Reducing microbial and human contamination in DNA extractions from ancient bones and teeth

Although great progress has been made in improving methods for generating DNA sequences from ancient biological samples, many, if not most, samples are still not amenable for analyses due to overwhelming contamination with microbial or modern human DNA. Here we explore different DNA decontamination procedures for ancient bones and teeth for use prior to DNA library preparation and high-throughput sequencing. Two procedures showed promising results: (i) the release of surface-bound DNA by phosphate buffer and (ii) the removal of DNA contamination by sodium hypochlorite treatment. Exposure to phosphate removes on average 64% of the microbial DNA from bone powder but only 37% of the endogenous DNA (from the organism under study), increasing the percentage of informative sequences by a factor of two on average. An average 4.6-fold increase, in one case reaching 24-fold, is achieved by sodium hypochlorite treatment, albeit at the expense of destroying 63% of the endogenous DNA preserved in the bone. While both pretreatment methods described here greatly reduce the cost of genome sequencing from ancient material due to efficient depletion of microbial DNA, we find that the removal of human DNA contamination remains a challenging problem

Nuclear and mitochondrial DNA sequences from two Denisovan individuals

Denisovans, a sister group of Neandertals, have been described on the basis of a nuclear genome sequence from a finger phalanx (Denisova 3) found in Denisova Cave in the Altai Mountains. The only other Denisovan specimen described to date is a molar (Denisova 4) found at the same site. This tooth carries a mtDNA sequence similar to that of Denisova 3. Here we present nuclear DNA sequences from Denisova 4 and a morphological description, as well as mitochondrial and nuclear DNA sequence data, from another molar (Denisova 8) found in Denisova Cave in 2010. This new molar is similar to Denisova 4 in being very large and lacking traits typical of Neandertals and modern humans. Nuclear DNA sequences from the two molars form a clade with Denisova 3. The mtDNA of Denisova 8 is more diverged and has accumulated fewer substitutions than the mtDNAs of the other two specimens, suggesting Denisovans were present in the region over an extended period. The nuclear DNA sequence diversity among the three Denisovans is comparable to that among six Neandertals, but lower than that among present-day humans

Inference of natural selection from ancient DNA.

Evolutionary processes, including selection, can be indirectly inferred based on patterns of genomic variation among contemporary populations or species. However, this often requires unrealistic assumptions of ancestral demography and selective regimes. Sequencing ancient DNA from temporally spaced samples can inform about past selection processes, as time series data allow direct quantification of population parameters collected before, during, and after genetic changes driven by selection. In this Comment and Opinion, we advocate for the inclusion of temporal sampling and the generation of paleogenomic datasets in evolutionary biology, and highlight some of the recent advances that have yet to be broadly applied by evolutionary biologists. In doing so, we consider the expected signatures of balancing, purifying, and positive selection in time series data, and detail how this can advance our understanding of the chronology and tempo of genomic change driven by selection. However, we also recognize the limitations of such data, which can suffer from postmortem damage, fragmentation, low coverage, and typically low sample size. We therefore highlight the many assumptions and considerations associated with analyzing paleogenomic data and the assumptions associated with analytical methods

Genetic insights into the social organization of Neanderthals

Genomic analyses of Neanderthals have previously provided insights into their population history and relationship to modern humans1–8, but the social organization of Neanderthal communities remains poorly understood. Here we present genetic data for 13 Neanderthals from two Middle Palaeolithic sites in the Altai Mountains of southern Siberia: 11 from Chagyrskaya Cave9,10 and 2 from Okladnikov Cave11—making this one of the largest genetic studies of a Neanderthal population to date. We used hybridization capture to obtain genome-wide nuclear data, as well as mitochondrial and Y-chromosome sequences. Some Chagyrskaya individuals were closely related, including a father–daughter pair and a pair of second-degree relatives, indicating that at least some of the individuals lived at the same time. Up to one-third of these individuals’ genomes had long segments of homozygosity, suggesting that the Chagyrskaya Neanderthals were part of a small community. In addition, the Y-chromosome diversity is an order of magnitude lower than the mitochondrial diversity, a pattern that we found is best explained by female migration between communities. Thus, the genetic data presented here provide a detailed documentation of the social organization of an isolated Neanderthal community at the easternmost extent of their known range

Expression of cyclin D1, D3, E, and p27 in human renal cell carcinoma analysed by tissue microarray

