DIP-STR: A new marker for resolving unbalanced DNA mixtures

The genetic characterization of unbalanced mixed stains remains an important area where improvementis imperative. Most cases of aggression, homicide and sexual assault produce biological traces withrelatively large amount of the victim's DNA and small amount of the aggressor's DNA. If this ratio issmaller than 1:10 it is currently not possible to obtain a conventional autosomal DNA profile of the minorcontributor, with potential loss of crucial DNA evidence. Y-STR analysis represents a solution for somecases but has several limitations. We propose here a method based on a new compound genetic markerformed by a Deletion/Insertion Polymorphism (DIP) linked to a Short Tandem Repeat polymorphism(STR), that we name DIP-STR. By means of allele-specific amplifications of DIP-STR haplotypes, we canproduce a high resolution autosomal DNA profile of a donor that contributes less than 0.1% to a DNAmixture. Based on these features DIP-STR markers may outperform conventional Y-STR markers inmixed stain analysis.

Análisis de la expresión de las moléculas relacionadas con el complejo principal de histocompatibilidad de clase I y de NKG2D como respuesta al daño oxidativo del DNA en el cáncer urotelial de vejiga. Relación con las características clínico patológicas y supervivencia

Es tracta d'un estudi retrospectiu de casos de 280 pacients diagnosticats de tumor vesical primari amb un seguiment mínim de 8 anys. S'ha construït un Tissue microarray i mitjançant mètodes semiquantitatius d’inmunohistoquímica es determinarà l'expressió de les molècules MICA (MHC class I chain-related gene A) i del seu receptor NKG2D (Natural-Killer group 2-member D) a nivell tissular, relacionant-lo amb variables anatomopatològiques segons els grups de risc, hàbit tabàquic i sexe. Finalment valorarem l'expressió de MICA/NKG2D com a factor independent de recidiva / progressió tumoral. En la literatura només existeixen 2 treballs que relacionin MICA amb el càncer vesical.

Restriction fragment length polymorphism of 195 bp repeated satellite dna of Trypanosoma cruzi supports the existence of two phylogenetic groups

The restriction fragment length polymorphism of the 195 bp repeated DNA sequence of Trypanosoma cruzi was analyzed among 23 T. cruzi stocks giving a reliable picture of the whole phylogenetic variability of the species. The profiles observed with the enzymes Hinf I and Hae III were linked together and supported the existence of two groups. Group 1 shows a 195 bp repeated unit (Hinf I) and high molecular weight DNA (Hae III), while group 2 presents a ladder profile for each enzyme, which is a characteristic of tandemly repeated DNA. The two groups, respectively, clustered stocks pertaining to the two principal lineages evidenced by isoenzyme and RAPD markers. The congruence among these three independent genomic markers corroborates the existence of two real phylogenetic lineages in T. cruzi. The specific monomorphic profiles for each major phylogenetic lineage suggest the existence of ancient sexuality and cryptic biological speciation.

A Mitochondrial DNA Phylogeny Indicates Close Relationships between Populations of Lutzomyia whitmani (Diptera: Psychodidae, Phlebotominae) from the Rain-forest Regions of Amazônia and Northeast Brazil

Phylogenetic analysis of all 31 described mitochondrial (cytochrome b) haplotypes of Lutzomyia whitmani demonstrated that new material from the State of Rondônia, in southwest Amazônia, forms a clade within a lineage found only in the rain-forest regions of Brazil. This rain-forest lineage also contains two other clades of haplotypes, one from eastern Amazônia and one from the Atlantic forest zone of northeast Brazil (including the type locality of the species in Ilhéus, State of Bahia). These findings do not favour recognizing two allopatric cryptic species of L. whitmani, one associated with the silvatic transmission of Leishmania shawi in southeast Amazônia and the other with the peridomestic transmission of Le. braziliensis in northeast Brazil. Instead, they suggest that there is (or has been in the recent past) a continuum of inter-breeding populations of L. whitmani in the rain-forest regions of Brazil.

Mitochondrial DNA sequence variation within the remnant populations of the endangered numbat (Marsupialia: Myrmecobiidae: Myrmecobius fasciatus).

