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Scanning Electrochemical Microscopy (SECM) has been used as a non-invasive electrochemical technique for studying cellular processes. SECM enables the quantification of cellular metabolites in real-time providing a deeper understanding of cellular responses to external stimuli. SECM imaging of living cells requires maintaining an ideal physiological environment to ensure reliable data collection on cellular reactivity. The cellular response can be directly influenced by physicochemical parameters including cell media composition, temperature and light exposure. This research demonstrates the effect of media composition on the electrochemical current signal of adenocarcinoma cervical cancer (HeLa) cells during SECM measurements using ferrocenemethanol as a redox mediator. Investigated media that are commonly used as electrolyte, are phosphate buffered saline (PBS), and Dulbecco's modified Eagle's medium (DMEM) in the absence and presence of fetal bovine serum (FBS). In addition, this research demonstrates that fluctuating light illumination impacts the stability of the cellular electrochemical current response. Our findings reveal that media composition and illumination are important parameters that must be carefully considered and monitored during SECM live cell imaging.

Endogenous H 2 S has been proposed to be a universal defense mechanism against different antibiotics. Here, we studied the role of H 2 S transiently generated during ciprofloxacin (CF) treatment in M9 minimal medium with sulfate or produced by E. coli when fed with cystine. The cysM and mstA mutants did not produce H 2 S, while gshA generated more H 2 S in response to ciprofloxacin in cystine-free media. All mutants showed a reduced ability to maintain cysteine homeostasis under these conditions. We found no relationship between H 2 S generation, cysteine concentration and sensitivity to ciprofloxacin. Excess cysteine, which occurred during E. coli growth in cystine-fed media, triggered continuous H 2 S production, accelerated glutathione synthesis and cysteine export. This was accompanied by a twofold increase in ciprofloxacin tolerance in all strains except gshA, whose sensitivity increased 5-8-fold at high CF doses, indicating the importance of GSH in restoring the intracellular redox situation during growth in cystine-fed media., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study presents comprehensive insights into the microbiological profile across all concentrated chicken broth processing stages, utilizing a combination of amplicon sequencing based on metataxonomic and culturing techniques. Samples were systematically collected throughout the production chain, with each batch yielding 10 samples per day across eight different dates. These samples underwent thorough analysis, including 16S rRNA and ITS sequencing (n = 30), culture-dependent microbiological tests (n = 40), and physical-chemical characterization (n = 10). Culturing analysis revealed the absence of Listeria monocytogenes and Salmonella spp. at any stage of processing, counts of various microorganisms such as molds, yeasts, Enterobacteria, and others remained below detection limits. Notably, spore counts of selected bacterial groups were observed post-processing, indicating the persistence of certain species, including Bacillus cereus and Clostridium perfringens, albeit in low counts. Furthermore, the study identified a diverse array of bacterial and fungal species throughout the processing chain, with notable occurrence of spore-forming bacteria. The presence of spore-forming bacteria in the final product, despite thermal processing, suggests the need for enhanced strategies to mitigate their introduction and persistence in the processing premises. Thus, this study offers valuable insights into microbial dynamics and diversity through processing concentrated chicken broth., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this work, we demonstrate the use of capillary electrophoresis and inductively coupled plasma mass spectrometry, as competitive methods primarily for ion chromatography, to determine changes in the concentration of small inorganic ions in the Nostoc sp. culture medium. Although macronutrients were analyzed by capillary electrophoresis with conductivity detection, micronutrients were analyzed by inductively coupled plasma mass spectrometry. The different light settings (light intensity and spectral composition) had a visible effect on the culture growth and depletion of calcium, magnesium, and phosphate ions, and iron and manganese elements when comparing the behavior under red or violet light with that under blue light., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Some fungi have demonstrated the ability to adapt rapidly to changing environments by exhibiting morphological plasticity, a trait influenced by species and environmental factors. Here, an anamorphic yeast strain IOJ-3 exhibited unique sectorization characteristics, naturally producing diverse filamentous sectors when cultivated on potato dextrose agar (PDA) medium or natural culture medium for durations exceeding 13 days. The strain IOJ-3 and its filamentous sectors were identified as Dothiora sorbi. The morphology of the sectors was consistent and heritable. The life cycle of strain IOJ-3 was investigated through microscopic observation, emphasizing the development of conidiogenous cells as a crucial stage, from which filamentous sectors originate. Some physiological characteristics of IOJ-3 and filamentous sectors are compared, and strain IOJ-3 has a higher antibiotic tolerance than two filamentous sectors, IOJ-3a expands faster on the culture medium, and IOJ-3b can penetrate cellophane. A transcriptomic analysis was conducted to investigate the differentially expressed genes between the yeast form IOJ-3 and its two filamentous sectors, revealing a total of 594 genes that exhibited consistent differential expression relative to IOJ-3, including 44 silencing genes in IOJ-3 that were activated. Gene Ontology analysis indicated that these differentially expressed genes were primarily associated with the cellular component category. Furthermore, adding 5-Azacytidine accelerated filamentous sectorization and increased the proportion of filamentous cells of strain IOJ-3 in PD liquid media, suggesting that the filamentous sectorization observed in strain IOJ-3 is linked to processes of DNA demethylation. In conclusion, this study sheds light on the biological characteristics of D. sorbi regarding morphological transitions and provides substantial direction for exploring genes related to fungal filamentous development., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jihong Jiang reports financial support was provided by National Natural Science Foundation of China. Yong Sun reports financial support was provided by Jiangsu Normal University, China., (Copyright © 2024 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Postbiotics are metabolic by-products from microorganisms that provide health benefits to the host. Their secretion can be influenced by various conditions affecting bacterial metabolism. This study presents a novel approach for producing potential postbiotics, specifically extracellular products (ECPs), from the probiotic strain Shewanella putrefaciens SpPdp11, grown under different culture conditions. These conditions include aquafeed media, with partial or total microalgae/cyanobacteria replacement as the microbial substrate, as well as variations in temperature and growth phase. The use of microalgae/cyanobacteria as substrates may represent a valuable strategy for generating novel postbiotics with unique properties. The ECPs assessed were evaluated for their in vitro cytotoxic, hydrolytic and antimicrobial activities. Three conditions (ECPs derived from aquafeed media with partial (FM2324 and FM1548) or total (M2324) microalgae/cyanobacteria replacement) were non-cytotoxic to various fish cell lines and hydrolysed key nutritional compounds (casein, lipids, amylase and gelatin). Proteomic analysis of these ECP conditions revealed common structural and regulatory DNA-associated proteins, while differentially expressed proteins were associated with amino acid metabolism and antioxidant system (FM2324 and FM1548) and chemotaxis system (M2324). The results highlight the potential of the selected postbiotics as feed additives for future in vivo studies, aligning with sustainable development for aquaculture., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Amino acids, including asparagine, aspartate, glutamine, and glutamate, play important roles in purine and pyrimidine biosynthesis as well as serve as anaplerotic sources fueling the tricarboxylic acid (TCA) cycle for mitochondrial energy generation. Despite extensive studies on glutamine and glutamate in CHO cell cultures, the roles of asparagine and aspartate, especially in feed media, remain underexplored. In this study, we utilized a CHO genome scale model to first deeply characterize the intracellular metabolic states of CHO cells cultured in different combinations of basal and feed media to understand the traits of asparagine/aspartate-dependent and glutamate-dependent feeds. Subsequently, we identified the critical role of asparagine and aspartate in the feed media as anaplerotic sources and conducted in silico simulations to ascertain their optimal ratios to improve cell culture performance. Finally, based on the model simulations, we reformulated the feed media by tailoring the concentrations of asparagine and aspartate. Our experimental data reveal a CHO cell preference for asparagine compared with aspartate, and thus maintaining an optimal ratio of these amino acids is a key factor for achieving optimal CHO cell culture performance in biopharmaceutical production., (© 2024 Wiley‐VCH GmbH.)

