Cell-free Extracts Research Articles

Autologous Cell-Free Fat Extract: A Novel Approach for Infraorbital Rejuvenation-A Pilot Study.

CEFFE (Cell-free fat extract) treatment for periocular fine lines requires thorough clinical evaluation to determine its efficacy and safety in enhancing skin quality. The research enrolled 10 healthy female participants aged 31-58, focusing on skin texture, elasticity, and barrier function. CEFFE treatment demonstrated significant benefits, with notable improvements observed as early as 3 months posttreatment, which continued throughout the 12-month follow-up period. Objective assessments revealed reductions in SEr% and SEw%, indicative of reduced skin roughness and wrinkles, particularly pronounced after the third month of treatment. Enhanced skin elasticity, as indicated by improvements in R2%, R5%, and R7%, was observed, with the most significant enhancements noted at the 6-month follow-up. Furthermore, TEWL decreased consistently, highlighting CEFFE's potential in maintaining the skin's barrier function and moisture retention. High patient satisfaction levels, with 70% expressing satisfaction ranging from satisfied to very satisfied, underscored CEFFE's clinical significance. CEFFE demonstrates potential as an effective and safe intervention for addressing periocular fine lines, providing a solution for fine lines while ensuring skin health (ChiCTR1900024329).

Engineered Biocorona on microplastics as a toxicity mitigation strategy in marine environment: Experiments with a marine crustacean Artemia salina

The marine environment has become a major sink for microplastics (MPs) wastes. When MPs interact with biological macromolecules, the biocorona forms on their surface, which can alter their biological reactivity and toxicity. In this study, we investigated the impact of biocorona formation on the toxicity of aminated (NH2) and carboxylated (COOH) polystyrene MPs towards the marine crustacean Artemia salina. Biocoronated MPs were prepared using cell-free extracts (CFEs) from microalgae Chlorella sp. (phytoplankton) and the brine shrimp Artemia salina (zooplankton). The results revealed that biocorona formation effectively reduced the toxicity of MPs. Pristine NH2-MPs exhibited higher reactive oxygen species production (ROS) (182%) compared to COOH-MPs (162%) in Artemia salina. Notably, NH2-MPs coronated with brine shrimp CFE exhibited a substantial reduction in ROS production (127%) than those coronated with algal CFE, with COOH-MPs showing a similar trend (120%). Biocorona formation also significantly decreased malondialdehyde (MDA) levels and antioxidant activity compared to pristine MPs. Molecular docking and dynamics simulations demonstrated a strong binding between polystyrene and acetylcholinesterase (AChE). In vitro studies indicated that pristine NH2-MPs exhibited more reduction in AChE activity (84%) compared to COOH-MPs (95%). However, no significant reduction in AChE activity was observed upon exposure to MPs coronated with either algal or brine shrimp cell-free extracts. Independent action modeling indicated an antagonistic interaction for MPs coronated with both the CFEs. Pearson correlation and cluster heatmap analysis suggested that the toxicity reduction in Artemia salina might be driven by decreased oxidative stress followed by the corona formation. Overall, this study provides valuable insights into the potential of biomolecules from phytoplankton and zooplankton to reduce MPs toxicity in Artemia salina, while highlighting their role in modulating the toxicity of other marine pollutants.

The potential correlations between cell-free extracts from Rhodobacter sphaeroides grown under low-oxygen conditions and volatile organic compounds in Chinese-style sausage

Limited research has explored the use of Rhodobacter sphaeroides cell-free extracts (RCFE) in meat processing. To examine the potential application of RCFE in improving the flavor quality of Chinese-style sausage, in this study, we investigated the effects and mechanisms of RCFE grown under low-oxygen conditions on the flavor development of Chinese-style sausage, using GC–MS and 4D label-free proteomics. The GC–MS analysis detected 60 volatile organic compounds, with significant increases in acids, esters, and alcohols following the addition of RCFE (p < 0.01). Fifteen differential flavor compounds were identified as potential biomarkers to distinguish sausages. From a total of 2689 proteins, 364 differentially expressed proteins were identified (p < 0.05, |Log2FC| > 1, and VIP > 1,) in RCFE grown under low- and high-oxygen conditions. KEGG pathway analysis suggested that the RCFE grown under low-oxygen conditions may enhance alcohol and acid levels by upregulating the expression of related enzymes, which subsequently increases ester levels in the sausage.

