Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to ^5′NGG^3′ protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5′ end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data.     

Modelling the Force of Infection for Hepatitis A in an Urban Population-Based Survey: A Comparison of Transmission Patterns in Brazilian Macro-Regions

Background

This study aimed to identify the transmission pattern of hepatitis A (HA) infection based on a primary dataset from the Brazilian National Hepatitis Survey in a pre-vaccination context. The national survey conducted in urban areas disclosed two epidemiological scenarios with low and intermediate HA endemicity.

Methods

A catalytic model of HA transmission was built based on a national seroprevalence survey (2005 to 2009). The seroprevalence data from 7,062 individuals aged 5–69 years from all the Brazilian macro-regions were included. We built up three models: fully homogeneous mixing model, with constant contact pattern; the highly assortative model and the highly assortative model with the additional component accounting for contacts with infected food/water. Curves of prevalence, force of infection (FOI) and the number of new infections with 99% confidence intervals (CIs) were compared between the intermediate (North, Northeast, Midwest and Federal District) and low (South and Southeast) endemicity areas. A contour plot was also constructed.

Results

The anti- HAV IgG seroprevalence was 68.8% (95% CI, 64.8%–72.5%) and 33.7% (95% CI, 32.4%–35.1%) for the intermediate and low endemicity areas, respectively, according to the field data analysis. The models showed that a higher force of infection was identified in the 10- to 19-year-old age cohort (∼9,000 infected individuals per year per 100,000 susceptible persons) in the intermediate endemicity area, whereas a higher force of infection occurred in the 15- to 29-year-old age cohort (∼6,000 infected individuals per year per 100,000 susceptible persons) for the other macro-regions.

Conclusion

Our findings support the shift of Brazil toward intermediate and low endemicity levels with the shift of the risk of infection to older age groups. These estimates of HA force of infection stratified by age and endemicity levels are useful information to characterize the pre-vaccination scenario in Brazil.     

An High-Throughput In Vivo Screening System to Select H3K4-Specific Histone Demethylase Inhibitors

Background

Histone demethylases (HDMs) have a prominent role in epigenetic regulation and are emerging as potential therapeutic cancer targets. The search for small molecules able to inhibit HDMs in vivo is very active but at the present few compounds were found to be specific for defined classes of these enzymes.

Methodology/Principal Findings

In order to discover inhibitors specific for H3K4 histone demethylation we set up a screening system which tests the effects of candidate small molecule inhibitors on a S.cerevisiae strain which requires Jhd2 demethylase activity to efficiently grow in the presence of rapamycin. In order to validate the system we screened a library of 45 structurally different compounds designed as competitive inhibitors of α -ketoglutarate (α-KG) cofactor of the enzyme, and found that one of them inhibited Jhd2 activity in vitro and in vivo. The same compound effectively inhibits human Jumonji AT-Rich Interactive Domain (JARID) 1B and 1D in vitro and increases H3K4 tri-methylation in HeLa cell nuclear extracts (NEs). When added in vivo to HeLa cells, the compound leads to an increase of tri-methyl-H3K4 (H3K4me3) but does not affect H3K9 tri-methylation. We describe the cytostatic and toxic effects of the compound on HeLa cells at concentrations compatible with its inhibitory activity.

Conclusions/Significance

Our screening system is proved to be very useful in testing putative H3K4-specific HDM inhibitors for the capacity of acting in vivo without significantly altering the activity of other important 2-oxoglutarate oxygenases.     

Osteoblast-Specific Krm2 Overexpression and Lrp5 Deficiency Have Different Effects on Fracture Healing in Mice

