Population Genetic Structure and Isolation by Distance of Helicobacter pylori in Senegal and Madagascar

Helicobacter pylori has probably infected the human stomach since our origins and subsequently diversified in parallel with their human hosts. The genetic population history of H. pylori can therefore be used as a marker for human migration. We analysed seven housekeeping gene sequences of H. pylori strains isolated from 78 Senegalese and 24 Malagasy patients and compared them with the sequences of strains from other geographical locations. H. pylori from Senegal and Madagascar can be placed in the previously described HpAfrica1 genetic population, subpopulations hspWAfrica and hspSAfrica, respectively. These 2 subpopulations correspond to the distribution of Niger-Congo speakers in West and most of subequatorial Africa (due to Bantu migrations), respectively. H. pylori appears as a single population in Senegal, indicating a long common history between ethnicities as well as frequent local admixtures. The lack of differentiation between these isolates and an increasing genetic differentiation with geographical distance between sampling locations in Africa was evidence for genetic isolation by distance. The Austronesian expansion that started from Taiwan 5000 years ago dispersed one of the 10 subgroups of the Austronesian language family via insular Southeast Asia into the Pacific and Madagascar, and hspMaori is a marker for the entire Austronesian expansion. Strain competition and replacement of hspMaori by hpAfrica1 strains from Bantu migrants are the probable reasons for the presence of hspSAfrica strains in Malagasy of Southeast Asian descent. hpAfrica1 strains appear to be generalist strains that have the necessary genetic diversity to efficiently colonise a wide host spectrum.     

Characteristics of a PCR-Based Assay for In Planta Detection of Xanthomonas campestris pv. pelargonii

Polymerase chain reaction (PCR) amplification was carried out with a primer pair targeting a sequence in the genome of Xanthomonas campestris pv. pelargonii , the causative agent of bacterial blight in geraniums. PCR amplification with the primer pair XcpMl/XcpM2 using total nucleic acid preparations from 22 geographicallydiverse isolates of X. campestris pv. pelargonii generated a major 197 bp DNA product. In contrast, no major amplification products were consistently generated from 12 other pathovars of X. campestris or from 19 isolates representing 10 different plant pathogenic bacteria, including two other bacterial pathogens of geraniums, Corynebacterium fascians and Pseudomonas cichorii . After PCR using this primer pair, between 1380 and 13800 copies of the X, campestris pv. pelargonii bacterial DNA target as template were detected by ethidium bromide staining of agarose gels, and between 13.8 and 138 copies by blot hybridization to a pathovar-specific biotinylated probe. Similarly, between 630 and 6300 colonyforming units (CFU) of X. campestris pv. pelargonii could be detected after ethidium bromide staining of agarose gels, and between 63 and 630 CFU after blot hybridization. The PCR-based assay was used to identify X. campestris pv. pelargonii in diseased geraniums; whereas discrete amplification products were not obtained with healthy plants.     

Meta-analysis of Dense Genecentric Association Studies Reveals Common and Uncommon Variants Associated with Height

Height is a classic complex trait with common variants in a growing list of genes known to contribute to the phenotype. Using a genecentric genotyping array targeted toward cardiovascular-related loci, comprising 49,320 SNPs across approximately 2000 loci, we evaluated the association of common and uncommon SNPs with adult height in 114,223 individuals from 47 studies and six ethnicities. A total of 64 loci contained a SNP associated with height at array-wide significance (p < 2.4 × 10⁻⁶), with 42 loci surpassing the conventional genome-wide significance threshold (p < 5 × 10⁻⁸). Common variants with minor allele frequencies greater than 5% were observed to be associated with height in 37 previously reported loci. In individuals of European ancestry, uncommon SNPs in IL11 and SMAD3, which would not be genotyped with the use of standard genome-wide genotyping arrays, were strongly associated with height (p < 3 × 10⁻¹¹). Conditional analysis within associated regions revealed five additional variants associated with height independent of lead SNPs within the locus, suggesting allelic heterogeneity. Although underpowered to replicate findings from individuals of European ancestry, the direction of effect of associated variants was largely consistent in African American, South Asian, and Hispanic populations. Overall, we show that dense coverage of genes for uncommon SNPs, coupled with large-scale meta-analysis, can successfully identify additional variants associated with a common complex trait.     

