An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional, and long-lasting T cell responses

The EuroVacc 02 phase I trial has evaluated the safety and immunogenicity of a prime-boost regimen comprising recombinant DNA and the poxvirus vector NYVAC, both expressing a common immunogen consisting of Env, Gag, Pol, and Nef polypeptide domain from human immunodeficiency virus (HIV)-1 clade C isolate, CN54. 40 volunteers were randomized to receive DNA C or nothing on day 0 and at week 4, followed by NYVAC C at weeks 20 and 24. The primary immunogenicity endpoints were measured at weeks 26 and 28 by the quantification of T cell responses using the interferon gamma enzyme-linked immunospot assay. Our results indicate that the DNA C plus NYVAC C vaccine regimen was highly immunogenic, as indicated by the detection of T cell responses in 90% of vaccinees and was superior to responses induced by NYVAC C alone (33% of responders). The vaccine-induced T cell responses were (a) vigorous in the case of the env response (mean 480 spot-forming units/10(6) mononuclear cells at weeks 26/28), (b) polyfunctional for both CD4 and CD8 T cell responses, (c) broad (the average number of epitopes was 4.2 per responder), and (d) durable (T cell responses were present in 70% of vaccinees at week 72). The vaccine-induced T cell responses were strongest and most frequently directed against Env (91% of vaccines), but smaller responses against Gag-Pol-Nef were also observed in 48% of vaccinees. These results support the development of the poxvirus platform in the HIV vaccine field and the further clinical development of the DNA C plus NYVAC C vaccine regimen

Alternative PCR protocol using a single primer set for assessing DNA quality in several tissues from a large variety of mammalian species living in areas endemic for leishmaniasis

The aim of this work was to establish a modified pre-diagnostic polymerase chain reaction (PCR) protocol using a single primer set that enables successful amplification of a highly conserved mammalian sequence in order to determine overall sample DNA quality for multiple mammalian species that inhabit areas endemic for leishmaniasis. The gene encoding interphotoreceptor retinoid-binding protein (IRBP), but not other conserved genes, was efficiently amplified in DNA samples from tail skin, ear skin, bone marrow, liver and spleen from all of the species tested. In tissue samples that were PCR-positive for Leishmania, we found that DNA from 100%, 55% and 22% of the samples tested resulted in a positive PCR reaction for the IRBP, beta-actin and beta-globin genes, respectively. Nucleotide sequencing of an IRBP amplicon resolved any questions regarding the taxonomical classification of a rodent, which was previously based simply on the morphological features of the animal. Therefore, PCR amplification and analysis of the IRBP amplicon are suitable for pre-diagnostically assessing DNA quality and identifying mammalian species living in areas endemic to leishmaniasis and other diseases.

Detection of human herpesvirus 6 and 7 DNA in saliva from healthy adults from Rio de Janeiro, Brazil

In this study, we aimed to evaluate virus shedding in the saliva of healthy adults from the metropolitan region of the city of Rio de Janeiro, Brazil, in order to verify the prevalence of both human herpesviruses 6 and 7 (HHV-6, HHV-7). The studied group comprised 182 healthy individuals at Pedro Ernesto University Hospital, who were being seen for annual odontologic revisions. Saliva specimens were subjected to a multiplex polymerase chain reaction (PCR) to detect the presence of HHV-6A, HHV-6B and HHV-7. The total Roseolovirus DNA prevalence was 22.4%. The PCR detected a HHV-6 prevalence of 9.8%, with HHV-6A detected in 7.1% of the samples and HHV-6B in 2.7%. HHV-7 DNA was revealed in 12.6% of the studied cases. Multiple infections caused by HHV-6A and 7 were found in 2.1% of the samples. No statistical differences were observed regarding age, but for HHV-7 infection, an upward trend was observed in female patients. Compared to studies from other countries, low prevalence rates of herpesvirus DNA were detected in saliva from the healthy individuals in our sample. PCR methodology thus proved to be a useful tool for Roseolovirus detection and it is important to consider possible geographic and populations differences that could explain the comparatively low prevalence rates described here.

Intragenomic variation in the second internal transcribed spacer of the ribosomal DNA of species of the genera Culex and Lutzia (Diptera: Culicidae)

Culex is the largest genus of Culicini and includes vectors of several arboviruses and filarial worms. Many species of Culex are morphologically similar, which makes their identification difficult, particularly when using female specimens. To aid evolutionary studies and species distinction, molecular techniques are often used. Sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) from 16 species of the genus Culex and one of Lutzia were used to assess their genomic variability and to verify their applicability in the phylogenetic analysis of the group. The distance matrix (uncorrected p-distance) that was obtained revealed intragenomic and intraspecific variation. Because of the intragenomic variability, we selected ITS2 copies for use in distance analyses based on their secondary structures. Neighbour-joining topology was obtained with an uncorrected p-distance. Despite the heterogeneity observed, individuals of the same species were grouped together and correlated with the current, morphology-based classification, thereby showing that ITS2 is an appropriate marker to be used in the taxonomy of Culex.

Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India

The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.

Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing the Plasmid Template.

We describe a new rapid and efficient polymerase chain reaction (PCR)-based site-directed mutagenesis method. This procedure is effective with any plasmid and it employs four oligonucleotide primers. One primer contains the desired mutation, the second is oriented in the opposite direction (one of these two primers should be phosphorylated), and the third and fourth should be coding in complementary fashion for a unique restriction site to be introduced in a nonessential region. The method consists of two simultaneous PCR reactions; the PCR products are digested with the enzyme that recognizes the newly introduced unique restriction site and then ligased and used to transform competent bacteria. Additionally, the use of Dpn I facilitates the elimination of template DNA. The newly introduced restriction site is essential for ligation in the correct orientation of the two-PCR products and is further used for mutant screening. Resulting plasmids carry both the new restriction site and the desired mutation. Using this method, more than 20 mutants have already been generated (using two different kinds of templates); all these mutants were sequenced for the desired mutation and transfected into AtT-20 cells and the expressed mutant proteins encoded by the vector were assayed.

DNA microarrays and plant defence

Recent progress in understanding plant defence has highlighted a complex, interacting network of signalling pathways leading to the induction of numerous genes. The advent of new technologies for the global analysis of gene expression is fundamentally affecting research in biology, and studies on plant defence should benefit from these new approaches. Genome-wide microarrays will provide a powerful tool for the discovery of all defence-related genes and should help in elucidating their function. The association of a particular signalling pathway with a defence response can be tested with microarrays and defined mutants. Comparison of transcript profiles after biotic and abiotic stresses reveals overlapping activation of defence-related genes and defines new concepts on how plants cope with multiple aggressions. The combination of expression data with other biochemical or metabolite measurements seems another promising approach. Finally, small-scale, dedicated microarrays containing sets of well-characterised genes might prove to be a very useful complement to more expensive, less accessible, large-scale arrays.

One person with two DNA profiles: a(nother) case of mosaicism or chimerism.

Nuclear DNA markers, such as short tandem repeats (STR), are widely used for crime investigation and paternity testing. STR were used to determine whether a piece of tissue regurgitated by a dog was part of the penis of a dead, emasculated, man. Unexpectedly, when analyzing the recovered material and a blood sample from the deceased, five out of the 18 loci differed. According to the results, one could have concluded that these samples originated from two different persons. However, taking into account contextual information and data from complementary genetic analyses, the most likely hypothesis was that the deceased was a genetic mosaic or a chimera. Within a forensic genetic context, such genetic peculiarities may prevent associating the perpetrator of an offense with a stain left at a crime scene or lead to false paternity exclusions. Fast recognition of mosaics or chimeras, adapted sampling scheme, as well as careful interpretation of the data should allow avoiding such pitfalls.

Sources of pre-analytical variations in yield of DNA extracted from blood samples: analysis of 50,000 DNA samples in EPIC.

