DNA profile match probabilities in a subdivided population: when can subdivision be ignored?

Li and Chakravarti [Li, C.C. & Chakravarti, A. (1994) Hum. Hered. 44, 100-109] compared the probability (MO) of a random match between the two DNA profiles of a pair of individuals drawn from a random-mating population to the probability (MF) of the match between a pair of random individuals drawn from a subdivided population. The level of heterogeneity in this subdivided population is measured by the parameter F, where there is no subdivision when F = 0 and increasing values of F indicate increasing subdivisions. Li and Chakravarti concluded that it is conservative to use the match probability MO, which is derived under the assumption that the two individuals are drawn from a homogeneous random-mating population without subdivision. However, MO may not be always greater than MF, even for biologically reasonable values of F. We explore here those mathematical conditions under which MO is less than MF, and we find that MO is not conservative mainly when there is an allele with a much higher frequency than all the other alleles. When empirical data for both variable number of tandem repeat (VNTR) and short tandem repeat (STR) systems are evaluated, we find that in the majority of cases MO represents a conservative probability of a match, and so the subdivision of human populations may usually be ignored for a random match, although not, of course, for relatives. Loci for which MO is not conservative should be avoided for forensic inference.

Down-regulation of NF-kappa B protein levels in activated human lymphocytes by 1,25-dihydroxyvitamin D3.

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2)D3], a steroid hormone with immunomodulating properties, on nuclear factor kappa B (NF-kappa B) proteins was examined in in vitro activated normal human lymphocytes by Western blot analysis. Over a 72-hr period of activation, the expression of the 50-kDa NF-kappa B, p50, and its precursor, p105, was increased progressively. When cells were activated in the presence of 1,25(OH)2D3, the levels of the mature protein as well as its precursor were decreased. The effect of the hormone on the levels of p50 was demonstrable in the cytosolic and nuclear compartments; it required between 4 and 8 hr and was specific, as 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were ineffective. Besides p50, 1,25(OH)2D3 decreased the levels of another NF-kappa B protein, namely c-rel. In addition, 1,25(OH)2D3 decreased the abundance of a specific DNA-protein complex formed upon incubation of nuclear extracts from activated lymphocytes with a labeled NF-kappa B DNA binding motif. Further, 1,25(OH)2D3 inhibited the transcriptional activity of NF-kappa B in Jurkat cells transiently transfected with a construct containing four tandem repeats of the NF-kappa B binding sequence of the immunoglobulin kappa light chain gene linked to the chloramphenicol acetyltransferase reporter gene. These observations demonstrate directly that there is de novo synthesis of NF-kappa B during human lymphocyte activation and suggest that this process is hormonally regulated.

Identification of a retroviroid-like element from plants.

The biological nature of carnation small viroid-like RNA (CarSV RNA), a 275-nt circular molecule with self-cleaving hammerhead structures in its strands of both polarities, was investigated. The lack of infectivity observed in a series of transmission assays in carnation indicates that CarSV RNA, in spite of sharing structural similarities with viroid and viroid-like satellite RNAs from plants, does not belong to either of these two groups. Additional evidence in this direction comes from the observation that CarSV RNA also exists in carnation plants as DNA tandem repeats. In this respect, CarSV RNA is similar to a small transcript of a tandemly repeated DNA sequence of the newt genome. Moreover, CarSV and newt RNAs have similarities in their sequences as well as in some characteristics of their corresponding hammerhead structures. Further analyses have revealed that CarSV DNA is found directly fused to DNA sequences of carnation etched ring caulimovirus, a pararetrovirus, most likely in the form of an extrachromosomal element. The properties of the CarSV RNA/DNA system are those of a retroviroid-like element having some features in common with viroid and viroid-like satellite RNAs from plants and others with the newt transcript.

Análise de frequências alélicas de 15 marcadores STR em alunos da Faculdade de Odontologia de Bauru

