Post-Translational Tubulin Modifications in Differentiated Human Neural Stem Cells

The tubulin protein fulfills a variety of cellular functions that range from chromosomal separation to locomotion. The functional diversity of tubulin is achieved through the expression of specific tubulin isotypes in different cell types or developmental time periods. Post-translational modifications (PTMs) of tubulin also are vital for specific intracellular tasks, such as binding and recruiting motor proteins. In neurons, the isotypic expression profile for tubulin is well characterized, and the importance of PTMs for proper neuronal function has gained recent attention due to their implication in neurodegenerative disorders. In contrast, the role of tubulin specializations in the specification of neural cell fate has received minimal attention and studies of tubulin PTMs and isotypes in neuroglia such as astrocytes are relatively few. To bridge this knowledge gap, we undertook an analysis of PTMs in neurons and astrocytes derived from the federally approved H9 hESC-derived human neural stem cell (hNSC) line. In hNSCs, basal cells can be directed to assume neural fate as neurons or astrocytes by specifying different media growth conditions. Immunocytochemical methods, fluorescent antibody probes, and confocal microscopy facilitated image acquisition of fluorescent signals from class III β- tubulin (βIII-tubulin), acetylated tubulin, and polyglutamylated tubulin. Fluorescent probe intensities were assessed with the EBImage package for the statistical programming language R and compared using Student's t-tests. Qualitative analysis indicated that βIII-tubulin, acetylated tubulin, and polyglutamylated tubulin were expressed to some degree in basal hNSCs and their media-differentiated hNSC neuronal and astroglial progeny. In media-differentiated hNSC astrocyte progeny, quantification and statistical analysis of fluorescence probe intensity showed that acetylated tubulin/ βIII-tubulin ratios were greater than the ratio for polyglutamylated tubulin/ βIII-tubulin. These findings represent a snapshot of the dynamic and varied changes tubulin expression profile during the specification of neural cell fate. Results imply that investigations of tubulin PTMs have the potential to advance our understanding of the generation and regeneration of nervous tissue.

Customizable Natural Language Processing Biomarker Extraction Tool

PURPOSE Natural language processing (NLP) in pathology reports to extract biomarker information is an ongoing area of research. MetaMap is a natural language processing tool developed and funded by the National Library of Medicine to map biomedical text to the Unified Medical Language System Metathesaurus by applying specific tags to clinically relevant terms. Although results are useful without additional postprocessing, these tags lack important contextual information. METHODS Our novel method takes terminology-driven semantic tags and incorporates those into a semantic frame that is task-specific to add necessary context to MetaMap. We use important contextual information to capture biomarker results to support Community Health System's use of Precision Medicine treatments for patients with cancer. For each biomarker, the name, type, numeric quantifiers, non-numeric qualifiers, and the time frame are extracted. These fields then associate biomarkers with their context in the pathology report such as test type, probe intensity, copy-number changes, and even failed results. A selection of 6,713 relevant reports contained the following standard-of-care biomarkers for metastatic breast cancer: breast cancer gene 1 and 2, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, and programmed death-ligand 1. RESULTS The method was tested on pathology reports from the internal pathology laboratory at Henry Ford Health System. A certified tumor registrar reviewed 400 tests, which showed > 95% accuracy for all extracted biomarker types. CONCLUSION Using this new method, it is possible to extract high-quality, contextual biomarker information, and this represents a significant advance in biomarker extraction.

