Who is the best surrogate for germ stem cell transplantation in fish?

Surrogate reproduction technology in fish has potential for aquaculture as well as endangered species preservation and propagation. Species with some unfavourable biological characteristics for culturing such as a late maturation or a large body size are ideal candidates for surrogate reproduction using smaller and faster-maturing host. One of the general prerequisites for the successful surrogacy and the pure donor-derived gamete production is the sterility of the host. Various sterilization methods have been developed and used in fish surrogacy; however, a direct comparison of available methods is missing. Such a knowledge gap hinders choice for the surrogate in various fish species, including those in high commercial demand such as tuna or sturgeons, where is a particular limitation from the point of the live material availability and difficulty to perform a high throughput assessment of different surrogates. Yet, large sturgeons or tuna species are one of the most prominent candidates for surrogacy. Zebrafish was utilized in this study as a model species to answer whether and to which extent different sterilization strategies can affect the surrogacy. Germ cell-depleted recipients (produced using knockdown of dead end gene), triploid recipients, and zebrafish x pearl danio hybrid recipients were tested as they represent the most frequently used types of surrogates. Spermatogonia isolated from vas::EGFP transgenic strain were intraperitoneally transplanted into swim-up 5-day old zebrafish. Transplantation success, survival, gonadal development, and reproductive output of the fish was analyzed. Germ cell-depleted recipients with empty gonads were identified as the most convenient among tested sterilization methods considering surrogacy induction success and reproductive output. The present study stands as significant aid for selecting suitable surrogates in various fish species.

Streblus asper Lour. exerts MAPK and SKN-1 mediated anti-aging, anti-photoaging activities and imparts neuroprotection by ameliorating Aβ in Caenorhabditis elegans

BACKGROUND: Streblus asper Lour., has been reported to have anti-aging and neuroprotective efficacies in vitro. OBJECTIVE: To analyze the anti-aging, anti-photoaging and neuroprotective efficacies of S. asper in Caenorhabditis elegans. METHODS: C. elegans (wild type and gene specific mutants) were treated with S. asper extract and analyzed for lifespan and other health benefits through physiological assays, fluorescence microscopy, qPCR and Western blot. RESULTS: The plant extract was found to increase the lifespan, reduce the accumulation of lipofuscin and modulate the expression of candidate genes. It could extend the lifespan of both daf-16 and daf-2 mutants whereas the pmk-1 mutant showed no effect. The activation of skn-1 was observed in skn-1::GFP transgenic strain and in qPCR expression. Further, the extract can extend the lifespan of UV-A exposed nematodes along with reducing ROS levels. Additionally, the extract also extends lifespan and reduces paralysis in Aβ transgenic strain, apart from reducing Aβ expression. CONCLUSIONS: S. asper was able to extend the lifespan and healthspan of C. elegans which was independent of DAF-16 pathway but dependent on SKN-1 and MAPK which could play a vital role in eliciting the anti-aging, anti-photoaging and neuroprotective effects, as the extract could impart oxidative stress resistance and neuroprotection.

Expression of Aspergillus niger glucose oxidase in Pichia pastoris and its antimicrobial activity against Agrobacterium and Escherichia coli

The gene encoding glucose oxidase from Aspergillus niger ZM-8 was cloned and transferred to Pichia pastoris GS115, a transgenic strain P. pastoris GS115-His-GOD constructed. The growth curve of P. pastoris GS115-His-GOD was consistent with that of Pichia pastoris GS115-pPIC9K under non-induced culture conditions. Under methanol induction conditions, the growth of the GOD-transgenic strain was significantly lowered than P. pastoris GS115-pPIC9K with the induced-culture time increase, and the optical densities of GOD-transgenic strain reached one-third of that of the P. pastoris GS115-pPIC9K at 51 h. The activity of glucose oxidase in the cell-free supernatant, the supernatant of cell lysate, and the precipitation of cell lysate was 14.3 U/mL, 18.2 U/mL and 0.48 U/mL, respectively. The specific activity of glucose oxidase was 8.3 U/mg, 6.52 U/mg and 0.73 U/mg, respectively. The concentration of hydrogen peroxide formed by glucose oxidase from supernatant of the fermentation medium, the supernatant of the cell lysate, and the precipitation of cell lysate catalyzing 0.2 M glucose was 14.3 μg/mL, 18.2 μg/mL, 0.48 μg/mL, respectively. The combination of different concentrations of glucose oxidase and glucose could significantly inhibit the growth of Agrobacterium and Escherichia coli in logarithmic phase. The filter article containing supernatant of the fermentation medium, supernatant of the cell lysate, and precipitation of cell lysate had no inhibitory effect on Agrobacterium and E. coli. The minimum inhibitory concentration of hydrogen peroxide on the plate culture of Agrobacterium and E. coli was 5.6 × 103 μg/mL and 6.0 × 103 μg/mL, respectively.

