Injected mitotic extracts induce condensation of interphase chromatin

Although extracts from mitotic cells have been shown to induce chromosome condensation when injected into amphibian oocytes, they have not as yet been shown to induce this response in somatic interphase cells. In the experiments reported here, when mitotic extracts were injected into syncytial frog embryos, whose somatic nuclei were arrested in interphase, chromosome condensation was observed. The inability of interphase extracts, injected at similar concentrations, to induce this event demonstrates the cell cycle-specific accumulation of the factors responsible.     

Purification and properties of chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from Alcaligenes eutrophus.

Chorismate mutase and prephenate dehydratase from Alcaligenes autophus H16 were purified 470-fold with a yield of 24%. During the course of purification, including chromatography on diethylaminoethyl (DEAE)-cellulose, phenylalanine-substituted Sepharose, Sephadex G-200 and hydrogyapatite, both enzymes appeared in association. The ratio of their specific activities remained almost constant. The molecular weight of chorismate mutase-prephenast dehydratase varied from 144,000 to 187,000 due to the three different determination methods used. Treatment of electrophoretically homogeneous mutase-dehydratase with sodium dodecyl sulfate dissociated the enzyme into a single component of molecular weight 47,000, indicating a tetramer of identical subunits. The isoelectric point of the bifunctional enzyme was 5.8. Prephenate dehydrogenase was not associated with other enzyme activities; it was separated from mutasedehydratase by DEAE-cellulose chromatgraphy. Chromatography on DEAE Sephadex, Sephadex G-200, and hydroxyapatite resulted in a 740-fold purification with a yield of 10%. The molecular weight of the enzyme was 55,000 as determined by sucrose gradient centrifugation and 65,000 as determined by gel filtration or electrophoresis. Its isoelectric point was pH 6.6. In the overall conversion of chorismate to phenylpyruvate, free prephenate was formed which accumulated in the reaction mixture. The dissociation of prephenate allowed prephenate dehydrogenase to compete with prephenate dehydratase for the substrate.     

Modelling biome shifts and tree cover change for 2050 in West Africa

Aim Africa is expected to face severe changes in climatic conditions. Our objectives are: (1) to model trends and the extent of future biome shifts that may occur by 2050, (2) to model a trend in tree cover change, while accounting for human impact, and (3) to evaluate uncertainty in future climate projections. Location West Africa. Methods We modelled the potential future spatial distribution of desert, grassland, savanna, deciduous and evergreen forest in West Africa using six bioclimatic models. Future tree cover change was analysed with generalized additive models (GAMs). We used climate data from 17 general circulation models (GCMs) and included human population density and fire intensity to model tree cover. Consensus projections were derived via weighted averages to: (1) reduce inter‐model variability, and (2) describe trends extracted from different GCM projections. Results The strongest predicted effect of climate change was on desert and grasslands, where the bioclimatic envelope of grassland is projected to expand into the desert by an area of 2 million km². While savannas are predicted to contract in the south (by 54 ± 22 × 10⁴ km²), deciduous and evergreen forest biomes are expected to expand (64 ± 13 × 10⁴ km² and 77 ± 26 × 10⁴ km²). However, uncertainty due to different GCMs was particularly high for the grassland and the evergreen biome shift. Increasing tree cover (1–10%) was projected for large parts of Benin, Burkina Faso, Côte d’Ivoire, Ghana and Togo, but a decrease was projected for coastal areas (1–20%). Furthermore, human impact negatively affected tree cover and partly changed the direction of the projected change from increase to decrease. Main conclusions Considering climate change alone, the model results of potential vegetation (biomes) show a ‘greening’ trend by 2050. However, the modelled effects of human impact suggest future forest degradation. Thus, it is essential to consider both climate change and human impact in order to generate realistic future tree cover projections.     

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.     

Cold chain and virus‐free chloroplast‐made booster vaccine to confer immunity against different poliovirus serotypes

The WHO recommends complete withdrawal of oral polio vaccine (OPV) type 2 by April 2016 globally and replacing with at least one dose of inactivated poliovirus vaccine (IPV). However, high‐cost, limited supply of IPV, persistent circulating vaccine‐derived polioviruses transmission and need for subsequent boosters remain unresolved. To meet this critical need, a novel strategy of a low‐cost cold chain‐free plant‐made viral protein 1 (VP1) subunit oral booster vaccine after single IPV dose is reported. Codon optimization of the VP1 gene enhanced expression by 50‐fold in chloroplasts. Oral boosting of VP1 expressed in plant cells with plant‐derived adjuvants after single priming with IPV significantly increased VP1‐IgG1 and VP1‐IgA titres when compared to lower IgG1 or negligible IgA titres with IPV injections. IgA plays a pivotal role in polio eradication because of its transmission through contaminated water or sewer systems. Neutralizing antibody titres (~3.17–10.17 log₂ titre) and seropositivity (70–90%) against all three poliovirus Sabin serotypes were observed with two doses of IPV and plant‐cell oral boosters but single dose of IPV resulted in poor neutralization. Lyophilized plant cells expressing VP1 stored at ambient temperature maintained efficacy and preserved antigen folding/assembly indefinitely, thereby eliminating cold chain currently required for all vaccines. Replacement of OPV with this booster vaccine and the next steps in clinical translation of FDA‐approved antigens and adjuvants are discussed.     