The first downstream processing step in the purification of a biopharmaceutical protein secreted into mammalian cell culture fluid is the primary clarification of the culture fluid. As cell densities in the fed-batch and increasingly more common perfusion bioreactors have increased over last two decades through intensified upstream bioreactor production processes, the traditional primary clarification unit operations of centrifugation and/or microfiltration become more challenging with issues like frequent desludging, cell disruption due to shear damage and quick fouling of membranes. We have developed a novel compact cell settler device exploiting the enhanced sedimentation on inclined surfaces and demonstrated that this settler device can be adapted easily to remove and contain cells or cell clumps from the clarified supernatant collected via the top effluent of the settler. In this work, we present high product recovery results during primary clarification of mammalian cell culture supernatant using our novel single-use disposable BioSettler150 while processing about 10 L of cell culture broth within short processing times of about 4 h., (© 2024 American Institute of Chemical Engineers.)

Self-healing cement takes advantage of microbial induced carbonate precipitation (MICP), a meritorious biological process, to achieve automatic healing of cement cracks. In this study, two beneficial factors, optimization of the bacteria culture medium and encapsulation of bacterial spores, were used to improve the MICP efficiency of Sporosarcina pasteurii in self-healing cement. On the one hand, in medium optimization, we compared the growth of Sporosarcina pasteurii fed with two generally used nitrogen sources, e.g., urea and ammonium chloride, and found that ammonium chloride can promote biomineralization more efficiently than urea. It was also confirmed that nickel (0.1 mg/l) and manganese ions (10 mg/l) benefit the MICP process through enhancement of urease activity and promotion of spore production. On the other hand, spores encapsulated in sodium alginate-gelatin gel beads prepared by using a flow nozzle device can have excellent swelling performance triggered by water. As an application demonstration, self-healing of cement cracks with consideration of the above beneficial factors was successfully verified without substantial influence on the cement compressive strength., (2024 Published under an exclusive license by the AVS.)

Due to its spore-forming ability, Bacillus coagulans has advantages over the other non-spore-forming probiotics. Among them, survival and stability during food processing and storage, resistance to acid pH, and digestive enzymes are important. However, there are few studies on the quality and amount of sporulation in B. coagulans. This study investigated the spore densities and formation efficiency of B. coagulans. The optimal medium formulation consisted of yeast extract (1.00 g L -1 ), potassium acetate (20.00 g L -1 ), and MnSO 4 (0.01 g L -1 and 0.03 g L -1 ). After reaching the optimal medium, a response surface regression equation was established based on the results of central composite design (CCD) experimental designs to optimize time, temperature, and pH parameters. The predicted results thus obtained were in good agreement (R 2  = 95.19%) with the results obtained by performing experiments. Multiple regression analysis and analysis of variance (ANOVA) showed that pH is negative, and temperature and time dose are positive factors. The maximum spore cell densities by optimization plots have obtained 9.80 log at temperature 83.77 °C, pH 3.05, and time 111.19 h, considering that B. coagulans needs special environmental and cellular conditions to enter the sporulation stage. In this study, the composition of the culture medium and factors such as temperature, time, and pH were considered influencing factors in B. coagulans sporulation., Competing Interests: Declarations. Ethics Approval and Consent to Participate: The study was approved by the ethics committee of Shiraz University of Medical Sciences, Shiraz, Iran (IR.SUMS.REC.1401.153). This article does not contain any studies with human participants or animals performed by any of the authors. Consent for Publication: Not applicable. Conflict of Interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Heavy metal Cd 2+ can easily be accumulated by fungi, causing significant stress, with the fungal cell membrane being one of the primary targets. However, the understanding of the mechanisms behind this stress remains limited. This study investigated the changes in membrane lipid molecules of Pleurotus ostreatus mycelia under Cd 2+ stress and the antagonistic effect of Ca 2+ on this stress. Cd 2+ in the growth media significantly inhibited mycelial growth, with increasing intensity at higher concentrations. The addition of Ca 2+ mitigated this Cd 2+ -induced growth inhibition. Lipidomic analysis showed that Cd 2+ reduced membrane lipid content and altered lipid composition, while Ca 2+ counteracted these changes. The effects of both Cd 2+ and Ca 2+ on lipids are dose dependent and phosphatidylethanolamine appeared most affected. Cd 2+ also caused a phosphatidylcholine/phosphatidylethanolamine ratio increase at high concentrations, but Ca 2+ helped maintain normal levels. The acyl chain length and unsaturation of lipids remained unaffected, suggesting Cd 2+ doesn't alter acyl chain structure of lipids. These findings suggest that Cd 2+ may affect the growth of mycelia by inhibiting the synthesis of membrane lipids, particular the synthesis of phosphatidylethanolamine, providing novel insights into the mechanisms of Cd 2+ stress in fungi and the role of Ca 2+ in mitigating the stress., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

A reduction in the cost of production and energy requirement is necessary for developing sustainable commercial bioprocesses. Bypassing sterilization, which is an energy and cost-intensive part of bioprocesses could be a way to achieve this. In this study, nonsterile cultivation of Yarrowia lipolytica was done on a synthetic medium containing acetic acid as the sole carbon source using two different strategies in the fed-batch mode. The contamination percentages throughout the process were measured using flow cytometry and complemented using brightfield microscopy. Maximum biomass and lipid yields of 0.57 (g biomass/g substrate) and 0.17 (g lipids/g substrate), respectively, and maximum biomass and lipid productivities of 0.085 and 0.023 g/L/h, respectively, were obtained in different fed-batch strategies. Feeding at the point of stationary phase resulted in better biomass yield and productivity with less than 2% contamination till 48 h. Feeding to maintain a minimum acetic level resulted in better lipid yield and productivity with less than 2% contamination during the complete process. The results of this study demonstrate the potential for cultivating Y. lipolytica in nonsterile conditions and monitoring the contamination throughout the process using flow cytometry., (© 2024 American Institute of Chemical Engineers.)

Recent mesoscopic characterization of nutrient-transporting channels in Escherichia coli has allowed the identification and measurement of individual channels in whole mature colony biofilms. However, their complexity under different physiological and environmental conditions remains unknown. Analysis of confocal micrographs of colony biofilms formed by cell shape mutants of E. coli shows that channels have high fractal complexity, regardless of cell phenotype or growth medium. In particular, colony biofilms formed by the mutant strain Δ ompR , which has a wide-cell phenotype, have a higher fractal dimension when grown on rich medium than when grown on minimal medium, with channel complexity affected by glucose and agar concentrations in the medium. Osmotic stress leads to a dramatic reduction in the Δ ompR cell size but has a limited effect on channel morphology. This work shows that fractal image analysis is a powerful tool to quantify the effect of phenotypic mutations and growth environment on the morphological complexity of internal E. coli biofilm structures. If applied to a wider range of mutant strains, this approach could help elucidate the genetic determinants of channel formation in E. coli colony biofilms.

Fusarioid fungi, particularly Neocosmospora solani and Fusarium oxysporum, are emerging as significant human pathogens, causing infections ranging from localized mycoses to life-threatening systemic diseases. Accurate identification and preservation of these fungi in clinical laboratories remain challenging because of their diverse morphologies and specific growth requirements. This study evaluated a novel milk-honey and malt agar (MHM) against conventional media for cultivating and preserving 60 clinical fusarioid isolates, including Neocosmospora spp. (n = 47), Bisifusarium spp. (n = 5), and Fusarium spp. (n = 8). Compared with Sabouraud dextrose 2 % agar (SDA) and malt extract agar (ME2), MHM significantly increased conidia production (p < 0.0001, mean = 3.4 × 10 3 , standard deviation (SD) = ±1.3 × 10 3 ), with results similar to those of carnation leaf agar (CLA). MHM facilitated superior preservation of fusarioid viability for up to one year at room temperature on slant cultures and over two years on swabs in Amies gel with charcoal, outperforming current methods such as Castellani (water) or cryopreservation. Morphological characterization of fusarioid fungi grown on MHM revealed distinct growth patterns and conidial structures for Neocosmospora, Bisifusarium, and Fusarium species, aiding in identifying these genera. The superior performance of MHM in stimulating conidiation, maintaining viability, and preserving morphology underscore its potential as a reference medium for medically relevant fusarioid fungi, with broad implications for clinical mycology laboratories and resource-limited settings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The fungus Phialomyces macrosporus was cultured using the One Strain Many Compounds (OSMAC) strategies to evaluate its metabolome. Variations in the nutrient culture media, culture regime, and cultivation parameters can significantly influence fungal extract quantity and chemical diversity. This study aimed to explore the mycobolome of P. macrosporus in five different culture media and two different cultivation conditions using NMR-based metabolomics. Principal component analysis (PCA) of 1 H-NMR spectra revealed clear differentiation between these samples, highlighting the rice dextrose agar medium (RDA) and potato dextrose broth (PDB) as standard complex media for conducting a fungal metabolite screening program., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)

