A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation

Iron absorption by the duodenal mucosa is initiated by uptake of ferrous Fe(II) iron across the brush border membrane and culminates in transfer of the metal across the basolateral membrane to the portal vein circulation by an unknown mechanism. We describe here the isolation and characterization of a novel cDNA (Ireg1) encoding a duodenal protein that is localized to the basolateral membrane of polarized epithelial cells. Ireg1 mRNA and protein expression are increased under conditions of increased iron absorption, and the 5' UTR of the Ireg1 mRNA contains a functional iron-responsive element (IRE). IREG1 stimulates iron efflux following expression in Xenopus oocytes. We conclude that IREG1 represents the long-sought duodenal iron export protein and is upregulated in the iron overload disease, hereditary hemochromatosis.     

Continent-wide tracking to determine migratory connectivity and tropical habitat associations of a declining aerial insectivore

North American birds that feed on flying insects are experiencing steep population declines, particularly long-distance migratory populations in the northern breeding range. We determine, for the first time, the level of migratory connectivity across the range of a songbird using direct tracking of individuals, and test whether declining northern populations have higher exposure to agricultural landscapes at their non-breeding grounds in South America. We used light-level geolocators to track purple martins, Progne subis, originating from North American breeding populations, coast-to-coast (n = 95 individuals). We show that breeding populations of the eastern subspecies, P. s. subis, that are separated by ca. 2000 km, nevertheless have almost completely overlapping non-breeding ranges in Brazil. Most (76%) P. s. subis overwintered in northern Brazil near the Amazon River, not in the agricultural landscape of southern Brazil. Individual non-breeding sites had an average of 91 per cent forest and only 4 per cent agricultural ground cover within a 50 km radius, and birds originating from declining northern breeding populations were not more exposed to agricultural landscapes than stable southern breeding populations. Our results show that differences in wintering location and habitat do not explain recent trends in breeding population declines in this species, and instead northern populations may be constrained in their ability to respond to climate change.     

Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.     

Wnt3 signaling in the epiblast is required for proper orientation of the anteroposterior axis

The establishment of anteroposterior (AP) polarity in the early mouse epiblast is crucial for the initiation of gastrulation and the subsequent formation of the embryonic (head to tail) axis. The localization of anterior and posterior determining genes to the appropriate region of the embryo is a dynamic process that underlies this early polarity. Several studies indicate that morphological and molecular markers which define the early AP axis are first aligned along the short axis of the elliptical egg cylinder. Subsequently, just prior to the time of primitive streak formation, a conformational change in the embryo realigns these markers with the long axis. We demonstrate that embryos lacking the signaling factor Wnt3 exhibit defects in this axial realignment. In addition, chimeric analyses and conditional removal of Wnt3 activity reveal that Wnt3 expression in the epiblast is required for induction of the primitive streak and mesoderm whereas activity in the posterior visceral endoderm is dispensable.     

Identification of putative ancestors of the multidrug-resistant <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar typhimurium DT104 clone harboring the<Emphasis Type="Italic"> Salmonella</Emphasis> genomic island 1

The origin of multidrug-resistant Salmonella enterica serovar typhimurium (S. typhimurium) harboring the Salmonella Genomic Island 1 (SGI1), which was detected for the first time in the mid-1980s is unknown. In this study, we performed microarray genomotyping of four multidrug-resistant SGI1 positive strains and found that unlike the S. typhimurium LT2 strain, the multidrug-resistant strains lacked genes STM0517-0529 allowing the utilization of allantoin as a sole nitrogen source. We extended this observation by PCR screening of additional 120 S. typhimurium field strains and found that this locus was absent in all SGI1 positive and also in 24% of SGI1 negative strains, which were proposed to be the original recipients of SGI1. To prove this hypothesis, we compared the STM0517-0529 negative strains (with or without the SGI1) by PFGE and PCR prophage typing and found that 8 out of 11 of the SGI1 negative strains and 17 out of 22 SGI1 positive strains were of identical PFGE pattern and PCR prophage pattern, while this specific pattern was never observed among STM0517-0529 positive strains. We therefore propose that a lineage of the S. typhimurium DT104 sensitive strain first lost the ability to metabolize allantoin and then acquired SGI1.     

Spatial and temporal variation in malaria transmission in a low endemicity area in northern Tanzania

Background

Current detection or screening for malaria infection necessitates drawing blood by fingerprick or venipuncture, which poses risks and limitations for repeated measurement. This study presents PCR detection of Plasmodium falciparum in human urine and saliva samples, and illustrates this potential application in genotyping malaria infections.

Methods

Urine and saliva were obtained from 47 thick film positive and 4 negative individuals one day after collection of blood slides and filter paper blood spots. P. falciparum DNA was extracted from blood, urine and saliva, in separate groups, using the Chelex method or Qiagen DNEasy^® kit (urine and saliva only). Blood, urine and saliva extracts were subjected to PCR in separate batches. Amplicons from the various sample types were examined for MSP2 polymorphisms and restriction fragment patterns on DHFR amino acid codon 59.