Entre as muitas aplicações das tecnologias de identificação biológica humana, estão as finalidades forenses. O objetivo desta pesquisa foi verificar frequências alélicas de Short Tandem Repeat (STR) e os parâmetros estatísticos de interesse em genética de populações e forense para desenvolver o primeiro banco de dados populacional de DNA na Faculdade de Odontologia de Bauru, Universidade de São Paulo, (FOB/USP) para futuros usos forenses. Frequências alélicas de 15 locos autossômicos e do marcador de gênero amelogenina foram determinadas utilizando amostras de 200 μL de saliva doados por 296 alunos de graduação da FOB/USP, com idade ≥ 18 anos, após aprovação ética. Os testes laboratoriais foram feitos com kits comerciais. Resultados e parâmetros estatísticos foram obtidos por meio de programas clássicos: GeneMapper-ID-X, MS Excel 2002 versão 10.6871.6870, GenAlEx 6.5 e Arlequin 3.5, comparando quatro populações (brasileira, portuguesa, norte-americana e a população deste estudo). Os locos mais polimórficos foram D18S51 (17 alelos) e FGA (15 alelos), seguidos pelo D21S11 (13 alelos) e os menos polimórficos foram D16S539 e TH01 (7 alelos cada). A análise comparativa com amostra da população brasileira proveniente de estudos anteriores (n > 100.000) pelo teste goodness of fit X2 index não mostrou diferenças significativas entre estes grupos (p = 0,9999). Outros parâmetros estatísticos foram calculados comparando as populações: local (deste estudo), portuguesa e norte-americana. A análise de variância molecular (AMOVA) entre as três populações, entre as pessoas da mesma população e para cada pessoa de cada população mostrou que existe uma elevada variância individual (99%), que esta variância é mantida uniformemente entre as pessoas da mesma amostra/região (1%) e entre as três populações estudadas (0%). O estudo confirmou o elevado grau de polimorfismo e a alta heterozigosidade (96,5%) da população. Houve diferença significativa quanto ao gênero (79,7% mulheres) quando comparado à população brasileira em geral (50,4%), explicada pelas características do corpo discente da FOB/USP composto por 80,6% de pessoas do gênero feminino. Interessante foi a observação de uma microvariante alélica no loco D18S51, fora da escada padrão e da escala de abrangência do kit, correspondente ao alelo 29, ainda não definida na base de dados internacional (STRBase, atualizada em 07/08/2015). Esta microvariante deverá ser confirmada por testes familiares e sequenciamento de DNA para verificar a possibilidade de outra ocorrência familiar ou duplicação de nucleotídeos. No futuro, os dados obtidos neste estudo devem ser incorporados ao banco de dados da população brasileira e podem ser considerados como referência genética da população regional, ajudando a elucidar casos forenses. Após a confirmação, a potencial nova microvariante alélica contribuirá para a base de dados internacional STRBase.

Empirical Evaluation of the Genetic Similarity of Samples From Twin Registries in Australia and the Netherlands Using 359 STRP Markers

One way to achieve the large sample sizes required for genetic studies of complex traits is to combine samples collected by different groups. It is not often clear, however, whether this practice is reasonable from a genetic perspective. To assess the comparability of samples from the Australian and the Netherlands twin studies, we estimated F,, (the proportion of total genetic variability attributable to genetic differences between cohorts) based on 359 short tandem repeat polymorphisms in 1068 individuals. IF,, was estimated to be 0.30% between the Australian and the Netherlands cohorts, a smaller value than between many European groups. We conclude that it is reasonable to combine the Australian and the Netherlands samples for joint genetic analyses.

Proinsulin is encoded by an RNA splice variant in human blood myeloid cells

Genes for peripheral tissue-restricted self-antigens are expressed in thymic and hematopoietic cells. In thymic medullary epithelial cells, self-antigen expression imposes selection on developing autoreactive T cells and regulates susceptibility to autoimmune disease in mouse models. Less is known about the role of self-antigen expression by hematopoietic cells. Here we demonstrate that one of the endocrine self-antigens expressed by human blood myeloid cells, proinsulin, is encoded by an RNA splice variant. The surface expression of immunoreactive proinsulin was significantly decreased after transfection of monocytes with small interfering RNA to proinsulin. Furthermore, analogous to proinsulin transcripts in the thymus, the abundance of the proinsulin RNA splice variant in blood cells corresponded with the length of the variable number of tandem repeats 5' of the proinsulin gene, known to be associated with type 1 diabetes susceptibility. Self-antigen expression by peripheral myeloid cells extends the umbrella of immunological self and, by analogy with the thymus, may be implicated in peripheral immune tolerance.