The Investigations of Nitric Oxide Influence on Lifespan of Fruit Fly D. melanogaster Transgenic Strain dNOS4

Introduction. Aging and longevity control are among the greatest problems in biology and medicine. The fruit fly Drosophila melanogaster is a nice model organism for longevity investigations because of its biological features. Many D. melanogaster genes have their orthologs, similar in other eukaryotes, including human. The role of nitric oxide (NO) in the D. melanogaster lifespan has been analyzed.Methods. Virgin flies of dNOS4 transgenic strain were used for the experiment. This strain contains non-functional additional copies of nitric oxide synthase (NOS) gene under heat shock promoter. For promoter activation, transgenic flies on their second day of life were exposed to heat shock (37°C) for an hour. After heat shock, flies were maintained on standard medium temperatures at 25°C, with females separate from males. Two types of control were used: Oregon R wild-type strain and Oregon R strain exposed to heat shock. The average lifespan was evaluated.Results. It was revealed that the longevity of females was significantly higher than males in each series of experiments (p < 0.05). The survival rate of females and males was similar in the first month of their life, but in the second month the mortality among males was much higher than among females in all series of experiments. The average lifespan of dNOS4 imago was 31 days (34 days for females and 28 days for males), maximum lifespan was 63 days. In controls, the average lifespan of Oregon R flies was 54 days (58 days for females and 50 days for males), and the maximum lifespan was 94 days. The average lifespan of Oregon R flies exposed to heat shock was 45 days (48 days for females and 41 days for males), and the maximum lifespan was 72 days. The difference between average lifespan in all studied groups is statistically significant (p < 0.05).Conclusion. Thus, NOS-transgene activation results in formation of non-functional dNOS4-transcripts and NO deficiency. In turn, NO deficiency decreases dNOS4 imago lifespan.

A comprehensive, non-invasive visualization of primordial germ cell development in mice by the Prdm1-mVenus and Dppa3-ECFP double transgenic reporter

The ability to monitor the development of a given cell lineage in a non-invasive manner by fluorescent markers bothin vivoandin vitroprovides a great advantage for the analysis of the lineage of interest. To date, a number of transgenic or knock-in mouse strains, in which developing germ cells are marked with fluorescent reporters, have been generated. We here describe a novel double transgenic reporter mouse strain that expresses membrane-targeted Venus (mVenus), a brighter variant of yellow fluorescent protein (YFP), under the control ofPrdm1(Blimp1) regulatory elements and enhanced cyan fluorescent protein (ECFP) under the control ofDppa3(Stella/Pgc7). The double transgenic strain unambiguously markedPrdm1expression in the lineage-restricted precursors of primordial germ cells (PGCs) in the proximal epiblast at embryonic day (E) 6.25 and specifically illuminatedPrdm1- andDppa3-positive migrating PGCs after E8.5. The double transgenic reporter also precisely recapitulated dynamic embryonic expression ofPrdm1outside the germ cell lineage. Moreover, we derived ES cells that bore both transgenes. These cells made a robust contribution both to the germ and somatic cell lineages in chimeras with accuratePrdm1-mVenus andDppa3-ECFP expression. The transgenic strain and the ES cells will serve as valuable experimental materials not only for analyzing the origin and properties of the germ cell lineagein vivo, but also for establishing a culture system to efficiently induce proper germ cells with temporally coordinatedPrdm1andDppa3expressionin vitro.