Cell-to-Cell Variation in Gene Expression for Cultured Human Cells Is Controlled in Trans by Diverse Genes: Implications for the Pathobiology of Aging.

Cell-to-cell variation in gene expression increases among homologous cells within multiple tissues during aging. We call this phenomenon variegated gene expression (VGE). Long, healthy life requires robust and coordinated gene expression. We posit that nature may have evolved VGE as a bet-hedging mechanism to protect reproductively active populations. The price we may pay is accelerated aging. That hypothesis will require the demonstration that genetic loci are capable of modulating degrees of VGE. While loci controlling VGE in yeast and genes controlling interindividual variation in gene expression in Caenorhabditis elegans have been identified, there has been no compelling evidence for the role of specific genetic loci in modulations of VGE of specific targets in humans. With the assistance of a core facility, we used a customized library of siRNA constructs to screen 1,195 human genes to identify loci contributing to the control of VGE of a gene with relevance to the biology of aging. We identified approximately 50 loci controlling VGE of the prolongevity gene, SIRT1. Because of its partial homology to FOXO3A, a variant of which is enriched in centenarians, our laboratory independently confirmed that the knockdown of FOXF2 greatly diminished VGE of SIRT1 but had little impact upon the VGE of WRN. While the role of these VGE-altering genes on aging in vivo remains to be determined, we hypothesize that some of these genes can be targeted to increase functionality during aging.

An integrated device for the rapid and sensitive detection of the influenza hemagglutinin.

Influenza is a viral respiratory tract infection responsible for up to 5 million cases of severe infection and nearly 600 000 deaths worldwide each year. While treatments for influenza exist, diagnostics for the virus at the point of care are limited in their sensitivity and ability to differentiate between subtypes. We have developed an integrated two-dimensional paper network (2DPN) for the detection of the influenza virus by the surface glycoprotein, hemagglutinin. The hemagglutinin assay was developed using proteins computationally designed to bind with high affinity to the highly-conserved sialic acid binding site. The integrated 2DPN uses a novel geometry that allows automated introduction of an enzymatic amplification reagent directly to the detection zone. This assay was integrated into a prototype device and demonstrated successful detection of clinically relevant virus concentrations spiked into 70 µL of virus-free pediatric nasal swab samples. Using this novel geometry, we found improved assay performance on the device (compared to a manually-operated dipstick method), with a sensitivity of 4.45 × 102 TCID50 per mL on device.

Rapid sample preparation with Lyse-It® for Listeria monocytogenes and Vibrio cholerae.

Sample preparation is a leading bottleneck in rapid detection of pathogenic bacteria. Here, we use Lyse-It® for bacterial cellular lysis, genomic DNA fragmentation, and protein release and degradation for both Listeria monocytogenes and Vibrio cholerae. The concept of Lyse-It® employs a conventional microwave and Lyse-It® slides for intensely focused microwave irradiation onto the sample. High microwave power and a <60 second irradiation time allow for rapid cellular lysis and subsequent intracellular component release. The pathogenic bacteria are identified by quantitative polymerase chain reaction (qPCR), which subsequently demonstrates the viability of DNA for amplification post microwave-induced lysis. Intracellular component release, degradation, and detection of L. monocytogenes and V. cholerae has been performed and shown in this paper. These results demonstrate a rapid, low-cost, and efficient way for bacterial sample preparation on both food and water-borne Gram-positive and -negative organisms alike.

Disposable Autonomous Device for Swab-to-Result Diagnosis of Influenza.

A prototype of a self-contained, automated, disposable device for chemically amplified protein-based detection of influenza virus from nasal swab specimens was developed and evaluated in a clinical setting. The device required only simple specimen manipulation without any dedicated instrumentation or specialized training by the operator for interpretation. The device was based on a sandwich immunoassay for influenza virus nucleoprotein; it used an enzyme-labeled antibody and a chromogenic substrate to provide an amplified visible signal, in a two-dimensional paper network format. All reagents were stored within the device. Device performance was assessed at Seattle Children's Hospital; clinical staff collected nasal swab samples from 25 patients and then operated test devices on site to detect influenza A and B in those specimens. The total test time from device initiation to result was approximately 35 min. Device performance for influenza A detection was ∼70% accurate using in-house qRT-PCR influenza A as a gold-standard comparison. The ratio of valid to total completed device runs yielded a success rate of 92%, and the negative predictive value for both the influenza A and B assay was 81%. The ability to diagnose respiratory infections rapidly and close to the patient was well received by hospital staff, inspiring further optimization of device function.

WRN Mutation Update: Mutation Spectrum, Patient Registries, and Translational Prospects.

