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The article explores the growth patterns of the cephalopod Sepia officinalis under long-term culture conditions. Researchers analyze body weight and specific growth rate data to identify growth trends and patterns. The study reveals different growth patterns, such as decreasing SGR followed by stabilization, and highlights the influence of temperature on growth rates. The findings suggest that growth models for Sepia officinalis may differ from commonly accepted cephalopod growth patterns, emphasizing the importance of considering various factors when assessing growth in cultured cephalopods. [Extracted from the article]

Microalgae are a promising source of natural antioxidants, but optimising culture conditions to enhance antioxidant production remains a significant challenge, as varying cultivation environments induce oxidative stress and promote antioxidant accumulation. This study aims to assess the production of enzymatic (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX; guaiacol peroxidase, GPX; glutathione reductase, GR) pigments and non-enzymatic antioxidants (chlorophylls, Chls; carotenoids, CAR; ascorbic acid, AsA; a-tocopherol, TOC; total glutathione, GSH) in Chlorella vulgaris (UMT-M1), Isochrysis galbana (SWC002), and Tetraselmis chui (SWC001) at different growth phases. Throughout the cultivation period, the early stationary phase was observed to be the most significant increase phase for most antioxidants. All tested species formed TOC in the largest amounts during this phase. In this phase, C. vulgaris and I. galbana had the highest CAT and GPX, whereas C. vulgaris and T. chui had the highest SOD and APX. By identifying the early stationary phase as a key growth phase for maximum antioxidant production, this research provides valuable insights into the cultivation strategies that can be employed to maximise the yield of these beneficial compounds. This study thus contributes to the growing body of knowledge necessary for commercialising microalgae-derived antioxidants, offering a sustainable and natural alternative to synthetic antioxidants. [ABSTRACT FROM AUTHOR]

Abstract The Rhizobium rhizogene-transformed root culture from Physalis peruviana L. (P. peruviana) may be a promising and novel source of valuable phenolics, including caffeoylputrescine (CP), which is known for antioxidant, antidiabetic, insect-resistant, disease-resistant, and neuroprotective properties. In this study, to improve the production efficiency of phytochemical components in P. peruviana hairy root cultures, we optimized various culture conditions, including the inoculum size, liquid volume, culture media type, carbon source, sucrose concentration, initial pH, and application of elicitors, to enhance the total phenolic content and CP yield in these hairy root cultures. The findings indicate that the use of sucrose as carbon source resulted in the highest biomass (13.28 g DW/L), total phenolic content (6.26 mg/g), and CP yield (2.40 mg/L). The White medium excelled in enhancing the total phenolic content (9.35 mg/g), whereas the B5 medium was most effective for the biomass (13.38 g DW/L) and CP yield (6.30 mg/L). A sucrose concentration of 5% was best for the biomass (18.40 g DW/L), whereas a sucrose concentration of 4% was ideal for the CP yield. Optimal culture conditions were as follows: an inoculum size of 0.5 g/100 mL, a liquid volume of 100 mL in a 250-mL flask, B5 medium, 4% sucrose, and a pH of 5.5. Among the tested elicitors, methyl jasmonate (MeJA) at 100 µM significantly increased the biomass (21.3 g/L), total phenolic content (23.34 mg/g), and CP yield (141.10 mg/L), which represent 0.96-, 2.12-, and 13.04-fold increases, respectively, over the control after 8 days. The optimized HR culture of P. peruviana provides a promising system to enhance the production of CP for pharmaceutical applications.

STUDY QUESTION Is there a difference in clinical pregnancy rates (CPRs) in good prognosis patients after single embryo transfer (SET) on Day 5, in case of stable culture at 36.6°C or 37.1°C? SUMMARY ANSWER CPR (with heartbeat at 7 weeks) after blastocyst transfer do not differ after culturing at 36.6°C or 37.1°C. WHAT IS KNOWN ALREADY Since the beginning of IVF, embryo culture has been performed at 37.0°C; however, the optimal culture temperature remains unknown. Changes in incubator types have led to significant improvements in temperature control. Stable temperature control, i.e. with temperature differences of max. 0.1°C between chambers, is possible in some incubators. A previous prospective pilot study showed that embryo development on Day 5/6 was not affected when embryos were cultured at a stable temperature of 36.6°C or 37.1°C, but culture at 37.1°C resulted in an increased CPR when compared to culture at 36.6°C (74.2% vs 46.4%). STUDY DESIGN, SIZE, DURATION A prospective randomized controlled trial was performed in a tertiary fertility centre between February 2017 and November 26, 2022. A sample size of 89/89 patients with fresh single embryo transfer (SET) was required to achieve 80% power to detect a difference of 0.22 between group proportions (0.43–0.65) at a significance level of 0.05 using a two-sided z -test with continuity correction. PARTICIPANTS/MATERIALS, SETTING, METHODS Patients were recruited on the day of oocyte retrieval based on inclusion criteria with final randomization after denudation once six mature oocytes were present. The primary endpoint was CPR (heartbeat at 7 weeks); secondary endpoints were fertilization rate, blastocyst development, biochemical pregnancy rate, live birth rate (LBR), and cumulative live birth rate (CLBR). MAIN RESULTS AND THE ROLE OF CHANCE A total of 304 patients were eligible for the study; of these 268 signed the consent, 234 (intention-to-treat) were randomized and 181 (per-protocol) received a SET on Day 5: 90 received culture at 36.6°C and 91 at 37.1°C. Patients were on average 32.4 ± 3.5 versus 32.5 ± 4.2 years old, respectively. No differences were observed in embryological outcomes per cycle between culture at 36.6°C versus 37.1°C: 12.0 ± 3.8 vs 12.1 ± 3.8 COCs retrieved (P  = 0.88), 10.0 ± 3.1 versus 9.9 ± 2.9 mature oocytes inseminated (P  = 0.68), with a maturation rate of 84.2% (901/1083) versus 83.5% (898/1104) (P  = 0.87); and 8.0 ± 3.1 versus 7.9 ± 2.7 normally fertilized oocytes with a fertilization rate of 79.7% (720/901) vs 80.5% (718/898) (P  = 0.96), respectively. On average 1.5 ± 1.7 versus 1.4 ± 1.9 (P  = 0.25) and 1.1 ± 1.1 versus 0.9 ± 1.0 (P  = 0.45) supernumerary blastocysts were vitrified on Day 5 and Day 6, respectively. The utilization rate per fertilized oocyte was 46.1% vs 41.5% (P  = 0.14). A SET was performed for 181 patients, leading to a biochemical pregnancy rate of 72.2% (65/90) versus 62.7% (57/91) (P  = 0.17), respectively. The CPR per fresh transfer cycle was 51.1% (46/90) versus 48.4% (44/91) [OR (95% CI) 1.11 (0.59–2.08), P  = 0.710]. To date, a CLBR of 73.3% (66/90) versus 67.0% (61/91) (P  = 0.354) has been observed, respectively. In each group, seven patients without live birth have remaining blastocysts frozen. The CPR for the intention-to-treat groups were 38.3% vs 38.6% [OR (95% CI) 0.98 (0.56–1.73), P  = 0.967], respectively, for culture at 36.6°C versus 37.1°C. LIMITATIONS, REASONS FOR CAUTION Only selected patients with expected good prognosis were eligible for the study. WIDER IMPLICATIONS OF THE FINDINGS Embryos tend to tolerate small changes in temperature deviations during culture to the blastocyst stage, as demonstrated by their similar implantation potential at two slightly different temperatures. STUDY FUNDING/COMPETING INTEREST(S) There is no funding or conflicts of interest to declare. TRIAL REGISTRATION NUMBER NCT03548532. TRIAL REGISTRATION DATE 23 October 2017 DATE OF FIRST PATIENT'S ENROLMENT 10 November 2017 [ABSTRACT FROM AUTHOR]

