UNASSIGNED: Amphiregulin (AR) is a growth factor that resembles the epidermal growth factor (EGF) and serves various functions in different cells. However, no systematic studies or reports on the role of AR in human oocytes have currently been performed or reported. This study aimed to explore the role of AR in human immature oocytes during in vitro maturation (IVM) and in vitro fertilization (IVF) in achieving better embryonic development and to provide a basis for the development of a pre-insemination culture medium specific for cumulus oocyte complexes (COCs).
UNASSIGNED: First, we examined the concentration of AR in the follicular fluid (FF) of patients who underwent routine IVF and explored the correlation between AR levels and oocyte maturation and subsequent embryonic development. Second, AR was added to the IVM medium to culture immature oocytes and investigate whether AR could improve the effects of IVM. Finally, we pioneered the use of a fertilization medium supplemented with AR for the pre-insemination culture of COCs to explore whether the involvement of AR can promote the maturation and fertilization of IVF oocytes, as well as subsequent embryonic development.
UNASSIGNED: A total of 609 FF samples were examined, and a positive correlation between AR levels and blastocyst formation was observed. In our IVM study, the development potential and IVM rate of immature oocytes, as well as the fertilization rate of IVM oocytes in the AR-added groups, were ameliorated significantly compared to the control group (All P < 0.05). Only the IVM-50 group had a significantly higher blastocyst formation rate than the control group (P < 0.05). In the final IVF study, the maturation, fertilization, high-quality embryo, blastocyst formation, and high-quality blastocyst rates of the AR-added group were significantly higher than those of the control group (All P < 0.05).
UNASSIGNED: AR levels in the FF positively correlated with blastocyst formation, and AR involvement in pre-insemination cultures of COCs can effectively improve laboratory outcomes in IVF. Furthermore, AR can directly promote the in vitro maturation and developmental potential of human immature oocytes at an optimal concentration of 50 ng/ml.

Pyrroloquinoline quinone (PQQ) is a redox cofactor with numerous important physiological functions, and the type VI secretion system (T6SS) is commonly found in Gram-negative bacteria and plays important roles in physiological metabolism of the bacteria. In this study, we found that the deletion of pqqF enhanced the secretion of Hcp-1 in Serratia marcesens FS14 in M9 medium. Transcriptional analysis showed that the deletion of pqqF almost had no effect on the expression of T6SS-1. Further study revealed that the increased secretion of Hcp-1 was altered by the pH changes of the culture medium through the reaction catalyzed by the glucose dehydrogenases in FS14. Finally, we demonstrated that decreased pH of culture medium has similar inhibition effects as PQQ induced on the secretion of T6SS-1. This regulation mode on T6SS by pH in FS14 is different from previously reported in other bacteria. Therefore, our results suggest a novel pH regulation mode of T6SS in S. marcesens FS14, and would broaden our knowledge on the regulation of T6SS secretion.

This study aimed to elucidate the influence of various culture medium components, including carbon sources, nitrogen sources, inorganic salts, suspension agents, and temperature, on the mycelial growth characteristics of Phallus dongsun. Employing single-factor experiments and response surface methodology within glass Petri dishes, the research identified that carrot powder, soybean powder, and ZnSO4 notably enhanced the proliferation of aerial mycelium, significantly augmenting the growth rate of P. dongsun mycelium. The resultant mycelium was observed to be dense, robust, and fluffy in texture. In particular, ZnSO4 markedly accelerated the mycelium growth rate. Furthermore, xanthan gum was found to effectively modulate the medium\'s viscosity, ensuring a stable suspension and facilitating nutrient equilibrium. The optimal cultivation temperature was determined to be 25°C, with mycelial growth ceasing below 5°C and mycelium perishing at temperatures exceeding 35°C. The optimal medium composition was established as follows: wheat starch 5 g/l, carrot powder 5 g/l, soybean powder 7.50 g/l, glucose 10 g/l, ZnSO4 0.71 g/l, NH4Cl 0.68 g/l, xanthan gum 0.5 g/l, and agar 20 g/l. Under these optimized conditions, the mycelium of P. dongsun exhibited a rapid growth rate (1.04 ± 0.14 mm/day), characterized by a thick, dense, and well-developed structure. This investigation provides a theoretical foundation for the conservation, strain selection, and breeding of P. dongsun.

Herpes simplex virus type 1 (HSV-1) plays an important role in the field of gene therapy and viral vaccines, especially as an oncolytic virus. However, the mass production of HSV-1 viral vectors remains a challenge in the industry. In this study, a microcarrier-mediated serum-reduced medium culture was used to improve the bioprocess of HSV-1 production and increase HSV-1 yields. The composition of the culture media, which included a basal medium, serum concentration, and glutamine additive, was optimized. The process was successfully conducted in a 1 L bioreactor, and virus production was threefold greater than that of conventional processes with a 10% serum medium. The bead-to-bead transfer process was also developed to further increase scalability. In spinner flasks, the detachment rate increased from 49.4 to 80.6% when combined agitation was performed during digestion; the overall recovery proportion increased from 37.9 to 71.1% after the operational steps were optimized. Specifically, microcarrier loss was reduced during aspiration and transfer, and microcarriers and detached cells were separated with filters. Comparable cell growth was achieved with the baseline process using 2D culture as the inoculum by exchanging the subculture medium. To increase virus production after bead-to-bead transfer, critical parameters, including shear stress during digestion, TrypLE and EDTA concentrations in the subculture, and the CCI, were identified from 47 parameters via correlation analysis and principal component analysis. The optimized bead-to-bead transfer process achieved an average of 90.4% overall recovery and comparable virus production compared to that of the baseline process. This study is the first to report the optimization of HSV-1 production in Vero cells cultured on microcarriers in serum-reduced medium after bead-to-bead transfer. KEY POINTS: • An HSV-1 production process was developed that involves culturing in serum-reduced medium, and this process achieved threefold greater virus production than that of traditional processes. • An indirect bead-to-bead transfer process was developed with over 90% recovery yield in bioreactors. • HSV-1 production after bead-to-bead transfer was optimized and was comparable to that achieved with 2D culture as inoculum.

2-Phenylethanol (2-PE) is an aromatic compound with a rose-like fragrance that is widely used in food and other industries. Yeasts have been implicated in the biosynthesis of 2-PE; however, few studies have reported the involvement of filamentous fungi. In this study, 2-PE was detected in Annulohypoxylon stygium mycelia grown in both potato dextrose broth (PDB) and sawdust medium. Among the 27 A. stygium strains investigated in this study, the strain \"Jinjiling\" (strain S20) showed the highest production of 2-PE. Under optimal culture conditions, the concentration of 2-PE was 2.33 g/L. Each of the key genes in Saccharomyces cerevisiae shikimate and Ehrlich pathways was found to have homologous genes in A. stygium. Upon the addition of L-phenylalanine to the medium, there was an upregulation of all key genes in the Ehrlich pathway of A. stygium, which was consistent with that of S. cerevisiae. A. stygium as an associated fungus provides nutrition for the growth of Tremella fuciformis and most spent composts of T. fuciformis contain pure A. stygium mycelium. Our study on the high-efficiency biosynthesis of 2-PE in A. stygium offers a sustainable solution by utilizing the spent compost of T. fuciformis and provides an alternative option for the production of natural 2-PE. KEY POINTS: • Annulohypoxylon stygium can produce high concentration of 2-phenylethanol. • The pathways of 2-PE biosynthesis in Annulohypoxylon stygium were analyzed. • Spent compost of Tremella fuciformis is a potential source for 2-phenylethanol.

Xenocoumacin 1 (Xcn 1), antibiotic discovered from secondary metabolites of Xenorhabdus nematophila, had the potential to develop into a new pesticide due to its excellent activity against bacteria, oomycetes and fungi. However, the current low yield of Xcn1 limits its development and utilization. To improve the yield of Xcn1, response surface methodology was used to determine the optimal composition of fermentation medium and one factor at a time approach was utilized to optimize the fermentation process. The optimal medium composed of in g/L: proteose peptone 20.8; maltose 12.74; K2HPO4 3.77. The optimal fermentation conditions were that 25 °C, initial pH 7.0, inoculum size 10%, culture medium 75 mL in a 250 mL shake flask with an agitation rate of 150 rpm for 48 h. Xenorhabdus nematophila YL001 was produced the highest Xcn1 yield (173.99 mg/L) when arginine was added to the broth with 3 mmol/L at the 12th h. Compared with Tryptic Soy Broth medium, the optimized fermentation process resulted in a 243.38% increase in Xcn1 production. The obtained results confirmed that optimizing fermentation technology led to an increase in Xcn1 yield. This work would be helpful for efficient Xcn1 production and lay a foundation for its industrial production.

