Growth Medium Research Articles

Exploring the Interplay between Nutrients, Bacteriophages, and Bacterial Lipases in Host- and Bacteria-mediated Pathogenesis.

Pathogenic bacteria and host cells counteract or neutralize each other's effect in two fundamental ways: Direct invasion and secretion of various substances. Among these, lipases secreted by pathogenic bacteria and host cell lysozyme are key actors. Secreted lipases from pathogenic bacterial are suggested as a key player in the pathogen-host interaction. Among the gut microbial energy sources, glucose and fats have been referred to as one of the best inducers and substrates for bacterial lipases. Enrichment of bacterial growth medium with extra glucose or oil has been shown to induce lipase production in pathogenic bacteria. More recently, research has focused on the role of human gut phage alterations in the onset of dysbiosis because the bacteria-phage interactions can be dramatically affected by the nutrient milieu of the gut. However, the reciprocal role of bacterial lipases and phages in this context has not been well studied and there is no data available about how high glucose or fat availability might modulate the cellular milieu of the pathogenic bacteria-phageeukaryotic host cell interface. The purpose of this study was to evaluate the immunologic outcome of pathogenic bacteria-phage interaction under normal, high glucose, and high butter oil conditions to understand how nutrient availability affects lipase activity in pathogenic bacteria and, ultimately, the eukaryotic host cell responses to pathogenic bacteria-phage interaction. 10 groups of co-cultured T84 and HepG2 cells were treated with Pseudomonas aeruginosa strain PAO1 (P.a PAO1) in the presence and absence of its KPP22 phage and incubated in three different growth media (DMEM, DMEM + glucose and DMEM + butter oil). Structural and physiological (barrier function and cell viability), inflammatory (IL-6 and IL-8), metabolic (glucose and triglycerides), and enzymatic (lipases and lysozyme) parameters were determined. Excess glucose or butter oil enhanced additively extracellular lipase activity of P.a PAO1. Excess glucose or butter oil treatments also magnified P. a PAO1- induced secretion of inflammatory signal molecules (IL-1β, IL-6) from co-cultured cells, concomitant with the enhancement of intracellular triglycerides in co-cultured HepG2 cells, these effects being abolished by phage KPP22. The results of the present study imply that KPP22 phage influences the interplay between food substances, gut bacterial lipases, and the gut cellular milieu. This can be applied in two-way interaction: by affecting the microbial uptake of excess free simple sugars and fats from the gut milieu leading to decreased bacterial lipases and by modulating the immune system of the intestinal -liver axis cells. Further studies are needed to see if the biological consequences of these effects also occur in vivo.

Temporary immersion bioreactor as an efficient method for in vitro propagation of Agave marmorata

Agave marmorata is an important wild species in Mexico for mezcal and pulque production. Propagation by suckers and seeds is not sufficient to restore wild populations. The aim of this study was to develop a protocol for large-scale micropropagation of A. marmorata using a temporary immersion bioreactor (TIB). The effect of liquid medium using a TIB and semi-solid medium as the control treatment was compared. Different immersion frequencies and culture medium volumes per explant were evaluated in the TIB. In all treatments, the percentage of hyperhydricity, number of shoots per explant, shoot length, and total chlorophyll and carotenoid content were evaluated. In addition, the survival rate during acclimatization was evaluated. The results obtained show that the TIB with an immersion of 2 min every 8 h and with a culture medium volume of 50 mL had the best developmental parameters, with 19.60 shoots per explant, a shoot length of 1.74 cm and a percentage of hyperhydricity of 3.33 %. The highest chlorophyll content was observed in temporary immersion every 8 h, with 100, 75 and 50 mL of culture medium per explant. Regarding carotenoid content, the highest content was found at the 8 and 12 h immersion frequencies, with the culture medium volumes of 100, 75 and 50 mL per explant. For acclimatization, no differences were found in the survival rate between the culture systems evaluated. In conclusion, this study demonstrates that the use of a TIB is an alternative for large-scale micropropagation of A. marmorata.

