To Stretch the Boundary of Secondary Metabolite Production in Plant Cell-Based Bioprocessing: Anthocyanin as a Case Study

Plant cells and tissue cultures hold great promise for controlled production of a myriad of useful secondary metabolites on demand. The current yield and productivity cannot fulfill the commercial goal of a plant cell-based bioprocess for the production of most secondary metabolites. In order to stretch the boundary, recent advances, new directions and opportunities in plant cell-based bioprocessing have been critically examined for the 10 years from 1992 to 2002. A review of the literature indicated that most of the R&D work was devoted predominantly to studies at an empirical level. A rational approach to molecular plant cell bioprocessing based on the fundamental understanding of metabolic pathways and their regulations is urgently required to stimulate further advances; however, the strategies and technical framework are still being developed. It is the aim of this review to take a step forward in framing workable strategies and technologies for molecular plant cell-based bioprocessing. Using anthocyanin biosynthesis as a case study, an integrated postgenomic approach has been proposed. This combines the functional analysis of metabolic pathways for biosynthesis of a particular metabolite from profiling of gene expression and protein expression to metabolic profiling. A global correlation not only can thus be established at the three molecular levels, but also places emphasis on the interactions between primary metabolism and secondary metabolism; between competing and/or complimentary pathways; and between biosynthetic and post-biosynthetic events

Purification, molecular cloning, and characterization of glutathione S-transferases (GSTs) from pigmented Vitis vinifera L. cell suspension cultures as putative anthocyanin transport proteins

The ligandin activity of specific glutathione S-transferases (GSTs) is necessary for the transport of anthocyanins from the cytosol to the plant vacuole. Five GSTs were purified from Vitis vinifera L. cv. Gamay Fréaux cell suspension cultures by glutathione affinity chromatography. These proteins underwent Edman sequencing and mass spectrometry fingerprinting, with the resultant fragments aligned with predicted GSTs within public databases. The corresponding coding sequences were cloned, with heterologous expression in Escherichia coli used to confirm GST activity. Transcriptional profiling of these candidate GST genes and key anthocyanin biosynthetic pathway genes (PAL, CHS, DFR, and UFGT) in cell suspensions and grape berries against anthocyanin accumulation demonstrated strong positive correlation with two sequences, VvGST1 and VvGST4, respectively. The ability of VvGST1 and VvGST4 to transport anthocyanins was confirmed in the heterologous maize bronze-2 complementation model, providing further evidence for their function as anthocyanin transport proteins in grape cells. Furthermore, the differential induction of VvGST1 and VvGST4 in suspension cells and grape berries suggests functional differences between these two proteins. Further investigation of these candidate ligandins may identify a mechanism for manipulating anthocyanin accumulation in planta and in vitro suspension cells

The RNA Binding Protein Quaking Regulates Formation of circRNAs

SummaryCircular RNAs (circRNAs), formed by non-sequential back-splicing of pre-mRNA transcripts, are a widespread form of non-coding RNA in animal cells. However, it is unclear whether the majority of circRNAs represent splicing by-products without function or are produced in a regulated manner to carry out specific cellular functions. We show that hundreds of circRNAs are regulated during human epithelial-mesenchymal transition (EMT) and find that the production of over one-third of abundant circRNAs is dynamically regulated by the alternative splicing factor, Quaking (QKI), which itself is regulated during EMT. Furthermore, by modulating QKI levels, we show the effect on circRNA abundance is dependent on intronic QKI binding motifs. Critically, the addition of QKI motifs is sufficient to induce de novo circRNA formation from transcripts that are normally linearly spliced. These findings demonstrate circRNAs are both purposefully synthesized and regulated by cell-type specific mechanisms, suggesting they play specific biological roles in EMT

Heterodimerization of Arabidopsis calcium/proton exchangers contributes to regulation of guard cell dynamics and plant defense responses.

Arabidopsis thaliana cation exchangers (CAX1 and CAX3) are closely related tonoplast-localized calcium/proton (Ca2+/H+) antiporters that contribute to cellular Ca2+ homeostasis. CAX1 and CAX3 were previously shown to interact in yeast; however, the function of this complex in plants has remained elusive. Here, we demonstrate that expression of CAX1 and CAX3 occurs in guard cells. Additionally, CAX1 and CAX3 are co-expressed in mesophyll tissue in response to wounding or flg22 treatment, due to the induction of CAX3 expression. Having shown that the transporters can be co-expressed in the same cells, we demonstrate that CAX1 and CAX3 can form homomeric and heteromeric complexes in plants. Consistent with the formation of a functional CAX1-CAX3 complex, CAX1 and CAX3 integrated into the yeast genome suppressed a Ca2+-hypersensitive phenotype of mutants defective in vacuolar Ca2+ transport, and demonstrated enzyme kinetics different from those of either CAX protein expressed by itself. We demonstrate that the interactions between CAX proteins contribute to the functioning of stomata, because stomata were more closed in cax1-1, cax3-1, and cax1-1/cax3-1 loss-of-function mutants due to an inability to buffer Ca2+ effectively. We hypothesize that the formation of CAX1-CAX3 complexes may occur in the mesophyll to affect intracellular Ca2+ signaling during defense responses