Phellodendron amurense is the main plant source of berberine, and over exploitation has led to the depletion of wild resources. Therefore, tissue culture technology is being used to produce berberine and reduce the pressure on the utilization of wild resources. In this study, based on the seedling of P. amurense used as the material, the callus induction rate was taken as the index to optimize callus culture conditions by single factor experiments and response surface methodology. The optimal conditions of callus culture were determined: stem segment as explants, sucrose concentration 18 g/L, amount of nitrogen 1.0 N, combination of exogenous plant hormone 2, 4-D 2.0 mg /L + 6-BA 1.4 mg/L, under these conditions, the callus induction rate of P. amurense was about 97.06%±8.69. Berberine was extracted from the callus that cultured under the optimal conditions and its content was6.14 ± 1.12 mg/g (DW), which met the berberine content standard of not less than 0.6% standard of the Chinese Pharmacopoeia. In vitro antioxidant and antibacterial activities were tested for the extracts of the callus and traditional Chinese medicine (TCM) “GuanHuangbai”. The results showed that the antioxidant activities of the two extracts were mainly related to the concentrations of berberine. The antioxidant activity of callus extracts (IC50: 0.2166 mg/ml ± 0.0387) was slightly higher than that of Guanhuangbai extracts (IC50: 0.2169 mg/ml ± 0.0392). The Guanhuangbai extracts of P. amurense exhibited much better antimicrobial activity than the callus extracts for gram-positive bacteria, but the similar potency for the gram-negative bacteria and fungi in the present study. The extracts from callus of P. amurense had better antimicrobial activity in the experiment.Key message: The present study demonstrates the optimization of callus culture from stem segment -derived in Phellodendron amurense and the antioxidative and bacteriostatic activities of callus were compared with those of Traditional Chinese Medicinal Guanhuangbai from P. amurense. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is one of the most common bacterial pathogens worldwide and the main etiological agent of numerous gastric diseases. The frequency of multidrug resistance of H. pylori is growing and the leading factor related to this phenomenon is its ability to form biofilm. Therefore, the establishment of a proper model to study this structure is of critical need. In response to this, the aim of this original article is to validate conditions of the optimal biofilm development of H. pylori in monoculture and co-culture with a gastric cell line in media simulating human fluids. Using a set of culture-based and microscopic techniques, we proved that simulated transcellular fluid and simulated gastric fluid, when applied in appropriate concentrations, stimulate autoaggregation and biofilm formation of H. pylori. Additionally, using a co-culture system on semi-permeable membranes in media imitating the stomach environment, we were able to obtain a monolayer of a gastric cell line with H. pylori biofilm on its surface. We believe that the current model for H. pylori biofilm formation in monoculture and co-culture with gastric cells in media containing host-mimicking fluids will constitute a platform for the intensification of research on H. pylori biofilms in in vitro conditions that simulate the human body. [ABSTRACT FROM AUTHOR]

In recent years, the importance of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) has increased significantly. For their widespread use, a standardized EV manufacturing is needed which often includes conventional, static 2D systems. For these system critical process parameters need to be determined. We studied the impact of process parameters on MSC proliferation, MSC-derived particle production including EVs, EV- and MSC-specific marker expression, and particle functionality in a HaCaT cell migration assay. We found that cell culture growth surface and media affected MSCs and their secretory behavior. Interestingly, the materials that promoted MSC proliferation did not necessarily result in the most functional MSC-derived particles. In addition, we found that MSCs seeded at 4 × 103 cells cm−2 produced particles with improved functional properties compared to higher seeding densities. MSCs in a highly proliferative state did not produce the most particles, although these particles were significantly more effective in promoting HaCaT cell migration. The same correlation was found when investigating the cultivation temperature. A physiological temperature of 37°C was not optimal for particle yield, although it resulted in the most functional particles. We observed a proliferation-associated particle production and found potential correlations between particle production and glucose consumption, enabling the estimation of final particle yields. Our findings suggest that parameters, which must be defined prior to each individual cultivation and do not require complex and expensive equipment, can significantly increase MSC-derived particle production including EVs. Integrating these parameters into a standardized EV process development paves the way for robust and efficient EV manufacturing for early clinical phases. [Display omitted] [ABSTRACT FROM AUTHOR]

Aquaculture is crucial for global food and nutrition security due to the inability of wild harvests to meet increasing demand. Africa’s contribution to aquaculture is generally low, despite its potential for economic development, food security, and reduced unemployment. The study focuses on the African catfish (Clarias gariepinus) a freshwater fish species that is widely farmed for food in Africa and other parts of the world. Proper nutrition is essential for the growth and development of the fish, and understanding their nutritional requirements is critical for producing healthy and high-quality fish. This review article provides an overview of the knowledge on the nutritional requirement of the African catfish, including protein, lipids, carbohydrates, vitamins, minerals, and amino acids. The recommended protein content for juvenile fish is between 40 and 50% and for adult fish is between 30 and 40%. Based on the reviewed studies, the recommended amount of methionine in C. gariepinus diets ranges from 18.7 to 29.7 g/kg of protein while the lysine requirement ranges from 44.9 to 62.2 g/kg protein). The recommended lipid content in the diet is between 5–15% for juvenile fish and 5–10% for adult fish. The African catfish requires a low-carbohydrate diet, with recommended carbohydrate content between 26 and 32%. They require a variety of vitamins, including vitamin A, vitamin D, vitamin E, and vitamin C, as well as minerals, such as calcium, phosphorus, and potassium. Clarias gariepinus also require a variety of essential and non-essential amino acids. Besides the nutritional requirements, culture conditions also have a significant effect on the feed performance. The recommended conditions include temperature ranging from 28 to 32 °C, Light intensity of 150 Lx, 12D:12L photoperiod, and stocking density in earthen of 7 fish m−3. Overall, understanding the nutritional requirements of C. gariepinus is crucial for the successful fish farming and sustainable aquaculture. Information in this review will be built to further guide the development of feeds for C. gariepinus.

Lutein is widely used in medicine, health care, and food processing due to its antioxidant effects; however, it is difficult for the traditional extraction of lutein using marigolds to meet the increasing market demand for lutein. To achieve high-efficiency lutein production, we investigated the effects of different conditions on the biomass accumulation and lutein yield of Chlorella vulgaris. The optimized cultivation conditions include mixotrophic cultivation using sodium nitrate as a nitrogen source, maintaining a total-organic-carbon-to-total-nitrogen ratio of 12:1, a total-nitrogen-to-total-phosphorus ratio of 10:1, and lighting duration of 24 h. The results of the study indicated that under these specific conditions, Chlorella vulgaris attained a final biomass concentration, biomass productivity, and growth yield of 6.08 g·L−1, 1.00 g·L−1·d−1, and 1.67 g biomass/g TOC, respectively. Additionally, the concentrations of total chlorophyll, carotenoid, lutein, and protein reached 139.20 mg·L−1, 31.87 mg·L−1, 15.02 mg·L−1, and 2.17 g·L−1, respectively, and the content of lutein reached 2.47 mg·g−1. This study supplies a theoretical basis for the industrial application of lutein production using Chlorella vulgaris. [ABSTRACT FROM AUTHOR]

This study aimed to optimize the production of keratinase by Bacillus tropicus KRKJVR 5 using response surface methodology (RSM). The factors considered were temperature (20 to 40°C), pH (5 to 9), inoculum size (0.5 to 2.5%), incubation period (24 to 120 hours) and agitation speed (RPM) (50 to 350). A total of 32 experimental runs were conducted based on the central composite design (CCD) model, with observed keratinase activity ranging from 2.00 to 13.33 U/mL. The highest keratinase activity (13.33 U/mL) was observed under the conditions of 30°C temperature, pH 7.0, 1.5% inoculum size, 72 hours incubation period and 150 RPM. The lowest activity (2.00 U/mL) was recorded at extreme conditions of low pH (3.0) and high temperature (50°C), highlighting the sensitivity of keratinase production to these factors. The regression analysis revealed a significant model with an R-squared value of 93.78%, indicating a strong fit to the data. The ANOVA results further supported the significance of these factors, with the overall model being highly significant (F-Value = 8.30, P-Value = 0.000). The contour plots demonstrated the optimal conditions for maximizing keratinase activity. The study effectively identified the optimal conditions for keratinase production by Bacillus tropicus KRKJVR 5 using response surface methodology. The results indicate that a temperature of 30°C, pH of 7.0, inoculum size of 1.5%, incubation period of 72 hours, and agitation speed of 150 RPM are ideal for producing high levels of keratinase. These findings provide valuable insights for scaling up the process for industrial applications, emphasizing the importance of precise control over culture conditions to achieve high enzyme yields. [ABSTRACT FROM AUTHOR]

