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This study describes the validation of a clinical RNA expression panel with evaluation of concordance between gene copy gain by a next-generation sequencing (NGS) assay and high gene expression by an RNA expression panel. The RNA Salah Targeted Expression Panel (RNA STEP) was designed with input from oncologists to include 204 genes with utility for clinical trial prescreening and therapy selection. RNA STEP was validated with the nanoString platform using remnant formalin-fixed, paraffin-embedded-derived RNA from 102 patients previously tested with a validated clinical NGS panel. The repeatability, reproducibility, and concordance of RNA STEP results with NGS results were evaluated. RNA STEP demonstrated high repeatability and reproducibility, with excellent correlation (r > 0.97, P < 0.0001) for all comparisons. Comparison of RNA STEP high gene expression (log2 ratio ≥ 2) versus NGS DNA-based gene copy number gain (copies ≥ 5) for 38 mutually covered genes revealed an accuracy of 93.0% with a positive percentage agreement of 69.4% and negative percentage agreement of 93.8%. Moderate correlation was observed between platforms (r = 0.53, P < 0.0001). Concordance between high gene expression and gene copy number gain varied by specific gene, and some genes had higher accuracy between assays. Clinical implementation of RNA STEP provides gene expression data complementary to NGS and offers a tool for prescreening patients for clinical trials., Competing Interests: Disclosure Statement T.A.B. and J.M.K. have a contract with Bristol Myers Squibb unrelated to this research. E.B.H. consults for Revolution Medicines, Janssen, Ellipses Pharma, Amgen, Kanaph Therapeutics, and ORI Capital, and holds a patent for protein-protein interactions as biomarkers; and research funding from Janssen, Novartis, Revolution Medicines, AstraZeneca, Spectrum Pharmaceuticals, and Genentech, all unrelated to this research. A.N.S. has clinical trial funding to his institution from AstraZeneca, BioAtla, Daiichi Sankyo, Eli Lily, Genentech, Genmab, Memgen, Mersana, Novartis, and Turning Point Therapeutics; and has participated in advisory boards for Daiichi Sankyo, Eli Lilly, and Zymeworks, all unrelated to this research., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social interactions, communication deficits and repetitive behaviors. A study of autistic human subjects has identified RFWD2 as a susceptibility gene for autism, and autistic patients have 3 copies of the RFWD2 gene. The role of RFWD2 as an E3 ligase in neuronal functions, and its contribution to the pathophysiology of ASD, remain unknown. We generated RFWD2 knockin mice to model the human autistic condition of high gene dosage of RFWD2. We found that heterozygous knockin (Rfwd2 +/- ) male mice exhibited the core symptoms of autism. Rfwd2 +/- male mice showed deficits in social interaction and communication, increased repetitive and anxiety-like behavior, and spatial memory deficits, whereas Rfwd2 +/- female mice showed subtle deficits in social communication and spatial memory but were normal in anxiety-like, repetitive, and social behaviors. These autistic-like behaviors in males were accompanied by a reduction in dendritic spine density and abnormal synaptic function on layer II/III pyramidal neurons in the prelimbic area of the medial prefrontal cortex (mPFC), as well as decreased expression of synaptic proteins. Impaired social behaviors in Rfwd2 +/- male mice were rescued by the expression of ETV5, one of the major substrates of RFWD2, in the mPFC. These findings indicate an important role of RFWD2 in the pathogenesis of autism., (© 2024. The Author(s).)

Cohesin, a chromatin-associated protein complex with four core subunits (Smc1a, Smc3, Rad21 and either Stag1 or 2), has a central role in cell proliferation and gene expression in metazoans. Human developmental disorders termed 'cohesinopathies' are characterized by germline variants of cohesin or its regulators that do not entirely eliminate cohesin function. However, it is not clear whether mutations in individual cohesin subunits have independent developmental consequences. Here, we show that zebrafish rad21 or stag2b mutants independently influence embryonic tailbud development. Both mutants have altered mesoderm induction, but only homozygous or heterozygous rad21 mutation affects cell cycle gene expression. stag2b mutants have narrower notochords and reduced Wnt signaling in neuromesodermal progenitors as revealed by single-cell RNA sequencing. Stimulation of Wnt signaling rescues transcription and morphology in stag2b, but not rad21, mutants. Our results suggest that mutations altering the quantity versus composition of cohesin have independent developmental consequences, with implications for the understanding and management of cohesinopathies., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Context.—: The American Society of Clinical Oncology/College of American Pathologists 2018 update of the human epidermal growth factor receptor 2 (HER2) testing guideline includes a fluorescence in situ hybridization (FISH) group with a HER2 to chromosome 17 centromere (CEP17) ratio less than 2.0 and HER2 copy number 6.0 or greater (group 3), which requires integrated review of HER2 immunohistochemistry (IHC)., Objective.—: To assess the clinicopathologic features of group 3 patients and determine features associated with HER2-positive status after workup., Design.—: Cases submitted for HER2 FISH between January 2019 and June 2022 were identified, and relevant clinicopathologic information was obtained., Results.—: One hundred forty-two HER2 FISH cases (1.6%) were group 3. In 52 cases (36.6%) IHC was negative (0/1+), in 3 (2.8%) IHC was positive (3+), and in 86 (60.6%) IHC was 2+. Annotated IHC 2+ slides were recounted by a second reviewer in targeted areas, where 16 of 86 (18.6%) had a HER2:CEP17 ratio less than 2.0 and a HER2 copy number of 4.0 or greater to less than 6.0 (HER2 negative). After combined IHC/FISH review, 74 of 142 (52.1%) were classified as HER2 positive. HER2 copy number/cell was higher in HER2-positive compared with HER2-negative cases after the workup. The extent and intensity of staining in IHC 2+ cases did not correlate with the level of gene amplification. Twenty percent of HER2-positive patients achieved pathologic complete response., Conclusions.—: About half of group 3 cases were classified as HER2 positive after additional workup. Pathologic complete response rates in HER2-positive cases were lower than expected for group 1 (HER2:CEP17 ratio ≥2.0; HER2 copy number ≥4.0) patients. IHC-targeted FISH recounts may be redundant and may potentially lead to classification of some patients as HER2 negative, resulting in withholding of targeted therapy., (© 2024 College of American Pathologists.)

The evolution of gene families is complex, involving gene-level evolutionary events such as gene duplication, horizontal gene transfer, and gene loss, and other processes such as incomplete lineage sorting (ILS). Because of this, topological differences often exist between gene trees and species trees. A number of models have been recently developed to explain these discrepancies, the most realistic of which attempts to consider both gene-level events and ILS. When unified in a single model, the interaction between ILS and gene-level events can cause polymorphism in gene copy number, which we refer to as copy number hemiplasy (CNH). In this paper, we extend the Wright-Fisher process to include duplications and losses over several species, and show that the probability of CNH for this process can be significant. We study how well two unified models-multilocus multispecies coalescent (MLMSC), which models CNH, and duplication, loss, and coalescence (DLCoal), which does not-approximate the Wright-Fisher process with duplication and loss. We then study the effect of CNH on gene family evolution by comparing MLMSC and DLCoal. We generate comparable gene trees under both models, showing significant differences in various summary statistics; most importantly, CNH reduces the number of gene copies greatly. If this is not taken into account, the traditional method of estimating duplication rates (by counting the number of gene copies) becomes inaccurate. The simulated gene trees are also used for species tree inference with the summary methods ASTRAL and ASTRAL-Pro, demonstrating that their accuracy, based on CNH-unaware simulations calibrated on real data, may have been overestimated., (© The Author(s) 2024. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For commercial re.use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site.for further information please contact journals.permissions@oup.com.)

