XÁC ĐỊNH MẬT ĐỘ NUÔI SINH KHỐI THÍCH HỢP VÀ ĐIỀU KIỆN THỨC ĂN TƯƠNG ỨNG CỦA TÔM TIÊN NƯỚC NGỌT, Branchinella thailandensis, LOÀI THỨC ĂN SỐNG TRIỂN VỌNG CHO ƯƠNG NUÔI THỦY SẢN Ở VIỆT NAM

Tôm tiên nước ngọt, Branchinella thailandensis, là một loài giáp xác nước ngọt có triển vọng sử dụng làm thức ăn cho con giống ở giai đoạn ấu trùng tại Việt Nam vì chúng có kích thước nhỏ, khả năng phát triển nhanh và hàm lượng carotenoid cao. Trong nghiên cứu này, B. thailandensis được nghiên cứu để xác định lượng thức ăn và mật độ nuôi thích hợp để nuôi sinh khối trong môi trường nước ngọt. Để đạt được mục tiêu này, 01 thí nghiệm 3 nhân tố được thực hiện: (i) mật độ nuôi tôm tiên với 3 mức (250, 500 và 1000 cá thể/L); (ii) mật độ thức ăn (tảo sống Spirulina platensis) với 3 mức(5 × 105 tế bào/mL, 1 × 106 tế bào/mL và 2 × 106 tế bào/mL); và (iii) mật độ vi khuẩn có lợi Bacillus subtilis với 2 mức (0 và 1 × 103 CFU/ mL). Kết quả nghiên cứu cho thấy, sau 72 giờ, để đạt được tỷ lệ sống cao nhất (68.33% ± 10.4%) và chiều dài tối đa (2.32 ± 0.23 mm), cần áp dụng mật độ thức ăn là 2 × 106 tế bào/mL với mật độ nuôi tối đa cần duy trì là 250 ấu trùng/L và có bổ sung vi khuẩn có lợi (1 × 103 CFU/ mL). Ở điều kiện nuôi này, 01 thí nghiệm 02 nhân tố được thực hiện để khảo sát nồng độ Carotenoid và khả năng lưu trữ vi khuẩn có lợi trong cơ thể của tôm tiên nước ngọt B. thailandensis. Nhân tố mật độ vi khuẩn có lợi B. subtilis với 2 mức (0 và 1 × 103 CFU/mL), nhân tố mật độ tảo với 3 mức: 5 × 105, 1 × 106, và 2 × 106 tế bào/mL. Kết quả nghiên cứu cho thấy B. thailandensis cũng có thể lưu giữ vi khuẩn có lợi trong cơ thể đến ít nhất là 48 giờ sau khi nở và đạt được hàm lượng carotenoid cao hơn hẳn lúc nuôi với nồng độ thức ăn thấp. ABSTRACT Freshwater fairy shrimp, Branchinella thailandensis, is a highly potential live feed for aquaculture species in Viet Nam, especially in their larval stages due to its small size, high growth and high carotenoid content. In this study, we aimed to identify the suitable feeding density and stocking density, which is inappropriate for mass production in freshwater. To attain these objectives, we conducted 01 three-factor experiment: (i) culture density with three levels (250, 500 and 1000 nauplii L-1); (ii) feed (live Spirulina platens) concentration with three levels (5 × 105 cells mL-1, 1 × 106 cells mL-1 and 2 × 106 cells mL-1); and (iii) beneficial bacteria Bacillus subtilis with two levels (0 and 1 × 103 CFU mL-1). The results indicated that after 72hrs, to the highest survival rate (68.33% ± 10.4%) and total length (2.32±0.23 mm) were obtained when shrimps were fed live S. platensis at 2 × 106 cells mL-1 with probiotics at 1 × 103 CFU/ mL, and density at 250 nauplii L-1. Under those culturing conditions, we conducted 01 two-factor experiment: feed (live Spirulina platens) concentration with three levels (5 × 105 cells mL-1, 1 × 106 cells mL-1 and 2 × 106 cells mL-1); and beneficial bacteria Bacillus subtilis with two levels (0 and 1 × 103 CFU mL-1). The results indicated that B. thailandensis could retain B. subtilis internally up to at least 48 hours and obtain significantly higher carotenoid content than at lower feed concentration.

