Deficiency of prohormone convertase dPC2 (AMONTILLADO) results in impaired production of bioactive neuropeptide hormones in Drosophila

Peptide hormones synthesized by secretory neurons in the CNS are important regulators of physiology, behavior, and development. Like other neuropeptides, they are synthesized from larger precursor molecules by a specific set of enzymes. Using a combination of neurogenetics, immunostainings, and direct mass spectrometric profiling, we show that the presence of Drosophila prohormone convertase 2 encoded by the gene amontillado (amon) is a prerequisite for the proper processing of neuropeptide hormones from the major neurohemal organs of the CNS. A loss of amon correlates with a loss of neuropeptide hormone signals from the larval ring gland and perisympathetic organs. Neuropeptide hormone signals were still detectable in the adult corpora cardiaca of older amon-deficient flies which were amon heat-shock-rescued until eclosion. A semiquantification by direct peptide profiling using stable isotopic standards showed, however, that their neuropeptide hormone levels are strongly reduced. Targeted expression of GFP under the control of amon regulatory regions revealed a co-localization with the investigated peptide hormones in secretory neurons of the brain and ventral nerve cord. The lack of AMON activity resulted in a deficiency of L3 larva to enter the wandering phase. In conclusion, our findings provide the first direct evidence that AMON is a key enzyme in the production of neuropeptides in the fruitfly.     

Seed planning,sourcing, and procurement

Ensuring the availability of adequate seed supplies of species and sources appropriate for restoration projects and programs necessitates extensive science‐based planning. The selection of target species requires a review of disturbance conditions and reference areas, development of a reference model, and consideration of specific objectives, timeframes, available resources, and budgets as well as the performance of prospective species in past restoration efforts. Identification of seed sources adapted to site conditions is critical to provide for short‐term establishment and long‐term sustainability. Seed zones and plant movement guidelines provide tools for sourcing plant materials with reduced risk of maladaptation. A seed zone framework also facilitates seed use planning and contributes to stability and predictability of the commercial market, thereby reducing costs and improving the availability of adapted seed supplies. Calculating the amount of seed required for each species is based on seed quality (viability, purity), seed weight, expected seedling establishment, and desired composition of the seeding. If adequate collections from wildland stands are not feasible, then seed increase in seed fields or use of nursery stock may be warranted. Adherence to seed collection and seed production protocols for conserving genetic diversity is critical to protect genetic resources and buffer new seedings and plantings against environmental stressors. Maintenance of genetic diversity becomes even more critical considering current or expected climate change impacts. Collaboration and partnerships can benefit seed selection and procurement programs through sharing of information, coordination in project planning, and increasing the availability of native seed.     

Artesunate inhibits the mevalonate pathway and promotes glioma cell senescence

Glioma is a common brain malignancy for which new drug development is urgently needed because of radiotherapy and drug resistance. Recent studies have demonstrated that artemisinin (ARS) compounds can display antiglioma activity, but the mechanisms are poorly understood. Using cell lines and mouse models, we investigated the effects of the most soluble ARS analogue artesunate (ART) on glioma cell growth, migration, distant seeding and senescence and elucidated the underlying mechanisms. Artemisinin effectively inhibited glioma cell growth, migration and distant seeding. Further investigation of the mechanisms showed that ART can influence glioma cell metabolism by affecting the nuclear localization of SREBP2 (sterol regulatory element‐binding protein 2) and the expression of its target gene HMGCR (3‐hydroxy‐3‐methylglutaryl coenzyme A reductase), the rate‐limiting enzyme of the mevalonate (MVA) pathway. Moreover, ART affected the interaction between SREBP2 and P53 and restored the expression of P21 in cells expressing wild‐type P53, thus playing a key role in cell senescence induction. In conclusion, our study demonstrated the new therapeutic potential of ART in glioma cells and showed the novel anticancer mechanisms of ARS compounds of regulating MVA metabolism and cell senescence.     

Allelopathic and intraspecific growth competition effects establishment of direct sown Miscanthus

High yielding perennial crops are being developed as a sustainable feedstock for renewable energy and bioproducts. Miscanthus is a leading biomass crop, but most plantations comprise a sterile hybrid Miscanthus × giganteus that is clonally propagated. To develop new varieties across large areas, rhizome cloning is inefficient, time consuming and expensive. Alternative approaches use seed, and in temperate regions, this has been successfully applied by raising seedlings as plug plants in glasshouses before transfer to the field. Direct sowing has yet to be proven commercially viable because poor germination has resulted in inconsistent stand establishment. Oversowing using seed clusters is a common approach to improve the establishment of crops and it was hypothesized that such an approach will improve uniformity of density in early Miscanthus stands and thereby improve yield. Sowing multiple seeds creates potential for new interactions, and we identified at least two inhibitory mechanisms related to seed numbers. Germinating seed produced allelopathic effects on nearby seed thereby inhibiting plant growth. The inhibitory effect of Miscanthus seed on germination percentages was related to seed number within clusters. An extract from germinating Miscanthus seed inhibited the germination of Miscanthus seed. The extract was analysed by HPLC, which identified a complex mixture including several known allelopathic compounds including proanthocyanidins and vanillic acid. There was also evidence of root competition in soil in a controlled environment experiment. When the experiment on competition was replicated at field scale, the establishment rates were much lower and there was evidence of shoot competition. We conclude that the numbers of seed required to ensure an acceptable level of establishment in the field may be economically impractical until other agronomic techniques are included either to reduce the inhibitory effects of higher seed numbers or to reduce oversowing rates.     

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein''s phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.     

