Evaluating ecohydrological theories of woody root distribution in the Kalahari

The contribution of savannas to global carbon storage is poorly understood, in part due to lack of knowledge of the amount of belowground biomass. In these ecosystems, the coexistence of woody and herbaceous life forms is often explained on the basis of belowground interactions among roots. However, the distribution of root biomass in savannas has seldom been investigated, and the dependence of root biomass on rainfall regime remains unclear, particularly for woody plants. Here we investigate patterns of belowground woody biomass along a rainfall gradient in the Kalahari of southern Africa, a region with consistent sandy soils. We test the hypotheses that (1) the root depth increases with mean annual precipitation (root optimality and plant hydrotropism hypothesis), and (2) the root-to-shoot ratio increases with decreasing mean annual rainfall (functional equilibrium hypothesis). Both hypotheses have been previously assessed for herbaceous vegetation using global root data sets. Our data do not support these hypotheses for the case of woody plants in savannas. We find that in the Kalahari, the root profiles of woody plants do not become deeper with increasing mean annual precipitation, whereas the root-to-shoot ratios decrease along a gradient of increasing aridity.     

DNA barcoding the native flowering plants and conifers of Wales

We present the first national DNA barcode resource that covers the native flowering plants and conifers for the nation of Wales (1143 species). Using the plant DNA barcode markers rbcL and matK, we have assembled 97.7% coverage for rbcL, 90.2% for matK, and a dual-locus barcode for 89.7% of the native Welsh flora. We have sampled multiple individuals for each species, resulting in 3304 rbcL and 2419 matK sequences. The majority of our samples (85%) are from DNA extracted from herbarium specimens. Recoverability of DNA barcodes is lower using herbarium specimens, compared to freshly collected material, mostly due to lower amplification success, but this is balanced by the increased efficiency of sampling species that have already been collected, identified, and verified by taxonomic experts. The effectiveness of the DNA barcodes for identification (level of discrimination) is assessed using four approaches: the presence of a barcode gap (using pairwise and multiple alignments), formation of monophyletic groups using Neighbour-Joining trees, and sequence similarity in BLASTn searches. These approaches yield similar results, providing relative discrimination levels of 69.4 to 74.9% of all species and 98.6 to 99.8% of genera using both markers. Species discrimination can be further improved using spatially explicit sampling. Mean species discrimination using barcode gap analysis (with a multiple alignment) is 81.6% within 10×10 km squares and 93.3% for 2×2 km squares. Our database of DNA barcodes for Welsh native flowering plants and conifers represents the most complete coverage of any national flora, and offers a valuable platform for a wide range of applications that require accurate species identification.     

Autophagy in idiopathic pulmonary fibrosis

Background

Autophagy is a basic cellular homeostatic process important to cell fate decisions under conditions of stress. Dysregulation of autophagy impacts numerous human diseases including cancer and chronic obstructive lung disease. This study investigates the role of autophagy in idiopathic pulmonary fibrosis.

Methods

Human lung tissues from patients with IPF were analyzed for autophagy markers and modulating proteins using western blotting, confocal microscopy and transmission electron microscopy. To study the effects of TGF-β₁ on autophagy, human lung fibroblasts were monitored by fluorescence microscopy and western blotting. In vivo experiments were done using the bleomycin-induced fibrosis mouse model.

Results

Lung tissues from IPF patients demonstrate evidence of decreased autophagic activity as assessed by LC3, p62 protein expression and immunofluorescence, and numbers of autophagosomes. TGF-β₁ inhibits autophagy in fibroblasts in vitro at least in part via activation of mTORC1; expression of TIGAR is also increased in response to TGF-β₁. In the bleomycin model of pulmonary fibrosis, rapamycin treatment is antifibrotic, and rapamycin also decreases expression of á-smooth muscle actin and fibronectin by fibroblasts in vitro. Inhibition of key regulators of autophagy, LC3 and beclin-1, leads to the opposite effect on fibroblast expression of á-smooth muscle actin and fibronectin.

Conclusion

Autophagy is not induced in pulmonary fibrosis despite activation of pathways known to promote autophagy. Impairment of autophagy by TGF-β₁ may represent a mechanism for the promotion of fibrogenesis in IPF.     

