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Cellular metabolism and behaviour is closely linked to cytoskeletal tension and scaffold mechanics. In the developing nervous system functional connectivity is controlled by the interplay between chemical and mechanical cues that initiate programs of cell behaviour. Replication of functional connectivity in neuronal populations in vitro has proven a technical challenge due to the absence of many systems of biomechanical regulation that control directional outgrowth in vivo. Here, a 3D culture system is explored by dilution of a type I collagen hydrogel to produce variation in gel stiffness. Hydrogel scaffold remodelling was found to be linked to gel collagen concentration, with a greater degree of gel contraction occurring at lower concentrations. Gel mechanics were found to evolve over the culture period according to collagen concentration. Less concentrated gels reduced in stiffness, whilst a biphasic pattern of increasing and then decreasing stiffness was observed at higher concentrations. Analysis of these cultures by PCR revealed a program of shifting integrin expression and highly variable activity in key morphogenic signal pathways, such as mitogen-associated protein kinase, indicating genetic impact of biomaterial interactions via mechano-regulation. Gel contraction at lower concentrations was also found to be accompanied by an increase in average collagen fibre diameter. Minor changes in biomaterial mechanics result in significant changes in programmed cell behaviour, resulting in adoption of markedly different cell morphologies and ability to remodel the scaffold. Advanced understanding of cell-biomaterial interactions, over short and long-term culture, is of critical importance in the development of novel tissue engineering strategies for the fabrication of biomimetic 3D neuro-tissue constructs. Simple methods of tailoring the initial mechanical environment presented to SH-SY5Y populations in 3D can lead to significantly different programs of network development over time.

Background Human bone marrow-derived mesenchymal stem/stromal cells (hBM MSCs) have multiple functions, critical for skeletal formation and function. Their functional heterogeneity, however, represents a major challenge for their isolation and in developing potency and release assays to predict their functionality prior to transplantation. Additionally, potency, biomarker profiles and defining mechanisms of action in a particular clinical setting are increasing requirements of Regulatory Agencies for release of hBM MSCs as Advanced Therapy Medicinal Products for cellular therapies. Since the healing of bone fractures depends on the coupling of new blood vessel formation with osteogenesis, we hypothesised that a correlation between the osteogenic and vascular supportive potential of individual hBM MSC-derived CFU-F (colony forming unit-fibroblastoid) clones might exist. Methods We tested this by assessing the lineage (i.e. adipogenic (A), osteogenic (O) and/or chondrogenic (C)) potential of individual hBM MSC-derived CFU-F clones and determining if their osteogenic (O) potential correlated with their vascular supportive profile in vitro using lineage differentiation assays, endothelial-hBM MSC vascular co-culture assays and transcriptomic (RNAseq) analyses. Results Our results demonstrate that the majority of CFU-F (95%) possessed tri-lineage, bi-lineage or uni-lineage osteogenic capacity, with 64% of the CFU-F exhibiting tri-lineage AOC potential. We found a correlation between the osteogenic and vascular tubule supportive activity of CFU-F clones, with the strength of this association being donor dependent. RNAseq of individual clones defined gene fingerprints relevant to this correlation. Conclusions This study identified a donor-dependent correlation between osteogenic and vascular supportive potential of hBM MSCs and important gene signatures that support these functions that are relevant to their bone regenerative properties. Electronic supplementary material The online version of this article (10.1186/s13287-018-1095-7) contains supplementary material, which is available to authorized users.

Articles free to read on publisher website Hereditary haemochromatosis (HHC) is an autosomal recessive disorder of iron metabolism common in populations of north-west European ancestry. The term haemochromatosis was first used by von Recklinghausen (1889) to describe his post-mortem findings in patients who had died from ‘bronzed diabetes’. The condition was first suggested to be familial by Sheldon (1935), although it was as late as 1975 before the genetic nature of the condition was proven, when Simon et al (1975 ) demonstrated association with the HLA-A3 allele in the major histocompatibility complex (MHC) on chromosome 6...

Hemochromatosis type 4 is an atypical hemochromatosis characterized by dominant inheritance, increased serum ferritin, normal transferrin saturation, and prevalent iron deposition in the reticuloendothelial (RE) cells rather than in hepatocytes. Heterozygous missense mutations of the iron export

Hereditary hemochromatosis is an iron overload disorder that can lead to the impairment of multiple organs and is caused by mutations in one or more different genes. Type 1 hemochromatosis is the most common form of the disease and results from mutations in the HFE gene. Juvenile hemochromatosis (JH) is the most severe form, usually caused by mutations in hemojuvelin (HJV) or hepcidin (HAMP). The autosomal dominant form of the disease, type 4, is due to mutations in the SLC40A1 gene, which encodes for ferroportin (FPN). Hereditary hemochromatosis is commonly found in populations of European origin. By contrast, hemochromatosis in Asia is rare and less well understood and can be masked by the presence of iron deficiency and secondary iron overload from thalassemia. Here, we provide a comprehensive report of hemochromatosis in a group of patients of Asian origin. We have identified novel mutations in HJV, HAMP, and SLC40A1 in countries not normally associated with hereditary hemochromatosis (Pakistan, Bangladesh, Sri Lanka, and Thailand). Our family studies show a high degree of consanguinity, highlighting the increased risk of iron overload in many countries of the developing world and in countries in which there are large immigrant populations from these regions.

Background. Vascular calcification occurs in the majority of patients with chronic kidney disease, but a subset of patients does not develop calcification despite exposure to a similar uraemic environment. Physiological inhibitors of calcification, fetuin-A, osteoprotegerin (OPG) and undercarboxylated-matrix Gla protein (uc-MGP) may play a role in preventing the development and progression of ectopic calcification, but there are scarce and conflicting data from clinical studies.Methods. We measured fetuin-A, OPG and uc-MGP in 61 children on dialysis and studied their associations with clinical, biochemical and vascular measures.Results. Fetuin-A and OPG were higher and uc-MGP lower in dialysis patients than controls. In controls, fetuin-A and OPG increased with age. Fetuin-A showed an inverse correlation with dialysis vintage (P = 0.0013), time-averaged serum phosphate (P = 0.03) and hs-CRP (P = 0.001). Aortic pulse wave velocity (PWV) and augmentation index showed a negative correlation with fetuin-A while a positive correlation was seen with PWV and OPG. Patients with calcification had lower fetuin-A and higher OPG than those without calcification. On multiple linear regression analysis Fetuin-A independently predicted aortic PWV (P = 0.004, ss = -0.45, model R-2 = 48%) and fetuin-A and OPG predicted cardiac calcification (P = 0.02, ss = -0.29 and P = 0.014, ss = 0.33, respectively, model R-2 = 32%).Conclusions. This is the first study to define normal levels of the calcification inhibitors in children and show that fetuin-A and OPG are associated with increased vascular stiffness and calcification in children on dialysis. Higher levels of fetuin-A in children suggest a possible protective upregulation of fetuin-A in the early stages of exposure to the pro-calcific and pro-inflammatory uraemic environment.

