Hierarchy of simulation models in predicting structure and energetics of the Src SH2 domain binding to tyrosyl phosphopeptides.

Structure and energetics of the Src Src Homology 2 (SH2) domain binding with the recognition phosphopeptide pYEEI and its mutants are studied by a hierarchical computational approach. The proposed structure prediction strategy includes equilibrium sampling of the peptide conformational space by simulated tempering dynamics with the simplified, knowledge-based energy function, followed by structural clustering of the resulting conformations and binding free energy evaluation of a single representative from each cluster, a cluster center. This protocol is robust in rapid screening of low-energy conformations and recovers the crystal structure of the pYEEI peptide. Thermodynamics of the peptide-SH2 domain binding is analyzed by computing the average energy contributions over conformations from the clusters, structurally similar to the predicted peptide bound structure. Using this approach, the binding thermodynamics for a panel of studied peptides is predicted in a better agreement with the experiment than previously suggested models. However, the overall correlation between computed and experimental binding affinity remains rather modest. The results of this study show that small differences in binding free energies between the Ala and Gly mutants of the pYEEI peptide are considerably more difficult to predict than the structure of the bound peptides, indicating that accurate computational prediction of binding affinities still remains a major methodological and technical challenge.     

Robust Detection of DNA Hypermethylation of ZNF154 as a Pan-Cancer Locus with in Silico Modeling for Blood-Based Diagnostic Development

Sites that display recurrent, aberrant DNA methylation in cancer represent potential biomarkers for screening and diagnostics. Previously, we identified hypermethylation at the ZNF154 CpG island in 15 solid epithelial tumor types from 13 different organs. In this study, we measure the magnitude and pattern of differential methylation of this region across colon, lung, breast, stomach, and endometrial tumor samples using next-generation bisulfite amplicon sequencing. We found that all tumor types and subtypes are hypermethylated at this locus compared with normal tissue. To evaluate this site as a possible pan-cancer marker, we compare the ability of several sequence analysis methods to distinguish the five tumor types (184 tumor samples) from normal tissue samples (n = 34). The classification performance for the strongest method, measured by the area under (the receiver operating characteristic) curve (AUC), is 0.96, close to a perfect value of 1. Furthermore, in a computational simulation of circulating tumor DNA, we were able to detect limited amounts of tumor DNA diluted with normal DNA: 1% tumor DNA in 99% normal DNA yields AUCs of up to 0.79. Our findings suggest that hypermethylation of the ZNF154 CpG island is a relevant biomarker for identifying solid tumor DNA and may have utility as a generalizable biomarker for circulating tumor DNA.CME Accreditation Statement: This activity (“JMD 2016 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“JMD 2016 CME Program in Molecular Diagnostics”) for a maximum of 36 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.One in four deaths in the United States is due to cancer, despite an emphasis on prevention, early detection, and treatment that has lowered cancer death rates by 20% in the past two decades.^{1, 2} Further improvements in survival rates are likely to come from improving the limits of detection sensitivity at earlier stages of cancer. Currently, a diagnosis results from a cadre of screening and diagnostic tools that may include physical examination, radiographic imaging, sputum cytologic testing, blood tests, endoscopy, and/or biopsies. However, new approaches that rely heavily on genomic information may change future testing strategies.The future looks bright because minimally invasive sampling techniques coupled with genomic features that distinguish tumor cells from normal cells have the potential to detect cancer at earlier stages. For example, circulating tumor cells or cell-free plasma DNA can be detected in venous peripheral blood and tested for the presence of common mutations.³ Cell-free tumor DNA can also be detected in buccal epithelium, saliva, urine, stools, and bronchial aspirates.⁴ Such DNA has been used to detect mutations in patients with both localized and metastatic cancers.⁵ Moreover, somatic mutations in ovarian and endometrial cancers can potentially be detected using Papanicolaou specimens.⁶In addition to genetic mutations, epigenetic markers are emerging as tools with discriminatory power for disease detection. For example, DNA methylation is a robust epigenetic marker for which a number of commercially available tests have been developed. These tests detect tissue-specific DNA methylation using clinical specimens and are used in colorectal cancer (SEPT9, blood; VIM, stool), lung cancer (SHOX2, bronchial fluid), and brain cancer (MGMT, tumor).⁷ One advantage of this approach is marker stability under common storage conditions.⁴ However, despite DNA methylation''s potential as a diagnostic marker, a general lack of consensus on the methods remains. This is the principal reason for its slow implementation in clinical diagnostics.^{4, 7}Previously, our laboratory reported a pan-cancer hypermethylation signal around a CpG island near human ZNF154.⁸ This signal was initially detected by us in ovarian and endometrial cancers and replicated by us in multiple, independent cohorts from The Cancer Genome Atlas (TCGA), incorporating a total of 15 distinct tumor collections from 13 different organs with almost 6000 samples.⁸ These previous analyses relied on data generated from Illumina Infinium methylation arrays (Illumina Inc, San Diego, CA) to detect the methylation levels at select CpG sites. In this study, we measure the ZNF154 methylation signal across five tumor types using bisulfite amplicon sequencing. With this method, individual sequence reads are used to quantitate methylation levels of all CpGs within the amplicon while providing quantitative data for each DNA molecule in the pooled sample. Furthermore, the approach provides an intrinsic measure of quality control by tracking bisulfite conversion efficiency at cytosines in the non-CpG context wherein extensive amounts of unconverted cytosines signal an incomplete conversion reaction. This procedure is both time efficient and cost-effective because multiple samples can be sequenced in parallel using a 96-well plate and, as we report, generate reproducible measurements when assayed in independent experiments. The amplicon sequencing provides greater resolution of a target region than a methylation array by covering all amplified CpGs, revealing patterns of DNA methylation useful for distinguishing tumor from normal samples.We report that the magnitude and reproducibility of the ZNF154 hypermethylation signal across five solid tumor types reinforces the potential of this site as a biomarker for circulating tumor DNA (ctDNA). Next, we assess the potential application of various computational data classification methods toward cancer screening. By investigating a variety of technical approaches to characterize methylated bases within the sequenced samples, we identify features useful for distinguishing tumor samples from normal samples. Finally, we use a computational simulation to demonstrate the utility of these features in classifying samples as tumor or normal tissue at various abundance levels; here, tumor DNA methylation patterns are compiled into a background of normal DNA methylation patterns, at limiting dilution levels, mimicking the fractions at which ctDNA is recovered from blood.     