R-locus for roaned coat is associated with a tandem duplication in an intronic region of USH2A in dogs and also contributes to Dalmatian spotting

Structural variations (SVs) represent a large fraction of all genetic diversity, but how this genetic diversity is translated into phenotypic and organismal diversity is unclear. Explosive diversification of dog coat color and patterns after domestication can provide a unique opportunity to explore this question; however, the major obstacle is to efficiently collect a sufficient number of individuals with known phenotypes and genotypes of hundreds of thousands of markers. Using customer-provided information about coat color and patterns of dogs tested on a commercial canine genotyping platform, we identified a genomic region on chromosome 38 that is strongly associated with a mottled coat pattern (roaning) by genome-wide association study. We identified a putative causal variant in this region, an 11-kb tandem duplication (11,131,835–11,143,237) characterized by sequence read coverage and discordant reads of whole-genome sequence data, microarray probe intensity data, and a duplication-specific PCR assay. The tandem duplication is in an intronic region of usherin gene (USH2A), which was perfectly associated with roaning but absent in non-roaned dogs. We detected strong selection signals in this region characterized by reduced nucleotide diversity (π), increased runs of homozygosity, and extended haplotype homozygosity in Wirehaired Pointing Griffons and Australian Cattle Dogs (typically roaned breeds), as well as elevated genetic difference (FST) between Wirehaired Pointing Griffon (roaned) and Labrador Retriever (non-roaned). Surprisingly, all Dalmatians (N = 262) carried the duplication embedded in identical or similar haplotypes with roaned dogs, indicating this region as a shared target of selection during the breed’s formation. We propose that the Dalmatian’s unique spots were a derived coat pattern by establishing a novel epistatic interaction between roaning “R-locus” on chromosome 38 and an uncharacterized modifier locus. These results highlight the utility of consumer-oriented genotype and phenotype data in the discovery of genomic regions contributing to phenotypic diversity in dogs.

14 Telomere length in cloned camels produced by somatic cell nuclear transfer is not different from that in their naturally produced counterparts

There are controversial reports on the restoration of eroded telomere length in offspring produced by somatic cell nuclear transfer (SCNT) in different animal species. To the best of our knowledge, no earlier studies report telomere length in naturally produced or cloned animals in any of the camelid species. Therefore, the present study was conducted to estimate the telomere length in dromedary camels produced by SCNT, and their age-matched naturally produced counterparts by terminal restriction fragment (TRF) analysis. Genomic DNA was extracted from venous blood collected from 6 cloned animals (aged 3, 12, and 24 months), and their age-matched counterparts by a conventional phenol/chloroform protocol. Denatured and neutralized DNA was blotted onto a positively charged nylon membrane and fixed by baking at 80°C for 3h. After washing in a prewarmed digoxigenin (DIG) easy hybridization solution at 42°C for 1h, DNA hybridization was carried out using a telomere (TTAGGG)n-specific, DIG-labelled hybridization probe (Roche Diagnostics, Germany) at 42°C for 4h. Stringent washes were carried out at the same temperature, followed by chemiluminescence reaction. The signals were captured using the Azure Biosystems C600 gel documentation system. Molecular weights of the unknown TRF bands on the gel were calculated using known molecular weight marker provided by Telo TAGGG Telomere Length Assay Kit (Roche Diagnostics). A TeloTool program from MATLAB software with a built-in probe intensity correction algorithm was used for TRF analysis. The experiment was replicated 3 times, and the data, presented as mean±s.e.m., were analysed using a 2-sample t-test (Minitab statistical software). No difference (P>0.05) was found in the mean telomere length of cloned camels compared with their naturally produced age-matched counterparts (21.7±0.3 vs. 22.1±0.3; 21.9±0.3 vs. 22.1±0.3; 22.2±0.5 vs. 22.0±0.1; 20.5±0.5 vs. 22.5±0.7; 20.1±0.1 vs. 22.5±0.7; 21.7±1.1 vs. 22.6±0.2), respectively. In conclusion, this is the first study where telomere length has been reported in naturally produced and cloned dromedary camels produced by SCNT. We found that telomere lengths in cloned camels were similar to those of their age-matched naturally produced counterparts, suggesting that the camel cytoplast reprograms the somatic cell nucleus and restores the telomere length to its totipotency stage.