Preparation of a novel enzyme-modified cheddar cheese: Molecular mechanism of cheese flavor compensation by synergistic action of cell-free extracts and enzyme systems

A novel enzyme-modified cheddar cheese was prepared and the molecular mechanism of cheese flavor compensation by synergistic action of cell-free extracts and enzyme systems was investigated. By comparing five different protease-peptidase combinations, the group of neutral protease and flavor protease was found to increase the total leucine, valine, and isoleucine content (17.056 ± 0.136 g/kg) and the soluble nitrogen content was up to the level of a 12-month-matured cheese. Molecular docking and molecular dynamics simulations demonstrated their mode of action on four monomeric caseins. Adding a cell-free extract resulted in volatile flavor substances in the enzyme-modified cheese that were closest to those in the 12-month-matured cheese. This might be due to the flavor compensation effect of the conversion of leucine to 3-methylbutyraldehyde by transaminases and decarboxylases, and the conversion of 3-methylbutyric acid to 3-methylbutyraldehyde by ketoacid dehydrogenase and aldehyde dehydrogenase. This is essential for the enzyme modified cheddar cheese preparation.

Potential Probiotic Bacillus Strains with Antioxidant and Antimutagenic Activity Increased Weight Gain and Altered hsp70, cxc, tnfα, il1β, and lysC Gene Expression in Clarias gariepinus

The potential probiotic properties of three Bacillus strains were studied. A probiotic supplement for the African catfish Clarias gariepinus was produced via the solid-state fermentation protocol and incorporated into the fish feed for a period of seven weeks. Since the 36th day of the experiment, all experimental groups had a statistically significant increase in their weight gain than the control group. The maximum weight gain observed in fish fed the probiotic-supplemented feed was 29.16% higher than that of the control group, and the maximum feed conversion rate improvement was 24%. Cell-free extracts from these strains showed antioxidant (11.55–27.40%) and DNA-protective (45.33–61.83%) activity in a series of in vitro biosensor tests. Further investigation into the antimutagenic activity of the strains revealed that two of them reduced the level of induced mutagenesis in an Escherichia coli model (by 33.58% and 54.35%, respectively). We also assessed the impact of probiotic strains on the expression of several key genes in the host (C. gariepinus), including hsp70, cxc, tnfα, il1β, and lysC. More than a 10-fold increase in expression rates was observed for hsp70 in gonads and liver; for cxc in muscles and gonads; for tnfα in brain, gills, and liver; for il1β in the brain, gills, gonads, and liver; and for lysC in gills, gonads, liver, and muscles. This study provides evidence that probiotics exhibiting antioxidant and antimutagenic properties can provide significant benefits in vivo within aquaculture systems. The molecular effects of these probiotics appear to be complex and tissue-specific, with both upregulation and downregulation of immune system genes observed. Nevertheless, at the organismal level, the impact was unequivocally positive in terms of aquaculture objectives, manifested as enhanced body weight gain in the fish. Consequently, these Bacillus strains warrant serious consideration as potential probiotics for this species.

Cell-Free Fat Extract with Anti-melanogenic Property Improves the Appearance of Infraorbital Dark Circles: A Preliminary Clinical Study.

The formation of infraorbital dark circles involves various factors including hyperpigmentation, skin aging and laxity. This study is to investigate whether cell-free fat extract (CEFFE) inhibits melanin synthesis and improves the appearance of infraorbital dark circles. Imaging systems analysed the melanin content in three groups of zebrafish embryos treated with 0 (CTRL), 100, and 200μg/mL of CEFFE. Eleven patients with infraorbital dark circles were enrolled and received intradermal injections of autologous CEFFE. Clinical trial assessments included subjective assessments (Global Aesthetic Improvement Scale and Likert satisfaction scale) and objective assessments (standard two-dimensional photographs, VISIA® photographs, and infraorbital skin colour measurement). In the experiment of zebrafish embryos, the melanin content of CEFFE treatment was significantly reduced. In clinical trial, 72.73% of patients considered CEFFE treatment for infraorbital dark circles to be effective, and more than half of the patients were satisfied with the treatment. In the evaluation of skin colour at the inner, middle, and outer edges of the infraorbital dark circles, there was a gradual improvement in infraorbital skin colour after each treatment and at the 3-month follow-up, with a statistically significant improvement in skin colour on the outer edge of the infraorbital dark circles (p < 0.05). Simultaneously, we observed a reduction in periocular wrinkles, with a significant difference at the 3-month follow-up (p < 0.05). The events observed included bruising, erythema, and oedema, which resolved spontaneously. Our study demonstrated that CEFFE inhibits melanogenesis and is a safe and effective treatment for infraorbital dark circles. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

An Expanded Substrate Spectrum of Sulfide:Quinone Oxidoreductase Found in Pollutant-Degrading Bacteria.

