bluuebunny/crossref_metadata_2025 huggingface dataset:
Genomic Stratification of Resectable Colorectal Liver Metastasis Patients and Implications for Adjuvant Therapy and Survival
Authors: Ecker Brett L. , Shin Paul , Saadat Lily V. , Court Colin M. , Balachandran Vinod P. , Chandwani Rohit , Drebin Jeffrey A. , Jarnagin William R. , Kingham T. Peter , Soares Kevin C. , Vakiani Efsevia , Wei Alice C. , Kemeny Nancy E. , Smith J. Joshua , Gonen Mithat , D’Angelica Michael I.
Published: November 2021

Objective:

To determine whether genomic risk groups identified by somatic mutation testing of colorectal liver metastasis (CRLM) can be used for “molecularly-guided” selection for adjuvant systemic chemotherapy and hepatic artery infusion of FUDR (SYS+HAI-FUDR).

Background:

Several genomic biomarkers have been associated with clinical phenotype and survival for patients with resectable CRLM. It is unknown whether prognostication afforded by genomic stratification translates into enhanced patient selection for adjuvant hepatic artery infusion therapy.

Methods:

Consecutive patients with resected CRLM and available mutational characterization via Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets were reviewed from a prospective institutional database. Patients were stratified into three genomic risk groups based on previously defined alterations in SMAD4, EGFR and the RAS/RAF pathway. The association between SYS+HAI-FUDR and overall survival, relative to adjuvant chemotherapy alone (SYS), was evaluated in each genomic risk group by Cox proportional hazard regression and propensity score matched analyses.

Results:

A total of 334 patients (SYS+HAI-FUDR 204; SYS 130) were identified; the rates of RAS/RAF alterations and SMAD4 inactivation were 47.4% and 11.7%, respectively. After a median follow-up of 58 months, adjuvant SYS+HAI-FUDR was independently associated with a reduced risk of death (HR 0.50, 95%CI 0.26–0.98, P = 0.045) in the low-risk genomic group, but not in the moderate-risk (HR 1.07, 95%CI 0.5–2.07, P = 0.749) or high-risk (HR 1.62, 95%CI 0.29–9.12, P = 0.537) cohorts. Following propensity score matching, adjuvant SYS+HAI-FUDR remained associated with significant improvements in long-term survival selectively in the low-risk genomic cohort (5-year actuarial survival: 89% vs. 68%, P = 0.019).

Conclusions:

Genomic alterations in RAS/RAF, SMAD4, and EGFR may be useful to guide treatment selection in resectable CRLM patients and warrant external validation and integration in future clinical trial design.
Coronary Artery Stent for Securing High-risk Pancreatico-jejunal Anastomosis After Pancreatoduodenectomy
Authors: Huscher Cristiano , Perri Giampaolo , Lazzarin Gianni , Marchegiani Giovanni , Malleo Giuseppe , Salvia Roberto , Bassi Claudio
Published: November 2021

Objective:

To assess the feasibility and clinical utility of coronary artery stent (CAS) in securing pancreatico-jejunal anastomosis (PJ) and avoid stent displacement after pancreatoduodenectomy (PD).

Summary of Background Data:

Externalized trans-anastomotic stent (ETS) is a standard mitigation strategy for postoperative pancreatic fistula (POPF) in high-risk patients. However, major morbidity remains extremely elevated, especially in case of ETS malfunction due to displacement.

Methods:

A pilot series of 72 patients underwent PD and PJ with CAS positioning between January 2016 and December 2019. All patients were at high-risk for POPF (soft pancreatic texture; main pancreatic duct diameter ≤ 3 mm) and underwent a CT-scan at postoperative day 5 and 10 to assess the correct CAS positioning. Postoperative outcomes were analyzed, and displacement rates were compared with a cohort of 141 patients with the same high-risk characteristics, undergoing PD with PJ and externalized trans-anastomotic stent (ETS).

Results:

No CAS-related complications were registered in the study group. In particular, no CAS displacement was registered, compared to a 28% ETS malfunction (either displacement or occlusion). The POPF rate, major morbidity, and mortality were 11%, 6%, and 0% respectively.

Conclusions:

The CAS positioning appears to be a feasible and safe mitigation strategy to secure PJ anastomosis after PD with high POPF risk avoiding stent displacement. Further validation and comparison with current standard of care is required in a prospective controlled setting.
Initiating early palliative care for older people with advanced cancer and its barriers
Authors: Bugaj Till J. , Oeljeklaus Lydia , Haun Markus W.
Published: November 2021

Purpose of review

Early palliative care (EPC) is known to generally improve both health-related quality of life (QoL) and symptom intensity at small effect sizes. However, it is unclear whether EPC is effective in older people, a population that is notoriously unaccounted for. This review summarizes the recent evidence concerning the efficacy of EPC in older patients with advanced cancer and delineates existing barriers to accessing respective services.

Recent findings

The search for studies published in MEDLINE from January 2020 to September 2021 yielded six relevant records. Data from a recent feasibility trial and subgroups from larger randomised trials point to a somewhat lesser decline in QoL for patients undergoing EPC compared to those receiving treatment as usual. However, enrolling older patients in such trials remains a major challenge mostly due to them feeling too ill to participate.

