Optimal Medium Research Articles

From Cell Clones to Recombinant Protein Product Heterogeneity in Chinese Hamster Ovary Cell Systems

Chinese hamster ovary (CHO) cells are commonly used to produce recombinant therapeutic proteins (RTPs). The yield of RTPs in CHO cells has been greatly improved through cell editing and optimization of culture media, cell culture processes, and expression vectors. However, the heterogeneity of cell clones and product aggregation considerably affect the yield and quality of RTPs. Recently, novel technologies such as semi-targeted and site-specific transgene integration, endoplasmic reticulum-residents, and cell culture process optimization have been used to address these issues. In this review, novel developments in the field of CHO cell expression system heterogeneity are summarized. Moreover, the advantages and limitations of the new strategies are discussed, and important methods for the control of RTP quality are outlined.

Improved efficiency of callus induction, bud differentiation, and plant regeneration in radish (Raphanus sativus L.)

ABSTRACT Radish (Raphanus sativus L.) is a recalcitrant variety for embryogenic callus induction that does not result in an efficient genetic transformation system. In this study, a highly efficient system for callus induction and bud differentiation in radish was established using orthogonal design tests with combinations of several synthetic auxins and cytokinins. Thirteen types of explants were tested; among them, cotyledon had the highest callus induction rate (91.01%) and was the most suitable explant. The optimal medium for callus induction was improved Murashige and Skoog medium (MS-1) supplemented with 0.2 mg/L 2,4-dichlorophenoxyacetic acid, 0.2 mg/L α-naphthalene acetic acid, 0.5 mg/L thidiazuron (TDZ), 4 mg/L 6-benzylaminopurine (BA), 0.1 mg/L kinetin, with a callus induction rate of 94.77%. The optimal medium for shoot regeneration was MS-1 medium supplemented with 1 mg/L TDZ, 1 mg/L BA, with a bud regeneration rate of 15.54%. The most suitable medium of rooting was 1/2 MS-1 medium supplemented with 0.2 mg/L NAA, 0.2 mg/L 3-indolebutyric acid, and 0.1% activated carbon, achieving a rooting ratio of 89.05% and transplant survival rate of 94.92%. This study lays a good foundation for future breeding and molecular studies on radishes. HIGHLIGHTS Cotyledon had the highest callus induction rate (91.01%) and were the optimal explants. Optimal medium for callus induction was MS-1 medium supplemented with 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg/L thidiazuron (TDZ), 0.2 mg/L α-naphthalene acetic acid (NAA), 4 mg/L 6-benzylaminopurine (BA), 0.1 mg/L kinetin, 8 g/L agar, and 3 g/L sugar, with a callus induction rate of 94.77%; Optimal medium for shoot regeneration was MS-1 medium supplemented with 1 mg/L TDZ, 1 mg/L BA, 8 g/L agar, and 3 g/L sugar, with a bud regeneration rate of 15.54%. An efficient regeneration system for callus induction and bud differentiation in radish has been established.

Optimized gene transduction in human lung organoids: A high-efficiency method for advanced research applications

Human induced pluripotent stem cell (iPSC)-derived lung organoids, engineered to carry targeted genes, offer a robust platform for investigating mechanistic insights in lung research. Although lentiviral vectors (LVVs) are highly effective for stable expression due to their integrative properties, achieving efficient transduction in human iPSC-derived lung organoids poses a significant technical challenge, likely due to the complex structure of these organoids. In this study, we optimized a method to enhance LVV transduction efficiency by physically disrupting the organoids to increase surface area, followed by spinoculation to apply shear force during cell dissociation. This approach, combined with the use of an optimized culture medium, significantly improved transduction efficiency. The success of this method was validated at both the gene and protein levels using single-cell RNA sequencing (scRNA-seq) and various cellular and molecular assays. Our optimized transduction protocol may provide a valuable tool for investigating specific cellular and molecular mechanisms in development and disease models using human iPSCs-derived lung organoids.

