Supplementation Of Medium Research Articles

Effect of lactose on the in vitro development of sheep secondary follicles

Considering that follicular development is an energy-dependent process, supplementation of the culture medium with energy substrates, such as lactose, would improve follicle viability and growth. Thus, the aim of this study was to evaluate the effect of lactose on morphology, development, glutathione (GSH) concentration, mitochondrial activity, DNA fragmentation, and meiotic resumption of oocytes from sheep secondary follicles cultured in vitro. Secondary follicles were isolated from the cortex of ovine ovaries and cultured individually for 18 days in α-MEM supplemented with bovine serum albumin (BSA), insulin, glutamine, hypoxanthine, transferrin, selenium and ascorbic acid (control medium: α-MEM+) or in α-MEM+ plus different concentrations of lactose (0.025, 0.05 and 0.1 M). After culture, some of the oocytes were subjected to TUNEL assay and in vitro maturation (IVM). Follicular morphology, glutathione (GSH) concentration and mitochondrial activity were evaluated at the end of the culture. At the day 18, the percentage of morphologically normal follicles was greater (P<0.05) in the treatment of 0.025 M lactose (92.5 %) compared to the control group (75.55 %). In addition, GSH concentrations increased (P<0.05) in treatment containing 0.025 M lactose compared to the other treatments. Furthermore, oocytes cultured in 0.025 M lactose had greater (P<0.05) mitochondrial activity levels than in α-MEM+ and 0.1 M lactose. The group α-MEM+ presented a increase of TUNEL-positive oocytes (35.09 %) compared to 0.025 lactose (9.09 %). The percentage of meiotic resumption was greater (P<0.05) in oocytes from secondary follicles cultured in 0.025 M lactose (54.5 %) than in α-MEM+ (45.5 %). In conclusion, 0.025 M lactose improved survival, GSH and active mitochondria levels and meiotic resumption of oocytes from in vitro cultured secondary follicles. Supplementation of the culture medium of preantral follicles with lactose can gradually provide energy to follicular cells, potentially enhancing the production of viable oocytes for biotechniques such as IVM and in vitro fertilization.

Sclerostin inhibits Protein kinase C inhibitor GÖ6976 induced osteogenic differentiation of dental follicle cells

Human dental follicle cells (DFCs) as multipotent stem cells are currently investigated within the field of regenerative medicine considering their potential for the regeneration of dental tissues, bone defects caused by periodontal or degenerative diseases and the treatment of craniofacial disorders. However, molecular mechanisms of the differentiation into mineralizing cells are still inadequately understood. Previous studies have shown that GÖ6976, an inhibitor of classical isoforms of protein kinase C (PKC), enhanced ostogenic differentiation of DFCs. A possible mechanism for increased osteogenic differentiation could be the regulation of ossification inhibitors. This study therefore investigated whether the osteogenic differentiation inhibitor sclerostin (SOST) is regulated by GÖ6976 and whether the addition of sclerostin attenuates the stimulating effect of the PKC inhibitor. We demonstrated that the expression of the sclerostin gene decreased after PKC inhibition by GÖ6976 and that its gene expression is likely maintained by PKC via the BMP signaling pathway. Furthermore, supplementation of osteogenic differentiation medium with sclerostin impairs GÖ6976-induced differentiation of DFCs. Our data suggest that regulation of sclerostin mediates PKC inhibition-induced mineralization of DFCs.