1. While the composition of communities of arbuscular mycorrhizal (AM) fungi can have a large effect on the performance of their plant hosts, but the role of individual fungal species in shaping this response is as yet unresolved. 2. We have used the fungicide benomyl to alter the community of AM fungi in undisturbed monoliths of soil in a natural community. Changes in the community were characterised by root colonisation (%RLC), cloning, sequencing and tRFLP of a partial SSUrDNA fragment. Eleven plant species were sufficiently abundant in the monoliths to be examined. 3. In the highly mycorrhiza-dependent perennial herb Ajuga reptans, phosphate concentration was significantly reduced after benomyl treatment over a full growing season. The other plant species showed low colonisation and no significant difference in phosphate concentration after benomyl treatment. 4. Although colonisation in A. reptans was reduced, many mycorrhizal fungi survived in the roots. Some became more abundant following fungicide treatment, suggesting competitive release. Fungi that increased were generalists that have been identified in field samples from published studies colonising a wide range of plant species. Those that declined were specialists with a narrow host range; five types had not been recorded previously in field samples. 5. AM fungi in this study differed greatly in their response to perturbation, independent of the identity of the host plant. If such functional diversity is widespread, then elucidating the part played by AM fungal diversity in regulating plant community structure will be key to our understanding and management of ecosystems.

Type IV hemochromatosis is associated with dominant mutations in the SLC40A1 gene encoding ferroportin (FPN). Known as the “ferroportin disease,” this condition is typically characterized by high serum ferritin, reduced transferrin saturation, and macrophage iron loading. Previously FPN expression in vitro has been shown to cause iron deficiency in human cell lines and mediate iron export from Xenopus oocytes. We confirm these findings by showing that expression of human FPN in a human cell line results in an iron deficiency because of a 3-fold increased export of iron. We show that FPN mutations A77D, V162Δ, and G490D that are associated with a typical pattern of disease in vivo cause a loss of iron export function in vitro but do not physically or functionally impede wild-type FPN. These mutants may, therefore, lead to disease by haploinsufficiency. By contrast the variants Y64N, N144D, N144H, Q248H, and C326Y, which can be associated with greater transferrin saturation and more prominent iron deposition in liver parenchyma in vivo, retained iron export function in vitro. Because FPN is a target for negative feedback in iron homeostasis, we postulate that the latter group of mutants may resist inhibition, resulting in a permanently “turned on” iron exporter.

Co-inheritance of HFE mutations has a substantial role in iron overload in beta-thalassaemia carriers in north European populations where two HFE mutations, C282Y and H63D, are prevalent. In Thailand, there was little information about the allele frequency of HFE mutations. It is of interest to determine whether such determinants represent a potential risk in developing iron overload as nearly 40% of the Thai population carry either one of thalassaemia or haemoglobinpathy alleles. A total of 380 normal controls from five different regions including Bangkok were screened for the HFE C282Y, H63D and IVS5+1 G-->A alleles. In addition, 70 individuals with homozygous haemoglobin E (Hb EE) were also tested and their genotypes were correlated with levels of serum ferritin. H63D is the major HFE mutation found in the Thai population with an average allele frequency of 3% (range 1-5%). One individual was heterozygous for the splice site mutation IVS5 + 1 G --> A, and the C282Y allele was not detected. In the Hb EE group, five individuals had iron deficiency (ferritin

Background: There is evidence that iron may play a role in the pathology of Alzheimer's disease (AD). There may be genetic factors that contribute to iron deposition resulting in tissue damage thus exacerbating AD. Methods: We have genotyped 269 healthy elderly controls, 191 cases with definite or probable AD, and 69 with mild cognitive impairment (MCI) from the OPTIMA cohort. Results: We have examined the interaction between the C2 variant of the transferrin (TF) gene and the C282Y allele of the haemochromatosis (HFE) gene as risk factors for developing AD. Our results showed that each of the two variants was associated with an increased risk of AD only in the presence of the other. Neither allele alone had any effect. Carriers of both variants were at 5 times greater risk of AD compared with all others. The interaction was significant by logistic regression (p = 0.014) and by synergy factor analysis (p = 0.015, synergy factor = 5.1). Further, carriers of these two alleles plus apolipoprotein E e4 (APOE4) were at still higher risk of AD: of the 14 tri-carriers of the three variants, identified in this study, 12 had AD and two MCI. Conclusion: We suggest that the combination of TF C2 and HFE C282Y may lead to an excess of redox-active iron and the induction of oxidative stress in neurones, which is exacerbated in carriers of APOE4. Since 4% of Northern Europeans carry the two iron-related variants and since iron overload is a treatable condition, these results merit replication.

Background: Patients with hereditary haemochromatosis (HH) are usually homozygous for the C282Y mutation in the HFE gene. They have variable expression of iron overload and present with a variety of complications, including liver disease, diabetes, arthropathy, fatigue, and cardiomyopathy. The mitochondrial 16189 variant is associated with diabetes, dilated cardiomyopathy, and low body fat at birth, and might contribute to genetic predisposition in further multifactorial disorders. The objective of this study was to determine the frequency of the 16189 variant in a range of patients with haemochromatosis, who had mutations in the HFE gene. Methods: Blood DNA was analysed for the presence of the 16189 variant in British, French, and Australian C282Y homozygotes and controls, with known iron status, and in birth cohorts. Results: The frequency of the mitochondrial 16189 variant was found to be elevated in individuals with haemochromatosis who were homozygous for the C282Y allele, compared with population controls and with C282Y homozygotes who were asymptomatic (42/292 (14.4%); 102/1186 (8.6%) (p = 0.003); and 2/64 (3.1%) (p = 0.023), respectively). Conclusions: Iron loading in C282Y homozygotes with HH was exacerbated by the presence of the mitochondrial 16189 variant.