For older patients, the efficacy of EPC, like many other medical interventions, has hardly been studied so far. Existing work yielded several specific barriers for older patients to access this type of care. Future research should prioritize efficacy trials of EPC tailored to the needs of older patients enabling clinicians to enter truly evidence-based shared decision-making with their patients.
Microglia do not restrict SARS-CoV-2 replication following infection of the central nervous system of K18-hACE2 transgenic mice
Authors: Olivarria Gema M. , Cheng Yuting , Furman Susana , Pachow Collin , Hohsfield Lindsay A. , Smith-Geater Charlene , Miramontes Ricardo , Wu Jie , Burns Mara S. , Tsourmas Kate I. , Stocksdale Jennifer , Manlapaz Cynthia , Yong William H. , Teijaro John , Edwards Robert , Green Kim N. , Thompson Leslie M. , Lane Thomas E.
Published: November 2021

Unlike SARS-CoV-1 and MERS-CoV, infection with SARS-CoV-2, the viral pathogen responsible for COVID-19, is often associated with neurologic symptoms that range from mild to severe, yet increasing evidence argues the virus does not exhibit extensive neuroinvasive properties. We demonstrate SARS-CoV-2 can infect and replicate in human iPSC-derived neurons and that infection shows limited anti-viral and inflammatory responses but increased activation of EIF2 signaling following infection as determined by RNA sequencing. Intranasal infection of K18 human ACE2 transgenic mice (K18-hACE2) with SARS-CoV-2 resulted in lung pathology associated with viral replication and immune cell infiltration. In addition, ∼50% of infected mice exhibited CNS infection characterized by wide-spread viral replication in neurons accompanied by increased expression of chemokine (Cxcl9, Cxcl10, Ccl2, Ccl5andCcl19) and cytokine (Ifn-λandTnf-α) transcripts associated with microgliosis and a neuroinflammatory response consisting primarily of monocytes/macrophages. Microglia depletion via administration of colony-stimulating factor 1 receptor inhibitor, PLX5622, in SARS-CoV-2 infected mice did not affect survival or viral replication but did result in dampened expression of proinflammatory cytokine/chemokine transcripts and a reduction in monocyte/macrophage infiltration. These results argue that microglia are dispensable in terms of controlling SARS-CoV-2 replication in in the K18-hACE2 model but do contribute to an inflammatory response through expression of pro-inflammatory genes. Collectively, these findings contribute to previous work demonstrating the ability of SARS-CoV-2 to infect neurons as well as emphasizing the potential use of the K18-hACE2 model to study immunological and neuropathological aspects related to SARS-CoV-2-induced neurologic disease.

Importance

Understanding the immunological mechanisms contributing to both host defense and disease following viral infection of the CNS is of critical importance given the increasing number of viruses that are capable of infecting and replicating within the nervous system. With this in mind, the present study was undertaken to evaluate the role of microglia in aiding in host defense following experimental infection of the central nervous system (CNS) of K18-hACE2 with SARS-CoV-2, the causative agent of COVID-19. Neurologic symptoms that range in severity are common in COVID-19 patients and understanding immune responses that contribute to restricting neurologic disease can provide important insight into better understanding consequences associated with SARS-CoV-2 infection of the CNS.
NUDT18 catalyzes the hydrolysis of active metabolites of the antivirals Remdesivir, Ribavirin and Molnupiravir
Authors: Jemth Ann-Sofie , Scaletti Emma Rose , Homan Evert , Stenmark Pål , Helleday Thomas , Michel Maurice
Published: November 2021

Remdesivir and Molnupiravir have gained considerable interest due to their activity against SARS-CoV-2. Cellular hydrolysis of their active triphosphate forms, Remdesivir-TP and Molnupiravir-TP, would decrease drug efficiency. We therefore tested Remdesivir-TP as a substrate against a panel of human hydrolases and found that NUDT18 catalyzes the hydrolysis of Remdesivir-TP. The kcat value of NUDT18 for Remdesivir-TP was determined to 2.6 s-1 and the Km value was 156 μM, suggesting that NUDT18 catalyzed hydrolysis of Remdesivir-TP occurs in cells. We demonstrate that the triphosphates of the antivirals Ribavirin and Molnupiravir are hydrolyzed by NUDT18, albeit with a lower efficiency compared to Remdesivir-TP. NUDT18 also hydrolyses the triphosphates of Sofosbuvir and Aciclovir although with significantly lower activity. These results suggest that NUDT18 can act as a cellular sanitizer of modified nucleotides and may influence the antiviral efficacy of Remdesivir, Molnupiravir and Ribavirin. NUDT18 is expressed in respiratory epithelial cells and may limit the antiviral efficacy of Remdesivir and Molnupiravir against SARS-CoV2 replication by decreasing the intracellular concentration of their active metabolites at their intended site of action.
Cell twisting during desiccation reveals axial asymmetry in wall organization
Authors: Keynia Sedighe , Davis Thomas C. , Szymanski Daniel B. , Turner Joseph A.
Published: November 2021

Plant cell size and shape are tuned to their function and specified primarily by cellulose microfibril (CMF) patterning of the cell wall. Arabidopsis thaliana leaf trichomes are responsible for protecting plants against environmental elements and are unicellular structures that employ a highly anisotropic cellulose wall to extend and taper, generating pointed branches. During elongation, the mechanisms by which shifts in fiber orientation generate cells with predictable sizes and shapes are unknown. Specifically, the axisymmetric growth of trichome branches is often thought result from axisymmetric CMF patterning. Here, we analyzed the direction and degree of twist of branches after desiccation to reveal the presence of an asymmetric cell wall organization with a left-hand bias. CMF organization, quantified using computational modeling, suggests a limited reorientation of microfibrils during growth and maximum branch length limited by the wall axial stiffness. The model provides a mechanism for CMF asymmetry, which occurs after the branch bending stiffness becomes low enough that ambient bending affects the principal stresses. After this stage, the CMF synthesis results in a constant bending stiffness for longer branches. The resulting natural frequency of branches after a length of 200 μm falls within the range of the sounds associated with many insects.