Cardiac autonomic balance in small-for-gestational-age neonates

The cardiac sympathetic nervous system is one putative key factor involved in the intrauterine programming of adult cardiovascular disease. We therefore analyzed cardiac autonomic system activity in small for gestational age (SGA) neonates. Heart rate variability (HRV) from 24-h ECG recordings were analyzed for time-domain and frequency-domain parameters in 27 SGA neonates [median 261 (240-283) days of gestation] compared with 27 appropriate for gestational age (AGA) neonates [median 270 (239-293) days of gestation]. In addition, salivary alpha-amylase levels were analyzed during resting conditions and in response to a pain-induced stress event in 18 SGA [median 266 (240-292) days of gestation] and 34 AGA [median 271 (240-294) days of gestation] neonates. Overall HRV was not significantly different in SGA neonates compared with AGA neonates (SD of all valid NN intervals: P = 0.14; triangular index: P = 0.29), and the sympathovagal balance [low frequency (LF)/high frequency (HF)] was similar (P = 0.62). Parameters mostly influenced by sympathetic activity did not reveal significant differences: (SD of the average of valid NN intervals: P = 0.27; average of the hourly means of SDs of all NN intervals: P = 0.66, LF: P = 0.83) as well as vagal tone-influenced parameters were unaltered (average of the hourly square root of the mean of the sum of the squares of differences between adjacent NN intervals: P = 0.59; proportion of pairs of adjacent NN intervals differing by >50 ms: P = 0.93; HF: P = 0.82). Median resting levels for alpha-amylase were not significantly different in SGA neonates (P = 0.13), and a neonatal stress stimulus revealed similar stress response patterns (P = 0.29). HRV and salivary alpha-amylase levels as indicators of cardiac autonomic activity were not altered in SGA neonates compared with AGA neonates. Thus, it appears that the intrauterine activation of the sympathetic system in SGA fetuses does not directly persist into postnatal life, and neonatal sympathovagal balance appears to be preserved.     

ARF1 regulates Nef-induced CD4 degradation

BACKGROUND: The HIV Nef protein downregulates CD4 through sequential connection with clathrin-coated pits and the COP1 coatomer, resulting in accelerated endocytosis and lysosomal targeting. RESULTS: Here we report that the small GTPase ARF1 controls the Nef-induced, COP-mediated late-endosomal targeting of CD4. We find that Nef binds ARF1 directly and can recruit the GTPase onto endosomal membranes. Furthermore, a complex comprising Nef, ARF1, and betaCOP can be immunoprecipitated from cells expressing the viral protein. Residues in a C-terminal loop of the viral protein facilitate both these interactions and the targeting of Nef and CD4 to acidic late endosomes, whereas other residues primarily involved in mediating CD4 endocytosis are dispensable for this process. Finally, a dominant-negative ARF1 mutant blocks the migration of the Nef-CD4 complex to lysosomes. CONCLUSIONS: Our results support a model in which ARF1 is the immediate downstream partner of Nef for CD4 lysosomal targeting.     

A fruiting body tissue method for efficient Agrobacterium-mediated transformation of Agaricus bisporus

We describe a modified Agrobacterium-mediated method for the efficient transformation of Agaricus bisporus. Salient features of this procedure include cocultivation of Agrobacterium and fruiting body gill tissue and use of a vector with a homologous promoter. This method offers new prospects for the genetic manipulation of this commercially important mushroom species.     

PCR-based genotyping of epidemic and preepidemic Trichoderma isolates associated with green mold of Agaricus bisporus.