Haemochromatosis (HH) is a clinically and genetically heterogeneous disease caused by inappropriate iron absorption. Most HH patients are homozygous for the C282Y mutation in the HFE gene. However, penetrance of the C282Y mutation is incomplete, and other genetic factors may well affect the HH phenotype. Ferroportin and TFR2 mutations also cause HH, and two HAMP mutations have recently been reported that causes juvenile haemochromatosis (JH) in the homozygous state. Here, we report evidence for digenic inheritance of HH. We have detected two new HAMP mutations in two different families, in which there is concordance between severity of iron overload and heterozygosity for HAMP mutations when present with the HFE C282Y mutation. In family A, the proband has a JH phenotype and is heterozygous for C282Y and a novel HAMP mutation Met50del IVS2+1(-G). This is a four nucleotide ATGG deletion which causes a frameshift. The proband's unaffected mother is also heterozygous for Met50del IVS2+1(-G), but lacks the C282Y mutation and is heterozygous for the HFE H63D mutation. Met50del IVS2+1(-G) was absent from 642 control chromosomes. In family B, a second novel, less severe HAMP mutation, G71D, was identified. This was detected in the general population at an allele frequency of 0.3%. We propose that the phenotype of C282Y heterozygotes and homozygotes may be modified by heterozygosity for mutations which disrupt the function of hepcidin in iron homeostasis, with the severity of iron overload corresponding to the severity of the HAMP mutation.

1 The arbuscular mycorrhizal (AM) fungi colonizing plants at a woodland site in North Yorkshire (UK) have been characterized from the roots of five plant species (Rubus fruticosus agg. L., Epilobium angustifolium L., Acer pseudoplatanus L., Ajuga reptans L. and Glechoma hederacea L.), and identified using small-subunit rRNA (SSUrRNA) gene amplification and sequencing. 2 Interactions between five plant species from the site and four co-occurring glomalean fungi were investigated in artificial one-to-one AM symbioses. Three of the fungi were isolated from the site; the fourth was a culture genetically similar to a taxon found at the site. Phosphorus uptake and growth responses were compared with non-mycorrhizal controls. 3 Individual fungi colonized each plant with different spatial distribution and intensity. Some did not colonize at all, indicating incompatibility under the conditions used in the experiments. 4 Glomus hoi consistently occupied a large proportion of root systems and outperformed the other fungi, improving P uptake and enhancing the growth of four out of the five plant species. Only G. hoi colonized and increased P uptake in Acer pseudoplatanus, the host plant with which it associates almost exclusively under field conditions. Colonization of all plant species by Scutellospora dipurpurescens was sparse, and beneficial to only one of the host plants (Teucrium scorodonia). Archaeospora trappei and Glomus sp. UY1225 had variable effects on the host plants, conferring a range of P uptake and growth benefits on Lysimachia nummularia and T. scorodonia, increasing P uptake whilst not affecting biomass in Ajuga reptans and Glechoma hederacea, and failing to form mycorrhizas with A. pseudoplatanus. 5 These experimental mycorrhizas show that root colonization, symbiont compatibility and plant performance vary with each fungus-plant combination, even when the plants and fungi naturally co-exist. 6 We provide evidence of physical and functional selectivity in AM. The small number of described AM fungal species (154) has been ascribed to their supposed lack of host specificity, but if the selectivity we have observed is the general rule, then we may predict that many more, probably hard-to-culture glomalean species await discovery, or that members of species as currently perceived may be physiologically or functionally distinct.

The role of infection in erythropoietic dysfunction is poorly understood. In children with P. falciparum malaria, the by-product of hemoglobin digestion in infected red cells (hemozoin) is associated with the severity of anemia which is independent of circulating levels of the inflammatory cytokine tumor necrosis alpha (TNF-α). To gain insight into the common and specific effects of TNF-α and hemozoin on erythropoiesis, we studied the gene expression profile of purified primary erythroid cultures exposed to either TNF-α (10 ng/ml) or to hemozoin (12.5 μg/ml heme units) for 24 hours. Perturbed gene function was assessed using co-annotation of associated gene ontologies and expression of selected genes representative of the profile observed was confirmed by real time PCR (rtPCR). The changes in gene expression induced by each agent were largely distinct; many of the genes significantly modulated by TNF-α were not affected by hemozoin. The genes modulated by TNF-α were significantly enriched for those encoding proteins involved in the control of type 1 interferon signalling and the immune response to viral infection. In contrast, genes induced by hemozoin were significantly enriched for functional roles in regulation of transcription and apoptosis. Further analyses by rtPCR revealed that hemozoin increases expression of transcription factors that form part of the integrated stress response which is accompanied by reduced expression of genes involved in DNA repair. This study confirms that hemozoin induces cellular stress on erythroblasts that is additional to and distinct from responses to inflammatory cytokines and identifies new genes that may be involved in the pathogenesis of severe malarial anemia. More generally the respective transcription profiles highlight the varied mechanisms through which erythropoiesis may be disrupted during infectious disease.

Membrane type 4 matrix metalloproteinase (MT4-MMP) shows the least sequence homology to the other MT-MMPs, suggesting a distinct function for this protein. We have isolated a complete cDNA corresponding to the mouse homologue which includes the signal peptide and a complete pro-domain, features that were lacking from the human form originally isolated. Mouse MT4-MMP (mMT4-MMP) expressed in COS-7 cells is located at the cell surface but does not show ability to activate pro-MMP2. The pro-catalytic domain was expressed in Escherichia coli as insoluble inclusions and active enzyme recovered after refolding. Activity of the isolated catalytic domain against synthetic peptides commonly used for MMP enzyme assays could be inhibited by TIMP1, -2, and -3. The recombinant mMT4-MMP catalytic domain was also unable to activate pro-MMP2 and was very poor at hydrolyzing components of the extracellular matrix with the exception of fibrinogen and fibrin. mMT4-MMP was able to hydrolyze efficiently a peptide consisting of the pro-tumor necrosis factor alpha (TNFalpha) cleavage site, a glutathione S-transferase-pro-TNFalpha fusion protein, and was found to shed pro-TNFalpha when co-transfected in COS-7 cells. MT4-MMP was detected by Western blot in monocyte/macrophage cell lines which in combination with its fibrinolytic and TNFalpha-converting activity suggests a role in inflammation.