An Exceptional Valorization of CuO Nanoparticles in Ionic Liquids as an Efficient Medium for the Electrophilic Substitution of Indole Towards the Formation of Bis(indolyl)methanes

Aim: Ionic liquids are promising green solvents with simple but unique structure-related physical properties such as negligible vapour pressure, exceptional thermal conductivity, remarkable thermal stability and their suitability and inertness towards a broad range of catalytic applications. CuO NPs have been addressed as a cost-effective and a reagent of a choice that necessitates only mild reaction conditions to offer a high yield of the desired products with exceptional selectivity in a short duration of time. Therefore, in the present work, attempts have been made to explore the catalytic potentials of CuO NPs in an ionic liquid medium to synthesize biologically important bis(indolyl)methanes. Background: Catalytic explorations of metal oxide nanoparticles in ionic liquids offers a cooperative effect that has a significant impact on the kinetics as well as on the outcome of the reaction. Therefore, such catalytic systems in the present times have seized the scientific community's interest from the perspectives of sustainable development in synthetic organic chemistry. The combination of metal oxide nanoparticles with highly tunable ionic liquids is not only used to synthesize simple organic molecules but also explored in the synthesis of complex organic molecules of high commercial and biological relevance. Objectives: The current work offers a rapid and robust protocol for synthesizing bis(indolyl)methanes via electrophilic substitution reaction between indole and various aldehydes in the presence of a CuO nanoparticles-ionic liquid system. The discussion focuses on the high tolerance of different functionalities by the catalytic system leading to the synthesis of bis(indolyl)methanes. Methods: CuO NPs have been synthesized via the co-precipitation method using ionic liquid. The applicability of metal oxide nanoparticles-IL matrix was further investigated in synthesizing bis(indolyl)methanes. Results: The FT-IR absorption below 600 cm-1 and the XRD pattern showing all the peaks in the diffraction diagram revealed the formation of CuO NPs. FESEM images show the flake-shaped morphology of CuO NPs and are found to be separated from the agglomerated clusters Conclusion: Ionic liquid-CuO NPs matrix reveals good to exceptional catalytic properties, and their advancements as a catalytic system at room temperature open new avenues for synthetic organic chemists.

Design approaches for 3D cell culture and 3D bioprinting platforms.

The natural habitat of most cells consists of complex and disordered 3D microenvironments with spatiotemporally dynamic material properties. However, prevalent methods of in vitro culture study cells under poorly biomimetic 2D confinement or homogeneous conditions that often neglect critical topographical cues and mechanical stimuli. It has also become increasingly apparent that cells in a 3D conformation exhibit dramatically altered morphological and phenotypical states. In response, efforts toward designing biomaterial platforms for 3D cell culture have taken centerstage over the past few decades. Herein, we present a broad overview of biomaterials for 3D cell culture and 3D bioprinting, spanning both monolithic and granular systems. We first critically evaluate conventional monolithic hydrogel networks, with an emphasis on specific experimental requirements. Building on this, we document the recent emergence of microgel-based 3D growth media as a promising biomaterial platform enabling interrogation of cells within porous and granular scaffolds. We also explore how jammed microgel systems have been leveraged to spatially design and manipulate cellular structures using 3D bioprinting. The advent of these techniques heralds an unprecedented ability to experimentally model complex physiological niches, with important implications for tissue bioengineering and biomedical applications.

Assessment of tepojal as a support medium in in vitro germination of Barkeria whartoniana (Orchidaceae) and subcultures of B. uniflora and B. scandens

Objective: We evaluated the use of tepojal as a substitute for bacteriological agar for in vitro culture of three species of Mexican orchids of the genus Barkeria. We had three objectives: (1) evaluate the use of tepojal for the in vitro germination of B. whartoniana seeds; (2) compare the growth and survival of B. uniflora and B. scandens plants in in vitro culture on bacteriological agar and tepojal, and (3) compare the early ex situ survival of the B. uniflora and B. scandens previously cultivated in vitro on bacteriological agar and tepojal. Design/Methodology/Approach: For the growth experiment, 1,050 seedlings of Barkeria scandens and B. uniflora were used. Two types of culture medium were prepared: (1) Liquid with tepojal and 40 % MS medium with dextrose, yeast, coconut water, activated carbon and refined sugar, and (2) solid at 40 %, with the same elements as the liquid one, but with 6 g of bacteriological agar. For the germination experiment of Barkeria whartoniana in tepojal we use seeds from a mature (open) capsule. After disinfection, seeds were sown in tepojal with liquid medium and in a solid (agar) culture using a syringe technique. All the seeds were sown in 100 % p668 MS medium with 100 mL/L of coconut water and 15 g/L of refined sugar and using in both the MS medium. Results: Barkeria uniflora seedlings had a greater growth than in B. scandens regardless of the type of treatment. When comparing within each species, we found that the two treatments (tepojal vs agar) did not produce differences in the growth of the shoot of both species. Roots growth was influenced by both the effect of the species and the treatment, as the longest roots were found in tepojal medium. Seed germination observed in the liquid medium with tepojal was not apparently different from that in agar. In both cases the entire surface of the jar was covered with green protocorms. Findings/Conclusions: Tepojal did function well as a substrate to germinate Barkeria whartoniana, but tests must continue to be carried out to evaluate its effectiveness. As no differences were found for aerial growth between agar and tepojal in Barkeria scandens and Barkeria uniflora, we consider this substrate as a good substitute for agar because it can produce more biomass per gram of culture medium used; therefore, if we consider the costs the use of tepojal is much cheaper and much easier to get.