Preprint: Empirically-based comparisons of the reliability and validity of common quantification approaches for eyeblink startle potentiation in humans

Startle potentiation is a well validated translational measure of negative affect. Startle potentiation is widely used in clinical and affective science and there are multiple approaches for its quantification. The three most commonly used approaches quantify startle potentiation as the increase in startle response from a neutral to threat condition based on 1) raw potentiation, 2) standardized potentiation or 3) percent-change potentiation. These three quantification approaches may yield qualitatively different conclusions about effects of independent variables on affect when within or between group differences exist for startle response in the neutralcondition. Accordingly, we directly compared these quantification approaches in a shock-threat task using four independent variables known to influence startle response in the no-threat condition: probe intensity, time (i.e., habituation), alcohol administration, and individual differences in general startle reactivity measured at baseline. We confirmed the expected effects of time, alcohol, and general startle reactivity on affect using self-reported fear/anxiety as a criterion. The percent-change approach displayed apparent artifact across all four independent variables, which raises substantial concerns about its validity. Both raw and standardized potentiation approaches were stable across probe intensity and time, which supports their validity. However, only raw potentiation displayed effects that were consistentwith a priori specifications and/or the self-report criterion for the effects of alcohol and general startle reactivity. Supplemental analyses of reliability and validity for each approach provided additional evidence in support of raw potentiation.

Direct-To-Consumer DNA testing of 6,000 dogs reveals 98.6-kb duplication causing blue eyes and heterochromia in Siberian Huskies

SummaryConsumer genomics enables genetic discovery on an unprecedented scale by linking very large databases of personal genomic data with phenotype information voluntarily submitted via web-based surveys1. These databases are having a transformative effect on human genomic research, yielding insights on increasingly complex traits, behaviors, and disease by including many thousands of individuals in genome-wide association studies (GWAS)2, 3. The promise of consumer genomic data is not limited to human research, however. Genomic tools for dogs are readily available, with hundreds of causal Mendelian variants already characterized4–6, because selection and breeding have led to dramatic phenotypic diversity underlain by a simple genetic structure7, 8. Here, we report the results of the first consumer genomics study ever conducted in a non-human model: a GWAS of blue eyes based on more than 3,000 customer dogs with a validation panel of nearly 3,000 more, the largest canine GWAS to date. We discovered a novel association with blue eyes on chromosome 18 (P = 1x10-65) and used both sequence coverage and microarray probe intensity data to identify the putative causal variant: a 98.6-kb duplication directly upstream of the hox gene ALX4, which plays an important role in mammalian eye development9, 10. This duplication was largely restricted to Siberian Huskies and is highly, but not completely, penetrant. These results underscore the power of consumer-data-driven discovery in nonhuman species, especially dogs, where there is intense owner interest in the personal genomic information of their pets, a high level of engagement with web-based surveys, and an underlying genetic architecture ideal for mapping studies.

Simplifying Electron Beam Channeling in Scanning Transmission Electron Microscopy (STEM)

Sub-angstrom scanning transmission electron microscopy (STEM) allows quantitative column-by-column analysis of crystalline specimens via annular dark-field images. The intensity of electrons scattered from a particular location in an atomic column depends on the intensity of the electron probe at that location. Electron beam channeling causes oscillations in the STEM probe intensity during specimen propagation, which leads to differences in the beam intensity incident at different depths. Understanding the parameters that control this complex behavior is critical for interpreting experimental STEM results. In this work, theoretical analysis of the STEM probe intensity reveals that intensity oscillations during specimen propagation are regulated by changes in the beam’s angular distribution. Three distinct regimes of channeling behavior are observed: the high-atomic-number (Z) regime, in which atomic scattering leads to significant angular redistribution of the beam; the low-Zregime, in which the probe’s initial angular distribution controls intensity oscillations; and the intermediate-Zregime, in which the behavior is mixed. These contrasting regimes are shown to exist for a wide range of probe parameters. These results provide a new understanding of the occurrence and consequences of channeling phenomena and conditions under which their influence is strengthened or weakened by characteristics of the electron probe and sample.