Optimization and characterization of bioactive secondary metabolites from Streptomyces sp CMSTAAHL-4 isolated from mangrove sediment

BackgroundTen morphologically different actinomycetes were isolated from mangrove sediments of Manakudy, Kanyakumari District, India. The potent strain was selected based on their primary screening against Gram positive Staphylococcus aureus, Enterococcus faecalis and Gram negative Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi bacterial pathogens. The selected strain was identified as Streptomyces sp CMSTAAHL-4 by 16S rRNA sequencing. The media optimization for secondary metabolites production was performed by One-Variable at a Time and Response Surface Methodology-Central Composite Design. Minimum inhibitory concentration and minimum bacterial concentration for the extracted secondary metabolites were determined. The antioxidant potential of the secondary metabolites showed that the concentration of the metabolites increases, with the percentage of inhibition. The anti-inflammatory activity of the secondary metabolites found that maximum activity was observed at 500 µg/ml of the metabolites. Alcohols, alkenes, alkynes, alkyl halides, carboxylic acids, aliphatic esters functional groups were identified by fourier transform infrared spectroscopy, gas chromatography and mass spectrometer analysis of the secondary metabolites revealed five bioactive compounds. The X-ray diffraction analysis revealed that the secondary metabolites are amorphous. The thermogravimetric analysis showed the thermal stability of secondary metabolites. Atomic force microscopy analysis revealed specific structural characteristics of the secondary metabolites, which may be associated with their potential biological activities.ConclusionsThe results showed that the antibacterial, antioxidant, and anti-inflammatory chemicals present in the isolated secondary metabolites give them therapeutic properties.

Model-driven engineering of Cutaneotrichosporon oleaginosus ATCC 20509 for improved microbial oil production.

Increasing demand for palm oil has drastic effects on the ecosystem as its production is unsustainable. C. oleaginosus is a yeast with great potential for microbial oil production and is a sustainable alternative to palm oil. Herein we deployed the Design-Build-Test-Learn approach to establish C. oleaginosus as an efficient fatty acid production platform. In the design step, we combined transcriptome data analysis and metabolic modeling and selected gene overexpression targets (ATP-citrate lyase, acetyl-CoA carboxylase, threonine synthase, and hydroxymethylglutaryl-CoA synthase) and media supplements (biotin, thiamine, threonine, serine, and aspartate). Characterization of transformants at various C/N ratios, and medium supplements provided up to 56% (w/w) lipid content and a 1.4-fold increase in lipid yield on glycerol (g/g). Additionally quadratic regressions suggested C/N ratio of 240 as the optimum value. These results and introduced pipeline for strain and medium optimization establish C. oleaginous as a sustainable alternative to palm as an oil source.

A Study on the Preparation of Multifunctional Bacillus spp. Composite Inoculants and Their Ability to Promote Watermelon Growth

The development of plant-growth-promoting bacteria (PGPB) is one of the important research topics in agricultural microbiology. Four bacterial isolates that exhibited nitrogen fixation, phosphate and potassium solubilization, as well as indole-3-acetic acid (IAA) and siderophore production abilities, were selected from biogas residue, namely BR-1, BR-16, BR-17, and BR-44. According to morphological and molecular biological identification, BR-1, BR-16, BR-17, and BR-44 belonged to Bacillus subtilis, Bacillus cereus, Bacillus megaterium, and Bacillus subtilis, respectively. The four functional strains were combined into a composite microbial community. The optimal media were peptone (15 g/L), sucrose (10 g/L), and KCl (2 g/L); the optimal incubation conditions were an initial pH of 7.0, a volume of 47 mL/250 mL, an inoculum size of 6% v/v, an incubation temperature of 31 °C, a shaking speed of 205 r/min, and an incubation period of 20 h, as determined through a single factor test and the response surface methodology (RSM). In the optimized process, a liquid bacterial inoculant with an OD600 of 1.866 was obtained, with an effective viable count of 8.955 × 109 CFU/mL. A solid bacterial inoculant was prepared by using bran as a substrate, and its effective viable count was 1.11 × 109 CFU/g. The application of the bacterial inoculants promoted the growth of watermelon, increased the photosynthetic efficiency and yield, and improved fruit quality. This study provides a theoretical basis and technical support for the development and application of microbial inoculants.