Comparative Characteristics of Sialoglycans Expression Disorders in the Placental Barrier Structures in Preeclampsia and Fetal Growth Restriction

Bulletin of Experimental Biology and Medicine - We compared the expression profiles of α2,3- and α2,6-sialoglycans in the glycocalyx of the placental barrier structures in early and late...     

Pathological upstaging of clinical T1 renal cell carcinoma: an analysis of 115,835 patients from National Cancer Data Base, 2004–2013

Purpose

Clinical staging is vital for treatment decision-making by renal cell carcinoma (RCC) patients. Some RCCs clinically appear T1 on CT, but are actually T3a due to extension into fat or renal vein, causing the tumor to be pathologically upstaged. The objective of this study to determine the rate, survival, and predictors of RCC upstaging, for patients with cT1 disease treated surgically.

Methods

Using the National Cancer Data Base Participant User File for RCC from 2004 to 2013, we selected AJCC cT1 patients, who underwent surgical resection and whose AJCC pathological T stage (pT) was available. Upstaging was characterized dichotomously—overall (any pT > T1) and pT3a-specific upstaging. Patient and tumor characteristics of those upstaged and not were compared using Chi-squared analyses. Multivariable logistic regression was used to analyze predictors of upstaging, and Cox proportional hazards regression was used to estimate overall survival hazards ratios.

Results

Overall upstaging (pT > T1) was observed in 8252 (7.1%) patients, and T3a-specific upstaging was observed in 3380 (5.4%) patients. Patients who were older, male, and had comorbidities, and tumors that were cT1b, underwent RN, and had high Fuhrman grade were at a higher risk of pathological upstaging. Upstaging led to a 40% increased risk of death compared to patients who were not upstaged.

Conclusion

The rate of upstaging is not negligible (5–7% of the time), negatively impacts survival, and various patient and tumor characteristics can be used to predict upstaging.

