Genetic interaction motif finding by expectation maximization – a novel statistical model for inferring gene modules from synthetic lethality

Background  

Synthetic lethality experiments identify pairs of genes with complementary function. More direct functional associations (for example greater probability of membership in a single protein complex) may be inferred between genes that share synthetic lethal interaction partners than genes that are directly synthetic lethal. Probabilistic algorithms that identify gene modules based on motif discovery are highly appropriate for the analysis of synthetic lethal genetic interaction data and have great potential in integrative analysis of heterogeneous datasets.     

Acute intermittent porphyria caused by a C→T mutation that produces a stop codon in the porphobilinogen deaminase gene

Summary A mutation of the porphobilinogen (PBG) deaminase gene that produces the cross-reacting immunological material (CRIM)-negative type of acute intermittent porphyria (AIP) has been identified in one of 43 unrelated patients with this form of the disorder. The mutation is a CT transition that abolishes a PstI recognition site in exon 9 of the gene and converts a codon for glutamine to a stop codon.     

ααααanti-3.7 type II: a new α-globin gene rearrangement suggesting that the α-globin gene duplication could be caused by intrachromosomal recombination

Summary We report here a new human -globin gene rearrangement carrying the two normal, 2 and 1, and two hybrid, 1/2, globin genes in the order 5-2-1/2-1/2-1-3. Both the hybrid genes, subtyped with ApaI and RsaI restriction enzymes, were found to be of the uncommon anti 3.7 type II. The hybrid genes were expressed at the biosynthetic level and their interaction with the -thalassaemia IVS 1 nt 1 GA mutation caused thalassaemia intermedia. We also report a case of an -globin gene rearrangement in the twin of one of the -globin gene carriers; the duplicated gene was of the anti 4.2 type and was associated with the absence of RsaI polymorphism. The singular finding of an -anti 3.7 cluster with two identical rare hybrid genes suggests that the reciprocal unequal recombination causing the -globin gene rearrangements could be of the intra-chromosomal rather than the interchromosomal type.     

Are stunted child – overweight mother pairs a real defined entity or a statistical artifact?

In a methodological contribution, Dieffenbach & Stein (DS) (The Journal of Nutrition, 142(4), 771–773.) concluded that the double burden of malnutrition (DBM), represented by stunted child – overweight mother pairs (SCOM), is a statistical artifact, meaning that SCOM does not describe a unique phenomenon because the observed rates of SCOM across a number of countries were not strongly different from the product of observed rates of maternal overweight (OM) and child stunting (SC), which DS referred to as the expected rate of SCOM. However, a growing literature continues to use SCOM as an indicator of the DBM. This study shows that the analysis by DS is not sufficient to conclude that SCOM can be explained by the co-occurrence of OM and SC due to chance alone because the analysis by DS was conducted at the country level, but applied to SCOM, which is a household-level variable. Using Demographic and Health Surveys data from 202 country-year data sets, we do not confirm important implicit assumptions that are required for the claim by DS to be supported. We also outline that comparing the expected to the observed rate of SCOM is primarily informative when putting it in relation to factors that influence the supply and demand of food consumed by households. When considering these factors, we find further evidence that it is misleading to consider SCOM as a statistical artifact, as the difference between the observed and the expected rate of SCOM significantly differs by household wealth. Recognizing that SCOM is a distinct phenomenon is important for policymakers who develop double-duty strategies that address malnutrition, and for researchers who need useful indicators to study the determinants of malnutrition at the household level.     

Dose–response relationships and statistical performance of a battery of bacterial gene profiling assays

Because of increasing awareness and legislative demands, there is a demand for the development and use of biological assays for the assessment of the toxicity of chemicals, environmental samples. Recently, a growing number of bacterial reporter assays have been developed and implemented. Nevertheless, little data is published on the performance of these assays in terms of analytical parameters. We present results on a battery of 14 transgenic Escherichia coli strains carrying different promoter::reporter fusions. Growth characteristics and basal expression levels were modeled and fitted, data show that growth curves should be taken into account during test development. Our study shows that the induction profiles reflect the mode of action, e.g., paraquat clearly induces the soxRS operon. The sensitivity of the assay compares well to that of whole organism tests, e.g., fish and Daphnia for polar organics. Metal toxicity is detected less efficiently, e.g., cadmium is detected near the LC50 of carp, considered a relatively insensitive species towards cadmium. The assay variability ranges from 10 to 40% depending on the strain, comparable to that of other bioassays. The variability was shown to be determined by the intrinsic traits of the promoter–strain combination, not by operating conditions. Electronic supplementary material Supplementary material is available for this article at and is accessible for authorized users.     