We used randomly amplified polymorphic DNA (RAPD)-PCR to estimate genetic variation among isolates of Trichoderma associated with green mold on the cultivated mushroom Agaricus bisporus. Of 83 isolates examined, 66 were sampled during the recent green mold epidemic, while the remaining 17 isolates were collected just prior to the epidemic and date back to the 1950s. Trichoderma harzianum biotype 4 was identified by RAPD analysis as the cause of almost 90% of the epidemic-related episodes of green mold occurring in the major commercial mushroom-growing region in North America. Biotype 4 was more closely allied to T. harzianum biotype 2, the predominant pathogenic genotype in Europe, than to the less pathogenic biotype 1 and Trichoderma atroviride (formerly T. harzianum biotype 3). No variation in the RAPD patterns was observed among the isolates within biotype 2 or 4, suggesting that the two pathogenic biotypes were populations containing single clones. Considerable genetic variation, however, was noted among isolates of biotype 1 and T. atroviride from Europe. Biotype 4 was not represented by the preepidemic isolates of Trichoderma as determined by RAPD markers and PCR amplification of an arbitrary DNA sequence unique to the genomes of biotypes 2 and 4. Our findings suggest that the onset of the green mold epidemic in North America resulted from the recent introduction of a highly virulent genotype of the pathogen into cultivated mushrooms.     

A polymerase chain reaction-based test for the identification of Trichoderma harzianum biotypes 2 and 4, responsible for the worldwide green mold epidemic in cultivated Agaricus bisporus

We describe a polymerase chain reaction (PCR)-based test that is specific for the pathogenic European biotype 2 (Th2) and North American biotype 4 (Th4) of Trichoderma harzianum, responsible for the green mold epidemic in the cultivated mushroom, Agaricus bisporus. A PCR primer pair was designed that targets a 444-bp arbitrary sequence in the genome of Th4. The primers also amplified the same product with Th2, but showed no reactivity with other biotypes of T. harzianum, several biocontrol Trichoderma, or with 31 other genera and species of fungi. The PCR-based test should have application in disease management programs, and in the evaluation of biocontrol Trichoderma for potential pathogenicity on mushrooms. Received: 23 November 1998 / Received revision: 19 February 1999 / Accepted: 5 March 1999     

Progenitors Mobilized by Gamma-Tocotrienol as an Effective Radiation Countermeasure

The purpose of this study was to elucidate the role of gamma-tocotrienol (GT3)-mobilized progenitors in mitigating damage to mice exposed to a supralethal dose of cobalt-60 gamma-radiation. CD2F1 mice were transfused 24 h post-irradiation with whole blood or isolated peripheral blood mononuclear cells (PBMC) from donors that had received GT3 72 h prior to blood collection and recipient mice were monitored for 30 days. To understand the role of GT3-induced granulocyte colony-stimulating factor (G-CSF) in mobilizing progenitors, donor mice were administered a neutralizing antibody specific to G-CSF or its isotype before blood collection. Bacterial translocation from gut to heart, spleen and liver of irradiated recipient mice was evaluated by bacterial culture on enriched and selective agar media. Endotoxin in serum samples also was measured. We also analyzed the colony-forming units in the spleens of irradiated mice. Our results demonstrate that whole blood or PBMC from GT3-administered mice mitigated radiation injury when administered 24 h post-irradiation. Furthermore, administration of a G-CSF antibody to GT3-injected mice abrogated the efficacy of blood or PBMC obtained from such donors. Additionally, GT3-mobilized PBMC inhibited the translocation of intestinal bacteria to the heart, spleen, and liver, and increased colony forming unit-spleen (CFU-S) numbers in irradiated mice. Our data suggests that GT3 induces G-CSF, which mobilizes progenitors and these progenitors mitigate radiation injury in recipient mice. This approach using mobilized progenitor cells from GT3-injected donors could be a potential treatment for humans exposed to high doses of radiation.