The European Prospective Investigation into Cancer and nutrition (EPIC) is a long-term, multi-centric prospective study in Europe investigating the relationships between cancer and nutrition. This study has served as a basis for a number of Genome-Wide Association Studies (GWAS) and other types of genetic analyses. Over a period of 5 years, 52,256 EPIC DNA samples have been extracted using an automated DNA extraction platform. Here we have evaluated the pre-analytical factors affecting DNA yield, including anthropometric, epidemiological and technical factors such as center of subject recruitment, age, gender, body-mass index, disease case or control status, tobacco consumption, number of aliquots of buffy coat used for DNA extraction, extraction machine or procedure, DNA quantification method, degree of haemolysis and variations in the timing of sample processing. We show that the largest significant variations in DNA yield were observed with degree of haemolysis and with center of subject recruitment. Age, gender, body-mass index, cancer case or control status and tobacco consumption also significantly impacted DNA yield. Feedback from laboratories which have analyzed DNA with different SNP genotyping technologies demonstrate that the vast majority of samples (approximately 88%) performed adequately in different types of assays. To our knowledge this study is the largest to date to evaluate the sources of pre-analytical variations in DNA extracted from peripheral leucocytes. The results provide a strong evidence-based rationale for standardized recommendations on blood collection and processing protocols for large-scale genetic studies.

NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies.

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

Recurrent de novo mitochondrial DNA mutations in respiratory chain deficiency.

Starting from a cohort of 50 NADH-oxidoreductase (complex I) deficient patients, we carried out the systematic sequence analysis of all mitochondrially encoded complex I subunits (ND1 to ND6 and ND4L) in affected tissues. This approach yielded the unexpectedly high rate of 20% mutation identification in our series. Recurrent heteroplasmic mutations included two hitherto unreported (T10158C and T14487C) and three previously reported mutations (T10191C, T12706C and A13514G) in children with Leigh or Leigh-like encephalopathy. The recurrent mutations consistently involved T-->C transitions (p<10(-4)). This study supports the view that an efficient molecular screening should be based on an accurate identification of respiratory chain enzyme deficiency.

Role of the HCF-1 basic region in sustaining cell proliferation.

BACKGROUND: The human herpes simplex virus-associated host cell factor 1 (HCF-1) is a conserved human transcriptional co-regulator that links positive and negative histone modifying activities with sequence-specific DNA-binding transcription factors. It is synthesized as a 2035 amino acid precursor that is cleaved to generate an amino- (HCF-1(N)) terminal subunit, which promotes G1-to-S phase progression, and a carboxy- (HCF-1(C)) terminal subunit, which controls multiple aspects of cell division during M phase. The HCF-1(N) subunit contains a Kelch domain that tethers HCF-1 to sequence-specific DNA-binding transcription factors, and a poorly characterized so called "Basic" region (owing to a high ratio of basic vs. acidic amino acids) that is required for cell proliferation and has been shown to associate with the Sin3 histone deacetylase (HDAC) component. Here we studied the role of the Basic region in cell proliferation and G1-to-S phase transition assays. METHODOLOGY/PRINCIPAL FINDINGS: Surprisingly, much like the transcriptional activation domains of sequence-specific DNA-binding transcription factors, there is no unique sequence within the Basic region required for promoting cell proliferation or G1-to-S phase transition. Indeed, the ability to promote these activities is size dependent such that the shorter the Basic region segment the less activity observed. We find, however, that the Basic region requirements for promoting cell proliferation in a temperature-sensitive tsBN67 cell assay are more stringent than for G1-to-S phase progression in an HCF-1 siRNA-depletion HeLa-cell assay. Thus, either half of the Basic region alone can support G1-to-S phase progression but not cell proliferation effectively in these assays. Nevertheless, the Basic region displays considerable structural plasticity because each half is able to promote cell proliferation when duplicated in tandem. Consistent with a potential role in promoting cell-cycle progression, the Sin3a HDAC component can associate independently with either half of the Basic region fused to the HCF-1 Kelch domain. CONCLUSIONS/SIGNIFICANCE: While conserved, the HCF-1 Basic region displays striking structural flexibility for controlling cell proliferation.

Comprehensive analysis of NRG1 common and rare variants in Hirschsprung patients.