Proteomic analysis of the 20S proteasome (PSMA3)-interacting proteins reveals a functional link between the proteasome and mRNA metabolism

The 26S proteasome is a large multi-subunit protein complex that exerts specific degradation of proteins in the cell. The 26S proteasome consists of the 20S proteolytic particle and the 19S regulator. In order to be targeted for proteasomal degradation most of the proteins must undergo the post-translational modification of poly-ubiquitination. However, a number of proteins can also be degraded by the proteasome via a ubiquitin-independent pathway. Such degradation is exercised largely through the binding of substrate proteins to the PSMA3 (alpha 7) subunit of the 20S complex. However, a systematic analysis of proteins interacting with PSMA3 has not yet been carried out. In this report, we describe the identification of proteins associated with PSMA3 both in the cytoplasm and nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and tandem mass-spectrometry revealed a large number of PSMA3-bound proteins that are involved in various aspects of mRNA metabolism, including splicing. In vitro biochemical studies confirmed the interactions between PSMA3 and splicing factors. Moreover, we show that 20S proteasome is involved in the regulation of splicing in vitro of SMN2 (survival motor neuron 2) gene, whose product controls apoptosis of neurons.     

Draft genome sequence of the anoxygenic filamentous phototrophic bacterium Oscillochloris trichoides subsp. DG-6

Oscillochloris trichoides is a mesophilic, filamentous, photoautotrophic, nonsulfur, diazotrophic bacterium which is capable of carbon dioxide fixation via the reductive pentose phosphate cycle and possesses no assimilative sulfate reduction. Here, we present the draft genome sequence of Oscillochloris trichoides subsp. DG-6, the type strain of the species, which has permitted the prediction of genes for carbon and nitrogen metabolism and for the light-harvesting apparatus.     

Glucose‐based microbial production of the hormone melatonin in yeast Saccharomyces cerevisiae

Melatonin is a natural mammalian hormone that plays an important role in regulating the circadian cycle in humans. It is a clinically effective drug exhibiting positive effects as a sleep aid and a powerful antioxidant used as a dietary supplement. Commercial melatonin production is predominantly performed by complex chemical synthesis. In this study, we demonstrate microbial production of melatonin and related compounds, such as serotonin and N‐acetylserotonin. We generated Saccharomyces cerevisiae strains that comprise heterologous genes encoding one or more variants of an L‐tryptophan hydroxylase, a 5‐hydroxy‐L‐tryptophan decarboxylase, a serotonin acetyltransferase, an acetylserotonin O‐methyltransferase, and means for providing the cofactor tetrahydrobiopterin via heterologous biosynthesis and recycling pathways. We thereby achieved de novo melatonin biosynthesis from glucose. We furthermore accomplished increased product titers by altering expression levels of selected pathway enzymes and boosting co‐factor supply. The final yeast strain produced melatonin at a titer of 14.50 ± 0.57 mg L^?1 in a 76h fermentation using simulated fed‐batch medium with glucose as sole carbon source. Our study lays the basis for further developing a yeast cell factory for biological production of melatonin.     

A Single Intramuscular Vaccination of Mice with the HSV-1 VC2 Virus with Mutations in the Glycoprotein K and the Membrane Protein UL20 Confers Full Protection against Lethal Intravaginal Challenge with Virulent HSV-1 and HSV-2 Strains

Herpes Simplex Virus type-1 (HSV-1) and type-2 (HSV-2) establish life-long infections and cause significant orofacial and genital infections in humans. HSV-1 is the leading cause of infectious blindness in the western world. Currently, there are no available vaccines to protect against herpes simplex infections. Recently, we showed that a single intramuscular immunization with an HSV-1(F) mutant virus lacking expression of the viral glycoprotein K (gK), which prevents the virus from entering into distal axons of ganglionic neurons, conferred significant protection against either virulent HSV-1(McKrae) or HSV-2(G) intravaginal challenge in mice. Specifically, 90% of the mice were protected against HSV-1(McKrae) challenge, while 70% of the mice were protected against HSV-2(G) challenge. We constructed the recombinant virus VC2 that contains specific mutations in gK and the membrane protein UL20 preventing virus entry into axonal compartments of neurons, while allowing efficient replication in cell culture, unlike the gK-null virus, which has a major defect in virus replication and spread. Intramuscular injection of mice with 10⁷ VC2 plaque forming units did not cause any significant clinical disease in mice. A single intramuscular immunization with the VC2 virus protected 100% of mice against lethal intravaginal challenge with either HSV-1(McKrae) or HSV-2(G) viruses. Importantly, vaccination with VC2 produced robust cross protective humoral and cellular immunity that fully protected vaccinated mice against lethal disease. Quantitative PCR did not detect any viral DNA in ganglionic tissues of vaccinated mice, while unvaccinated mice contained high levels of viral DNA. The VC2 virus may serve as an efficient vaccine against both HSV-1 and HSV-2 infections, as well as a safe vector for the production of vaccines against other viral and bacterial pathogens.