Aggressive forms of periodontitis, especially in young patients, are often associated with an increased proportion of the Gram-negative bacterium Aggregatibacter actinomycetemcomitans of the microbiota of the affected periodontal sites. One of the virulence factors of A. actinomycetemcomitans is a leukotoxin (LtxA) that induces a pro-inflammatory cell death process in leukocytes. A. actinomycetemcomitans exhibits a large genetic diversity and different genotypes vary in LtxA production capacity. The genotype JP2 is a heavy LtxA producer due to a 530-base pair deletion in the promoter for the toxin genes, and this trait has been associated with an increased pathogenic potential. The present study focused on the production and release of LtxA by different A. actinomycetemcomitans genotypes and serotypes under various growth conditions. Four different strains of this bacterium were cultured in two different culture broths, and the amount of LtxA bound to the bacterial surface or released into the broths was determined. The cultures were examined during the logarithmic and the early stationary phases of growth. The JP2 genotype exhibited the highest LtxA production among the strains tested, and production was not affected by the growth phase. The opposite was observed with the other strains. The composition of the culture broth had no effect on the growth pattern of the tested strains. However, the abundant release of LtxA from the bacterial surface into the culture broth was found in the presence of horse serum. Besides confirming the enhanced leucotoxicity of the JP2 genotype, the study provides new data on LtxA production in the logarithmic and stationary phases of growth and the effect of media composition on the release of the toxin from the bacterial membrane. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs – being small and non‐living – are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC‐EVs is increasingly investigated. However, due to variations in MSC‐EV manufacturing strategies, MSC‐EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC‐EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC‐EV researchers to characterise MSC‐EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter‐laboratory assessment using a novel multiplex bead‐based EV flow cytometry assay panel. This assessment involved 11 different MSC‐EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC‐related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC‐EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC‐derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC‐EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre‐clinical and clinical research, enhances the quality control of MSC‐EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC‐EV field. [ABSTRACT FROM AUTHOR]

Simple Summary: Simple Summary: In this study, a new cell line derived from red hybrid tilapia brain tissue, RHTiB, was established. This fibroblast-like cell line was maintained for over 50 passages with optimal growth at 25 °C in Leibovitz-15 medium with 10% fetal bovine serum at pH 7.4. Genetic and chromosomal analyses confirmed its origin from red hybrid tilapia. Additionally, immunofluorescence and RT-qPCR tests revealed successful TiLV propagation in RHTiB cell lines. The development of this novel cell line offers valuable prospects in enhancing diagnostic techniques in red hybrid tilapia. Tilapia lake virus (TiLV) presents a substantial threat to global tilapia production. Despite the development of numerous cell lines for TiLV isolation and propagation, none have been specifically derived from red hybrid tilapia (Oreochromis spp.). In this study, we successfully established a new cell line, RHTiB, from the red hybrid tilapia brain. RHTiB cells were cultured for 1.5 years through over 50 passages and demonstrated optimal growth at 25 °C in Leibovitz-15 medium supplemented with 10% fetal bovine serum at pH 7.4. Morphologically, RHTiB cells displayed a fibroblast-like appearance, and cytochrome oxidase I gene sequencing confirmed their origin from Oreochromis spp. Mycoplasma contamination testing yielded negative results. The revival rate of the cells post-cryopreservation was observed to be between 75 and 80% after 30 days. Chromosomal analysis at the 25th passage revealed a diploid count of 22 pairs (2n = 44). While no visible cytopathic effects were observed, both immunofluorescence microscopy and RT-qPCR analysis demonstrated successful TiLV propagation in the RHTiB cell line, with a maximum TiLV concentration of 107.82 ± 0.22 viral copies/400 ng cDNA after 9 days of incubation. The establishment of this species-specific cell line represents a valuable advancement in the diagnostic and isolation tools for viral diseases potentially impacting red hybrid tilapia. [ABSTRACT FROM AUTHOR]

Hydrogen is ideal for replacing fossil fuels because upon combustion it generates only water. Dark fermentation (DF) from lignocellulose might be a competitive process for hydrogen production at the industrial scale. However, lignocellulose must be pretreated to obtain fermentable sugars, which is costly and creates pollution. Microorganisms from bovine rumen efficiently degrade lignocellulose. Unfortunately, they have scarcely been explored for the production of hydrogen. Therefore, deeper studies on the culture conditions have to be undertaken to understand the behavior of microbial consortia from the rumen of bovines (MCRB) during hydrogen production. In this work, we evaluated the production of hydrogen by DF with MCRB by varying the incubation time, two culture media (MB and Rhodospirillaceae), headspace (40 and 80 mL), and thermal treatment. It was found that the production of hydrogen was maximum at 16 h MCRB incubation in MB. An amount of 80 mL headspace resulted in a threefold production of hydrogen as compared to 40 mL; the MCRB without heat treatment had a higher H2 yield. The production of hydrogen with 32 MCRB was highly variable, ranging between 21 and 696 mL. Our findings show a different perspective on the treatment of MCRB for the production of hydrogen and give insights on the impact of the culture conditions for increasing hydrogen production. [ABSTRACT FROM AUTHOR]

In order to screen a strain of Aspergillus niger with high L-malic acid production ability, a wild-type strain was mutagenized into a strain with high L-malic acid production ability by iterative mutagenesis with UV mutagenesis and high concentration of actinomycinone, and the composition of its seed or fermentation medium was optimised. It showed that the optimal glucose concentration of the seed medium was 60 g/L, the optimal nitrogen source was (NH4)2SO4 with 4.95 g/L, and the optimal rotational speed for the formation of A. niger spheres was 220 r/min. The optimal concentration of glucose and (NH4)2SO4 of the fermentation medium were 180 g/L and 4.95 g/L, respectively. CuSO4·5H2O was the best trace element for the production of malic acid, with the optimal addition amount of 0.065 g/L. The optimized medium was more conducive to the synthesis of L-malic acid through the optimization of the one-way experiments. The results presented that the production of L-malic acid at 96 h reached 18.15 g/L , which was significantly improved by 245%, and the percentage of L-malic acid in the total acid reached 71.69%. This study lays the foundation for the industrial production of L-malic acid.

Abstract Background Schistosomiasis is a disease primarily caused by eggs laid by pathogens called schistosomes. Among the schistosome species infecting humans, Schistosoma japonicum possesses the largest fecundity; each adult female produces an average of 3500 eggs per day. The lack of proper culture conditions supporting continuous oviposition in vitro has precluded detailed investigation of mechanisms regulating sexual maturation and egg production in Schistosoma japonicum. Methods We optimized in vitro culture conditions by replacing reagents that are part of the classical ABC169 medium. Fast Blue BB staining and 4′,6-diamidino-2-phenylindole (DAPI) labeling were applied to observe the sexual development status of the females. In vitro RNA interference (RNAi) technology was used to validate the capability of the modified medium. The detection of male β-alanyl-tryptamine (BATT) was conducted using liquid chromatography–mass spectrometry (LC–MS). Results Both m-AB169 (1640) and AB169 (1640) media are capable of facilitating the sexual development of paired virgin female S. japonicum, as well as sustaining the mature reproductive organs and egg production of adult S. japonicum for at least 22 days in vitro. M-AB169 (1640) provided a more stable condition for supporting the sexual maturity of female S. japonicum, as evidenced by the consistent initiation of egg production compared with AB169 (1640). Through a comparative analysis of S. japonicum and S. mansoni in diverse media, we demonstrated that these closely related species display distinct demands for their sexual development and egg production, suggesting a potential influence of nutritional factors on the observed variations in host ranges among different schistosome species. Importantly, we successfully identified the presence of the pheromone β-alanyl-tryptamine (BATT) in S. japonicum, previously identified in S. mansoni, highlighting its conserved role in schistosome reproductive development. Through the employment of double-stranded RNA (dsRNA) treatment to silence two genes that are involved in either the male (gli1, glioma-associated oncogene homolog 1) or female (vf1, vitellogenic factor 1) side in male-induced female reproductive development of S. mansoni, we confirmed that the combination of m-AB169 (1640) and RNAi technology has the capacity to facilitate in vitro studies of S. japonicum’s reproductive and oviposition processes. Conclusions We developed a novel medium, m-AB169 (1640), that not only maintains the mature reproductive organs and continuous oviposition of adult female Schistosoma japonicum for up to 22 days but also supports the reproductive development and subsequent egg-laying of virgin females after pairing with male worms. This study provides a valuable in vitro platform for functional studies of the mechanisms underlying the fascinating biology of the female sexual development and egg production of S. japonicum, which may accelerate the development of new strategies targeting schistosome egg production. Graphical Abstract