Wolfiporia cocos, a versatile fungus acclaimed for its nutritional and therapeutic benefits in Traditional Chinese Medicine, holds immense potential for pharmaceutical and industrial applications. In this study, we aimed to optimize liquid fermentation techniques and culture medium composition to maximize mycelial biomass (MB) yield, pachymic acid (PA) concentration, and overall PA production. Additionally, we investigated the molecular basis of our findings by quantifying the expression levels of genes associated with PA and MB biosynthesis using quantitative real-time polymerase chain reaction. Under the optimized fermentation conditions, significant results were achieved, with maximum MB reaching 6.68 g l-1, PA content peaking at 1.25 mg g-1, and a total PA yield of 4.76 g l-1. Notably, among the four examined genes, squalene monooxygenase, exhibited enhanced expression at 0.06 ratio under the optimized conditions. Furthermore, within the realm of carbohydrate-active enzymes, the glycoside hydrolases 16 family displayed elevated expression levels at 21 ratios, particularly during MB production. This study enhances understanding of genetic mechanism governing MB and PA production in W. cocos, highlighting the roles of squalene monooxygenase and glycoside hydrolases 16 carbohydrate-active enzymes.

In this research, to provide an optimal growth medium for the production of iturin A, the concentrations of key amino acid precursors were optimized in shake flask cultures using the response surface method. The optimized medium were applied in a biofilm reactor for batch fermentation, resulting in enhanced production of iturin A. On this basis, a step-wise pH control strategy and a combined step-wise pH and temperature control strategy were introduced to further improve the production of iturin A. Finally, the fed-batch fermentation was performed based on combined step-wise pH and temperature control. The titer and productivity of iturin A reached 7.86 ± 0.23 g/L and 65.50 ± 1.92 mg/L/h, respectively, which were 37.65 and 65.20% higher than that before process optimization.

This study investigates the influence of four culture media pre-equilibration methods on embryo development and clinical pregnancy outcomes. The methods are as follows: Method A involved covering media with fresh mineral oil in humid-type incubators for 24 h. Method B replicated Method A in dry-type incubators. Method C utilized pre-equilibrated (humidified) mineral oil to cover the media, also in humid-type incubators for 24 h. Method D followed the same process as Method C but in dry-type incubators. Subsequently, media from all groups were transferred to dry-type incubators for 72 h. Osmolality was measured at 24, 48, 72, and 96 h. For G1 PLUS, no significant differences were observed among groups at 24, 48, and 72 h. However, at 96 h, Groups B and D exhibited significantly higher osmolality than Groups A and C (A vs B, p = 0.043; A vs D: p = 0.046; B vs C, p = 0.043; C vs D, p = 0.046). No significant variations were found between Groups A and C or B and D. Similar results were obtained for G2 PLUS. A retrospective analysis of embryo development and clinical outcomes using Methods A revealed significant improvements in good blastocysts and available embryos compared with Method B for all (p = 0.005 and 0.004) and IVF cycles (p = 0.025 and 0.017). Method A also enhanced blastocyst formation in ICSI cycles (p = 0.017). However, clinical pregnancy outcomes did not significantly differ between Methods A and B. Pre-equilibrating culture media overnight in humid-type incubators, even when covered with fresh mineral oil, significantly mitigates osmolality rise and improves embryo development potential during embryo culture in dry-type incubators.

Ralstonia solanacearum is a devastating phytopathogen infecting a broad range of economically important crops. Phosphate (Pi) homeostasis and assimilation play a critical role in the environmental adaptation and pathogenicity of many bacteria. However, the Pi assimilation regulatory mechanism of R. solanacearum remains unknown. This study revealed that R. solanacearum pstSCAB-phoU-phoBR operon expression is sensitive to extracellular Pi concentration, with higher expression under Pi-limiting conditions. The PhoB-PhoR fine-tunes the Pi-responsive expression of the Pho regulon genes, demonstrating its pivotal role in Pi assimilation. By contrast, neither PhoB, PhoR, PhoU, nor PstS was found to be essential for virulence on tomato plants. Surprisingly, the PhoB regulon is activated in a Pi-abundant rich medium. Results showed that histidine kinase VsrB, which is known for the exopolysaccharide production regulation, partially mediates PhoB activation in the Pi-abundant rich medium. The 271 histidine of VsrB is vital for this activation. This cross-activation mechanism between the VsrB and PhoB-PhoR systems suggests the carbohydrate-Pi metabolism coordination in R. solanacearum. Overall, this research provides new insights into the complex regulatory interplay between Pi metabolism and growth in R. solanacearum.