Neuroinflammation is a prominent feature of Alzheimer's disease (AD). Activated microglia undergo a reprogramming of cellular metabolism necessary to power their cellular activities during disease. Thus, selective targeting of microglial immunometabolism might be of therapeutic benefit for treating AD. In the AD brain, the levels of microglial hexokinase 2 (HK2), an enzyme that supports inflammatory responses by promoting glycolysis, are significantly increased. In addition, HK2 displays non-metabolic activities that extend its inflammatory role beyond glycolysis. The antagonism of HK2 affects microglial phenotypes and disease progression in a gene-dose-dependent manner. HK2 complete loss fails to improve pathology by exacerbating inflammation, while its haploinsufficiency reduces pathology in 5xFAD mice. We propose that the partial antagonism of HK2 is effective in slowing disease progression by modulating NF-κB signaling through its cytosolic target, IKBα. The complete loss of HK2 affects additional inflammatory mechanisms related to mitochondrial dysfunction., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: High-grade aneuploidies of X and Y sex chromosomes (HGAs) are exceedingly rare and complex conditions. We aimed to investigate the effect of supernumerary X chromosomes (extra-Xs) on the clinical, hormonal, metabolic, and echocardiographic features of patients with HGAs., Design and Methods: In a cross-sectional study, we compared 23 subjects with HGAs and 46 age-matched subjects with 47,XXY Klinefelter syndrome (KS), according to the number of extra-Xs: two (47,XXY and 48,XXYY), three (48,XXXY and 49,XXXYY), or four supernumerary Xs (49,XXXXY). A second cohort consisting of 46 pubertal stage-matched KS subjects was employed for validation. Clinical, hormonal, metabolic and ultrasonographic parameters were collected and analyzed., Results: The increase in the number of extra-Xs was associated with a progressive adverse effect on height, pubertal development, testicular volume and function, adrenal steroidogenesis, and thyroid function. A progressive linear increase in ACTH and a decrease in cortisol/ACTH ratios were found. Weight and body mass index, Sertoli cell function, lipid profile, and glucose tolerance post-oral glucose tolerance test were all worse in the HGA cohort compared to KS. Cardiac evaluation revealed a linear association with reduced left and right end-diastolic diameters and reduced ejection fraction., Conclusion: The increase in the number of extra-Xs is associated with a "dose-dependent" progressive impairment in steroid producing glands, thyroid function, cardiac structure, and performance., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

Even with advanced plasmid and viral vectors, attaining copy numbers of multiple genes among different transfected cells is challenging. We achieved one gene expression from a single-copy gene in one cell using a transgene competition system, a combination of the Kazusa cDNA clones and our dual recombinase-mediated cassette exchange system. All 48 nuclear receptors were simultaneously expressed in one dish at the same expression level in HEK293 using this system, and the cell proliferation rate was compared. Significant differences were observed between cells transfected with CMV- or EF1 promoter-driven expression of the 48 nuclear receptors after 8 weeks. The EF1-NR1I2 cell line, which exhibited the highest increase from 2 to 8 weeks, showed 1.13-fold higher proliferation than the EF1-DsRed line. On the other hand, the EF1-NR4A1 cell line, which showed the maximum decrease at 8 weeks, showed 0.88-fold lower proliferation than the EF1-DsRed line. The results were confirmed in both our transgene competition system and long-term growth experiments. Our transgene competition system offers a wide-range, simple, and accurate cell competition method., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hasegawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Down syndrome (DS) is the most common chromosomal disorder and a major cause of intellectual disability. The genetic etiology of DS is the extra copy of chromosome 21 (HSA21)-encoded genes; however, the contribution of specific HSA21 genes to DS pathogenesis remains largely unknown. Here, we identified ZBTB21, an HSA21-encoded zinc-finger protein, as a transcriptional repressor in the regulation of synaptic function. We found that normalization of the Zbtb21 gene copy number in DS mice corrected deficits in cognitive performance, synaptic function, and gene expression. Moreover, we demonstrated that ZBTB21 binds to canonical cAMP-response element (CRE) DNA and that its binding to CRE could be competitive with CRE-binding factors such as CREB. ZBTB21 represses CRE-dependent gene expression and results in the negative regulation of synaptic plasticity, learning and memory. Together, our results identify ZBTB21 as a CRE-binding protein and repressor in cAMP-dependent gene regulation, contributing to cognitive defects in DS.

The model haloarchaeon Haloferax volcanii is polyploid with about 20 copies of its major chromosome. Recently it has been described that highly efficient intermolecular gene conversion operates in H. volcanii to equalize the chromosomal copies. In the current study, 24 genes were selected that encode proteins with orthologs involved in gene conversion or homologous recombination in archaea, bacteria, or eukaryotes. Single gene deletion strains of 22 genes and a control gene were constructed in two parent strains for a gene conversion assay; only radA and radB were shown to be essential. Protoplast fusions were used to generate strains that were heterozygous for the gene HVO&#95;2528 , encoding an enzyme for carotinoid biosynthesis. It was revealed that a lack of six of the proteins did not influence the efficiency of gene conversion, while sixteen mutants had severe gene conversion defects. Notably, lack of paralogous proteins of gene families had very different effects, e.g., mutant Δrad25b had no phenotype, while mutants Δrad25a, Δrad25c, and Δrad25d were highly compromised. Generation of a quadruple rad25 and a triple sph deletion strain also indicated that the paralogs have different functions, in contrast to sph2 and sph4 , which cannot be deleted simultaneously. There was no correlation between the severity of the phenotypes and the respective transcript levels under non-stressed conditions, indicating that gene expression has to be induced at the onset of gene conversion. Phylogenetic trees of the protein families Rad3/25, MutL/S, and Sph/SMC/Rad50 were generated to unravel the history of the paralogous proteins of H. volcanii . Taken together, unselected intermolecular gene conversion in H. volcanii involves at least 16 different proteins, the molecular roles of which can be studied in detail in future projects.

Human epidermal growth factor receptor 2 (HER)-positive breast cancer (BC) is characterized by an aggressive clinical course. In the case of HER2 overexpression/amplification, patients benefit from HER2-targeting therapies. Standardized diagnostic HER2 assessment includes immunohistochemistry (IHC) and/or in situ hybridization (ISH). The aim of this study was to compare this "gold standard" with the Droplet Digital™ polymerase chain reaction (ddPCR), a method that allows sensitive and precise detection of copy number variations (CNV) in FFPE (formalin-fixed, paraffin-embedded) DNA samples. Partitioning of the PCR reaction into 20,000 droplets enables a precise quantitative "CN" discrimination also in heterogeneous samples. FFPE breast cancer samples (n = 170) with routinely assessed HER2 status by IHC/ISH were retrospectively analyzed using the ddPCR CNV ERBB2 assay. Comparison of HER2 status assessment by the two methods revealed concordant results in 92.9% (158/170) of the cases. Discrepant cases were verified and interpreted. For ddPCR, a cut off value of 3 HER2 copies was set to distinguish between HER2-negative and HER2-positive BC. Results obtained with the ddPCR CNV ERBB2 assay were consistent and reproducible, and serial dilutions demonstrated a high stability and sensitivity of the method. The ddPCR CNV ERBB2 assay may be a specific and convenient tool to quantify HER2 copy numbers in BC samples. In our study, this method showed high reproducibility in accuracy of HER2 assessment compared to IHC/ISH analysis., (© 2023. The Author(s).)