The periodontal ligament (PDL) is a highly specialized fibrous tissue comprising heterogeneous cell populations of an intricate nature. These complexities, along with challenges due to cell culture, impede a comprehensive understanding of periodontal pathophysiology. This study aims to address this gap, employing single-cell RNA sequencing (scRNA-seq) technology to analyze the genetic intricacies of PDL both in vivo and in vitro. Primary human PDL samples (n = 7) were split for direct in vivo analysis and cell culture under serum-containing and serum-free conditions. Cell hashing and sorting, scRNA-seq library preparation using the 10x Genomics protocol, and Illumina sequencing were conducted. Primary analysis was performed using Cellranger, with downstream analysis via the R packages Seurat and SCORPIUS. Seven distinct PDL cell clusters were identified comprising different cellular subsets, each characterized by unique genetic profiles, with some showing donor-specific patterns in representation and distribution. Formation of these cellular clusters was influenced by culture conditions, particularly serum presence. Furthermore, certain cell populations were found to be inherent to the PDL tissue, while others exhibited variability across donors. This study elucidates specific genes and cell clusters within the PDL, revealing both inherent and context-driven subpopulations. The impact of culture conditions—notably the presence of serum—on cell cluster formation highlights the critical need for refining culture protocols, as comprehending these influences can drive the creation of superior culture systems vital for advancing research in PDL biology and regenerative therapies. These discoveries not only deepen our comprehension of PDL biology but also open avenues for future investigations into uncovering underlying mechanisms. [ABSTRACT FROM AUTHOR]

Pseudoxanthomonas species are candidate microorganisms with potential for biocontrol, therefore this study aimed to isolate and characterize a strain of Pseudoxanthomonas sp. by morphological, physiological and biochemical analysis. In addition, Pseudoxanthomonas sp. growth conditions were also optimized. The effects of different culture mediums, carbon sources, nitrogen sources and pH on the growth of Pseudoxanthomonas sp. were studied by a combination of univariate and orthogonal experiments to determine its optimal growth kinetics. The results showed that the isolated strains were gram-negative, pale yellow, round, raised, with clean edges, translucent and shiny. One of the best - growing representative strains was selected for further physiological and biochemical characterization and was identified as Pseudoxanthomonas sp. The optimal growth conditions for the representative strain were reported. It was found that using LB medium as the base medium, adding 1.5% glucose as the carbon source, 1.5% yeast extract as the nitrogen source, adjusting the medium pH to 7.5, and incubating at a temperature of 37? yielded the best growth kinetics. These findings hold significance for subsequent research endeavors. Specifically, they are pivotal for advancing the biotechnological development of Pseudoxanthomonas sp. [ABSTRACT FROM AUTHOR]

Worldwide, bacterial resistance is one of the most severe public health problems. Currently, the failure of antibiotics to counteract superbugs highlights the need to search for new molecules with antimicrobial potential to combat them. The objective of this research was to evaluate the antimicrobial activity of Bacillus amyloliquefaciens BS4 against Gram-negative bacteria. Thirty yeasts and thirty-two Bacillus isolates were tested following the agar well-diffusion method. Four Bacillus sp. strains (BS3, BS4, BS17, and BS21) showed antagonistic activity against E. coli ATCC 25922 using bacterial culture (BC) and the cell-free supernatant (CFS), where the BS4 strain stood out, showing inhibitory values of 20.50 ± 0.70 mm and 19.67 ± 0.58 mm for BC and CFS, respectively. The Bacillus sp. BS4 strain can produce antioxidant, non-hemolytic, and antimicrobial metabolites that exhibit activity against several microorganisms such as Salmonella enterica, Klebsiella pneumoniae, Shigella flexneri, Enterobacter aerogenes, Proteus vulgaris, Yersinia enterocolitica, Serratia marcescens, Aeromonas sp., Pseudomonas aeruginosa, Candida albicans, and Candida tropicalis. According to the characterization of the supernatant, the metabolites could be proteinaceous. The production of these metabolites is influenced by carbon and nitrogen sources. The most suitable medium to produce antimicrobial metabolites was TSB broth. The one-factor-at-a-time method was used to standardize parameters such as pH, agitation, temperature, carbon source, nitrogen source, and salts, resulting in the best conditions of pH 7, 150 rpm, 28 °C, starch (2.5 g/L), tryptone (20 g/L), and magnesium sulfate (0.2 g/L), respectively. Moreover, the co-culture was an excellent strategy to improve antimicrobial activity, achieving maximum antimicrobial activity with an inhibition zone of 21.85 ± 1.03 mm. These findings position the Bacillus amyloliquefaciens BS4 strain as a promising candidate for producing bioactive molecules with potential applications in human health. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis is a disease primarily caused by eggs laid by pathogens called schistosomes. Among the schistosome species infecting humans, Schistosoma japonicum possesses the largest fecundity; each adult female produces an average of 3500 eggs per day. The lack of proper culture conditions supporting continuous oviposition in vitro has precluded detailed investigation of mechanisms regulating sexual maturation and egg production in Schistosoma japonicum. Methods: We optimized in vitro culture conditions by replacing reagents that are part of the classical ABC169 medium. Fast Blue BB staining and 4′,6-diamidino-2-phenylindole (DAPI) labeling were applied to observe the sexual development status of the females. In vitro RNA interference (RNAi) technology was used to validate the capability of the modified medium. The detection of male β-alanyl-tryptamine (BATT) was conducted using liquid chromatography–mass spectrometry (LC–MS). Results: Both m-AB169 (1640) and AB169 (1640) media are capable of facilitating the sexual development of paired virgin female S. japonicum, as well as sustaining the mature reproductive organs and egg production of adult S. japonicum for at least 22 days in vitro. M-AB169 (1640) provided a more stable condition for supporting the sexual maturity of female S. japonicum, as evidenced by the consistent initiation of egg production compared with AB169 (1640). Through a comparative analysis of S. japonicum and S. mansoni in diverse media, we demonstrated that these closely related species display distinct demands for their sexual development and egg production, suggesting a potential influence of nutritional factors on the observed variations in host ranges among different schistosome species. Importantly, we successfully identified the presence of the pheromone β-alanyl-tryptamine (BATT) in S. japonicum, previously identified in S. mansoni, highlighting its conserved role in schistosome reproductive development. Through the employment of double-stranded RNA (dsRNA) treatment to silence two genes that are involved in either the male (gli1, glioma-associated oncogene homolog 1) or female (vf1, vitellogenic factor 1) side in male-induced female reproductive development of S. mansoni, we confirmed that the combination of m-AB169 (1640) and RNAi technology has the capacity to facilitate in vitro studies of S. japonicum's reproductive and oviposition processes. Conclusions: We developed a novel medium, m-AB169 (1640), that not only maintains the mature reproductive organs and continuous oviposition of adult female Schistosoma japonicum for up to 22 days but also supports the reproductive development and subsequent egg-laying of virgin females after pairing with male worms. This study provides a valuable in vitro platform for functional studies of the mechanisms underlying the fascinating biology of the female sexual development and egg production of S. japonicum, which may accelerate the development of new strategies targeting schistosome egg production. [ABSTRACT FROM AUTHOR]