The Effect of Hypoxic and Normoxic Culturing Conditions in Different Breast Cancer 3D Model Systems

The field of 3D cell cultures is currently emerging, and material development is essential in striving toward mimicking the microenvironment of a native tissue. By using the response of reporter cells to a 3D environment, a comparison between materials can be assessed, allowing optimization of material composition and microenvironment. Of particular interest, the response can be different in a normoxic and hypoxic culturing conditions, which in turn may alter the conclusion regarding a successful recreation of the microenvironment. This study aimed at determining the role of such environments to the conclusion of a better resembling cell culture model to native tissue. Here, the breast cancer cell line MCF7 was cultured in normoxic and hypoxic conditions on patient-derived scaffolds and compared at mRNA and protein levels to cells cultured on 3D printed scaffolds, Matrigel, and conventional 2D plastics. Specifically, a wide range of mRNA targets (40), identified as being regulated upon hypoxia and traditional markers for cell traits (cancer stem cells, epithelial–mesenchymal transition, pluripotency, proliferation, and differentiation), were used together with a selection of corresponding protein targets. 3D cultured cells were vastly different to 2D cultured cells in gene expression and protein levels on the majority of the selected targets in both normoxic and hypoxic culturing conditions. By comparing Matrigel and 3DPS-cultured cells to cells cultured on patient-derived scffolds, differences were also noted along all categories of mRNA targets while specifically for the GLUT3 protein. Overall, cells cultured on patient-derived scaffolds closely resembled cells cultured on 3D printed scaffolds, contrasting 2D and Matrigel-cultured cells, regardless of a normoxic or hypoxic culturing condition. Thus, these data support the use of either a normoxic or hypoxic culturing condition in assays using native tissues as a blueprint to optimize material composition.

Towards Biomanufacturing of Cell-Derived Matrices

Cell-derived matrices (CDM) are the decellularised extracellular matrices (ECM) of tissues obtained by the laboratory culture process. CDM is developed to mimic, to a certain extent, the properties of the needed natural tissue and thus to obviate the use of animals. The composition of CDM can be tailored for intended applications by carefully optimising the cell sources, culturing conditions and decellularising methods. This unique advantage has inspired the increasing use of CDM for biomedical research, ranging from stem cell niches to disease modelling and regenerative medicine. However, while much effort is spent on extracting different types of CDM and exploring their utilisation, little is spent on the scale-up aspect of CDM production. The ability to scale up CDM production is essential, as the materials are due for clinical trials and regulatory approval, and in fact, this ability to scale up should be an important factor from the early stages. In this review, we first introduce the current CDM production and characterisation methods. We then describe the existing scale-up technologies for cell culture and highlight the key considerations in scaling-up CDM manufacturing. Finally, we discuss the considerations and challenges faced while converting a laboratory protocol into a full industrial process. Scaling-up CDM manufacturing is a challenging task since it may be hindered by technologies that are not yet available. The early identification of these gaps will not only quicken CDM based product development but also help drive the advancement in scale-up cell culture and ECM extraction.

Identifying sources of D-serine in Caenorhabditis elegans and their impact on behavior

Free D-serine (D-Ser) is a potent co-agonist of the N-methyl-D-aspartate receptor (NMDAR) in glutamate neurotransmission and regulates NMDAR functions in the nervous system. Serine racemases convert L-serine to D-Ser and are believed to be the major source of D-Ser in animals. In Caenorhabditis elegans, a knockout of the serine racemase serr-1 results in behavioral changes, but the level of D-Ser is unaffected. By growing C. elegans on peptone-free nematode growth medium (PF-NGM), we delineated the sources of D-Ser, both exogenous from peptone in culturing media and endogenous from the serine racemase serr-1, and a potential serine/aspartate racemase candidate, Y51H7C.9, identified by sequence similarity network analysis. We also discovered a new serine dehydratase (aka serine ammonia-lyase), K01C8.1, in C. elegans. We identified the serr-1 knockout and PF-NGM culturing conditions as two independent factors that impact C. elegans locomotion behavior after off-food, both short-term and long-term, and no interactions were found between the two factors.

Study of the Seasonal Variations of the Fatty Acid Profiles of Selected Macroalgae

Due to the high consumption of fat-rich processed foods, efforts are being done to reduce their saturated fat (SFA) contents and replace it with polyunsaturated fatty acids (PUFA), creating a necessity to find alternative PUFA sources. Macroalgae, being a promising natural source of healthy food, may be such an alternative. The fatty acid (FA) profile of Fucus spiralis, Bifurcaria bifurcata, Ulva lactuca, and Saccorhiza polyschides were determined through direct transesterification and their seasonal variation was studied. F. spiralis showed the highest FA content overall, B. bifurcata presented the higher PUFA amounts, and U. lactuca and S. polyschides the higher SFA. The production of FA was shown to be influenced by the seasons. Spring and summer seemed to induce the FA production in F. spiralis and B. bifurcata while in U. lactuca the same was verified in winter. U. lactuca presented a ω6/ω3 ratio between 0.59 and 1.38 while B. bifurcata presented a ratio around 1.31. The study on the seasonal variations of the macroalgal FA profile can be helpful to understand the best season to yield FA of interest, such as ALA, EPA, and DHA. It may also provide valuable information on the best culturing conditions for the production of desired FAs.