Background: The mutational status of ovarian cancer cell line IGROV-1 is inconsistent across the literature, suggestive of multiple clonal populations of the cell line. IGROV-1 has previously been categorised as an inappropriate model for high-grade serous ovarian cancer., Methods: IGROV-1 cells were obtained from the Netherlands Cancer Institute (IGROV-1-NKI) and the MD Anderson Cancer Centre (IGROV-1-MDA). Cell lines were STR fingerprinted and had their chromosomal copy number analysed and BRCA1/2 genes sequenced. Mutation status of ovarian cancer-related genes were extracted from the literature., Results: The IGROV-1-NKI cell line has a tetraploid chromosomal profile. In contrast, the IGROV-1-MDA cell line has pseudo-normal chromosomes. The IGROV-1-NKI and IGROV-MDA are both STR matches (80.7% and 84.6%) to the original IGROV-1 cells isolated in 1985. However, IGROV-1-NKI and IGROV-1-MDA are not an STR match to each other (78.1%) indicating genetic drift. The BRCA1 and BRCA2 gene sequences are 100% identical between IGROV-1-MDA and IGROV-1-NKI, including a BRCA1 heterozygous deleterious mutation. The IGROV-1-MDA cells are more resistant to cisplatin and olaparib than IGROV-1-NKI. IGROV-1 has a mutational profile consistent with both Type I (PTEN, PIK3CA and ARID1A) and Type II ovarian cancer (BRCA1, TP53) and is likely to be a Type II high-grade serous carcinoma of the SET (Solid, pseudo-Endometroid and Transitional cell carcinoma-like morphology) subtype., Conclusions: Routine testing of chromosomal copy number as well as the mutational status of ovarian cancer related genes should become the new standard alongside STR fingerprinting to ensure that ovarian cancer cell lines are appropriate models., (© 2024. The Author(s).)

Background: Predictive markers for fecal microbiota transplantation (FMT) outcomes in patients with active ulcerative colitis (UC) are poorly defined. We aimed to investigate changes in gut microbiota pre- and post-FMT and to assess the potential value in determining the total copy number of fecal bacterial siderophore genes in predicting FMT responsiveness., Methods: Patients with active UC (Mayo score ≥ 3) who had undergone two FMT procedures were enrolled. Fecal samples were collected before and 8 weeks after each FMT session. Patients were classified into clinical response and non-response groups, based on their Mayo scores. The fecal microbiota profile was accessed using metagenomic sequencing, and the total siderophore genes copy number via quantitative real-time polymerase chain reaction. Additionally, we examined the association between the total siderophore genes copy number and FMT efficacy., Results: Seventy patients with UC had undergone FMT. The clinical response and remission rates were 50% and 10% after the first FMT procedure, increasing to 72.41% and 27.59% after the second FMT. The cumulative clinical response and clinical remission rates were 72.86% and 25.71%. Compared with baseline, the response group showed a significant increase in Faecalibacterium, and decrease in Enterobacteriaceae, consisted with the changes of the total bacterial siderophore genes copy number after the second FMT (1889.14 vs. 98.73 copies/ng, P < 0.01). Virulence factor analysis showed an enriched iron uptake system, especially bacterial siderophores, in the pre-FMT response group, with a greater contribution from Escherichia coli. The total baseline copy number was significantly higher in the response group than non-response group (1889.14 vs. 94.86 copies/ng, P < 0.01). A total baseline copy number cutoff value of 755.88 copies/ng showed 94.7% specificity and 72.5% sensitivity in predicting FMT responsiveness., Conclusions: A significant increase in Faecalibacterium, and decrease in Enterobacteriaceae and the total fecal siderophore genes copy number were observed in responders after FMT. The siderophore genes and its encoding bacteria may be of predictive value for the clinical responsiveness of FMT to active ulcerative colitis., (© 2024. The Author(s).)

Culture-independent 16S rRNA gene metabarcoding is a commonly used method for microbiome profiling. To achieve more quantitative cell fraction estimates, it is important to account for the 16S rRNA gene copy number (hereafter 16S GCN) of different community members. Currently, there are several bioinformatic tools available to estimate the 16S GCN values, either based on taxonomy assignment or phylogeny. Here we present a novel approach ANNA16, Artificial Neural Network Approximator for 16S rRNA gene copy number, a deep learning-based method that estimates the 16S GCN values directly from the 16S gene sequence strings. Based on 27,579 16S rRNA gene sequences and gene copy number data from the rrnDB database, we show that ANNA16 outperforms the commonly used 16S GCN prediction algorithms. Interestingly, Shapley Additive exPlanations (SHAP) shows that ANNA16 can identify unexpected informative positions in 16S rRNA gene sequences without any prior phylogenetic knowledge, which suggests potential applications beyond 16S GCN prediction., (© 2024. The Author(s).)

Excess gene dosage from chromosome 21 (chr21) causes Down syndrome (DS), spanning developmental and acute phenotypes in terminal cell types. Which phenotypes remain amenable to intervention after development is unknown. To address this question in a model of DS neurogenesis, we derived trisomy 21 (T21) human induced pluripotent stem cells (iPSCs) alongside, otherwise, isogenic euploid controls from mosaic DS fibroblasts and equipped one chr21 copy with an inducible XIST transgene. Monoallelic chr21 silencing by XIST is near-complete and irreversible in iPSCs. Differential expression reveals that T21 neural lineages and iPSCs share suppressed translation and mitochondrial pathways and activate cellular stress responses. When XIST is induced before the neural progenitor stage, T21 dosage correction suppresses a pronounced skew toward astrogenesis in neural differentiation. Because our transgene remains inducible in postmitotic T21 neurons and astrocytes, we demonstrate that XIST efficiently represses genes even after terminal differentiation, which will empower exploration of cell type-specific T21 phenotypes that remain responsive to chr21 dosage.

Plasmids play important roles in microbial ecosystems, serving as carriers of antibiotic resistance and virulence. In the laboratory, they are essential tools for genetic manipulation and recombinant protein expression. We uncovered an intriguing survival phenotype in a fraction of the bacterial population while using plasmid-mediated arabinose-inducible gene expression to monitor the production of toxic ParE proteins. This phenotype was not correlated with changes to the plasmid sequence and could not be rescued by increasing arabinose uptake. Instead, survival correlates with a marked reduction in plasmid copy number (PCN). Reduced PCN is reproducible, not a function of the pre-existing population, and can be sequentially enriched by continual passage with induction. The reduction in PCN appears to allow mitigation of toxicity from the expression of ParE proteins while balancing the need to maintain a threshold PCN to withstand selection conditions. This indicates an adaptive cellular response to stressful conditions, likely by altering the regulation of plasmid replication. Furthermore, this survival mechanism appears to not be limited to a specific bacterial strain of Escherichia coli or ParE toxin family member, suggesting a generalized response. Finally, bacterial whole genome sequencing indicated an N845S residue substitution in DNA polymerase I, which correlates with the observed reduction in PCN and has been previously reported to impact plasmid replication. Further understanding this molecular mechanism has broader implications for this adaptive response of the dynamics of plasmid-mediated gene expression, microbial adaptation, and genetic engineering methodologies., Importance: This research has increased our understanding of how bacteria respond to the pressure from plasmid-borne toxic genes, such as those found in toxin-antitoxin systems. Surprisingly, we found that bacteria survived toxic ParE protein expression by reducing the number of these plasmids in the cells. This discovery reveals another way in which bacteria can balance toxin expression with antibiotic selection to attenuate the effects of deleterious genes. This insight is not only valuable for understanding bacterial survival strategies but may also influence the development of better tools in biotechnology, where plasmids are often used to study the functional roles of genes., Competing Interests: The authors declare no conflict of interest.