Oxophilic gallium single atoms bridged ruthenium clusters for practical anion-exchange membrane electrolyzer

The development of highly efficient and durable alkaline hydrogen evolution reaction (HER) catalysts is crucial for achieving high-performance practical anion exchange membrane water electrolyzer (AEMWE) at ampere-level current density. Herein, we report a design concept by employing Ga single atoms as an electronic bridge to stabilize the Ru clusters for boosting alkaline HER performance in practical AEMWE. Experimental and theoretical results collectively reveal that the bridged Ga sites trigger strong metal-support interaction for the homogeneous distribution of Ru clusters with high density, as well as optimize the Ru–H bond strength due to the electron transfer between Ru and Ga for enhanced intrinsic HER activity. Moreover, the oxophilic Ga sites near the Ru clusters tend to adsorb the hydroxyl species and accelerate the water dissociation for sufficient proton supplement in an alkaline medium. The Ru–GaSA/N–C catalyst exhibits a low overpotential of 4 ± 1 mV (10 mA cm−2) and high mass activity of 9.3 ± 0.5 A mg−1Ru at −0.05 V vs RHE. In particular, the Ru–GaSA/N–C-based AEMWE in 1 M KOH delivers a voltage of only 1.74 V to reach an industrial current density of 1 A cm−2, and can steadily operate at 1 A cm−2 for more than 170 h.

Study of Shoot Tip Necrosis Problems of Fegra Fig (Ficus palmata Forssk.) In Vitro in Saudi Arabia

This work studied the micropropagation of fegra fig (F. palmata Forssk.) during which we experienced the incidence of shoot-tip necrosis (STN). STN was evident during the shoot elongation stage, which was regenerated on Murashige and Skoog (MS) medium supplemented with 2 mg/L 6-benzylaminopurine. To alleviate SNT, we conducted a series of experiments and supplemented the medium with calcium chloride (40, 80, and 120 mg/L), ascorbic acid (50, 100, and 150 mg/L), silver nitrate (1, 2, and 3 mg/L), and boric acid (9.3, 12.4, and 15.5 mg/L). Results showed that all the treatments controlled STN at varying levels, and supplementation of medium with 3 mg/L silver nitrate reduced the incidence of STN from 80% to 24%. The regenerated shoots were rooted on the same medium with incubation of cultures in the dark for 3 weeks and subsequent 4 weeks of incubation under 16/8-hour light/dark photoperiod. The growth parameters (number of shoots and roots, length of the main shoot and root, fresh and dry weights), photosynthetic pigments (chlorophylls and carotenoids), and relative water content of plantlets were restored with the application of 3 mg/L silver nitrate to the medium. Incubation of cultures initially in the dark followed by 16/8-hour light incubation facilitated axillary shoot elongation. On the basis of our findings, it is recommended to culture the regenerated axillary shoots of fegra fig onto MS medium containing 3% sucrose, 1.5 mg/L activated charcoal, and 3 mg/L silver nitrate to manage STN effectively.

Media Pembelajaran Video Simulasi Perubahan Wujud Zat dan Bentuk Energi: Penelitian dan Pengembangan

IPAS (Social and Exact Science Education) is a subject in the Kurikulum Merdeka. This subject invite student to recognize natural anomalies by facts. However, results of the researcher's observations on the interest of 4th-grade elementary school students in learning science showed that 93.75% of students experienced difficulties in learning science, especially in the matter of changes in the state of matter and energy forms. So, based on the results of the theoretical formulation and potential problems, the researcher is interested in proposing the development of a simulation video product. This type of research is Research and Development (R&amp;D) based on 7 stages, namely, potential and problems, data collection, product design, design validation, design revision, product testing, and product revision due to limitations of materials, time, and manpower. After the prototype product is completed, the researcher validates the content, media, and language with 9 experts, with each having 3 validations: (Content validators, Media validators, Language validators) The test results show that 86% of the material validation, 92% of the media expert validation, and 96% of the language validation, it can be concluded that the simulation video product is very feasible to use with minor revisions. Furthermore, the researchers carried out a teacher and student readability test on the simulation video media. The scores obtained were 91,2% for teachers and 89,2% for students. So that simulation video products able as media supplements in learning.