     

Mosaic retroposon insertion patterns in placental mammals

One and a half centuries after Charles Darwin and Alfred Russel Wallace outlined our current understanding of evolution, a new scientific era is dawning that enables direct observations of genetic variation. However, pure sequence-based molecular attempts to resolve the basal origin of placental mammals have so far resulted only in apparently conflicting hypotheses. By contrast, in the mammalian genomes where they were highly active, the insertion of retroelements and their comparative insertion patterns constitute a neutral, virtually homoplasy-free archive of evolutionary histories. The “presence” of a retroelement at an orthologous genomic position in two species indicates their common ancestry in contrast to its “absence” in more distant species. To resolve the placental origin controversy we extracted ∼2 million potentially phylogenetically informative, retroposon-containing loci from representatives of the major placental mammalian lineages and found highly significant evidence challenging all current single hypotheses of their basal origin. The Exafroplacentalia hypothesis (Afrotheria as the sister group to all remaining placentals) is significantly supported by five retroposon insertions, the Epitheria hypothesis (Xenarthra as the sister group to all remaining placentals) by nine insertion patterns, and the Atlantogenata hypothesis (a monophyletic clade comprising Xenarthra and Afrotheria as the sister group to Boreotheria comprising all remaining placentals) by eight insertion patterns. These findings provide significant support for a “soft” polytomy of the major mammalian clades. Ancestral successive hybridization events and/or incomplete lineage sorting associated with short speciation intervals are viable explanations for the mosaic retroposon insertion patterns of recent placental mammals and for the futile search for a clear root dichotomy.Genomic variation is based on genetic convertibility and is amplified by gene flow and sexual recombination. While only a small fraction of genetic variation is directly exposed to the natural selection driving the evolution of species, the majority of changes are neutral (Kimura 1968). In the molecular Darwinian and post-genomic age when such variations are now directly observable in prodigious numbers, one still needs only compare the volumes of controversy in scientific reports to dispel the notion that all the mysteries of evolutionary history are now an open book. The basal node of the placental mammals is a prime example of such a controversy. After continuous morphological inconsistencies and a lack of resolution due to narrow temporal speciation events at the deeper divergences of the placental mammalian tree, the hope was that large-scale DNA sequencing and an increasing number of sampled species might resolve the higher-level mammalian relationships by reducing systematic biases. But the extraction and analysis of molecular traces of evolutionary history has been no less sensitive to misinterpretation than was the understanding of dusty specimens 150 years ago. The reliability of sequence data in genome-scale phylogenetic approaches is subject to unequal evolutionary rates among lineages, regional- or lineage-specific compositional biases, shifts in site-specific evolutionary rates over time and sequence regions (e.g., Nishihara et al. 2007), variable accuracies of multisequence alignments and analytical tools, and last but not least, the general liability of such data to homoplasy.In a seminal work, Murphy et al. (2001a) investigated ∼10 kb of sequence information to substantiate the previously proposed (Waddell et al. 1999) major mammalian groups Afrotheria, Xenarthra, Laurasiatheria, and Euarchontoglires. Moderate support was found for Afrotheria as the basal split of placentals (Exafroplacentalia; Fig. 1A). A subsequent combination and expansion of previously published data (Madsen et al. 2001; Murphy et al. 2001a) found significant support for a basal split between Afrotheria and other placentals (Murphy et al. 2001b). Nikolaev et al. (2007) also found support for the Afrotheria as the first placental split, drawing on ∼200 kb of protein-coding sequences from the 1% of the human genome studied in the ENCODE pilot project (The ENCODE Project Consortium 2007). However, other studies of large-scale sequences (e.g., Hallström et al. 2007) and previous studies of smaller data sets could not significantly confirm an Afrotherian root (e.g., Madsen et al. 2001; Waddell et al. 2001; Delsuc et al. 2002; Amrine-Madsen et al. 2003). A sister-group relationship of Afrotheria and Xenarthra (Atlantogenata) also received significant support in analyses of sequences from ∼1700 conserved genome loci (Wildman et al. 2007), 2840 protein-coding genes (Hallström et al. 2007), and mitochondrial genomes (Kjer and Honeycutt 2007); and this was recently confirmed by large-scale analyses of 2.8 Mbp of protein-coding data (Hallström and Janke 2008) and large-scale genomic sequences (Prasad et al. 2008). Thus, despite many different molecular studies, the basal origin of placental mammals remains controversial and unresolved. Unfortunately, pure sequence data cannot resolve these apparent contradictions.Open in a separate windowFigure 1.Different hypotheses of the placental origin. (A) The Exafroplacentalia hypothesis proposes a mammalian clade merging Boreotheria (Supraprimates plus Laurasiathera) and Xenarthra, with Afrotheria as the sister group. (B) The Epitheria hypothesis merges Boreotheria and Afrotheria, with Xenarthra as the sister group. (C) The Atlantogenata hypothesis proposes Xenarthra and Afrotheria in one clade.By contrast, the presence/absence patterns of inserted retroelements constitute a virtually homoplasy-free marker system with theoretically infinite character states (Steel and Penny 2000). Specific genomic insertions of such elements in the ancestor of two species reliably document their common ancestry. The very few known examples of discordance in retroelement presence/absence data can be explained by deletion via illegitimate recombination between perfect direct repeats flanking each insertion (van de Lagemaat et al. 2005), exact parallel insertions (Cantrell et al. 2001), or lineage sorting, a phenomenon related to incomplete allele fixation and frequent intervals of speciation events (Shedlock et al. 2004; Ray et al. 2006).Analyses of a smaller number of retroelements as phylogenetic markers in mammals validated the four superordinal mammalian clades (Nishihara et al. 2005; Kriegs et al. 2006; Möller-Krull et al. 2007). However, in support of Shoshani and McKenna (1998), but in contradiction to most other molecular investigations, we found the first evidence, two L1MB5 retroelements, that placed Xenarthra (Epitheria hypothesis) (Fig. 1B) instead of Afrotheria at the base of the placental tree (Kriegs et al. 2006). Meanwhile, Murphy et al. (2007) presented two L1MB5 retroelements and four additional indels that favored a sister group relationship of Afrotheria and Xenarthra (Atlantogenata) (Fig. 1C) at the base of the placental tree. Thus, to date, even retroposon insertion patterns have not satisfactorily resolved the basal split of placental mammals.With the intention of resolving the conflicting topologies of placental lineages, we aimed to examine a much larger sample of retroposed elements and in more species. For this study we took a slightly different tack and aligned whole genomes of three representative placental species, including additional species for informative loci to represent a maximum divergence within each of the groups, and scanned them for diagnostic retroelement insertions. In an analysis of ∼2 million potential phylogenetically informative loci, we found multiple retroposon insertion patterns significantly supporting all three placental speciation hypotheses, indicating a complex ancestral speciation scenario including successive early divergences in close temporal proximity and hybridization and/or lineage sorting events as viable sources for an effective “soft” polytomy. This very rare example of multiple retroposon incongruence goes a long way toward explaining the decades-long stream of apparently conflicting evidence for one or the other placental evolutionary history based on multiple marker systems including both morphological and molecular sequence evidence.     