Pseudomonas putida is widely used in industrial applications, including the recombinant proteins production, because of its natural advantageous properties. In this study, the gene encoding FleQ, the primary regulator of flagellar synthesis, was deleted to construct a new non-motile P. putida KT2440-derived strain (ΔfleQ). The non-motile cells showed reduced biofilm formation and enhanced expression of a heterologous gene in nutrient-rich media compared with the wild-type (WT) strain, attributed to the reallocation of cellular resources from flagellar synthesis and cellular motility. Additionally, the ΔfleQ strain exhibited enhanced tolerance to chloramphenicol, indicating higher ribosome production, confirmed by a higher RNA/protein ratio relative to the WT. While the WT strain showed decreased growth and a three-fold increase in reporter gene activity in minimal media, the ΔfleQ strain maintained consistent reporter gene expression and exhibited a relatively higher growth rate. This suggests that the FleQ is involved in modulating proteome allocation based on nutrient quality. The removal of FleQ allows for more flexible resource allocation, creating a chassis strain with nutrient quality-independent gene expression capacity, which could be valuable in industrial applications where consistent output is essential., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

The extremophilic nature and metabolic flexibility of Galdieria spp. highlights their potential for biotechnological application. However, limited research into continuous cultivation of Galdieria spp. has slowed progress towards the commercialization of these algae. The objective of this research was to investigate biomass productivity and growth yields during continuous photoautotrophic, mixotrophic and heterotrophic cultivation of Galdieria sp. RTK371; a strain recently isolated from within the Taupō Volcanic Zone in Aotearoa-New Zealand. Results indicate Galdieria sp. RTK371 grows optimally at pH 2.5 under warm white LED illumination. Photosynthetic O 2 production was dependent on lighting intensity with a maximal value of (133.5 ± 12.1 nmol O 2 mg biomass -1  h -1 ) achieved under 100 μmol m -2  s -1 illumination. O 2 production rates slowed significantly to 42 ± 1 and <0.01 nmol O 2 mg biomass -1  h -1 during mixotrophic and heterotrophic growth regimes respectively. Stable, long-term chemostat growth of Galdieria sp. RTK371 was achieved during photoautotrophic, mixotrophic and heterotrophic growth regimes. During periods of ammonium limitation, Galdieria sp. RTK371 increased its intracellular carbohydrate content (up to 37% w/w). In contrast, biomass grown in ammonium excess was composed of up to 65% protein (w/w). Results from this study demonstrate that the growth of Galdieria sp. RTK371 can be manipulated during continuous cultivation to obtain desired biomass and product yields over long cultivation periods., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Short-chain esters, particularly isobutyl acetate and isoamyl acetate, hold significant industrial value due to their wide-ranging applications in flavors, fragrances, solvents, and biofuels. In this study, we demonstrated the biosynthesis of acetate esters using Yarrowia lipolytica as a host by feeding alcohols to the yeast culture. Initially, we screened for optimal alcohol acyltransferases for ester biosynthesis in Y. lipolytica. Strains of Y. lipolytica expressing atf1 from Saccharomyces cerevisiae, produced 251 or 613 mg/L of isobutyl acetate or of isoamyl acetate, respectively. We found that introducing additional copies of ATF1 enhanced ester production. Furthermore, by increasing the supply of acetyl-CoA and refining the culture conditions, we achieved high production of isoamyl acetate, reaching titers of 3404 mg/L. We expanded our study to include the synthesis of a range of acetate esters, facilitated by enriching the culture medium with various alcohols. This study underscores the versatility and potential of Y. lipolytica in the industrial production of acetate esters., (© 2024 American Institute of Chemical Engineers.)

This study compared the influence of growth conditions on the composition and acidogenicity of saliva-derived microcosm biofilms and enamel demineralization. Biofilms grown in sucrose-supplemented modified McBain medium, containing 25/50 mmol/L PIPES (buffer), under anaerobiosis/microaerophilia, for 3 and 7 days were evaluated for their acidogenicity, microbial composition, matrix, and enamel mineral content. The viability of total lactobacilli was higher in the group containing 25 mmol/L PIPES grown under anaerobiosis, which also showed lower pH values. The viability of total streptococci and total microorganisms was significantly higher at 7 days in the groups with 50 mmol/L PIPES than at 3 days, regardless of the incubation atmosphere. No significant differences were observed in lactic acid, calcium, superficial hardness loss, or lesion depth. In conclusion, the incubation atmosphere, buffer content in the growth media, and duration of biofilm formation displayed species-varied influence on microcosm biofilms, without causing significant changes in acid metabolism or enamel demineralization.

Bile salt hydrolase (BSH), a pivotal enzyme in cholesterol management, holds significant promise in both human and animal subjects. This study investigated the effect of fermentation dynamics in Heyndrickxia coagulans ATCC 7050 and Lactiplantibacillus plantarum ATCC 10012 to enhance BSH production. Cultivation of cultures in MRS and M17 media revealed that MRS medium enhanced BSH production by 235.98 % in H. coagulans ATCC 7050 and 147.37 % in L. plantarum ATCC 10012, compared to M 17 medium. Additionally, varying oxygen concentration levels indicated that H. coagulans ATCC 7050 exhibited its minimum doubling time of 79.8 ± 0.64 min in anaerobic conditions, whereas L.plantarum ATCC 10012 demonstrated its minimum doubling time of 85.5 ± 1.2 min under microaerophilic conditions. However, their highest BSH activity was observed during the stationary phase under anaerobic conditions, yielding 17.14 ± 0.78 U/mL by H. coagulans ATCC 7050 and 19.04 ± 0.81 U/mL by L.plantarum ATCC 10012. Furthermore, it was observed that both organisms did not retain BSH within their cells. BSH activity was assessed using ninhydrin assay that detected free taurine liberated from sodium taurocholate. However, ninhydrin can yield false-positive results owing to its interaction with other free amino acids. To subjugate this limitation, the study introduced a novel and sensitive HPTLC-MS method capable of accurately detecting taurine. By comprehending fermentation dynamics and selecting appropriate conditions, BSH production increased 2.1-fold in both organisms. These findings illuminate critical insights, offering a pathway for novel strategies to enhance the BSH-producing capabilities of these LAB strains., Competing Interests: Declaration of competing interest The authors state that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in the paper titled “ Fermentation dynamics of bile salt hydrolase production in Heyndrickxia coagulans ATCC 7050 and Lactiplantibacillus plantarum ATCC 10012: Addressing ninhydrin assay limitations with a novel HPTLC-MS method”., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The influence of the initial pH on the morphology and secondary metabolite production in cocultures and axenic cultures of Aspergillus terreus and Streptomyces rimosus was investigated. The detected secondary metabolites (6 of bacterial and 4 of fungal origin) were not found in the cultures initiated at pH values less than or equal to 4.0. The highest mean levels of oxytetracycline were recorded in S. rimosus axenic culture at pH 5.0. Initiating the axenic culture at pH 5.9 led to visibly lower product levels, yet the presence of A. terreus reduced the negative effect of non-optimal pH and led to higher oxytetracycline titer than in the corresponding S. rimosus axenic culture. The cocultivation initiated at pH 5.0 or 5.9 triggered the formation of oxidized rimocidin. The products of A. terreus were absent in the cocultures. At pH 4.0, the striking morphological differences between the coculture and the axenic cultures were recorded., (© 2024. The Author(s).)