Statement of significance

The growth of plant cell walls is governed by the direction of cellulose synthesis but the factors that influence the overall wall anisotropy are only partially understood. The twist of leaf trichome branches after desiccation reveals a left-handed asymmetry in cell wall organization even though the geometry is axisymmetric. This surprising behavior provides information about the directionality of cellulose synthesis control in plant cells.
SARS-CoV-2 variants of concern remain dependent on IFITM2 for efficient replication in human lung cells
Authors: Nchioua Rayhane , Schundner Annika , Kmiec Dorota , Prelli-Bozzo Caterina , Zech Fabian , Koepke Lennart , Graf Alexander , Krebs Stefan , Blum Helmut , Frick Manfred , Sparrer Konstantin M. J. , Kirchhoff Frank
Published: November 2021

It has recently been shown that an early SARS-CoV-2 isolate (NL-02-2020) hijacks interferon-induced transmembrane proteins (IFITMs) for efficient replication in human cells. To date, several “Variants of Concern” (VOCs) showing increased infectivity and resistance to neutralization have emerged and globally replaced the early viral strains. Here, we determined whether the four SARS-CoV-2 VOCs (Alpha, Beta, Gamma and Delta) maintained the dependency on IFITM proteins for efficient replication. We found that depletion of IFITM2 strongly reduces viral RNA production by all four VOCs in the human epithelial lung cancer cell line Calu-3. Silencing of IFITM1 had little effect, while knock-down of IFITM3 resulted in an intermediate phenotype. Strikingly, depletion of IFITM2 generally reduced infectious virus production by more than four orders of magnitude. In addition, an antibody directed against the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells thought to represent major viral target cells in the lung. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are critical cofactors for efficient replication of genuine SARS-CoV-2 VOCs, including the currently dominating Delta variant.

IMPORTANCE

Recent results showed that an early SARS-CoV-2 isolate requires endogenously expressed IFITM proteins for efficient infection. However, whether IFITMs are also important cofactors for infection of emerging SARS-CoV-2 VOCs that out-competed the original strains and currently dominate the pandemic remained to be determined. Here, we demonstrate that depletion of endogenous IFITM2 expression almost entirely prevents the production of infectious Alpha, Beta, Gamma and Delta VOC SARS-CoV-2 virions in a human lung cell line. In comparison, silencing of IFITM1 had little impact, while knock-down of IFITM3 had intermediate effects on viral replication. Finally, an antibody targeting the N-terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells. Our results show that SARS-CoV-2 VOCs including the currently dominant Delta variant are dependent on IFITM2 for efficient replication suggesting that IFITM proteins play a key role in viral transmission and pathogenicity.
Upregulation of CREB1 and FOXO1 transcription factor pathways in Neuregulin-1 mediated neuroprotection following ischemic stroke
Authors: Bennett Kimberly R. , Surles-Zeigler Monique C. , Augello Catherine J. , Ako Etchi , Rodgers Victor G. J. , Ford Byron D.
Published: November 2021

Neuregulin-1 (NRG-1) is growth factor that has been investigated for its neuroprotective properties following ischemic stroke. While NRG-1 has shown significant promise in preventing neuronal damage following stroke, the mechanisms behind its neuroprotective effects are unclear. The goal of this research was to investigate the effects of NRG-1 treatment on ischemia-induced gene expression profiles following a permanent middle cerebral artery occlusion (MCAO) in rats. Rats were sacrificed twelve hours following MCAO and either vehicle or NRG-1 treatment. RNA extracted from the peri-infarct cortex of the brain was hybridized to an Affymetrix Rat Genome 2.0st Microarray Gene Chip. Data were analyzed using the Affymetrix Transcriptome Analysis Console (TAC) 4.0 software and the STRING Protein-Protein Interaction Networks database. Our results showed that NRG-1 delivery increased the regulation of pro-survival genes. Most notably, NRG-1 treatment upregulated the CREB1 and FOXO1 transcription factor pathways which are involved in increasing anti-inflammatory and cell proliferation responses and decreasing apoptosis and oxidative stress responses, respectively. Luminex multiplex transcription factor assays demonstrated that the activities of CREB1 and FOXO1 were increased by NRG-1 treatment with MCAO. These findings provide novel insight into the molecular mechanisms involved in NRG-1 mediated neuroprotection.
Selective YAP activation in Procr cells is essential for ovarian stem/progenitor expansion and epithelium repair
Authors: Wang Jingqiang , He Lingli , Xie Zhiyao , Yu Wentao , Bai Lanyue , Wang Zuoyun , Lu Yi , Liu Chunye , Fu Junfen , Zhang Lei , Zeng Yi Arial
Published: November 2021