Susceptibility of cell substrates to PrPSc infection and safety control measures related to biological and biotherapeutical products

Concerns over the potential for infectious prion proteins to contaminate human biologics and biotherapeutics have been raised from time to time. Transmission of the pathogenic form of prion protein (PrPSc) through veterinary vaccines has been observed, yet no human case through the use of vaccine products has been reported. However, iatrogenic transmissions of PrPSc in humans through blood components, tissues, and growth hormone have been reported. These findings underscore the importance of reliable detection or diagnostic methods to prevent the transmission of prion diseases, given that the number of asymptomatic infected individuals remains unknown, the perceived incubation time for human prion diseases could be decades, and no cure of the diseases has been found yet. A variety of biochemical and molecular methods can selectively concentrate PrPSc to facilitate its detection in tissues and cells. Furthermore, some methods routinely used in the manufacturing process of biological products have been found to be effective in reducing PrPSc from the products. Questions remain unanswered as to the validation criteria of these methods, the minimal infectious dose of the PrPSc required to cause infection and the susceptibility of cells used in gene therapy or the manufacturing process of biological products to PrPSc infections. Here, we discuss some of these challenging issues.     

Interactions between tomato volatile organic compounds and aphid behaviour

Abstract

In the tritrophic system consisting of tomato, Solanum lycopersicum (L.), the aphid Macrosiphum euphorbiae (Thomas) and its natural enemy, the parasitoid Aphidius ervi (Haliday), it has been shown that the release of volatile organic compounds following aphid attack is responsible for attracting aphid parasitoids in wind tunnel experiments. The main compounds involved in these multitrophic interactions have been characterized and quantified. In this work, the possible activity of such compounds on plant direct defences against the aphid M. euphorbiae was assessed in laboratory tests. The selected compounds were applied to uninfested tomato plants, either by evaporation or contact, and performance of aphids, in terms of plant acceptance, fixing behaviour and aphid development, calculated in standard conditions. The results showed that two compounds, namely methyl salicylate and cis-hex-3-en-1-ol, alter aphid performance. These two compounds have been reported to be those eliciting the best response by A. ervi in terms of flight behavior (wind tunnel bioassay) and antennal stimulation (EAG bioassay).     

Effects of the observation method (direct v. from video) and of the presence of an observer on behavioural results in veal calves

This study aimed at assessing the effect of the observation method (direct or from video) and the effect of the presence of an observer on the behavioural results in veal calves kept on a commercial farm. To evaluate the effect of the observation method, 20 pens (four to five calves per pen) were observed by an observer for 60 min (two observation sessions of 30 min) and video-recorded at the same time. To evaluate the effect of the presence of the observer in front of the pen, 24 pens were video-recorded on 4 consecutive days and an observer was present in front of each pen for 60 min (two observation sessions of 30 min) on the third day. Behaviour was recorded using instantaneous scan sampling. For the study of the observer's effect, the analysis was limited to the posture, abnormal oral behaviour and manipulation of substrates. The two observation methods gave similar results for the time spent standing, but different results for all other behaviours. The presence of an observer did not affect the behaviour of calves at day level; however, their behaviour was affected when the observer was actually present in front of the pens. A higher percentage of calves were standing and were manipulating substrate in the presence of the observer, but there was no effect on abnormal oral behaviour. In conclusion, direct observations are a more suitable observation method than observations from video recordings for detailed behaviours in veal calves. The presence of an observer has a short-term effect on certain behaviours of calves that will have to be taken into consideration when monitoring these behaviours.     

Ecological changes in the Loch Lomond region

Summary

The Lichen flora of Rhum is described with emphasis on its ecology. In general, the 387 species represent a fine example of eu-oceanic assemblages and contain many taxa typical of western Britain. The margins of freshwater lochs hold particularly rich communities; the flora of the serpentine is singular for its narrow range of species. There is some evidence that existing fragments of woodland have continuity with the ancient forest. Lecanactis plocina is reported new to Scotland.     

Hepatocyte nuclear factor 4α regulates the expression of the murine pyruvate carboxylase gene through the HNF4-specific binding motif in its proximal promoter

Pyruvate carboxylase (PC) is the first regulatory enzyme of gluconeogenesis. Here we report that the proximal promoter of the murine PC gene contains three binding sites for hepatocyte nuclear factor 4α (HNF4α). These sites include the classical direct repeat 1 (DR1) (− 386/− 374), non-perfect DR1 (− 118/− 106) and HNF4α-specific binding motif (H4-SBM) (− 26/− 14). Under basal conditions, mutation of the non-perfect DR1 decreased promoter activity by 50%, whereas mutation of neither the DR1 nor the H4-SBM had any effect. In marked contrast, only mutation of the H4-SBM decreased HNF4α-transactivation of the promoter activity by 65%. EMSA revealed that HNF4α binds to the DR1site and H4-SBM with similar affinity while it binds poorly to the non-perfect DR1. Interestingly, this non-perfect DR1 also coincides with two E-boxes. Mutation of the non-perfect DR1 together with the nearby E-box reduced USF1- but not USF2-transactivation of promoter activity, suggesting that USF1 partly contributes to the basal activity of the promoter. Substitution of the H4-SBM with the DR1 marginally reduced the basal promoter activity but did not eliminate HNF4α-transactivation, suggesting that HNF4α can exert its effect via DR1 within this promoter context. ChIP-assay confirmed that HNF4α is associated with the H4-SBM. Suppression of HNF4α expression in AML12 cells down-regulated PC mRNA and PC protein by 60% and 50%, respectively, confirming that PC is a target of HNF4α. We also propose a model for differential regulation of P1 promoter of PC gene in adipose tissue and liver.