We have retrospectively analyzed 837 random anonymized dried blood spot (DBS) samples from neonatal screening programs in Scandinavia for mutations in HFE, the candidate gene for hemochromatosis. We have found C282Y allele frequencies of 2.3% (+2.0%) (-1.3%) in Greenland, 4.5%+/-1.9% in Iceland, 5.1%+/-2.3% in the Faeroe Islands, and 8.2%+/-2.7% in Denmark. The high prevalence of HFE mutations in Denmark suggests that population screening for the C282Y mutation could be highly advantageous in terms of preventive health care. Long-term follow-up evaluation of C282Y homozygotes and H63D/C282Y compound heterozygotes will give an indication of the penetrance of the mutations.

Early-season colonisation of new roots of Hyacinthoides non-scripta (L.) Chouard ex Rothm. was investigated to determine how arbuscular mycorrhizal symbiosis is re-established after the annual root system is shed. During the rootless phase in summer, colonies of bulbs were removed and replanted after the soil around and below the bulb had been mixed (major disturbance) so as to disrupt the external mycelium of arbuscular mycorrhizal (AM) fungi. As a minor disturbance treatment, top soil was removed, bulbs were turned or not in their growth position with as little other disturbance as possible, and the top soil replaced. Control plants were left undisturbed. Half of the plants were harvested 3–4 weeks after the onset of root emergence. Populations of all AM fungi in roots were greatly reduced by major disturbance, whilst those in other treatments and controls were unaffected. At the second harvest, in spring, when shoots had emerged, root colonisation by fine endophytes and Scutellospora morphotypes developed in all treatments, whereas that of Acaulospora morphotypes remained low after major disturbance. Disturbance treatments delayed the appearance, at the second harvest, of mycorrhizas with degenerate arbuscules. Leaf phosphorus concentration was unaffected by soil disturbance, possibly due to partial recovery of AM fungal populations or buffering by resources stored in the bulb.

summary Roots of bluebell (Hyacinthoides non-scripta (L.) Chouard ex Rothm.) are colonized by a range of fungal symbionts from several genera of the order Glomales. Using the identification scheme described in Merryweather & Fitter (1998), arbuscular mycorrhizal (AM) fungi in bluebell roots were quantified throughout the single growing season of 1994‐5 and compared with populations of spores found in the soil around the roots. In the early part of the growing season, when its activity is entirely subterranean (autumn and winter), bluebell habitually associates with a Scutellospora morphotype which is almost certainly S. dipurpurescens Morton & Koske (emend. Walker, 1993) whose spores occur in the root zone. This is the time of maximum phosphorus inflow and bulb-stored carbohydrate utilization by this mycorrhiza. A diverse flora of other AM fungal morphotypes (Acaulospora and Glomus), which might also form mycorrhizas with other plants in the vicinity of bluebells, invade the roots later in the season (spring), when P inflow is reduced and carbohydrate is available as fresh photosynthate. Their contribution to the mycorrhiza might be less than that of Scutellospora, particularly in terms of P assimilation. Both AM fungi in roots and glomalean spores recovered from soil around bluebell roots showed a significant degree of correlation with the vegetation within which the test plants grew. In the case of AM fungi in roots, Scutellospora showed no special preference for either, but Glomus correlated with a canopy of sycamore (Acer pseudoplatanus L.) and Acaulospora with oak (Quercus petraea (Mattuschka) Liebl.). Spores which most closely resembled S. dipurpurescens and Acaulospora gerdemannii Schenck & Nicolson were significantly more numerous under sycamore, but a spore like Acaulospora koskei Blaskowski, the most numerous and frequently encountered glomalean spore in the system, showed no preference for areas dominated by either tree. There was no significant relationship between AM fungal populations in bluebell roots and glomalean spores recovered from associated soil. The two spore taxa most frequently found in the vicinity of bluebell roots (A. koskei and S. dipurpurescens) were also found in lower numbers in soil from a region of the field site in which bluebell was absent, indicating that the main bluebell AM fungi do not exclusively associate with that host.

The arbuscular mycorrhizas of bluebell (Hyacinthoides non-scripta (L.) Chouard ex Rothm.) involve several symbiotic fungi of the order Glomales. We have previously simplified the system by ignoring the taxonomic diversity of the fungi, but it is unlikely that all fungal species contribute in the same manner or to the same extent to the functioning of the symbiosis. To discover how many and which fungi take part in the bluebell mycorrhiza we sought to identify the range of arbuscular mycorrhizal (AM) fungi found in bluebell roots sampled during a complete growing season, September to June. Although the taxonomy of the Glomales by their spores is not yet fully understood, identification is, to a large extent, possible. Arbuscular mycorrhizal communities are usually characterized by their spores but, since spores can rarely be directly associated with individual plants or plant species, a more satisfactory approach would be to identify fungal symbionts where they interact with the host plant, in the roots. Unfortunately, the intraradical mycelia of the fungi are less easily distinguished than the spores and, as yet, classification is possible only to the family level. We have developed a method whereby different AM fungal taxa in the roots of bluebell can be distinguished by objective assessment. A large suite of morphological characteristics of the fungi in roots was recorded in samples taken at monthly intervals. Hierarchical cluster analysis of the resulting data separated six distinct AM fungal morphotypes (Scutellospora type, Acaulospora type, three Glomus types and fine endophytes) and a classification system created by which identification by eye was possible. We compared the fungi in roots with glomalean spores in soil from the root zone of the same bluebell plants. Two species occurred in most samples, Scutellospora dipurpurescens Morton & Koske (emend. Walker, 1993) and Acaulospora koskei Blaskowski. A further six occurred sporadically, five Acaulospora spp. and Glomus rubiforme Gerdemann & Trappe) Almeida & Schenck (=Sclerocystis rubiformis). The presence of a single species of Scutellospora was consistent with a Scutellospora root morphotype which varied little. By contrast, the diversity of Acaulospora in the spore assemblage was reflected by variation within the Acaulospora morphotype. Glomus spores were very rarely found in field collections, yet Glomus morphotypes were found to be an important component of the bluebell mycorrhiza. Because some important species are not represented in spore assemblages in the field and those that are found can only be associated with vegetational groups, not individual plants or single species, glomalean spore populations provide only a partial account of the fungi which contribute to arbuscular mycorrhizas. Although it is still not possible to identify AM fungi in roots with the same precision as their spores, the method reported here permits assessment of diversity in the roots of individual plant species, which may be applied to the investigation of mycorrhizal function and demography in natural ecosystems.