Sulfide:quinone oxidoreductase (Sqr) catalyzes the initial procedure on sulfide transformation, alongside sulfide (H2S, S2-) oxidization coupled with coenzyme Q (CoQ) reducing and reactive sulfur species (RSS) production. Here, we assessed the reactivity of propanethiol (PT) as an alternative substrate for Sqr to maintain intracellular homeostasis in strain S-1 capable of degrading emerging sulfur-containing pollutants. We deleted a gene encoding Sqr, and serial transcriptional difference induced by RSS dynamics was therefore revealed. Next, the reaction properties of two Sqr homologs from strains JMP134 and S-1 were comparatively characterized, respectively. As a result, an additional role of Sqr in yielding RSS from PT was found in reaction mixture prepared by cell-free extracts or purified enzymes. Interestingly, the transformation velocity of PT by Sqr was slower than that of sulfides. From this scenario, it was a rate-determining step that PT as a nucleophilic compound can be added into Sqr cysteine to form disulfide bond and likely serve nonoptimal sulfur recipient. In addition, the role of persulfidation driven by RSS in combating oxidative and sulfur stresses required to be further clarified. Nevertheless, this promiscuity of Sqr-binding organosulfur compounds and its catalytic modulation underscored that expanded substrates might benefit sulfide homeostasis in thiol-degrading bacteria.

Enzyme Activity and Biodegradation Potentials of Serratia marcescens Cell-Free Extract on Polyethylene Terephthalate Sheet

Enzyme Activity and Biodegradation Potentials of Serratia marcescens Cell-Free Extract on Polyethylene Terephthalate Sheet

Anti-inflammatory, cytotoxic and morphological impact of phycocyanin on ultraviolet radiation irradiated human fibroblast cells

Background Recently, the use of natural products as skin photoprotective agents has been in increasing demand. This study investigated the bioactivity of phycocyanin (PC) extracted from Spirulina sp. on human skin fibroblast cell line (CCD-966SK), specifically focusing on apoptosis, necrosis, anti-inflammatory effects, and enzymatic reactions. Methods The first step of this study was cyanobacterial cell culture and the extraction and purification of PC. After that, CCD-966SK cell line was cultivated under normal and UV irradiation. The bioassays included the cytotoxicity measurement, cell viability assay, morphology determination, tumour necrosis factor-α and Interleukin 6 release assays, enzyme activity for superoxide dismutase and glutathione peroxidase as well as malondialdehyde content and the cell-free extract of cyanobacterial stains were assessed. Results The cell viability results showed that as the concentration of the PC increased, the viability of CCD-966SK cell line was reduced, which suggested that the effect of PC on the growth of fibroblast cells was dose dependent. The morphological results indicated that presence of PC in the fibroblast cell culture medium led to a transformation in cell morphology from spindle-shaped to spherical. PC released anti-inflammatory IL-6 and TNF-a cytokines, indicating high inflammation resistance. Furthermore, the findings revealed that PC dramatically reduced the release of superoxide dismutase, glutathione peroxidase, and malondialdehyde from inflammatory cells, with the reduction being more apparent at increasing doses. Conclusions In conclusion, the results indicated that PC inhibit the CCD-966SK cell line by membrane destructor, which led to the increase the leakage of cell constituent and increase enzymes activities.

