Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic viruslike particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody

Anhydrobiosis-Associated Nuclear DNA Damage and Repair in the Sleeping Chironomid: Linkage with Radioresistance

Anhydrobiotic chironomid larvae can withstand prolonged complete desiccation as well as other external stresses including ionizing radiation. To understand the cross-tolerance mechanism, we have analyzed the structural changes in the nuclear DNA using transmission electron microscopy and DNA comet assays in relation to anhydrobiosis and radiation. We found that dehydration causes alterations in chromatin structure and a severe fragmentation of nuclear DNA in the cells of the larvae despite successful anhydrobiosis. Furthermore, while the larvae had restored physiological activity within an hour following rehydration, nuclear DNA restoration typically took 72 to 96 h. The DNA fragmentation level and the recovery of DNA integrity in the rehydrated larvae after anhydrobiosis were similar to those of hydrated larvae irradiated with 70 Gy of high-linear energy transfer (LET) ions (4He). In contrast, low-LET radiation (gamma-rays) of the same dose caused less initial damage to the larvae, and DNA was completely repaired within within 24 h. The expression of genes encoding the DNA repair enzymes occurred upon entering anhydrobiosis and exposure to high- and low-LET radiations, indicative of DNA damage that includes double-strand breaks and their subsequent repair. The expression of antioxidant enzymes-coding genes was also elevated in the anhydrobiotic and the gamma-ray-irradiated larvae that probably functions to reduce the negative effect of reactive oxygen species upon exposure to these stresses. Indeed the mature antioxidant proteins accumulated in the dry larvae and the total activity of antioxidants increased by a 3–4 fold in association with anhydrobiosis. We conclude that one of the factors explaining the relationship between radioresistance and the ability to undergo anhydrobiosis in the sleeping chironomid could be an adaptation to desiccation-inflicted nuclear DNA damage. There were also similarities in the molecular response of the larvae to damage caused by desiccation and ionizing radiation

MicroRNAs play critical roles during plant development and in response to abiotic stresses

MicroRNAs (miRNAs) have been identified as key molecules in regulatory networks. The fine-tuning role of miRNAs in addition to the regulatory role of transcription factors has shown that molecular events during development are tightly regulated. In addition, several miRNAs play crucial roles in the response to abiotic stress induced by drought, salinity, low temperatures, and metals such as aluminium. Interestingly, several miRNAs have overlapping roles with regard to development, stress responses, and nutrient homeostasis. Moreover, in response to the same abiotic stresses, different expression patterns for some conserved miRNA families among different plant species revealed different metabolic adjustments. The use of deep sequencing technologies for the characterisation of miRNA frequency and the identification of new miRNAs adds complexity to regulatory networks in plants. In this review, we consider the regulatory role of miRNAs in plant development and abiotic stresses, as well as the impact of deep sequencing technologies on the generation of miRNA data

Contingent and non-contingent recreational-like exposure to ethanol alters BDNF expression and signaling in the cortico-accumbal network differently

Rationale: Although brain-derived neurotrophic factor (BDNF) is part of a homeostatic pathway involved in the development of alcohol dependence, it is not clear whether this is also true after recreational ethanol consumption. Objectives: We examined BDNF expression and signaling in the cortico-striatal network immediately and 24 h after either a single intravenous (i.v.) ethanol operant self-administration session or the last of 14 sessions. Methods: To compare contingent and non-contingent ethanol exposure, we incorporated the “yoked control-operant paradigm” in which rats actively taking ethanol (S-Et) were paired with two yoked controls receiving passive infusions of ethanol (Y-Et) or saline. Results: A single ethanol exposure transiently reduced BDNF mRNA levels in the medial prefrontal cortex (mPFC) of Y-Et. Immediately after the last of 14 sessions, mRNA and mature BDNF protein levels (mBDNF) were reduced in the mPFC in both S-Et and Y-Et while mBDNF expression was raised in the nucleus accumbens (NAc), suggesting enhanced anterograde transport from the mPFC. Conversely, 24 h later mBDNF expression and signaling were raised in the mPFC and NAc of S-Et rats but reduced in the NAc of Y-Et rats, with concomitant reduction of downstream signaling pathways. Conclusions: Our findings indicate that recreational-like i.v. doses of ethanol promote early changes in neurotrophin expression, depending on the length and modality of administration, the brain region investigated, and the presence of the drug. A rapid intervention targeting the BDNF system might be useful to prevent escalation to alcohol abuse

Differential responses to drought stress in leaves and roots of wild jujube, Ziziphus lotus

The aim of this study was to investigate the effects of drought stress induced by polyethylene glycol-6000 in wild jujube, Ziziphus lotus. One-month-old, hydroponically grown seedlings were subjected to three treatments, i.e. normal watering (-0.2 MPa), moderate (-1.2 MPa) and severe (-2.1 MPa) drought stress for 14 days under controlled climatic conditions. Plant growth was markedly reduced with increasing osmotic stress. The shoot water potential (I-w) and leaf relative water content followed similar patterns and significantly decreased with increasing osmolality of solutions. As a consequence of drought, contents in proline and soluble sugars were found to be more elevated in leaves than in roots. The level of lipid peroxidation in terms of malonyldialdehyde contents increased in both leaves and roots of drought-stressed plants. Wild jujube displayed higher activities of antioxidant enzymes in the roots than in the leaves. Catalase and guaiacol peroxidase activities increased significantly in drought-stressed roots, whereas ascorbate peroxidase activity showed a slight decline with no significant changes. These findings suggest that Z. lotus was able to adapt to severe drought stress by accumulation of compatible solutes and by activation of free radical-scavenging enzymes. Overall, defence mechanisms in Z. lotus against oxidative stress are organized differently in plant tissues, with higher solute accumulation in leaves and increased activity of antioxidants in roots, during drought stress