Annotation of a serum N-glycan library for rapid identification of structures

Glycosylation is one of the most common post-translational modifications of proteins and has been shown to change with various pathological states including cancer. Global glycan profiling of human serum based on mass spectrometry has already led to several promising markers for diseases. The changes in glycan structure can result in altered monosaccharide composition as well as in the linkages between the monosaccharides. High-throughput glycan structural elucidation is not possible because of the lack of a glycan template to expedite identification. In an effort toward rapid profiling and identification of glycans, we have constructed a library of structures for the serum glycome to aid in the rapid identification of serum glycans. N-Glycans from human serum glycoproteins are used as a standard and compiled into a library with exact structure (composition and linkage), liquid chromatography retention time, and accurate mass. Development of the library relies on highly reproducible nanoLC-MS retention times. Tandem MS and exoglycosidase digestions were used for structural elucidation. The library currently contains over 300 entries with 50 structures completely elucidated and over 60 partially elucidated structures. This database is steadily growing and will be used to rapidly identify glycans in unknown biological samples.     

Sites of genetic instability in mitosis and cancer

Certain chromosomal regions called common fragile sites are prone to difficulty during replication. Many tumors have been shown to contain alterations at fragile sites. Several models have been proposed to explain why these sites are unstable. Here we describe work to investigate models of fragile site instability using a yeast artificial chromosome carrying human DNA from a common fragile site region. In addition, we describe a yeast system to investigate whether repair of breaks at a naturally occurring fragile site in yeast, FS2, involves mitotic recombination between homologous chromosomes, leading to loss of heterozygosity (LOH). Our initial evidence is that repair of yeast fragile site breaks does lead to LOH, suggesting that human fragile site breaks may similarly contribute to LOH in cancer. This work is focused on gaining understanding that may enable us to predict and prevent the situations and environments that promote genetic changes that contribute to tumor progression.     

Fasting Reduces the Incidence of Delayed-Type Vomiting Associated with Doxorubicin Treatment in Dogs with Lymphoma

Fasting reduces gastrointestinal cellular proliferation rates through G₁ cycle blockade and can promote cellular protection of normal but not cancer cells through altered cell signaling including down-regulation of insulin-like growth factor 1 (IGF-1). Consequently, the purpose of this study was to determine the effects of fasting on delayed-type chemotherapy-induced nausea and vomiting in dogs receiving doxorubicin. This prospective randomized crossover study involved intended administration of two doses of doxorubicin. Cancer-bearing dogs were randomized to be fasted for 24 hours beginning at 6 P.M. the night before the first or second doxorubicin administration, and all treatments were administered within an hour before or after 12 P.M. Dogs were fed normally before the alternate dose. Circulating IGF-1 concentrations were determined from serum samples obtained immediately before each doxorubicin treatment. Data from 35 doses were available from 20 dogs enrolled. Dogs that were fasted exhibited a significantly lower incidence of vomiting, when compared to fed dogs (10% compared to 67%, P = .020). Furthermore, among the 15 dogs that completed crossover dosing, vomiting was abrogated in four of five dogs that experienced doxorubicin-induced vomiting when fed normally (P = .050). No differences in other gastrointestinal, constitutional, or bone marrow toxicities or serum IGF-1 levels were observed.     

A homozygous mutation in the tight-junction protein JAM3 causes hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts

The tight junction, or zonula occludens, is a specialized cell-cell junction that regulates epithelial and endothelial permeability, and it is an essential component of the blood-brain barrier in the cerebrovascular endothelium. In addition to functioning as a diffusion barrier, tight junctions are also involved in signal transduction. In this study, we identified a homozygous mutation in the tight-junction protein gene JAM3 in a large consanguineous family from the United Arab Emirates. Some members of this family had a rare autosomal-recessive syndrome characterized by severe hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. Their clinical presentation overlaps with some reported cases of pseudo-TORCH syndrome as well as with cases involving mutations in occludin, another component of the tight-junction complex. However, massive intracranial hemorrhage distinguishes these patients from others. Homozygosity mapping identified the disease locus in this family on chromosome 11q25 with a maximum multipoint LOD score of 6.15. Sequence analysis of genes in the candidate interval uncovered a mutation in the canonical splice-donor site of intron 5 of JAM3. RT-PCR analysis of a patient lymphoblast cell line confirmed abnormal splicing, leading to a frameshift mutation with early termination. JAM3 is known to be present in vascular endothelium, although its roles in cerebral vasculature have not been implicated. Our results suggest that JAM3 is essential for maintaining the integrity of the cerebrovascular endothelium as well as for normal lens development in humans.     