We describe the rapid single-step detection, by mutagenically separated polymerase chain reaction (MS-PCR), of the HLE C282Y mutation, for which > 90% of haemochromatosis patients in the U.K. are homozygous. In addition to using purified DNA as a template, whole blood and lysed buccal cell extracts from mouthwash samples can be used. Therefore sample collection may be non-invasive and purification steps kept to a minimum.

The successful expression of animal or human virus epitopes on the surface of plant viruses has recently been demonstrated. These chimeric virus particles (CVPs) could represent a cost-effective and safe alternative to conventional animal cell-based vaccines. We report the insertion of oligonucleotides coding for a short linear epitope from the VP2 capsid protein of mink enteritis virus (MEV) into an infectious cDNA clone of cowpea mosaic virus and the successful expression of the epitope on the surface of CVPs when propagated in the black-eyed bean, Vigna unguiculata. The efficacy of the CVPs was established by the demonstration that one subcutaneous injection of 1 mg of the CVPs in mink conferred protection against clinical disease and virtually abolished shedding of virus after challenge with virulent MEV, demonstrating the potential utility of plant CVPs as the basis for vaccine development. The epitope used occurs in three different virus species-MEV, canine parvovirus, and feline panleukopenia virus- and thus the same vaccine could be used in three economically important viral hosts-mink, dogs, and cats, respectively.

Simon et al 1 in 1976 first reported the association between the leucocyte antigens HLA-A3 and HLA-B14 and genetic haemochromatosis (GH). Now, 20 years later, Feder et al 2 report the cloning of what is the strongest candidate gene for this disorder to have been identified so far. Formal proof from functional studies is now required to confirm that mutations in this gene are responsible for causing haemochromatosis. The question now arises as to why this gene has proved so elusive.

Insecticidal effects of three plant-derived genes, those encoding snowdrop lectin (GNA), bean (Phaseolus vulgaris) chitinase (BCH) and wheat α-amylase (WAI), were investigated and compared with effects of the cowpea trypsin inhibitor gene (CpTI). Transgenic potato plants containing each of the three genes singly, and in pairwise combinations were produced. All the introduced genes were driven by the CaMV 35S promoter; expression was readily detectable at the RNA level in transformants, but not detectable accumulation of WAI could be detected in transgenic potatoes containing its encoding gene. GNA and BCH were accumulated at levels up to 2.0% of total soluble protein; both proteins were expressed in a functional form, and GNA was shown to undergo 'correct' N-terminal processing. Accumulation levels of individual proteins were higher in plants containing a single foreign gene than in plants containing two foreign genes.

Potato plants (Solanum tuberosum) cv. Desiree were transformed with the genes encoding the proteins bean chitinase (BCH), snowdrop lectin (GNA) and wheat α-amylase inhibitor (WAI) under the control of the constitutive CaMV 35S promoter. Transgenic plants with detectable levels of foreign RNA were then selected for further characterisation with respect to protein expression levels by immunodot blot analysis using polyclonal antibodies raised against the respective protein. With the exception of WAI, plants expressing high levels of RNA, expressed correspondingly high levels of the foreign protein (1.5–2.0% of the total soluble protein). Although high levels of WAI mRNA were detected in some of the transformants, the protein could not be detected. On the bases of expression levels, two lines, designated PWG6#85 (transformed with the double construct WAI/GNA) and PBG6#47 (transformed with the double construct BCH/GNA), were selected for testing in aphid trials for enhanced levels of resistance. Both transgenic lines had a marked and significant effect on fecundity. The number of nymphs produced per female per day peaked at 4.1 and 4.2 for lines PBG6#47 and PWG6#85 respectively, compared to a value of 5.4 on control plants. Total nymphal production was also significantly lower on either of the transgenic lines compared to control plants (P

myb-related transcription factors contain highly conserved DNA-binding domains. Using a mixture of degenerate oligonucleotides derived from the highly conserved region as probe, 14 myb-related clones were isolated from a cDNA library constructed using tomato hypocoyl mRNA. The expression of these clones was studied by northern blot analysis using poly(A)+ RNA from 7 tissue types (hypocotyl, leaf, root, green and red fruit, immature and mature flower). This study has revealed a wide range of expression patterns which include multiple and single transcripts, some of which show marked tissue specificity. Two clones showing different expression patterns have been fully sequenced. The DNA-binding domains of these two tomato myb clones are compared with myb genes from other plant species and organisms. Of the three clones analysed so far by Southern hybridization, two are single-copy genes and one has multiple genomic copies.

summary We controlled arbuscular mycorrhizal (AM) fungi in the routs of bluebell (Hyacinthoides non-scripta L. Chouard ex Rothm.) in the field by immersina otherwise undisturbed colonies in a fungicide suspension. Monthly application of benomyl successfully reduced root colonization by AM fungi throughout a 2 yr experimental period. Benonlyl had lit) effect on the availability of soil phosphorus (P), but reduced the P concentration of all parts of the plant (bulb, roots, leaves and inflorescences). Unlike the vegetative parts, flowers and seed of benomyl-treated plants had the same P concentration as untreated plants at the end of the first season. However, at the final harvest after two growing seasons flower P concentration had been reduced by treatment. H. non-scripta appears to protect reproductive structures from P deficiency when the plant is deprived of P, suggesting that A.M plays an important role in fitness determination. This is the first demonstration that depriving a plant growing in its natural environment of mycorrhiza on a long-term basis can reduce P acquisition. Taken with data from experiments in controlled conditions, it confirms that H. non-scripta is an obligately mycorrhizal species.

summary The arbuscular mycorrhizal (AM) association between fungi in the order Glomales and the roots of a very wide range of vascular plants is of global ecological significance but has proved particularly intractable to study in the field. We have developed a reliable technique to identify the fungal symbionts in roots taken directly from natural communities. Selective Enrichment of Amplified DNA combines the use of recently-developed specific DNA primers with a novel method based on the principle of subtractive hybridization to remove interfering plant-derived DNA after amplification with the polymerase chain reaction. Using this technique we have shown that endomycorrhizas of bluebells (Hyacinthoides non-scripta) sampled directly from a woodland habitat are multispceies communities of varying composition which contain at least three genera of mycorrhizal fungi. The technique works well on a range of plant species and should have wide application to the identification of other symbionts, including pathogens. A spore survey has indicated that two particular AM types are associated with bluebells and this observation corroborates the molecular data. The presence of a Glomus species in bluebell roots was not expected from the spore data.

