A Lentiviral Gene Therapy Strategy for the In Vitro Production of Feline Erythropoietin

Nonregenerative anemia due to chronic renal failure is a common problem in domestic cats. Unfortunately, administration of recombinant human erythropoietin often only improves anemia temporarily due to antibody development. In this in vitro study, feline erythropoietin cDNA was cloned from feline renal tissue and utilized in the construction of a replication-defective lentiviral vector. The native recombinant feline erythropoietin (rfEPO) sequence was confirmed by sequencing. Upon viral vector infection of human 293H cells, Crandall Renal Feline Kidney cell line and primary feline peripheral blood mononuclear cells, bioactive rfEPO protein was produced. The presence of cellular rfEPO cDNA was confirmed by standard PCR, production of abundant rfEPO mRNA was confirmed by real-time PCR, and secretion of rfEPO protein was demonstrated by Western blot analyses, while rfEPO protein bioactivity was confirmed via an MTT proliferation bioassay. This in vitro study demonstrates the feasibility of a replication-defective lentiviral vector delivery system for the in vitro production of biologically active feline erythropoietin. Anemic cats with chronic renal failure represent a potential in vivo application of a lentiviral gene therapy system.     

Methyl-compounds driven benthic carbon cycling in the sulfate-reducing sediments of South China Sea

Methane is a potent greenhouse gas; methane production and consumption within seafloor sediments has generated intense interest. Anaerobic oxidation of methane (AOM) and methanogenesis (MOG) primarily occur at the depth of the sulfate–methane transition zone or underlying sediment respectively. Methanogenesis can also occur in the sulfate-reducing sediments through the utilization of non-competitive methylated compounds; however, the occurrence and importance of this process are not fully understood. Here, we combined a variety of data, including geochemical measurements, rate measurements and molecular analyses to demonstrate the presence of a cryptic methane cycle in sulfate-reducing sediments from the continental shelf of the northern South China Sea. The abundance of methanogenic substrates as well as the high MOG rates from methylated compounds indicated that methylotrophic methanogenesis was the dominant methanogenic pathway; this conclusion was further supported by the presence of the methylotrophic genus Methanococcoides. High potential rates of AOM were observed in the sediments, indicating that methane produced in situ could be oxidized simultaneously by AOM, presumably by ANME-2a/b as indicated by 16S rRNA gene analysis. A significant correlation between the relative abundance of methanogens and methanotrophs was observed over sediment depth, indicating that methylotrophic methanogenesis could potentially fuel AOM in this environment. In addition, higher potential rates of AOM than sulfate reduction rates at in situ methane conditions were observed, making alternative electron acceptors important to support AOM in sulfate-reducing sediment. AOM rates were stimulated by the addition of Fe/Mn oxides, suggesting AOM could be partially coupled to metal oxide reduction. These results suggest that methyl-compounds driven methane production drives a cryptic methane cycling and fuels AOM coupled to the reduction of sulfate and other electron acceptors.     

Intraspecific genetic variations, fitness cost and benefit of RPW8, a disease resistance locus in Arabidopsis thaliana

The RPW8 locus of Arabidopsis thaliana confers broad-spectrum resistance to powdery mildew pathogens. In many A. thaliana accessions, this locus contains two homologous genes, RPW8.1 and RPW8.2. In some susceptible accessions, however, these two genes are replaced by HR4, a homolog of RPW8.1. Here, we show that RPW8.2 from A. lyrata conferred powdery mildew resistance in A. thaliana, suggesting that RPW8.2 might have gained the resistance function before the speciation of A. thaliana and A. lyrata. To investigate how RPW8 has been maintained in A. thaliana, we examined the nucleotide sequence polymorphisms in RPW8 from 51 A. thaliana accessions, related disease reaction phenotypes to the evolutionary history of RPW8.1 and RPW8.2, and identified mutations that confer phenotypic variations. The average nucleotide diversities were high at RPW8.1 and RPW8.2, showing no sign of selective sweep. Moreover, we found that expression of RPW8 incurs fitness benefits and costs on A. thaliana in the presence and absence of the pathogens, respectively. Our results suggest that polymorphisms at the RPW8 locus in A. thaliana may have been maintained by complex selective forces, including those from the fitness benefits and costs both associated with RPW8.     

Ten di- and trinucleotide microsatellite loci in the Caribbean spiny lobster, Panulirus argus, for studies of regional population connectivity

We describe 10 microsatellite loci for Panulirus argus (Caribbean spiny lobster). The number of alleles at each locus ranged from four to 39 (mean = 21.8) in 89 juvenile specimens collected at two different times at a recruitment site in south Florida. Levels of expected and observed heterozygosities ranged from 0.48 to 0.96 (mean = 0.83) and from 0.32 to 0.98 (mean = 0.71), respectively. Significant departures from Hardy–Weinberg equilibrium were observed at two loci. There was no evidence of genotypic disequilibrium for any pair of loci. Overall, the loci were well resolved, highly polymorphic and independently segregating, confirming their utility for population genetic studies.     

Identification of an iron-hepcidin complex

Following its identification as a liver-expressed antimicrobial peptide, the hepcidin peptide was later shown to be a key player in iron homoeostasis. It is now proposed to be the 'iron hormone' which, by interacting with the iron transporter ferroportin, prevents further iron import into the circulatory system. This conclusion was reached using the corresponding synthetic peptide, emphasizing the functional importance of the mature 25-mer peptide, but omitting the possible functionality of its maturation. From urine-purified native hepcidin, we recently demonstrated that a proportion of the purified hepcidin had formed iron-hepcidin complexes. This interaction was investigated further by computer modelling and, based on the sequence similarity of hepcidin with metallothionein, a three-dimensional model of hepcidin, containing one atom of iron, was constructed. To characterize these complexes further, the interaction with iron was analysed using different spectroscopic methods. Monoferric hepcidin was identified by MS, as were possibly other complexes containing two and three atoms of iron respectively, although these were present only in minor amounts. UV/visible absorbance and CD studies identified the iron-binding events which were facilitated at a physiological pH. EPR spectroscopy identified the ferric state of the bound metal, and indicated that the iron-hepcidin complex shares some similarities with the rubredoxin iron-sulfur complex, suggesting the presence of Fe(3+) in a tetrahedral sulfur co-ordination. The potential roles of iron binding for hepcidin are discussed, and we propose either a regulatory function in the maturation of pro-hepcidin into active hepcidin or as the necessary link in the interaction between hepcidin and ferroportin.     

High diversity of pipistrelloid bats (Vespertilionidae: Hypsugo,Neoromicia, and Pipistrellus) in a West African rainforest with the description of a new species

The pipistrelloid bats (genera Hypsugo, Neoromicia, and Pipistrellus) of Africa have been poorly studied, partly as a result of problems associated with species identification. This paper examines the diversity of pipistrelloid bats from Mount Nimba, a biodiversity hotspot in the Upper Guinean rainforest zone. Traditional morphometrics, the structure of the baculum, and sequences of the cytochrome oxidase subunit I (COI) gene were used to identify taxa. Species richness was exceptionally high and included at least ten taxa identifiable on molecular grounds. Of these, existing names could be assigned to six taxa. A seventh taxon was described as a species new to science, Neoromicia roseveari sp. nov. , and was distinguished on molecular grounds, craniodental morphology, and baculum structure. The remaining taxa may refer to as‐yet undescribed species but we lacked sufficient material to formally describe them here. The high species richness of pipistrelloid bats on Mount Nimba may be associated with the transition zone from lowland rainforest to moist savannah. © 2013 The Linnean Society of London