TMEM8 – a non-globin gene entrapped in the globin web

For more than 30 years it was believed that globin gene domains included only genes encoding globin chains. Here we show that in chickens, the domain of α-globin genes also harbor the non-globin gene TMEM8. It was relocated to the vicinity of the α-globin cluster due to inversion of an ∼170-kb genomic fragment. Although in humans TMEM8 is preferentially expressed in resting T-lymphocytes, in chickens it acquired an erythroid-specific expression profile and is upregulated upon terminal differentiation of erythroblasts. This correlates with the presence of erythroid-specific regulatory elements in the body of chicken TMEM8, which interact with regulatory elements of the α-globin genes. Surprisingly, TMEM8 is not simply recruited to the α-globin gene domain active chromatin hub. An alternative chromatin hub is assembled, which includes some of the regulatory elements essential for the activation of globin gene expression. These regulatory elements should thus shuttle between two different chromatin hubs.     

Midkine is a NF-κB-inducible gene that supports prostate cancer cell survival

Background

Atherosclerotic peripheral arterial disease (PAD) affects 8–10 million people in the United States and is associated with a marked impairment in quality of life and an increased risk of cardiovascular events. Noninvasive assessment of PAD is performed by measuring the ankle-brachial index (ABI). Complex traits, such as ABI, are influenced by a large array of genetic and environmental factors and their interactions. We attempted to characterize the genetic architecture of ABI by examining the main and interactive effects of individual single nucleotide polymorphisms (SNPs) and conventional risk factors.

Methods

We applied linear regression analysis to investigate the association of 435 SNPs in 112 positional and biological candidate genes with ABI and related physiological and biochemical traits in 1046 non-Hispanic white, hypertensive participants from the Genetic Epidemiology Network of Arteriopathy (GENOA) study. The main effects of each SNP, as well as SNP-covariate and SNP-SNP interactions, were assessed to investigate how they contribute to the inter-individual variation in ABI. Multivariable linear regression models were then used to assess the joint contributions of the top SNP associations and interactions to ABI after adjustment for covariates. We reduced the chance of false positives by 1) correcting for multiple testing using the false discovery rate, 2) internal replication, and 3) four-fold cross-validation.

Results

When the results from these three procedures were combined, only two SNP main effects in NOS3, three SNP-covariate interactions (ADRB2 Gly 16 – lipoprotein(a) and SLC4A5 – diabetes interactions), and 25 SNP-SNP interactions (involving SNPs from 29 different genes) were significant, replicated, and cross-validated. Combining the top SNPs, risk factors, and their interactions into a model explained nearly 18% of variation in ABI in the sample. SNPs in six genes (ADD2, ATP6V1B1, PRKAR2B, SLC17A2, SLC22A3, and TGFB3) were also influencing triglycerides, C-reactive protein, homocysteine, and lipoprotein(a) levels.

Conclusion

We found that candidate gene SNP main effects, SNP-covariate and SNP-SNP interactions contribute to the inter-individual variation in ABI, a marker of PAD. Our findings underscore the importance of conducting systematic investigations that consider context-dependent frameworks for developing a deeper understanding of the multidimensional genetic and environmental factors that contribute to complex diseases.     