Antimicrobial activities and bacteriocins of lactic acid bacteria isolated from Nigeria indigenous fermented cowmilk &amp;ldquo;nunu&amp;rdquo;

Nunu is a nutritious fermented yoghurt-like milk beverage that can serve as a source of lactic acid bacteria (LAB) with antimicrobial and bacteriocin potentials. Nunu samples were screened for LAB with antimicrobial activity against test organisms. Three LAB, namely Lactobacillus parafarraginis OCLP1, L. fermentum CSL58 and L. buchneri MZE12 out of a total of 14 isolates produced antimicrobial activity against Bacillus anthracis CHF-2011-32b, B. cereus DHM5-1, Providencia vermicola KSVDL-09-71600, P. rettgeri SIRT3, P. stuartti SILT11 and other test organisms. Antimicrobial factors were assayed in LAB cell-free extracts (CFE), neutralized CFE, and ammonium sulphate precipitants of the CFE, and Lactobacillus parafarraginis OCLP1 demonstrated highest activity with zone of inhibition = 20.33±3.51 mm­, 13.33±1.09 mm and 25.33±2.52 mm respectively. The CFE of the LAB especially L. parafarraginis OCLP1 demonstrated wide activity spectra by inhibiting both the gram-positive and gram-negative bacteria tested. Result of plasmid curing of the LAB indicate that the bacteriocins of L. parafarraginis OCLP1 was plasmid-encoded while the others have their gene clusters on the chromosome. This study concluded that bacteriocin-producing LAB can be found in “nunu” and recommend the use of the isolated LAB as starter in fermented food production after ensuring their safety through toxicological studies.

Eco-toxicological impact of biologically synthesized FeONPs on rice plants and PGP bacteria

Nanoformulations of plant micronutrients can improve nutrient use efficiency and availability in plants compared to conventional fertilizers and, therefore, may prove helpful in addressing the widespread challenge of nutrient deficiency in agricultural soils. In this study, iron-oxide nanoparticles (FeONPs) were bio-synthesized using Aspergillus terreus cell-free extract and tested for bio-efficacy in rice (Oryza sativa L., ssp. japonica, T309) in situ. Physicochemical characteristics of biosynthesized FeONPs (B-FeONPs) were analyzed through UV-visible spectroscopy (UV-Vis), dynamic light scattering (DLS), X-ray diffraction (XRD), Fourier-transform infrared (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray (EDX). Bio-efficacy was tested at both environmentally relevant concentrations (0-50 mg.kg−1) as well as supra-environmental concentrations (100 mg.kg−1). Germination, wet and dry biomass, root length, shoot length, chlorophyll and carotenoid content, and iron fortification in rice plants were evaluated under greenhouse conditions. Potential eco-toxic effects of B-FeONPs were investigated in multiple strains of plant growth-promoting (PGP) bacteria. A significant increment in plant growth parameters (p < 0.05), including wet biomass, dry biomass, root length, and chlorophyll and carotenoid content was observed in B-FeONPs treated rice plants as compared to those treated with bulk Fe-fertilizer FeSO4. B-FeONPs caused no oxidative stress and increased the % Fe content in the plants. Uptake and accumulation of the B-FeONPs were also confirmed in the roots and leaves of plants using TEM. The B-FeONPs did not demonstrate bactericidal activity against any of the tested strains of PGP bacteria at both environmentally relevant and supra-environmental concentrations. In conclusion, the data demonstrate that B-FeONPs exhibit significant potential in enhancing nutrient use efficiency and promoting growth in rice plants. These findings underscore the potential of B-FeONPs as sustainable alternatives to conventional Fe fertilizers, offering sustainable solutions for addressing nutrient deficiencies in agricultural soils.

Cyanobacterial consortia as biostimulant for germination of Zea mays seeds

Cyanobacterial extracts are known to be rich in several plant growth promoting metabolites as well as phytohormones. Indole 3 acetic acid is one of the phytohormones that are known to be promoting seed germination. On the basis of the ability of three cyanobacterial species Westiellopsis sp. TPR-29, Hapalosiphon sp. Ryu2-7DN_D3 and Chlorogloeopsis fritschii PCC 6912 to produce IAA, their cell free supernatant were tested for induction of germination in Zea mays (Maize) seeds. The seeds were treated with cell free extracts of 15 days old cyanobacterial cultures for 24 and 48 hours.