The canonical Wnt/β-catenin pathway plays a key role in the regulation of bone remodeling in mice and humans. Two transmembrane proteins that are involved in decreasing the activity of this pathway by binding to extracellular antagonists, such as Dickkopf 1 (Dkk1), are the low-density lipoprotein receptor related protein 5 (Lrp5) and Kremen 2 (Krm2). Lrp 5 deficiency (Lrp5^−/−) as well as osteoblast-specific overexpression of Krm2 in mice (Col1a1-Krm2) result in severe osteoporosis occurring at young age. In this study, we analyzed the influence of Lrp5 deficiency and osteoblast-specific overexpression of Krm2 on fracture healing in mice using flexible and semi-rigid fracture fixation. We demonstrated that fracture healing was highly impaired in both mouse genotypes, but that impairment was more severe in Col1a1-Krm2 than in Lrp5^−/− mice and particularly evident in mice in which the more flexible fixation was used. Bone formation was more reduced in Col1a1-Krm2 than in Lrp5^−/− mice, whereas osteoclast number was similarly increased in both genotypes in comparison with wild-type mice. Using microarray analysis we identified reduced expression of genes mainly involved in osteogenesis that seemed to be responsible for the observed stronger impairment of healing in Col1a1-Krm2 mice. In line with these findings, we detected decreased expression of sphingomyelin phosphodiesterase 3 (Smpd3) and less active β-catenin in the calli of Col1a1-Krm2 mice. Since Krm2 seems to play a significant role in regulating bone formation during fracture healing, antagonizing KRM2 might be a therapeutic option to improve fracture healing under compromised conditions, such as osteoporosis.     

Political Systems Affect Mobile and Sessile Species Diversity – A Legacy from the Post-WWII Period

Political ideologies, policies and economy affect land use which in turn may affect biodiversity patterns and future conservation targets. However, few studies have investigated biodiversity in landscapes with similar physical properties but governed by different political systems. Here we investigate land use and biodiversity patterns, and number and composition of birds and plants, in the borderland of Austria, Slovenia and Hungary. It is a physically uniform landscape but managed differently during the last 70 years as a consequence of the political “map” of Europe after World War I and II. We used a historical map from 1910 and satellite data to delineate land use within three 10-kilometre transects starting from the point where the three countries meet. There was a clear difference between countries detectable in current biodiversity patterns, which relates to land use history. Mobile species richness was associated with current land use whereas diversity of sessile species was more associated with past land use. Heterogeneous landscapes were positively and forest cover was negatively correlated to bird species richness. Our results provide insights into why landscape history is important to understand present and future biodiversity patterns, which is crucial for designing policies and conservation strategies across the world.     

Spinosad and the Tomato Borer Tuta absoluta: A Bioinsecticide,an Invasive Pest Threat,and High Insecticide Resistance

The introduction of an agricultural pest species into a new environment is a potential threat to agroecosystems of the invaded area. The phytosanitary concern is even greater if the introduced pest’s phenotype expresses traits that will impair the management of that species. The invasive tomato borer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), is one such species and the characterization of the insecticide resistance prevailing in the area of origin is important to guide management efforts in new areas of introduction. The spinosad is one the main insecticides currently used in Brazil for control of the tomato borer; Brazil is the likely source of the introduction of the tomato borer into Europe. For this reason, spinosad resistance in Brazilian populations of this species was characterized. Spinosad resistance has been reported in Brazilian field populations of this pest species, and one resistant population that was used in this study was subjected to an additional seven generations of selection for spinosad resistance reaching levels over 180,000-fold. Inheritance studies indicated that spinosad resistance is monogenic, incompletely recessive and autosomal with high heritability (h ² = 0.71). Spinosad resistance was unstable without selection pressure with a negative rate of change in the resistance level ( = −0.51) indicating an associated adaptive cost. Esterases and cytochrome P450-dependent monooxygenases titration decreased with spinosad selection, indicating that these detoxification enzymes are not the underlying resistance mechanism. Furthermore, the cross-resistance spectrum was restricted to the insecticide spinetoram, another spinosyn, suggesting that altered target site may be the mechanism involved. Therefore, the suspension of spinosyn use against the tomato borer would be a useful component in spinosad resistance management for this species. Spinosad use against this species in introduced areas should be carefully monitored to prevent rapid selection of high levels of resistance and the potential for its spread to new areas.     

