) using renewable resource cell-free yeast extract. These extracts serve as a sustainable, biogenic route for both reducing the metal precursor and stabilizing the surface of AgNPs. This method offers several advantages such as cost-effectiveness, environment-friendliness, ease of synthesis, and scalability. HR-TEM imaging of the biosynthesized AgNPs revealed an isotropic growth route, resulting in an average size of about ~ 18nm and shapes ranging from spherical to oval. Further characterization by FTIR and XPS results revealed various functional groups, including carboxyl, hydroxyl, and amide contribute to enhanced colloidal stability. AgNPs exhibited potent antibacterial activity against tested MDR strains, showing particularly high efficacy against Gram-negative bacteria. These findings suggest their potential role in developing alternative treatments to address the growing threat of antimicrobial resistance. Additionally, seed priming experiments demonstrated that pre-sowing treatment with AgNPs improves both the germination rate and survival of Sorghum jowar and Zea mays seedlings. KEY POINTS: •Yeast extract enables efficient, cost-effective, and eco-friendly AgNP synthesis. •Biosynthesized AgNPs showed strong antibacterial activity against MDR bacteria. •AgNPs boost seed germination and protect against seed-borne diseases.

Staurosporine-producing Streptomyces sp. strain 11 × 1 cell-free culture filtrates control diseases caused by the oomycete plant pathogens Pythium myriotylum and Phytophthora sojae

ABSTRACT Oomycetes are devastating soil-borne plant pathogens, and cause numerous losses on diverse crops worldwide. In this study, our aim was to identify actinomycetes with antimicrobial activity towards oomycetes from the rhizosphere of soybean. Strong growth inhibition of P. myriotylum was found in dual culture with a strain called 11 × 1, and also from using the 11 × 1 cell-free culture filtrate and EtOAc extract. The P. myriotylum growth-inhibiting activity of the 11 × 1 culture filtrates was stable at high temperatures, and acidic and alkaline conditions. The 11 × 1 strain was identified as a Streptomyces strain with the highest 16S rDNA sequence identity to S. scabiei, and phylogenomic analysis also showed clustering with S. scabiei strains. Applying the 11 × 1 culture filtrates to ginger and soybean leaves inhibited the necrosis caused by P. myriotylum and P. sojae, respectively. Pot assay experiments showed a strong control effect of the 11 × 1 culture filtrate against P. myriotylum-caused soft-rot disease of ginger, and against P. sojae caused root-rot of soybean. Analysis of the biosynthetic gene clusters from the genome of 11 × 1 predicted the production of staurosporine, and staurosporine was subsequently identified from LC-MS analysis of the 11 × 1 extract. The concentrations of staurosporine quantified in the 11 × 1 extracts were in the range of the staurosporine minimum inhibitory concentrations for P. myriotylum, supporting the contribution of staurosporine to the biocontrol effect of 11 × 1. The use of 11 × 1 culture filtrate to control both ginger and soybean diseases could be beneficial in an intercropping system of ginger and the leguminous nitrogen-fixing soybean.

Characteristics of Lactic Acid Bacteria as Potential Probiotic Starters and Their Effects on the Quality of Fermented Sausages.

The aim of this study was to explore the potential of commercial lactic acid bacteria (LAB) as probiotic starters in fermented sausages. We initially investigated the growth activity, acid production capability, and tolerance to fermentation conditions of Lactobacillus sakei, Lactiplantibacillus plantarum, and Pediococcus pentosaceus. All three LAB strains proved viable as starters for fermented sausages. Subsequently, we explored their potential as probiotics based on their antibacterial and antioxidant capabilities. L. plantarum exhibited stronger inhibition against Escherichia coli and Staphylococcus aureus. All three strains displayed antioxidant abilities, with cell-free supernatants showing a higher antioxidant activity compared to intact cells and cell-free extracts. Moreover, the activities of superoxide dismutase, glutathione peroxidase, and catalase were stronger in the cell-free supernatant, cell-free extract, and intact cell, respectively. Finally, we individually and collectively inoculated these three LAB strains into sausages to investigate their impact on quality during the fermentation process. External starters significantly reduced pH, thiobarbituric acid reactive substances, and sodium nitrite levels. The improvements in color and texture had positive effects, with the L. plantarum inoculation achieving higher sensory scores. Overall, all three LAB strains show promise as probiotic fermentation starters in sausage production.

Antifungal Activity Enhancement of Cell-Free Streptomyces griseus Extract Obtained by Fermentation with Magnetic Manganese Ferrite Nanoparticles.

The present study aims to obtain manganese ferrite nanoparticles functionalized with chitosan (C-MNP) or ethylenediamine (E-MNP) by coprecipitation and polyol one-step methods, characterize their interaction with S. griseus demonstrating cell immobilization, and evaluate the antimicrobial activity of the free cell extracts obtained from immobilized S. griseus fermentation in the presence of different concentrations of MNP. The adsorption isotherms were analyzed mathematically using Langmuir and Freundlich models. The highest coefficient of determination (R2) for the S. griseus cell adsorption isotherm with C-MNP was observed with a linear function of the Langmuir model. The adsorption isotherm of S. griseus cells with E-MNP was better fitted to the Freundlich model. Cell immobilization by adsorption on magnetic nanoparticles was demonstrated in both cases. Different concentrations of C-MNP and E-MNP were used in fermentations to prepare cell-free extracts with antifungal activity. The best results were obtained with E-MNP, with a 91.5% inhibition of radial fungal growth. Magnetic nanoparticles offer potential applications in different fields and easy biomass separation.

Annexin A5 Derived from Cell-free Fat Extract Attenuates Osteoarthritis via Macrophage Regulation.

Osteoarthritis (OA) is a challenging degenerative joint disease to manage. Previous research has indicated that cell-free fat extract (CEFFE) may hold potential for OA treatment. This study investigated the role of Annexin A5 (AnxA5) within CEFFE in regulating macrophage polarization and protecting chondrocytes. In vitro experiments demonstrated that AnxA5 effectively inhibited M1 macrophage polarization by facilitating toll-like receptor (TLR) 4 internalization and lysosomal degradation through calcium-dependent endocytosis. This process decreased TLR4 expression, suppressed pro-inflammatory mediator release, and reduced the production of reactive oxygen species. Furthermore, AnxA5 displayed protective effects against chondrocyte necrosis and apoptosis. In vivo, studies revealed that intra-articular administration of AnxA5 ameliorated pain symptoms in a monosodium iodoacetate-induced osteoarthritis rat model. Histological analyses indicated a decrease in synovial inflammation and mitigation of cartilage damage following AnxA5 treatment. These results underscored the potential of AnxA5 as a therapeutic option for OA due to its capacity to regulate macrophage polarization and maintain chondrocyte viability. Further investigation into the specific mechanisms and clinical applications of AnxA5 may help improve the management of OA.

Characterization of the Endophytic Bacillus subtilis KRS015 Strain for Its Biocontrol Efficacy Against Verticillium dahliae.

Endophytes play important roles in promoting plant growth and controlling plant diseases. Verticillium wilt is a vascular wilt disease caused by Verticillium dahliae, a widely distributed soilborne pathogen that causes significant economic losses on cotton each year. In this study, an endophyte KRS015, isolated from the seed of the Verticillium wilt-resistant Gossypium hirsutum 'Zhongzhimian No. 2', was identified as Bacillus subtilis by morphological, phylogenetic, physiological, and biochemical analyses. The volatile organic compounds (VOCs) produced by KRS015 or its cell-free fermentation extract had significant antagonistic effects on various pathogenic fungi, including V. dahliae. KRS015 reduced Verticillium wilt index and colonization of V. dahliae in treated cotton seedlings significantly; the disease reduction rate was ∼62%. KRS015 also promoted plant growth, potentially mediated by the growth-related cotton genes GhACL5 and GhCPD-3. The cell-free fermentation extract of KRS015 triggered a hypersensitivity response, including reactive oxygen species (ROS) and expression of resistance-related plant genes. VOCs from KRS015 also inhibited germination of conidia and the mycelial growth of V. dahliae, and were mediated by growth and development-related genes such as VdHapX, VdMcm1, Vdpf, and Vel1. These results suggest that KRS015 is a potential agent for controlling Verticillium wilt and promoting growth of cotton.

Studying Translesion DNA Synthesis Using Xenopus In Vitro Systems.

Cell-free extracts derived from Xenopus eggs have been widely used to decipher molecular pathways involved in several cellular processes including DNA synthesis, the DNA damage response, and genome integrity maintenance. We set out assays using Xenopus cell-free extracts to study translesion DNA synthesis (TLS), a branch of the DNA damage tolerance pathway that allows replication of damaged DNA. Using this system, we were able to recapitulate TLS activities that occur naturally in vivo during early embryogenesis. This chapter describes protocols to detect chromatin-bound TLS factors by western blotting and immunofluorescence microscopy upon induction of DNA damage by UV irradiation, monitor TLS-dependent mutagenesis, and perform proteomic screening.

Addition of α-1,3-glucan-binding domains to α-1,3-glucanase Agn1p from Schizosaccharomyces pombe enhances hydrolytic activity of insoluble α-1,3-glucan.

The glycoside hydrolase (GH) 71 α-1,3-glucanase (Agn1p) from Schizosaccharomyces pombe consists of an N-terminal signal sequence and a catalytic domain. Meanwhile, the GH87 α-1,3-glucanase (Agl-KA) from Bacillus circulans KA-304 consists of an N-terminal signal sequence, a first discoidin domain (DS1), a carbohydrate-binding module family 6 (CBM6), a threonine and proline repeat linker (TP), a second discoidin domain (DS2), an uncharacterized domain, and a catalytic domain. DS1, CBM6, and DS2 exhibit α-1,3-glucan binding activity. This study involved genetically fusing TP, DS1, CBM6, TP, and DS2 to the C-terminus of Agn1p, generating the fusion enzyme Agn1p-DCD. The fusion enzyme was then expressed in Escherichia coli and purified from the cell-free extract. Agn1p-DCD and Agn1p exhibited similar characteristics, such as optimal pH, optimal temperature, pH stability, and thermostability. Insoluble α-1,3-glucan (1%) hydrolyzing assay showed that Agn1p-DCD and Agn1p released approximately 7.6 and 5.0 mM of reducing sugars, respectively, after 48 h of reaction. Kinetic analysis and an α-1,3-glucan binding assay indicated that the addition of DS1, CBM6, and DS2 enhanced the affinity of Agn1p for α-1,3-glucan. Moreover, Agn1p-DCD contributed to enhancing the fungal growth inhibition activity when combined with a mixture of GH19 chitinase and GH16 β-1,3-glucanase.

Measuring Molecular Diffusion in Self-Organizing Xenopus Extracts by Fluorescence Correlation Spectroscopy.

The cytoplasm is densely packed with macromolecules and organelles, displaying viscoelastic properties at various scales. How biochemical reactions function efficiently enough in a seemingly jammed environment remains elusive. Cell-free Xenopus laevis extracts represent a powerful system for investigating the biochemistry and biophysics of living systems. Here we present a protocol for characterizing macromolecular diffusion in self-organizing cytoplasmic extracts using fluorescence correlation spectroscopy (FCS), which measures the motions on a distance scale of ~200nm. The method can also be used to characterize diffusion in the cytoplasm as it progresses through different phases of the cell cycle.

Measuring protein stoichiometry with single-molecule imaging in Xenopus egg extracts.

In human cells, DNA double-strand breaks are rapidly bound by the highly abundant non-homologous end joining (NHEJ) factor Ku70/Ku80 (Ku). Cellular imaging and structural data revealed a single Ku molecule is bound to a free DNA end and yet the mechanism regulating Ku remains unclear. Here, we describe how to utilize the cell-free Xenopus laevis egg extract system in conjunction with single-molecule microscopy to investigate regulation of Ku stoichiometry during non-homologous end joining. Egg extract is an excellent model system to study DNA repair as it contains the soluble proteome including core and accessory NHEJ factors, and efficiently repairs double-strand breaks in an NHEJ-dependent manner. To examine the Ku stoichiometry in the extract system, we developed a single-molecule photobleaching assay, which reports on the number of stable associated Ku molecules by monitoring the intensity of fluorescently labeled Ku molecules bound to double-stranded DNA over time. Photobleaching is distinguishable as step decreases in fluorescence intensity and the number of photobleaching events indicate fluorophore stoichiometry. In this paper we describe sample preparation, experimental methodology, and data analysis to discern Ku stoichiometry and the regulatory mechanism controlling its loading. These approaches can be readily adopted to determine stoichiometry of molecular factors within other macromolecular complexes.

Detection of Metabolic Gene of Interest (MGOI) in WGS of Microbacterium barkeri (LMA4)

This communication reports about detection of metabolically active genes of interest cell free extract of actinomycetal isolates from Pond soil. The strain LMA4 previously identified as Microbacterium barkeri, based on biotyping as well as 16 s r RNA sequencing, was observed with moderate bioactivity against test strains of Staphylococcus aureus and Escherichia coli. The Actinomycetal strain was Gram positive, aerial profuse branching bearing rounded spores. The whole genome sequencing was carried using Next Generation Sequencing tools and techniques. The FASTA sequence received from sequencing farm had been subjected to BLASTn analysis and aligned with sequences of databse. The sequence similarity was checked considering percentage of identity, as inferred from online portal. The whole genome sequence of LMA4 had inferred about elution of antibiotic producing genes namely [i] Thiazole synthase protein, [ii] Indole-3-glycerol phosphate synthase Protein, [iii] Antibiotic synthesis MbtH protein, [iv] Antibiotic biosynthesis monooxygenase protein, [v] class II terpene cyclase protein and [vi] YcaO-like family protein in the sequence profile. The genes were identified, ORFs were predicted and more over conserved domain was spotted. The genes were structurally represented using modelling approaches and more so ligand based (target oriented) molecular simulation study had predicted affirmative interactions between receptor and ligand molecule. The receptors were WGS detected gene products, and the ligands were Sortase of Staphylococcus aureus and D-ala-ala ligase of Escherichia coli. The study may pave a way towards extracting the metabolites of interest and implementing as natural cost effective drugs.

In vitro translation in yeast extract to study interactions with cytosolic chaperones.

Saccharomyces cerevisiae, a well-established model organism, serves as a valuable tool for unraveling various eukaryotic cellular processes. Its utility extends to studying protein folding and interactions with chaperones, as it demonstrates a capability to produce and process proteins in a manner closely resembling that of higher eukaryotes. To gain insights into the events taking place following protein synthesis in the cytosol, an in vitro translation system is essential-one that mirrors in vivo processes yet allows for easy manipulation. To address this need, multiple protocols for preparation of cell-free translation lysates from S. cerevisiae have been documented. This chapter introduces an optimized and modified in vitro pull-down approach following protein translation in yeast cell-free extract. The advantages of this system in investigating the interactions of newly synthesized mitochondrial proteins with cytosolic chaperones are described. This procedure exploits the dual advantages of yeast cell-free lysates, serving as both a protein synthesis tool, and a reservoir for cytosolic factors and chaperones. In summary, the depicted approach provides a versatile platform that deepens our understanding of the early cytosolic events in the biogenesis of nascent proteins before reaching their ultimate organelle.