Assessment of DNA and mtDNA Degradation in Sperm Cells Collected by Laser Micro-dissection

Sexual assault samples are among the most frequently analyzed in a forensic laboratory. These accounts for almost half of all samples processed routinely and a large portion of these cases remain unsolved. These samples often pose problems to traditional analytic methods of identification because they consist most frequently of cell mixtures from at least two contributors: the victim (usually female) and the perpetrator (usually male). In this study we prepared simulated sexual assault samples and conducted amplification and DNA genotyping from sperm cells collected with laser microdissection at different time intervals to assess the DNA degradation at those intervals. Furthermore, we then investigated the possibility of recovering mtDNA from the collected sperm cells, particularly in cases where autosomal DNA was not suitably amplified. With this work we determined that it becomes possible to extend the timeframe for performing an analysis, by researching other sources of DNA, namely mtDNA.info:eu-repo/semantics/publishedVersio

Mitochondrial DNA studies of Lisbon immigrants from Portuguese speaking African countries

Since the end of the 1970s, Portugal has had an important role in migratory movements, becoming a destination for immigrants of a wide range of nationalities, mainly from African countries. According to PORDATA, until the end of 2014 there were 40,000 immigrants from Cape Verde, 20,000 from Angola, 18,000 from Guinea-Bissau, and 3,000 from Mozambique living in Portugal, and of those, >80 per cent live in the Lisbon region. This may be one of the main contributors to genetic variation of Lisbon residents in the present and the future. Mitochondrial DNA (mtDNA) has features that make it desirable for forensics, namely, high copy number, lack of recombination, and matrilineal inheritance. These features are also important in evolutionary and population studies. We aim to characterize mtDNA diversity in immigrants from Portuguese Speaking African Countries (PALOP) living in Lisbon and their potential contribution to genetic variation of Lisbon population. Blood samples were collected from 439 PALOP immigrants living in Lisbon, of which 173 immigrants from Angola, 103 immigrants from Cape Verde, eighty-three immigrants from Mozambique and eighty immigrants from Guinea-Bissau, from January 2000 to December 2016. The control region of the mtDNA was amplified using two pairs of primers—L15971/H016 and L16555/H639, and sequenced by BigDye Terminator v.3.1 Cycle Sequence (AB). Sequenced products were detected in a sequencer Genetic Analyzer 3130 (AB). Finally the results were analysed by Sequencing Analysis v.5.2 software and also compared with Revised Cambridge Reference Sequence (rCRS) using SeqScape v.3 (AB) software. The haplogroups were determined based on Phylotree, build 17. Genetic distances and other genetic parameters were calculated with Arlequin software ver.3.5 and analysed and represented with PhyML 3.0. For each sample, the complete sequence of the control region was obtained. The comparison of the sequences obtained with the rCRS, among the 439 analysed individuals, allowed the identification of 319 different haplotypes, corresponding to 164 different haplogroups distributed by ten macrohaplogroups. Macrohaplogroup L was the most common with 386 haplotypes followed by U with fifteen haplotypes, H with twelve haplotypes, M and T with six haplotypes, K with five, R with four, X and J with two and HV with one. PALOP’s immigrants presented a high number of unique haplotypes, most of them belonging to macrohaplogroup L, originating from sub-Saharan regions of Africa.This macrohaplogroup is uncommon in European and Portuguese populations. Consistent with this, phylogenetic analysis showed the establishment of two distinct groups, one composed of the Portuguese population and another of the African populations. In comparing the different immigrant populations living in Lisbon, the genetically closest community to the Portuguese population is Mozambique and the furthest is Cape Verde, followed by Guinea-Bissau and Angola. Our results show that the PALOP immigrants living in Lisbon are genetically heterogeneous. The increase in genetic diversity in Lisbon due to immigrants from PALOP countries may have a major impact on haplotypic and allelic frequencies, on which all forensic and medico-legal investigations are based

Factors associated with delayed diagnosis of tuberculosis in hospitalized patients in a high TB and HIV burden setting: a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>The most essential components of TB control are early diagnosis and adequate treatment. Delay in the diagnosis and treatment of tuberculosis may result in more extensive disease and more complications, increase severity of the disease and is associated with higher risk of mortality. The purpose of this study was to identify factors associated with delayed diagnosis of TB in hospitalized patients.</p> <p>Methods</p> <p>We conducted a cross-sectional study in a general, tertiary care, university-affiliated hospital. Adult patients with TB that were hospitalized were identified retrospectively, and risk factors for delayed diagnosis were collected.</p> <p>Results</p> <p>The median delay until diagnosis was 6 days (IQR: 2-12 days). One hundred and sixty six (54.4%) patients were diagnosed ≤ 6 days, and 139 (45.6%) > 6 days after admission. The main factors associated with diagnostic delay (> 6 days) were extra-pulmonary TB and negative sputum smear.</p> <p>Conclusions</p> <p>Although hospitalization permits a rapid management of the patient and favors a faster diagnosis, we found an unacceptable time delay before the diagnosis of pulmonary TB was made. Future studies should focus on attempt to explain the reasons of diagnostic retard in the patients with the characteristics related to delay in this study.</p