Transgenic sweet potato plants were obtained following transformation with Agrobacterium tumefaciens LBA4404. Discs cut from freshly-harvested storage roots of the sweet potato cultivar ‘Jewel’, were inoculated and cultured on various media to induce regeneration. Regeneration occurred via somatic embryogenesis, but somatic embryos were only successfully induced to germinate and produce shoots after removal of antibiotics from the tissue culture medium. Kanamycin monosulphate was used in the initial stages of culture as a selective agent for transformed tissue. Plants were confirmed as transgenic by Southern analysis of DNA, RNA dot blot analysis, and protein analysis using enhanced chemiluminescence detection methods. Transgenic plants have been obtained that express the β-glucuronidase enzyme, the cowpea trypsin inhibitor and the snowdrop lectin.

summary The extent of host benefit from arbuscular mycorrhiza (AM) in terms of phosphorus (P) assimilation under natural conditions has rarely been demonstrated or quantified. An apparently obligately mycorrhizal host, Hyacinthoides non-scripta (L.) Chouard ex Rothm., was selected and, throughout the season September-July 1989–90, entire clones were collected from a field site at frequent intervals without experimental modification of the host, its mycosymbiont or their environment. In a concurrent glasshouse experiment, sand-cultured non-mycorrhizal plants given a nutrient solution containing a supply of P were unable to assimilate sufficient to maintain tissue P content. We provide correlative evidence of mycorrhizal behaviour in an undisturbed, natural ecosystem by demonstrating the temporal coincidence of root colonization by mycorrhizal fungi and the inflow of phosphorus in adult H. non-scripta. Annual carbon (biomass) and phosphorus budgets are described. Carbon was reallocated from the resting bulb, first for root production and, later, to developing leaves and inflorescences. A similar pattern was observed in P allocation. A substantial amount of P was lost in seed, leaves, roots and scapes shed at the end of a season, when bulb biomass had increased, but P content had not.

summary This investigation aimed to assess the degree of dependence of young plants of Hyacinthoides non-scripta (L.) Chouard ex Rothm. on the formation of arbuscular mycorrhiza (AM) at different stages of its life cycle. H. non-scripta is a bulbous perennial with a restricted root system of coarse, unbranched elements which are plainly inadequate for the acquisition of phosphate (P) from soil. In a glasshouse experiment non-mycorrhizal H. non-scripta were unable to take up sufficient P to maintain a positive P budget, confirming that the species is obligately mycorrhizal when adult. We needed to be able to assess the ages of field-collected plants. The bulb of H. non-scripta, which is totally rebuilt annually, provides no indicator of age other than size. The development of individual bulbs was monitored in reconstructed soil profiles for several years and the relationship of size and age calculated in order to determine the age structure of a population in the field and to quantify the mycorrhizal colonization of their roots according to age and depth in the soil. Seedlings of H. non-scripta are supplied with a P-rich endosperm and initially inhabit upper soil horizons where P is plentiful. However, they are at risk from winter frosts, summer drought and, possibly, predators. During their first four to five seasons they descend in the soil to a depth of approximately 20 cm, down a gradient of decreasing soil phosphorus concentration; simultaneously, their roots become increasingly colonized by arbuscular mycorrhizal fungi. Because of the change in environment, they appear to become increasingly dependent on the symbiosis, suggesting that the association changes from facultative to obligate during the plant's life history.

The range of sap-sucking insect pests to which GNA, (the mannose specific lectin from snowdrops (Galanthus nivalis) has been shown to be insecticidal in artificial diets has been extended to include the peach potato aphid (Myzus persicae). A gene construct for constitutive expression of GNA from the CaMV35S gene promoter has been introduced into tobacco plants. A transgenic tobacco line which expresses high levels of GNA has been shown to have enhanced resistance toM. persicae in leaf disc and whole plant bioassays,demonstrating the potential for extending transgenic plant technology to the control of sap-sucking insect pests.

This article is available open access through the publisher’s website. Copyright @ 2011 American Society of Hematology. This article has an erratum: http://bloodjournal.hematologylibrary.org/content/118/26/6993.3. Understanding the pattern of gene expression during erythropoiesis is crucial for a synthesis of erythroid developmental biology. Here, we isolated 4 distinct populations at successive erythropoietin-dependent stages of erythropoiesis, including the terminal, pyknotic stage. The transcriptome was determined using Affymetrix arrays. First, we demonstrated the importance of using defined cell populations to identify lineage and temporally specific patterns of gene expression. Cells sorted by surface expression profile not only express significantly fewer genes than unsorted cells but also demonstrate significantly greater differences in the expression levels of particular genes between stages than unsorted cells. Second, using standard software, we identified more than 1000 transcripts not previously observed to be differentially expressed during erythroid maturation, 13 of which are highly significantly terminally regulated, including RFXAP and SMARCA4. Third, using matched filtering, we identified 12 transcripts not previously reported to be continuously up-regulated in maturing human primary erythroblasts. Finally, using transcription factor binding site analysis, we identified potential transcription factors that may regulate gene expression during terminal erythropoiesis. Our stringent lists of differentially regulated and continuously expressed transcripts containing many genes with undiscovered functions in erythroblasts are a resource for future functional studies of erythropoiesis. Our Human Erythroid Maturation database is available at https://cellline.molbiol.ox.ac.uk/eryth/index.html. National Health Service Blood and Transplant, National Institute for Health Research Biomedical Research Center Program, and National Institute for Health Research.