Adipo-Derived Stem Cell Features and MCF-7

Human adipose tissue-derived stem cells (hADSCs) are highly suitable for regeneration therapies being easily collected and propagated in vitro. The effects of different external factors and culturing conditions are able to affect hADSC proliferation, senescence, differentiation, and migration, even at the molecular level. In the present paper, we exposed hADSCs to an exhausted medium from the breast cancer cell line (MCF-7) to evaluate whether the soluble factors released by these cells may be able to induce changes in stem cell behavior. In particular, we investigated the expression of stemness-related genes (OCT4; Sox 2; Nanog), the cell-cycle regulators p21 (WAF1/CIP1) p53, epigenetic markers (DNMT1 and Sirt1), and autophagy-related proteins. From our results, we can infer that the exhausted medium from MCF-7 is able to influence the hADSCs behavior increasing the expression of stemness-related genes, cell proliferation, and autophagy. Polyamines detectable in MCF-7 exhausted medium could be related to the higher proliferation capability observed in hADSCs, suggesting direct crosstalk between these molecules and the observed changes in stem cell potency.

Culturable and Non-Culturable Blood Microbiota of Healthy Individuals

Next-generation sequencing (NGS) and metagenomics revolutionized our capacity for analysis and identification of the microbial communities in complex samples. The existence of a blood microbiome in healthy individuals has been confirmed by sequencing, but some researchers suspect that this is a cell-free circulating DNA in blood, while others have had isolated a limited number of bacterial and fungal species by culture. It is not clear what part of the blood microbiota could be resuscitated and cultured. Here, we quantitatively measured the culturable part of blood microbiota of healthy individuals by testing a medium supplemented with a high concentration of vitamin K (1 mg/mL) and culturing at 43 °C for 24 h. We applied targeted sequencing of 16S rDNA and internal transcribed spacer (ITS) markers on cultured and non-cultured blood samples from 28 healthy individuals. Dominant bacterial phyla among non-cultured samples were Proteobacteria 92.97%, Firmicutes 2.18%, Actinobacteria 1.74% and Planctomycetes 1.55%, while among cultured samples Proteobacteria were 47.83%, Firmicutes 25.85%, Actinobacteria 16.42%, Bacteroidetes 3.48%, Cyanobacteria 2.74%, and Fusobacteria 1.53%. Fungi phyla Basidiomycota, Ascomycota, and unidentified fungi were 65.08%, 17.72%, and 17.2% respectively among non-cultured samples, while among cultured samples they were 58.08%, 21.72%, and 20.2% respectively. In cultured and non-cultured samples we identified 241 OTUs belonging to 40 bacterial orders comprising 66 families and 105 genera. Fungal biodiversity accounted for 272 OTUs distributed in 61 orders, 105 families, and 133 genera. Bacterial orders that remained non-cultured, compared to blood microbiota isolated from fresh blood collection, were Sphingomonadales, Rhizobiales, and Rhodospirillales. Species of orders Bacillales, Lactobacillales, and Corynebacteriales showed the best cultivability. Fungi orders Tremellales, Polyporales, and Filobasidiales were mostly unculturable. Species of fungi orders Pleosporales, Saccharomycetales, and Helotiales were among the culturable ones. In this study, we quantified the capacity of a specific medium applied for culturing of blood microbiota in healthy individuals. Other culturing conditions and media should be tested for optimization and better characterization of blood microbiota in healthy and diseased individuals.

Integrated multi-omics analyses on patient-derived CRC organoids highlight altered molecular pathways in colorectal cancer progression involving PTEN