Neodymium (III)-containing poly(lactide-co-glycolide)-coated robocast bioactive glass scaffolds for photothermal therapy and bone regeneration

In this study, trivalent neodymium-doped silicate-based 13–93 bioactive glass scaffolds were prepared by the robocasting method using sol-gel-derived bioactive glass powders for tissue engineering applications. Sintered scaffolds were coated by borate-based 13-93B3 bioactive glass-containing polylactide-co-glycolide solution. The produced composite scaffolds’ mechanical, morphological, and structural characteristics were thoroughly examined, as their in vitro bioactivity in cell culture media and simulated body fluid. Furthermore, the scaffolds’ amoxicillin adsorption and release behavior was examined over time. The outcomes demonstrated that it was feasible to effectively create periodic, mesh-like-patterned robocast glass scaffolds utilizing Nd3+-doped sol-gel-derived bioactive glass powders. The scaffolds’ compressive strengths ranged from 10.02 MPa to 18.6 MPa, with the PLGA-coated scaffolds exhibiting the highest strength values. All of the scaffolds that were submerged in simulated body fluid for 28 days showed hydroxyapatite formation. The presence of borate glass on the surface of the silicate-based glass scaffolds improved the hydroxyapatite formation ability. The quantity of drug adsorption for all types of scaffolds was measured to be between 4 and 9% whereas the cumulative drug release was in the range of 58 to 96%. Borate glass particle-containing PLGA coating enhanced the drug delivery behavior.

Isolation of the inorganic phosphorus-solubilizing bacteria Lysinibacillus sphaericus and assessing its role in promoting rice growth.

Soluble phosphorus scarcity severely limits plant growth and crop yield. In this study, a strain of inorganic phosphorus-solubilizing bacteria, Lysinibacillus sphaericus, was isolated from rice rhizosphere soil. The available phosphorus content in liquid inorganic phosphorus identification medium and in L. sphaericus-inoculated soil increased from 204.28mg/L to 1124.68mg/L and from 4.75mg/kg to 7.04mg/kg, respectively. The pH decreased significantly from 6.87 to 6.14. Incubation with L. sphaericus significantly increased malic and succinic acid content in the liquid inorganic phosphorus identification medium and increased acid phosphatase and alkaline phosphatase activity in the soil. Inoculation with L. sphaericus significantly increased rice growth, chlorophyll a/b content, and photosynthesis by increasing the soluble phosphorus content in the rice rhizosphere soil under phosphorus-deficient conditions. Further analysis revealed that L. sphaericus improved soil phosphorus release by decreasing soil pH and promoting acid phosphatase and alkaline phosphatase activity. This study supports the production of microbial fertilizers to improve rice yield in phosphorus-deficient conditions.

Bioprocess monitoring applications of an innovative ATR-FTIR spectroscopy platform

Pharmaceutical manufacturing is reliant upon bioprocessing approaches to generate the range of therapeutic products that are available today. The high cost of production, susceptibility to process failure, and requirement to achieve consistent, high-quality product means that process monitoring is paramount during manufacturing. Process analytic technologies (PAT) are key to ensuring high quality product is produced at all stages of development. Spectroscopy-based technologies are well suited as PAT approaches as they are non-destructive and require minimum sample preparation. This study explored the use of a novel attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy platform, which utilises disposable internal reflection elements (IREs), as a method of upstream bioprocess monitoring. The platform was used to characterise organism health and to quantify cellular metabolites in growth media using quantification models to predict glucose and lactic acid levels both singularly and combined. Separation of the healthy and nutrient deficient cells within PC space was clearly apparent, indicating this technique could be used to characterise these classes. For the metabolite quantification, the binary models yielded R2 values of 0.969 for glucose, 0.976 for lactic acid. When quantifying the metabolites in tandem using a multi-output partial least squares model, the corresponding R2 value was 0.980. This initial study highlights the suitability of the platform for bioprocess monitoring and paves the way for future in-line developments.

