Differentiating Ovine BSE from CH1641 Scrapie by Serial Protein Misfolding Cyclic Amplification

Whilst ovine BSE displays distinct pathological characteristics to ovine CH1641-like scrapie upon passage in rodents, they have very similar molecular phenotypes. As such, the in vitro differentiation of these strains in routine surveillance programmes presents a significant diagnostic challenge. In this study, using serial protein-misfolding cyclic amplification (sPMCA), ovine BSE was readily amplified in vitro in brain substrates from sheep with V???R???Q???/V???R???Q??? or AHQ/AHQ PRNP genotypes. In contrast, the CH1641 strain was refractory to such amplification. This method allowed for complete and unequivocal differentiation of experimental BSE from CH1641 prion strains within an ovine host.     

Epidermal and cortical roles of NFP and DMI3 in coordinating early steps of nodulation in Medicago truncatula

Legumes have evolved the capacity to form a root nodule symbiosis with soil bacteria called rhizobia. The establishment of this symbiosis involves specific developmental events occurring both in the root epidermis (notably bacterial entry) and at a distance in the underlying root cortical cells (notably cell divisions leading to nodule organogenesis). The processes of bacterial entry and nodule organogenesis are tightly linked and both depend on rhizobial production of lipo-chitooligosaccharide molecules called Nod factors. However, how these events are coordinated remains poorly understood. Here, we have addressed the roles of two key symbiotic genes of Medicago truncatula, the lysin motif (LysM) domain-receptor like kinase gene NFP and the calcium- and calmodulin-dependent protein kinase gene DMI3, in the control of both nodule organogenesis and bacterial entry. By complementing mutant plants with corresponding genes expressed either in the epidermis or in the cortex, we have shown that epidermal DMI3, but not NFP, is sufficient for infection thread formation in root hairs. Epidermal NFP is sufficient to induce cortical cell divisions leading to nodule primordia formation, whereas DMI3 is required in both cell layers for these processes. Our results therefore suggest that a signal, produced in the epidermis under the control of NFP and DMI3, is responsible for activating DMI3 in the cortex to trigger nodule organogenesis. We integrate these data to propose a new model for epidermal/cortical crosstalk during early steps of nodulation.     

Cancer-related mutations in BRCA1-BRCT cause long-range structural changes in protein-protein binding sites: a molecular dynamics study

Cancer-associated mutations in the BRCT domain of BRCA1 (BRCA1-BRCT) abolish its tumor suppressor function by disrupting interactions with other proteins such as BACH1. Many cancer-related mutations do not cause sufficient destabilization to lead to global unfolding under physiological conditions, and thus abrogation of function probably is due to localized structural changes. To explore the reasons for mutation-induced loss of function, the authors performed molecular dynamics simulations on three cancer-associated mutants, A1708E, M1775R, and Y1853ter, and on the wild type and benign M1652I mutant, and compared the structures and fluctuations. Only the cancer-associated mutants exhibited significant backbone structure differences from the wild-type crystal structure in BACH1-binding regions, some of which are far from the mutation sites. Backbone differences of the A1708E mutant from the liganded wild type structure in these regions are much larger than those of the unliganded wild type X-ray or molecular dynamics structures. These BACH1-binding regions of the cancer-associated mutants also exhibited increases in their fluctuation magnitudes compared with the same regions in the wild type and M1562I mutant, as quantified by quasiharmonic analysis. Several of the regions of increased fluctuation magnitude correspond to correlated motions of residues in contact that provide a continuous path of fluctuating amino acids in contact from the A1708E and Y1853ter mutation sites to the BACH1-binding sites with altered structure and dynamics. The increased fluctuations in the disease-related mutants suggest an increase in vibrational entropy in the unliganded state that could result in a larger entropy loss in the disease-related mutants upon binding BACH1 than in the wild type. To investigate this possibility, vibrational entropies of the A1708E and wild type in the free state and bound to a BACH1-derived phosphopeptide were calculated using quasiharmonic analysis, to determine the binding entropy difference DeltaDeltaS between the A1708E mutant and the wild type. DeltaDeltaS was determined to be -4.0 cal mol(-1) K(-1), with an uncertainty of 2 cal mol(-1) K(-1); that is, the entropy loss upon binding the peptide is 4.0 cal mol(-1) K(-1) greater for the A1708E mutant, corresponding to an entropic contribution to the DeltaDeltaG of binding (-TDeltaDeltaS) 1.1 kcal mol(-1) more positive for the mutant. The observed differences in structure, flexibility, and entropy of binding likely are responsible for abolition of BACH1 binding, and illustrate that many disease- related mutations could have very long-range effects. The methods described here have potential for identifying correlated motions responsible for other long-range effects of deleterious mutations.     