Adaptive Event‐Triggered Finite‐Time Control of Affine Cyber‐Physical Systems Under Denial‐of‐Service (DoS) and Deception Attacks

ABSTRACTAiming at sustainable development to meet the requirements of Industry 5.0/6.0 revolutions, integrating a resilient, decentralized cyber‐tolerant control framework for large‐scale affine systems is indispensable. This work introduces an intelligent finite‐time cyber‐resilient control framework for an affine system under sensor/actuator deception and denial‐of‐service attacks. First, an adaptive disturbance observer employing a radial basis function neural network (RBF‐NN) has been formulated for smooth and fast estimations of unknown aggregated mismatched uncertainties, which include exogenous disturbance, sensor deception attack, and parametric perturbation. Later, an adaptive finite‐time control law integrated with RBF‐NN is formulated under event‐triggered action for the investigated cyber‐physical affine system. A suitable triggering condition for data communication through the sensor‐to‐controller channel has been derived using the Lyapunov approach, and input‐to‐state stability has been guaranteed under the designed control framework. Numerical simulations with in‐depth qualitative analysis have been showcased to validate the effectiveness and feasibility of the applied control methodology. The suggested control methodology ensures optimal communication medium utilization and is superior in mitigating cyber intrusions.

The Establishment of a Highly Efficient In Vitro Regeneration System for Viburnum opulus L. ‘Roseum’

Viburnum opulus L. ‘Roseum’ is a highly valuable ornamental plant for landscaping, but it has a long propagation cycle and low propagation coefficient. In this study, stem segments with axillary buds from Viburnum opulus L. ‘Roseum’ were used as explants. We systematically analyzed the use of sodium hypochlorite for the sterilization of explants, as well as the effects of different plant growth regulator combinations and concentrations on shoot bud induction, shoot proliferation, the rooting of tissue-cultured shoots, and the transplanting of the tissue-cultured shoots. A complete rapid propagation technology system for Viburnum opulus L. ‘Roseum’ was established. The results showed that a disinfection method using 75% ethanol for 30 s and soaking in 5% sodium hypochlorite for 5 min was the most suitable for disinfecting the stem segments of Viburnum opulus L. ‘Roseum’, which showed low contamination and a 73.33% survival rate. The ideal medium for primary bud induction was WPM (Woody Plant Basal Medium) + 2.0 mg·L−1 6-benzylaminopurine (6-BA) + 0.15 mg·L−1 indole-3-butyric acid solution (IBA) + 25 g·L−1 sucrose. The optimal medium for shoot proliferation was WPM + 1.0 mg·L−1 6-BA + 0.15 mg·L−1 IBA + 25 g·L−1 sucrose, achieving an induction rate of 7.17. For the rooting of tissue-cultured shoots, the most suitable formulation was 1/2 WPM + 0.3 mg·L−1 naphthaleneacetic acid (NAA) + 0.3 mg·L−1 activated charcoal (AC) + 25 g·L−1 sucrose, which induced robust and developed root systems. This study provides a technical basis for the establishment of a fast propagation system for the industrial production of Viburnum opulus L. ‘Roseum’.

Corn or Soybean Oil as the Sole Carbon Source for Polyhydroxybutyrate Production in a Biofuel Biorefinery Concept

The use of polyhydroxybutyrate (PHB) can help diminish fossil chemical dependency because it can partially replace petrochemical plastics due to its biodegradability and similar mechanical properties. However, its production costs are high compared with fossil-based plastics. Alternative carbon sources can be used in the fermentation media because they are renewable and low-cost. Vegetable oils are especially attractive due to their high carbon content, contributing to high production rates per gram of substrate. This work aimed to produce PHB from Cupriavidus necator LPB1421 using either corn or soybean oil as the sole carbon source. Urea was the best nitrogen source, enabling a DCW production of 4.35 g/L (corn oil) and 10.4 g/L (soybean oil). After media optimization, the DCW of corn oil reached 22.13 g/L, with 57.46% PHB accumulation (12.71 g PHB/L), whereas soybean oil led to a DCW of 19.83 g/L, with 54.91% PHB accumulation (10.89 g PHB/L). This media composition was employed in a kinetics assay, revealing similar fermentation parameters among both oils and a yield of 0.2118 g PHB/g for corn oil and 0.1815 g PHB/g for soybean oil. These results open the possibility of integrating PHB production with biofuel manufacturing in a bioethanol/biodiesel biorefinery concept.