The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating plants with a substantially reduced plastid genome size. We show that the lack of the IR results in a mildly reduced plastid ribosome number, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, the IR deletion plants contain an increased number of plastid genomes, suggesting that genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (1) demonstrate that the IR can enhance the translation capacity of the plastid, (2) reveal the relationship between genome size and genome copy number, and (3) provide a simplified plastid genome structure that will facilitate future synthetic biology applications., (© 2024. The Author(s).)

There are sex-dependent differences in the prevalence, age of onset, and course of psychiatric disorders and cognitive abilities. Although it has been assumed that the direct effect of gonadal hormones in sensitive periods leads to sexually dimorphic brain development, current evidence suggests that another possible factor may be sex-specific regulations at the gene level. Understanding the sex differences at the gene level can be promising to identify the mechanisms that predispose or trigger psychiatric disorders, and may provide new prevention or treatment strategies. This paper aims to review the findings on the mechanisms that affect the sex-specific differences in brain development at the gene level and to discuss the relationship of these findings with different cognitive characteristics of the sexes and psychiatric disorders. [ABSTRACT FROM AUTHOR]

An oxygen requirement for de novo biotin synthesis in Saccharomyces cerevisiae precludes the application of biotin-prototrophic strains in anoxic processes that use biotin-free media. To overcome this issue, this study explores introduction of the oxygen-independent Escherichia coli biotin-biosynthesis pathway in S. cerevisiae. Implementation of this pathway required expression of seven E. coli genes involved in fatty-acid synthesis and three E. coli genes essential for the formation of a pimelate thioester, key precursor of biotin synthesis. A yeast strain expressing these genes readily grew in biotin-free medium, irrespective of the presence of oxygen. However, the engineered strain exhibited specific growth rates 25% lower in biotin-free media than in biotin-supplemented media. Following adaptive laboratory evolution in anoxic cultures, evolved cell lines that no longer showed this growth difference in controlled bioreactors, were characterized by genome sequencing and proteome analyses. The evolved isolates exhibited a whole-genome duplication accompanied with an alteration in the relative gene dosages of biosynthetic pathway genes. These alterations resulted in a reduced abundance of the enzymes catalyzing the first three steps of the E. coli biotin pathway. The evolved pathway configuration was reverse engineered in the diploid industrial S. cerevisiae strain Ethanol Red. The resulting strain grew at nearly the same rate in biotin-supplemented and biotin-free media non-controlled batches performed in an anaerobic chamber. This study established an unique genetic engineering strategy to enable biotin-independent anoxic growth of S. cerevisiae and demonstrated its portability in industrial strain backgrounds. [Display omitted] • Biotin biosynthesis is oxygen dependent in S. cerevisiae. • Expression of E.coli KAPA pathway enables yeast to grow without biotin and O 2. • Evolution for fast anaerobic growth without biotin involved large genomic remodeling. • Gene dosage alteration of fabB, D , bioC was critical to restore fast biotin-independent growth. • FabB, FabD and BioC protein abundances were deemed critical for fast growth in anoxic conditions. [ABSTRACT FROM AUTHOR]

Obesity is linked to type 2 diabetes (T2D) and cardiovascular diseases; however, the underlying molecular mechanisms remain unclear. We aimed to identify obesity-associated molecular features that may contribute to obesity-related diseases. Using circulating monocytes from 1,264 Multi-Ethnic Study of Atherosclerosis (MESA) participants, we quantified the transcriptome and epigenome. We discovered that alterations in a network of coexpressed cholesterol metabolism genes are a signature feature of obesity and inflammatory stress. This network included 11 BMI-associated genes related to sterol uptake (↑LDLR, ↓MYLIP), synthesis (↑SCD, FADS1, HMGCS1, FDFT1, SQLE, CYP51A1, SC4MOL), and efflux (↓ABCA1, ABCG1), producing a molecular profile expected to increase intracellular cholesterol. Importantly, these alterations were associated with T2D and coronary artery calcium (CAC), independent from cardiometabolic factors, including serum lipid profiles. This network mediated the associations between obesity and T2D/CAC. Several genes in the network harbored C-phosphorus-G dinucleotides (e.g., ABCG1/cg06500161), which overlapped Encyclopedia of DNA Elements (ENCODE)-annotated regulatory regions and had methylation profiles that mediated the associations between BMI/inflammation and expression of their cognate genes. Taken together with several lines of previous experimental evidence, these data suggest that alterations of the cholesterol metabolism gene network represent a molecular link between obesity/inflammation and T2D/CAC.

Ionizing radiation (IR) is a mutagen that promotes tumorigenesis in multiple exposure contexts. One severe consequence of IR is the development of second malignant neoplasms (SMNs), a radiotherapy-associated complication in survivors of cancers, particularly pediatric cancers. SMN genomes are poorly characterized, and the influence of genetic background on genotoxin-induced mutations has not been examined. Using our mouse models of SMNs, we performed whole exome sequencing of neoplasms induced by fractionated IR in wild-type and Nf1 mutant mice. Using non-negative matrix factorization, we identified mutational signatures that did not segregate by genetic background or histology. Copy-number analysis revealed recurrent chromosomal alterations and differences in copy number that were background dependent. Pathway analysis identified enrichment of non-synonymous variants in genes responsible for cell assembly and organization, cell morphology, and cell function and maintenance. In this model system, ionizing radiation and Nf1 heterozygosity each exerted distinct influences on the mutational landscape.

Intellectual disability (ID) affects approximately 1%-3% of humans with a gender bias toward males. Previous studies have identified mutations in more than 100 genes on the X chromosome in males with ID, but there is less evidence for de novo mutations on the X chromosome causing ID in females. In this study we present 35 unique deleterious de novo mutations in DDX3X identified by whole exome sequencing in 38 females with ID and various other features including hypotonia, movement disorders, behavior problems, corpus callosum hypoplasia, and epilepsy. Based on our findings, mutations in DDX3X are one of the more common causes of ID, accounting for 1%-3% of unexplained ID in females. Although no de novo DDX3X mutations were identified in males, we present three families with segregating missense mutations in DDX3X, suggestive of an X-linked recessive inheritance pattern. In these families, all males with the DDX3X variant had ID, whereas carrier females were unaffected. To explore the pathogenic mechanisms accounting for the differences in disease transmission and phenotype between affected females and affected males with DDX3X missense variants, we used canonical Wnt defects in zebrafish as a surrogate measure of DDX3X function in vivo. We demonstrate a consistent loss-of-function effect of all tested de novo mutations on the Wnt pathway, and we further show a differential effect by gender. The differential activity possibly reflects a dose-dependent effect of DDX3X expression in the context of functional mosaic females versus one-copy males, which reflects the complex biological nature of DDX3X mutations.