Werner syndrome (WS) is a rare autosomal recessive disorder characterized by a constellation of adult onset phenotypes consistent with an acceleration of intrinsic biological aging. It is caused by pathogenic variants in the WRN gene, which encodes a multifunctional nuclear protein with exonuclease and helicase activities. WRN protein is thought to be involved in optimization of various aspects of DNA metabolism, including DNA repair, recombination, replication, and transcription. In this update, we summarize a total of 83 different WRN mutations, including eight previously unpublished mutations identified by the International Registry of Werner Syndrome (Seattle, WA) and the Japanese Werner Consortium (Chiba, Japan), as well as 75 mutations already reported in the literature. The Seattle International Registry recruits patients from all over the world to investigate genetic causes of a wide variety of progeroid syndromes in order to contribute to the knowledge of basic mechanisms of human aging. Given the unusually high prevalence of WS patients and heterozygous carriers in Japan, the major goal of the Japanese Consortium is to develop effective therapies and to establish management guidelines for WS patients in Japan and elsewhere. This review will also discuss potential translational approaches to this disorder, including those currently under investigation.

A rapid, instrument-free, sample-to-result nucleic acid amplification test.

The prototype demonstrated here is the first fully integrated sample-to-result diagnostic platform for performing nucleic acid amplification tests that requires no permanent instrument or manual sample processing. The multiplexable autonomous disposable nucleic acid amplification test (MAD NAAT) is based on two-dimensional paper networks, which enable sensitive chemical detection normally reserved for laboratories to be carried out anywhere by untrained users. All reagents are stored dry in the disposable test device and are rehydrated by stored buffer. The paper network is physically multiplexed to allow independent isothermal amplification of multiple targets; each amplification reaction is also chemically multiplexed with an internal amplification control. The total test time is less than one hour. The MAD NAAT prototype was used to characterize a set of human nasal swab specimens pre-screened for methicillin-resistant Staphylococcus aureus (MRSA) bacteria. With qPCR as the quantitative reference method, the lowest input copy number in the range where the MAD NAAT prototype consistently detected MRSA in these specimens was ∼5 × 10(3) genomic copies (∼600 genomic copies per biplexed amplification reaction).

Comparison of point-of-care-compatible lysis methods for bacteria and viruses.

Nucleic acid sample preparation has been an especially challenging barrier to point-of-care nucleic acid amplification tests in low-resource settings. Here we provide a head-to-head comparison of methods for lysis of, and nucleic acid release from, several pathogenic bacteria and viruses-methods that are adaptable to point-of-care usage in low-resource settings. Digestion with achromopeptidase, a mixture of proteases and peptidoglycan-specific hydrolases, followed by thermal deactivation in a boiling water bath, effectively released amplifiable nucleic acid from Staphylococcus aureus, Bordetella pertussis, respiratory syncytial virus, and influenza virus. Achromopeptidase was functional after dehydration and reconstitution, even after eleven months of dry storage without refrigeration. Mechanical lysis methods proved to be effective against a hard-to-lyse Mycobacterium species, and a miniature bead-mill, the AudioLyse, is shown to be capable of releasing amplifiable DNA and RNA from this species. We conclude that point-of-care-compatible sample preparation methods for nucleic acid tests need not introduce amplification inhibitors, and can provide amplification-ready lysates from a wide range of bacterial and viral pathogens.

CTCF regulates ataxin-7 expression through promotion of a convergently transcribed, antisense noncoding RNA.

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder caused by CAG/polyglutamine repeat expansions in the ataxin-7 gene. Ataxin-7 is a component of two different transcription coactivator complexes, and recent work indicates that disease protein normal function is altered in polyglutamine neurodegeneration. Given this, we studied how ataxin-7 gene expression is regulated. The ataxin-7 repeat and translation start site are flanked by binding sites for CTCF, a highly conserved multifunctional transcription regulator. When we analyzed this region, we discovered an adjacent alternative promoter and a convergently transcribed antisense noncoding RNA, SCAANT1. To understand how CTCF regulates ataxin-7 gene expression, we introduced ataxin-7 mini-genes into mice, and found that CTCF is required for SCAANT1 expression. Loss of SCAANT1 derepressed ataxin-7 sense transcription in a cis-dependent fashion and was accompanied by chromatin remodeling. Discovery of this pathway underscores the importance of altered epigenetic regulation for disease pathology at repeat loci exhibiting bidirectional transcription.

An antisense transcript spanning the CGG repeat region of FMR1 is upregulated in premutation carriers but silenced in full mutation individuals.