Ovarian surface epithelium (OSE) undergoes recurring ovulatory rupture and OSE stem cells rapidly generate new cells for the repair. How the stem cell senses the rupture and promptly turns on proliferation is unclear. Our previous study has identified that Protein C Receptor (Procr) marks OSE progenitors. In this study, we observed decreased adherent junction and selective activation of YAP signaling in Procr progenitors at OSE rupture site. OSE repair is impeded upon deletion of Yap in these progenitors. Interestingly, Procr+ progenitors show lower expression of Vgll4, an antagonist of YAP signaling. Overexpression of Vgll4 in Procr+ cells hampers OSE repair and progenitor proliferation, indicating that selective low Vgll4 expression in Procr+ progenitors is critical for OSE repair. In addition, YAP activation promotes transcription of the OSE stemness gene Procr. The combination of increased cell division and Procr expression leads to expansion of Procr+ progenitors surrounding the rupture site. These results illustrate a YAP- dependent mechanism by which the stem/progenitor cells recognize the ovulatory rupture, and rapidly multiply their numbers, highlighting a YAP- induced stem cell expansion strategy.
Single nucleus RNA-seq in the hippocampus of a Down syndrome mouse model reveals new key players in memory
Authors: Sierra Cesar , De Toma Ilario , Dierssen Mara
Published: November 2021

Down syndrome (DS) is the most common genetic cause of intellectual disability. Even though great advances in the last decades have allowed better delineation of its pathogenetic mechanisms, its cellular and molecular bases are still poorly understood. To evaluate the consequences of chromosome aneuploidy on the hippocampus, we analyzed single-nucleus transcriptional profiles of the DS mouse model Ts65Dn. Our data revealed abnormal cell composition specifically of the Ts65Dn dentate gyrus and of specific subtypes of interneurons, without major changes in CA1 or CA3. We found that trisomy results in a highly cell-type specific global alteration of the transcriptome and detected previously undefined differentially expressed genes in specific neuronal populations. We identified the long-non-coding gene Snhg11 to be specifically downregulated in the trisomic dentate gyrus and provide evidence for its involvement in hippocampal-dependent cognitive phenotypes, possibly contributed by impaired adult neurogenesis.
Natural Genomic Design in Sinorhizobium meliloti: Novel Genomic Architectures
Authors: Guo Xianwu , Flores Margarita , Mavingui Patrick , Fuentes Sara Isabel , Hernández Georgina , Dávila Guillermo , Palacios Rafael
Published: November 2021

The complete nucleotide sequence of the genome of Sinorhizobium meliloti, the symbiont of alfalfa, was reported in 2001 by an international consortium of laboratories. The genome comprises a chromosome of 3.65 megabases (Mb) and two megaplasmids, pSymA and pSymB, of 1.35 Mb and 1.68 Mb, respectively. Based on the nucleotide sequence of the whole genome, we designed a pathway of consecutive rearrangements leading to novel genomic architectures. In a first step we obtained derivative strains containing two replicons; in a second step we obtained a strain containing the genetic information in one single replicon of 6.68 MB. From this last architecture we isolated revertants containing two replicons, and from these we could return to the original architecture showing the three replicons. We found that the relative frequency of excision of cointegrated replicons is higher at the site used for the cointegration than at other sites. This might conciliate two apparently opposed facts: the highly dynamic state of genomic architecture in S. meliloti and the common observation that different isolates and derived cellular clones of S. meliloti usually present the architecture of one chromosome and two distinct megaplasmids. Different aspects that must be considered to obtain full advantage of the strategy of natural genomic design are discussed.
Theories and Applications for Sequencing Randomly Selected Clones
Authors: Wendl Michael C. , Marra Marco A. , Hillier LaDeana W. , Chinwalla Asif T. , Wilson Richard K. , Waterston Robert H.
Published: November 2021

Theory is developed for the process of sequencing randomly selected large-insert clones. Genome size, library depth, clone size, and clone distribution are considered relevant properties and perfect overlap detection for contig assembly is assumed. Genome-specific and nonrandom effects are neglected. Order of magnitude analysis indicates library depth is of secondary importance compared to the other variables, especially as clone size diminishes. In such cases, the well-known Poisson coverage law is a good approximation. Parameters derived from these models are used to examine performance for the specific case of sequencing random human BAC clones. We compare coverage and redundancy rates for libraries possessing uniform and nonuniform clone distributions. Results are measured against data from map-based human-chromosome-2 sequencing. We conclude that the map-based approach outperforms random clone sequencing, except early in a project. However, simultaneous use of both strategies can be beneficial if a performance-based estimate for halting random clone sequencing is made. Results further show that the random approach yields maximum effectiveness using nonbiased rather than biased libraries.
Microarray Expression Profiling Identifies Genes with Altered Expression in HDL-Deficient Mice
Authors: Callow Matthew J. , Dudoit Sandrine , Gong Elaine L. , Speed Terence P. , Rubin Edward M.
Published: November 2021

Based on the assumption that severe alterations in the expression of genes known to be involved in high-density lipoprotein (HDL) metabolism may affect the expression of other genes, we screened an array of >5000 mouse expressed sequence tags for altered gene expression in the livers of two lines of mice with dramatic decreases in HDL plasma concentrations. Labeled cDNA from livers of apolipoprotein AI (apoAI)-knockout mice, scavenger receptor BI (SR-BI) transgenic mice, and control mice were cohybridized to microarrays. Two-samplet statistics were used to identify genes with altered expression levels in the knockout or transgenic mice compared with control mice. In the SR-BI group we found nine array elements representing at least five genes that were significantly altered on the basis of an adjusted P value < 0.05. In the apoAI-knockout group, eight array elements representing four genes were altered compared with the control group (adjustedP < 0.05). Several of the genes identified in the SR-BI transgenic suggest altered sterol metabolism and oxidative processes. These studies illustrate the use of multiple-testing methods for the identification of genes with altered expression in replicated microarray experiments.
Identification, Characterization, and Mapping of Expressed Sequence Tags from an Embryonic Zebrafish Heart cDNA Library
Authors: Ton Christopher , Hwang David M. , Dempsey Adam A. , Tang Hong-Chang , Yoon Jennifer , Lim Mindy , Mably John D. , Fishman Mark C. , Liew Choong-Chin
Published: November 2021