Aberrations in the GI/S transition of the cell cycle have been observed in many malignancies and seem to be critical in the transformation process. Few studies have delineated the presence of GI/S regulatory defects and their clinical relevance in renal cell carcinoma (RCC). Therefore, we have examined the protein contents of cyclin D 1, D3, E, and p27 in 218 RCCs, using tissue microarray and immunohistochemistry. The results from a subset of tumours were confirmed by Western blotting and immunohistochemical staining of regular tissue sections. Interestingly, low protein contents of cyclin D I and p27 were associated with high nuclear grade, large tumour size, and poor prognosis for patients with conventional tumours. We further observed substantial differences in the pattern of GI/S regulatory defects between the different RCC subtypes. The majority of both conventional and papillary cases expressed p27; however, chromophobe tumours generally lacked p27 staining. In addition, conventional RCCs often expressed high cyclin DI protein levels, while papillary RCCs exhibited high cyclin E. In summary, we have shown that GI/S regulatory defects are present in RCC and are associated with clinico-pathological parameters. The pattern of cell cycle regulatory defects also differed between RCC subtypes. (C) 2003 Cancer Research UK

Modified Epidermal Growth Factor Receptor (EGFR)-Bearing Liposomes (MRBLs) Are Sensitive to EGF in Solution

Cancers often overexpress EGF and other growth factors to promote cell replication and migration. Previous work has not produced targeted drug carriers sensitive to abnormal amounts of growth factors. This work demonstrates that liposomes bearing EGF receptors covalently crosslinked to p-toluic acid or methyl-PEO4-NHS ester (or, in short, MRBLs) exhibit an increased rate of release of encapsulated drug compounds when EGF is present in solution. Furthermore, the modified EGF receptors retain the abilities to form dimers in the presence of EGF and bind specifically to EGF. These results demonstrate that MRBLs are sensitive to EGF in solution and indicate that MRBL-reconstituted modified EGF receptors, in the presence of EGF in solution, form dimers which increase MRBL permeability to encapsulated compounds

Expression and subcellular localization of cyclin D1 protein in epithelial ovarian tumour cells

The expression of cyclin D1 protein in tumour sections from 81 patients with epithelial ovarian cancer was analysed using immunohistochemistry. The tumours that overexpressed cyclin D1 in more than 10% of neoplastic cells were considered positive. Thus overexpression of cyclin D1 was observed in 72/81 (89%) of the cases examined. Protein was detected in both the nucleus and the cytoplasm in 24/81 (30%) and localized exclusively in the cytoplasm in 48/81 (59%) of the tumours. Cyclin D1 was overexpressed in both borderline and invasive tumours. There was no association between protein overexpression and tumour stage and differentiation. Furthermore, no correlation between cyclin D1 expression and clinical outcome was observed. However, in tumours overexpressing cyclin D1 (n = 72), the proportion displaying exclusively cytoplasmic localization of protein was higher in those with serous compared with non-serous histology (P = 0.004, odds ratio 4.8, 95% confidence interval 1.4–19.1). Western analysis using a monoclonal antibody to cyclin D1 identified a 36 kDa protein in homogenates from seven tumours displaying cytoplasmic only and one tumour demonstrating both nuclear and cytoplasmic immunostaining. Using restriction fragment length polymorphism polymerase chain reaction and PCR-multiplex analysis, amplification of the cyclin D1 gene (CCNDI) was detected in 1/29 of the tumours demonstrating overexpression of cyclin D1 protein. We conclude that deregulation of CCND1 expression leading to both cytoplasmic and nuclear protein localization is a frequent event in ovarian cancer and occurs mainly in the absence of gene amplification. © 1999 Cancer Research Campaig

Validation study of the prognostic value of cyclin-dependent kinase (CDK)-based risk in Caucasian breast cancer patients

In a Japanese study, cyclin-dependent kinase (CDK) based risk determined by CDK 1 and 2 activities was associated with risk of distance recurrence in early breast cancer patients. The aim of our study was to validate this risk categorization in European early breast cancer patients. We retrospectively analyzed frozen breast cancer specimens of 352 Dutch patients with histologically confirmed primary invasive early breast cancer. CDK-based risk was determined in tumour tissues by calculating a risk score (RS) according to kinases activity and protein mass concentration assay without the knowledge of outcome. Determination of CDK-based risk was feasible in 184 out of 352 (52%) tumours. Median follow-up of these patients was 15 years. In patients not receiving systemic treatment, the proportions of risk categories were 44% low, 16% intermediate, and 40% high CDK-based risk. These groups remained significant after univariate and multivariate Cox-regression analysis. Factors associated with a shorter distant recurrence-free period were positive lymph nodes, mastectomy with radiotherapy, and high CDK-based risk. There was no significant correlation with overall survival (OS). CDK-based risk is a prognostic marker of distance recurrence of patients with early breast cancer. More validation would be warranted to use of CDK-based risk into clinical practice