Results and discussion

Malaria infections exhibited primarily low-grade parasite densities, with a geometric mean of 775 asexual parasites/μl. Regularly matching polymorphic MSP2 genotypes were found between the corresponding urine, saliva and peripheral blood amplicons of each individual, with different inter-individual polymorphic genotypes. Amplicon yields were significantly dependent on DNA extraction method, parasite density and primer set (p < 0.001). A Qiagen^® kit extraction had more than 2× higher amplicon yield than the Chelex method, for both urine and saliva. Amplicon yields were 1.6 fold higher from saliva than urine. For each unit increase in log parasite density, the probability of amplicon enhanced 1.8 fold. Highest amplicon yields were obtained from the primer set with the shortest PCR product.

Conclusion

P. falciparum infection is detectable by PCR on human urine and saliva samples. Subject to further refinement of extraction technique and amplicon yields, large-scale malaria parasite screening and epidemiological surveys could be possible without the need to collect blood and use of needles or sharps.     

Vasoactive intestinal peptide (VIP) prevents killing of virulent and phoP mutant Salmonella typhimurium by inhibiting IFN-gamma stimulated NADPH oxidative pathways in murine macrophages

Vasoactive intestinal peptide is an immunomodulator with great potential in the treatment of inflammatory pathology. In this study, we have examined the effect of VIP on the growth dynamics of virulent Salmonella enterica. Serovar typhimurium (S. typhimurium) 14028 and 4/74 and an avirulent mutant (14028 phoP) in a murine, macrophage cell line (J774.2). In contrast to standard growth dynamics, in which phoP mutants do not survive in macrophages, we show that VIP (10(-10) M) significantly enhances phoP growth over a 24 h post-infection period even when the cells are co-cultured with IFN-gamma. We examined the effect of VIP on the generation of NADPH-induced reactive oxygen species (ROS) in Salmonella-infected/IFN-gamma cultured J774 cells. VIP inhibited gp91 mRNA levels, gp91 protein and subsequent ROS. The importance of ROS in killing of Salmonella by J774 cells was highlighted by experiments in which ROS production by J774 cells was inhibited using a conventional inhibitor, N-acetyl-L-cysteine captopril (ACC) and in which Salmonella growth significantly increased. Our findings suggest that although VIP inhibits inflammatory pathways in myeloid cells it also promotes the growth of avirulent (phoP) mutants.     

Wnt/PCP signaling: a veritable polar star in establishing patterns of polarity in embryonic tissues

Embryonic patterning has traditionally been viewed as the establishment of spatially significant gene expression in response to secreted signals. Recent work has highlighted the role of the Wnt/planar cell polarity (PCP) pathway in patterning tissues. Rather than establishing characteristic arrays of gene expression, however, this pathway functions to institute uniform polarity of cells within a tissue. Cells thus polarized can undergo directed migrations, cell divisions, etc., which are essential for normal morphogenesis. In this review, I will highlight the similarities between mechanisms that establish patterns of polarity between Drosophila and vertebrates. Further, I will discuss recent advances with regard to Wnt/PCP signaling in vertebrates.     

BIGSdb: Scalable analysis of bacterial genome variation at the population level

Background  

The opportunities for bacterial population genomics that are being realised by the application of parallel nucleotide sequencing require novel bioinformatics platforms. These must be capable of the storage, retrieval, and analysis of linked phenotypic and genotypic information in an accessible, scalable and computationally efficient manner.     

Positive Allosteric Interaction of Structurally Diverse T-Type Calcium Channel Antagonists

Low-voltage-activated (T-type) calcium channels play a role in diverse physiological responses including neuronal burst firing, hormone secretion, and cell growth. To better understand the biological role and therapeutic potential of the target, a number of structurally diverse antagonists have been identified. Multiple drug interaction sites have been identified for L-type calcium channels, suggesting a similar possibility exists for the structurally related T-type channels. Here, we radiolabel a novel amide T-type calcium channel antagonist (TTA-A1) and show that several known antagonists, including mibefradil, flunarizine, and pimozide, displace binding in a concentration-dependent manner. Further, we identify a novel quinazolinone T-type antagonist (TTA-Q4) that enhanced amide radioligand binding, increased affinity in a saturable manner and slowed dissociation. Functional evaluation showed these compounds to be state-dependent antagonists which show a positive allosteric interaction. Consistent with slowing dissociation, the duration of efficacy was prolonged when compounds were co-administered to WAG/Rij rats, a genetic model of absence epilepsy. The development of a T-type calcium channel radioligand has been used to demonstrate structurally distinct TTAs interact at allosteric sites and to confirm the potential for synergistic inhibition of T-type calcium channels with structurally diverse antagonists.