Myasthenia gravis accompanied by thymomas not related to foamy virus genome in Belarusian's patients

The spectrum and features of neurological disorders have been changed due to the Chernobyl catastrophe in the Republic of Belarus. More recently neurologists in Belarus have noted a significant increase in the frequency of myasthenia gravis (MG) with concomitant rise in the thymomas. There is some evidence suggesting that retroviruses play a key role in the development and pathogenesis of autoimmune diseases. This study analyzed thymomas from 45 MG patients from the Republic of Belarus by using PCR and primers for two regions of FV--gag and bel-2 genes. The results showed that none of the varied thymuses from the 45 MG patients contained FV genome. No relationship can be confirmed between FV and this disease and the results suggest that no pathological link between FV and MG exists.     

Motivation, emotion, and their inhibitory control mirrored in brain oscillations

Recent studies suggest brain oscillations as a mechanism for cerebral integration. Such integration can exist across a number of functional domains, with different frequency rhythms associated with each domain. Here, evidence is summarized which shows that delta oscillations depend on activity of motivational systems and participate in salience detection. Theta oscillations are involved in memory and emotional regulation. Alpha oscillations participate in inhibitory processes which contribute to a variety of cognitive operations such as attention and memory. The importance of inhibitory functions associated with alpha oscillations increases during the course of evolution. In ontogenesis, these functions develop later and may be more sensitive to a variety of detrimental environmental influences. In a number of developmental stages and pathological conditions, a deficient alpha and/or increased slow-wave activity are associated with cognitive deficits and a lack of inhibitory control. It is shown that slow-wave and alpha oscillations are reciprocally related to each other. This reciprocal relationship may reflect an inhibitory control over motivational and emotional drives which is implemented by the prefrontal cortex.     