Reformulation of a thermostable broadly protective recombinant vaccine against human papilloma virus

The causal relationship between Human Papilloma Virus (HPV) infection and cervical cancer has motivated the development, and further improvement, of prophylactic vaccines against this virus. 70% of cervical cancers, 80% of which in low-resources countries, are associated to HPV16 and HPV18 infection, with 13 additional HPV types, classified as high-risk, responsible for the remaining 30% of tumors. Current vaccines, Cervarix® (GlaxoSmithKline) and Gardasil®(Merk), are based on virus-like particles (VLP) obtained by self-assembly of the major capsid protein L1. Despite their undisputable immunogenicity and safety, the fact that protection afforded by these vaccines is largely limited to the cognate serotypes included in the vaccine (HPV 16 and 18, plus five additional viral types incorporated into a newly licensed nonavalent vaccine) along with high production costs and reduced thermal stability, are pushing the development of 2nd generation HPV vaccines based on minor capsid protein L2. The increase in protection broadness afforded by the use of L2 cross-neutralizing epitopes, plus a marked reduction of production costs due to bacterial expression of the antigens and a considerable increase in thermal stability could strongly enhance vaccine distribution and usage in low-resource countries. Previous studies from our group identified three tandem repeats of the L2 aa. 20-38 peptide as a strongly immunogenic epitope if exposed on the scaffold protein thioredoxin (Trx). The aim of this thesis work is the improvement of the Trx-L2 vaccine formulation with regard to cross-protection and thermostability, in order to identify an antigen suitable for a phase I clinical trial. By testing Trx from different microorganisms, we selected P. furiosus thioredoxin (PfTrx) as the optimal scaffold because of its sustained peptide epitope constraining capacity and striking thermal stability (24 hours at 100°C). Alternative production systems, such as secretory Trx-L2 expression in the yeast P. pastoris, have also been set-up and evaluated as possible means to further increase production yields, with a concomitant reduction of production costs. Limitations in immune-responsiveness caused by MHC class II polymorphisms –as observed, for example, in different mouse strains- have been overcome by introducing promiscuous T-helper (Th) epitopes, e.g., PADRE (Pan DR Epitope), at both ends of PfTrx. This allowed us to obtain fairly strong immune responses even in mice (C57BL/6) normally unresponsive to the basic Trx-L2 vaccine. Cross-protection was not increased, however. I thus designed, produced and tested a novel multi-epitope formulation consisting of 8 and 11 L2(20-38) epitopes derived from different HPV types, tandemly joined into a single thioredoxin molecule (“concatemers”). To try to further increase immunogenicity, I also fused our 8X and 11X PfTrx-L2 concatemers to the N-terminus of an engineered complement-binding protein (C4bp), capable to spontaneously assemble into ordered hepatmeric structures, previously validated as a molecular adjuvant. Fusion to C4bp indeed improved antigen presentation, with a fairly significant increase in both immunogenicity and cross-protection. Another important issue I addressed, is the reduction of vaccine doses/treatment, which can be achieved by increasing immunogenicity, while also allowing for a delayed release of the antigen. I obtained preliminary, yet quite encouraging results in this direction with the use of a novel, solid-phase vaccine formulation, consisting of the basic PfTrx-L2 vaccine and its C4bp fusion derivative adsorbed to mesoporus silica-rods (MSR).

Substructuring in four populations of African descent

When a suspect's DNA profile is admitted into court as a match to evidence the probability of the perpetrator being another individual must be calculated from database allele frequencies. The two methods used for this calculation are phenotypic frequency and likelihood ratio. Neither of these calculations takes into account substructuring within populations. In these substructured populations the frequency of homozygotes increases and that of heterozygotes usually decreases. The departure from Hardy- Weinberg expectation in a sample population can be estimated using Sewall Wright's Fst statistic. Fst values were calculated in four populations of African descent by comparing allele frequencies at three short tandem repeat loci. This was done by amplifying the three loci in each sample using the Polymerase Chain Reaction and separating these fragments using polyacrylamide gel electrophoresis. The gels were then silver stained and autoradiograms taken, from which allele frequencies were estimated. Fst values averaged 0.007+- 0.005 within populations of African descent and 0.02+- 0.01 between white and black populations.

Characterization of ALDH1A1 as a novel tumorigenic target mediating TAZ-induced lung tumorigenesis and stem cell phenotypes

Recent studies suggest that lung cancer stem cells (CSCs) may play major roles in lung cancer development, metastasis and drug resistance. Therefore, identification of lung CSC drivers may provide promising targets for lung cancer. TAZ (transcriptional co-activator with PDZ-binding motif) is a transcriptional co-activator and key downstream effector of the Hippo pathway, which plays critical roles in various biological processes. TAZ has been shown to be overexpressed in non-small cell lung cancer (NSCLC) and involved in tumorigenicity of lung epithelial cells. However, whether TAZ is a driver for lung CSCs and tumor formation in vivo is unknown. In addition, the molecular mechanism underlying TAZ-induced lung tumorigenesis remains to be determined. In this study, we provided evidence that constitutively active TAZ (TAZ-S89A) is a driver for lung tumorigenesis in vivo in mice and formation of lung CSC. Oncogenes upregulated in TAZ-overexpressing cells were identified with further validation. The most dramatically activated gene, Aldh1a1 (Aldehyde dehydrogenase 1 family member a1), a well-established CSC marker, showed that TAZ induces Aldh1a1 transcription by activating its promoter activity through interaction with the transcription factor TEA domain (TEAD) family member. Most significantly, inhibition of ALDH1A1 with its inhibitor A37 or CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene knockout in lung cancer cells suppressed lung tumorigenic and CSC phenotypes in vitro, and tumor formation in mice in vivo. In conclusion, this study identified TAZ as a novel inducer of lung CSCs and the first transcriptional activator of the stem cell marker ALDH1A1. Most significantly, we identified ALDH1A1 as a critical meditator of TAZ-induced tumorigenic and CSC phenotypes in lung cancer. Our studies provided preclinical data for targeting of TAZ-TEAD-ALDH1A1 signaling to inhibit CSC-induced lung tumorigenesis and drug resistance in the future.

High hydrostatic pressure adaptive strategies in an obligate piezophile Pyrococcus yayanosii

Pyrococcus yayanosii CH1, as the first and only obligate piezophilic hyperthermophilic microorganism discovered to date, extends the physical and chemical limits of life on Earth. It was isolated from the Ashadze hydrothermal vent at 4,100 m depth. Multi-omics analyses were performed to study the mechanisms used by the cell to cope with high hydrostatic pressure variations. In silico analyses showed that the P. yayanosii genome is highly adapted to its harsh environment, with a loss of aromatic amino acid biosynthesis pathways and the high constitutive expression of the energy metabolism compared with other non-obligate piezophilic Pyrococcus species. Differential proteomics and transcriptomics analyses identified key hydrostatic pressure-responsive genes involved in translation, chemotaxis, energy metabolism (hydrogenases and formate metabolism) and Clustered Regularly Interspaced Short Palindromic Repeats sequences associated with Cellular apoptosis susceptibility proteins.

Imunidade celular: caracterização das proteínas celulares que inibem a infecção viral

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz

Whole genome sequence of Klebsiella pneumoniae U25, a hypermucoviscous, multidrug resistant, biofilm producing isolate from India

Klebsiella pneumoniae U25 is a multidrug resistant strain isolated from a tertiary care hospital in Chennai, India. Here, we report the complete annotated genome sequence of strain U25 obtained using PacBio RSII. This is the first report of the whole genome of K. pneumoniae species from Chennai. It consists of a single circular chromosome of size 5,491,870-bp and two plasmids of size 211,813 and 172,619-bp. The genes associated with multidrug resistance were identified. The chromosome of U25 was found to have eight antibiotic resistant genes [blaOXA-1, blaSHV-28, aac(6’)1b-cr, catB3, oqxAB, dfrA1]. The plasmid pMGRU25-001 was found to have only one resistant gene (catA1) while plasmid pMGRU25-002 had 20 resistant genes [strAB, aadA1, aac(6’)-Ib, aac(3)-IId, sul1,2, blaTEM-1A,1B, blaOXA-9, blaCTX-M-15, blaSHV-11, cmlA1, erm(B), mph(A)]. A mutation in the porin OmpK36 was identified which is likely to be associated with the intermediate resistance to carbapenems in the absence of carbapenemase genes. U25 is one of the few K. pneumoniae strains to harbour clustered regularly interspaced short palindromic repeats (CRISPR) systems. Two CRISPR arrays corresponding to Cas3 family helicase were identified in the genome. When compared to K. pneumoniae NTUHK2044, a transposase gene InsH of IS5-13 was found inserted.

Tandem electroabsorption modulators integrated with DFB laser by ultra-low-pressure selective-area-growth MOCVD for 10 GHz optical short pulse generation

　

10 GHz optical short pulse generation using tandem electroabsorption modulators monolithically integrated with distributed feedback laser by ultra-low-pressure selective area growth

In this work, a novel light source of tandem InGaAsP/InGaAsP multiple quantum well electroabsoption modulator( EAM ) monolithically integrated with distributed feedback laser is fabricated by ultra-low-pressure ( 22 x 10(2) Pa ) selective area growth metal-organic chemical vapor diposition technique. Superior device performances have been obtained, such as low threshold current of 19 mA, output light power of 4.5 mW, and over 20 dB extinction ratio at 5 V applied voltage when coupled into a single mode fiber. Over 10 GHz 3dB bandwidth in EAM part is developed with a driving voltage of 2 V. Using this sinusoidal voltage driven integrated device, 10 GHz repetition rate pulse with an actual width of 13.7 ps without any compression elements is obtained due to the gate operation effect of tandem EAMs.