High-Throughput Chemotherapeutic Drug Screening System for Gastric Cancer (Cure-GA).

Three dimensional (3D) cell cultures can be effectively used for drug discovery and development but there are still challenges in their general application to high-throughput screening. In this study, we developed a novel high-throughput chemotherapeutic 3D drug screening system for gastric cancer, named 'Cure-GA', to discover clinically applicable anticancer drugs and predict therapeutic responses. Primary cancer cells were isolated from 143 fresh surgical specimens by enzymatic treatment. Cell-Matrigel mixtures were automatically printed onto the micropillar surface then stabilized in an optimal culture medium for 3 days to form tumoroids. These tumoroids were exposed in the drug-containing media for 7 days. Cell viability was measured by fluorescence imaging and adenosine triphosphate assays. On average, 0.31±0.23g of fresh tumor tissue yielded 4.05×106 ± 4.38×106 viable cells per sample. Drug response results were successfully acquired from 103 gastric cancer tissues (success rate = 72%) within 13±2 days, averaging 6.4±2.7 results per sample. Pearson correlation analysis showed viable cell numbers significantly impacted drug data acquisition (p<0.00001). Tumoroids retained immunohistochemical characteristics, mutation signatures, and gene expression consistent with primary tumors. Drug reactivity data enabled prediction of synergistic drug correlations. Additionally, a multiparameter index-based prognosis model for patients undergoing gastrectomy followed by adjuvant XELOX was developed, showing significant differences in 1-year recurrence-free survival rates between drug responders and non-responders (p<0.0001). The Cure-GA platform enables rapid evaluation of chemotherapeutic responses using patient-derived tumoroids, providing clinicians with crucial insights for personalized treatment strategies and improving therapeutic outcomes.

Numerical Study on the Mechanism of Gas Explosion Suppression by Different Arrangement Forms of Staggered Cylinders

ABSTRACT In order to study the suppression mechanism of porous media on gas explosion, the gas explosion behavior in a pipeline with one end closed and the other end temporarily closed by a thin film was numerically simulated in this paper, in which the porous media structure was characterized by “pseudo-porous media” composed of staggered cylinders. The RANS method is used to simulate turbulence in numerical calculation, and the coupling effect between turbulence and combustion is described by the reaction progress variable equation. The calculation results show that, compared with porosity, hole spacing is a more critical parameter affecting gas explosion. The function of pores is mainly reflected in the initial stage of explosion flame development, and the function of pore spacing is reflected after the flame reaches the maximum instantaneous displacement velocity. When the Pe number calculated based on local aperture and laminar flame velocity is less than 72, the flame cannot pass through porous media, and the influence of porous media on gas explosion can be shown. When the Pe number is greater than this value, the influence of porous media on gas explosion is not obvious; The hole spacing has a significant effect on the flame shape, bifurcation number, and flame thickness. When the hole spacing is larger, the flame front is more slender and bifurcated, while when the hole spacing is smaller, the flame presents a more blunt shape, and its expansion is delayed to a greater extent. This law provides a strong basis for the optimization of porous media in burner design, especially in adjusting the flame shape and improving combustion efficiency.

Enhancing Bioactive Cordycepin Production via Precision Fermentation with an Engineered Aspergillus oryzae

An optimal culture medium and highly stable biometabolites are important in industrial production processes. The response surface methodology with a Box–Behnken design was performed to determine the optimal culture medium of an engineered Aspergillus oryzae strain for cordycepin production by submerged fermentation. The influences of glucose, yeast extract, and adenine concentrations on cordycepin production were explored, and their concentrations were used for experimental design. The results reveal that the optimal culture components involved 30.0 g/L of glucose, 9.8 g/L of yeast extract, and 1.5 g/L of adenine. As predicted, the maximum cordycepin concentration (1724.53 ± 18.30 mg/L) was obtained with a short fermentation time (2 days). A significant increase in cordycepin yield (&gt;50% increase) was observed in the culture grown in the optimized culture medium compared to that grown in the basal medium. A xanthine oxidase inhibitory activity assay demonstrated that the cordycepin product had a pharmacological function. It exhibited strong stability under high thermal and acidic conditions, with over 95% product recovery. The findings of this study are valuable for developing cost-effective processes for producing health-benefiting products.

Study on the Establishment of Efficient Leaf Regeneration System in ‘Yuluxiang’ Pear

The ‘Yuluxiang’ pear is a key cultivated variety in China, celebrated for its high quality. However, it exhibits a low leaf regeneration frequency of only 35.0%, which hinders its transgenic breeding process. To establish an efficient regeneration system, we utilized tissue culture seedlings of the ‘Yuluxiang’ pear and investigated various factors influencing leaf regeneration: plant growth regulators, natural organic materials, leaf wounding and positioning methods, duration of dark culture, ages and lines of plantlets, as well as culture containers. Our results indicated that the optimal medium for leaf regeneration consisted of MS supplemented with 6-BA (6-Benzyl Aminopurine) 1.5 mg/L, NAA (α-Naphthalene acetic acid) 0.4 mg/L along with 10% (v/v) coconut water. Suitable wounding involved ensuring no damage to leaves while placing the abaxial side facing down on the medium; the ideal duration for dark culture was determined to be 21 days; optimal plantlet age was found to be 20 days; both plantlet line 1 and line 2 demonstrated effectiveness; triangle bottles were identified as appropriate culture containers. In summary, we successfully established an efficient leaf regeneration system for the ‘Yuluxiang’ pear that achieved a maximum regeneration frequency of 96.70% with an average bud number of 5.15 per explant. This system also proved effective for the ‘Qiuyue’ pear, yielding a regeneration frequency of 88.89% and an average bud number of 3.44 per explant. After investigating the germination methods of 200 leaves from this screened leaf regeneration system of ‘Yuluxiang’, it was found that there were both direct and indirect regeneration methods, and the germination rate of direct and indirect regeneration was 68.00% and 70.50%, respectively. Therefore, this study also laid a solid foundation for the future genetic transformation of ‘Yuluxiang’ pear.

Evaluation of the effectiveness of communications between the Department of transport and Development of road transport infrastructure of Moscow and the population within the framework of the implementation of strategic projects

The article examines the assessment of the effectiveness of communications between the Moscow Department of Transport and Development of Road Transport Infrastructure (DTRDTI) and the population in the context of implementing strategic projects. The purpose of the study is to determine the level of information transparency and accessibility of data on the department’s key initiatives for citizens. The materials and methods include the comparative and content analysis as well as statistical processing of data from DTRDTI’s official websites, publications in the media, social networks and other platforms for the period from 2018 to 2022. The methods of content analysis, comparative analysis and statistical data processing are used. The sample covers 1,520 news stories and information messages. The results show that DTRDTI actively informs Muscovites about the progress of strategic projects. Over the study period, the number of publications increased by 67%, and audience reach, and coverage increased by 84%. A special section “Strategic Projects” has been created on the official website of the department, which is updated weekly. Press conferences, briefings and meetings with journalists are regularly held. In 2022, the Moscow Transport mobile application was launched, allowing to track the work on projects and diverse activities of DTRDTI in real time. The use of VR and 3D modeling technologies for visualization of future objects is noted. At the same time, some problems were identified: uneven presentation of information on different projects, insufficient adaptation of content for perception by non-specialists, lack of versions in foreign languages. It is concluded that DTRDTI demonstrates a high level of information openness. At the same time, it is necessary to ensure a more balanced and accessible coverage of the projects being implemented for a wider audience, as well as to develop new communication channels and formats. The experience of the DTRDTI can be of value not only for the relevant agencies of other regions, but also for the scientific community. The approaches and analysis algorithms proposed in the article are applicable for media audit and optimisation of information activities of authorities of different levels.

Peningkatan Produktivitas UMKM Mille Crepes “Kirimi Dessert”

The COVID-19 pandemic has had a significant impact on the Small and Medium Enterprises (SMEs) sector, including Kirimi dessert, a home store located on Jalan Noja I, Kesiman Petilan Village, Denpasar. This study aims to identify problems and provide solutions for Kirimi dessert MSMEs in facing challenges during the pandemic. The business, which focuses on making mille crepes and various desserts, faces several main obstacles such as limited production space, inability to fulfil orders when demand is high, and lack of promotion and personal branding. The research methodology used a qualitative approach through interviews and discussions with the business owner. The solutions offered include the development of MSME innovation, business expansion, and increased promotion through logo design, packaging, and social media optimisation. The implementation of this solution is expected to help MSMEs increase market reach and sales during the COVID-19 pandemic.

Enhancement Strategy for Protocatechuic Acid Production Using Corynebacterium glutamicum with Focus on Continuous Fermentation Scale-Up and Cytotoxicity Management.

Protocatechuate acid (PCA) is a phenolic acid naturally synthesized by various organisms. Protocatechuic acid is synthesized by plants for physiological, metabolic functions, and self-defense, but extraction from plants is less efficient compared to the microbial culture process. The microbial synthesis of protocatechuic acid is sustainable and, due to its high yield, can save energy consumption when producing the same amount. To enhance PCA production using Corynebacterium glutamicum, a statistical optimization of the production medium was performed using full factorial design, the steepest ascent method, and the response surface method. The optimized production medium enabled a PCA production of over 5 g/L in a 72 h batch culture. However, PCA cytotoxicity affected the strain growth and PCA production rate, with an inhibitory concentration of approximately 5 g/L in the fermentation broth. Finally, continuous fermentation was operated for 150 h in the steady-state mode, maintaining the concentration of PCA below 5 g/L. The optimization method established in this study successfully increased PCA production levels, and the findings presented herein are anticipated to contribute to the industrialization of PCA production using C. glutamicum.

Optimization of Submerged Fermentation Conditions for Polysaccharide Production in Species of the Genus Ganoderma (Agaricomycetes) and Comparative Analysis of the Antioxidant Activities of Different Strains

Species of the genus &lt;i&gt;Ganoderma &lt;/i&gt;are important medicinal and edible mushrooms, with polysaccharides being its main active ingredient. The submerged fermentation of &lt;i&gt;Ganoderma&lt;/i&gt; is an important source of polysaccharides in the mycelia. In this study, a single factor test and an orthogonal test were employed to optimize the medium and fermentation conditions with the objective of obtaining a high content of polysaccharide in the liquid submerged fermentation culture mycelia of &lt;i&gt;Ganoderma&lt;/i&gt; species. The results demonstrated that the optimal culture medium formula for polysaccharide accumulation in mycelia was: soluble starch, 25 g/L; wheat bran, 3 g/L; and KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 4.5 g/L, with a pH of 4.0, and the optimal culture conditions were a temperature of 27&amp;deg;C, a rotational speed of 90 r/min, and an incubation period of 11 d. Furthermore, the mycelia of 20 strains from nine species of &lt;i&gt;Ganoderma &lt;/i&gt;were cultured under optimal fermentation conditions, and the content of polysaccharide in mycelia and its antioxidant activity were analyzed. The findings revealed that the polysaccharide content in the mycelia of strains GD and Du996 was notably elevated, at 56.97 and 53.22 mg/g, respectively. The polysaccharide content was found to be high in the mycelia of &lt;i&gt;G. sichuanense&lt;/i&gt; and &lt;i&gt;G. lucidum&lt;/i&gt;, with an average of 35 mg/g. Nevertheless, no notable discrepancy was observed in the polysaccharide content of the mycelia of the cultivated and wild strains. Moreover, the antioxidant activity analysis revealed that the DPPH clearance rate of strains Du1320, Du280, and GD was 92.85&amp;#37;, 90.86&amp;#37;, and 89.06&amp;#37;, respectively. The ABTS clearance rate of strain GSD was the highest (75.39&amp;#37;), and the hydroxyl radical clearance rate of strain GD was the highest (81.98&amp;#37;). In conclusion, this study optimized the medium and conditions for high polysaccharide yield in the liquid submerged fermentation culture of &lt;i&gt;Ganoderma&lt;/i&gt; and identified the strains with the highest yield and antioxidant activity. The present results may provide a theoretical basis for the future production of polysaccharides in mycelia by liquid fermentation of &lt;i&gt;Ganoderma&lt;/i&gt; as well as for the development and utilization of strains with high polysaccharide yield and antioxidant activity.