Predicting and probing the local temperature rise around plasmonic core-shell nanoparticles to study thermally activated processes.

Ultrafast spectroscopy can be used to study dynamic processes on femtosecond to nanosecond timescales, but is typically used for photoinduced processes. Several materials can induce ultrafast temperature rises upon absorption of femtosecond laser pulses, in principle allowing to study thermally activated processes, such as (catalytic) reactions, phase transitions, and conformational changes. Gold-silica core-shell nanoparticles are particularly interesting for this, as they can be used in a wide range of media and are chemically inert. Here we computationally model the temporal and spatial temperature profiles of gold nanoparticles with and without silica shell in liquid and gas media. Fast rises in temperature within tens of picoseconds are always observed. This is fast enough to study many of the aforementioned processes. We also validate our results experimentally using a poly(urethane-urea) exhibiting a temperature-dependent hydrogen bonding network, which shows local temperatures above 90 ◦C are reached on this timescale. Moreover, this experimentally shows the hydrogen bond breaking in such polymers occurs within tens of picoseconds.

Crosstalk among miR-29, α-SMA, and TGFβ1/β3 in melatonin-induced exosome (Mel-prExo) treated human limbal mesenchymal stem cells (hLMSCs): An insight into scarless healing of the cornea.

Inflammatory mediators that infiltrate the corneal stroma after corneal infections, trauma or refractive surgery can trigger the transformation of corneal keratocytes into myofibroblasts, resulting in highly irregular collagen deposition and subsequently corneal scarring. Mesenchymal stem cells (MSCs) can be used as therapeutic agents to regenerate corneal and conjunctival tissue damage, regulate inflammation, and reduce the development of limbal stem cell failure. The use of MSC-derived exosomes as a cell-free therapeutic vector is a novel therapeutic approach. This study aimed to assess the effect of exosomes obtained from melatonin (Mel)-treated human limbal mesenchymal stem cells (hLMSCs) on naïve hLMSCs and to determine their influence on the antifibrotic and pro-regenerative pathways involved in corneal scarring. hLMSCs were treated with varying concentrations of Mel, followed by isolation and characterization of the procured exosomes (Mel-prExos). These exosomes were added to the cell culture media of naïve hLMSCs to examine their antifibrotic and pro-regenerative effects. The expression of miR-155, miR-29, TGFβ1, TGFβ3, PPARγ, and α-SMA miRNAs and genes were compared between Mel-treated hLMSCs and Mel-prExo-treated hLMSCs by using real-time PCR. We found that at 1 μM Mel and in the presence of Mel-prExos, TGFβ1 was expressed 0.001-fold, while TGFβ3 was expressed 0.6-fold. miR-29 expression was increased 38-fold in the control-Exo group compared to that in the control group. Changes in TGFβ1/β3 and α-SMA expression are associated with miR-29 and miR-155. This approach could prove beneficial for ocular surface tissue engineering applications.

Study on the Mechanism of Action of the Traditional Chinese Medical Prescription Gushukang in Treating Osteoporosis Based on Network Pharmacology and Experimental Verification.

