CRISPR/Cas9 activity in the rice <Emphasis Type="Italic">OsBEIIb</Emphasis> gene does not induce off-target effects in the closely related paralog <Emphasis Type="Italic">OsBEIIa</Emphasis>

Genome editing with the CRISPR/Cas9 system allows mutations to be induced at any 20-bp target site in the genome preceded by the short protospacer adjacent motif (PAM) 5′-NGG-3′. The brevity and degeneracy of the PAM ensures that the motif occurs every ~10 bp in plant genomes, and all plant genes therefore contain many targetable sites. However, the CRISPR/Cas9 system tolerates up to three mismatches in the target site, so the ability to target genes in a specific manner requires the design of synthetic guide RNAs (sgRNAs) that do not bind off-target sites anywhere else in the genome. This is straightforward for single-copy genes but more challenging if a target gene has one or more paralogs because the principles that balance targeting efficiency (the frequency of on-target mutations) and accuracy (the absence of off-target mutations) are not fully understood and may be partially species-dependent. To investigate this phenomenon in rice, we targeted the rice starch branching enzyme IIb gene (OsBEIIb) with two sgRNAs designed to differ at two and six positions, respectively, from corresponding sites in the close paralog OsBEIIa. In each case, half of the mismatches were in the essential seed region immediately upstream of the PAM, where exact pairing is thought to be necessary, and the other half were in the distal part of the target. The sgRNAs also differed in predicted targeting efficiency (39 and 96 %, respectively). We found that the sgRNA with the low predicted efficiency was actually the most efficient in practice, achieving a mutation frequency of 5 % at the target site, whereas the sgRNA with the high predicted efficiency generated no mutations at the second target site. Furthermore, neither of the sgRNAs induced an off-target mutation in the OsBEIIa gene. Our data indicate that efficiency predictions should be tested empirically because they do not always reflect the experimental outcome and that a 1-bp mismatch in the seed region of a sgRNA is sufficient to avoid off-target effects even in closely related rice genes.     

Effects of climate change and horticultural use on the spread of naturalized alien garden plants in Europe

Climate warming is supposed to enlarge the area climatically suitable to the naturalization of alien garden plants in temperate regions. However, the effects of a changing climate on the spread of naturalized ornamentals have not been evaluated by spatially and temporarily explicit range modelling at larger scales so far. Here, we assess how climate change and the frequency of cultivation interactively determine the spread of 15 ornamental plants over the 21st century in Europe. We coupled species distribution modelling with simulations of demography and dispersal to predict range dynamics of these species in annual steps across a 250 × 250 m raster of the study area. Models were run under four scenarios of climate warming and six levels of cultivation intensity. Cultivation frequency was implemented as size of the area used for planting a species. Although the area climatically suitable to the 15 species increases, on average, the area predicted to be occupied by them in 2090 shrinks under two of the three climate change scenarios. This contradiction obviously arises from dispersal limitations that were pronounced although we assumed that cultivation is spatially adapting to the changing climate. Cultivation frequency had a much stronger effect on species spread than climate change, and this effect was non‐linear. The area occupied increased sharply from low to moderate levels of cultivation intensity, but levelled off afterwards. Our simulations suggest that climate warming will not necessarily foster the spread of alien garden plants in Europe over the next decades. However, climatically suitable areas do increase and hence an invasion debt is likely accumulating. Restricting cultivation of species can be effective in preventing species spread, irrespective of how the climate develops. However, for being successful, they depend on high levels of compliance to keep propagule pressure at a low level.     

Effect of Retreatment with Interferon Alone or Interferon plus Ribavirin on Hepatitis C Virus Quasispecies Diversification in Nonresponder Patients with Chronic Hepatitis C

Alpha interferon (IFN-alpha) treatment is effective on a long-term basis in only 15 to 25% of patients with chronic hepatitis C. The results of recent trials indicate that response rates can be significantly increased when IFN-alpha is given in combination with ribavirin. However, a large number of patients do not respond even to combination therapy. Nonresponsiveness to IFN is characterized by evolution of the hepatitis C virus (HCV) quasispecies. Little is known about the changes occurring within the HCV genomes when nonresponder patients are retreated with IFN or with IFN plus ribavirin. In the present study we have examined the genetic divergence of HCV quasispecies during unsuccessful retreatment with IFN or IFN plus ribavirin. Fifteen nonresponder patients with HCV-1 (4 patients with HCV-1a and 11 patients with HCV-1b) infection were studied while being retreated for 2 months (phase 1) with IFN-alpha (6 MU given three times a week), followed by IFN plus ribavirin or IFN alone for an additional 6 months (phase 2). HCV quasispecies diversification in the E2 hypervariable region-1 (HVR1) and in the putative NS5A IFN sensitivity determining region (ISDR) were analyzed for phase 1 and phase 2 by using the heteroduplex tracking assay and clonal frequency analysis techniques. A major finding of this study was the relatively rapid evolution of the HCV quasispecies observed in both treatment groups during the early phase 1 compared to the late phase 2 of treatment. The rate of quasispecies diversification in HVR1 was significantly higher during phase 1 versus phase 2 both in patients who received IFN plus ribavirin (P = 0.017) and in patients who received IFN alone (P = 0. 05). A trend toward higher rates of quasispecies evolution in the ISDR was also observed during phase 1 in both groups, although the results did not reach statistical significance. However, the NS5A quasispecies appeared to be rather homogeneous and stable in most nonresponder patients, suggesting the presence of a single well-fit major variant, resistant to antiviral treatment, in agreement with published data which have identified an IFN sensitivity determinant region within the NS5A. During the entire 8 months of retreatment, there was no difference in the rate of fixation of mutation between patients who received combination therapy and patients who were treated with IFN alone, suggesting that ribavirin had no major effects on the evolution of the HCV quasispecies after the initial 2 months of IFN therapy.     

Evidence of doubly uniparental inheritance of the mitochondrial DNA in Polititapes rhomboides (Bivalvia,Veneridae): Evolutionary and population genetic analysis of F and M mitotypes

Doubly uniparental inheritance (DUI) is a particular mitochondrial DNA inheritance mode reported in a number of bivalves. DUI species show two types of mtDNA, one transmitted from females to daughters and sons (F mitotype) and another one from males to sons (M mitotype). In Veneridae, the existence of DUI has been investigated in several species but it was found in only two of them. In this study, we obtained partial sequences of rrnL, cytb and cox1 genes of males and females of Polititapes rhomboides from NW Spain and we demonstrated the existence of heteroplasmy in males, as expected under DUI. F and M mitotypes showed a taxon-specific phylogenetic pattern and similar evolutionary rates. We focused on cox1 for population genetic analysis, examining separately F and M mitotypes, but also F mitotypes from females (F_♀) and males (F_♂). In all cases, cox1 bears signs of strong purifying selection, with no apparent evidence of relaxed selection in the M genome, while the divergence between F and M genomes is in agreement with the neutral model of evolution. The cox1 polymorphism, higher at the M than at the F genome, also shows clear footprints of genetic hitchhiking with favourable mutations at other mtDNA loci, except for F_♂. In terms of population structure, results suggest that the pattern depends on the examined mitotype (F, F_♀, F_♂ or M).     

Human Papillomavirus Infection in a Male Population Attending a Sexually Transmitted Infection Service

Objective

Human Papillomavirus (HPV) infection in men may produce cancer and other major disorders. Men play an important role in the transmission of the virus and act as a reservoir. The aim of this study was to determine the HPV-genotypes and their prevalence in a group of men attending a Sexually Transmitted Infection service.

Patients and Samples

Between July 2002 and June 2011, 1392 balanopreputial, 435 urethral, 123 anal, and 67 condyloma lesions from 1551 men with a mean age of 35.8±11.3 years old (range: 17–87) were collected for HPV-DNA testing.

Methods

A fragment of the L1-gene and a fragment of the E6/E7-genes were amplified by PCR. Positive samples were typed by hybridization.

Results

The HPV genome was detected in 36.9% (486/1318) balanopreputial and in 24.9% (101/405) urethral (p<0.0001) swabs from 38.1% (538) of 1469 men. Co-infections were present in 5.4% (80/1469) of cases. HPV was found in 43.9% (373/850) of men younger than 35 vs. 31.7% (187/589) of men aged >35. HPV was found in 59.4% (104) of 165 men with lesions (macroscopic or positive peniscopy), and in 22.8% (61/267) without clinical alterations. HPV was also detected in 71.4% (40/56) men with condylomata and in 58.7% (64/109) of men with positive peniscopy.

Conclusions

HPV prevalence in men was high and decreased with age. HPV was found more frequently in balanopreputial than in urethral swabs. There was a low rate of co-infections. Low-risk HPV vaccine genotypes were the most recurrent especially in younger. Although HPV has been associated with clinical alterations, it was also found in men without any clinical presentation. Inclusion of men in the national HPV vaccination program may reduce their burden of HPV-related disease and reduce transmission of the virus to non-vaccinated women.     