Comparative Salivary Proteome of Hepatitis B- and C-Infected Patients

Hepatitis B and C virus (HBV and HCV) infections are an important cause of cirrhosis and hepatocellular carcinoma. The natural history has a prominent latent phase, and infected patients may remain undiagnosed; this situation may lead to the continuing spread of these infections in the community. Compelling reasons exist for using saliva as a diagnostic fluid because it meets the demands of being an inexpensive, noninvasive and easy-to-use diagnostic method. Indeed, comparative analysis of the salivary proteome using mass spectrometry is a promising new strategy for identifying biomarkers. Our goal is to apply an Orbitrap-based quantitative approach to explore the salivary proteome profile in HBV- and HCV-infected patients. In the present study, whole saliva was obtained from 20 healthy, (control) 20 HBV-infected and 20 HCV-infected subjects. Two distinct pools containing saliva from 10 subjects of each group were obtained. The samples were ultracentrifuged and fractionated, and all fractions were hydrolyzed (trypsin) and injected into an LTQ-VELOS ORBITRAP. The identification and analyses of peptides were performed using Proteome Discoverer1.3 and ScaffoldQ + v.3.3.1. From a total of 362 distinct proteins identified, 344 proteins were identified in the HBV, 326 in the HCV and 303 in the control groups. Some blood proteins, such as flavin reductase (which converts biliverdin to bilirubin), were detected only in the HCV group. The data showed a reduced presence of complement C3, ceruloplasmin, alpha(1)-acid glycoprotein and alpha(2)-acid glycoprotein in the hepatitis-infected patients. Peptides of serotransferrin and haptoglobin were less detected in the HCV group. This study provides an integrated perspective of the salivary proteome, which should be further explored in future studies targeting specific disease markers for HBV and HCV infection.     

Immunogenicity of a Killed Bivalent (O1 and O139) Whole Cell Oral Cholera Vaccine,Shanchol, in Haiti

Background

Studies of the immunogenicity of the killed bivalent whole cell oral cholera vaccine, Shanchol, have been performed in historically cholera-endemic areas of Asia. There is a need to assess the immunogenicity of the vaccine in Haiti and other populations without historical exposure to Vibrio cholerae.

Methodology/Principal Findings

We measured immune responses after administration of Shanchol, in 25 adults, 51 older children (6–17 years), and 47 younger children (1–5 years) in Haiti, where cholera was introduced in 2010. A≥4-fold increase in vibriocidal antibody titer against V. cholerae O1 Ogawa was observed in 91% of adults, 74% of older children, and 73% of younger children after two doses of Shanchol; similar responses were observed against the Inaba serotype. A≥2-fold increase in serum O-antigen specific polysaccharide IgA antibody levels against V. cholerae O1 Ogawa was observed in 59% of adults, 45% of older children, and 61% of younger children; similar responses were observed against the Inaba serotype. We compared immune responses in Haitian individuals with age- and blood group-matched individuals from Bangladesh, a historically cholera-endemic area. The geometric mean vibriocidal titers after the first dose of vaccine were lower in Haitian than in Bangladeshi vaccinees. However, the mean vibriocidal titers did not differ between the two groups after the second dose of the vaccine.

Conclusions/Significance

A killed bivalent whole cell oral cholera vaccine, Shanchol, is highly immunogenic in Haitian adults and children. A two-dose regimen may be important in Haiti, and other populations lacking previous repeated exposures to V. cholerae.     

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.     

The function of glutaredoxin GRXS15 is required for lipoyl-dependent dehydrogenases in mitochondria

Iron–sulfur (Fe–S) clusters are ubiquitous cofactors in all life and are used in a wide array of diverse biological processes, including electron transfer chains and several metabolic pathways. Biosynthesis machineries for Fe–S clusters exist in plastids, the cytosol, and mitochondria. A single monothiol glutaredoxin (GRX) is involved in Fe–S cluster assembly in mitochondria of yeast and mammals. In plants, the role of the mitochondrial homolog GRXS15 has only partially been characterized. Arabidopsis (Arabidopsis thaliana) grxs15 null mutants are not viable, but mutants complemented with the variant GRXS15 K83A develop with a dwarf phenotype similar to the knockdown line GRXS15^amiR. In an in-depth metabolic analysis of the variant and knockdown GRXS15 lines, we show that most Fe–S cluster-dependent processes are not affected, including biotin biosynthesis, molybdenum cofactor biosynthesis, the electron transport chain, and aconitase in the tricarboxylic acid (TCA) cycle. Instead, we observed an increase in most TCA cycle intermediates and amino acids, especially pyruvate, glycine, and branched-chain amino acids (BCAAs). Additionally, we found an accumulation of branched-chain α-keto acids (BCKAs), the first degradation products resulting from transamination of BCAAs. In wild-type plants, pyruvate, glycine, and BCKAs are all metabolized through decarboxylation by mitochondrial lipoyl cofactor (LC)-dependent dehydrogenase complexes. These enzyme complexes are very abundant, comprising a major sink for LC. Because biosynthesis of LC depends on continuous Fe–S cluster supply to lipoyl synthase, this could explain why LC-dependent processes are most sensitive to restricted Fe–S supply in grxs15 mutants.