Polyphenols are abundant natural plant micronutrients that commonly contribute to human health due to their anti-inflammatory, antioxidant, antiviral, anti-carcinogenic, anti-aging, anti-allergic, and other biological activities. Their therapeutic benefits mainly depend on the structure, stability, chemical interactions, and absorption, which ultimately affect the bioavailability of these compounds. The bioactivity of polyphenols is evaluated by in vitro and in vivo studies, sometimes yielding inconsistent results due to numerous differences between used models. Among the main differences is the production of reactive oxygen species (ROS) in cultured cell models, potentially leading to misinterpretation of the effects of polyphenolic compounds. Little attention is paid to the polyphenol stability in cell culture medium and the potential generation of artifacts due to their chemical instability. Stability tests of polyphenols are strongly advised to be performed in parallel with cell culture, to help avoid misleading conclusions. This review highlights the existing challenges with cell-based research, focusing on polyphenols' stability in the cell culture media. We also emphasize that new methods analyzing the molecular interactions of compounds with cell culture media supplements are essential to provide a comprehensive understanding of the polyphenols in in vitro models., Competing Interests: Declaration of competing interest Una Glamočlija is employee of pharmaceutical company Bosnalijek d.d. The authors declare that there is no other conflict of interest with this manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Considering that follicular development is an energy-dependent process, supplementation of the culture medium with energy substrates, such as lactose, would improve follicle viability and growth. Thus, the aim of this study was to evaluate the effect of lactose on morphology, development, glutathione (GSH) concentration, mitochondrial activity, DNA fragmentation, and meiotic resumption of oocytes from sheep secondary follicles cultured in vitro. Secondary follicles were isolated from the cortex of ovine ovaries and cultured individually for 18 days in α-MEM supplemented with bovine serum albumin (BSA), insulin, glutamine, hypoxanthine, transferrin, selenium and ascorbic acid (control medium: α-MEM + ) or in α-MEM + plus different concentrations of lactose (0.025, 0.05 and 0.1 M). After culture, some of the oocytes were subjected to TUNEL assay and in vitro maturation (IVM). Follicular morphology, glutathione (GSH) concentration and mitochondrial activity were evaluated at the end of the culture. At the day 18, the percentage of morphologically normal follicles was greater (P<0.05) in the treatment of 0.025 M lactose (92.5 %) compared to the control group (75.55 %). In addition, GSH concentrations increased (P<0.05) in treatment containing 0.025 M lactose compared to the other treatments. Furthermore, oocytes cultured in 0.025 M lactose had greater (P<0.05) mitochondrial activity levels than in α-MEM+ and 0.1 M lactose. The group α-MEM + presented a increase of TUNEL-positive oocytes (35.09 %) compared to 0.025 lactose (9.09 %). The percentage of meiotic resumption was greater (P<0.05) in oocytes from secondary follicles cultured in 0.025 M lactose (54.5 %) than in α-MEM + (45.5 %). In conclusion, 0.025 M lactose improved survival, GSH and active mitochondria levels and meiotic resumption of oocytes from in vitro cultured secondary follicles. Supplementation of the culture medium of preantral follicles with lactose can gradually provide energy to follicular cells, potentially enhancing the production of viable oocytes for biotechniques such as IVM and in vitro fertilization., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bacterial cellulose (BC) is an emerging material for high-end applications due to its biocompatibility and physicochemical characteristics. However, the scale-up production of this material is still expensive, with the culture medium constituting one-third of the total cost. Herein, four different media (yeast nitrogen base, YNB; Murashige and Skoog, MSO; black tea; and NPK fertilizer solution) were compared while using sucrose as an additional carbon source. The yields of BC were best for YNB and fertilizer with 0.37 and 0.34 g BC /g C respectively. These two were then compared using glucose as a carbon source, with improvements in the production of 29% for the fertilizer, while only an 8% increase for YNB was seen; however, as the carbon concentration increased with a fixed N concentration, the yield was lower but the rate of production of BC increased. The obtained BC films were sanitized and showed low molecular weight and all the expected cellulose characteristic FT-IR bands while SEM showed nanofibers around 0.1 μm. Compared to traditional methods for lab-scale production, the use of the fertilizer and the consortium represent benefits compared to traditional lab-scale BC culture methods such as a competitive cost (two times lower) while posing resilience and tolerance to stress conditions given that it is produced by microbial communities and not with a single strain. Additionally, the low molecular weight of the films could be of interest for certain coating formulations., Competing Interests: Declarations. Ethical Approval: Not applicable Consent to Participate: Not applicable Consent for Publication: All authors have agreed to the publication of this work. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mammalian embryos often suffer from oxidative stress in vitro, as the oxygen in the atmosphere is higher than that in the oviductal environment. Vitamin C (Vc) has been proven to enhance early embryonic development in vitro , but the underlying mechanism remains unclear. In this study, we investigated the pathways of action by which Vc promotes the in vitro development of porcine embryos. Comparative analysis of in vitro and in vivo gene expression profiles of morula found that most of the differentially expressed genes were enriched in pathways related to mitochondrial function. The addition of 12.5 μg/mL Vc to the culture medium significantly increased blastocyst production in a dose- and duration-dependent manner. Moreover, ROS levels were significantly higher in embryos cultured in the air (21% oxygen) than cultured in a hypoxic condition (5% oxygen) and were reduced by Vc supplementation. Vc also significantly increased the mitochondrial membrane potential levels and the expression levels of mitochondrial function-related genes ( MFN1 and OPA1 ) and TCA cycle-related genes ( PDHA1 and OGDH ) in embryos cultured in vitro . These results suggest that the addition of Vc to the in vitro culture medium can increase the developmental potential and improve the mitochondrial function of early porcine embryos.

Raffinose, a trisaccharide abundantly found in soybeans, is a potential alternative carbon source for biorefineries. Nevertheless, residual intermediate di- or monosaccharides and low catabolic efficiency limit raffinose use through conventional microbial hosts. This study presents a Vibrio-based platform to convert raffinose efficiently. Vibrio sp. dhg was selected as the starting strain for the Adaptive Laboratory Evolution (ALE) strategy to leverage its significantly higher metabolic efficiency. We conducted ALE on a solid minimal medium supplemented with raffinose to prevent the enrichment of undesired phenotypes due to the shared effect of extracellular raffinose hydrolysis among multiple strains. As a result, we generated the VRA10 strain that efficiently utilizes raffinose without leaving behind degraded di- or monosaccharides, achieving a notable growth rate (0.40 h -1 ) and raffinose consumption rate (1.2 g/g dcw /h). Whole genome sequencing and reverse engineering identified that a missense mutation in the melB gene (encoding a melibiose/raffinose:sodium symporter) and the deletion of the two galR genes (encoding transcriptional repressors for galactose catabolism) facilitated rapid raffinose utilization. The further engineered strain produced 6.2 g/L of citramalate from 20 g/L of raffinose. This study will pave the way for the efficient utilization of diverse raffinose-rich byproducts and the expansion of alternative carbon streams in biorefinery applications., Competing Interests: Declaration of interest The authors declare no competing interests., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Yeast extract (YE) is a complex nutritional source associated with high performance on microbial production processes. However, its inherent compositional variability challenges its scalability. While prior efforts have focused on growth-associated products, the dynamics of growth-uncoupled production, which leads to higher production rates and conversion yields, still need to be explored. This production scenario is common in large-scale applications. This study presents a systematic approach to replace YE for the production of the terpene amorpha-4,11-diene in Escherichia coli. Sequential processing was successfully applied to identify glutamic acid, alanine, leucine, valine, isoleucine and glycine as the key amino acids (AAs) under slow-growth conditions. Thoroughly applying biomass retention as part of sequential processing increased production capacity by 45% using these AAs instead of YE. Further studies, including flux balance analyses, targeted pyruvate as the common AA precursor. The optimized fed-batch process feeding pyruvate with 0.09 g Pyr h -1 enhanced amorpha-4,11-diene production by 37%, although adding only 1% carbon via pyruvate. Flux balance analysis revealed the criteria for optimum pyruvate feeding, for example, to prevent succinate secretion and maintain the NADH/NAD + balance. These findings illustrate the interplay between media composition and metabolic activity and provide a successful guideline for identifying lean, best-performing media for industrial applications., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Background: Recombinant myofibril-bound serine proteinase (rMBSP) was successfully expressed in Pichia pastoris GS115 in our laboratory. However, low production of rMBSP in shake flask constraints further exploration of properties., Methods: A 5-L high cell density fermentation was performed and the fermentation medium was optimized. Response surface methodology (RSM) was used to optimize the culture condition through modeling three selected parameter., Results: Under the optimized culture medium (LBSM, 1% yeast powder and 1% peptone) and culture conditions (induction pH 5.5, temperature 29 °C, time 40 h), the yield of rMBSP was 420 mg/L in a 5-L fermenter, which was a 6-fold increase over thar, expressed in flask cultivation. The desired enzyme was purified by two-step, which yielded a 33.7% recovery of a product that had over 85% purity. The activity of purified rMBSP was significantly inhibited by Ca 2+ , Mg 2+ , SDS, guanidine hydrochloeide, acetone, isopropanol, chloroform, n -hexane and n -heptane. Enzymatic analysis revealed a K m of 2.89 ± 0.09 μM and a V max of 14.20 ± 0.12 nM•min -1 for rMBSP. LC-MS/MS analysis demonstrated the specific cleavage of bovine serum albumin by rMPSP., Conclusion: These findings suggest that rMPSP has potential as a valuable enzyme for protein science research.