Anion-polarisation--directed short-range-order in antiperovskite Li2_2FeSO

Short-range ordering in cation-disordered cathodes can have a significant effect on their electrochemical properties. Here, we characterise the cation short-range order in the antiperovskite cathode material Li$_2$FeSO, using density functional theory, Monte Carlo simulations, and synchrotron X-ray pair-distribution-function data. We predict partial short-range cation-ordering, characterised by favourable OLi$_4$Fe$_2$ oxygen coordination with a preference for polar cis-OLi$_4$Fe$_2$ over non-polar trans-OLi$_4$Fe$_2$ configurations. This preference for polar cation configurations produces long-range disorder, in agreement with experimental data. The predicted short-range-order preference contrasts with that for a simple point-charge model, which instead predicts preferential trans-OLi$_4$Fe$_2$ oxygen coordination and corresponding long-range crystallographic order. The absence of long-range order in Li$_2$FeSO can therefore be attributed to the relative stability of cis-OLi$_4$Fe$_2$ and other non-OLi$_4$Fe$_2$ oxygen-coordination motifs. We show that this effect is associated with the polarisation of oxide and sulfide anions in polar coordination environments, which stabilises these polar short-range cation orderings. We propose similar anion-polarisation-directed short-range-ordering may be present in other heterocationic materials that contain cations with different formal charges. Our analysis also illustrates the limitations of using simple point-charge models to predict the structure of cation-disordered materials, where other factors, such as anion polarisation, may play a critical role in directing both short- and long-range structural correlations

Dynamics in the satellite system of Triangulum: Is AndXXII a dwarf satellite of M33?

We present results from a spectroscopic survey of the dwarf spheroidal And XXII and the two extended clusters EC1 and EC2. These three objects are candidate satellites of the Triangulum galaxy, M33, which itself is likely a satellite of M31. We use the DEep Imaging Multi-Object Spectrograph mounted on the Keck-II telescope to derive radial velocities for candidate member stars of these objects and thereby identify the stars that are most likely actual members. Eleven most probable stellar members (of 13 candidates) are found for AndXXII. We obtain an upper limit of sigma_v < 6.0 km s-1 for the velocity dispersion of AndXXII, [Fe/H] ~ -1.6 for its metallicity, and 255pc for the Plummer radius of its projected density profile. We construct a colour magnitude diagram for AndXXII and identify both the red giant branch and the horizontal branch. The position of the latter is used to derive a heliocentric distance to And XXII of 853 pm 26 kpc. The combination of the radial velocity, distance, and angular position of AndXXII indicates that it is a strong candidate for being the first known satellite of M33 and one of the very few examples of a galactic satellite of a satellite. N-body simulations imply that this conclusion is unchanged even if M31 and M33 had a strong encounter in the past few Gyr. We test the hypothesis that the extended clusters highlight tidally stripped galaxies by searching for an excess cloud of halo-like stars in their vicinity. We find such a cloud for the case of EC1 but not EC2. The three objects imply a dynamical mass for M33 that is consistent with previous estimates.Comment: 14 pages, 14 figures, revised for MNRAS publicatio

The Human–Nature Relationship and Its Impact on Health: A Critical Review

Within the past four decades, research has been increasingly drawn toward understanding whether there is a link between the changing human–nature relationship and its impact on people’s health. However, to examine whether there is a link requires research of its breadth and underlying mechanisms from an interdisciplinary approach. This article begins by reviewing the debates concerning the human–nature relationship, which are then critiqued and redefined from an interdisciplinary perspective. The concept and chronological history of “health” is then explored, based on the World Health Organization’s definition. Combining these concepts, the human–nature relationship and its impact on human’s health are then explored through a developing conceptual model. It is argued that using an interdisciplinary perspective can facilitate a deeper understanding of the complexities involved for attaining optimal health at the human–environmental interface

Week 96 Extension Results of a Phase 3 Study Evaluating Long-Acting Cabotegravir with Rilpivirine for HIV-1 Treatment

BACKGROUND: ATLAS (NCT02951052), a phase 3, multicenter, open-label study, demonstrated that switching to injectable cabotegravir (CAB) with rilpivirine (RPV) long-acting dosed every 4 weeks was noninferior at week (W) 48 to continuing three-drug daily oral current antiretroviral therapy (CAR). Results from the W 96 analysis are presented. METHODS AND DESIGN: Participants completing W 52 of ATLAS were given the option to withdraw, transition to ATLAS-2M (NCT03299049), or enter an Extension Phase to continue long-acting therapy (Long-acting arm) or switch from CAR to long-acting therapy (Switch arm). Endpoints assessed at W 96 included proportion of participants with plasma HIV-1 RNA less than 50 copies/ml, incidence of confirmed virologic failure (CVF; two consecutive HIV-1 RNA ≥200 copies/ml), safety and tolerability, pharmacokinetics, and patient-reported outcomes. RESULTS: Most participants completing the Maintenance Phase transitioned to ATLAS-2M (88%, n = 502/572). Overall, 52 participants were included in the W 96 analysis of ATLAS; of these, 100% (n = 23/23) and 97% (n = 28/29) in the Long-acting and Switch arms had plasma HIV-1 RNA less than 50 copies/ml at W 96, respectively. One participant had plasma HIV-1 RNA 50 copies/ml or higher in the Switch arm (173 copies/ml). No participants met the CVF criterion during the Extension Phase. No new safety signals were identified. All Switch arm participants surveyed preferred long-acting therapy to their previous daily oral regimen (100%, n = 27/27). CONCLUSION: In this subgroup of ATLAS, 98% (n = 51/52) of participants at the Extension Phase W 96 analysis maintained virologic suppression with long-acting therapy. Safety, efficacy, and participant preference results support the therapeutic potential of long-acting CAB+RPV treatment for virologically suppressed people living with HIV-1