ObjectiveThe molecular mechanisms underlying sex differences in dyslipidemia are poorly understood. We aimed to distinguish genetic and hormonal regulators of sex differences in plasma lipid levels.Approach and resultsWe assessed the role of gonadal hormones and sex chromosome complement on lipid levels using the four core genotypes mouse model (XX females, XX males, XY females, and XY males). In gonadally intact mice fed a chow diet, lipid levels were influenced by both male-female gonadal sex and XX-XY chromosome complement. Gonadectomy of adult mice revealed that the male-female differences are dependent on acute effects of gonadal hormones. In both intact and gonadectomized animals, XX mice had higher HDL cholesterol (HDL-C) levels than XY mice, regardless of male-female sex. Feeding a cholesterol-enriched diet produced distinct patterns of sex differences in lipid levels compared with a chow diet, revealing the interaction of gonadal and chromosomal sex with diet. Notably, under all dietary and gonadal conditions, HDL-C levels were higher in mice with 2 X chromosomes compared with mice with an X and Y chromosome. By generating mice with XX, XY, and XXY chromosome complements, we determined that the presence of 2 X chromosomes, and not the absence of the Y chromosome, influences HDL-C concentration.ConclusionsWe demonstrate that having 2 X chromosomes versus an X and Y chromosome complement drives sex differences in HDL-C. It is conceivable that increased expression of genes escaping X-inactivation in XX mice regulates downstream processes to establish sexual dimorphism in plasma lipid levels.

eIF4E, the major cap-binding protein, has long been considered limiting for translating the mammalian genome. However, the eIF4E dose requirement at an organismal level remains unexplored. By generating an Eif4e haploinsufficient mouse, we found that a 50% reduction in eIF4E expression, while compatible with normal development and global protein synthesis, significantly impeded cellular transformation. Genome-wide translational profiling uncovered a translational program induced by oncogenic transformation and revealed a critical role for the dose of eIF4E, specifically in translating a network of mRNAs enriched for a unique 5' UTR signature. In particular, we demonstrate that the dose of eIF4E is essential for translating mRNAs that regulate reactive oxygen species, fueling transformation and cancer cell survival in vivo. Our findings indicate eIF4E is maintained at levels in excess for normal development that are hijacked by cancer cells to drive a translational program supporting tumorigenesis.

UnlabelledDespite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1. The construct that ultimately endowed robust growth on chlorate included cld, a cytochrome c gene, clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream of cld enabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence between narQP and nrfA that enhanced growth.ImportanceThe ability of chlorate reduction composite transposons to form functional metabolisms after transfer to a new host is an important part of their propagation. To study this phenomenon, we engineered Shewanella oneidensis MR-1 into a chlorate reducer. We defined a set of genes sufficient to endow growth on chlorate from a plasmid, but found that chromosomal insertion of these genes was nonfunctional. Evolution of this inoperative strain into a chlorate reducer showed that tandem duplication was a dominant mechanism of activation. While copy number changes are a relatively rapid way of increasing gene dosage, replicating almost 1 megabase of extra DNA is costly. Mutations that alleviate the need for high copy number are expected to arise and eventually predominate, and we identified a single nucleotide polymorphism (SNP) that relieved the copy number requirement. This study uses both rational and evolutionary approaches to gain insight into the evolution of a fascinating respiratory metabolism.

Although the diagnosis of autism spectrum disorder (ASD) is based on behavioral signs and symptoms, the evaluation of a child with ASD has become increasingly focused on the identification of the genetic etiology of the disorder. In this review, we begin with a clinical overview of ASD, highlighting the heterogeneity of the disorder. We then discuss the genetics of ASD and present updated guidelines on genetic testing. We then consider the insights gained from the identification of both single gene disorders and rare variants, with regard to clinical phenomenology and potential treatment targets.

Esophageal adenocarcinoma (EAC) develops from Barrett's esophagus (BE), wherein normal squamous epithelia is replaced by specialized intestinal metaplasia in response to chronic gastroesophageal acid reflux. BE can progress to low- and high-grade dysplasia, intramucosal, and invasive carcinoma. Both BE and EAC are characterized by loss of heterozygosity, aneuploidy, specific genetic mutations, and clonal diversity. Given the limitations of histopathology, genomic and epigenomic analyses may improve the precision of risk stratification. Assays to detect molecular alterations associated with neoplastic progression could be used to improve the pathologic assessment of BE/EAC and to select high-risk patients for more intensive surveillance.

The Breakage Fusion Bridge (BFB) process is a key marker for genomic instability, producing highly rearranged genomes in relatively small numbers of cell cycles. While the process itself was observed during the late 1930s, little is known about the extent of BFB in tumor genome evolution. Moreover, BFB can dramatically increase copy numbers of chromosomal segments, which in turn hardens the tasks of both reference-assisted and ab initio genome assembly. Based on available data such as Next Generation Sequencing (NGS) and Array Comparative Genomic Hybridization (aCGH) data, we show here how BFB evidence may be identified, and how to enumerate all possible evolutions of the process with respect to observed data. Specifically, we describe practical algorithms that, given a chromosomal arm segmentation and noisy segment copy number estimates, produce all segment count vectors supported by the data that can be produced by BFB, and all corresponding BFB architectures. This extends the scope of analyses described in our previous work, which produced a single count vector and architecture per instance. We apply these analyses to a comprehensive human cancer dataset, demonstrate the effectiveness and efficiency of the computation, and suggest methods for further assertions of candidate BFB samples. Source code of our tool can be found online.

Ependymal tumors across age groups are currently classified and graded solely by histopathology. It is, however, commonly accepted that this classification scheme has limited clinical utility based on its lack of reproducibility in predicting patients' outcome. We aimed at establishing a uniform molecular classification using DNA methylation profiling. Nine molecular subgroups were identified in a large cohort of 500 tumors, 3 in each anatomical compartment of the CNS, spine, posterior fossa, supratentorial. Two supratentorial subgroups are characterized by prototypic fusion genes involving RELA and YAP1, respectively. Regarding clinical associations, the molecular classification proposed herein outperforms the current histopathological classification and thus might serve as a basis for the next World Health Organization classification of CNS tumors.

In Saccharomyces cerevisiae, absence of the checkpoint kinase Mec1 (ATR) is viable upon mutations that increase the activity of the ribonucleotide reductase (RNR) complex. Whether this pathway is conserved in mammals remains unknown. Here we show that cells from mice carrying extra alleles of the RNR regulatory subunit RRM2 (Rrm2(TG)) present supraphysiological RNR activity and reduced chromosomal breakage at fragile sites. Moreover, increased Rrm2 gene dosage significantly extends the life span of ATR mutant mice. Our study reveals the first genetic condition in mammals that reduces fragile site expression and alleviates the severity of a progeroid disease by increasing RNR activity.

Parasympathetic innervation is critical for submandibular gland (SMG) development and regeneration. Parasympathetic ganglia (PSG) are derived from Schwann cell precursors that migrate along nerves, differentiate into neurons, and coalesce within their target tissue to form ganglia. However, signals that initiate gangliogenesis after the precursors differentiate into neurons are unknown. We found that deleting negative regulators of FGF signaling, Sprouty1 and Sprouty2 (Spry1/2DKO), resulted in a striking loss of gangliogenesis, innervation, and keratin 5-positive (K5+) epithelial progenitors in the SMG. Here we identify Wnts produced by K5+ progenitors in the SMG as key mediators of gangliogenesis. Wnt signaling increases survival and proliferation of PSG neurons, and inhibiting Wnt signaling disrupts gangliogenesis and organ innervation. Activating Wnt signaling and reducing FGF gene dosage rescues gangliogenesis and innervation in both the Spry1/2DKO SMG and pancreas. Thus, K5+ progenitors produce Wnt signals to establish the PSG-epithelial communication required for organ innervation and progenitor cell maintenance.