An In Vitro Cord Formation Assay Identifies Unique Vascular Phenotypes Associated with Angiogenic Growth Factors

Vascular endothelial growth factor (VEGF) plays a dominant role in angiogenesis. While inhibitors of the VEGF pathway are approved for the treatment of a number of tumor types, the effectiveness is limited and evasive resistance is common. One mechanism of evasive resistance to inhibition of the VEGF pathway is upregulation of other pro-angiogenic factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF). Numerous in vitro assays examine angiogenesis, but many of these assays are performed in media or matrix with multiple growth factors or are driven by VEGF. In order to study angiogenesis driven by other growth factors, we developed a basal medium to use on a co-culture cord formation system of adipose derived stem cells (ADSCs) and endothelial colony forming cells (ECFCs). We found that cord formation driven by different angiogenic factors led to unique phenotypes that could be differentiated and combination studies indicate dominant phenotypes elicited by some growth factors. VEGF-driven cords were highly covered by smooth muscle actin, and bFGF-driven cords had thicker nodes, while EGF-driven cords were highly branched. Multiparametric analysis indicated that when combined EGF has a dominant phenotype. In addition, because this assay system is run in minimal medium, potential proangiogenic molecules can be screened. Using this assay we identified an inhibitor that promoted cord formation, which was translated into in vivo tumor models. Together this study illustrates the unique roles of multiple anti-angiogenic agents, which may lead to improvements in therapeutic angiogenesis efforts and better rational for anti-angiogenic therapy.     

The E1 copper binding domain of full-length amyloid precursor protein mitigates copper-induced growth inhibition in brain metastatic prostate cancer DU145 cells

Copper plays an important role in the aetiology and growth of tumours and levels of the metal are increased in the serum and tumour tissue of patients affected by a range of cancers including prostate cancer (PCa). The molecular mechanisms that enable cancer cells to proliferate in the presence of elevated copper levels are, therefore, of key importance in our understanding of tumour growth progression. In the current study, we have examined the role played by the amyloid precursor protein (APP) in mitigating copper-induced growth inhibition of the PCa cell line, DU145. A range of APP molecular constructs were stably over-expressed in DU145 cells and their effects on cell proliferation in the presence of copper were monitored. Our results show that endogenous APP expression was induced by sub-toxic copper concentrations in DU145 cells and over-expression of the wild-type protein was able to mitigate copper-induced growth inhibition via a mechanism involving the cytosolic and E1 copper binding domains of the full-length protein. APP likely represents one of a range of copper binding proteins that PCa cells employ in order to ensure efficient proliferation despite elevated concentrations of the metal within the tumour microenvironment. Targeting the expression of such proteins may contribute to therapeutic strategies for the treatment of cancers.     

Silver spoons in the rough: can environmental enrichment improve survival of hatchery Atlantic salmon Salmo salar in the wild?

This study tested the ‘silver spoon’ hypothesis which posits that individuals that develop under favourable conditions should enjoy a fitness advantage later in life because they are more likely to recognize and settle in high‐quality habitats. Atlantic salmon Salmo salar of two age classes (0+ and 1+ years) were reared in environmentally enriched or standard hatchery tanks for a short period (c. 10 weeks), were then released into a natural river and sampled on repeated occasions to test for silver‐spoon effects. Compared with controls, enriched fish had a 6·4% higher recapture rate and settled in higher velocity habitats when they were stocked as 0+ year fry, but not when they were stocked as 1+ year parr. The opportunity for selection was generally higher for environmentally enriched fish than for controls, and also higher for 0+ than for 1+ year fish. Selection favoured individuals with high condition factor, extensive fat reserves and longer than average pectoral fins in both age classes but favoured a small body size in 1+ year and a large body size in 0+ year releases. Stomach analysis showed that enriched fish ate more, and adapted quicker to natural prey than controls. These results provide support for silver‐spoon effects in fish and indicate that enrichment can improve post‐release performance in conservation programmes, but seemingly only if fish are not kept in captivity for too long.     