Nematicidal activity of Pleurotus djamor metabolites on Meloidogyne incognita under greenhouse conditions

This study evaluated the nematicidal activity of the cell-free crude extract (CFCE) of Pleurotus djamor on Meloidogyne incognita under greenhouse conditions. The fungus was grown in a solid medium for 14 days in the dark. Distilled water was added to the medium, filtered, and centrifuged, and the proteolytic activity of the CFCE produced was measured. Three experimental groups were established: control group (distilled water), CFCE (denatured enzymes), and CFCE (active enzymes). The nematicidal activity of the extracts was assessed in vitro on second-stage juveniles (J2) and eggs of M. incognita, separately. Under greenhouse conditions, CFCEs were applied to tomato seedlings grown on substrate infested with M. incognita eggs. About the in vitro tests, the active CFCE showed a significant difference concerning the control group (p&lt;0.05) according to the t-test, with a 22% reduction in eggs. On the other hand, about J2, both treatments showed a significant difference compared to the control (p&lt;0.01) with a 100% reduction, but no significant difference between themselves (p&gt;0.05). Similarly, the treatments in the greenhouse showed the same statistical behavior as the control (p&lt;0.01). However, there was no significant difference between them (p&gt;0.05) regarding the number of eggs and the number of galls. The reduction in the number of eggs was 94 and 97 % and in the number of galls was 71 and 67% for denatured and active CFCE, respectively. This study points to the nematicidal action of metabolites on J2 and the activity of enzymes on M. incognita eggs.

Discovery, characterization, and synthetic potential of two novel bacterial aryl-alcohol oxidases

The search for novel synthetic tools to prepare industrial chemicals in a safer and greener manner is a continuing challenge in synthetic chemistry. In this manuscript, we report the discovery, characterization, and synthetic potential of two novel aryl-alcohol oxidases from bacteria which are able to oxidize a variety of aliphatic and aromatic alcohols with efficiencies up to 4970 min−1 mM−1. Both enzymes have shown a reasonable thermostability (thermal melting temperature values of 50.9 and 48.6 °C for ShAAO and SdAAO, respectively). Crystal structures revealed an unusual wide-open entrance to the active-site pockets compared to that previously described for traditional fungal aryl-alcohol oxidases, which could be associated with differences observed in substrate scope, catalytic efficiency, and other functional properties. Preparative-scale reactions and the ability to operate at high substrate loadings also demonstrate the potential of these enzymes in synthetic chemistry with total turnover numbers > 38000. Moreover, their availability as soluble and active recombinant proteins enabled their use as cell-free extracts which further highlights their potential for the large-scale production of carbonyl compounds.Key points• Identification and characterization of two novel bacterial aryl-alcohol oxidases• Crystal structures reveal wide-open active-site pockets, impacting substrate scope• Total turnover numbers and cell-free extracts demonstrate the synthetic potentialGraphical

Anti-quorum sensing and anti-biofilm activities of Pasteurella multocida strains

A total of 52 Pasteurella multocida strains of capsular serogroups (A, B and D) were screened for anti-quorum sensing activity against Chromobacterium violaceum. Of which, 12 strains of serogroups A were found to possess anti-quorum sensing activity. Inhibition activity was highest for strain NIVEDIPm9 and lowest for strain NIVEDIPm30 based on zone of pigment inhibition. Further, cell free extract of NIVEDIPm9 strain showed highest anti-biofilm activity in reference E. coli strain and concentration dependent degradation activity of C6-AHL molecule. In whole genome sequence annotation of NIVEDIPm9 strain predicted the presence of four metallo-β-lactamases (MBL) fold metallo-hydrolase proteins. In docking studies, MBL1 and MBL3 proteins showed high binding affinity with autoinduce signalling molecules AHL compound of OH-C10, binding energy value were −6.3 and −6.2 kcal/mol. Interaction study of VAF and quorum sensing molecules showed that OmpA and HgbA proteins were stimulated by all the ten molecules (C4-AHLs, C6-AHLs, C10-AHLs, C14-AHLs, 3-oxo-C10-AHLs, 3OH-C10-HSL, C8-HSL, C10-HSL, C12-HSL, C14-HSL), while toxA gene was stimulated by OH-C10-AHL molecule, sodC gene was stimulated by none. In conclusion, we described the anti-quorum sensing activities of diverse P. multocida strains causing Pasteurellosis in livestock.