The numbat has been reduced to two populations in Western Australia. To better understand the effects of range reduction on gene flow and genetic variation, and to address questions crucial for the species' management, we analysed mitochondrial DNA (mtDNA) sequences of free-ranging individuals and museum specimens. The results suggest recent connectivity between the remnant populations, although one of those may have lost significant amounts of genetic diversity during the recent population size reduction. We propose that for management purposes the remnant populations should be treated as a single historical lineage and that, subject to certain caveats, consideration should be given to population augmentation by translocation.

The likelihood approach to compare populations : a study on DNA evidence and pitfalls of intuitions

The paper follows on from earlier work [Taroni F and Aitken CGG. Probabilistic reasoning in the law, Part 1: assessment of probabilities and explanation of the value of DNA evidence. Science & Justice 1998; 38: 165-177]. Different explanations of the value of DNA evidence were presented to students from two schools of forensic science and to members of fifteen laboratories all around the world. The responses were divided into two groups; those which came from a school or laboratory identified as Bayesian and those which came from a school or laboratory identified as non-Bayesian. The paper analyses these responses using a likelihood approach. This approach is more consistent with a Bayesian analysis than one based on a frequentist approach, as was reported by Taroni F and Aitken CGG. [Probabilistic reasoning in the law, Part 1: assessment of probabilities and explanation of the value of DNA evidence] in Science & Justice 1998.

Effects of physiological self-crowding of DNA on shape and biological properties of DNA molecules with various levels of supercoiling.

DNA in bacterial chromosomes and bacterial plasmids is supercoiled. DNA supercoiling is essential for DNA replication and gene regulation. However, the density of supercoiling in vivo is circa twice smaller than in deproteinized DNA molecules isolated from bacteria. What are then the specific advantages of reduced supercoiling density that is maintained in vivo? Using Brownian dynamics simulations and atomic force microscopy we show here that thanks to physiological DNA-DNA crowding DNA molecules with reduced supercoiling density are still sufficiently supercoiled to stimulate interaction between cis-regulatory elements. On the other hand, weak supercoiling permits DNA molecules to modulate their overall shape in response to physiological changes in DNA crowding. This plasticity of DNA shapes may have regulatory role and be important for the postreplicative spontaneous segregation of bacterial chromosomes.

A 35.7 kb DNA fragment from the Bacillus subtilis chromosome containing a putative 12.3 kb operon involved in hexuronate catabolism and a perfectly symmetrical hypothetical catabolite-responsive element.

The Bacillus subtilis strain 168 chromosomal region extending from 109 degrees to 112 degrees has been sequenced. Among the 35 ORFs identified, cotT and rapA were the only genes that had been previously mapped and sequenced. Out of ten ORFs belonging to a single putative transcription unit, seven are probably involved in hexuronate catabolism. Their sequences are homologous to Escherichia coli genes exuT, uidB, uxaA, uxaB, uxaC, uxuA and uxuB, which are all required for the uptake of free D-glucuronate, D-galacturonate and beta-glucuronide, and their transformation into glyceraldehyde 3-phosphate and pyruvate via 2-keto-3-deoxygluconate. The remaining three ORFs encode two dehydrogenases and a transcriptional regulator. The operon is preceded by a putative catabolite-responsive element (CRE), located between a hypothetical promoter and the RBS of the first gene. This element, the longest and the only so far described that is fully symmetrical, consists of a 26 bp palindrome matching the theoretical B. subtilis CRE sequence. The remaining predicted amino acid sequences that share homologies with other proteins comprise: a cytochrome P-450, a glycosyltransferase, an ATP-binding cassette transporter, a protein similar to the formate dehydrogenase alpha-subunit (FdhA), protein similar to NADH dehydrogenases, and three homologues of polypeptides that have undefined functions.

Mitochondrial DNA variation of Triatoma infestans populations and its implication on the specific status of T. melanosoma

DNA sequence comparison of 412 base-pairs fragments of the mitochondrial cytochrome B gene was used to infer the genetic structure of nine geographical Triatoma infestans populations and their phylogenetic relationship with T. melanosoma and T. brasiliensis. T. infestans and T. melanosoma were compared by morphometry, allozyme and cytogenetic analyses, as well as subjected to reciprocal crosses, in order to clarify the taxonomic status of the latter. No differences were found to distinguish the two species and the crosses between them yielded progeny. T. infestans populations presented four haplotypes that could be separated in two clusters: one formed by the samples from Bolivia (Andes and Chaco) and the other formed by samples from Argentina and Brazil. Silvatic and domestic T. infestans populations from Bolivia (Andes) were genetically identical.

