The synergetic effect from the combination of different adsorption resins in batch and semi-continuous cultivations of S. Cerevisiae cell factories to produce acetylated Taxanes precursors of the anticancer drug Taxol

In situ product recovery is an efficient way to intensify bioprocesses as it can perform adsorption of the desired natural products in the cultivation. However, it is common to use only one adsorbent (liquid or solid) to perform the product recovery. For this study, the use of an in situ product recovery method with three combined commercial resins (HP-20, XAD7HP, and HP-2MG) with different chemical properties was performed. A new yeast strain of Saccharomyces cerevisiae was engineered using CRISPR Cas9 (strain EJ2) to deliver heterologous expression of oxygenated acetylated taxanes that are precursors of the anticancer drug Taxol ® (paclitaxel). Microscale cultivations using a definitive screening design (DSD) were set to get the best resin combinations and concentrations to retrieve high taxane titers. Once the best resin treatment was selected by the DSD, semi-continuous cultivation in high throughput microscale was performed to increase the total taxanes yield up to 783 ± 33 mg/L. The best T5α-yl Acetate yield obtained was up to 95 ± 4 mg/L, the highest titer of this compound ever reported by a heterologous expression. It was also observed that by using a combination of the resins in the cultivation, 8 additional uncharacterized taxanes were found in the gas chromatograms compared to the dodecane overlay method. Lastly, the cell-waste reactive oxygen species concentrations from the yeast were 1.5-fold lower in the resin's treatment compared to the control with no adsorbent aid. The possible future implications of this method could be critical for bioprocess intensification, allowing the transition to a semi-continuous flow bioprocess. Further, this new methodology broadens the use of different organisms for natural product synthesis/discovery benefiting from clear bioprocess intensification advantages

Increased paclitaxel recovery from Taxus baccata vascular stem cells using novel in situ product recovery approaches

In this study, several approaches were tested to optimise the production and recovery of the widely used anticancer drug Taxol® (paclitaxel) from culturable vascular stem cells (VSCs) of Taxus baccata, which is currently used as a successful cell line for paclitaxel production. An in situ product recovery (ISPR) technique was employed, which involved combining three commercial macro-porous resin beads (HP-20, XAD7HP and HP-2MG) with batch and semi-continuous cultivations of the T. baccata VSCs after adding methyl jasmonate (Me-JA) as an elicitor. The optimal resin combination resulted in 234 ± 23 mg of paclitaxel per kg of fresh-weight cells, indicating a 13-fold improved yield compared to the control (with no resins) in batch cultivation. This resin treatment was further studied to evaluate the resins’ removal capacity of reactive oxygen species (ROS), which can cause poor cell growth or reduce product synthesis. It was observed that the ISPR cultivations had fourfold less intracellular ROS concentration than that of the control; thus, a reduced ROS concentration established by the resin contributed to increased paclitaxel yield, contrary to previous studies. These paclitaxel yields are the highest reported to date using VSCs, and this scalable production method could be applied for a diverse range of similar compounds utilising plant cell culture. Graphical Abstract: [Figure not available: see fulltext.]

Transport of Live Cells under Sterile Conditions Using a Chemotactic Droplet

© 2018 The Author(s). 1-Decanol droplets, formed in an aqueous medium containing decanoate at high pH, become chemotactic when a chemical gradient is placed in the external aqueous environment. We investigated if such droplets can be used as transporters for living cells. We developed a partially hydrophobic alginate capsule as a protective unit that can be precisely placed in a droplet and transported along chemical gradients. Once the droplets with cargo reached a defined final destination, the association of the alginate capsule and decanol droplet was disrupted and cargo deposited. Both Escherichia coli and Bacillus subtilis cells survived and proliferated after transport even though transport occurred under harsh and sterile conditions

Influence of training status on high-intensity intermittent performance in response to β-alanine supplementation

Recent investigations have suggested that highly trained athletes may be less responsive to the ergogenic effects of β-alanine (BA) supplementation than recreationally active individuals due to their elevated muscle buffering capacity. We investigated whether training status influences the effect of BA on repeated Wingate performance. Forty young males were divided into two groups according to their training status (trained: T, and non-trained: NT cyclists) and were randomly allocated to BA and a dextrose-based placebo (PL) groups, providing four experimental conditions: NTPL, NTBA, TPL, TBA. BA (6.4 g day-1 ) or PL was ingested for 4 weeks, with participants completing four 30-s lower-body Wingate bouts, separated by 3 min, before and after supplementation. Total work done was significantly increased following supplementation in both NTBA (p = 0.03) and TBA (p = 0.002), and it was significantly reduced in NTPL (p = 0.03) with no difference for TPL (p = 0.73). BA supplementation increased mean power output (MPO) in bout 4 for the NTBA group (p = 0.0004) and in bouts 1, 2 and 4 for the TBA group (p ≤ 0.05). No differences were observed in MPO for NTPL and TPL. BA supplementation was effective at improving repeated high-intensity cycling performance in both trained and non-trained individuals, highlighting the efficacy of BA as an ergogenic aid for high-intensity exercise regardless of the training status of the individual

RNA Interference of Four Genes in Adult Bactrocera dorsalis by Feeding Their dsRNAs

BACKGROUND: RNA interference (RNAi) is a powerful method to inhibit gene expression in a sequence specific manner. Recently silencing the target gene through feeding has been successfully carried out in many insect species. METHODOLOGY/PRINCIPAL FINDINGS: Escherichia coli strain HT115 was genetically engineered to express dsRNA targeting genes that encode ribosomal protein Rpl19, V type ATPase D subunit, the fatty acid elongase Noa and a small GTPase Rab11. qRT-PCR showed that mRNA level of four target genes was reduced compared to ds-egfp control by feeding either engineered bacteria or dsRNAs. The maximum down-regulation of each gene varied from 35% to 100%. Tissue specific examination indicated that RNAi could be observed not only in midgut but also in other tissues like the ovary, nervous system and fat body. Silencing of rab11 through ingestion of dsRNA killed 20% of adult flies. Egg production was affected through feeding ds-noa and ds-rab11 compared to ds-egfp group. Adult flies were continuously fed with dsRNA and bacteria expressing dsRNA for 14 days and up-regulations of target genes were observed during this process. The transcripts of noa showed up-regulation compared to ds-egfp control group in four tissues on day 7 after continuous feeding either dsRNA or engineered bacteria. The maximum over-expression is 21 times compared to ds-egfp control group. Up-regulation of rab11 mRNA level could be observed in testes on day 7 after continuous bacteria treatment and in midgut on day 2 after ds-rab11 treatment. This phenomenon could also be observed in rpl19 groups. CONCLUSIONS: Our results suggested that it is feasible to silence genes by feeding dsRNA and bacteria expressing dsRNA in Bactrocera dorsalis. Additionally the over-expression of the target gene after continuously feeding dsRNA or bacteria was observed

Degradation behaviors and mechanisms of MoS2 crystals relevant to bioabsorbable electronics

Monolayer molybdenum disulfide (MoS2) exhibits unique semiconducting and bioresorption properties, giving this material enormous potential for electronic/biomedical applications, such as bioabsorbable electronics. In this regard, understanding the degradation performance of monolayer MoS2 in biofluids allows modulation of the properties and lifetime of related bioabsorbable devices and systems. Herein, the degradation behaviors and mechanisms of monolayer MoS2 crystals with different misorientation angles are explored. High-angle grain boundaries (HAGBs) biodegrade faster than low-angle grain boundaries (LAGBs), exhibiting degraded edges with wedge and zigzag shapes, respectively. Triangular pits that formed in the degraded grains have orientations opposite to those of the parent crystals, and these pits grow into larger pits laterally. These behaviors indicate that the degradation is induced and propagated based on intrinsic defects, such as grain boundaries and point defects, because of their high chemical reactivity due to lattice breakage and the formation of dangling bonds. High densities of dislocations and point defects lead to high chemical reactivity and faster degradation. The structural cause of MoS2 degradation is studied, and a feasible approach to study changes in the properties and lifetime of MoS2 by controlling the defect type and density is presented. The results can thus be used to promote the widespread use of two-dimensional materials in bioabsorption applications

Genomic HIV RNA Induces Innate Immune Responses through RIG-I-Dependent Sensing of Secondary-Structured RNA

Contains fulltext : 108031.pdf (publisher's version ) (Open Access)BACKGROUND: Innate immune responses have recently been appreciated to play an important role in the pathogenesis of HIV infection. Whereas inadequate innate immune sensing of HIV during acute infection may contribute to failure to control and eradicate infection, persistent inflammatory responses later during infection contribute in driving chronic immune activation and development of immunodeficiency. However, knowledge on specific HIV PAMPs and cellular PRRs responsible for inducing innate immune responses remains sparse. METHODS/PRINCIPAL FINDINGS: Here we demonstrate a major role for RIG-I and the adaptor protein MAVS in induction of innate immune responses to HIV genomic RNA. We found that secondary structured HIV-derived RNAs induced a response similar to genomic RNA. In primary human peripheral blood mononuclear cells and primary human macrophages, HIV RNA induced expression of IFN-stimulated genes, whereas only low levels of type I IFN and tumor necrosis factor alpha were produced. Furthermore, secondary structured HIV-derived RNA activated pathways to NF-kappaB, MAP kinases, and IRF3 and co-localized with peroxisomes, suggesting a role for this organelle in RIG-I-mediated innate immune sensing of HIV RNA. CONCLUSIONS/SIGNIFICANCE: These results establish RIG-I as an innate immune sensor of cytosolic HIV genomic RNA with secondary structure, thereby expanding current knowledge on HIV molecules capable of stimulating the innate immune system

Host Cell Invasion and Virulence Mediated by Candida albicans Ssa1

Candida albicans Ssa1 and Ssa2 are members of the HSP70 family of heat shock proteins that are expressed on the cell surface and function as receptors for antimicrobial peptides such as histatins. We investigated the role of Ssa1 and Ssa2 in mediating pathogenic host cell interactions and virulence. A C. albicans ssa1Δ/Δ mutant had attenuated virulence in murine models of disseminated and oropharyngeal candidiasis, whereas an ssa2Δ/Δ mutant did not. In vitro studies revealed that the ssa1Δ/Δ mutant caused markedly less damage to endothelial cells and oral epithelial cell lines. Also, the ssa1Δ/Δ mutant had defective binding to endothelial cell N-cadherin and epithelial cell E-cadherin, receptors that mediate host cell endocytosis of C. albicans. As a result, this mutant had impaired capacity to induce its own endocytosis by endothelial cells and oral epithelial cells. Latex beads coated with recombinant Ssa1 were avidly endocytosed by both endothelial cells and oral epithelial cells, demonstrating that Ssa1 is sufficient to induce host cell endocytosis. These results indicate that Ssa1 is a novel invasin that binds to host cell cadherins, induces host cell endocytosis, and is critical for C. albicans to cause maximal damage to host cells and induce disseminated and oropharyngeal disease