Jing Yang, Yan Ma, Bo Li, Zhiqin Xi, Li Zhang, Yuxi Wang, Wenli Feng Department of Dermatovenereology, The Second Hospital of Shanxi Medical University, Taiyuan, Shanxi, 030001, People’s Republic of ChinaCorrespondence: Wenli Feng; Jing Yang, Department of Dermatovenereology, The Second Hospital of Shanxi Medical University, No. 382, Wuyi Road, Taiyuan, Shanxi, 030001, People’s Republic of China, Tel +86-351-3365410, Email Wenlifeng2010@163.com; yang_jing@sxmu.edu.cnPurpose: RNA terminal phosphate cyclase like 1 (RCL1) undergoes overexpression during the immune response of Candida albicans following drug treatment. This study aims to investigate the expression levels of RCL1 in C. albicans under various stress conditions.Methods: Fifteen itraconazole (ITR)-resistant strains of clinical C. albicans, and one standard strain were employed for RCL1 sequencing, and mutations in RCL1 were analyzed. Subsequently, 14 out of the 15 ITR-resistant clinical strains and 14 clinical strains sensitive to ITR, fluconazole (FCA) as well as voriconazole (VRC) were cultured under diverse conditions. The expression of RCL1 ITR-resistant and sensitive C. albicans was then assessed using real-time quantitative PCR (RT-qPCR) assays.Results: Compared to the standard strain, three missense mutations (C6A, G10A, and A11T) were identified in the RCL1 gene of ITR-resistant C. albicans through successful forward sequencing. Additionally, using successful reverse sequencing, one synonymous mutation (C1T) and four missense mutations (C1T, A3T, A7G, and T8G) were found in the RCL1 gene of ITR-resistant C. albicans. RCL1 expression was significantly higher in ITR-resistant C. albicans than in sensitive strains under standard conditions (37°C, 0.03% CO2, pH 4.0). Low temperature (25°C) increased RCL1 expression in sensitive C. albicans while decreasing it in ITR-resistant strains. Elevated CO2 concentrations (5% CO2) had a negligible effect on RCL1 expression in sensitive C. albicans, but effectively reduced RCL1 level in ITR-resistant strains. Furthermore, a medium with a pH of 7 decreased the expression of RCL1 in both resistant and sensitive C. albicans.Conclusion: This study demonstrated that RCL1 mutations in ITR-resistant C. albicans, and variations in culture conditions significantly influence RCL1 expression in both ITR-resistant and sensitive C. albicans, thereby inducing alterations in the dimorphism of C. albicans.Keywords: Candida albicans, itraconazole resistance, RCL1, culture conditions

To further enhance the industrial utilization value of Haematococcus lacustris, the plasma mutagenesis of Haematococcus lacustris was carried out by an atmospheric and room temperature plasma (ARTP) mutagenesis equipment. The optimum input power and mutagenesis time for plasma mutagenesis were determined with lethal rate of algal cells as the index. After mutagenesis, high-yield astaxanthin mutant algae strains were obtained through primary screening of solid plate culture and secondary screening of liquid culture. Then, the culture conditions of high yield algal plants at vegetative growth stage were optimized by single-factor and orthogonal experiment with algae cell density as the index, and the suitable high light conditions for astaxanthin accumulation during astaxanthin induction stage were selected. The genetic stability of the high yielding mutant algae strains was observed after multiple subcultures under the optimized culture conditions. The results showed that the optimum conditions for plasma mutagenesis of Haematococcus lacustris were 240 W for 150 s or 400 W for 120 s. 11 Mutant alga strains with fast growth and high astaxanthin yield were obtained through primary screening and rescreening, wherein the strain HP3 grew fastest and had the highest astaxanthin yield. After culture, its cell density and astaxanthin yield were increased by 25.5% and 61.6% respectively compared with the original strain. After two-stage optimization, the cell density and astaxanthin yield of HP3 increased by 14.3% and 19.3% respectively, reaching 7.2×105 cell/mL and 31.264 mg/L. HP3 showed good growth and stable heredity. Its cell density and astaxanthin yield were similar to those of primary culture. The results have practical significance for the breeding of industrial algal strains producing astaxanthin from Haematococcus lacustris.

The composition of the vaginal microbiota is known to be influenced by various factors and to be associated with several disorders affecting women’s health. Although metagenomics is currently a widely used method for studying the human microbiota, it has certain limitations, such as a lack of information on bacterial viability. It is therefore important to use culture-based methods such as culturomics. Here, we used 35 different culture conditions to comprehensively characterize the vaginal bacterial diversity of a single woman's flora. A total of 206 bacterial species, belonging to six phyla (for a little more than half to Firmicutes, followed mainly by Actinobacteria, Bacteroidetes, and Proteobacteria) and 45 families, and 2 fungal species were cultivated. While several species of lactobacilli have been isolated, a wide variety of other bacteria were also separated, including 65 never reported before in vaginal flora, including a new bacterial species, Porphyromonas vaginalis sp. nov. Extensive culture-based methods are essential to establish a comprehensive, evidence-based repertoire of bacterial viability. If combined with molecular methods, they can provide a much more thorough understanding of the vaginal microbiota and fulfil the unknown part of metagenomic studies. [ABSTRACT FROM AUTHOR]

Objective　To investigate the effects of different culture conditions（RPMI-1 640，DMEM and DMEM/F12 medium） on the passage of MPM cells isolated from the tissues of Malignant pleural mesothelioma（MPM），and to study the effects of CDKN2B on the proliferation，invasion and apoptosis of MPM cells. Methods　MPM cells were isolated from MPM tissues and cultured in RPMI-1 640，DMEM and DMEM/F12 medium，respectively. Cell proliferation was examined by CCK-8，and the nuclei and chromosomes were observed by Wright-Giemsa staining. Fluorescence intensities of Calretinin， CD141， CK5， EMA and WT-1 were conducted by immunofluorescence assay. The mRNA and protein expression of CDKN2B were detected by RT-qPCR and Western blot，respectively. Transwell was used to detect cell invasion and flow cytometry was used to detect cell apoptosis. Results　The established MPM cells showed good viability when passaged to the 10th generation in RPMI-1 640，DMEM and DMEM/F12 cultures，and the MPM markers Calretinin，CD141，CK5，EMA and WT1 were all expressed in the cells. The viability of MPM cells in RPMI-1 640 culture medium was relatively stable. CDKN2B was downregulated in MPM cells（P < 0.05），and overexpression of CDKN2B significantly suppressed the proliferation（P < 0.05），invasion（P < 0.05） and epithelial interstitial transformation of MPM cells（P < 0.01），and promoted the apoptosis（P < 0.01）. Conclusion　The established MPM cells were stably passaged in RPMI-1 640 culture medium，and CDKN2B may be a potential target for the diagnosis and treatment of MPM. [ABSTRACT FROM AUTHOR]

The study aimed to investigate the growth rate and the population size of the copepod Apocyclops dengizicus under different culture conditions of salinity, temperature, and diet. Five salinities, four levels of temperature, and four diet ratios of baker’s yeast and the microalgae were employed for a 20-day experiment. The results showed that A. dengizicus could survive in every experimental concentration of salinity after 20 days of culture, of which the highest growth rate and the largest population size were recorded at a salinity of 15 ppt with values of 0.26 ± 0.01d-1 and 1221 ± 270 individuals, respectively. As for temperature, the A. dengizicus population thrived best at 34°C, with a growth rate reaching 0.29 ± 0.01d-1 and a maximum population size of 2097.0 ± 193.29 individuals obtained at the end of the experiment. Moreover, a diet with 25% Saccharomyces cerevisiae and 75% Chlorella vulgaris showed the best population development (0.25 ± 0.01d-1, 997.5 ± 1 92.09 individuals).

Lutein is widely used in medicine, health care, and food processing due to its antioxidant effects; however, it is difficult for the traditional extraction of lutein using marigolds to meet the increasing market demand for lutein. To achieve high-efficiency lutein production, we investigated the effects of different conditions on the biomass accumulation and lutein yield of Chlorella vulgaris. The optimized cultivation conditions include mixotrophic cultivation using sodium nitrate as a nitrogen source, maintaining a total-organic-carbon-to-total-nitrogen ratio of 12:1, a total-nitrogen-to-total-phosphorus ratio of 10:1, and lighting duration of 24 h. The results of the study indicated that under these specific conditions, Chlorella vulgaris attained a final biomass concentration, biomass productivity, and growth yield of 6.08 g·L−1, 1.00 g·L−1·d−1, and 1.67 g biomass/g TOC, respectively. Additionally, the concentrations of total chlorophyll, carotenoid, lutein, and protein reached 139.20 mg·L−1, 31.87 mg·L−1, 15.02 mg·L−1, and 2.17 g·L−1, respectively, and the content of lutein reached 2.47 mg·g−1. This study supplies a theoretical basis for the industrial application of lutein production using Chlorella vulgaris.

Aggressive forms of periodontitis, especially in young patients, are often associated with an increased proportion of the Gram-negative bacterium Aggregatibacter actinomycetemcomitans of the microbiota of the affected periodontal sites. One of the virulence factors of A. actinomycetemcomitans is a leukotoxin (LtxA) that induces a pro-inflammatory cell death process in leukocytes. A. actinomycetemcomitans exhibits a large genetic diversity and different genotypes vary in LtxA production capacity. The genotype JP2 is a heavy LtxA producer due to a 530-base pair deletion in the promoter for the toxin genes, and this trait has been associated with an increased pathogenic potential. The present study focused on the production and release of LtxA by different A. actinomycetemcomitans genotypes and serotypes under various growth conditions. Four different strains of this bacterium were cultured in two different culture broths, and the amount of LtxA bound to the bacterial surface or released into the broths was determined. The cultures were examined during the logarithmic and the early stationary phases of growth. The JP2 genotype exhibited the highest LtxA production among the strains tested, and production was not affected by the growth phase. The opposite was observed with the other strains. The composition of the culture broth had no effect on the growth pattern of the tested strains. However, the abundant release of LtxA from the bacterial surface into the culture broth was found in the presence of horse serum. Besides confirming the enhanced leucotoxicity of the JP2 genotype, the study provides new data on LtxA production in the logarithmic and stationary phases of growth and the effect of media composition on the release of the toxin from the bacterial membrane.

Autoimmune conditions, such as rheumatoid arthritis, are characterised by a loss of immune tolerance, whereby the immune cells attack self-antigens causing pain and inflammation. These conditions can be brought into remission using pharmaceutical treatments, but often have adverse side effects and some patients do not respond favourably to them. Human umbilical cord mesenchymal stromal cells (UCMSCs) present a promising alternative therapeutic due to their innate anti-inflammatory properties which can be strengthened using pro-inflammatory conditions. Their therapeutic mechanism of action has been attributed to paracrine signalling, by which nanosized acellular particles called 'extracellular vesicles' (EVs) are one of the essential components. Therefore, this research analysed the anti-inflammatory properties of UCMSC-EVs 'primed' with pro-inflammatory cytokines and at baseline with no inflammatory cytokines (control). Both control and primed EVs were co-cultured with un-pooled peripheral blood mononuclear cells (PBMCs; n = 6) from healthy donors. Neither control nor primed EVs exerted a pro-inflammatory effect on PBMCs. Instead, the primed EVs showed the immunosuppressive potential by increasing the expression of the anti-inflammatory protein FoxP3 in PBMCs. This may be attributed to the upregulated miRNAs identified in primed EVs in comparison to control EVs (miR-139-5p, miR-140-5p, miR-214-5p). These findings aid in understanding how UCMSC-EVs mediate immunosuppression and support their potential use in treating autoimmune conditions. [ABSTRACT FROM AUTHOR]

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) is an excellent tool for bacterial identification. It allows high throughput, sensitive and specific applications in clinical diagnostics and environmental research. Currently, there is no optimal standardized protocol for sample preparation and culture conditions to profile bacteria. The performance of MALDI‐TOF MS is affected by several variables, such as sample preparation, culture media and culture conditions, incubation time/growth stage, incubation temperature, high salt content, blood in the culture media, and others. This review thus aims to clarify why a uniformed protocol is not plausible, to assess the effects these factors have on MALDI‐TOF MS identification score, and discuss possible optimizations for its methodology, in relation to specific bacterial representatives and strain requirements. [ABSTRACT FROM AUTHOR]

Biochemical profile of five marine microalgae Isochrysis galbana, Chaetoceros calcitrans, Tetraselmis suecica, Nannochloropsis oculata and Aphanocapsa sp. was studied to understand the changes of the biochemical profile under different culture conditions, i.e. Conway and f/2 media at 20 and 30ppt salinities (‰). The microalgae culture was undertaken in the indoor facility with 24±1° C temperature, PH 8.7±1, 24-hour illumination. Biochemical constituents of five algal species showed variations, the highest protein (26.86%) was recorded in C. calcitrans in f/2 medium at 30‰, lipid (32.00%) in N. oculata in Conway medium at 30‰, carbohydrate (32.06%) in T. suecica in Conway medium at 30‰ and ash (54.71%) in C. calcitrans in f/2 medium at 30‰. Among macro minerals, a high concentration of sodium (1.05g/100g) was recorded in C. calcitrans in Conway medium-20‰, potassium (4.12g/100g) in C. calcitrans in f/2 medium-30‰, calcium (1.57g/100g) in C. calcitrans in Conway medium-20‰, phosphorus (8.53g/100g) in C. calcitrans in Conway medium-20‰, magnesium (4.10g/100g) in N. oculata in f/2 medium-20‰. Among micro minerals, a high concentration of iron (1.44g/100g) was recorded in C. calcitrans in f/2 medium-30‰, zinc(0.95g/100g) in I. galbana in Conway medium20‰, manganese(0.02g/100g) in N. oculata in Conway medium-30‰, copper(0.04g/100g) in Aphanocapsa sp. in Conway medium-30‰. Among fatty acids, C16:0 become the dominant saturated fatty acid (SFA) found in I. galbana, C16:1 become the dominant monounsaturated fatty acid (MUFA) found in N. oculate and C18:2 become the dominant polyunsaturated fatty acid (PUFA) found in I. galbana cultured in Conway medium-20‰. The biochemical composition of five microalgae indicated that these are potential food sources for humans, cattle and aquaculture industries. [ABSTRACT FROM AUTHOR]

The biofilm produced by 26 strains of Vibrio vulnificus (Vv) and the factors affecting biofilm formation were studied to provide theoretical basis for effective control of biofilm formation by Vv. In this study, Congo red plate method, improved test tube method and improved microplate method were used to analyze the ability of 25 isolated Vv strains and 1 standard strain to form biofilm, and one strain with the strongest ability to produce biofilm was selected from them. The effects of different initial bacterial concentration, temperature, culture time, pH, NaCl concentration, metal cations and contact materials on the formation of biofilm were investigated. The results showed that 25 strains (96.15%) had biofilm forming ability among the selected strains and VvK had the strongest ability to produce biofilm. At 25 ℃, the initial bacterial concentration was 108 CFU/mL, 3% NaCl and pH8~9 for 24 h, the biofilm quantity was the largest. However, the formation of biofilm was inhibited by certain concentration of metal cations (Cu2+, Mn2+, Ca2+, Mg2+) definitely, and the inhibition ability of above cations decreases in turn. When VvK contacted hydrophilic surfaces (stainless steel and glass), the amount of biofilm formation was significantly higher than hydrophobic surfaces (polystyrene), and the amount of biofilm formation on stainless steel was the largest. The biofilm forming ability of different Vv was quite different, and had specific rules under different cultivation conditions, which should be paid more attention.

Abstract Background Biofilm-associated infections are a global threat to our economy and human health; as such, development of antibiofilm compounds is an urgent need. Our previous study identified eleven environmental isolates of endophyte bacteria, actinomycetes, and two strains of Vibrio cholerae as having strong antibiofilm activity, but only tested crude extracts from liquid culture. Here we grew the same bacteria in solid culture to induce the formation of colony biofilms and the expression of genes that may ultimately produce antibiofilm compounds. This research aimed to compare antibiofilm inhibition and destruction activities between liquid and solid cultures of these eleven environmental isolates against the biofilms of representative pathogenic bacteria. Results We measured antibiofilm activity using the static antibiofilm assay and crystal violet staining. The majority of our isolates exhibited higher inhibitory antibiofilm activity in liquid media, including all endophyte bacteria, V. cholerae V15a, and actinomycetes strains (CW01, SW03, CW17). However, for V. cholerae strain B32 and two actinomycetes bacteria (TB12 and SW12), the solid crude extracts showed higher inhibitory activity. Regarding destructive antibiofilm activity, many endophyte isolates and V. cholerae strains showed no significant difference between culture methods; the exceptions were endophyte bacteria isolate JerF4 and V. cholerae B32. The liquid extract of isolate JerF4 showed higher destructive activity relative to the corresponding solid culture extract, while for V. cholerae strain B32 the solid extract showed higher activity against some biofilms of pathogenic bacteria. Conclusions Culture conditions, namely solid or liquid culture, can influence the activity of culture extracts against biofilms of pathogenic bacteria. We compared the antibiofilm activity and presented the data that majority of isolates showed a higher antibiofilm activity in liquid culture. Interestingly, solid extracts from three isolates (B32, TB12, and SW12) have a better inhibition or/and destruction antibiofilm activity compared to their liquid culture. Further research is needed to characterize the activities of specific metabolites in solid and liquid culture extracts and to determine the mechanisms of their antibiofilm actions.

Objective: This study aimed to develop a low cost and high density lactobacillus starter. Methods: The Lactobacillus fermentum BLHN3 isolated from chopped pepper was used as material, on the basis of MRS medium, the high density fermentation medium and culture conditions were optimized. Results: The optimal carbon source, nitrogen source, buffer salt, growth-promoting factor of L. fermentum BLHN3 were as follows: 30.0 g/L Trehalose, 34.0 g/L Soybean peptone, 2.0 g/L Ammonium citrate, 5.0 g/L Sodium acetate, 2.0 g/L Dipotassium hydrogen phosphate, 10% Carrot juice. The viable count of L. fermentum BLHN3 using the optimized medium was 6.05×109 CFU/mL. We determined the optimal fermentation process using this medium as follows: Initial culture pH was 6, and culture temperature was 37 ℃， inoculum was 3%, and loading liquid was 30 mL. Based on these results, a study of semi-continuous high-density culture was carried out, preferably three centrifugation cultures. Conclusion: The optimization of medium and culture conditions can significantly improve the growth activity of L. fermentum BLHN3, which was significantly higher than that of MRS medium.

Introduction: Exploring endophytic fungi isolated from medicinal herbs could be a turning point in the research of secondary metabolites biosynthesis, as these endophytic fungi are capable of synthesizing the similar compounds as their host plant. The advantages of manipulating endophytic fungi for bioactive compound production are the reduction of dependency rate on slow-growing and rare plants, cost-effective, continuous process, environmentally friendly and high yield in a short period. Thus, the current study envisages investigating the influence of culture conditions against the anti-MRSA potential production of the endophytic fungal isolate, Ceratobasidium ramicola IBRLCM127 isolated from the local medicinal plant Curcuma mangga Valeton & Zijp. Methods: The endophytic fungal isolate was used to produce fungal metabolites through submerged fermentation. The physical parameter improvement was investigated using the 'one-factor-at-atime' technique. The fungal fermentative broth was subjected to an anti-MRSA assay using Lorian method, whereas the growth of a fungus was determined based on the cell growth weight. Results: The highest anti-MRSA potential of 42.50±0.1 U/ml and 5.49±0.1 g/L of mycelial growth was observed after improving the basal medium containing yeast extract sucrose broth incorporated with water extract from the host plant, 6 days old of inoculum age, 2 agar plugs of mycelia, incubation temperature of 25 0C and 12 days of cultivation 12 days of cultivation shaken at 120 rpm in the absence of light. Conclusion: The improved culture conditions shorten the incubation period and yield a significant enhancement of anti-MRSA potential and fungal growth with 13.27% and 10.91%, respectively. [ABSTRACT FROM AUTHOR]

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Manglietiastrum sinicum Y.W. Law is a critically endangered species with great ornamental and commercial value, which urgently requires protection. We tested different combinations of basal media and plant growth regulators to determine (i) the optimal conditions for bud induction and proliferation of explants and (ii) optimal rooting conditions. RAPD- and ISSR-PCR were used to assess the genetic fidelity of regenerated plantlets. Murashige and Skoog medium (MS) supplemented with 0.5 mg/L 6-benzyladenine (BA) and 0.05 mg/L indole-3-butyric acid (IBA) is the optimal medium for bud induction (100% induction). MSM medium (a special basal medium for M. sinicum) was more suitable for the efficient proliferation and rooting of M. sinicum. Maximum bud proliferation rate (446.20%) was obtained on MSM, with 0.4 mg/L BA, 0.5 mg/L kinetin, and 0.06 mg/L IBA, while maximum root induction rate (88.89%) was obtained on MSM supplemented with 0.4 mg/L 1-naphthylacetic acid and 1.0 mg/L IBA with a 7-day initial darkness treatment. The rooted plantlets were transferred to a substrate containing peat soil, perlite, coconut chaff, and bark (volume ratio 2:1:1:1), with a resulting survival rate of 92.2%. RAPD and ISSR markers confirmed the genetic uniformity and stability of regenerated plants. [ABSTRACT FROM AUTHOR]

Hydrogen is ideal for replacing fossil fuels because upon combustion it generates only water. Dark fermentation (DF) from lignocellulose might be a competitive process for hydrogen production at the industrial scale. However, lignocellulose must be pretreated to obtain fermentable sugars, which is costly and creates pollution. Microorganisms from bovine rumen efficiently degrade lignocellulose. Unfortunately, they have scarcely been explored for the production of hydrogen. Therefore, deeper studies on the culture conditions have to be undertaken to understand the behavior of microbial consortia from the rumen of bovines (MCRB) during hydrogen production. In this work, we evaluated the production of hydrogen by DF with MCRB by varying the incubation time, two culture media (MB and Rhodospirillaceae), headspace (40 and 80 mL), and thermal treatment. It was found that the production of hydrogen was maximum at 16 h MCRB incubation in MB. An amount of 80 mL headspace resulted in a threefold production of hydrogen as compared to 40 mL; the MCRB without heat treatment had a higher H2 yield. The production of hydrogen with 32 MCRB was highly variable, ranging between 21 and 696 mL. Our findings show a different perspective on the treatment of MCRB for the production of hydrogen and give insights on the impact of the culture conditions for increasing hydrogen production.

Worldwide, bacterial resistance is one of the most severe public health problems. Currently, the failure of antibiotics to counteract superbugs highlights the need to search for new molecules with antimicrobial potential to combat them. The objective of this research was to evaluate the antimicrobial activity of Bacillus amyloliquefaciens BS4 against Gram-negative bacteria. Thirty yeasts and thirty-two Bacillus isolates were tested following the agar well-diffusion method. Four Bacillus sp. strains (BS3, BS4, BS17, and BS21) showed antagonistic activity against E. coli ATCC 25922 using bacterial culture (BC) and the cell-free supernatant (CFS), where the BS4 strain stood out, showing inhibitory values of 20.50 ± 0.70 mm and 19.67 ± 0.58 mm for BC and CFS, respectively. The Bacillus sp. BS4 strain can produce antioxidant, non-hemolytic, and antimicrobial metabolites that exhibit activity against several microorganisms such as Salmonella enterica, Klebsiella pneumoniae, Shigella flexneri, Enterobacter aerogenes, Proteus vulgaris, Yersinia enterocolitica, Serratia marcescens, Aeromonas sp., Pseudomonas aeruginosa, Candida albicans, and Candida tropicalis. According to the characterization of the supernatant, the metabolites could be proteinaceous. The production of these metabolites is influenced by carbon and nitrogen sources. The most suitable medium to produce antimicrobial metabolites was TSB broth. The one-factor-at-a-time method was used to standardize parameters such as pH, agitation, temperature, carbon source, nitrogen source, and salts, resulting in the best conditions of pH 7, 150 rpm, 28 °C, starch (2.5 g/L), tryptone (20 g/L), and magnesium sulfate (0.2 g/L), respectively. Moreover, the co-culture was an excellent strategy to improve antimicrobial activity, achieving maximum antimicrobial activity with an inhibition zone of 21.85 ± 1.03 mm. These findings position the Bacillus amyloliquefaciens BS4 strain as a promising candidate for producing bioactive molecules with potential applications in human health.

Maroteaux—Lamy syndrome (mucopolysaccharidosis type VI) is an orphan genetic disease caused by mutations in the arylsulfatase B gene (ARSB), which encodes the lysosomal enzyme arylsulfatase B (ASB). The relevance of the study lies in the need of a Russian recombinant ASB product for patients with the disease in the Russian Federation. Previously, the authors have developed producer lines coexpressing the target ASB enzyme with an auxiliary formylglycine-generating enzyme (FGE), based on Chinese hamster ovary (CHO) cells. Further development of the recombinant ASB preparation places priority on increasing the enzyme yield. The aim of this study was to increase the productivity of producer clones by optimising the culture process and adding calcium chloride and copper sulfate to the culture medium. Materials and methods: a suspension-adapted CHO cell line was used. Monoclonal cell lines were developed using Cell Metric and ClonePix FL systems. The concentration of ASB in the culture liquid was determined using the enzyme-linked immunosorbent assay (ELISA). The authors analysed batch culture and/or fed-batch culture in media supplemented with various concentrations of copper sulfate and calcium chloride. Results: the combined addition of copper sulfate and calcium chloride at concentrations of 300 μM during batch culture of producer clones coexpressing ASB and FGE increases viability and specific productivity of the cells up to 4.58±1.62 pg/ (cell×day). The cultivation of the lead producer clone coexpressing ASB and FGE under fed-batch conditions for 12 days and the addition of copper sulfate to the growth medium at the concentration of 300 μM allow for increasing the yield of the active lysosomal enzyme, arylsulfatase B, to 420 mg/L. Conclusions: the cultivation of producer clones coexpressing ASB and FGE under fed-batch conditions with copper sulfate added to the medium significantly improves cell line growth properties and the ASB yield. This approach to the selection of culture conditions for producer cell lines can be applied to other enzymes of the sulfatase family.

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Aims: A suitable medium and cultivation parameters have an important role in the improvement of the production of pyocyanin pigment by Pseudomonas aeruginosa microorganism. The study aimed to optimize culture conditions and medium components for maximal pyocyanin production in a recombinant strain P. aeruginosa PS39-phzMS created in previous research. In addition, the process of extraction of pyocyanin was also investigated to select a proper applied solvent for recovering a high amount of pyocyanin as well as its quality. Methodology and results: The pyocyanin purification has based on solvent. Among six tested solvents for extracting pyocyanin out of bacterial broth, two out of six recovered a significant amount of pyocyanin, namely dichloromethane and chloroform, in which chloroform showed a higher pyocyanin yield (25.27 ± 1.02 µg/mL) than dichloromethane (20.26 ± 0.88 µg/mL). The thin-layer chromatography (TLC) of the extracted pyocyanin illustrated a similar to pure pyocyanin with Rf of 0.72 and no mark of other impurity metabolites. The UV-Vis spectra showed a similar peak at 520 nm with pure pyocyanin and the highest peak at 274 nm. Each single culture parameter was studied for the maximal production of pyocyanin. Next, a pyocyanin-producing GM medium was modified on the base of the KingA to find the relative capacity to biosynthesize high pyocyanin yield in P. aeruginosa PS39-phzMS. The results showed that pyocyanin production was the highest in optimal culture conditions, at 30 °C, pH 8, 120 h and agitation of 200 rpm. In the combination of culture condition with the GM, pyocyanin was created at the highest amount of 49.57 µg/mL. Conclusion, significance and impact of study: Based on the obtained results of the study, a pyocyanin-producing procedure was optimized, which suggests a promising application to scale-up pyocyanin production by the P. aeruginosa PS39-phzMS. [ABSTRACT FROM AUTHOR]

Dunaliella tertiolecta RCC6 was cultivated indoors in glass bubble column photobioreactors operated under batch and semi‐continuous regimens and using two different conditions of light and temperature. Biomass was harvested by centrifugation, frozen, and then lyophilized. The soluble material was obtained by sequential extraction of the lyophilized biomass with solvents with a gradient of polarity (hexane, ethyl acetate, and methanol) and its metabolic composition was investigated through nuclear magnetic resonance (NMR) spectroscopy. The effect of light on chlorophyll biosynthesis was clearly shown through the relative intensities of the 1H NMR signals due to pheophytins. The highest signal intensity was observed for the biomasses obtained at lower light intensity, resulting in a lower light availability per cell. Under high temperature and light conditions, the 1H NMR spectra of the hexane extracts showed an incipient accumulation of triacylglycerols. In these conditions and under semi‐continuous regimen, an enhancement of β‐carotene and sterols production was observed. The antibacterial and antibiofilm activities of the extracts were also tested. Antibacterial activity was not detected, regardless of culture conditions. In contrast, the minimal biofilm inhibitory concentrations (MBICs) against Escherichia coli for the hexane extract obtained under semi‐continuous regimen using high temperature and irradiance conditions was promising. [ABSTRACT FROM AUTHOR]

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Adult human Schwann cells represent a relevant tool for studying peripheral neuropathies and developing regenerative therapies to treat nerve damage. Primary adult human Schwann cells are, however, difficult to obtain and challenging to propagate in culture. One potential solution is to generate Schwann cells from human induced pluripotent stem cells (hiPSCs). Previously published protocols, however, in our hands did not deliver sufficient viable cell numbers of hiPSC-derived Schwann cells (hiPSC-SCs). We present here, two modified protocols from two collaborating laboratories that overcome these challenges. With this, we also identified the relevant parameters to be specifically considered in any proposed differentiation protocol. Furthermore, we are, to our knowledge, the first to directly compare hiPSC-SCs to primary adult human Schwann cells using immunocytochemistry and RT-qPCR. We conclude the type of coating to be important during the differentiation process from Schwann cell precursor cells or immature Schwann cells to definitive Schwann cells, as well as the amounts of glucose in the specific differentiation medium to be crucial for increasing its efficiency and the final yield of viable hiPSC-SCs. Our hiPSC-SCs further displayed high similarity to primary adult human Schwann cells. [ABSTRACT FROM AUTHOR]

Exopolysaccharides are important metabolites of Monascus with healthy activities. However, the low production level limits their applications. Hence, the aim of this work was to increase the yield of exopolysaccharides (EPS) and optimize liquid fermentation by adding flavonoids. The EPS yield was optimized via both medium composition and culture conditions. The optional fermentation conditions achieved for EPS production of 7.018 g/L were 50 g/L sucrose, 3.5 g/L yeast extract, 1.0 g/L MgSO4·7H2O, 0.9 g/L KH2PO4, 1.8 g/L K2HPO4·3H2O, 1 g/L quercetin, and 2 mL/L Tween-80, with pH 5.5, inoculum size 9%, seed age 52 h, shaking speed 180 rpm, and fermentation culture 100 h, respectively. Furthermore, the addition of quercetin increased EPS production by 11.66%. The results also showed little citrinin residue in the EPS. The exopolysaccharides' composition and antioxidant capacity of quercetin-modified exopolysaccharides were then preliminarily investigated. The addition of quercetin changed the composition of the exopolysaccharides and the molecular weight (Mw). In addition, the antioxidant activity of Monascus exopolysaccharides was monitored using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS+), and -OH. Monascus exopolysaccharides have good scavenging ability of DPPH and -OH. Furthermore, quercetin increased the scavenging ABTS+ ability. Overall, these findings provide a potential rationale for the application of quercetin in improving the EPS yield. [ABSTRACT FROM AUTHOR]

The maturation of oocytes external to the body is referred to IVM and is utilised by many different species. IVM bypasses traditional superovulation methods, where several hormonal injections are administered. Unfortunately, the pregnancy rates using IVM oocytes is less than those matured in vivo. This has impeded the uptake of the technology in ART laboratories. This review aims to explore the effects of multiple elements during IVM, discussing not only those which improve and impact on oocyte viability but those that can have deleterious effects on the oocyte.

In this study, the liquid culture system of Pleurotus nebrodensis mycelium with high yield were established by using orthogonal experiments. Results indicated a 58.08 % increase in mycelium biomass and a 2.22 % increase in polysaccharide content after condition optimization. Experiments showed that the extracted polysaccharides have significant antioxidant and anti-exercise fatigue activities. They could effectively scavenge DPPH, ABTS, hydroxyl free radicals and superoxide anaion, prolong weight-loaded swimming time in mice, reduce levels of MDA, LD, and LDH in serum, enhance SOD activity, as well as increase hepatic and muscle glycogen reserves. The mechanism may be attributed to the activation of the Nrf2/Keap1 signaling pathway, inhibition of oxidative stress, and subsequent exerting anti-fatigue effects. The results in this work provides new avenue for easily accessible natural polysaccharide resources with a very convenient approach, which is beneficial for the development of anti-fatigue functional foods for public consumption. [ABSTRACT FROM AUTHOR]