Expansion of the polymorphic CGG repeats within the 5'-UTR of the FMR1 gene is associated with variable transcriptional regulation of FMR1. Here we report a novel gene, ASFMR1, overlapping the CGG repeat region of FMR1 and transcribed in the antisense orientation. The ASFMR1 transcript is spliced, polyadenylated and exported to the cytoplasm. Similar to FMR1, ASFMR1 is upregulated in individuals with premutation alleles and is not expressed from full mutation alleles. Moreover, it exhibits premutation-specific alternative splicing. Taken together, these observations suggest that in addition to FMR1, ASFMR1 may contribute to the variable phenotypes associated with the CGG repeat expansion.

Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus.

Rac2 is a Rho family GTPase expressed specifically in hematopoietic cells. The 4.5 kb proximal Rac2 gene promoter exhibits strong but promiscuous activity following either transient or stable transfection into tissue culture cells, indicating that additional cis-elements are required to silence Rac2 expression in non-hematopoietic cells. A bacterial artificial chromosome (BAC) containing the human Rac2 gene, including as little as 1.6 kb of upstream and 8 kb of downstream sequence, exhibits hematopoietic-restricted expression in transgenic mice. The Rac2 genomic locus exhibits distinct patterns of DNA methylation in expressing versus non-expressing cells. Cells that lack Rac2 expression exhibit increased cytosine methylation in the sequences flanking the gene, whereas cells that express Rac2 exhibit increased cytosine methylation within the body of the Rac2 gene. Treatment of non-expressing cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine reduces cytosine methylation of the Rac2 gene locus and is sufficient to induce Rac2 gene expression. Conversely, treatment with the histone deacetylase (HDAC) inhibitor trichostatin A fails to induce Rac2 gene expression. These findings define a genomic fragment sufficient to direct hematopoietic-specific expression of Rac2, and reveal the importance of cytosine methylation in the repression of Rac2 expression in non-hematopoietic cells.

Identification of the human HEX1/hExo1 gene promoter and characterization of elements responsible for promoter activity.

HEX1/hExo1 is a Class III nuclease of the RAD2 family with 5' to 3' exonuclease and flap structure-specific endonuclease activities. HEX1/hExo1 is expressed at low levels in a wide variety of tissues, but at higher levels in fetal liver and adult bone marrow, suggesting HEX1/hExo1 is important for hematopoietic stem cell development. A putative HEX1/hExo1 promoter fragment extending from -6240 to +1600bp exhibits cell-type specific activity in transient transfection assays. This fragment directs high luciferase reporter gene expression in the hematopoietic cell line K562, chronic myelogenous leukemia cells, but low luciferase expression in the non-hematopoietic cell line HeLa, human cervical carcinoma cells. Deletion studies identified a fragment spanning -688 to +1600bp that exhibits full transcriptional activity while a slightly shorter fragment from -658 to +1600bp exhibits significantly decreased promoter activity. In vitro binding assays revealed DNA-binding activities that interact with -687 to -681bp and -665 to -658bp elements. Oligonucleotide competition and antibody disruption studies determined that the transcription factor CREB-1 recognizes the -687 to -681bp element, while transcription factors Sp1 and Sp3 recognize the -665 to -658bp element. Mutation of either the CREB-1 or Sp1/Sp3 binding sites dramatically reduces HEX1/hExo1 promoter activity and elimination of both elements abolishes promoter activity.

Cloning and characterization of the gene encoding the mouse homologue of CpG binding protein.

Human CpG binding protein (CGBP) is a ubiquitously-expressed transcriptional activator that binds specifically to unmethylated CpG motifs. Several protein domains have been identified within CGBP including two plant homeodomains (PHD), acidic and basic regions, a coiled-coil domain, as well as a CXXC DNA-binding domain. The global function of CGBP remains unclear, although failure to express CGBP results in embryonic lethality in mice. This study reports the identification and characterization of the murine CGBP gene locus. A 2509 bp murine CGBP cDNA was cloned and nucleotide sequence determined. Comparison of the mouse and human CGBP sequences revealed 86% identity at the nucleotide level and 96% identity at the amino acid level. Examination of the deduced translation product revealed that the PHD, CXXC, coiled-coil, and basic domains are identical between mouse and human, while the acidic region exhibits approximately 90% identity with its human counterpart. A single murine CGBP transcript of approximately 2.6 kb was detected in a wide variety of adult tissues as well as embryonic stem cells. Analysis of the mouse gene locus revealed a relatively small gene spanning approximately 5 kb and comprised of 15 exons. Examination of the human CGBP gene showed a similar size and structure with identical intronic splice sites. In contrast to the human CGBP gene, which is located 800 bp upstream of the MBD1 gene, analysis of the murine CGBP gene locus failed to detect the murine MBD1 gene within several kilobases of the CGBP coding region.