Accurate masses and radii of normal stars: modern results and applications

This paper presents and discusses a critical compilation of accurate, fundamental determinations of stellar masses and radii. We have identified 95 detached binary systems containing 190 stars (94 eclipsing systems, and alpha Centauri) that satisfy our criterion that the mass and radius of both stars be known to 3% or better. To these we add interstellar reddening, effective temperature, metal abundance, rotational velocity and apsidal motion determinations when available, and we compute a number of other physical parameters, notably luminosity and distance. We discuss the use of this information for testing models of stellar evolution. The amount and quality of the data also allow us to analyse the tidal evolution of the systems in considerable depth, testing prescriptions of rotational synchronisation and orbital circularisation in greater detail than possible before. The new data also enable us to derive empirical calibrations of M and R for single (post-) main-sequence stars above 0.6 M(Sun). Simple, polynomial functions of T(eff), log g and [Fe/H] yield M and R with errors of 6% and 3%, respectively. Excellent agreement is found with independent determinations for host stars of transiting extrasolar planets, and good agreement with determinations of M and R from stellar models as constrained by trigonometric parallaxes and spectroscopic values of T(eff) and [Fe/H]. Finally, we list a set of 23 interferometric binaries with masses known to better than 3%, but without fundamental radius determinations (except alpha Aur). We discuss the prospects for improving these and other stellar parameters in the near future.Comment: 56 pages including figures and tables. To appear in The Astronomy and Astrophysics Review. Ascii versions of the tables will appear in the online version of the articl

Defining an Essence of Structure Determining Residue Contacts in Proteins

The network of native non-covalent residue contacts determines the three-dimensional structure of a protein. However, not all contacts are of equal structural significance, and little knowledge exists about a minimal, yet sufficient, subset required to define the global features of a protein. Characterisation of this “structural essence” has remained elusive so far: no algorithmic strategy has been devised to-date that could outperform a random selection in terms of 3D reconstruction accuracy (measured as the Ca RMSD). It is not only of theoretical interest (i.e., for design of advanced statistical potentials) to identify the number and nature of essential native contacts—such a subset of spatial constraints is very useful in a number of novel experimental methods (like EPR) which rely heavily on constraint-based protein modelling. To derive accurate three-dimensional models from distance constraints, we implemented a reconstruction pipeline using distance geometry. We selected a test-set of 12 protein structures from the four major SCOP fold classes and performed our reconstruction analysis. As a reference set, series of random subsets (ranging from 10% to 90% of native contacts) are generated for each protein, and the reconstruction accuracy is computed for each subset. We have developed a rational strategy, termed “cone-peeling” that combines sequence features and network descriptors to select minimal subsets that outperform the reference sets. We present, for the first time, a rational strategy to derive a structural essence of residue contacts and provide an estimate of the size of this minimal subset. Our algorithm computes sparse subsets capable of determining the tertiary structure at approximately 4.8 Å Ca RMSD with as little as 8% of the native contacts (Ca-Ca and Cb-Cb). At the same time, a randomly chosen subset of native contacts needs about twice as many contacts to reach the same level of accuracy. This “structural essence” opens new avenues in the fields of structure prediction, empirical potentials and docking

Trypanosoma cruzi Epimastigotes Are Able to Store and Mobilize High Amounts of Cholesterol in Reservosome Lipid Inclusions

Reservosomes are lysosome-related organelles found in Trypanosoma cruzi epimastigotes. They represent the last step in epimastigote endocytic route, accumulating a set of proteins and enzymes related to protein digestion and lipid metabolism. The reservosome matrix contains planar membranes, vesicles and lipid inclusions. Some of the latter may assume rectangular or sword-shaped crystalloid forms surrounded by a phospholipid monolayer, resembling the cholesterol crystals in foam cells.Using Nile Red fluorimetry and fluorescence microscopy, as well as electron microscopy, we have established a direct correlation between serum concentration in culture medium and the presence of crystalloid lipid inclusions. Starting from a reservosome purified fraction, we have developed a fractionation protocol to isolate lipid inclusions. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed that lipid inclusions are composed mainly by cholesterol and cholesterol esters. Moreover, when the parasites with crystalloid lipid-loaded reservosomes were maintained in serum free medium for 48 hours the inclusions disappeared almost completely, including the sword shaped ones.Taken together, our results suggest that epimastigote forms of T. cruzi store high amounts of neutral lipids from extracellular medium, mostly cholesterol or cholesterol esters inside reservosomes. Interestingly, the parasites are able to disassemble the reservosome cholesterol crystalloid inclusions when submitted to serum starvation