Preserving Nature’s Treasure: A Journey into the In Vitro Conservation and Micropropagation of the Endangered Medicinal Marvel—Podophyllum hexandrum Royle

Podophyllum hexandrum Royle, also known as Podophyllum emodi Wall, holds significant ecological, ornamental, and medicinal values. However, it has become endangered due to overexploitation, prolonged seed dormancy, slow natural regeneration, and climate change. This study developed an efficient in vitro protocol for callogenesis and micropropagation of P. hexandrum to conserve germplasm in in vitro conditions. Callus formation from various plant parts, including the leaf, stem, rhizome, radicle, and cotyledon, was induced using Murashige and Skoog (MS) medium supplemented with different plant growth regulators. The combination of benzyladenine at 1 mg L−1 and 4-dichlorophenoxy acetic acid at 3 mg L−1 was optimal for biomass production, yielding 215.88 ± 0.31 mg, with growth per gram at 8.32 ± 0.32 and a growth rate of 13.62 ± 0.25 mg/day on MS medium. For shoot proliferation, benzyladenine (3.5 mg L−1) and naphthalene acetic acid (0.5 mg L−1) combined with activated charcoal showed the highest shoot induction percentage per explant. For shoot regeneration from calluses, 6-benzylaminopurine (0.5 mg L−1) and thidiazuron (2 mg L−1) were most effective, producing superior shoot length, number of regenerations, and regeneration percentage. Root induction was successful with α-naphthalene acetic acid supplementation (0.5 to 1.5 mg L−1) in MS medium, resulting in the highest number per explant (4.08 ± 0.08), length (5.45 ± 0.15 cm), and rooting rate (87.00 ± 1.66%) of roots in plantlets. Subculturing for callus culture was performed every 28 days for up to four subcultures to prevent nutrient depletion and toxic metabolite accumulation, ensuring tissue health and viability. Continuous subculturing of callus on MS medium maintained healthy P. hexandrum germplasm in vitro. Overall, this micropropagation protocol provides a rapid system for conserving P. hexandrum germplasm.

Driving Osteocytogenesis from Mesenchymal Stem Cells in Osteon-like Biomimetic Nanofibrous Scaffolds.

The treatment of critical-sized bone defects caused by tumor removal, skeletal injuries, or infections continues to pose a major clinical challenge. A popular potential alternative solution to autologous bone grafts is a tissue-engineered approach that utilizes the combination of mesenchymal stromal/stem cells (MSCs) with synthetic biomaterial scaffolds. This approach aims to support new bone formation by mimicking many of the biochemical and biophysical cues present within native bone. Regrettably, osteocyte cells, crucial for bone maturation and homeostasis, are rarely produced within MSC-seeded scaffolds, thereby restricting the development of fully mature cortical bone from these synthetic implants. In this work, we have constructed a multimodal scaffold by combining electrospun poly(lactic-co-glycolic acid) (PLGA) fibrous scaffolds with poly(ethylene glycol) (PEG)-based hydrogels that mimic the functional unit of cortical bone, osteon (osteon-mimetic) scaffolds. These scaffolds were decorated with a novel bone morphogenic protein-6 (BMP6) peptide (BMP6p) after our findings revealed that the BMP6p drives higher levels of Smad signaling than the full-length protein counterpart, soluble or when bound to the PEG hydrogel backbone. We show that our osteon-mimetic scaffolds, in presenting concentric layers of BMP6p-PEG hydrogel overlaid on MSC-seeded PLGA nanofibers, promoted the rapid formation of osteocyte-like cells with a phenotypic dendritic morphology, producing early osteocyte markers, including E11/gp38 (E11). Maturation of these osteocyte-like cells was further confirmed by the observation of significant dentin matrix protein 1 (DMP1) throughout our bilayered scaffolds after 3 weeks, even when cultured in a medium without dexamethasone (DEX) or any other osteogenic supplements. These results demonstrate that these osteon-mimetic scaffolds, in presenting biochemical and topographical cues reminiscent of the forming osteon, can drive the formation of osteocyte-like cells in vitro from hBMSCs without the need for any osteogenic factor media supplementation.

Sialyllactose supplementation enhances sialylation of Fc-fusion glycoprotein in recombinant Chinese hamster ovary cell culture

Sialylation during N-glycosylation plays an important role in the half-life of therapeutic glycoproteins in vivo and has sparked interest in the production of therapeutic proteins using recombinant Chinese hamster ovary (rCHO) cells. To improve the sialylation of therapeutic proteins, we examined the effect of sialyllactose supplementation on sialylation of Fc-fusion glycoproteins produced in rCHO cells. Two enzymatically-synthesized sialyllactoses, 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL), were administered separately to two rCHO cell lines producing the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44, respectively. Two sialyllactoses successfully increased sialylation of Fc-fusion glycoprotein in both cell lines, as evidenced by isoform distribution, sialylated N-glycan formation, and sialic acid content. Increased sialylation by adding sialyllactose was likely the result of increased amount of intracellular CMP-sialic acid (CMP-SA), the direct nucleotide sugar for sialylation. Furthermore, the degree of sialylation enhanced by sialyllactoses was slightly effective or nearly similar compared with the addition of N-acetylmannosamine (ManNAc), a representative nucleotide sugar precursor, to increase sialylation of glycoproteins. The effectiveness of sialyllactose was also confirmed using three commercially available CHO cell culture media. Taken together, these results suggest that enzymatically-synthesized sialyllactose represents a promising candidate for culture media supplementation to increase sialylation of glycoproteins in rCHO cell culture.

Towards a circular economy - Repurposing side streams from the potato processing industry by Chlorella vulgaris

Common wastewater treatment strategies in the food industry do not include efficient remediation strategies for nitrogen, phosphorous and organic carbon. Incorporating microalgae in water treatment plants is rising in popularity because of their high nutrient and trace element uptake driven by light. In this study, four different side streams from an Austrian potato processing company have been screened for their applicability of microalgal cultivation. The side streams were assessed for Chlorella vulgaris growth and their requirement of any additional pretreatment or media supplementation. One side stream specifically, called blanching water II, a stream generated by boiling the potatoes for ease of peeling, turned out very useful to cultivate Chlorella vulgaris and concomitantly remedy the wastewater. Compared to a state-of-the-art cultivation in BG11, cultivating Chlorella vulgaris in blanching water II led to a 45 % increase in specific growth rate of 1.29 day−1 and a 48% increase in biomass productivity to 294.6 mg/L/day, while all nitrogen and phosphate present in the side stream were metabolized. Overall, the results demonstrate that the water remediation process for blanching water II shows vast potential in regard to water purification and waste to value approaches.

Bioengineered niches that recreate physiological extracellular matrix organisation to support long-term haematopoietic stem cells

Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via stem cell transplantation. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs and progenitor populations at the expense of naive LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1α expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF-1α expressing PerSCs, LT-HSC numbers were maintained with normal clonal and in vivo reconstitution potential, without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders.

Broadening the Scope of Sapofection: Cationic Peptide-Saponin Conjugates Improve Gene Delivery In Vitro and In Vivo.

Gene therapies represent promising new therapeutic options for a variety of indications. However, despite several approved drugs, its potential remains untapped. For polymeric gene delivery, endosomal escape represents a bottleneck. SO1861, a naturally occurring triterpene saponin with endosomal escape properties isolated from Saponaria officinalis L., has been described as additive agent to enhance transfection efficiency (sapofection). However, the challenge to synchronize the saponin and gene delivery system in vivo imposes limitations. Herein, we address this issue by conjugating SO1861 to a peptide-based gene vector using a pH-sensitive hydrazone linker programmed to release SO1861 at the acidic pH of the endosome. Nanoplexes formulated with SO1861-equipped peptides were investigated for transfection efficiency and tolerability in vitro and in vivo. In all investigated cell lines, SO1861-conjugated nanoplexes have shown superior transfection efficiency and cell viability over supplementation of transfection medium with free SO1861. Targeted SO1861-equipped nanoplexes incorporating a targeting peptide were tested in vitro and in vivo in an aggressively growing neuroblastoma allograft model in mice. Using a suicide gene vector encoding the cytotoxic protein saporin, a slowed tumor growth and improved survival rate were observed for targeted SO1861-equipped nanoplexes compared to vehicle control.

Characterization and Enhanced Production of Fungal Melanin from Aspergillus Species Isolated from Soil

This study aimed to isolate and identify fungi from soil samples, focusing on their ability to produce melanin. Eleven different genera of fungi were isolated and identified using lactophenol cotton blue staining and microscopic examination, referencing H.L. Barnett and Barry B. Hunter. Among these, only Aspergillus was found to produce melanin. Optimal melanin production conditions were determined to be a 1.5% tyrosine supplement in the growth medium, under shaking conditions (120 rpm) and dark incubation for three weeks, resulting in a yield of 21.08 mg/100 ml. Physiochemical characterization revealed that the extracted melanin was insoluble in organic solvents but soluble in alkaline solutions (NaOH, KOH), and partially soluble in DMSO. Spectrometric analysis using UV-Visible spectroscopy showed characteristic absorption peaks. FTIR indicated functional groups and Scanning Electron Microscopy (SEM) images displayed a granular and heterogeneous surface topology. The study also assessed the effects of different carbon and nitrogen sources, and trace elements on melanin production. Maltose and sucrose were the most effective carbon sources, while peptone was the most effective nitrogen source. Among trace elements, calcium significantly enhanced melanin yield, while copper and zinc had moderate effects. These findings provide valuable insights into optimizing fungal melanin production and its potential industrial applications. The study highlights the sustainable and scalable production of melanin from Aspergillus, contributing to the broader understanding of fungal metabolites and their commercial exploitation. Future research should focus on genetic and metabolic pathways to further enhance melanin biosynthesis and explore its diverse applications.

Multi‐omics reveal the molecular basis of LrAN2 overexpression in black goji callus promoting the excessive accumulation of petanin, a promising food blue colorant

AbstractLycium ruthenicum fruit (LRF) is a potential source of natural colorant in a wide pH range due to richness in monoacylated anthocyanin petanin. However, the yield of LRF largely lags behind the market demand in food industry. In this study, overexpressing LrAN2 (LrAN2OE), an anthocyanin activator in LRF, produced excessive acylated anthocyanins including petanin in L. ruthenicum callus (LRC). Physiological analysis indicates that LrAN2OE extensively orchestrate the redox homeostasis, including antioxidant enzymes system and antioxidants with low molecular weight. KEGG enrichment analysis indicates that LrAN2 hierarchically orchestrate the acylated anthocyanin biosynthesis in LRC at multi‐omics level, such as small RNAome, transcriptome, and metabolome. The anthocyanin yield of LrAN2OE callus cultured by 5% sucrose or glucose is significantly enhanced. Furthermore, the anthocyanin yield approach to 96.23 mg/g dry weight, approximately 3.5 folds of that in LRF, when LrAN2OE suspension cells are cultured in liquid MS medium supplement with 5% sucrose and 100 mM NaCl. Notably, anthocyanins extracted from LrAN2OE callus are stable in pH 1.0–2.0, pH 7.0, and pH 9.0, showing red or blue color within 30 days at 4°C. Our findings suggest that LrAN2 is perfect gene resource for metabolic engineering of acylated anthocyanins in LRC, a promising chassis producing acylated anthocyanins. Taken together, LRC metabolically engineered by LrAN2 is a potential source of natural food color, which facilitates to make up the shortfall of LRF as source of natural colorant in future food industry.

Comparative Analysis of the Mass Spectra of Mycobacterium abscessus Complex Strains Isolated on Various Nutrient Media.

Mycobacterium abscessus complex (MABSc) causes chronic infection in patients with concomitant structural changes in the respiratory tract, which is especially important for patients with cystic fibrosis. To isolate an MABSc culture from clinical material, a variety of nutrient media are used. For species determination of microorganisms isolated on these media, additional identification methods are used, for example, polymerase chain reaction, sequencing, or mass spectrometry. The latter method is relatively easy to implement but requires improvement, due to the identification inaccuracy of nontuberculosis mycobacterias in general. Consequently, a set of nutrient media may be important for subsequent identification by mass spectrometry. The study was conducted on 64 strains of MABSc representatives: 56 strains were obtained from patients with cystic fibrosis and 8 strains from patients with pulmonary pathology unrelated to cystic fibrosis. The obtained MABSc strains were transplanted to the universal chromogenic medium and the selective medium for the Burkholderia cepacia complex (BCC) isolation. Species identification was carried out by mass spectrometry based on matrix-activated laser time-of-flight desorption/ionization (MALDI-ToF MS). Microbial identification is based on a comparison of the obtained mass spectra with reference spectra from the database. Microorganisms were identified based on the coincidence degree (Score value). Sample preparation for microbial identification by mass spectrometry was carried out by an extended direct application method. Fragments of the rpoB and hsp65 genes with lengths of 752 bp and 441 bp, respectively, were used as molecular markers for subspecific identification of MABSc strains. A comparison of the peaks obtained after mass spectrometry of MABSc strains isolated on the studied nutrient media showed significant differences between these indicators selective medium for the BCC isolation with the supplement of iron polymaltose hydroxide (III) and universal chromogenic medium (P < 0.001) and selective medium for the BCC isolation with universal chromogenic medium (P < 0.001). Twenty-five strains of MABSc representatives were sequenced: results of subspecies determination in strains isolated on the universal chromogenic medium coincided with the results sequencing in 13 (86.6%) strains out of 15. MALDI-ToF mass spectrometry allows microbial identification in a short time and with minimal cost, but it does not yet allow the proper identification of the subspecies of certain microbial groups, such as MABSc. Cultivation methods need optimization and new approaches to the extraction process of the bacterial protein fraction.

Human platelet lysate supports SH-SY5Y neuroblastoma cell proliferation and differentiation into a dopaminergic-like neuronal phenotype under xenogeneic-free culture conditions.

SH-SY5Y is a human neuroblastoma cell line that can be differentiated into several neuronal phenotypes, depending on culture conditions. For this reason, this cell line has been widely used as an in vitro model of neurodegenerative conditions, such as Parkinson's disease (PD). However, most studies published to date used fetal bovine serum (FBS) as culture medium supplement for SH-SY5Y cell differentiation. We report on the testing of human platelet lysate (hPL) as a culture medium supplement to support SH-SY5Y cell culture. Both standard hPL and a fibrinogen-depleted hPL (FD-hPL) formulation, which does not require the addition of anticoagulants to culture media, promoted an increase in SH-SY5Y cell proliferation in comparison to FBS, without compromising metabolic activity. SH-SY5Y cells cultured in hPL or FD-hPL also displayed a higher number of neurite extensions and stained positive for MAP2 and synaptophysin, in the absence of differentiation stimuli; reducing hPL or FD-hPL concentration to 1% v/v did not affect cell proliferation or metabolic activity. Furthermore, following treatment with retinoic acid (RA) and further stimulation with brain-derived neurotrophic factor (BDNF) and nerve growth factor beta (NGF-β), the percentage of SH-SY5Y cells stained positive for dopaminergic neuronal differentiation markers (tyrosine hydroxylase [TH] and Dopamine Transporter [DAT]) was higher in hPL or FD-hPL than in FBS, and gene expression of dopaminergic markers TH, DAT, and DR2 was also detected. Overall, the data herein presented supports the use of hPL to differentiate SH-SY5Y cells into a neuronal phenotype with dopaminergic features, and the adoption of FD-hPL as a fully xenogeneic free alternative to FBS to support the use of SH-SY5Y cells as a neurodegeneration model.