Have p53 gene mutations and protein expression a different biological significance in colorectal cancer?*

p53 alterations are considered the most common genetic events in many types of neoplasms, including colorectal carcinoma (CRC). These alterations include mutations of the gene and/or overexpression of the protein. The aim of our study was to assess whether in 160 patients undergoing resective surgery for primary operable CRC there was an association between p53 mutations and protein overexpression and between these and other biological variables, such as cell DNA content (DNA-ploidy) and S-phase fraction (SPF), and the traditional clinicopathological variables. p53 mutations, identified by PCR-SSCP-sequencing analysis, were found in 68/160 patients (43%) and positive staining for p53 protein, detected with the monoclonal antibody DO-7, was present in 48% (77/160) of the cases, with agreement of 57% (91/160). In particular, a significant association was found between increased p53 expression and genetic alterations localized in the conserved regions of the gene or in the L3 DNA-binding domain and the specific type of mutation. Furthermore, both overexpression of p53 and mutations in the conserved areas of the gene were found more frequently in distal than in proximal CRCs, suggesting that they might be "biologically different diseases." Although p53 mutations in conserved areas were associated with flow cytometric variables, overexpression of p53 and mutations in its L3 domain were only related respectively to DNA-aneuploidy and high SPF. These data may reflect the complex involvement of p53 in the different pathways regulating cell-cycle progression. In conclusion, the combination of the mutational status and immunohistochemistry of p53, and flow cytometric data may provide an important insight into the biological features of CRCs.     

The significance of calcium ions on Pseudomonas fluorescens biofilms – a structural and mechanical study

The purpose of this study was to investigate the effects of calcium ions on the structural and mechanical properties of Pseudomonas fluorescens biofilms grown for 48?h. Advanced investigative techniques such as confocal laser scanning microscopy and atomic force spectroscopy were employed to characterize biofilm structure as well as biofilm mechanical properties following growth at different calcium concentrations. The presence of calcium during biofilm development led to higher surface coverage with distinct structural phenotypes in the form of a granular and heterogeneous surface, compared with the smoother and homogeneous biofilm surface in the absence of calcium. The presence of calcium also increased the adhesive nature of the biofilm, while reducing its elastic properties. These results suggest that calcium ions could have a functional role in biofilm development and have practical implications, for example, in analysis of biofouling in membrane-based water-treatment processes such as nanofiltration or reverse osmosis where elevated calcium concentrations may occur at the solid–liquid interface.     

Attenuator LRR – a regulatory tool for modulating gene expression in Gram-positive bacteria

With the rapid development of synthetic biology in recent years, particular attention has been paid to RNA devices, especially riboswitches, because of their significant and diverse regulatory roles in prokaryotic and eukaryotic cells. Due to the limited performance and context-dependence of riboswitches, only a few of them (such as theophylline, tetracycline and ciprofloxacin riboswitches) have been utilized as regulatory tools in biotechnology. In the present study, we demonstrated that a ribosome-dependent ribo-regulator, LRR, discovered in our previous work, exhibits an attractive regulatory performance. Specifically, it offers a 60-fold change in expression in the presence of retapamulin and a low level of leaky expression of about 1–2% without antibiotics. Moreover, LRR can be combined with different promoters and performs well in Bacillus thuringiensis, B. cereus, B. amyloliquefaciens, and B. subtilis. Additionally, LRR also functions in the Gram-negative bacterium Escherichia coli. Furthermore, we demonstrate its ability to control melanin metabolism in B. thuringiensis BMB171. Our results show that LRR can be applied to regulate gene expression, construct genetic circuits and tune metabolic pathways, and has great potential for many applications in synthetic biology.     

Whole-exome sequencing reveals a recurrent mutation in the cathepsin C gene that causes Papillon–Lefevre syndrome in a Saudi family

Papillon–Lefevre syndrome (PALS) is a rare, autosomal recessive disorder characterized by periodontitis and hyperkeratosis over the palms and soles. Mutations in the cathepsin C gene (CTSC) have been recognized as the cause of PALS since the late 1990s. More than 75 mutations in CTSC have been identified, and phenotypic variability between different mutations has been described. Next generation sequencing is widely used for efficient molecular diagnostics in various clinical practices. Here we investigated a large consanguineous Saudi family with four affected and four unaffected individuals. All of the affected individuals suffered from hyperkeratosis over the palms and soles and had anomalies of both primary and secondary dentition. For molecular diagnostics, we combined whole-exome sequencing and genome-wide homozygosity mapping procedures, and identified a recurrent homozygous missense mutation (c.899G>A; p.Gly300Asp) in exon 7 of CTSC. Validation of all eight family members by Sanger sequencing confirmed co-segregation of the pathogenic variant (c.899G>A) with the disease phenotype. This is the first report of whole-exome sequencing performed for molecular diagnosis of PALS in Saudi Arabia. Our findings provide further insights into the genotype–phenotype correlation of CTSC pathogenicity in PALS.     