Crops resistant to insect attack offer a different strategy of pest control to indiscriminate pesticide usage, which has undesirable effects on both the environment and humans. Transgenic plant technology can be a useful tool in producing resistant crops, by introducing entirely novel resistance genes into a plant species. Although most work in this area has focused on the use of genes encoding insecticidalBacillus thuringiensisδ-endotoxins in transgenic plants, an alternative approach is to use plant genes which encode proteins with insecticidal properties. Protease inhibitors are involved in endogenous plant defence against insects. Over-expression of several inhibitors from constitutive promoters has been shown to afford protection in transgenic tobacco plants against attack by lepidopteran larvae. However, the degree of protection is not sufficiently high, and shows species- and inhibitor-specific effects. By assaying the interactions of protease inhibitors with insect gut proteasesin vitro, the most effective inhibitor can be selected for a particular insect species. Data from bioassays of insects using artificial diets, and with transgenic plants, suggest that thein vitroassay of relative inhibitor effectiveness is consistent with the effects of different inhibitors on insect development and survivalin vivo. Development of this techniology is considered. A different approach must be taken with sucking insect pests, as they do not rely on proteolysis for nutrition, and asBttoxins effective against hom opterans have not been reported to date. Bioassay in artificial diet was used to identify plant proteins with insecticidal effects on the rice brown planthopper (a model homopteran). The lectin from snowdrop (GNA) was found to be the most effective of the proteins tested. GNA was shown to be present in the phloem sap of a transgenic tobacco plant transformed with a chimeric gene construct, containing the rice sucrose synthase-1 gene promoter and the GNA coding sequence, by immunoassay of honeydew produced by aphids feeding on it. GNA is also insecticidal to the aphidMyzus persicae, which will feed on tobacco, and thus a bioassay of transgenic tobacco, to ‘prove’ the technology, can be carried out. The effects of combining different resistance genes in the same transgenic plant to improve the effectiveness of protection are discussed, and exemplified.

We have investigated the efficacy of using the Escherichia coli lac operator-repressor system to control plant gene expression. The lacI gene was modified to allow optimal expression in plant cells and then placed downstream of the cauliflower mosaic virus (CaMV) 35S RNA promoter. This construct was introduced into tobacco plants by leaf disc transformation. Transgenic tobacco plants synthesized significant quantities of LacI protein (up to 0.06% of total soluble protein). We have used the E.coli beta-glucuronidase gene (gus) as the reporter gene by placing it downstream of the maize chlorophyll a/b binding protein (CAB) gene promoter. Lac operators were introduced into several positions within the CAB promoter and operator-free plasmid was used as control. Repression was assessed by comparing the transient expression from CAB-operator-gus reporter constructs in protoplasts expressing lac protein, with that in control cells not expressing the repressor. Repression varied between 10 and 90% with different operator positions. Transient assays were also performed in the presence of the inducer, isopropyl-beta-D-thiogalactoside (IPTG). In lacI protoplasts the presence of IPTG manifested itself in a 4.2-fold relief of repression. The study was extended to show regulation of expression in stable transformants. Tobacco transformants harbouring a CAB-operator-gus reporter construct and the lacI gene were shown to have repressed GUS levels, but in the presence of IPTG, repression was relieved 15-fold. We conclude that the lac repressor can enter the plant cell nucleus, find its cognate operator sequence in the chromatin to form a repressor--operator complex and effectively block transcription of a downstream gene.

Previous studies with purified variants of the 434 repressor having different operator-binding specificities have demonstrated the interactions of a heterodimeric repressor with a hybrid operator site. The present study investigates the interactions between the 434 repressor and its operator site. The optimum 434 operator half-site is used with a P22 operator half-site to create a hybrid 434/P22 operator. We show that this hybrid operator can be efficiently bound by a heterodimeric repressor, consisting of one wild-type 434 repressor monomer and one 434 repressor monomer with the binding specificity of the P22 repressor, to bring about repression in Escherichia coli.

We have isolated four insulin-like growth factor binding proteins (IGFBPs) from adult human serum by insulin-like growth factor (IGF) I affinity chromatography and high performance liquid chromatography. A 36-kDa binding protein (BP), not digestible with N-glycanase, is increased in patients with extrapancreatic tumor hypoglycemia and during IGF I administration in healthy adults. Its 38 NH2-terminal amino acids are identical to those of an IGFBP sequence derived from a human cDNA that cross-hybridizes with the rat IGFBP-2 cDNA. With probes encoding a NH2-terminal, COOH-terminal, and a middle region of this protein we have obtained three cDNA clones from a Hep G2 cDNA library; one encodes human IGFBP-2, and the other two presumably represent unspliced heteronuclear and alternatively spliced mRNA, respectively. A 28-30-kDa IGFBP represents a novel BP species in human serum. Its 30 NH2-terminal amino acids are not homologous to IGFBP-1, -2, or -3. It is not digestible with N-glycanase and does not bind 125I-IGF I. The NH2-terminal sequences of a 42/45- and a 31-kDa IGFBP are identical to that of human IGFBP-3. The 42/45-kDa proteins are two glycosylation variants of BP-3. The 31-kDa protein presumably is a degradation product of BP-3 that lacks the COOH terminus. It is likely that the different IGFBPs modulate auto-/paracrine and endocrine effects of IGFs on growth and metabolism in a different and specific manner.

Organophosphate-resistant and -susceptible strains of Culex quinquefasciatus (mosquito) have been compared on the basis of their esterase activities. The homozygous resistant strain (Dar) shows two highly active esterases after starch-gel electrophoresis, of Rm 0.2 and 0.4, which are absent from susceptible strains (Apo, Mon), and which previous selection studies have shown to be inseparable from organophosphate resistance. After SDS/polyacrylamide-gel electrophoresis and silver staining of total C. quinquefasciatus proteins, a 62 kDa band is observed in strain Dar at high concentrations, and in susceptible strains in trace amounts. After Western blotting, this 62 kDa protein is recognized by antisera raised against the two esterases eluted from starch gels. After chromatofocusing of Dar proteins, the 62 kDa protein is seen to be associated with esterase activity, and of a similar pI to that observed for esterases after isoelectric focusing. Post-translational modification is not required for recognition of the 62 kDa putative esterase, since the protein is immunoprecipitated by the anti-esterase serum from products of translation of Dar mRNA in vitro.

Mutations in the hepcidin gene HAMP and the hemojuvelin gene HJV have recently been shown to result in juvenile haemochromatosis (JH). Hepcidin is an antimicrobial peptide that plays a key role in regulating intestinal iron absorption. Hepcidin levels are reduced in patients with haemochromatosis due to mutations in the HFE and HJV genes. Digenic inheritance of mutations in HFE and HAMP can result in either JH or hereditary haemochromatosis (HH) depending upon the severity of the mutation in HAMP. Here we review these findings and discuss how understanding the different types of haemochromatosis and our increasing knowledge of iron metabolism may help to elucidate the host's response to infection.