Aberrant activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) 3 has been implicated in cell proliferation and survival of many cancers including head and neck squamous cell carcinoma (HNSCC). AZD1480, an orally active pharmacologic inhibitor of JAK1/JAK2, has been tested in several cancer models. In the present study, the in vitro and in vivo effects of AZD1480 were evaluated in HNSCC preclinical models to test the potential use of JAK kinase inhibition for HNSCC therapy. AZD1480 treatment decreased HNSCC proliferation in HNSCC cell lines with half maximal effective concentration (EC50) values ranging from 0.9 to 4 μM in conjunction with reduction of pSTAT3(Tyr705) expression. In vivo antitumor efficacy of AZD1480 was demonstrated in patient-derived xenograft (PDX) models derived from two independent HNSCC tumors. Oral administration of AZD1480 reduced tumor growth in conjunction with decreased pSTAT3(Tyr705) expression that was observed in both PDX models. These findings suggest that the JAK1/2 inhibitors abrogate STAT3 signaling and may be effective in HNSCC treatment approaches.

The progression and clonal development of tumors often involve amplifications and deletions of genomic DNA. Estimation of allele-specific copy number, which quantifies the number of copies of each allele at each variant loci rather than the total number of chromosome copies, is an important step in the characterization of tumor genomes and the inference of their clonal history. We describe a new method, falcon, for finding somatic allele-specific copy number changes by next generation sequencing of tumors with matched normals. falcon is based on a change-point model on a bivariate mixed Binomial process, which explicitly models the copy numbers of the two chromosome haplotypes and corrects for local allele-specific coverage biases. By using the Binomial distribution rather than a normal approximation, falcon more effectively pools evidence from sites with low coverage. A modified Bayesian information criterion is used to guide model selection for determining the number of copy number events. Falcon is evaluated on in silico spike-in data and applied to the analysis of a pre-malignant colon tumor sample and late-stage colorectal adenocarcinoma from the same individual. The allele-specific copy number estimates obtained by falcon allows us to draw detailed conclusions regarding the clonal history of the individual's colon cancer.

Changes in gene copy number are among the most frequent mutational events in all genomes and were among the mutations for which a physical basis was first known. Yet mechanisms of gene duplication remain uncertain because formation rates are difficult to measure and mechanisms may vary with position in a genome. Duplications are compared here to deletions, which seem formally similar but can arise at very different rates by distinct mechanisms. Methods of assessing duplication rates and dependencies are described with several proposed formation mechanisms. Emphasis is placed on duplications formed in extensively studied experimental situations. Duplications studied in microbes are compared with those observed in metazoan cells, specifically those in genomes of cancer cells. Duplications, and especially their derived amplifications, are suggested to form by multistep processes often under positive selection for increased copy number.

We describe the pathology of 5 patients with germline PRKACA copy number gain and Cushing syndrome: 4 males and 1 female, aged 2 to 43 years, including a mother and son. Imaging showed normal or slightly enlarged adrenal glands in 4 patients and a unilateral mass in the fifth. Biochemically, the patients had corticotropin-independent hypercortisolism. Four underwent bilateral adrenalectomy; unilateral adrenalectomy was performed in the patient with the adrenal mass. Pathologically, 3 patients, including the 1 with the tumor (adenoma), had primary pigmented nodular adrenocortical disease with extranodular cortical atrophy and mild intracapsular and extracapsular extension of cortical cells. The other 2 patients had cortical hyperplasia and prominent capsular and extracapsular micronodular cortical hyperplasia. Immunoperoxidase staining revealed differences for synaptophysin, inhibin-A, and Ki-67 (nuclei) in the atrophic cortices (patients 1, 2, and 3) and hyperplastic cortices (patients 4 and 5) and for Ki-67 (nuclei) and vimentin in the extracortical nodules in the 2 groups of patients. β-Catenin stained the cell membrane, cytoplasm, and nuclei of the adenoma. The patients were well at follow-up (1-23 years); 24-hour urinary cortisol excretion was elevated in the patient who had unilateral adrenalectomy.

The use of genome-wide copy-number analysis and massive parallel sequencing has revolutionized the understanding of the clonal architecture of pediatric acute lymphoblastic leukemia (ALL) by demonstrating that this disease is composed of highly variable clonal ancestries following the rules of Darwinian selection. The current study aimed to analyze the molecular composition of childhood ALL biopsies and patient-derived xenografts with particular emphasis on mechanisms associated with acquired chemoresistance. Genomic DNA from seven primary pediatric ALL patient samples, 29 serially passaged xenografts, and six in vivo selected chemoresistant xenografts were analyzed with 250K single-nucleotide polymorphism arrays. Copy-number analysis of non-drug-selected xenografts confirmed a highly variable molecular pattern of variegated subclones. Whereas primary patient samples from initial diagnosis displayed a mean of 5.7 copy-number alterations per sample, serially passaged xenografts contained a mean of 8.2 and chemoresistant xenografts a mean of 10.5 copy-number alterations per sample, respectively. Resistance to cytarabine was explained by a new homozygous deletion of the DCK gene, whereas methotrexate resistance was associated with monoallelic deletion of FPGS and mutation of the remaining allele. This study demonstrates that selecting for chemoresistance in xenografted human ALL cells can reveal novel mechanisms associated with drug resistance.

Here we present the results of a large-scale bioinformatics annotation of non-coding RNA loci in 48 avian genomes. Our approach uses probabilistic models of hand-curated families from the Rfam database to infer conserved RNA families within each avian genome. We supplement these annotations with predictions from the tRNA annotation tool, tRNAscan-SE and microRNAs from miRBase. We identify 34 lncRNA-associated loci that are conserved between birds and mammals and validate 12 of these in chicken. We report several intriguing cases where a reported mammalian lncRNA, but not its function, is conserved. We also demonstrate extensive conservation of classical ncRNAs (e.g., tRNAs) and more recently discovered ncRNAs (e.g., snoRNAs and miRNAs) in birds. Furthermore, we describe numerous "losses" of several RNA families, and attribute these to either genuine loss, divergence or missing data. In particular, we show that many of these losses are due to the challenges associated with assembling avian microchromosomes. These combined results illustrate the utility of applying homology-based methods for annotating novel vertebrate genomes.

The function of synapses depends on spatially and temporally controlled molecular interactions between synaptic components that can be described in terms of copy numbers, binding affinities, and diffusion properties. To understand the functional role of a given synaptic protein, it is therefore crucial to quantitatively characterise its biophysical behaviour in its native cellular environment. Single molecule localisation microscopy (SMLM) is ideally suited to obtain quantitative information about synaptic proteins on the nanometre scale. Molecule counting of recombinant proteins tagged with genetically encoded fluorophores offers a means to determine their absolute copy numbers at synapses due to the known stoichiometry of the labelling. As a consequence of its high spatial precision, SMLM also yields accurate quantitative measurements of molecule concentrations. In addition, live imaging of fluorescently tagged proteins at synapses can reveal diffusion dynamics and local binding properties of behaving proteins under normal conditions or during pathological processes. In this perspective, it is argued that the detailed structural information provided by super-resolution imaging can be harnessed to gain new quantitative information about the organisation and dynamics of synaptic components in cellula. To illustrate this point, I discuss the concentration-dependent aggregation of α-synuclein in the axon and the concomitant changes in the dynamic equilibrium of α-synuclein at synapses in quantitative terms.

PurposeTo investigate the molecular events associated with the activation of androgen receptor (AR) as a potential therapeutic target in patients with salivary duct carcinoma (SDC).Experimental designComprehensive molecular and expression analysis of the AR gene in 35 tumor specimens (20 males and 15 females) and cell lines derived from SDC using Western blotting and RT-PCR, FISH analysis, and DNA sequencing was conducted. In vitro and in vivo animal studies were also performed.ResultsAR expression was detected in 70% of the tumors and was mainly nuclear and homogenous in both male and female SDCs, although variable cytoplasmic and/or nuclear localization was also found. We report the identification of ligand-independent AR splice variants, mutations, and extra AR gene copy in primary untreated SDC tumors. In contrast to prostate cancer, no AR gene amplification was observed. In vitro knockdown of AR in a female derived SDC cell line revealed marked growth inhibition in culture and in vivo androgen-independent tumor growth.ConclusionsOur study provides new detailed information on the molecular and structural alterations associated with AR gene activation in SDC and sheds more light on the putative functional role of AR in SDC cells. On the basis of these data, we propose that patients with SDC (male and female) can be stratified for hormone-based therapy in future clinical trials.