Macropore effects on phosphorus acquisition by wheat roots – a rhizotron study

Background and aims

Macropores may be preferential root pathways into the subsoil. We hypothesised that the presence of macropores promotes P-uptake from subsoil, particularly at limited water supply in surface soil. We tested this hypothesis in a rhizotron experiment with spring wheat (Triticum aestivum cv. Scirocco) under variation of fertilisation and irrigation.

Methods

Rhizotrons were filled with compacted subsoil (bulk density 1.4 g cm^?3), underneath a P-depleted topsoil. In half of these rhizotrons the subsoil contained artificial macropores. Spring wheat was grown for 41 days with and without irrigation and ³¹P–addition. Also, a ³³P–tracer was added at the soil surface to trace P-distribution in plants using liquid scintillation counting and radioactive imaging.

Results

Fertilisation and irrigation promoted biomass production and plant P-uptake. Improved growing conditions resulted in a higher proportion of subsoil roots, indicating that the topsoil root system additionally promoted subsoil nutrient acquisition. The presence of macropores did not improve plant growth but tended to increase translocation of ³³P into both above- and belowground biomass. ³³P–imaging confirmed that this plant-internal transport of topsoil-P extended into subsoil roots.

Conclusions

The lack of penetration resistance in macropores did not increase plant growth and nutrient uptake from subsoil here; however, wheat specifically re-allocated topsoil-P for subsoil root growth.

     

Transpiration and metabolisation of TCE by willow plants – a pot experiment

Willows were grown in glass cylinders filled with compost above water-saturated quartz sand, to trace the fate of TCE in water and plant biomass. The experiment was repeated once with the same plants in two consecutive years. TCE was added in nominal concentrations of 0, 144, 288, and 721 mg l^?1. Unplanted cylinders were set-up and spiked with nominal concentrations of 721 mg l^?1 TCE in the second year. Additionally, ¹³C-enriched TCE solution (δ¹³C = 110.3 ‰) was used. Periodically, TCE content and metabolites were analyzed in water and plant biomass. The presence of TCE-degrading microorganisms was monitored via the measurement of the isotopic ratio of carbon (¹³C/¹²C) in TCE, and the abundance of ¹³C-labeled microbial PLFAs (phospholipid fatty acids). More than 98% of TCE was lost via evapotranspiration from the planted pots within one month after adding TCE. Transpiration accounted to 94 to 78% of the total evapotranspiration loss. Almost 1% of TCE was metabolized in the shoots, whereby trichloroacetic acid (TCAA) and dichloroacetic acid (DCAA) were dominant metabolites; less trichloroethanol (TCOH) and TCE accumulated in plant tissues. Microbial degradation was ruled out by δ¹³C measurements of water and PLFAs. TCE had no detected influence on plant stress status as determined by chlorophyll-fluorescence and gas exchange.     

Insertion of a foreign gene into the β-casein locus by Cre-mediated site-specific recombination

The expression of foreign genes in transgenic animals is generally unpredictable as transgenes are integrated at random after pro-nuclear injection into fertilized oocytes. In many cases, transgene expression is inhibited by neighbouring chromatin structures or by the repeated nature of the multiple transgene copies present at the integration site. A strategy involving homologous and site-specific recombination has been devised by which single copies of a foreign gene can be inserted specifically into the locus of a highly expressed gene. As a first step, a loxP recombination target site is introduced by homologous recombination into a predetermined gene locus such that the loxP sequence is placed next to the promoter region and replaces the translational initiation signal. In a subsequent site-specific recombination reaction, a gene of interest can be integrated into the pre-existing loxP site. This biphasic recombination strategy was used to integrate a luciferase reporter gene into the locus of the murine β-casein gene in embryonic stem cells.