The sea urchin embryo, an invertebrate model for mammalian developmental neurotoxicity, reveals multiple neurotransmitter mechanisms for effects of chlorpyrifos: therapeutic interventions and a comparison with the monoamine depleter, reserpine

Lower organisms show promise for the screening of neurotoxicants that might target mammalian brain development. Sea urchins use neurotransmitters as embryonic growth regulatory signals, so that adverse effects on neural substrates for mammalian brain development can be studied in this simple organism. We compared the effects of the organophosphate insecticide, chlorpyrifos in sea urchin embryos with those of the monoamine depleter, reserpine, so as to investigate multiple neurotransmitter mechanisms involved in developmental toxicity and to evaluate different therapeutic interventions corresponding to each neurotransmitter system. Whereas reserpine interfered with all stages of embryonic development, the effects of chlorpyrifos did not emerge until the mid-blastula stage. After that point, the effects of the two agents were similar. Treatment with membrane permeable analogs of the monoamine neurotransmitters, serotonin and dopamine, prevented the adverse effects of either chlorpyrifos or reserpine, despite the fact that chlorpyrifos works simultaneously through actions on acetylcholine, monoamines and other neurotransmitter pathways. This suggests that different neurotransmitters, converging on the same downstream signaling events, could work together or in parallel to offset the developmental disruption caused by exposure to disparate agents. We tested this hypothesis by evaluating membrane permeable analogs of acetylcholine and cannabinoids, both of which proved effective against chlorpyrifos- or reserpine-induced teratogenesis. Invertebrate test systems can provide both a screening procedure for mammalian neuroteratogenesis and may uncover novel mechanisms underlying developmental vulnerability as well as possible therapeutic approaches to prevent teratogenesis.     

Improved sentinel node identification by SPECT/CT in overweight patients with breast cancer.

Overweight has been reported as a cause for the nonvisualization of sentinel nodes (SNs) on preoperative planar lymphoscintigraphy in patients with breast cancer. The purpose of this study was to assess whether SPECT/CT may improve SN identification in overweight patients. METHODS: Lymphoscintigraphy was performed in 220 consecutive patients with breast cancer. Body mass index (BMI) was calculated for each. A total of 122 patients were overweight or obese (BMI, > or = 25). Planar images and SPECT/CT images were interpreted separately, and SN identification on each of the modalities was related to BMI and to findings at surgery. RESULTS: Planar imaging identified SNs in 171 patients (78%) with a BMI (mean +/- SD) of 25.2 +/- 4 kg/m2 and failed to do so in 49 patients (22%) with a BMI of 28 +/- 8 kg/m2. In 29 of the latter patients (59%), SNs were identified on SPECT/CT. SPECT/CT detected "hot" nodes in 200 patients (91%) and failed to do so in 20 patients with a BMI of 29.2 +/- 6.6 kg/m2. For the 122 overweight or obese patients, planar assessment failed to identify SNs in 34 patients (28%) and SPECT/CT failed to do so in 13 patients (11%) (P < 0.001). For 116 patients, surgery took place in our hospital (Tel-Aviv Sourasky Medical Center). An intraoperative blue dye technique failed to detect SNs in 48 patients (41%) with a BMI of 28.2 +/- 7 kg/m2. SPECT/CT localized hot nodes in 36 (75%) of the latter patients, and planar imaging did so in 22 (46%) of those patients. Of 19 patients for whom scintigraphy failed, 6 (32%) had nodal metastatic involvement. CONCLUSION: The addition of SPECT/CT to lymphoscintigraphy improved SN identification in overweight patients with breast cancer. Moreover, SPECT/CT accurately identified SNs in 75% of patients for whom the identification of SNs by the intraoperative blue dye technique failed.     

Surgical management of large renal tumors

The surgical management of patients with renal cell carcinoma has undergone many changes. With equivalent oncologic outcomes and appreciation of the importance of renal functional preservation, the utilization of nephron-sparing partial nephrectomy has increased in recent years. Nevertheless, tumors of larger size continue to be preferentially treated with radical nephrectomy. Here, we present evidence that improvements in techniques and durability of oncologic outcomes has justified the use of nephron sparing to accomplish renal functional preservation even in patients with large renal tumors. In addition, surgical technical considerations when managing such tumors are discussed. Finally, we discuss cytoreductive surgery and the evolving role of systemic targeted therapies in the management of advanced metastatic disease.