Pistil-derived lipids influence pollen tube growth and male fertility in Arabidopsis thaliana.

Pollen germination and pollen tube elongation require rapid phospholipid production and remodeling in membrane systems that involve both de novo synthesis and turnover. Phosphatidic acid phosphohydrolase (PAH) and lysophosphatidylcholine acyltransferase (LPCAT) are 2 key enzymes in membrane lipid maintenance. PAH generates diacylglycerol (DAG), a necessary precursor for the de novo synthesis of phosphatidylcholine (PC), while LPCAT reacylates lysophosphatidylcholine to PC and plays an essential role in the remodeling of membrane lipids. In this study, we investigated the synthetic defects of pah and lpcat mutations in sexual reproduction of Arabidopsis (Arabidopsis thaliana) and explored the prospect of pistil lipid provision to pollen tube growth. The combined deficiencies of lpcat and pah led to decreased pollen tube growth in the pistil and reduced male transmission. Interestingly, pistils of the lipid mutant dgat1 ameliorated the male transmission deficiencies of pah lpcat pollen. In contrast, pollination with a nonspecific phospholipase C (NPC) mutant exacerbated the fertilization impairment of the pah lpcat pollen. Given the importance of DAG in lipid metabolism and its contrasting changes in the dgat1 and npc mutants, we further investigated whether DAG supplement in synthetic media could influence pollen performance. DAG was incorporated into phospholipids of germinating pollen and stimulated pollen tube growth. Our study provides evidence that pistil-derived lipids contribute to membrane lipid synthesis in pollen tube growth, a hitherto unknown role in synergistic pollen-pistil interactions.

Engineering EPS in Halomonas bluephagenesis leads to self-flocculation and filamentation for convenient downstream processing

The halophile Halomonas bluephagenesis has established itself as a cost-effective chassis in industrial bioproduction. However, downstream processing (DSP) expense is a persistent challenge. This study uses genetic knockout of exopolysaccharides (EPS-KO) in six industrially relevant H. bluephagenesis strains, each producing distinct polyhydroxyalkanoate (PHA), to evaluate their impact on reducing DSP costs. Remarkably, EPS-KO cells exhibited an unprecedented filamentous morphology, reaching lengths over 50 times (10–75 μm) that of standard cells. This unexpected behavior initially posed a challenge to industrial applicability due to rapid cell lysis under fermentation agitation. Systematic media supplementation of salt, carbon source, nitrogen, and essential minerals significantly influenced morphology, and tailored nutrient schemes that mitigated filamentation while maintaining production levels were designed. Scaled production in a 5000 L bioreactor demonstrated 85 % PHA and 96 g/L cell dry weight after 40 h fermentation, consistent with non-KO cells. During fermentation, EPS-KO enhanced cell-wall penetrability, reducing the need for high-cost γ-butyrolactone by 40 %. Additionally, a new innovative 5000 L FlipFlow bioreactor design cut rotation speed by 50 % and air input by 25 %. During DSP, centrifugation time decreased tenfold due to rapid cell self-flocculation (15–30 min), and the water and enzyme required for PHA purification was reduced by 30 % and 35 %, respectively. This study highlights the use of EPS-KO cells, a controlled media strategy, and low shear force FlipFlow bioreactor to successfully reduce DSP costs without sacrificing productivity. These novel findings represent a notable advancement in cost-saving strategies while presenting the intriguing discovery of media-controlled filamentous cell behavior.

Enhanced Recombinant Protein Expression in Insect Cells by Natural and Recombinant Components of Lepidoptera Hemolymph.

