In vivo self-assembled small RNAs as a new generation of RNAi therapeutics

RNAi therapy has undergone two stages of development, direct injection of synthetic siRNAs and delivery with artificial vehicles or conjugated ligands; both have not solved the problem of efficient in vivo siRNA delivery. Here, we present a proof-of-principle strategy that reprogrammes host liver with genetic circuits to direct the synthesis and self-assembly of siRNAs into secretory exosomes and facilitate the in vivo delivery of siRNAs through circulating exosomes. By combination of different genetic circuit modules, in vivo assembled siRNAs are systematically distributed to multiple tissues or targeted to specific tissues (e.g., brain), inducing potent target gene silencing in these tissues. The therapeutic value of our strategy is demonstrated by programmed silencing of critical targets associated with various diseases, including EGFR/KRAS in lung cancer, EGFR/TNC in glioblastoma and PTP1B in obesity. Overall, our strategy represents a next generation RNAi therapeutics, which makes RNAi therapy feasible.Subject terms: RNAi, siRNAs     

Influence of different Sinorhizobium meliloti inocula on abundance of genes involved in nitrogen transformations in the rhizosphere of alfalfa (Medicago sativa L.)

Inoculation of leguminous seeds with selected rhizobial strains is practised in agriculture to ameliorate the plant yield by enhanced root nodulation and nitrogen uptake of the plant. However, effective symbiosis between legumes and rhizobia does not only depend on the capacity of nitrogen fixation but also on the entire nitrogen turnover in the rhizosphere. We investigated the influence of seed inoculation with two indigenous Sinorhizobium meliloti strains exhibiting different efficiency concerning plant growth promotion on nitrogen turnover processes in the rhizosphere during the growth of alfalfa. Quantification of six target genes (bacterial amoA, nirK, nirS, nosZ, nifH and archaeal amoA) within the nitrogen cycle was performed in rhizosphere samples before nodule formation, at bud development and at the late flowering stage. The results clearly demonstrated that effectiveness of rhizobial inocula is related to abundance of nifH genes in the late flowering phase of alfalfa. Moreover, other genes involved in nitrogen turnover had been affected by the inocula, e.g. higher numbers of amoA copies were observed during flowering when the more effective strain had been inoculated. However, the respective gene abundances differed overall to a greater extent between the three plant development stages than between the inoculation variants.     

An immunocytochemical investigation with monoclonal antibodies to somatostatin

Summary Four monoclonal antibodies specific for somatostatin have been produced and characterized. These antibodies were used to assess the anatomical relationship of somatostatin-containing cells in the pancreas and gastrointestinal tract of man, baboon and rat with ten other peptide-containing endocrine cells. The peptides investigated were gastrin, cholecystokinin, motilin, secretin, neurotensin, gastric inhibitory polypeptide, gut-glucagon, pancreatic glucagon, pancreatic polypeptide and insulin.The only regions in which somatostatin cells were seen in close contact with another endocrine cell were in the pancreas and the gastric antrum. In the pancreas somatostatin cells were commonly seen in close contact with insulin, glucagon and pancreatic polypeptide cells and infrequent contact was demonstrable with the gastrin-immunoreactive cells in the antrum of both rat and man. In all other cases no evidence was obtained for a close anatomical relationship between somatostatin cells and the other enteroendocrine cells.     

Metformin and its sulphonamide derivative simultaneously potentiateanti-cholinesterase activity of donepezil and inhibit beta-amyloid aggregation

The aim of this study was to assess in vitro the effects of sulphenamide and sulphonamide derivatives of metformin on the activity of human acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), establish the type of inhibition, and assess the potential synergism between biguanides and donepezil towards both cholinesterases (ChEs) and the effects on the β-amyloid aggregation. Sulphonamide 5 with para-trifluoromethyl- and ortho-nitro substituents in aromatic ring inhibited AChE in a mixed-type manner at micromolar concentrations (IC₅₀?=?212.5?±?48.3?µmol/L). The binary mixtures of donepezil and biguanides produce an anti-AChE effect, which was greater than either compound had alone. A combination of donepezil and sulphonamide 5 improved the IC₅₀ value by 170 times. Compound 5 at 200?µmol/L inhibited Aβ aggregation by ～20%. In conclusion, para-trifluoromethyl-ortho-nitro-benzenesulphonamide presents highly beneficial anti-AChE and anti-Aβ aggregation properties which could serve as a promising starting point for the design and development of novel biguanide-based candidates for AD treatment.     

Ancient pathogen DNA in human teeth and petrous bones

Recent ancient DNA (aDNA) studies of human pathogens have provided invaluable insights into their evolutionary history and prevalence in space and time. Most of these studies were based on DNA extracted from teeth or postcranial bones. In contrast, no pathogen DNA has been reported from the petrous bone which has become the most desired skeletal element in ancient DNA research due to its high endogenous DNA content. To compare the potential for pathogenic aDNA retrieval from teeth and petrous bones, we sampled these elements from five ancient skeletons, previously shown to be carrying Yersinia pestis. Based on shotgun sequencing data, four of these five plague victims showed clearly detectable levels of Y. pestis DNA in the teeth, whereas all the petrous bones failed to produce Y. pestis DNA above baseline levels. A broader comparative metagenomic analysis of teeth and petrous bones from 10 historical skeletons corroborated these results, showing a much higher microbial diversity in teeth than petrous bones, including pathogenic and oral microbial taxa. Our results imply that although petrous bones are highly valuable for ancient genomic analyses as an excellent source of endogenous DNA, the metagenomic potential of these dense skeletal elements is highly limited. This trade‐off must be considered when designing the sampling strategy for an aDNA project.     