IPH response to the Joint Committee on Health and Children and members of the Northern Ireland Assembly on “Cross-border cooperation on healthcare”

The Institute of Public Health in Ireland were asked to submit a paper on 'Cross-border cooperation on healthcare' for a joint meeting between the Oireachtas Joint Committee on Health and Children and the Northern Ireland Assembly Health Committee which took place in Leinster House on 1 March 2012. Key points from the submission included: o    The Institute of Public Health in Ireland (IPH) is an all-island organisation which promotes cooperation between the Republic of Ireland and Northern Ireland with the aim of improving population health on the island and tackling health inequalities. IPH work is focused on addressing the causes of ill health rather than the design and delivery of treatment services. o    North/South cooperation on health was mandated under the Belfast Agreement in 1998 in five domains, including health promotion. IPH has supported the North South Ministerial Council (NSMC) in respect of the health promotion strand since inception. o    The Department of Health and Department of Health, Social Services and Public Safety North-South Feasibility Study (December 2011) states that mutual benefits are most evident from cooperation in the areas of (i) anticipating trends and illnesses in a collective manner (ii) public health issues (iii) specialised services where the population or activity required to sustain the service cannot be met by either jurisdiction alone and (iv) in relation to those areas adjacent to the border. o    The European Directive on Cross-Border Healthcare will be implemented in the next few years which will have implications in relation to patients travelling for healthcare across the Republic of Ireland/Northern Ireland border. o    IPH is supporting the development of new public health strategies in the Republic of Ireland and Northern Ireland which are both due for publication this year. o    There are tangible benefits from cross-border cooperation in the health sector, both in public health and in health service planning and delivery and there are many examples of successful initiatives. However, developments are not occurring in the context of an agreed plan or overall strategic context and tend to be project-based and concentrated in border counties. o    Successful cross-border cooperation requires high level support and integration into departmental policy cycles. The provision of data on an all-island basis supports cross-border cooperation as does the operation of sustainable all-island organisations which can support research, evaluations and programmes. o    In the future, cross-border cooperation in health will be more effective if developed with a strategic planning process intrinsically linked to Departmental priorities. o    North-South cooperation in the areas of alcohol, obesity, tobacco health surveys and rare diseases will be particularly beneficial.

IPH responds to Northern Ireland Assembly Inquiry into Sustainable Transport

The Institute of Public Health in Ireland (IPH) aims to improve health on the island of Ireland by working to combat health inequalities and influence public policies in favour of health.  The Institute promotes cooperation between Northern Ireland and the Republic of Ireland in public health research, training and policy advice. IPH commends the Regional Development Committee on selecting sustainable transport as its subject for inquiry and welcomes the inquiry’s focus on identifying a move to more sustainable transport in Northern Ireland. IPH thanks the Committee for the opportunity to contribute views and experience

IPH gives evidence to NI Assembly Health Committee Inquiry into Obesity

IPH Chief Executive, Dr Jane Wilde gave evidence to the Northern Ireland Assembly Health Committee inquiry into obesity. Dr Wilde recommended the following: Supporting the Department of Health’s strategic approach based on an understanding of the nature and complexity of obesity. Urgent and short term action to coordinate current activities and ensure focus on the most vulnerable. Exploring new forms and incentives to promote cross departmental work. Setting intermediate outcomes and targets Building stronger links between research, policy and practice, for example asking the Health Committee to set up a round table of researchers and policy makers Working systematically and transparently to identify key areas for cooperation with UK, Ireland and Europe Drawing from IPH work and other research, Dr Wilde briefed the Health Committee on the extent and impact of obesity, reasons for rising levels of obesity and the need for a stronger strategic response. She highlighted the importance of cross government action, the responsibilities of those beyond the health sector and the need for stronger evidence-informed policy and practice.

NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly.

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.

A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis

Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100°C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure.