Examining peak height ratios in low template DNA samples with and without sampling using a single-tube extraction protocol

The developments of the polymerase chain reaction (PCR) and the short tandem repeat multiplex kits increased the ease and lowered the time and sample quantity required for deoxyribonucleic acid (DNA) typing compared to previous methods. However the amplification of low mass of DNA can lead to increased stochastic effects, such as allele drop-out (ADO) and heterozygous peak height (PH) imbalance, which make it difficult to determine the true donor profile. These stochastic effects are believed to be due to: 1) pre-PCR sampling from pipetting and sample transferal of dilute samples prior to amplification resulting in unbalanced heterozygous allele templates in the amplification reaction, and 2) the kinetics of the PCR process where, when few target templates are available, there is uneven amplification of heterozygous alleles during early PCR cycles. This study looks to examine the contribution of PCR chemistry and pre-PCR sampling errors on stochastic effects by utilizing a single-tube DNA extraction and direct amplification method. Cells were collected into tubes using the McCrone and Associates, Inc. cell transfer method, which allowed for approximation of DNA mass without quantification. The forensicGEM® Saliva Kit was used to lyse the cells and inactivate nucleases without inhibiting downstream amplification. The samples were then directly amplified with the AmpFLSTR® Identifiler® Plus PCR Amplification Kit. These samples should only show the effects of PCR chemistry since pipetting and tube transferal steps prior to amplification were removed with the expectation that equal numbers of heterozygous alleles are present in the sample pre-amplification. Comparisons of PH imbalance were made to samples extracted with forensicGEM® but had one or more pipetting and tube transferal steps prior to amplification. These samples were either created through the dilution of stock DNA or from the cell transfer method where aliquots were then taken for amplification; thus these samples would exhibit the effects of both pre-PCR sampling and PCR chemistry errors and inefficiencies. The use of carrier ribonucleic acid (cRNA) was also added to cell transfer samples prior to the amplification of samples to see if it assisted with amplification and increased signal. Results show that the samples with only PCR chemistry generally have significantly higher mean peak height ratios (PHRs) than samples with both pre-PCR sampling and PCR chemistry except in cases where there were large numbers of ADOs. When compared to the diluted samples, the cell transfer samples had significantly higher mean PHR at 0.0625 ng and 0.125 ng, and higher mean PHR at 0.0375 ng when PHs from ADOs are included. Average peak heights (APHs) in the cell transfer samples were also significantly higher in these comparisons. When compared to aliquots taken from cell transfer samples, mean PHR was significantly higher at 0.0625 ng in cell transfer samples with only PCR chemistry than cell transfer samples with both pre-PCR sampling and PCR chemistry; however APH for the samples with only PCR chemistry was also significantly higher in one experiment and not significantly different in another. In a third experiment, the difference in mean PHR was not significant while APH was significantly higher in the samples with pre-PCR sampling and PCR chemistry; however there were also a large numbers of ADOs. Our results also found quantification of dilute samples unreliable but cell counting through the cell transfer method is an appropriate alternative for DNA mass approximation. Also there were no significant changes in PHR or APH in the presence or absence of cRNA

Target enrichment using parallel nanoliter quantitative PCR amplification

Background: Next generation targeted resequencing is replacing Sanger sequencing at high pace in routine genetic diagnosis. The need for well validated, high quality enrichment platforms to complement the bench-top next generation sequencing devices is high. Results: We used the WaferGen Smartchip platform to perform highly parallelized PCR based target enrichment for a set of known cancer genes in a well characterized set of cancer cell lines from the NCI60 panel. Optimization of PCR assay design and cycling conditions resulted in a high enrichment efficiency. We provide proof of a high mutation rediscovery rate and have included technical replicates to enable SNP calling validation demonstrating the high reproducibility of our enrichment platform. Conclusions: Here we present our custom developed quantitative PCR based target enrichment platform. Using highly parallel nanoliter singleplex PCR reactions makes this a flexible and efficient platform. The high mutation validation rate shows this platform’s promise as a targeted resequencing method for multi-gene routine sequencing diagnostics

microRNA expression in peripheral blood cells following acute ischemic stroke and their predicted gene targets.

BackgroundmicroRNA (miRNA) are important regulators of gene expression. In patients with ischemic stroke we have previously shown that differences in immune cell gene expression are present. In this study we sought to determine the miRNA that are differentially expressed in peripheral blood cells of patients with acute ischemic stroke and thus may regulate immune cell gene expression.MethodsmiRNA from peripheral blood cells of forty-eight patients with ischemic stroke and vascular risk factor controls were compared. Differentially expressed miRNA in patients with ischemic stroke were determined by microarray with qRT-PCR confirmation. The gene targets and pathways associated with ischemic stroke that may be regulated by the identified miRNA were characterized.ResultsIn patients with acute ischemic stroke, miR-122, miR-148a, let-7i, miR-19a, miR-320d, miR-4429 were decreased and miR-363, miR-487b were increased compared to vascular risk factor controls. These miRNA are predicted to regulate several genes in pathways previously identified by gene expression analyses, including toll-like receptor signaling, NF-κβ signaling, leukocyte extravasation signaling, and the prothrombin activation pathway.ConclusionsSeveral miRNA are differentially expressed in blood cells of patients with acute ischemic stroke. These miRNA may regulate leukocyte gene expression in ischemic stroke including pathways involved in immune activation, leukocyte extravasation and thrombosis

Aquaporin 5 Interacts with Fluoride and Possibly Protects Against Caries

Aquaporins (AQP) are water channel proteins and the genes coding for AQP2, AQP5, and AQP6 are clustered in 12q13. Since AQP5 is expressed in serous acinar cells of salivary glands, we investigated its involvement in caries. DNA samples from 1,383 individuals from six groups were studied. Genotypes of eight single nucleotide polymorphisms covering the aquaporin locus were tested for association with caries experience. Interaction with genes involved in enamel formation was tested. The association between enamel microhardness at baseline, after creation of artificial caries lesion, and after exposure to fluoride and the genetic markers in AQP5 was tested. Finally, AQP5 expression in human whole saliva, after exposure to fluoride in a mammary gland cell line, which is known to express AQP5, and in Wistar rats was also verified. Nominal associations were found between caries experience and markers in the AQP5 locus. Since these associations suggested that AQP5 may be inhibited by levels of fluoride in the drinking water that cause fluorosis, we showed that fluoride levels above optimal levels change AQP5 expression in humans, cell lines, and rats. We have shown that AQP5 is involved in the pathogenesis of caries and likely interact with fluoride.Fil: Anjomshoaa, Ida. University of Pittsburgh; Estados UnidosFil: Briseño Ruiz, Jessica. University of Pittsburgh; Estados UnidosFil: Deeley, Kathleen. University of Pittsburgh; Estados UnidosFil: Poletta, Fernando Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. CEMIC-CONICET. Centro de Educaciones Médicas e Investigaciones Clínicas "Norberto Quirno". CEMIC-CONICET.; ArgentinaFil: Mereb, Juan C.. Provincia de Río Negro. Ministerio de Salud. Hospital de Área El Bolsón ; ArgentinaFil: Leite, Aline L.. Universidade de Sao Paulo; BrasilFil: Barreta, Priscila A. T.. Universidade de Sao Paulo; BrasilFil: Silva, Thelma L.. Universidade de Sao Paulo; BrasilFil: Dizak, Piper. University of Pittsburgh; Estados UnidosFil: Ruff, Timothy. University of Pittsburgh; Estados UnidosFil: Patir, Asli. İstanbul Medipol Üniversitesi; TurquíaFil: Koruyucu, Mine. İstanbul Üniversitesi; TurquíaFil: Abbasoğlu, Zerrin. Yeditepe Üniversitesi; TurquíaFil: Casado, Priscila L.. Universidade Federal Fluminense; BrasilFil: Brown, Andrew. University of Pittsburgh; Estados UnidosFil: Zaky, Samer H.. University of Pittsburgh; Estados UnidosFil: Bayram, Merve. İstanbul Medipol Üniversitesi; TurquíaFil: Küchler, Erika C.. University of Pittsburgh; Estados UnidosFil: Cooper, Margaret E.. University of Pittsburgh; Estados UnidosFil: Liu, Kai. University of Pittsburgh; Estados UnidosFil: Marazita, Mary L.. University of Pittsburgh; Estados UnidosFil: Tanboğa, İlknur. Marmara Üniversitesi; TurquíaFil: Granjeiro, José M.. Universidade Federal Fluminense; Brasil. Instituto Nacional de Metrologia, Qualidade e Tecnologia; BrasilFil: Seymen, Figen. İstanbul Üniversitesi; TurquíaFil: Castilla, Eduardo Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. CEMIC-CONICET. Centro de Educaciones Médicas e Investigaciones Clínicas "Norberto Quirno". CEMIC-CONICET.; Argentina. Fundación Oswaldo Cruz; BrasilFil: Orioli, Iêda M.. Universidade Federal do Rio de Janeiro; BrasilFil: Sfeir, Charles. University of Pittsburgh; Estados UnidosFil: Owyang, Hongjiao. Marmara Üniversitesi; TurquíaFil: Rabelo Buzalaf, Marilia Afonso. Universidade de Sao Paulo; BrasilFil: Vieira, Alexandre R.. University of Pittsburgh; Estados Unido

Characterization of spotted hyena, Crocuta crocuta microsatellite loci

We have isolated 10 polymorphic microsatellite loci in the spotted hyena,Crocuta crocuta.The loci displayed between eight and 14 alleles in a minimum of 12 individuals tested. These loci will be used to investigate relatedness within social groups, the genetic structure of populations, sexual selection, and mate choice in spotted hyenas

Circumpolar Diversity and Geographic Differentiation of mtDNA in the Critically Endangered Antarctic Blue Whale (Balaenoptera musculus intermedia)

To the best of our knowledge, one or more authors of this paper were federal employees when contributing to this work.\ud This is the publisher’s final pdf. The published article is copyrighted by the Public Library of Science and can be found at: http://www.plosone.org/home.action.The Antarctic blue whale (Balaenoptera musculus intermedia) was hunted to near extinction between 1904 and 1972, declining from an estimated initial abundance of more than 250,000 to fewer than 400. Here, we describe mtDNA control region diversity and geographic differentiation in the surviving population of the Antarctic blue whale, using 218 biopsy samples collected under the auspices of the International Whaling Commission (IWC) during research cruises from 1990-2009. Microsatellite genotypes and mtDNA sequences identified 166 individuals among the 218 samples and documented movement of a small number of individuals, including a female that traveled at least 6,650 km or 131 degrees longitude over four years. mtDNA sequences from the 166 individuals were aligned with published sequences from 17 additional individuals, resolving 52 unique haplotypes from a consensus length of 410 bp. From this minimum census, a rarefaction analysis predicted that only 72 haplotypes (95% CL, 64, 86) have survived in the contemporary population of Antarctic blue whales. However, haplotype diversity was relatively high (0.968 +/- 0.004), perhaps as a result of the longevity of blue whales and the relatively recent timing of the bottleneck. Despite the potential for circumpolar dispersal, we found significant differentiation in mtDNA diversity (F-ST = 0.032, p<0.005) and microsatellite alleles (F-ST = 0.005, p<0.05) among the six Antarctic Areas historically used by the IWC for management of blue whales

Identification and Characterization of White Grape Varieties Autochthonous of aWarm Climate Region (Andalusia, Spain)

The high sensitivity of one of the most important crops in the world, such as vine (Vitis vinifera L.), to particular changes caused by the phenomena associated with global warming, is encouraging the wine industry to place value on grape varieties that are autochthonous to each production area. These are generally conserved in germplasm banks and may pose a useful tool to counteract the effects of climate change. In order to determine the actual resource that such varieties constitute, this research has carried out a genetic identification, a morphological characterization, and an analysis of the grape musts obtained from four autochthonous varieties (Cañocazo, Castellano, Mantúo de Pilas, and Palomino Fino). This genetic analysis has allowed the identification of autochthonous varieties with different genotypes. However, all of them had similar phenotypic characteristics in terms of high hair density in adult leaves. With respect to the physicochemical composition of the musts, significant differences have been observed between the autochthonous varieties, with respect to the control variety of Palomino Fino. Nevertheless, all of them have exhibited an adequate physicochemical composition to produce quality white wines. For all of the above reasons, these local varieties should be considered suitable for cultivation in areas with warmer and drier climates, such as Andalusia (Spain)

Brucella abortus Infection of Placental Trophoblasts Triggers Endoplasmic Reticulum Stress-Mediated Cell Death and Fetal Loss via Type IV Secretion System-Dependent Activation of CHOP.

Subversion of endoplasmic reticulum (ER) function is a feature shared by multiple intracellular bacteria and viruses, and in many cases this disruption of cellular function activates pathways of the unfolded protein response (UPR). In the case of infection with Brucella abortus, the etiologic agent of brucellosis, the unfolded protein response in the infected placenta contributes to placentitis and abortion, leading to pathogen transmission. Here we show that B. abortus infection of pregnant mice led to death of infected placental trophoblasts in a manner that depended on the VirB type IV secretion system (T4SS) and its effector VceC. The trophoblast death program required the ER stress-induced transcription factor CHOP. While NOD1/NOD2 expression in macrophages contributed to ER stress-induced inflammation, these receptors did not play a role in trophoblast death. Both placentitis and abortion were independent of apoptosis-associated Speck-like protein containing a caspase activation and recruitment domain (ASC). These studies show that B. abortus uses its T4SS to induce cell-type-specific responses to ER stress in trophoblasts that trigger placental inflammation and abortion. Our results suggest further that in B. abortus the T4SS and its effectors are under selection as bacterial transmission factors.IMPORTANCE Brucella abortus infects the placenta of pregnant cows, where it replicates to high levels and triggers abortion of the calf. The aborted material is highly infectious and transmits infection to both cows and humans, but very little is known about how B. abortus causes abortion. By studying this infection in pregnant mice, we discovered that B. abortus kills trophoblasts, which are important cells for maintaining pregnancy. This killing required an injected bacterial protein (VceC) that triggered an endoplasmic reticulum (ER) stress response in the trophoblast. By inhibiting ER stress or infecting mice that lack CHOP, a protein induced by ER stress, we could prevent death of trophoblasts, reduce inflammation, and increase the viability of the pups. Our results suggest that B. abortus injects VceC into placental trophoblasts to promote its transmission by abortion