Gushukang (GSK), a traditional Chinese medical prescription, has made a great and extensive contribution to the treatment of different forms of osteoporosis, but polypharmacology studies of its mechanism of action are lacking. This study investigates the pharmacological mechanism of osteoporosis using network pharmacology and molecular docking. Experimental verification was carried out to confirm the efficacy of GSK on RANKLinduced osteoclast differentiation in RAW264.7 cells to verify the network pharmacology studies. The effective chemical components and corresponding targets of osteoporosis with oral bioavailability of more than 30% and drug-like properties greater than 0.18 were searched in the TCMSP and TCM-ID databases. DrugBank, GeneCards, OMIM, TTD, and other databases were examined for targets related to osteoporosis. Using Cytoscape software, a network of possible TCM-active ingredient-osteoporosis targets was created. STRING software was used to create the networks of protein-protein interactions. The DAVID program was carried out to conduct GO and KEGG pathway enrichment analyses of the targets. Molecular docking and pattern of action analysis were carried out using software like AutoDock Vina and Discovery Studio Visualizer. The growth media for RAW264.7 cells contained varying doses of GSK serum and 50 ng/mL RANKL. The activity of TRAP was altered. Additionally, genes related to osteoclasts were examined using an RT-PCR assay. Network pharmacological analysis revealed that the primary efficacy targets of osteoporosis were PTGS2, PTGS1, HSP90AA1, NCOA2, ADRB2, ESR1, NCOA1, and AR. The pharmacological targets of osteoporosis may be mediated by substances including quercetin, kaempferol, luteolin, naringenin, icariin, anthocyanin, tanshinone IIA, and cryptotanshinone. GSK markedly inhibited RANKL-induced TRAP activity. qRT-PCR results revealed decreased expression of the PTGS2 and ADRB2 genes upon GSK treatment. The findings of network pharmacology, molecular docking, as well as experimental verification provide a new further study for elucidating the pharmacodynamic substance basis and polypharmacology mechanism of GSK in treating osteoporosis.

Characteristics of Aqueous Chemical Species Generation in Plasma-Facing Liquid Systems Using Helium Jet Plasma.

Plasma-facing liquids (PFLs) facilitate the storage of reactive O and N species (RONS), including H2O2 and NO2 -, which remain in the PFL after plasma treatment, and they can continuously influence the target immersed in the liquid. However, their behaviors and levels of generation and extinction depend strongly on the plasma characteristics and liquid condition. Therefore, understanding the effects of the liquid type on the plasma discharge characteristics and the RONS generated via plasma discharge is necessary. We compared the RONS generation and storage trends of deionized H2O and a high-conductivity PFL, RPMI 1640, which is a well-known cell culture medium commonly used to culture mammalian cells. RPMI 1640 acted as an electrode and enhanced the plasma discharge power by supplying abundant radicals and RONS. The production of gaseous hydroxyl radicals and NO markedly increased, which facilitated H2O2 and NO2 - production in the PFL for the first 200 s, and then the increase in the RONS concentration stagnated. With respect to storage, as the components within RMPI 1640 exhibited high reaction constants for their reactions with H2O2, H2O2 elimination was completed in <30 min. Unlike H2O2, the concentration of NO2 - in the PFL was unchanged.

Effects of 6PPD-Quinone on Human Liver Cell Lines as Revealed with Cell Viability Assay and Metabolomics Analysis

N-(1,3-Dimethyl butyl)-N′-phenyl-phenylenediamine-quinone (6PPD-Q) is a derivative of the widely used rubber tire antioxidant 6PPD, which was first found to be acutely toxic to coho salmon. Subsequent studies showed that 6PPD-Q had species-specific acute toxicity in fishes and potential hepatotoxicity in mice. In addition, 6PPD-Q has been reported in human urine, demonstrating the potential widespread exposure of humans to this chemical. However, whether 6PPD-Q poses a higher risk to humans than its parent compound, 6PPD, and could cause adverse effects in humans is still unclear. In this study, we utilized two human liver cell models (the human proto-hepatocyte model L02 and the human hepatocellular carcinoma cell line HepG2) to investigate the potentially differential effects of these two chemicals. Cell viability curve analysis showed that 6PPD-Q had lower IC50 values than 6PPD for both liver cell lines, suggesting higher toxicity of 6PPD-Q to human liver cells than 6PPD. In addition, L02 cells are more sensitive to 6PPD-Q exposure, which might be derived from its weaker metabolic transformation of 6PPD-Q, since significantly lower levels of phase I and phase II metabolites were detected in 6PPD-Q-exposed L02 cell culture medium. Furthermore, pathway analysis showed that 6PPD-Q exposure induced changes in phenylalanine, tyrosine, and tryptophan biosynthesis and tyrosine metabolism pathways in L02 cells, which might be the mechanism underlying its liver cell toxicity. Gene expression analysis revealed that exposure to 6PPD-Q induced excessive ROS production in L02 cells. Our results further supported the higher risk of 6PPD-Q than 6PPD and provided insights for understanding the effects of 6PPD-Q on human health.

Quality Control of L-Ascorbic Acid 2-Phosphate Magnesium Using Reversed-Phase Liquid Chromatography.

L-Ascorbic acid 2-phosphate magnesium (AP-Mg) salt is a Vitamin C derivative frequently used as a raw material in cell culture media for research purposes as well as for Good Manufacturing Practice (GMP)-manufacturing of cell and tissue advanced therapy medicinal products. A selective reversed-phase HPLC (RP-LC) method was developed and validated. Commercially available AP-Mg products from different suppliers were analyzed. Various new impurities were found using this newly developed RP-LC method. Using quantitative nuclear magnetic resonance spectroscopy, a mass balance of roughly 99.9% was obtained; the total numbers of impurities detected in both methods are also identical. The values of the relative ultraviolet (UV) response factors at λ = 210nm of the impurities in this RP-LC method were discussed. When equaling the overall mean relative response factor of the impurities to 0.6 (estimated central value), the mass balance in the RP-LC method was nearly 100%. The structures of the new impurities are proposed as ethylation derivatives of open-ring AP-Mg products as well as phosphorylated derivatives of ascorbic acid.

A new hiPSC-based viral expression system to study virus host-cell interaction in hiPSC-derived cardiomyocytes

Abstract Coxsackievirus B3 (CVB3) belongs to the Picornaviridae family and is a member of the enterovirus genus. CVB3 is the most frequently detected pathogen in viral myocarditis, which leads to acute heart infections in 20-40% of cases and to dilated cardiomyopathy (DCM) in cases of prolonged viral persistence. Complex arrhythmias and sudden deaths due to CVB infections have also been reported, particularly in newborns and children. Observed arrhythmias include bradycardia, AV block and junctional ectopic tachycardia, suggesting that in addition to the functioning myocardium, the pacemaker cells and cardiac conduction system may also be impaired by CVB3 infection. The symptomatic phenotype is highly variable and depends on unclear host factors. It ranges from inconspicuous inflammation and rapid healing to acute congestive heart failure or viral persistence with progressive chronic disease. Despite the increasing and profound knowledge about viral myocarditis, it is still a challenge to identify further host factors that contribute to acute and chronic myocarditis, as science lacks sensitive and controllable disease models. The presented project shows the generation of a controlled, inducible virus-expressing hiPSC line that provides a solution for the beforementioned problems. The new human induced pluripotent stem cell line (hiPSC) i can be used to carry out virologic experiments in a controlled manner and without risk to the user. For this purpose, the modified and non-infectious genome of the enterovirus Coxsackievirus B3 (CVB3) is stably integrated into the genome of a wild-type hiPSC line. The CVB3 genome to be stably integrated was modified to prevent the formation of functional capsids by a DVP0 mutation. In addition, the 3'UTR and 5'UTR regions of the CVB3 genome were removed, which prevents uncontrolled self-replication of viral RNA. As a result, work with this CVB3DVP0-UTR genome is classified as S1 compliant. The expression of the CVB3DP0-UTR virus genome can be controlled by adding doxycycline to the cell culture medium by a TeT-On System. The transfected cells are then cultivated as clones and tested for virus expression. The dose-dependent CVB3DVP0 expression is verified using qPCR and FACS after 1 day of doxycycline application in the cell culture medium. Clones isolated and positively tested for CVB3 expression are used to carry out initial experiments on the interaction of the expressed viral proteins with the cell. Particular focus is placed on the induction of cell death, remodelling of the cytoskeleton and increase of reactive oxygen species (ROS) in mitochondria after CVB3DVP0-UTR induction in order to validate the cell system for application. Differentiation of the CVB3-expressing hiPSC-line into cardiomyocytes with subsequent virus induction results in the elevation of mitochondrial ROS, degradation of the cytoskeleton and arrhythmic behaviour of the cells.

Discovery, characterization, and engineering of an advantageous Streptomyces host for heterologous expression of natural product biosynthetic gene clusters

BackgroundStreptomyces is renowned for its robust biosynthetic capacity in producing medically relevant natural products. However, the majority of natural products biosynthetic gene clusters (BGCs) either yield low amounts of natural products or remain cryptic under standard laboratory conditions. Various heterologous production hosts have been engineered to address these challenges, and yet the successful activation of BGCs has still been limited. In our search for a valuable addition to the heterologous host panel, we identified the strain Streptomyces sp. A4420, which exhibited rapid initial growth and a high metabolic capacity, prompting further exploration of its potential.ResultsWe engineered a polyketide-focused chassis strain based on Streptomyces sp. A4420 (CH strain) by deleting 9 native polyketide BGCs. The resulting metabolically simplified organism exhibited consistent sporulation and growth, surpassing the performance of most existing Streptomyces based chassis strains in standard liquid growth media. Four distinct polyketide BGCs were chosen and expressed in various heterologous hosts, including the Streptomyces sp. A4420 wild-type and CH strains, alongside Streptomyces coelicolor M1152, Streptomyces lividans TK24, Streptomyces albus J1074, and Streptomyces venezuelae NRRL B-65442. Remarkably, only the Streptomyces sp. A4420 CH strain demonstrated the capability to produce all metabolites under every condition outperforming its parental strain and other tested organisms. To enhance visualization and comparison of the tested strains, we developed a matrix-like analysis involving 15 parameters. This comprehensive analysis unequivocally illustrated the significant potential of the new strain to become a popular heterologous host.ConclusionOur engineered Streptomyces sp. A4420 CH strain exhibits promising attributes for the heterologous expression of natural products with a focus on polyketides, offering an alternative choice in the arsenal of heterologous production strains. As genomics and cloning strategies progress, establishment of a diverse panel of heterologous production hosts will be crucial for expediting the discovery and production of medically relevant natural products derived from Streptomyces.

Fabrication of 3D engineered intestinal tissue producing abundant mucus by air-liquid interface culture using paper-based dual-layer scaffold.

Fabrication of engineered intestinal tissues with the structures and functions as humans is crucial and promising as the tools for developing drugs and functional foods. The aim of this study is to fabricate an engineered intestinal tissue from Caco-2 cells by air-liquid interface culture using a paper-based dual-layer scaffold and analyze its structure and functions. Just by simply placing on a folded paper soaked in the medium, the electrospun gelatin microfiber mesh as the upper cell adhesion layer of the dual-layer scaffold was exposed to the air, while the lower paper layer worked to preserve and supply the cell culture medium to achieve stable culture over several weeks. Unlike the flat tissue produced using the conventional commercial cultureware Transwell, the engineered intestinal tissue fabricated in this study formed three-dimensional villous architectures. Microvilli and tight junction structures characteristic of epithelial tissue were also formed at the apical side. Furthermore, compared to the tissue prepared by Transwell, mucus production was significantly larger and the enzymatic activities of drug metabolism and digestion were almost equivalent. In conclusion, the air-liquid interface culture using the paper-based dual-layer scaffold developed in this study was simple but effective in fabricating the engineered intestinal tissue with superior structures and functions.

Rice Weevil (Sitophilus oryzae L.) Gut Bacteria Inhibit Growth of Aspergillus flavus and Degrade Aflatoxin B1

In this study, bacteria residing in the gut of the rice weevils (Sitophilus oryzae L.) (Coleoptera: Curculionidae) feeding on aflatoxin-contaminated corn kernels were isolated and evaluated for their ability to suppress Aspergillus flavus and to remove/degrade aflatoxin B1 (AFB1). Four morphologically distinct S. oryzae gut-associated bacterial isolates were isolated and identified as Bacillus subtilis (RWGB1), Bacillus oceanisediminis (RWGB2), Bacillus firmus (RWGB3), and Pseudomonas aeruginosa (RWGB4) based on 16S rRNA gene sequence analysis. These bacterial isolates inhibited A. flavus growth in the dual culture assay and induced morphological deformities in the fungal hyphae, as confirmed by scanning electron microscopy. All four bacterial isolates were capable of removing AFB1 from the nutrient broth medium. In addition, culture supernatants of these bacterial isolates degraded AFB1, and the degradation of toxin molecules was confirmed by liquid chromatography-mass spectrometry. The bacterial isolates, B. subtilis RWGB1, B. oceanisediminis RWGB2, and P. aeruginosa RWGB4, were capable of producing antifungal volatile organic compounds that inhibited A. flavus growth. These results suggest that the bacterial isolates from S. oryzae gut have the potential to bind and/or degrade AFB1. Further research on their application in the food and feed industries could enhance the safety of food and feed production.

Study of metal organic framework with siRNA for overcoming matrix barrierin breast cancer

Objective: This study aimed to develop a new delivery strategy that utilized metal organic framework (MOF) loaded with small-interfering RNA (siRNA) targeting ITGAV to overcome tumor matrix barrier, and thus enhance drug penetration and immune accessibility in breast cancer. Methods: MOF@siITGAV particles were constructed and characterized. The uptake of MOF@siITGAV in breast cancer cell line 4T1 was observed by the cellular uptake assay. The toxicity of MOF@siITGAV was detected by cell counting kit 8 (CCK-8). The blank control group, naked siITGAV group and MOF@siITGAV group were set. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to detect the expressions of ITGAV. The level of transforming growth factor β1 (TGF-β1) in the cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA). The penetration of MOF@siITGAV in 4T1 cells was tested by constructing 3D spheroids. Mouse models of triple negative breast cancer were established. The effect of MOF@siITGAV on the growth of transplanted tumors and main organs was verified. Imminohistochemical (IHC) was used to test the expression of collagen and CD8. Results: MOF@siITGAV particles were constructed with sizes of (198.0±3.3) nm and zeta potential of -(20.2±0.4) mV. MOF@siITGAV could be engulfed by 4T1 cells and triggered to release siRNA. Compared to the blank control group, the expression of ITGAV in the MOF@siITGAV group [(46.5±11.3)%] and the naked siITGAV group [(109.9±19.0)%] was lower. TGF-β1 in the cell culture medium of the blank control group, naked siITGAV group, and MOF@siITGAV group was (474.5±34.4) pg/ml, (437.2±16.5) pg/ml, and (388.4±14.4) pg/ml, respectively. MOF@siITGAV could better penetrate into 4T1 spheroids and exhibit no obvious toxicity. The cell viability was (99.7±3.5)%, (98.2±5.2)%, (97.3±6.6)%, (92.1±8.1)%, and (92.4±4.1)%, respectively, after MOF@siITGAV treatment with the concentration of 0, 10, 20, 40, 80, and 160 μg/ml, respectively, for 24 h. The tumor growth in the MOF@siITGAV group was suppressed significantly. After 15-day treatment, the tumor volume of the MOF@siITGAV group was (135.3±41.9) mm3, smaller than that of the blank control group [(691.1±193.0) mm3] (P=0.025). The expression of collagen and the number of CD8 positive cells of the MOF@siITGAV group were lower than those of the other two groups. No significant abnormalities were observed in the main organs of mice. Conclusions: Targeting the integrinαv on the surface of cancer cells could destroy extracellular matrix, improve drug delivery, and increase immune infiltration.

Cartilage-Specific Gene Expression and Extracellular Matrix Deposition in the Course of Mesenchymal Stromal Cell Chondrogenic Differentiation in 3D Spheroid Culture

Articular cartilage damage still remains a major problem in orthopedical surgery. The development of tissue engineering techniques such as autologous chondrocyte implantation is a promising way to improve clinical outcomes. On the other hand, the clinical application of autologous chondrocytes has considerable limitations. Mesenchymal stromal cells (MSCs) from various tissues have been shown to possess chondrogenic differentiation potential, although to different degrees. In the present study, we assessed the alterations in chondrogenesis-related gene transcription rates and extracellular matrix deposition levels before and after the chondrogenic differentiation of MSCs in a 3D spheroid culture. MSCs were obtained from three different tissues: umbilical cord Wharton’s jelly (WJMSC—Wharton’s jelly mesenchymal stromal cells), adipose tissue (ATMSC—adipose tissue mesenchymal stromal cells), and the dental pulp of deciduous teeth (SHEDs—stem cells from human exfoliated deciduous teeth). Monolayer MSC cultures served as baseline controls. Newly formed 3D spheroids composed of MSCs previously grown in 2D cultures were precultured for 2 days in growth medium, and then, chondrogenic differentiation was induced by maintaining them in the TGF-β1-containing medium for 21 days. Among the MSC types studied, WJMSCs showed the most similarities with primary chondrocytes in terms of the upregulation of cartilage-specific gene expression. Interestingly, such upregulation occurred to some extent in all 3D spheroids, even prior to the addition of TGF-β1. These results confirm that the potential of Wharton’s jelly is on par with adipose tissue as a valuable cell source for cartilage engineering applications as well as for the treatment of osteoarthritis. The 3D spheroid environment on its own acts as a trigger for the chondrogenic differentiation of MSCs.