Glycocalyx restricts adenoviral vector access to apical receptors expressed on respiratory epithelium in vitro and in vivo: role for tethered mucins as barriers to lumenal infection

Inefficient adenoviral vector (AdV)-mediated gene transfer to the ciliated respiratory epithelium has hindered gene transfer strategies for the treatment of cystic fibrosis lung disease. In part, the inefficiency is due to an absence of the coxsackie B and adenovirus type 2 and 5 receptor (CAR) from the apical membranes of polarized epithelia. In this study, using an in vitro model of human ciliated airway epithelium, we show that providing a glycosylphosphatidylinositol (GPI)-linked AdV receptor (GPI-CAR) at the apical surface did not significantly improve AdV gene transfer efficiency because the lumenal surface glycocalyx limited the access of AdV to apical GPI-CAR. The highly glycosylated tethered mucins were considered to be significant glycocalyx components that restricted AdV access because proteolytic digestion and inhibitors of O-linked glycosylation enhanced AdV gene transfer. To determine whether these in vitro observations are relevant to the in vivo situation, we generated transgenic mice expressing GPI-CAR at the surface of the airway epithelium, crossbred these mice with mice that were genetically devoid of tethered mucin type 1 (Muc1), and tested the efficiency of gene transfer to murine airways expressing apical GPI-human CAR (GPI-hCAR) in the presence and absence of Muc1. We determined that AdV gene transfer to the murine airway epithelium was inefficient even in GPI-hCAR transgenic mice but that the gene transfer efficiency improved in the absence of Muc1. However, the inability to achieve a high gene transfer efficiency, even in mice with a deletion of Muc1, suggested that other glycocalyx components, possibly other tethered mucin types, also provide a significant barrier to AdV interacting with the airway lumenal surface.     

R. Vanbreuseghem 1909–1993

Editor's Note. Dr Vanbreuseghem was a member of the Mycopathologia Educational Board from 1959 to 1960.     

Genome‐wide association study identifies loci associated with liability to alcohol and drug dependence that is associated with variability in reward‐related ventral striatum activity in African‐ and European‐Americans

Genetic influences on alcohol and drug dependence partially overlap, however, specific loci underlying this overlap remain unclear. We conducted a genome‐wide association study (GWAS) of a phenotype representing alcohol or illicit drug dependence (ANYDEP) among 7291 European‐Americans (EA; 2927 cases) and 3132 African‐Americans (AA: 1315 cases) participating in the family‐based Collaborative Study on the Genetics of Alcoholism. ANYDEP was heritable (h ² in EA = 0.60, AA = 0.37). The AA GWAS identified three regions with genome‐wide significant (GWS; P < 5E‐08) single nucleotide polymorphisms (SNPs) on chromosomes 3 (rs34066662, rs58801820) and 13 (rs75168521, rs78886294), and an insertion‐deletion on chromosome 5 (chr5:141988181). No polymorphisms reached GWS in the EA. One GWS region (chromosome 1: rs1890881) emerged from a trans‐ancestral meta‐analysis (EA + AA) of ANYDEP, and was attributable to alcohol dependence in both samples. Four genes (AA: CRKL, DZIP3, SBK3; EA: P2RX6) and four sets of genes were significantly enriched within biological pathways for hemostasis and signal transduction. GWS signals did not replicate in two independent samples but there was weak evidence for association between rs1890881 and alcohol intake in the UK Biobank. Among 118 AA and 481 EA individuals from the Duke Neurogenetics Study, rs75168521 and rs1890881 genotypes were associated with variability in reward‐related ventral striatum activation. This study identified novel loci for substance dependence and provides preliminary evidence that these variants are also associated with individual differences in neural reward reactivity. Gene discovery efforts in non‐European samples with distinct patterns of substance use may lead to the identification of novel ancestry‐specific genetic markers of risk.