BackgroundThere was ancient human selection on the wild progenitor of modern maize, Balsas teosinte, for decreased shoot branching (tillering), in order to allow more nutrients to be diverted to grain. Mechanistically, the decline in shoot tillering has been associated with selection for increased expression of the major domestication gene Teosinte Branched 1 (Tb1) in shoot primordia. Therefore, TB1 has been defined as a repressor of shoot branching. It is known that plants respond to changes in shoot size by compensatory changes in root growth and architecture. However, it has not been reported whether altered TB1 expression affects any plant traits below ground. Previously, changes in dosage of a well-studied mutant allele of Tb1 in modern maize, called tb1-ref, from one to two copies, was shown to increase tillering. As a result, plants with two copies of the tb1-ref allele have a larger shoot biomass than heterozygotes. Here we used aeroponics to phenotype the effects of tb1-ref copy number on maize roots at macro-, meso- and micro scales of development.ResultsAn increase in the tb1-ref copy number from one to two copies resulted in: (1) an increase in crown root number due to the cumulative initiation of crown roots from successive tillers; (2) higher density of first and second order lateral roots; and (3) reduced average lateral root length. The resulting increase in root system biomass in homozygous tb1-ref mutants balanced the increase in shoot biomass caused by enhanced tillering. These changes caused homozygous tb1-ref mutants of modern maize to more closely resemble its ancestor Balsas teosinte below ground.ConclusionWe conclude that a decrease in TB1 function in maize results in a larger root system, due to an increase in the number of crown roots and lateral roots. Given that decreased TB1 expression results in a more highly branched and larger shoot, the impact of TB1 below ground may be direct or indirect. We discuss the potential implications of these findings for whole plant coordination of biomass accumulation and maize domestication.

Recurrent deletions of chromosome 15q13.3 associate with intellectual disability, schizophrenia, autism and epilepsy. To gain insight into the instability of this region, we sequenced it in affected individuals, normal individuals and nonhuman primates. We discovered five structural configurations of the human chromosome 15q13.3 region ranging in size from 2 to 3 Mb. These configurations arose recently (∼0.5-0.9 million years ago) as a result of human-specific expansions of segmental duplications and two independent inversion events. All inversion breakpoints map near GOLGA8 core duplicons-a ∼14-kb primate-specific chromosome 15 repeat that became organized into larger palindromic structures. GOLGA8-flanked palindromes also demarcate the breakpoints of recurrent 15q13.3 microdeletions, the expansion of chromosome 15 segmental duplications in the human lineage and independent structural changes in apes. The significant clustering (P = 0.002) of breakpoints provides mechanistic evidence for the role of this core duplicon and its palindromic architecture in promoting the evolutionary and disease-related instability of chromosome 15.

BackgroundThere is an urgent need for better therapeutics in head and neck squamous cell cancer (HNSCC) to improve survival and decrease treatment morbidity. Recent advances in high-throughput drug screening techniques and next-generation sequencing have identified new therapeutic targets in other cancer types, but an HNSCC-specific study has not yet been carried out. We have exploited data from two large-scale cell line projects to clearly describe the mutational and copy number status of HNSCC cell lines and identify candidate drugs with elevated efficacy in HNSCC.MethodsThe genetic landscape of 42 HNSCC cell lines including mutational and copy number data from studies by Garnett et al., and Barretina et al., were analyzed. Data from Garnett et al. was interrogated for relationships between HNSCC cells versus the entire cell line pool using one- and two-way analyses of variance (ANOVAs). As only seven HNSCC cell lines were tested with drugs by Barretina et al., a similar analysis was not carried out.ResultsRecurrent mutations in human papillomavirus (HPV)-negative patient tumors were confirmed in HNSCC cell lines, however additional, recurrent, cell line-specific mutations were identified. Four drugs, Bosutinib, Docetaxel, BIBW2992, and Gefitinib, were found via multiple-test corrected ANOVA to have lower IC50 values, suggesting higher drug sensitivity, in HNSCC lines versus non-HNSCC lines. Furthermore, the PI3K inhibitor AZD6482 demonstrated significantly higher activity (as measured by the IC50) in HNSCC cell lines harbouring PIK3CA mutations versus those that did not.ConclusionHNSCC-specific reanalysis of large-scale drug screening studies has identified candidate drugs that may be of therapeutic benefit and provided insights into strategies to target PIK3CA mutant tumors. PIK3CA mutations may represent a predictive biomarker for response to PI3K inhibitors. A large-scale study focused on HNSCC cell lines and including HPV-positive lines is necessary and has the potential to accelerate the development of improved therapeutics for patients suffering with head and neck cancer. This strategy can potentially be used as a template for drug discovery in any cancer type.

Background and Objectives: Monosomy of chromosome 5 associated with utilization of non-canonical sugar L-sorbose is one of the well-studied aneuploidies in Candida albicans. Stress-induced ploidy changes are crucial determinants for pathogenicity and genetic diversity in C. albicans. The five scattered regulatory regions (A, B, C, 135, and 139) comprising of two functionally redundant pathways (SUR1 and SUR2) were found to be responsible for the growth on L-sorbose. So far, three genes such as CSU51, CSU53 and CSU57 have been identified in region A, region 135 and region C, respectively. In this study we have verified the role of region B in this regulatory pathway. Materials and Methods: We employed a combinatorial gene deletion approach to verify the role of region B followed by co-over expression studies and qRT-PCR to identify the regulatory role of this region. Results: We confirmed the role of region B in the regulation of SOU1 gene expression. The qRT-PCR results showed that regulation occurs at transcriptional level along with other two regions in SUR1 pathway. A previously uncharacterized open reading frame in region B has been implicated in this regulation and designated as CSU52. Integrating multiple copies of CSU52 in the genome at tandem, suppresses the growth of recipient strain on L-sorbose, establishing it as a repressor of SOU1 gene. Conclusion: This finding completes the identification of regulators in SUR1 pathway. This result paves the way to study the underlying molecular mechanisms of SOU1 gene regulation that in-turn helps to understand stress induced aneuploidy.

