Scalable in vitro production of defined mouse erythroblasts

Mouse embryonic stem cells (mESCs) can be manipulated in vitro to recapitulate the process of erythropoiesis, during which multipotent cells undergo lineage specification, differentiation and maturation to produce erythroid cells. Although useful for identifying specific progenitors and precursors, this system has not been fully exploited as a source of cells to analyse erythropoiesis. Here, we establish a protocol in which characterised erythroblasts can be isolated in a scalable manner from differentiated embryoid bodies (EBs). Using transcriptional and epigenetic analysis, we demonstrate that this system faithfully recapitulates normal primitive erythropoiesis and fully reproduces the effects of natural and engineered mutations seen in primary cells obtained from mouse models. We anticipate this system to be of great value in reducing the time and costs of generating and maintaining mouse lines in a number of research scenarios.

The Interactions among Isolates of Lactiplantibacillus plantarum and Dairy Yeast Contaminants: Towards Biocontrol Applications

Yeast diversity in the cheese manufacturing process and in the cheeses themselves includes indispensable species for the production of specific cheeses and undesired species that cause cheese defects and spoilage. The control of yeast contaminants is problematic due to limitations in sanitation methods and chemicals used in the food industry. The utilisation of lactic acid bacteria and their antifungal products is intensively studied. Lactiplantibacillus plantarum is one of the most frequently studied species producing a wide spectrum of bioactive by-products. In the present study, twenty strains of L. plantarum from four sources were tested against 25 species of yeast isolated from cheeses, brines, and dairy environments. The functional traits of L. plantarum strains, such as the presence of class 2a bacteriocin and chitinase genes and in vitro production of organic acids, were evaluated. The extracellular production of bioactive peptides and proteins was tested using proteomic methods. Antifungal activity against yeast was screened using in vitro tests. Testing of antifungal activity on artificial media and reconstituted milk showed significant variability within the strains of L. plantarum and its group of origin. Strains from sourdoughs (CCDM 3018, K19-3) and raw cheese (L12, L24, L32) strongly inhibited the highest number of yeast strains on medium with reconstituted milk. These strains showed a consistent spectrum of genes belonging to class 2a bacteriocins, the gene of chitinase and its extracellular product 9 LACO Chitin-binding protein. Strain CCDM 3018 with the spectrum of class 2a bacteriocin gene, chitinase and significant production of lactic acid in all media performed significant antifungal effects in artificial and reconstituted milk-based media.

Micropropagation of Cassava (Manihot esculenta Crantz): Review

Cassava is a vital crop to the food security of millions of people worldwide, particularly in Sub-Saharan Africa. Because the crop produced a reasonable yield on marginal soils, it could help relieve global hunger. As a result, increasing cassava output and its quality attributes are significant. However, the low multiplication rate of this main crop has resulted in the delayed dissemination of improved varieties among farmers. As a result, tissue culture techniques may be a feasible solution for overcoming these challenges. Cassava in vitro propagation had done using either the shoots multiplication technique or somatic embryogenesis. However, the shoot multiplication approach is preferable since it retains clonal fidelity. Plant regeneration via somatic embryogenesis or organogenesis entailed the use of numerous basal media containing various plant growth hormones. Several studies found that each type of cassava clone required a unique protocol to achieve optimal shoot initiation, shoot multiplication, root induction, and elongation. This review describes recent research on cassava micropropagation that makes use of a variety of experimental systems. While each of these systems focuses on a different aspect of technique, they can be significant in understanding the in vitro production of cassava planting material.

In vitro assessment of histamine and lactate production by a multi-strain synbiotic

Recent studies suggest histamine and d-lactate may negatively impact host health. As excess histamine is deleterious to the host, the identification of bacterial producers has contributed to concerns over the consumption of probiotics or live microorganisms in fermented food items. Some probiotic products have been suspected of inducing d-lactic-acidosis; an illness associated with neurocognitive symptoms such as ataxia. The goals of the present study were to test the in vitro production of histamine and d-lactate by a 24-strain daily synbiotic and to outline methods that others can use to test for their production. Using enzymatic based assays, no significant production of histamine was observed compared to controls (P > 0.05), while d-lactate production was comparable to a commercially available probiotic with no associated health risk. These assays provide a means to add to the safety profile of synbiotic and probiotic products.

Sustainable and efficient protocols for in vitro germination and antioxidants production from seeds of the endangered species Araucaria araucana

Background The Pehuén or Monkey puzzle tree (Araucaria araucana) is an evergreen coniferous tree, which has been historically used for social, medicinal, and nutritional purposes. We have recently showed the value of A. araucana seeds as a rich source of micronutrients and antioxidants. This endemic species present in a reduced area in Argentina and Chile is endangered because of the low germination rate and the overexploitation of its edible seeds. Thus, the massive extraction of its seeds is ecologically non-viable resulting in limited availability of its active metabolites. However, biotechnological approaches are attractive strategies of production of valuable metabolites and healthy specimens of endangered plants. The aim of this work was to develop a protocol for in vitro production of antioxidants derived from A. araucana seeds and to obtain healthy plants by optimized seed germination. Results Calli of Pehuén seeds were induced in Murashige and Skoog medium with different combinations of auxins and cytokinins, in light and dark conditions. Callus from embryonic axes developed in medium with 1 mg/l α-naphthaleneacetic acid and 1.5 mg/l 6-benzylaminopurine in light conditions had efficient biomass production, antioxidant activity, high phenolic, and flavonoid content and no cytotoxicity on mammalian cells. Additionally, 100 % germination was obtained in vitro and healthy plants were acclimatized to non-sterile conditions. Conclusion In conclusion, in vitro culture of A. araucana could provide new and sustainable options for production of its valuable metabolites with possible therapeutic and nutritional uses. Also, optimized plant germination and acclimatization of endangered species can contribute to the preservation of pristine environments.

Biological and Inflammatory Effects of Antigen 5 from Polybia paulista (Hymenoptera, Vespidae) Venom in Mouse Intraperitoneal Macrophages

The social wasp Polybia paulista (Hymenoptera, Vespidae) is highly aggressive, being responsible for many medical occurrences. One of the most allergenic components of this venom is Antigen 5 (Poly p 5). The possible modulation of the in vitro immune response induced by antigen 5 from P. paulista venom, expressed recombinantly (rPoly p 5), on BALB/c mice peritoneal macrophages, activated or not with LPS, was assessed. Here, we analyzed cell viability changes, expression of the phosphorylated form of p65 NF-κB subunit, nitric oxide (NO), proinflammatory cytokines production, and co-stimulatory molecules (CD80, CD86). The results suggest that rPoly p 5 does not affect NO production nor the expression of co-stimulatory molecules in mouse peritoneal macrophages. On the other hand, rPoly p 5 induced an increase in IL-1β production in non-activated macrophages and a reduction in the production of TNF-α and MCP-1 cytokines in activated macrophages. rPoly p 5 decreased the in vitro production of the phosphorylated p65 NF-κB subunit in non-activated macrophages. These findings suggest an essential role of this allergen in the polarization of functional M2 macrophage phenotypes, when analyzed in previously activated macrophages. Further investigations, mainly in in vivo studies, should be conducted to elucidate Polybia paulista Ag5 biological role in the macrophage functional profile modulation.

​Effect of Season on Quantity and Competence of Oocytes Recovered Transvaginally from Holstein Cows for In vitro Fertilization

Background: Season of the year can affect the reproductive behavior in Holstein cows, altering the competition of the oocytes, reflecting a reduced production of embryos. The objective of this study was to evaluate the average of total oocytes, competition of oocytes and embryos in the in vitro production process at different season of the year in Holstein cows. Methods: During the four seasons of the year, was performed on each of the oocyte donor cows (winter, n = 957; spring, n = 1571; summer, n = 1776; autumn, n = 1128), by in vivo transvaginal follicular aspiration technique after the collection were subjected to the embryos production in vitro. Result: The highest number of total embryos were produced in winter and autumn, compared to spring and summer (3.76±0.16 and 3.54±0.18 vs. 2.73±0.11 and 2.45±0.10; respectively, P less than 0.05). During winter, a higher percentage of oocyte competition was observed, followed by autumn and spring and less competition shown in summer (26.03±0.39, 19.08±0.29, respectively, P less than 0.05). The quantity and competence of the oocytes collected and in vitro embryo production were drastically reduced during the hottest months of the year in this area of intense heat.

Resveratrol-supplemented holding or re-culture media improves viability of fresh or vitrified-warmed in vitro-derived bovine embryos

The effect of resveratrol supplementation on fresh (E1) or vitrified/warmed (E2) in vitro produced bovine embryos was investigated by evaluating the time-dependent response. After in vitro production, resveratrol (0.5 µM) was added to the incubation media and after two incubation periods with or without resveratrol, blastocysts were re-cultured for 24h. The rates of re-expansion, hatching, total cell number (TCN), apoptotic cells (ACN), reactive oxygen species (ROS) and intracellular glutathione (GSH) content were evaluated. For E1, the re-expansion rate differed at 6 and 10h within and between treatments (P<0.05), as did the re-expansion rate after 24h (P<0.01). The hatching rate increased after 10h with resveratrol (P<0.01) with differences within (P<0.05), but not between treatments after 24h of re-cultivation. At E2, hatching rate differed between treatments at 24h (P<0.01), with higher TCN in resveratrol-treated blastocysts after 10h (P<0.01). Resveratrol supplementation reduced ROS generation in E1 and E2 after 10h of incubation and increased GSH content (P<0.01). These results indicate that supplementation of holding re-cultivation medium with resveratrol for treatment of fresh or vitrified/warmed in vitro produced bovine embryos has a positive and time-dependent effect. The reduction of ROS content, the increase of GSH and the anti-apoptotic ability of resveratrol are responsible for its protective effects, allowing an extension of embryo storage time before transfer to recipients.