Microalgae are recognized as a sustainable resource to produce biofertilizers, biofuels, and pigments, with the added benefits of environmental sustainability, such as carbon sequestration and pollutant removal. However, traditional cultivation methods face challenges like low biomass productivity and high operational costs. This review focuses on the innovative use of hydrogels as a medium for microalgae cultivation, which addresses these challenges by enhancing nutrient permeability, light distribution, and overall growth efficiency. Hydrogels provide a three-dimensional matrix that not only supports higher biomass yields but also facilitates the removal of pollutants from wastewater, contributing to circular economy goals. The review also explores the environmental benefits, challenges, and prospects of integrating hydrogel technology into microalgae cultivation systems. By highlighting influencing factors through which hydrogels improve microalgal productivity and environmental outcomes, this work aims to provide insights into the potential of hydrogel-based systems for sustainable development.

Background: The importance of embryonic stem cells (ESCs) in chickens is undeniable, as they can be applied across various fields, including animal modeling, developmental biology, cell fate research, drug screening, toxicity testing, and gene function studies. However, a widely applicable culture system for chicken ESCs has yet to be developed., Objectives: This study aimed to investigate the effects of different culture systems on the derivation and maintenance of chicken ESCs, with a focus on optimizing the selected culture conditions., Methods: To achieve this, we tested the effectiveness of various species-specific ESC media in the derivation and culture of chicken PGCs, while incorporating different small molecule compounds to optimize the process. The pluripotency and differentiation potential of the resulting ESC-like cells were also evaluated., Results: The combination of PD0325901, SB431542, and LIF (R2i+LIF system) was found to be effective in generating chicken ESC-like clones. Further experiments showed that enhancing the R2i+LIF system with cytokines such as SCF and FGF2 significantly extended the culture period and increased the passage number of chicken ESC-like cells. These ESC-like cells were characterized through positive alkaline phosphatase staining and the expression of pluripotency markers POUV, NANOG, and SOX2. Additionally, differentiation assays confirmed their ability to form the three germ layers., Conclusions: The newly developed culture system provides suitable conditions for the short-term culture of chicken ESCs. However, further optimization is required to establish a system that can sustain long-term maintenance.

Background: Natural pigments are becoming more significant because of the rising cost of raw materials, pollution, and the complexity of synthetic pigments. Compared to synthetic pigments, natural pigments exhibit antimicrobial properties and is less allergic. Pigments from microbial sources could easily be obtained in an inexpensive culture media, produced in high yields, and microbes are capable of producing different colored pigments. Searching for new sources for natural pigments to replace synthetic ones in food applications has become an urgent necessity, but the instability of these compounds is sometimes considered one of the obstacles that reduce their application. Encapsulation provides an ideal solution for natural dye protection through a controlled release strategy. Thus, this study aims at isolation of several soil fungi and subsequent screening their pigment production ability. The chosen pigment-producing fungal strain underwent full identification. The produced pigment was extracted with ethyl acetate and estimated spectrophotometrically. As there is a necessity to obtain a high pigment yield for efficient industrial application, the best production medium was tested, optimum conditions for maximum dye production were also investigated through the response surface methodology, and gamma irradiation was also employed to enhance the fungal productivity. Encapsulation of the produced pigment into chitosan microsphere was tested. The pigment release under different pH conditions was also investigated., Results: A new strain, Fusarium verticillioides AUMC 15934 was chosen and identified for a violet pigment production process. Out of four different media studied, the tested strain grew well on potato dextrose broth medium. Optimum conditions are initial medium pH 8, 25 °C-incubation temperature, and for 15-day incubation period under shaking state. Moreover, a 400 Gy irradiation dose enhanced the pigment production. Chitosan microsphere loaded by the pigment was successfully prepared and characterized by infrared spectroscopy and scanning electron microscopy., Conclusion: This irradiated Fusarium strain provides a more economically favorable source for production of a natural violet dye with an optimum productivity, enhanced yield, and improved properties (such as, enhanced stability, controlled release, and bioaccessibility) by encapsulation with chitosan for efficient application in food industry., (© 2024. The Author(s).)

This study aimed to optimize the culture conditions of the termite-derived fungus Aspergillus chevalieri BYST01 for the production of physcion, a characteristic component of the traditional herb rhubarb, which has been commercially approved as a botanical fungicide in China. First, potato dextrose broth was screened as the suitable basal medium for further optimization, with an initial yield of 28.0 mg/L. Then, the suitable carbon source, fermentation time, temperature, pH value, and the rotary shaker speed for physcion production were determined using the one-variable-at-a-time method. Based on the results of single factors experiments, the variables with statistically significant effects on physcion production were further confirmed using the Plackett-Burman design (PBD). Among the five variables, temperature, initial pH, and rotary shaker speed were identified as significant factors ( P < 0.05) for physcion productivity in the PDB and were further analyzed by response surface methodology (RSM). Finally, we found that the maximum physcion production (82.0 mg/L) was achieved under the following optimized conditions:initial pH 6.6, rotary shaker speed of 177 rpm, temperature of 28 °C, and glucose concentration of 30 g/L in PDB medium after 11 d of fermentation. The yield of physcion under the optimized culture conditions was approximately threefold higher than that obtained using the basal culture medium. Furthermore, the optimum fermentation conditions in the 5-L bioreactor achieved a maximal physcion yield of 85.2 mg/L within 8 d of fermentation. Hence, response surface methodology proved to be a powerful tool for optimizing physcion production by A. chevalieri BYST01. This study may be helpful in promoting the application of physcion produced by A. chevalieri BYST01 to manage plant diseases., Competing Interests: The authors declare that they have no competing interests., (© 2024 Zhang et al.)