Prior research has established the anti-apoptotic effects in insect cell cultures of Bombyx mori (B. mori) hemolymph, as well as the heightened production yields of recombinant proteins facilitated by baculovirus vectors in insect cells cultivated in media supplemented with this hemolymph. In this study, we investigated the hemolymph of another Lepidoptera species, Trichoplusia ni (T. ni), and observed similar beneficial effects in insect cells cultivated in media supplemented with this natural substance. We observed enhancements in both production yield (approximately 1.5 times higher) and late-stage cell viabilities post-infection (30-40% higher). Storage-protein 2 from B. mori (SP2Bm) has previously been identified as one of the abundant hemolymph proteins potentially responsible for the beneficial effects observed after the use of B. mori hemolymph-supplemented cell culture media. By employing a dual baculovirus vector that co-expresses the SP2Bm protein alongside the GFP protein, we achieved a threefold increase in reporter protein production compared to a baculovirus vector expressing GFP alone. This study underscores the potential of hemolymph proteins sourced from various Lepidoptera species as biotechnological tools to augment baculovirus vector productivities, whether utilized as natural supplements in cell culture media or as hemolymph-derived recombinant proteins co-expressed by baculovirus vectors.

Optimization of the replication of hepatitis E virus genotype 3 in vitro.

Hepatitis E virus (HEV) is responsible for ∼20 million human infections worldwide every year. The genotypes HEV-3 and HEV-4 are zoonotic and are responsible for most of the autochthonous HEV cases in high-income countries. There are several cell culture systems that allow for propagation of different HEV genotypes in vitro. One of these systems uses human lung carcinoma cells (A549), and was further optimized for propagation of HEV-3 47832c strain. In this study, we investigated the effect of different media supplements as well as microRNA-122 (miR-122) on improving the replication of HEV-3 47832c in A549 cells. We observed that supplementation of maintenance media with 5% fetal bovine serum was sufficient for efficient replication of HEV-3, and verified the positive effect of media supplementation with Amphotericin B, MgCl2, and dimethyl sulfoxide on replication of HEV-3. We have also demonstrated that adding miR-122 mimics to the culture media does not have any significant effect on the replication of HEV-3 47832c. Herein, we detected over a 6-fold increase in HEV-3 replication in A549/D3 cells by adding all three supplements: Amphotericin B, MgCl2, and dimethyl sulfoxide to the culture media, while demonstrating that miR-122 might not play a key role in replication of HEV-3 47832c.

Chemical reversion of age-related oocyte dysfunction fails to enhance embryo development in a bovine model of postovulatory aging

PurposeThere are no clinical treatments to prevent/revert age-related alterations associated with oocyte competence decline in the context of advanced maternal age. Those alterations have been attributed to oxidative stress and mitochondrial dysfunction. Our study aimed to test the hypothesis that in vitro maturation (IVM) medium supplementation with antioxidants (resveratrol or phloretin) may revert age-related oocyte competence decline.MethodsBovine immature oocytes were matured in vitro for 23 h (young) and 30 h (aged). Postovulatory aged oocytes (control group) and embryos obtained after fertilization were examined and compared with oocytes supplemented with either 2 μM of resveratrol or 6 μM phloretin (treatment groups) during IVM.ResultsAged oocytes had a significantly lower mitochondrial mass and proportion of mitochondrial clustered pattern, lower ooplasmic volume, higher ROS, lower sirtuin-1 protein level, and a lower blastocyst rate in comparison to young oocytes, indicating that postovulatory oocytes have a lower quality and developmental competence, thus validating our experimental model. Supplementation of IVM medium with antioxidants prevented the generation of ROS and restored the active mitochondrial mass and pattern characteristic of younger oocytes. Moreover, sirtuin-1 protein levels were also restored but only following incubation with resveratrol. Despite these findings, the blastocyst rate of treatment groups was not significantly different from the control group, indicating that resveratrol and phloretin could not restore the oocyte competence of postovulatory aged oocytes.ConclusionResveratrol and phloretin can both revert the age-related oxidative stress and mitochondrial dysfunction during postovulatory aging but were insufficient to enhance embryo developmental rates under our experimental conditions.