Development of a novel encystment medium: Enhancing diagnostic potential of Acanthamoeba spp.

Background and Aim: Acanthamoeba spp. are pathogenic microorganisms linked to severe infections in humans and animals, requiring a deeper understanding of their encystation process for effective diagnostics and research. This study focused on developing a novel encystment medium to induce synchronized encystation of Acanthamoeba spp. efficiently and rapidly. Materials and Methods: The study employed response surface methodology with a central composite design to optimize the encystment medium formulation. The key components included Tris-HCl, NaCl, glucose, and MgCl2. The optimized liquid medium was spray-dried to produce a dehydrated powder for practical application. The encystation efficiency of different Acanthamoeba strains was assessed using hemocytometry and fluorescence microscopy. Results: The optimized medium, comprising 3.152 g/L Tris-HCl, 5.55 g/L NaCl, 8% (w/v) glucose, and 5.0 mM MgCl2 at pH 9.0, demonstrated exceptional encystation efficiency with rates ranging from 99% to 100%. A spray-dried powdered version of this medium was equally effective, achieving a 98.77% encystation rate for A. castellanii American Type Culture Collection 50739 in glucose-free conditions. Notably, optimal glucose concentrations varied among Acanthamoeba strains, with certain strains reaching maximum encystation at 6–8% glucose. Conclusion: This study successfully developed an innovative encystment medium that promotes rapid and efficient cyst production in Acanthamoeba spp. The medium enhances laboratory research and diagnostic capabilities, paving the way for future advancements in understanding and managing Acanthamoeba infections. Keywords: Acanthamoeba, diagnostic tools, encystation, medium optimization, response surface methodology.

Response surface methodology and artificial neural network based media optimization for pullulan production in Aureobasidium pullulans.

The selection and optimization of carbon and nitrogen sources are essential for enhancing pullulan production in Aureobasidium pullulans. In this study, combinations of carbon (sucrose, fructose, glucose) and nitrogen sources ((NH4)2SO4, urea, NaNO3) were screened, where sucrose and NaNO3 offered the highest pullulan yield (9.33gL-1). Plackett-Burman design of experiment identified KH2PO4, NaCl, and sucrose as significant factors, which were further optimized using a central composite design. A hyperparameter-optimized artificial neural network (ANN) model with a 3-6-2-1 architecture demonstrated superior predictive accuracy (R2: 0.96) and generalizability (R2CV: 0.74) over a reduced quadratic model (R2: 0.82). The predicted pullulan yield (31.9gL-1) under ANN model optimized conditions (sucrose: 79.9gL-1, KH2PO4: 0.25gL-1, NaCl: 4.3gL-1) closely matched with the observed yield (30.17gL-1), while quadratic model showed a significant deviation (39.7gL-1 vs. 21.0gL-1), highlighting the reliability of the ANN model.

Selection of the Best Performing Media for the Growth and Development of Cordyceps militaris Mycelia

Since ages, the genus Cordyceps has been used as a medicinal fungus for augmenting endurance, longevity and vitality. This fungus commands a high commercial value due to scarcity in its availability and high global market demand. In the present investigations different defined media viz, agar, optimised nutrient broth, chitosan + malt extract agar, chitosan + agar + optimised nutrient broth, Kenknights and Munnaiers, malt extract agar, malt extract agar + optimised nutrient broth, oat meal agar, rice extract agar, rice extract agar + optimised nutrient broth and rose bengal agar have been used to identify the optimal media supporting the growth of Cordyceps militaris mycelia. After 14-days of culture, chitosan + agar + optimised nutrient broth, Kenknights and Munnaiers media, oat meal agar media and rice extract agar media recorded better mycelial growth, in comparison to agar, that was used as control. Maximal mycelial growth was recorded on Kenknights and Munnaiers media, with a 20% higher growth than the agar media. Even after 21 days of culture, Kenknights and Munnaiers media recorded best growth characteristics with a 25% higher mycelial growth. It was also recorded that the mycelial growth rate on Kenknights and Munnaiers media was highest on the 14th day, in comparison to 7th and 21st day after inoculation. Thus, our results provide valuable scientific information towards in-vitro culture of Cordyceps with optimal growth dynamics, for sustaining market demand of this important mushroom.