The anti-obesity effects of postbiotics: A systematic review of pre-clinical and clinical studies

BackgroundThe growing prevalence of obesity has become a major concern worldwide, therefore a great number of studies are conducted every day in the field of obesity. Since postbiotics are a newly introduced term, there is not much systematic evidence about their function and impact on obesity. We designed this study to systematically review the effect of different types of postbiotics on obesity. MethodsA systematic search was conducted using PubMed, SCOPUS, and Web of Science databases up to August 2023. Both human and animal interventional studies that investigated the effects of any type of postbiotic on obesity and obesity-related factors were eligible. Screening, data extraction, and quality assessment were conducted independently by two researchers. The quality of the studies was appraised using Cochrane and Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE’s) risk of bias tool. ResultsOf the 19373 retrieved studies, finally, 49 studies were included (9 human studies and 40 animal studies). Short-chain fatty acids and heat-killed (inactivated) bacteria were the most used postbiotics. In human clinical trials, inactivated Lactobacillus amylovorus (CP1563), Bifidobacterium animalis subsp. lactis (CECT 8145) and Pediococcus pentosaceus (LP28) were administered orally as postbiotics which improved body composition and anthropometric indices. Animal studies evaluated other types of postbiotics including muramyl dipeptide, cell-free extracts, urolithin A&B, extracellular Vesicles, exopolysaccharides, and surface Layer Proteins, supporting the anti-obesity effects of postbiotics. ConclusionPostbiotics seem to be a safe intervention and the results were in favor of a reduction in adipogenesis as well as an increase in energy expenditure. Further high-quality studies are required in this relatively new topic.

Biosynthesis of selenium nanoparticles as a potential therapeutic agent in breast cancer: G2/M arrest and apoptosis induction

The drawbacks and adverse reactions of conventional breast cancer (BC) medications have prompted researchers to seek novel therapeutic approaches. This study aimed to study the impact of biosynthesized selenium nanoparticles by yeast on breast cancer (MCF-7) cells and to find potential underlying mechanisms. Therefore, marine yeast isolates were screened for their ability to biosynthesis selenium nanoparticles (SeNPs). The most potent isolate was identified as Candida pseudojiufengensis based on 18 S rRNA gene sequencing. Incubation of cell-free extract with 0.8 mM of SeO2 for 48 h at 40°C in pH of 7.0 were optimal conditions for the biosynthesis of SeNPs. The biosynthesized SeNPs were characterized by UV–Vis spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), and dynamic light scattering (DLS) measurements including average particle size distribution and average zeta potential. The results showed that the biosynthesized SeNPs displayed a maximum absorbance peak in the UV-Vis spectrum at 560 nm due to surface plasmon resonance. TEM image elevated spherical shape particles with an average size of 12 nm. SRB assay, flow cytometry, and other biochemical methods were employed to assess SeNPs anti-proliferative effects on MCF-7 cells. SeNPs showed superior anticancer efficacy against MCF-7 cells compared to colon (HCT-116) and liver (HepG2) cancer cells, as evidenced by lower IC50 values (19.59 µg/ml) against 36.36 µg/ml and 27.81 ±1.4 µg/ml, respectively. However, SeNPs demonstrated no cytotoxic effects against HSF cells. Moreover, treatment with SeNPs induces G2/M arrest along with triggering apoptosis in MCF-7 cells. Furthermore, MCF-7 cells treated with SeNPs showed increased oxidative stress, as indicated by observable rises in LPO and 8-OHDG, accompanied by considerable exhaustion in antioxidant enzyme activity. These findings demonstrated that Se nanoparticles synthesized from yeast have therapeutic promise in BC treatment.

Detection of presence of Bioactive metabolites in CCFE (Co cultured Cell Free Extracts) of Microbacterium barkeri (LMA4), Corynebacterium argentoratense (LMA5) and Streptomyces shenzhenensis (LMA6)

The members of actinomycetes, versatile microspecies with dynamic source of bioactive molecules, are the nodal attraction of researchers. Continuous efforts are streaming to detect and launch new or derivetatised antibiotics to defend against the reemerged drug resistant infectious agents. This communication is dealt with detection of bioactive molecules from three strains of novel actinomycetal strains, namely, Microbacterium barkeri (LMA4), Corynebacterium argentoratense (LMA5) (Gene bank No. OP023130) and Streptomyces shenzhenensis (LMA6) (Gene bank No. OQ092768) isolated from pond soil of near by locality. The strains were cocultured with laboratory maintained bacteria, Staphylococcus aureus (BMS4) and a Gram negative, Escherichia coli (BME4) and the co cultured cell free extract (CCFE) of respective actinobacterial strains were subjected to UV-visible and LC MS analysis. The preliminary observation could note about similar peak pattern with Streptomycin, Doxorubicin, Pyrazine, Pyrrolizidines, Oxacillin, Ciprofloxacin, Allistatin, Gentamycin, Chlorellin, Penicillin, Penicillin G, Kanamycin, Levofloxacin, Amikacin, Ofloxacin, Imipenem and more over Ampicillin, as inferred from UV visible, followed by Liquid chromatography. The conclusive result was inferred from the LC-MS (m/z) spectrum analysis. It was noted that the peak with ID-53, eluted from the CFE, of co-culture broth of LMA4 with BMS4, with retention time (RT) 26.99 (min), of m/z 751.3, carried out with Electrospray Ionisation (ES) in +ve mode (ES+), Peaks with IDs, 45, 54 (24.65 and 27.11respective RTs), having resultant m/z, 749.8and 751.7 in ES-, and the CFE containing LMA6 and BMS4, with eluted peak Ids 12, 42 (RTs, 5.71, 20.55), having m/z 752.4 and 749.8 and 749.8 in ES+ mode, which could be assigned with structure of Azithromycin.