Antrodia gossypium,Phlebiopsis gigantea and Heterobasidion parviporum: in vitro growth and Norway spruce wood block decay

Polymerase chain reaction-amplified and sequenced isolates of Antrodia gossypium, Phlebiopsis gigantea and Heterobasidion parviporum from decaying Norway spruce wood blocks after three and six months, which exhibited linear growth, were investigated. P. gigantea strains showed the fastest growth, whereas A. gossypium growth was five times slower. The differences between the mean daily increment of A. gossypium and the other examined isolates (except Hp2) were statistically significant. There were also significant differences in wood decay between densities over time. These results were confirmed by the decay acceleration index (DAI) and decay activity index, which were positively correlated with wood density regardless of the fungus species. The registered P. gigantea strains (Rotstop and PG Suspension) exhibited a strong decomposition ability (28% after six months); the weight loss caused by A. gossypium after six months of decay (15.2%) was similar to the results of P. gigantea (GB) after just three months (13.2%). All tested H. parviporum isolates showed rather rapid growth and equally strong wood decay (20–25%) compared to those of P. gigantea. DAI showed that A. gossypium may significantly contribute to wood decomposition over time, particularly in less dense wood samples. The use of both saprotrophs as biological agents against root pathogens is discussed.     

Neurological effects of recombinant human interferon.

Ten women with advanced locally recurrent breast cancer who had failed to respond to radiation and hormonal and cytotoxic agents were given up to 12 weeks of recombinant leucocyte interferon 20 X 10(6) U/m2 daily or 50 X 10(6) U/m2 three times a week. Within one hour of administration influenza-like symptoms began, which one week later were superseded by lethargy, anorexia, and nausea, with a consequent loss of weight in most patients. Other side effects included profound somnolence, confusion, paraesthesia, and (in one patient) signs of an upper motor neurone lesion in the legs. All these effects together with increased slow wave activity in electroencephalograms from all patients during treatment disappeared when interferon was withdrawn and did not recur on reintroducing the drug at a lower dosage. Studies are continuing to determine the mechanisms of these effects.     

Targeting an Essential GTPase Obg for the Development of Broad-Spectrum Antibiotics

A promising new drug target for the development of novel broad-spectrum antibiotics is the highly conserved small GTPase Obg (YhbZ, CgtA), a protein essential for the survival of all bacteria including Neisseria gonorrhoeae (GC). GC is the agent of gonorrhea, a prevalent sexually transmitted disease resulting in serious consequences on reproductive and neonatal health. A preventive anti-gonorrhea vaccine does not exist, and options for effective antibiotic treatments are increasingly limited. To address the dire need for alternative antimicrobial strategies, we have designed and optimized a 384-well GTPase assay to identify inhibitors of Obg using as a model Obg protein from GC, Obg_GC. The assay was validated with a pilot screen of 40,000 compounds and achieved an average Z’ value of 0.58 ± 0.02, which suggests a robust assay amenable to high-throughput screening. We developed secondary assessments for identified lead compounds that utilize the interaction between Obg_GC and fluorescent guanine nucleotide analogs, mant-GTP and mant-GDP, and an Obg_GC variant with multiple alterations in the G-domains that prevent nucleotide binding. To evaluate the broad-spectrum potential of Obg_GC inhibitors, Obg proteins of Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus were assessed using the colorimetric and fluorescence-based activity assays. These approaches can be useful in identifying broad-spectrum Obg inhibitors and advancing the therapeutic battle against multidrug resistant bacteria.     

Genome-resolved metagenomics to study co-occurrence patterns and intraspecific heterogeneity among plant pathogen metapopulations

Assessment of pathogen diversity in agricultural fields is essential for informing management decisions and the development of resistant plant varieties. However, many population genomic studies have relied on culture-based approaches that do not provide quantitative assessment of pathogen populations at the field-level or the associated host microbiome. Here, we applied whole-genome shotgun sequencing of microbial DNA extracted directly from the washings of pooled leaf samples, collected from individual tomato and pepper fields in Alabama that displayed the classical symptoms of bacterial spot disease caused by Xanthomonas spp. Our results revealed that while the occurrence of both X. perforans and X. euvesicatoria within fields was limited, evidence of co-occurrence of up to three distinct X. perforans genotypes was obtained in 7 of 10 tomato fields sampled. These population dynamics were accompanied by the corresponding type 3 secreted effector repertoires associated with the co-occurring X. perforans genotypes, indicating that metapopulation structure within fields should be considered when assessing the adaptive potential of X. perforans. Finally, analysis of microbial community composition revealed that co-occurrence of the bacterial spot pathogens Pseudomonas cichorii and Xanthomonas spp. is common in Alabama fields and provided evidence for the non-random association of several other human and plant opportunists.