Hirschsprung disease (HSCR, OMIM 142623) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract, which results in tonic contraction of the aganglionic gut segment and functional intestinal obstruction. The RET proto-oncogene is the major gene for HSCR with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. Many other genes have been described to be associated with the pathology, as NRG1 gene (8p12), encoding neuregulin 1, which is implicated in the development of the enteric nervous system (ENS), and seems to contribute by both common and rare variants. Here we present the results of a comprehensive analysis of the NRG1 gene in the context of the disease in a series of 207 Spanish HSCR patients, by both mutational screening of its coding sequence and evaluation of 3 common tag SNPs as low penetrance susceptibility factors, finding some potentially damaging variants which we have functionally characterized. All of them were found to be associated with a significant reduction of the normal NRG1 protein levels. The fact that those mutations analyzed alter NRG1 protein would suggest that they would be related with HSCR disease not only in Chinese but also in a Caucasian population, which reinforces the implication of NRG1 gene in this pathology.

Prevalence of human papillomavirus and Epstein-Barr virus DNA in penile cancer cases from Brazil

Penile cancer is a potentially mutilating disease. Although its occurrence is relatively rare worldwide, penile cancer rates can be high in developing countries. A few studies have been conducted on the involvement of human papillomavirus (HPV) in penile carcinoma, which have found HPV present in 30-70% of penile malignant lesions, with a higher prevalence of HPV 16 and 18. It has been assumed that cofactors, such as Epstein-Barr virus (EBV) infections, may play a role in the progression of penile neoplasia. The aim of this study was to determine HPV and EBV prevalence in 135 penile malignant lesions from Brazilian men through the use of MY09/11 polymerase chain reaction (PCR), type-specific PCR and restriction fragment length polymorphism analysis. HPV prevalence among the men tested was 60.7%. Of the men who tested positive, 27 presented with HPV 16 (29.7%), five with HPV 18 (5.5%), 21 with HPV 45 (23.1%) and nine with HPV 6 (9.9%). Seven mixed infections were detected (9.2%), while 11 cases remained untyped (13.4%). Regarding EBV positivity, 46.7% of the samples contained EBV DNA with EBV-1 as the most prevalent type (74.6%). More than 23% of the men were co-infected with both HPV and EBV, while 35% presented exclusively with HPV DNA and 20% presented only with EBV DNA. Penile carcinoma aetiology has not been fully elucidated and the role of HPV and EBV infections individually or synergistically is still controversial. Hence, more studies are needed to determine their possible role in carcinogenesis.

DNA multigene sequencing of topotypic specimens of the fascioliasis vector Lymnaea diaphana and phylogenetic analysis of the genus Pectinidens (Gastropoda)

Freshwater lymnaeid snails are crucial in defining transmission and epidemiology of fascioliasis. In South America, human endemic areas are related to high altitudes in Andean regions. The species Lymnaea diaphana has, however, been involved in low altitude areas of Chile, Argentina and Peru where human infection also occurs. Complete nuclear ribosomal DNA 18S, internal transcribed spacer (ITS)-2 and ITS-1 and fragments of mitochondrial DNA 16S and cytochrome c oxidase (cox)1 genes of L. diaphana specimens from its type locality offered 1,848, 495, 520, 424 and 672 bp long sequences. Comparisons with New and Old World Galba/Fossaria, Palaearctic stagnicolines, Nearctic stagnicolines, Old World Radix and Pseudosuccinea allowed to conclude that (i) L. diaphana shows sequences very different from all other lymnaeids, (ii) each marker allows its differentiation, except cox1 amino acid sequence, and (iii) L. diaphana is not a fossarine lymnaeid, but rather an archaic relict form derived from the oldest North American stagnicoline ancestors. Phylogeny and large genetic distances support the genus Pectinidens as the first stagnicoline representative in the southern hemisphere, including colonization of extreme world regions, as most southern Patagonia, long time ago. The phylogenetic link of L. diaphana with the stagnicoline group may give light to the aforementioned peculiar low altitude epidemiological scenario of fascioliasis.