Establishing suitable in vitro culture conditions for microorganisms is crucial for dissecting their biology and empowering potential applications. However, a significant number of bacterial and fungal species, including Pneumocystis jirovecii, remain unculturable, hampering research efforts. P. jirovecii is a deadly pathogen of humans that causes life-threatening pneumonia in immunocompromised individuals and transplant patients. Despite the major impact of Pneumocystis on human health, limited progress has been made in dissecting the pathobiology of this fungus. This is largely due to the fact that its experimental dissection has been constrained by the inability to culture the organism in vitro. We present a comprehensive in silico genome-scale metabolic model of Pneumocystis growth and metabolism, to identify metabolic requirements and imbalances that hinder growth in vitro. We utilise recently published genome data and available information in the literature as well as bioinformatics and software tools to develop and validate the model. In addition, we employ relaxed Flux Balance Analysis and Reinforcement Learning approaches to make predictions regarding metabolic fluxes and to identify critical components of the Pneumocystis growth medium. Our findings offer insights into the biology of Pneumocystis and provide a novel strategy to overcome the longstanding challenge of culturing this pathogen in vitro., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Nev et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

WNT3A is an intestinal ligand triggering the Wnt/β-catenin (Wnt) pathway, which can be enhanced by R-spondin 1 (RSPO1) through the RSPO1-LGR axis or antagonized by the adenomatous polyposis coli (APC) protein supporting β-catenin-degradation. Wnt interplays with several pathways including PI3K/mTOR (mTOR). In this study, we evaluated the influence of WNT3A-commercial and home-made culture media and RSPO1 protein on the Wnt and mTOR interplay in non-APC and APC-mutated intestinal patient-derived organoids (PDOs). Normal mucosa (NM) of sporadic CRC and FAP PDOs were cultured with: WNT3A-lacking/containing commercial (A/A+B) or home-made (BASAL/WNT3A-conditioned medium (CM)±RSPO1) media. In non-APC-mutated-PDOs (CRC-NM), WNT3A-CM, over commercial A+B, strongly activated Wnt-target-genes CCND1 and c-MYC . Most importantly, the addition of RSPO1 to home-made WNT3A-CM or A+B led to the downregulation of the mTOR-downstream-effector phospho-S6 ribosomal protein (p-S6R), highlighting the activation of the RSPO1-pS6R in both non-APC (CRC-NM) and APC-mutated (FAP-NM) PDOs, independently from LGR5 gene expression modulation. Our work demonstrates that home-made WNT3A-CM strongly impacts the crosstalk between Wnt and mTOR over commercial media, and proposes RSPO1 as a key regulator of the RSPO1-p-S6R axis in both non-APC and APC-mutated PDOs. Together, these findings represent an important methodological guide for scientists working in these fields to select the most appropriate intestinal PDO media.

In order to obtain more effective cell culture parameters of Taxus anticancer plants, we optimized the callus induction and subculture conditions of the explants (stem segments with buds) of the anticancer medicinal plant Taxus media by using the plant tissue culture technology and orthogonal test. Furthermore, we studied the method to inhibit browning in the culture. The results indicated that the optimal conditions for inducing callus was culture in the medium composed of B 5 +0.25 mg/L 2, 4-D+1.5 mg/L NAA+0.5 mg/L KT in the dark, which showed short induction time and a high induction rate (86.7%). The formula of the optimal medium for subculture was B 5 +0.5 mg/L 2, 4-D+2.0 mg/L NAA+1.5 mg/L KT.The proliferation multiple of callus cultured by subculture on the 10th day of callus growth was the highest. Activated carbon inhibited the browning in callus subculture, with the optimal inhibitory concentration of 0.8 g/L. The results of this study lay a foundation for the production of taxol by suspension culture of T . media cells.

The study aimed to enhance exopolysaccharides (EPSs) production by the bacterial strain Bacillus subtilis ES (OR501464) isolated from sugar cane juice. Spoiled grape and fig extract were utilized as cost-effective substrates for EPS synthesis by B. subtilis ES (OR501464), and the impact of nutritional factors on EPS synthesis was assessed. Among nineteen bacterial isolates evaluated for EPS production, the isolate with the highest EPS yield was identified through a combination of phenotypic and genotypic analyses. The optimization process revealed that the highest EPS yield of 4.7 g/L was achieved in a production medium containing 4% sucrose, 0.1% NaNO 3 , 0.002% Na 2 SO4, and 2% NaCl at 30 °C and pH 9. Additionally, the study explored EPS generation by B. subtilis ES (OR501464) using spoiled grape and fig extract as substrates. The addition of 2% NaCl to spoiled grape extract increased EPS production to 4.357 mg/mL compared to 3.977 mg/mL with grape alone. However, 2% NaCl did not enhance EPSs production in fig waste. Supplementing spoiled fig or grape extract with 0.2 g/L Na 2 SO 4 and 1 g/L NaNO 3 increased EPS production by B. subtilis ES (OR501464). The EPS was analyzed using GC-MS and FTIR spectroscopy for partial characterization. The study found that spoiled figs and grapes can be used as effective substrates for EPS production. The highest yield was achieved by adding 0.2 g/L Na 2 SO 4 and 1 g/L NaNO 3 . This study highlights the use of spoiled figs and grapes to produce valuable biopolymers, promoting sustainable and eco-friendly bioprocessing technologies., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Accessibility of paclitaxel and other taxoids from natural resources is restricted. Endophytic fungi are novel, rapidly growing resources for producing these compounds. Neopestalotiopsis vitis (N. vitis) has been recently isolated from Corylus avellana, and its ability to produce a variety of taxoids has been detected and confirmed by analytical methods. Simultaneous growth and high production of taxoids by application of different sorts and concentrations of carbon and nitrogen were targeted in the present research. These criteria were assessed in different acidities (pH 4.0-7.0), carbon sources (sucrose, fructose, glucose, mannitol, sorbitol, and malt extract), and nitrogen forms (urea, ammonium nitrate, potassium nitrate, ammonium phosphate, and ammonium sulfate) by testing one parameter at a time approach. The first analysis introduced pH 7.0 as the best acidity of the medium for N. vitis, where the highest paclitaxel yield was generated. Further analysis introduced 3% Malt extract as the best carbon-providing medium. In the next step, the effects of nitrogen forms on the growth rate, paclitaxel yield, alkaloids, and amino acid contents were evaluated. Based on the results of this experiment, 5 mM ammonium sulfate was selected as the best nitrogen source to obtain the maximum biomass and paclitaxel yield. Overall, the results introduce a medium containing 3% (w/v) malt extract and 5 mM ammonium sulfate at pH 7.0 as the best medium in which N. vitis produces the highest paclitaxel yield coincident with rapid and sustainable growth. The findings pave the way for industrial manufacturing of taxoids., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rezazadeh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rationale: Natural variations in the abundance of the stable isotopes of nitrogen (δ 15 N) and carbon (δ 13 C) offer valuable insights into metabolic fluxes. In the wake of strong interest in cancer metabolism, recent research has revealed δ 15 N and δ 13 C variations in cancerous compared to non-cancerous tissues and cell lines. However, our understanding of natural isotopic variations in cultured mammalian cells, particularly in relation to metabolism, remains limited. This study aims to start addressing this gap using metabolic modulations in cells cultured under controlled conditions., Methods: Prostate cancer cells (PC3) were cultured in different conditions and their δ 15 N and δ 13 C were measured using isotope ratio mass spectrometry. Isotopic variations during successive cell culture passages were assessed and two widely used cell culture media (RPMI and DMEM) were compared. Metabolism was modulated through glutamine deprivation and hypoxia., Results: Successive cell culture passages generally resulted in reproducible δ 15 N and δ 13 C values. The impact of culture medium composition on δ 15 N and δ 13 C of the cells highlights the importance of maintaining a consistent medium composition across conditions whenever possible. Glutamine deprivation and hypoxia induced a lower δ 13 C in bulk cell samples, with only the former affecting δ 15 N. Gaps between theory and experiments were bridged and the lessons learned throughout the process are provided., Conclusions: Exposing cultured cancer cells to hypoxia allowed us to further investigate the relation between metabolic modulations and natural isotopic variations, while mitigating the confounding impact of changing culture medium composition. This study highlights the potential of natural δ 13 C variations for studying substrate fluxes and nutrient allocation in reproducible culture conditions. Considering cell yield and culture medium composition is pivotal to the success of this approach., (© 2024 The Author(s). Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

The secretion rate of albumin is a key indicator of function in liver tissue models used for hepatotoxicity and pharmacokinetic testing. However, it is not generally clear how to determine molecular secretion rates from measurements of the molecular concentration in supernatant media. Here, we develop computational and analytical models of molecular transport in an experimental system that enable determination of albumin secretion rates based on measurements of albumin concentration in supernatant media. The experimental system is a 3D-bioprinted human liver tissue construct embedded in a 3D culture environment made from packed microgel particles swollen in liquid growth media. The mathematical models reveal that the range of albumin synthesis rates necessary to match experimentally measured albumin concentrations corresponds to reaction-limited conditions, where a steady state of albumin spatial distribution is rapidly reached between media exchanges. Our results show that temporally resolved synthesis rates can be inferred from serial concentration measurements of collected supernatant media. This link is critical to confidently assessing in vitro tissue performance in applications where critical quality attributes must be quantified, like in drug development and screening.

Background: Hepatitis B virus (HBV) clearance depends on an effective adaptive immune response, especially HBV-specific T cell-mediated cellular immunity; however, it is difficult to produce enough HBV-specific T cells effectively., Results: In this work, we investigated the proportions of stimulated cells, serum, and culture media as the three primary factors to determine the most effective procedure and applied it to HLA-A2 (+) people. In parallel, we also examined the correlation between clinical parameters and HBV-specific immunity. Concerning amplification efficiency, 4 × 10 5 cells stimulation was superior to 2 × 10 6 cells stimulation, AIM-V medium outperformed 1640 medium, and fetal bovine serum (FBS) exceeded human AB serum under comparable conditions. As expected, this procedure is also suitable for developing HBV-specific CD8 + T cells in HLA-A2(+) individuals. Expanded HBV-specific T cell responses decreased with treatment time and were negatively correlated with HBV DNA and HBsAg. Furthermore, the number of HBV-specific IFN-γ + SFCs was strongly correlated with the ALT level and negatively correlated with the absolute lymphocyte count and the ALB concentration., Conclusions: We confirm that stimulating 4 × 10 5 PBMCs in AIM-V medium supplemented with 10% FBS is the best approach and that HBeAg, HBsAg, and ALB are independent predictors of HBV-specific T-cell responses., (© 2024. The Author(s).)

We propose a simple tool for liquid static culture using a copolymer film with high gas permeability. The film bags were successfully used to culture microorganisms Escherichia coli, Komagataella phaffii (methylotrophic) and Bacillus sp. (biofilm-forming), with cells cultured under physical stress-free conditions with sufficient oxygen supply. Similar growth curves and plasmid productivity were observed for liquid shake and film bag E. coli cultures. The early growth response of the film bag culture following colony inoculation of liquid media differed from conventional shake cultures. Our results indicate that a gas-permeable film bag is a promising liquid culture tool and provides novel microbiology materials., (© 2024. The Author(s).)

The human gut microbiota is increasingly being recognised to play an important role in maintaining health. The families Lachnospiraceae and Oscillospiraceae in particular, are often reduced in disease states but are relatively poorly represented in culture collections. Cultured representatives are required to investigate the physiology and host interactions of gut microbes. Establishing cultured isolate collections can be laborious and expensive owing to the fastidious growth requirements of these organisms and the costs associated with taxonomic classification. This study proposes a culturomics platform combining a single basal culture medium with matrix-assisted laser adsorption/ionisation coupled to time-of-flight mass spectrometry (MALDI-TOF MS) for fast and reliable isolation and identification of hundreds of novel isolates. In this study, basal YCFA medium supplemented with either glucose, apple pectin, or porcine mucin was used to cultivate a total of 724 different isolates derived from only 11 different faecal samples from healthy volunteers, of which 389 isolates belonged to the Lachnospiraceae and Oscillospiraceae families. Moreover, 27 isolates could not be assigned to known species based on their 16S rRNA gene, 17 of which may even represent novel genera. To aid MALDI-TOF MS identification of gut bacteria, the commercial database was complemented with the MaldiGut database presented here, containing a collection of 132 different Main Spectrum Profiles, including the profiles of 125 Firmicutes species, 3 Bacteroidetes species, 3 Actinobacteria species, and one Verrucomicrobia species. The culturomics platform and MaldiGut database presented here will enable further expansion of the gut culturome, especially within the understudied Lachnospiraceae and Oscillospiraceae families.

The prevalence of human Demodex mites has surged in recent years, prompting significant concern among both patients and the medical community. This study aimed to investigate the survival duration and morphological alterations of Demodex folliculorum under diverse temperature conditions and in various culture media. We employed the eyelash sampling technique to procure the mites. The collected specimens were then subjected to culture at two distinct temperature ranges (16-22 °C and 4 °C) across a spectrum of media, including 30% tea tree oil (TTO), phosphate-buffered saline (PBS), pure water, 0.9% physiological saline, 5 µg/ml propidium iodide (PI), liquid paraffin, glycerol, and a blank culture medium. Post-administration, the mites' activity and morphological changes were meticulously documented. Our findings indicate that the survival span of Demodex mites within the same medium was notably extended at 4 °C compared to room temperature. Specifically, under 4 °C, the use of liquid paraffin as a culture medium yielded the longest survival time of 12 days, surpassing all other conditions. Remarkably, the morphological integrity of the mites in this group remained largely unaltered. These results suggest that 4 °C is the optimal temperature for the in vitro cultivation of Demodex mites, offering insights into the environmental preferences of these organisms and potentially informing future therapeutic strategies., (© 2024. The Author(s).)

Mammalian cells are suitable hosts for producing recombinant therapeutic proteins, with Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells being the most commonly used cell lines. Mammalian cell expression system includes stable and transient gene expression (TGE) system, with the TGE system having the advantages of short cycles and simple operation. By optimizing the TGE system, the expression of recombinant proteins has been significantly improved. Here, the TGE system and the detailed and up-to-date improvement strategies of mammalian cells, including cell line, expression vector, culture media, culture processes, transfection conditions, and co-expression of helper genes, are reviewed. KEY POINTS: • Detailed improvement strategies of transient gene expression system of mammalian cells are reviewed • The composition of transient expression system of mammalian cell are summarized • Proposed optimization prospects for transient gene expression systems., (© 2024. The Author(s).)

Chemical screens across hundreds of cell lines have shown that the drug sensitivities of human cancers can vary by genotype or lineage. However, most drug discovery studies have relied on culture media that poorly reflect metabolite levels in human blood. Here, we perform drug screens in traditional and Human Plasma-Like Medium (HPLM). Sets of compounds that show conditional anticancer activity span different phases of global development and include non-oncology drugs. Comparisons of the synthetic and serum-derived components that comprise typical media trace sets of conditional phenotypes to nucleotide synthesis substrates. We also characterize a unique dual mechanism for brivudine, a compound approved for antiviral use. Brivudine selectively impairs cell growth in low folate conditions by targeting two enzymes involved in one-carbon metabolism. Cataloged gene essentiality data further suggest that conditional phenotypes for other compounds are linked to off-target effects. Our findings establish general strategies for identifying drug-nutrient interactions and mechanisms of action by exploiting conditional lethality in cancer cells.

Timely and accurate identification of yeasts is essential for adequate treatment, considering the increase in antifungal resistance of some species, particularly for C. auris . Current matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) manufacturer's protocol for identification of yeasts requires 24- to 72-h cultivation on Sabouraud dextrose media (SAB), but not some of the mainstay primary culture media used in mycology such as inhibitory mold agar (IMA), Mycosel, CHROMagar Candida Plus, and CHROMagar Candida. As culture media can influence MALDI-TOF MS identification results, this study evaluated the accuracy and performance of identification of clinically relevant yeasts on these first-line media using the VITEK-MS MALDI-TOF MS system.IMPORTANCEIn this study, a panel of 140 strains (21 species) was used to assess the performance of the selected media. Although not in the manufacturer's list of accepted media, IMA and chromogenic media are suitable for the identification of yeasts on the VITEK-MS systems. CHROMagar Candida Plus allowed the identification of 135/140 isolates tested after 24-h incubation similar to SAB reference media (137/140). Yeast isolates that grew on Mycosel selective media were also reliably identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. VITEK-MS system with IVD database V3.2 correctly identified C. auris strains to the species level on CHROMagar Candida Plus alleviating the need for subcultivation and reduced turnaround time (24-72 h) to identification for patient screening., Competing Interests: The authors declare no conflict of interest.

Pseudomonas aeruginosa , a pathogen capable of causing diseases ranging from mild to life-threatening, has a large arsenal of virulence factors. Notably, extracellular vesicles have emerged as significant players in the pathogenesis of this organism. However, the full range of their functions is still being studied, and difficulties related to vesicle purification (long protocols, low yields, and specialized instruments) have become a major obstacle for their characterization. In this context, the utility of rapid new methods of vesicle isolation from clinical strains is still unknown. Here, we analyze the utility of the ExoBacteria OMV isolation kit for a collection of clinical strains of P. aeruginosa . We first phenotypically characterized 15 P . aeruginosa strains to ensure that our samples were heterogeneous. We then determined the best conditions for purifying vesicles from P. aeruginosa PAO1 reference strain by the rapid method and used them to isolate vesicles from clinical strains. Our results indicated that M9 minimal medium is the best for obtaining high purity with the rapid isolation kit. Although we were able to isolate vesicles from at least four strains, the low yield and the large number of strains with unpurifiable vesicles showed that the kit was not practical or convenient for clinical strains. Our findings suggest that although fast procedures for vesicle purification can be of great utility for Escherichia coli , the more complex phenotypes of clinical isolates of P. aeruginosa are a challenge for these protocols and new alternatives/optimizations need to be developed.IMPORTANCE Pseudomonas aeruginosa is recognized as an opportunistic pathogen in humans and animals. It can effectively colonize various environments thanks to a large set of virulence factors that include extracellular vesicles. Different methods were recently developed to reduce the time and effort associated with vesicle purification. However, the utility of rapid vesicle isolation methods for clinical strains of P. aeruginosa (which are recognized as being highly diverse) is not yet known. In this context, we analyzed the utility of the ExoBacteria OMV Isolation kit for vesicle purification in P. aeruginosa clinical strains. Our findings showed that the kit does not seem to be convenient for research on clinical strains due to low vesicle recovery. Our results underscore the importance of developing new rapid vesicle purification protocols/techniques for specific clinical phenotypes., Competing Interests: The authors declare no conflict of interest.

The performance of a novel selective agar was evaluated against the performance of conventional mycobacterial cultures, i.e., a combination of the mycobacterial growth indicator tube (MGIT) with Löwenstein-Jensen (LJ), for the detection of nontuberculous mycobacteria (NTM) in sputum samples from people with cystic fibrosis (pwCF). Two hundred eighty-three sputum samples (231 fresh sputum and 52 spiked sputum) from 143 pwCF were collected. They were inoculated without prior decontamination on NTM Elite agar (30°C ± 2°C for 28 days) and inoculated on both MGIT and LJ (35°C-37°C for 6-8 weeks) after N-acetyl-L-cysteine-2% sodium hydroxide decontamination. NTM were identified by Matrix-Assisted Laser Desorption Ionization/Time of Flight Mass Spectrometry and/or PCR, and whole-genome sequencing. A total of 67 NTM were recovered overall by the combination of all culture media. NTM Elite agar allowed the recovery of 65 NTM (97%), compared to 22 for the conventional MGIT and LJ media combination (32.8%), including 22 NTM for MGIT (32.8%) and 3 NTM with the LJ medium (4.5%). For Mycobacterium abscessus complex, the sensitivity of NTM Elite agar was 95% compared with a sensitivity of 30% for the conventional MGIT and LJ media combination. Overall, 17.3% of cultures on NTM Elite agar were contaminated with other micro-organisms vs 46.3% on MGIT and 77% on LJ. This study shows that the novel selective agar (NTM Elite agar) significantly outperforms the conventional MGIT and LJ media combination in terms of sensitivity, selectivity, and ease of culture, without the requirement of an L3 laboratory.IMPORTANCENontuberculous mycobacteria (NTM) are significant pulmonary pathogens in patients with pre-existing structural lung conditions such as cystic fibrosis, bronchiectasis, or chronic obstructive pulmonary disease. Mycobacterium avium complex and Mycobacterium abscessus complex (MABSC) are the most frequently isolated organisms. Compared to the recommended culture method for NTM, which combines solid and liquid culture media, NTM Elite agar enables a faster/easier diagnosis and speeds up identification and susceptibility testing as the final reading is at 28 days instead of 6-8 weeks for the conventional mycobacterial cultures. In addition, for the NTM Elite agar, no decontamination stage before inoculation is necessary, unlike the conventional mycobacterial cultures. NTM Elite agar is derived from a formulation of medium adapted to rapidly growing mycobacteria (RGM). The medium enables the growth of RGM while suppressing other flora. It is supported with published clinical data showing the benefits of this medium., Competing Interests: J.G. is an employee of bioMérieux. The Newcastle upon Tyne Hospitals, UK, represented by John Perry, receive ongoing funding from bioMérieux, France, for the development and evaluation of culture media. UZ Leuven and Munster University Hospital received funding from bioMérieux, France, for the evaluation of NTM culture media.

The performance of BACT/ALERT FA/FN Plus (France) blood culture containing a novel resin, DL (China) blood culture containing common resin, and adsorbent-free REDOX (USA) blood culture relying on dilution for antimicrobial neutralization at %peak serum concentration was evaluated by measuring the recovery of organisms and time to detection (TTD) in nine simulated microorganism-antimicrobial combination blood cultures. Significant differences were observed in the recovery rates among the aerobic media: 87.5% for BACT/ALERT media, 42.9% for DL media, and 12.5% for REDOX media. In contrast, no statistical difference was found in the TTD between FA Plus media and DL aerobic media. For the anaerobic media, the recovery rates were 91.4% for BACT/ALERT media, 2.9% for DL media, and 14.3% for REDOX media, with significant differences only between BACT/ALERT FN Plus media and the others. Among the seven main antimicrobial categories, only BACT/ALERT FA/FN Plus culture media demonstrated high recovery of microorganisms, with the exception of carbapenems. The DL culture media exhibited a relatively high recovery rate of microorganisms in the presence of piperacillin/tazobactam, levofloxacin, and gentamicin, but only in aerobic conditions. Conversely, REDOX media displayed microorganism recovery solely in the presence of gentamicin. BACT/ALERT FA/FN Plus culture media with novel resin showed absolute advantages over DL and REDOX culture media and can, therefore, be selectively applied in clinical settings when antimicrobials are used prior to blood collection. DL culture media, containing common resin, outperformed adsorbent-free dilution-based REDOX culture media, making it a viable backup option. There is a need to focus on improving the neutralization of carbapenems with current inefficiency in all three medias., Importance: We present a study on performance comparison of three different commercial culture media for neutralization of antibiotic effects in simulated blood cultures. BACT/ALERT (FA Plus and FN Plus) culture media with novel resin showed absolute advantages over DL and REDOX culture media at %PSL concentration of antimicrobials., Competing Interests: The authors declare no conflict of interest.

Penicillium exopolysaccharide (EPS) inhibits galactose lectins and enhances immunity. However, EPS production is low and its synthesis mechanism remains unclear. Penicillium EF-2 strains with high EPS production were selected for this study, and Penicillium fermentation conditions were subsequently improved. The optimal culture conditions were 30 g/L lactose, 6 g/L yeast extract powder, 4 d seed age, 10 % inoculation amount, 3 d of secondary fermentation time, and the final EPS yield was 3.97 g/L. UHPLC-Q-TOF-MS/MS was used to explore the mechanism of EPS synthesis at the metabolic level. Optimal carbon source: lactose and optimal nitrogen source: yeast extract can provide precursors for EPS synthesis through related metabolic pathways. Moreover, regulating the energy, vitamin, and lipid metabolic pathways created favourable conditions for EPS synthesis and secretion. These findings explain the mechanism of EPS synthesis at the metabolic level and provide a theoretical basis for optimising and industrialising EPS production., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)