MecE, MecB, and MecC proteins orchestrate methyl group transfer during dichloromethane fermentation.

Dichloromethane (DCM), a common hazardous industrial chemical, is anaerobically metabolized by four bacterial genera: Dehalobacter, Dehalobacterium, Ca. Dichloromethanomonas, and Ca. Formimonas. However, the pivotal methyltransferases responsible for DCM transformation have remained elusive. In this study, we investigated the DCM catabolism of Dehalobacterium formicoaceticum strain EZ94, contained in an enriched culture, using a combination of biochemical approaches. Initially, enzymatic assays were conducted with cell-free protein extracts, after protein separation by blue native polyacrylamide gel electrophoresis. In the slices with the highest DCM transformation activity, a high absolute abundance of the methyltransferase MecC was revealed by mass spectrometry. Enzymatic activity assays with heterologously expressed MecB, MecC, and MecE from strain EZ94 showed complete DCM transformation only when all three enzymes were present. Our experimental results, coupled with the computational analysis of MecB, MecC, and MecE sequences, enabled us to assign specific roles in DCM transformation to each of the proteins. Our findings reveal that both MecE and MecC are zinc-dependent methyltransferases responsible for DCM demethylation and re-methylation of a product, respectively. MecB functions as a cobalamin-dependent shuttle protein transferring the methyl group between MecE and MecC. This study provides the first biochemical evidence of the enzymes involved in the anaerobic metabolism of DCM.IMPORTANCEDichloromethane (DCM) is a priority regulated pollutant frequently detected in groundwater. In this work, we identify the proteins responsible for the transformation of DCM fermentation in Dehalobacterium formicoaceticum strain EZ94 using a combination of biochemical approaches, heterologous expression of proteins, and computational analysis. These findings provide the basis to apply these proteins as biological markers to monitor bioremediation processes in the field.

Sodium alginate hydrogels co-encapsulated with cell free fat extract-loaded core-shell nanofibers and menstrual blood stem cells derived exosomes for acceleration of articular cartilage regeneration

This study presents a novel scaffold system comprising sodium alginate hydrogels (SAh) co-encapsulated with cell-free fat extract (CEFFE)-loaded core-shell nanofibers (NFs) and menstrual blood stem cell-derived exosomes (EXOs). The scaffold integrates the regenerative potential of EXOs and CFFFE, offering a multifaceted strategy for promoting articular cartilage repair. Coaxially electrospun core-shell NFs exhibited successful encapsulation of CEFFE and seamless integration into the SAh matrix. Structural modifications induced by the incorporation of CEFFE-NFs enhanced hydrogel porosity, mechanical strength, and degradation kinetics, facilitating cell adhesion, proliferation, and tissue ingrowth. The release kinetics of growth factors from the composite scaffold demonstrated sustained and controlled release profiles, essential for optimal tissue regeneration. In vitro studies revealed high cell viability, enhanced chondrocyte proliferation, and migration in the presence of EXOs/CEFFE-NFs@SAh composite scaffolds. Additionally, in vivo experiments demonstrated significant cartilage regeneration, with the composite scaffold outperforming controls in promoting hyaline cartilage formation and defect bridging. Overall, this study underscores the potential of EXOs and CEFFE-NFs integrated into SAh matrices for enhancing chondrocyte viability, proliferation, migration, and ultimately, articular cartilage regeneration. Future research directions may focus on elucidating underlying mechanisms and conducting long-term in vivo studies to validate clinical applicability and scalability.