Prions Are Secreted in Milk from Clinically Normal Scrapie-Exposed Sheep

The potential spread of prion infectivity in secreta is a crucial concern for prion disease transmission. Here, serial protein misfolding cyclic amplification (sPMCA) allowed the detection of prions in milk from clinically affected animals as well as scrapie-exposed sheep at least 20 months before clinical onset of disease, irrespective of the immunohistochemical detection of protease-resistant PrP^Sc within lymphoreticular and central nervous system tissues. These data indicate the secretion of prions within milk during the early stages of disease progression and a role for milk in prion transmission. Furthermore, the application of sPMCA to milk samples offers a noninvasive methodology to detect scrapie during preclinical/subclinical disease.PrP^Sc, a disease-specific marker for prion diseases and the likely infectious agent, is widely distributed within the central nervous system (CNS) and lymphoreticular tissues (LRS) in ovine scrapie, human variant Creutzfeldt-Jakob disease (vCJD), and cervine chronic wasting disease (CWD) during both clinical and preclinical stages (4, 11, 25). Furthermore, while the LRS distribution of PrP^Sc is much more restricted in bovine spongiform encephalopathy (BSE), sheep experimentally infected with BSE display a PrP^Sc tissue distribution more akin to that of ovine scrapie (11).For rodent-adapted scrapie and cervine CWD, the disease agent is detected in excreta when animals are in the clinical stages of disease, a process likely to contribute to environmental reservoirs of infectivity and lateral disease transmission (5, 13, 21). Within an experimental rodent model, it has also been demonstrated that the shedding of PrP^Sc and concomitant infectivity in feces occurs during preclinical scrapie (21).Evidence now also demonstrates that milk provides a vehicle for the transmission for prion diseases. Scrapie-free lambs fed milk from clinical scrapie-affected ewes propagate PrP^Sc within their LRS (8). Additionally, a recent study using a transgenic mouse bioassay demonstrated the secretion of infectivity in milk from preclinical animals where scrapie infectivity was found in milk months before the onset of clinical signs in animals with an ARQ/VRQ PrP genotype (10). The presence of scrapie infectivity within milk was irrespective of mammary gland pathology or PrP^Sc accumulation, and these animals were estimated to have considerable accumulation of immunohistochemically (IHC) detectable PrP^Sc within the LRS at the time of sampling.Here, we applied serial protein misfolding cyclic amplification (sPMCA) to the in vitro detection of PrP^Sc within sheep milk (Fig. ​(Fig.1)1) (Table ​(Table11).Open in a separate windowFIG. 1.sPMCA analysis of ovine milk samples. Milk was clarified and seeded into brain homogenate from sheep unexposed to the scrapie agent. Samples underwent sPMCA, and products were digested with proteinase K before analysis of 10 μl of each sample. PrP was detected with monoclonal antibodies SHA31 and P4; molecular mass markers are indicated (kDa). Milk was sampled from animals not exposed to the scrapie agent (U), those displaying clinical signs of scrapie (C), or those exposed to a scrapie-positive farm environment but not displaying clinical disease (S). NS, non-seeded PMCA brain substrate subjected to identical sPMCA conditions at the same time as positive samples were analyzed. Samples from the four nonexposed animals were analyzed 18 to 20 times each by sPMCA. Samples from clinically affected or clinically normal scrapie-exposed animals were analyzed in triplicate. For this triplicate analysis of each sample, the sPMCA round at which samples became positive is indicated under the appropriate lane. n, negative at round 12. Each sample was PrP^Sc negative until the stated round and thereafter was positive.

TABLE 1.

Timeline of exposure of animals to a scrapie-positive farm environment, sample collection, and scrapie status

A phylogenomic profile of globins

Background

Globins occur in all three kingdoms of life: they can be classified into single-domain globins and chimeric globins. The latter comprise the flavohemoglobins with a C-terminal FAD-binding domain and the gene-regulating globin coupled sensors, with variable C-terminal domains. The single-domain globins encompass sequences related to chimeric globins and «truncated» hemoglobins with a 2-over-2 instead of the canonical 3-over-3 α-helical fold.

Results

A census of globins in 26 archaeal, 245 bacterial and 49 eukaryote genomes was carried out. Only ~25% of archaea have globins, including globin coupled sensors, related single domain globins and 2-over-2 globins. From one to seven globins per genome were found in ~65% of the bacterial genomes: the presence and number of globins are positively correlated with genome size. Globins appear to be mostly absent in Bacteroidetes/Chlorobi, Chlamydia, Lactobacillales, Mollicutes, Rickettsiales, Pastorellales and Spirochaetes. Single domain globins occur in metazoans and flavohemoglobins are found in fungi, diplomonads and mycetozoans. Although red algae have single domain globins, including 2-over-2 globins, the green algae and ciliates have only 2-over-2 globins. Plants have symbiotic and nonsymbiotic single domain hemoglobins and 2-over-2 hemoglobins. Over 90% of eukaryotes have globins: the nematode Caenorhabditis has the most putative globins, ~33. No globins occur in the parasitic, unicellular eukaryotes such as Encephalitozoon, Entamoeba, Plasmodium and Trypanosoma.

Conclusion

Although Bacteria have all three types of globins, Archaeado not have flavohemoglobins and Eukaryotes lack globin coupled sensors. Since the hemoglobins in organisms other than animals are enzymes or sensors, it is likely that the evolution of an oxygen transport function accompanied the emergence of multicellular animals.     

Genomic scale sub-family assignment of protein domains

Many classification schemes for proteins and domains are either hierarchical or semi-hierarchical yet most databases, especially those offering genome-wide analysis, only provide assignments to sequences at one level of their hierarchy. Given an established hierarchy, the problem of assigning new sequences to lower levels of that existing hierarchy is less hard (but no less important) than the initial top level assignment which requires the detection of the most distant relationships. A solution to this problem is described here in the form of a new procedure which can be thought of as a hybrid between pairwise and profile methods. The hybrid method is a general procedure that can be applied to any pre-defined hierarchy, at any level, including in principle multiple sub-levels. It has been tested on the SCOP classification via the SUPERFAMILY database and performs significantly better than either pairwise or profile methods alone. Perhaps the greatest advantage of the hybrid method over other possible approaches to the problem is that within the framework of an existing profile library, the assignments are fully automatic and come at almost no additional computational cost. Hence it has already been applied at the SCOP family level to all genomes in the SUPERFAMILY database, providing a wealth of new data to the biological and bioinformatics communities.