Background and Objectives: Monosomy of chromosome 5 associated with utilization of non-canonical sugar L-sorbose is one of the well-studied aneuploidies in Candida albicans. Stress-induced ploidy changes are crucial determinants for pathogenicity and genetic diversity in C. albicans. The five scattered regulatory regions (A, B, C, 135, and 139) comprising of two functionally redundant pathways (SUR1 and SUR2) were found to be responsible for the growth on L-sorbose. So far, three genes such as CSU51, CSU53 and CSU57 have been identified in region A, region 135 and region C, respectively. In this study we have verified the role of region B in this regulatory pathway. Materials and Methods: We employed a combinatorial gene deletion approach to verify the role of region B followed by co-over expression studies and qRT-PCR to identify the regulatory role of this region. Results: We confirmed the role of region B in the regulation of SOU1 gene expression. The qRT-PCR results showed that regulation occurs at transcriptional level along with other two regions in SUR1 pathway. A previously uncharacterized open reading frame in region B has been implicated in this regulation and designated as CSU52. Integrating multiple copies of CSU52 in the genome at tandem, suppresses the growth of recipient strain on L-sorbose, establishing it as a repressor of SOU1 gene. Conclusion: This finding completes the identification of regulators in SUR1 pathway. This result paves the way to study the underlying molecular mechanisms of SOU1 gene regulation that in-turn helps to understand stress induced aneuploidy. [ABSTRACT FROM AUTHOR]

The origin of mutations under selection has been intensively studied using the Cairns-Foster system, in which cells of an Escherichia coli lac mutant are plated on lactose and give rise to 100 Lac+ revertants over several days. These revertants have been attributed variously to stress-induced mutagenesis of nongrowing cells or to selective improvement of preexisting weakly Lac+ cells with no mutagenesis. Most revertant colonies (90%) contain stably Lac+ cells, while others (10%) contain cells with an unstable amplification of the leaky mutant lac allele. Evidence is presented that both stable and unstable Lac+ revertant colonies are initiated by preexisting cells with multiple copies of the F'lac plasmid, which carries the mutant lac allele. The tetracycline analog anhydrotetracycline (AnTc) inhibits growth of cells with multiple copies of the tetA gene. Populations with tetA on their F'lac plasmid include rare cells with an elevated plasmid copy number and multiple copies of both the tetA and lac genes. Pregrowth of such populations with AnTc reduces the number of cells with multiple F'lac copies and consequently the number of Lac+ colonies appearing under selection. Revertant yield is restored rapidly by a few generations of growth without AnTc. We suggest that preexisting cells with multiple F'lac copies divide very little under selection but have enough energy to replicate their F'lac plasmids repeatedly until reversion initiates a stable Lac+ colony. Preexisting cells whose high-copy plasmid includes an internal lac duplication grow under selection and produce an unstable Lac+ colony. In this model, all revertant colonies are initiated by preexisting cells and cannot be stress induced.

In addition to the cyanobacterial N2-fixers (diazotrophs), there is a high nifH gene diversity of non-cyanobacterial groups present in marine environments, yet quantitative information about these groups is scarce. N2 fixation potential (nifH gene expression), diversity and distributions of the uncultivated diazotroph phylotype γ-24774A11, a putative gammaproteobacterium, were investigated in the western South Pacific Ocean. γ-24774A11 gene copies correlated positively with diazotrophic cyanobacteria, temperature, dissolved organic carbon and ambient O2 saturation, and negatively with depth, chlorophyll a and nutrients, suggesting that carbon supply, access to light or inhibitory effects of DIN may control γ-24774A11 abundances. Maximum nifH gene-copy abundance was 2 × 10(4) l(-1), two orders of magnitude less than that for diazotrophic cyanobacteria, while the median γ-24774A11 abundance, 8 × 10(2) l(-1), was greater than that for the UCYN-A cyanobacteria, suggesting a more homogeneous distribution in surface waters. The abundance of nifH transcripts by γ-24774A11 was greater during the night than during the day, and the transcripts generally ranged from 0-7%, but were up to 26% of all nifH transcripts at each station. The ubiquitous presence and low variability of γ-24774A11 abundances across tropical and subtropical oceans, combined with the consistent nifH expression reported in this study, suggest that γ-24774A11 could be one of the most important heterotrophic (or photoheterotrophic) diazotrophs and may need to be considered in future N budget estimates and models.

Point mutations in the zone of polarizing activity regulatory sequence (ZRS) are known to cause human limb malformations. Although most mutations cause preaxial polydactyly (PPD), triphalangeal thumb (TPT) or both, a mutation in position 404 of the ZRS causes more severe Werner mesomelic syndrome (WMS) for which malformations include the distal arm or leg bones in addition to the hands and/or feet. Of more than 15 reported families with ZRS mutations, only one homozygous individual has been reported, with no change in phenotype compared with heterozygotes. Here, we describe a novel point mutation in the ZRS, 402C>T (AC007097.4:g.105548C>T), that is transmitted through two Mexican families with one homozygous individual. The homozygous phenotype for this mutation, WMS, is more severe than the numerous heterozygous individuals genotyped from both families who have TPT and PPD. A mouse transgenic enhancer assay shows that this mutation causes an expansion of the enhancer's expression domain in the developing mouse limb, confirming its pathogenicity. Combined, our results identify a novel ZRS mutation in the Mexican population, 402C>T, and suggest that a dosage effect exists for this ZRS mutation.

Evaluation of prostate cancer prognosis after surgery is increasingly relying upon genomic analyses of tumor DNA. We assessed the ability of the biomarker panel Genomic Evaluators of Metastatic Prostate Cancer (GEMCaP) to predict biochemical recurrence in 33 European American and 28 African American prostate cancer cases using genome-wide copy number data from a previous study. "Biomarker positive" was defined as ≥20% of the 38 constituent copy number gain/loss GEMCaP loci affected in a given tumor; based on this threshold, the frequency of a positive biomarker was significantly lower in African Americans (n = 2; 7%) than European Americans (n = 11; 33%; P = 0.013). GEMCaP positivity was associated with risk of recurrence [hazard ratio (HR), 5.92; 95% confidence interval (CI), 2.32-15.11; P = 3 × 10(-4)] in the full sample and among European Americans (HR, 3.45; 95% CI, 1.13-10.51; P = 0.032) but was not estimable in African Americans due to the low rate of GEMCaP positivity. Overall, the GEMCaP recurrence positive predictive value (PPV) was 85%; in African Americans, PPV was 100%. When we expanded the definition of loss to include copy-neutral loss of heterozygosity (i.e., loss of one allele with concomitant duplication of the other), recurrence PPV was 83% for European American subjects. Under this definition, 5 African American subjects had a positive GEMCaP test value; 4 went on to develop biochemical recurrence (PPV = 80%). Our results suggest that the GEMCaP biomarker set could be an effective predictor for both European American and African American men diagnosed with localized prostate cancer who may benefit from immediate aggressive therapy after radical prostatectomy.

Allohexaploid bread wheat (Triticum aestivum L.) provides approximately 20% of calories consumed by humans. Lack of genome sequence for the three homeologous and highly similar bread wheat genomes (A, B, and D) has impeded expression analysis of the grain transcriptome. We used previously unknown genome information to analyze the cell type-specific expression of homeologous genes in the developing wheat grain and identified distinct co-expression clusters reflecting the spatiotemporal progression during endosperm development. We observed no global but cell type- and stage-dependent genome dominance, organization of the wheat genome into transcriptionally active chromosomal regions, and asymmetric expression in gene families related to baking quality. Our findings give insight into the transcriptional dynamics and genome interplay among individual grain cell types in a polyploid cereal genome.

The centromere, responsible for chromosome segregation during mitosis, is epigenetically defined by CENP-A containing chromatin. The amount of centromeric CENP-A has direct implications for both the architecture and epigenetic inheritance of centromeres. Using complementary strategies, we determined that typical human centromeres contain ∼400 molecules of CENP-A, which is controlled by a mass-action mechanism. This number, despite representing only ∼4% of all centromeric nucleosomes, forms a ∼50-fold enrichment to the overall genome. In addition, although pre-assembled CENP-A is randomly segregated during cell division, this amount of CENP-A is sufficient to prevent stochastic loss of centromere function and identity. Finally, we produced a statistical map of CENP-A occupancy at a human neocentromere and identified nucleosome positions that feature CENP-A in a majority of cells. In summary, we present a quantitative view of the centromere that provides a mechanistic framework for both robust epigenetic inheritance of centromeres and the paucity of neocentromere formation.DOI: http://dx.doi.org/10.7554/eLife.02137.001.