Lack of molecular relationships between lipid peroxidation and mitochondrial DNA single strand breaks in isolated rat hepatocytes and mitochondria

We investigated the molecular relationships between lipid peroxidation and mitochondrial DNA (mtDNA) single strand breaks (ssb) in isolated rat hepatocytes and mitochondria exposed to tert-butylhydroperoxide (TBH). Our results show that mtDNA ssb induced by TBH are independent of lipid peroxidation and dependent on the presence of iron and of hydroxyl free radicals. These data contribute to the definition of the mechanisms whereby mtDNA ssb are induced and provide possible molecular targets for the prevention of this kind of damage in vivo.     

Consequences of the iron-dependent formation of ferredoxin and flavodoxin on photosynthesis and nitrogen fixation on Anabaena strains

Iron-dependent formation of ferredoxin and flavodoxin was determined in Anabaena ATCC 29413 and ATCC 29211 by a FPLC procedure. In the first species ferredoxin is replaced by flavodoxin at low iron levels in the vegetative cells only. In the heterocysts from Anabaena ATCC 29151, however, flavodoxin is constitutively formed regardless of the iron supply.Replacement of ferredoxin by flavodoxin had no effect on photosynthetic electron transport, whereas nitrogen fixation was decreased under low iron conditions. As ferredoxin and flavodoxin exhibited the same K_m values as electron donors to nitrogenase, an iron-limited synthesis of active nitrogenase was assumed as the reason for inhibited nitrogen fixation. Anabaena ATCC 29211 generally lacks the potential to synthesize flavodoxin. Under iron-starvation conditions, ferredoxin synthesis is limited, with a negative effect on photosynthetic oxygen evolution.     

An erythrocyte vesicle protein exported by the malaria parasite promotes tubovesicular lipid import from the host cell surface

Plasmodium falciparum is the protozoan parasite that causes the most virulent of human malarias. The blood stage parasites export several hundred proteins into their host erythrocyte that underlie modifications linked to major pathologies of the disease and parasite survival in the blood. Unfortunately, most are 'hypothetical' proteins of unknown function, and those that are essential for parasitization of the erythrocyte cannot be 'knocked out'. Here, we combined bioinformatics and genome-wide expression analyses with a new series of transgenic and cellular assays to show for the first time in malaria parasites that microarray read out from a chemical perturbation can have predictive value. We thereby identified and characterized an exported P. falciparum protein resident in a new vesicular compartment induced by the parasite in the erythrocyte. This protein, named Erythrocyte Vesicle Protein 1 (EVP1), shows novel dynamics of distribution in the parasite and intraerythrocytic membranes. Evidence is presented that its expression results in a change in TVN-mediated lipid import at the host membrane and that it is required for intracellular parasite growth, but not invasion. This exported protein appears to be needed for the maintenance of an essential tubovesicular nutrient import pathway induced by the pathogen in the host cell. Our approach may be generalized to the analysis of hundreds of 'hypothetical' P. falciparum proteins to understand their role in parasite entry and/or growth in erythrocytes as well as phenotypic contributions to either antigen export or tubovesicular import. By functionally validating these unknowns, one may identify new targets in host-microbial interactions for prophylaxis against this major human pathogen.     

Circulating miRNA‐375 as a potential novel biomarker for active Kaposi’s sarcoma in AIDS patients

The aim of this study was to identify circulating microRNAs (miRNAs) that could be used as biomarkers in patients at risk for or affected by AIDS‐Kaposi's sarcoma (KS). Screening of 377 miRNAs was performed using low‐density arrays in pooled plasma samples of 10 HIV/human herpesvirus 8 (HHV8)‐infected asymptomatic and 10 AIDS‐KS patients before and after successful combined antiretroviral therapy (cART). MiR‐375 was identified as a potential marker of active KS, being the most down‐regulated in AIDS‐KS patients after cART and the most up‐regulated in naïve AIDS‐KS patients compared to naïve asymptomatic subjects. Validation on individual plasma samples confirmed that miR‐375 levels were higher in AIDS‐KS compared to asymptomatic patients, decreased after cART‐induced remission in most AIDS‐KS patients and increased in patients with active KS. In asymptomatic patients miR‐375 was up‐regulated after cART in both screening and validation. Statistical analyses revealed an association between miR‐375 changes and CD4 cell counts, which could explain the discordant cases and the opposite trend between asymptomatic and AIDS‐KS patients. These data suggest that circulating miR‐375 might be a good indicator of active AIDS‐KS. Moreover, changes in miR‐375 levels may have a prognostic value in HIV/HHV8‐infected patients undergoing treatment. Further large‐scale validation is needed.