Extensive investigation into the molecular basis of iron overload disorders has provided new insights into the complexity of iron metabolism and related cellular pathways. The possible involvement of genes affecting iron homeostasis, including HFE, SLC40A1, HAMP and CYBRD1, was investigated in individuals who were referred for confirmation or exclusion of a diagnosis of haemochromatosis, but who tested negative or were heterozygous for the causative HFE mutation, C282Y. Denaturing high performance liquid chromatography analysis of these genes revealed a unique spectrum of mutations in the South African study population, including 67 unrelated patients and 70 population-matched controls. Two novel CYBRD1 gene mutations, R226H and IVS1-4C--G, were identified in 11% of South African Caucasian patient referrals. We identified a novel D270V mutation in the SLC40A1 gene in a Black South African female with iron overload. These mutations were absent in the control population. In Africans with iron overload not related to the HFE gene, the possible involvement of the SLC40A1 and CYBRD1 genes was demonstrated for the first time. This study confirms the genetic heterogeneity of haemochromatosis and highlights the significance of CYBRD1 mutations in relation to iron overload.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism. Iron absorption from the gut is inappropriately high, resulting in increasing iron overload. The hemochromatosis gene (HFE) was identified in 1996 by extensive positional cloning by many groups over a period of about 20 years. Two missense mutations were identified. Homozygosity for one of these, a substitution of a tyrosine for a conserved cysteine (C282Y), has now clearly been shown to be associated with HH in 60-100% of patients. The role of the second mutation, the substitution of an aspartic acid for a histidine (H63D), is not so clear but compound heterozygotes for both these mutations have a significant risk of developing HH. Here we review other putative mutations in the HFE gene and document a number of diallelic polymorphisms in HFE introns.

The resolution of intraradical mycelium, vesicles and arbuscules of vesicular-arbuscular mycorrhizal fungi, stained with acid fuchsin, is enhanced by epifluorescence illumination.

Summary Genetic hemochromatosis (GH) is believed to be a disease restricted to those of European ancestry. In northwestern Europe, >80% of GH patients are homozygous for one mutation, the substitution of tyrosine for cysteine at position 282 (C282Y) in the unprocessed protein. In a proportion of GH patients, two mutations are present, C282Y and H63D. The clinical significance of this second mutation is such that it appears to predispose 1%–2% of compound heterozygotes to expression of the disease. The distribution of the two mutations differ, C282Y being limited to those of northwestern European ancestry and H63D being found at allele frequencies >5%, in Europe, in countries bordering the Mediterranean, in the Middle East, and in the Indian subcontinent. The C282Y mutation occurs on a haplotype that extends ≤6 Mb, suggesting that this mutation has arisen during the past 2,000 years. The H63D mutation is older and does not occur on such a large extended haplotype, the haplotype in this case extending ≤700 kb. Here we report the finding of the H63D and C282Y mutations on new haplotypes. In Sri Lanka we have found H63D on three new haplotypes and have found C282Y on one new haplotype, demonstrating that these mutations have arisen independently on this island. These results suggest that the HFE gene has been the subject of selection pressure. These selection pressures could be due to infectious diseases, environmental conditions, or other genetic disorders such as anemia.

High frequencies of the haemochromatosis-related HFE C282Y mutation have been reported in North European populations, in which a high proportion of patients with the disease are homozygotes. However, the degree of penetrance of this genotype is unknown. We determined the HFE C282Y and H63D genotypes of 411 consenting volunteer blood donors on Jersey, and the serum ferritin and transferrin saturation levels of 204 of these volunteers. The C282Y allele frequency was found to be 8.3% in 822 chromosomes, indicating a homozygote frequency of 1/145. Consistent with this, four C282Y homozygotes were detected in 411 volunteers. As there are only 18 patients presently receiving treatment for haemochromatosis on Jersey, out of a total population of about 85000, there is a large discrepancy between the number of haemochromatosis patients and the number of C282Y homozygotes in this population. In a preliminary study of 204 consenting volunteers we found a correlation between transferrin saturation and HFE H63D/ C282Y genotype (P=0.017) and between serum ferritin and genotype (P = 0.056). We also observed elevated values of transferrin saturation in the two C282Y homozygotes assayed. These results suggest that a large proportion of the many undetected C282Y homozygotes on Jersey and in similar populations could be in the preclinical stages of haemochromatosis, and warrant investigation. However, there may be a wide variation in the expression of the condition, and a more extensive study of the level of disease penetrance encompassing a large number of hitherto undetected C282Y homozygotes is therefore imperative.

We have generated a detailed physical map of the 6p21.3/p22.1 boundary, using a combination of yeast artificial chromosome (YAC) fragmentation and high-resolution sequence tagged site (STS) content mapping. YACs from the CEPH, St. Louis, and ICRF libraries have been used to construct a 4.5-Mb contig spanning the markers D6S306 to D6S1571. YAC insert sizes were determined by pulsed field gel electrophoresis (PFGE). Chimerism of YACs was determined by fluorescent in situ hybridization (FISH), and their integrity was determined by fingerprinting with Alu-PCR. We have identified 10 new CA repeat loci in this region as well as over 50 novel STSs, several tRNA genes, a new histone H2B gene and the phospholipase D gene. Using these new markers, we have rapidly generated a bacterial clone contig of over 250 kb, spanning the markers D6S1260 to D6S1918 (WI-3111) with STSs spaced on average every 6 kb.

Haemochromatosis is a genetic disease associated with progressive iron overload, and is common among populations of northern European origin. HLA-H is a recently reported candidate gene for this condition. Two mutations have been identified, a substitution of cysteine for tyrosine at amino acid 282 (C282Y, nucleotide 845) and of histidine for aspartate at amino acid 63 (H63D, nucleotide 187). Over 90% of UK haemochromatosis patients are homozygous for the C282Y mutation. We have examined 5956 chromosomes (2978 people) for the presence of HLA-H C282Y and H63D by PCR followed by restriction enzyme analysis. We have found world wide allele frequencies of 1.9% for C282Y and 8.1% for H63D. The highest frequencies were 10% for C282Y in 90 Irish chromosomes and 30.4% for H63D in 56 Basque chromosomes. C282Y was most frequent in northern European populations and absent from 1042 African chromosomes, 484 Asian chromosomes, and 644 Australasian chromosomes. The distribution of the C282Y mutation coincides with that of populations in which haemochromatosis has been reported and is consistent with the theory of a north European origin for the mutation. The H63D polymorphism is more widely distributed and its connection with haemochromatosis remains unclear.