Aberrant Patterns of Alternative Splicing Are Frequent Events and Harbor Prognostic Significance in Patients with Myelodysplastic Syndrome

Background Aberrant patterns of alternative splicing (AS) are reported in acute myeloid leukemia and other malignances, but have not yet been investigated in myelodysplastic syndrome (MDS). In addition, mutations in spliceosome genes are especially common in MDS. Therefore, it would be highly interesting to analyze the clinical and biological significance of aberrant AS in MDS. Materials and Methods A total of 243 newly diagnosed MDS patients, including 78 with refractory anemia (RA), 22 with RA with ring sideroblasts(RARS), 76 with RA with excess blasts (RAEB), 24 with RAEB in transformation (RAEBT), and 43 with chronic myelomonocyticleukemia (CMMoL), who had complete clinical and genetic information were enrolled. We also included 20 healthy donors of hematopoietic stem cell transplantation for comparison. We extracted RNA from mononuclear cells (MNC) isolated from the bone marrow (BM), followed by hybridization on the microarrays of AffymetrixHuman Transcriptome Array 2.0, which contained > 6 million distinct probes covering coding, non-coding transcripts, and the exon-exon junctions among 67539 genes (44710 coding genesand 22829 noncoding genes). This comprehensive coverage of exons and junctions facilitated investigation of the splicing patterns of the genes. The aberrant AS was analyzed with the splicing index (SI) method using the default setting of AffymetrixTranscriptome Analysis Console version 3.0 software. The SI of a probe in a gene was derived from the ratio between MDS patients and normal controls in terms of the probe intensity divided by the gene expression level, ie. [intensityof probe A in gene A /gene A expression level]MDS/[intensity of probe A in gene A /gene A expression level]normal control. SI had to be less than -2 or more than 2 to be defined as an aberrant AS event. Results Totally 52730 of 67539 genes (78.1%) on the array were expressed in BM from both MDS patients and normal donor controls. Approximately 17565 of the 52730 expressed genes (33.3%) were differentially spliced between the MDS and normal marrow MNC (P < 0.05). As a whole, 88.9% of these aberrant spliced events were mapped to coding genome and 11.1% to noncoding regions. These aberrant AS exons fell into five common patterns, including exon skipping (57.8%), alternative donor sites (18.1%), alternative acceptor sites (17.8%), intron retention (5.6%) and mutually exclusive exons (0.7%). In average, when compared with the normal marrow MNC, there were 6.4 (4.8-12.0) more aberrant AS events in each AS gene (ASG) in the marrow MNC of 243 MDS patients.More aberrant AS events per ASG implied a more complex aberrant AS pattern.The numbers of aberrant AS events per ASG were not significantly associated with MDS subtypes and frequencies of spliceosome gene mutations. However, with a median follow up of 48.4 months, the patients with more aberrant AS events per ASG had a trend of shorter overall survival (OS) (median 20.1 vs 29.6 months, P=0.074) and a trend of shorter time to acute leukemic transformation (P=0.212) than the others (Fig.1). Further subgroup analysis showed a significantly shorter OS in the patients with more aberrant AS events per ASG than those with less events among RAEB patients (median OS: 10.8 months vs 16.9 months, P=0.007, Fig.2A). In addition, RA patients with more aberrant AS events per ASG had shorter time to acute leukemic transformation than RA patients with fewer aberrant AS events per ASG (P=0.006, Fig.2B). Among the patients without detectable mutations in spliceosome genes or genes related to epigenetic modification, those with more aberrant AS events per ASG also had a shorter OS than the others (median OS: 27.9 months vs not reached, P=0.013, Fig.3). Multivariate analysis in our cohort of 243 MDS patients revealed that higher aberrant AS events per ASG was an unfavorable prognostic factor for OS (P=0.016) independent of age, IPSS-R risk score, and mutations of SF3B1, ASXL1 and TP53. Conclusion To our knowledge, we are the first to present the prognostic impact of aberrant AS pattern in MDS patients. Large prospective cohorts are needed to confirm our observations. Disclosures No relevant conflicts of interest to declare.