Background Colorectal cancer (CRC) represents the fourth leading cause of cancer-related deaths. The heterogeneity of CRC identity limits the usage of cell lines to study this type of tumor because of the limited representation of multiple features of the original malignancy. Patient-derived colon organoids (PDCOs) are a promising 3D-cell model to study tumor identity for personalized medicine, although this approach still lacks detailed characterization regarding molecular stability during culturing conditions. Correlation analysis that considers genomic, transcriptomic, and proteomic data, as well as thawing, timing, and culturing conditions, is missing. Methods Through integrated multi–omics strategies, we characterized PDCOs under different growing and timing conditions, to define their ability to recapitulate the original tumor. Results Whole Exome Sequencing allowed detecting temporal acquisition of somatic variants, in a patient-specific manner, having deleterious effects on driver genes CRC-associated. Moreover, the targeted NGS approach confirmed that organoids faithfully recapitulated patients’ tumor tissue. Using RNA-seq experiments, we identified 5125 differentially expressed transcripts in tumor versus normal organoids at different time points, in which the PTEN pathway resulted of particular interest, as also confirmed by further phospho-proteomics analysis. Interestingly, we identified the PTEN c.806_817dup (NM_000314) mutation, which has never been reported previously and is predicted to be deleterious according to the American College of Medical Genetics and Genomics (ACMG) classification. Conclusion The crosstalk of genomic, transcriptomic and phosphoproteomic data allowed to observe that PDCOs recapitulate, at the molecular level, the tumor of origin, accumulating mutations over time that potentially mimic the evolution of the patient’s tumor, underlining relevant potentialities of this 3D model.

How To Culture Limnoterrestrial Heterotardigrades: A Case Study In The Echiniscus, Pseudechiniscus And Viridiscus Genera

Background: Tardigradologists have long tried to culture limnoterrestrial heterotardigrades without success. This is likely because they depend on specific conditions in their microhabitats. Most limnoterrestrial heterotardigrades are found associated with bryophytes and lichens (collectively cryptogams). In contrast to eutardigrades, many of which are easily cultured, heterotardigrades are found in cryptogams that prefer drier and sunnier habitats and that tend to grow on the vertical surfaces of boulders, cliffs, or tree trunks. By carefully observing heterotardigrades in the Echniscus and Viridiscus genera over many months, we determined that these organisms feed on chloroplasts and cytoplasm of both moss cells (typically moss protonema) and single-celled green algae associated with the moss (typically Chlorella vulgaris). We also determined that the cryptogams they associate with, and hence the heterotardigrades themselves, spend more time in a dried state than most eutardigrade species. Taking these observations into account we varied food, water, and desiccation cycle conditions with the aim of developing a culturing protocol for heterotardigrades. Results: We have maintained laboratory cultures of several generations and counting of Echiniscus, Pseudechniscus and Viridiscus genera tardigrades using the following experimentally derived protocol: Both moss and algae from their natural habitats are required as food sources and a small layer of rain or spring water is added every morning and allowed to evaporate some overnight. Furthermore, the organisms are more likely to lay eggs on a dense mat of moss protonema, grown by inserting tips of moss branches into a solidified KCM / agar medium. The medium also provides a walking substrate for the tardigrades, and possibly a source of cations. Crucially, the cultures must be allowed to dry out completely every ten days for a period of at least three days. Moss in the culture dish significantly improves the chances of the tardigrades coming out of their desiccation-resistant states successfully. Conclusions: We conclude that periodic drying out, moss and algae are all required to successfully culture heterotardigrades. Furthermore, drying must occur slowly, and the moss protonema enables this by retaining moisture thereby slowing evaporation. We suspect our protocol will work for most limnoterrestrial heterotardigrades with minor tweaking of culturing conditions.

Ultraviolet B irradiation-induced keratinocyte senescence and impaired development of 3D epidermal reconstruct

Ultraviolet B (UVB) induces morphological and functional changes of the skin. This study investigated the effect of UVB on keratinocyte senescence and the development of reconstructed human epidermis (RHE). Primary normal human keratinocytes (NHK) from juvenile foreskin were irradiated with UVB (30 mJ cm−2) and these effects were compared to NHK that underwent senescence in the late passage. UVB enhanced the accumulation of reactive oxygen species (ROS) and halted cell replication as detected by BrdU cell proliferation assay. The senescence phenotype was evaluated by beta-galactosidase (β-gal) staining and qPCR of genes related to senescent regulation, i.e. p16INK4a, cyclin D2, and IFI27. Senescence induced by high dose UVB resulted in morphological changes, enhanced β-gal activity, elevated cellular ROS levels and reduced DNA synthesis. qPCR revealed differential expression of the genes regulated senescence. p16INK4a expression was significantly increased in NHK exposed to UVB whereas enhanced IFI27 expression was observed only in cultural senescence. The levels of cyclin D2 expression were not significantly altered either by UVB or long culturing conditions. UVB significantly induced the aging phenotype in keratinocytes and impaired epidermal development. RHE generated from UVB-irradiated keratinocytes showed a thinner cross-sectional structure and the majority of keratinocytes in the lower epidermis were degenerated. The 3D epidermis model is useful in studying the skin aging process.