The generation of expressed sequence tags (ESTs) has proven to be a rapid and economical approach by which to identify and characterize expressed genes. We generated 5102 ESTs from a 3-d-old embryonic zebrafish heart cDNA library. Of these, 57.6% matched to known genes, 14.2% matched only to other ESTs, and 27.8% showed no match to any ESTs or known genes. Clustering of all ESTs identified 359 unique clusters comprising 1771 ESTs, whereas the remaining 3331 ESTs did not cluster. This estimates the number of unique genes identified in the data set to be approximately 3690. A total of 1242 unique known genes were used to analyze the gene expression patterns in the zebrafish embryonic heart. These were categorized into seven categories on the basis of gene function. The largest class of genes represented those involved in gene/protein expression (25.9% of known transcripts). This class was followed by genes involved in metabolism (18.7%), cell structure/motility (16.4%), cell signaling and communication (9.6%), cell/organism defense (7.1%), and cell division (4.4%). Unclassified genes constituted the remaining 17.91%. Radiation hybrid mapping was performed for 102 ESTs and comparison of map positions between zebrafish and human identified new synteny groups. Continued comparative analysis will be useful in defining the boundaries of conserved chromosome segments between zebrafish and humans, which will facilitate the transfer of genetic information between the two organisms and improve our understanding of vertebrate evolution.

[The sequence data described in this paper have been submitted to the GenBank data library under accession nos.BE693120–BE693210 and BE704450.]
Identification and Characterization of the Potential Promoter Regions of 1031 Kinds of Human Genes
Authors: Suzuki Yutaka , Tsunoda Tatsuhiko , Sese Jun , Taira Hirotoshi , Mizushima-Sugano Junko , Hata Hiroko , Ota Toshio , Isogai Takao , Tanaka Toshihiro , Nakamura Yusuke , Suyama Akira , Sakaki Yoshiyuki , Morishita Shinichi , Okubo Kousaku , Sugano Sumio
Published: November 2021

To understand the mechanism of transcriptional regulation, it is essential to identify and characterize the promoter, which is located proximal to the mRNA start site. To identify the promoters from the large volumes of genomic sequences, we used mRNA start sites determined by a large-scale sequencing of the cDNA libraries constructed by the “oligo-capping” method. We aligned the mRNA start sites with the genomic sequences and retrieved adjacent sequences as potential promoter regions (PPRs) for 1031 genes. The PPR sequences were searched to determine the frequencies of major promoter elements. Among 1031 PPRs, 329 (32%) contained TATA boxes, 872 (85%) contained initiators, 999 (97%) contained GC box, and 663 (64%) contained CAAT box. Furthermore, 493 (48%) PPRs were located in CpG islands. This frequency of CpG islands was reduced in TATA+/Inr+PPRs and in the PPRs of ubiquitously expressed genes. In the PPRs of the CGM2 gene, the DRA gene, and theTM30pl genes, which showed highly colon specific expression patterns, the consensus sequences of E boxes were commonly observed. The PPRs were also useful for exploring promoter SNPs.

[The nucleotide sequences described in this paper have been deposited in the DDBJ, EMBL, and GenBank data libraries under accession nos.AU098358–AU100608.]
Homogeneous Assays for Single-Nucleotide Polymorphism Typing Using AlphaScreen
Authors: Beaudet Lucille , Bédard Julie , Breton Billy , Mercuri Roberto J. , Budarf Marcia L.
Published: November 2021

AlphaScreen technology allows the development of high-throughput homogeneous proximity assays. In these assays, signal is generated when 680 nm laser light irradiates a donor bead in close proximity to an acceptor bead. For the detection of nucleic acids, donor and acceptor beads are brought into proximity by two bridging probes that hybridize simultaneously to a common target and to the generic oligonucleotides attached covalently to the beads. This method allows the detection of as little as 10 amole of a single-stranded DNA target. The combination of AlphaScreen with allele-specific amplification (ASA) and allele-specific hybridization (ASH) has allowed the development of two homogenous single-nucleotide polymorphism (SNP) genotyping platforms. Both types of assay are very robust, routinely giving accurate genotyping results with < 2 ng of genomic DNA per genotype. An AlphaScreen validation study was performed for 12 SNPs by using ASA assays and seven SNPs by using ASH assays. More than 580 samples were genotyped with accuracy >99%. The two assays are remarkably simple, requiring no post-PCR manipulations. Genotyping has been performed successfully in 96- and 384-well formats with volumes as small as 2 μL, allowing a considerable reduction in the amount of reagents and genomic DNA necessary for genotyping. These results show that the AlphaScreen technology can be successfully adapted to high-throughput genotyping.
Arrays of Arrays for High-Throughput Gene Expression Profiling
Authors: Zarrinkar Patrick P. , Mainquist James K. , Zamora Matthew , Stern David , Welsh John B. , Sapinoso Lisa M. , Hampton Garret M. , Lockhart David J.
Published: November 2021

Gene expression profiling using DNA arrays is rapidly becoming an essential tool for research and drug discovery and may soon play a central role in disease diagnosis. Although it is possible to make significant discoveries on the basis of a relatively small number of expression profiles, the full potential of this technology is best realized through more extensive collections of expression measurements. The generation of large numbers of expression profiles can be a time-consuming and labor-intensive process with current one-at-a-time technology. We have developed the ability to obtain expression profiles in a highly parallel yet straightforward format using glass wafers that contain 49 individual high-density oligonucleotide arrays. This arrays of arrays concept is generalizable and can be adapted readily to other types of arrays, including spotted cDNA microarrays. It is also scalable for use with hundreds and even thousands of smaller arrays on a single piece of glass. Using the arrays of arrays approach and parallel preparation of hybridization samples in 96-well plates, we were able to determine the patterns of gene expression in 27 ovarian carcinomas and 4 normal ovarian tissue samples, along with a number of control samples, in a single experiment. This new approach significantly increases the ease, efficiency, and throughput of microarray-based experiments and makes possible new applications of expression profiling that are currently impractical.
The Nuclear Receptor Superfamily Has Undergone Extensive Proliferation and Diversification in Nematodes
Authors: Sluder Ann E. , Mathews Siuyien Wong , Hough David , Yin Viravuth P. , Maina Claude V.
Published: November 2021

The nuclear receptor (NR) superfamily is the most abundant class of transcriptional regulators encoded in the Caenorhabditis elegans genome, with >200 predicted genes revealed by the screens and analysis of genomic sequence reported here. This is the largest number of NR genes yet described from a single species, although our analysis of available genomic sequence from the related nematode Caenorhabditis briggsae indicates that it also has a large number. Existing data demonstrate expression for 25% of theC. elegans NR sequences. Sequence conservation and statistical arguments suggest that the majority represent functional genes. An analysis of these genes based on the DNA-binding domain motif revealed that several NR classes conserved in both vertebrates and insects are also represented among the nematode genes, consistent with the existence of ancient NR classes shared among most, and perhaps all, metazoans. Most of the nematode NR sequences, however, are distinct from those currently known in other phyla, and reveal a previously unobserved diversity within the NR superfamily. In C. elegans, extensive proliferation and diversification of NR sequences have occurred on chromosome V, accounting for > 50% of the predicted NR genes.

[The sequence data described in this paper have been submitted to the GenBank data library under accession nos.AF083222–AF083225 and AF083251–AF083234.]
Isolation of Zebrafish gdf7 and Comparative Genetic Mapping of Genes Belonging to the Growth/Differentiation Factor 5, 6, 7 Subgroup of the TGF-β Superfamily
Authors: Davidson Alan J. , Postlethwait John H. , Yan Yi-Lin , Beier David R. , van Doren Cherie , Foernzler Dorothee , Celeste Anthony J. , Crosier Kathryn E. , Crosier Philip S.
Published: November 2021

The Growth/differentiation factor (Gdf)5, 6, 7 genes form a closely related subgroup belonging to the TGF-β superfamily. In zebrafish, there are three genes that belong to the Gdf5, 6, 7subgroup that have been named radar, dynamo, andcontact. The genes radar and dynamo both encode proteins most similar to mouse GDF6. The orthologous identity of these genes on the basis of amino acid similarities has not been clear. We have identified gdf7, a fourth zebrafish gene belonging to the Gdf5, 6, 7 subgroup. To assign correct orthologies and to investigate the evolutionary relationships of the human, mouse, and zebrafish Gdf5, 6, 7subgroup, we have compared genetic map positions of the zebrafish and mammalian genes. We have mapped zebrafish gdf7 to linkage group (LG) 17, contact to LG9, GDF6 to human chromosome (Hsa) 8 and GDF7 to Hsa2p. The radar anddynamo genes have been localized previously to LG16 and LG19, respectively. A comparison of syntenies shared among human, mouse, and zebrafish genomes indicates that gdf7 is the ortholog of mammalian GDF7/Gdf7. LG16 shares syntenic relationships with mouse chromosome (Mmu) 4, including Gdf6. Portions of LG16 and LG19 appear to be duplicate chromosomes, thus suggesting thatradar and dynamo are both orthologs of Gdf6. Finally, the mapping data is consistent with contact being the zebrafish ortholog of mammalian GDF5/Gdf5.

[The sequence data described in this paper have been submitted to the GenBank data library under accession numbers AF113022 and AF113023.]
The MouseAireGene: Comparative Genomic Sequencing, Gene Organization, and Expression
Authors: Blechschmidt Karin , Schweiger Michal , Wertz Karin , Poulson Richard , Christensen Hoang-My , Rosenthal Andre , Lehrach Hans , Yaspo Marie-Laure
Published: November 2021

Mutations in the humanAIREgene (hAIRE) result in the development of an autoimmune disease named APECED (autoimmunepolyendocrinopathycandidiasisectodermaldystrophy; OMIM 240300). Previously, we have cloned hAIREand shown that it codes for a putative transcription-associated factor. Here we report the cloning and characterization ofAire, the murine ortholog of hAIRE. Comparative genomic sequencing revealed that the structure of theAIREgene is highly conserved between human and mouse. The conceptual proteins share 73% homology and feature the same typical functional domains in both species. RT–PCR analysis detected three splice variant isoforms in various mouse tissues, and interestingly one isoform was conserved in human, suggesting potential biological relevance of this product. In situ hybridization on mouse and human histological sections showed that AIRE expression pattern was mainly restricted to a few cells in the thymus, calling for a tissue-specific function of the gene product.
An Effective Approach for Analyzing “Prefinished” Genomic Sequence Data
Authors: Kuehl Peter M. , Weisemann Jane M. , Touchman Jeffrey W. , Green Eric D. , Boguski Mark S.
Published: November 2021

Ongoing efforts to sequence the human genome are already generating large amounts of data, with substantial increases anticipated over the next few years. In most cases, a shotgun sequencing strategy is being used, which rapidly yields most of the primary sequence in incompletely assembled sequence contigs (“prefinished” sequence) and more slowly produces the final, completely assembled sequence (“finished” sequence). Thus, in general, prefinished sequence is produced in excess of finished sequence, and this trend is certain to continue and even accelerate over the next few years. Even at a prefinished stage, genomic sequence represents a rich source of important biological information that is of great interest to many investigators. However, analyzing such data is a challenging and daunting task, both because of its sheer volume and because it can change on a day-by-day basis. To facilitate the discovery and characterization of genes and other important elements within prefinished sequence, we have developed an analytical strategy and system that uses readily available software tools in new combinations. Implementation of this strategy for the analysis of prefinished sequence data from human chromosome 7 has demonstrated that this is a convenient, inexpensive, and extensible solution to the problem of analyzing the large amounts of preliminary data being produced by large-scale sequencing efforts. Our approach is accessible to any investigator who wishes to assimilate additional information about particular sequence data en route to developing richer annotations of a finished sequence.

[Our software system is available via an extensive web supplement to this article at http://www.ncbi.nlm.nih.gov/Kuehl/prefinished.]
The Genexpress IMAGE Knowledge Base of the Human Brain Transcriptome: A Prototype Integrated Resource for Functional and Computational Genomics
Authors: Piétu Geneviève , Mariage-Samson Régine , Fayein Nicole-Adeline , Matingou Christiane , Eveno Eric , Houlgatte Rémi , Decraene Charles , Vandenbrouck Yves , Tahi Fariza , Devignes Marie-Dominique , Wirkner Ute , Ansorge Wilhelm , Cox David , Nagase Takahiro , Nomura Nobuo , Auffray Charles
Published: November 2021

Expression profiles of 5058 human gene transcripts represented by an array of 7451 clones from the first IMAGE Consortium cDNA library from infant brain have been collected by semiquantitative hybridization of the array with complex probes derived by reverse transcription of mRNA from brain and five other human tissues. Twenty-one percent of the clones corresponded to transcripts that could be classified in general categories of low, moderate, or high abundance. These expression profiles were integrated with cDNA clone and sequence clustering and gene mapping information from an upgraded version of the Genexpress Index. For seven gene transcripts found to be transcribed preferentially or specifically in brain, the expression profiles were confirmed by Northern blot analyses of mRNA from eight adult and four fetal tissues, and 15 distinct regions of brain. In four instances, further documentation of the sites of expression was obtained by in situ hybridization of rat-brain tissue sections. A systematic effort was undertaken to further integrate available cytogenetic, genetic, physical, and genic map informations through radiation-hybrid mapping to provide a unique validated map location for each of these genes in relation to the disease map. The resulting Genexpress IMAGE Knowledge Base is illustrated by five examples presented in the printed article with additional data available on a dedicated Web site at the addresshttp://idefix.upr420.vjf.cnrs.fr/EXPR/welcome.html.
The Relative Power of Family-Based and Case-Control Designs for Linkage Disequilibrium Studies of Complex Human Diseases. II. Individual Genotyping
Authors: Teng Jun , Risch Neil
Published: November 2021

In this paper we consider test statistics based on individual genotyping. For sibships without parents, but with unaffected as well as affected sibs, we introduce a new test statistic (referred to asTDS), which contrasts the allele frequency in affected sibs versus that estimated for the parents from the entire sibship. For sibships without parents, this test is analogous to the TDT and is completely robust to nonrandom mating patterns. The efficiency of the TDS test is comparable to that of the THS test (which compares affected vs. unaffected sibs and was based on DNA pooling), for sibships with one affected child. However, as the number of affected sibs in the sibship grows, the relative efficiency of the TDS test versus theTHS test also increases. For example, for sibships with three affected, one-third fewer families are required; for families with four affected, nearly half as many are required. Thus, when sibships contain multiple affected individuals, theTDS test provides both an increase in power and robustness to nonrandom mating.
Analysis of 148 kb of Genomic DNA Around thewnt1Locus ofFugu rubripes
Authors: Gellner Klaus , Brenner Sydney
Published: November 2021

The analysis of the sequence of ∼150 kb of a genomic region corresponding to thewnt1gene of the Japanese pufferfishFugu rubripesconfirms the compact structure of the genome. Fifteen genes were found in this region, and 26.6% of the analyzed sequence is coding sequence. With an average intergenic distance of <5 kb, this gene density is comparable to that ofCaenorhabditis elegans. The compactness of this region corresponds to the reduction of the overall size of the genome, consistent with the conclusion that the gene number inFuguand human genomes is approximately the same. Eight of the genes have been mapped in the human genome and all of them are found in the chromosomal band 12q13, indicating a high degree of synteny in both species,Fuguand human. Comparative sequence analysis allows us to identify potential regulatory elements forwnt1andARF3, which are common to fish and mammals.

[The sequence data described in this paper have been submitted to GenBank under accession no.AF056116.]
DNA Sequence Chromatogram Browsing Using JAVA and CORBA
Authors: Parsons Jeremy D. , Buehler Eugen , Hillier LaDeana
Published: November 2021

DNA sequence chromatograms (traces) are the primary data source for all large-scale genomic and expressed sequence tags (ESTs) sequencing projects. Access to the sequencing trace assists many later analyses, for example contig assembly and polymorphism detection, but obtaining and using traces is problematic. Traces are not collected and published centrally, they are much larger than the base calls derived from them, and viewing them requires the interactivity of a local graphical client with local data. To provide efficient global access to DNA traces, we developed a client/server system based on flexible Java components integrated into other applications including an applet for use in a WWW browser and a stand-alone trace viewer. Client/server interaction is facilitated by CORBA middleware which provides a well-defined interface, a naming service, and location independence.

[The software is packaged as a Jar file available from the following URL: http://www.ebi.ac.uk/∼jparsons. Links to working examples of the trace viewers can be found athttp://corba.ebi.ac.uk/EST. All the Washington University mouse EST traces are available for browsing at the same URL.]
An Efficient DNA Sequencing Strategy Based on the Bacteriophage Mu in Vitro DNA Transposition Reaction
Authors: Haapa Saija , Suomalainen Sini , Eerikäinen Simo , Airaksinen Matti , Paulin Lars , Savilahti Harri
Published: November 2021

A highly efficient DNA sequencing strategy was developed on the basis of the bacteriophage Mu in vitro DNA transposition reaction. In the reaction, an artificial transposon with a chloramphenicol acetyltransferase (cat) gene as a selectable marker integrated into the target plasmid DNA containing a 10.3-kb mouse genomic insert to be sequenced. Bacterial clones carrying plasmids with the transposon insertions in different positions were produced by transforming transposition reaction products into Escherichia coli cells that were then selected on appropriate selection plates. Plasmids from individual clones were isolated and used as templates for DNA sequencing, each with two primers specific for the transposon sequence but reading the sequence into opposite directions, thus creating a minicontig. By combining the information from overlapping minicontigs, the sequence of the entire 10,288-bp region of mouse genome including six exons of mouse Kcc2 gene was obtained. The results indicated that the described methodology is extremely well suited for DNA sequencing projects in which considerable sequence information is on demand. In addition, massive DNA sequencing projects, including those of full genomes, are expected to benefit substantially from the Mu strategy.

[The sequence data reported in this paper have been submitted to the GenBank data library under accession no. AJ011033.]
Panning for Genes—A Visual Strategy for Identifying Novel Gene Orthologs and Paralogs
Authors: Retief Jacques D. , Lynch Kevin R. , Pearson William R.
Published: November 2021

We have developed a rapid visual method for identifying novel members of gene families. Starting with an evolutionary tree, 20–50 protein query sequences for a gene family are selected from different branches of the tree. These query sequences are used to search the GenBank and expressed sequence tag (EST) DNA databases and their nightly updates using the tfastx3 or tfasty3 programs. The results of all 20–50 searches are collated and resorted to highlight EST or genomic sequences that share significant similarity with the query sequences. The statistical significance of each DNA/protein alignment is plotted, highlighting the portion of the query sequence that is present in the database sequence and the percent identity in the aligned region. The collated results for database sequences are linked using the WWW to the underlying scores and alignments; these links can also be used to perform additional searches to characterize the novel sequence further. With traditional “deep” scoring matrices (BLOSUM50) one can search for previously unrecognized families of large protein superfamilies. Alternatively, by using query sequences and EST libraries from the same species (e.g., human or mouse) together with “shallow” scoring matrices and filters that remove high-identity sequences, one can highlight new paralogs of previously described subfamilies. Using query sequences from the glutathione transferase superfamily, we identified two novel mammalian glutathione transferase families that were recognized previously only in plants. Using query sequences from known mammalian glutathione transferase subfamilies, we identified new candidate paralogs from the mouse class-mu, class-pi, and class-theta families.
Genome-Scale Cloning and Expression of Individual Open Reading Frames Using Topoisomerase I-Mediated Ligation
Authors: Heyman John A. , Cornthwaite Jeremiah , Foncerrada Luis , Gilmore Jeremiah R. , Gontang Erin , Hartman Kristen J. , Hernandez Cathy L. , Hood Rhiannon , Hull Heather M. , Lee Wai-Yee , Marcil Robert , Marsh Ed J. , Mudd Kevin M. , Patino Mario J. , Purcell Thomas J. , Rowland Jon J. , Sindici Michelle L. , Hoeffler James P.
Published: November 2021

The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends. We have used the properties of Vaccinia DNA topoisomerase I to develop a ligase-free technology for the covalent joining of DNA fragments to suitable plasmid vectors. This system is much more efficient than cloning methods that require ligase because the rapid DNA rejoining activity of Vaccinia topoisomerase I allows ligation in only 5 min at room temperature, whereas the enzyme’s high substrate specificity ensures a low rate of vector-alone transformants. We have used this topoisomerase I-mediated cloning technology to develop a process for accelerated cloning and expression of individual ORFs. Its suitability for genome-scale molecular cloning and expression is demonstrated in this report.