Region In Human Cells Research Articles

Advances in RNA therapeutics for modulation of 'undruggable' targets.

Over the past decades, drug discovery utilizing small pharmacological compounds, fragment-based therapeutics, and antibody therapy have significantly advanced treatment options for many human diseases. However, a major bottleneck has been that>70% of human proteins/genomic regions are 'undruggable' by the above-mentioned approaches. Many of these proteins constitute essential drug targets against complex multifactorial diseases like cancer, immunological disorders, and neurological diseases. Therefore, alternative approaches are required to target these proteins or genomic regions in human cells. RNA therapeutics is a promising approach for many of the traditionally 'undruggable' targets by utilizing methods such as antisense oligonucleotides, RNA interference, CRISPR/Cas-based genome editing, aptamers, and the development of mRNA therapeutics. In the following chapter, we will put emphasis on recent advancements utilizing these approaches against challenging drug targets, such as intranuclear proteins, intrinsically disordered proteins, untranslated genomic regions, and targets expressed in inaccessible tissues.

Condensed but liquid-like domain organization of active chromatin regions in living human cells.

In eukaryotes, higher-order chromatin organization is spatiotemporally regulated as domains, for various cellular functions. However, their physical nature in living cells remains unclear (e.g., condensed domains or extended fiber loops; liquid-like or solid-like). Using novel approaches combining genomics, single-nucleosome imaging, and computational modeling, we investigated the physical organization and behavior of early DNA replicated regions in human cells, which correspond to Hi-C contact domains with active chromatin marks. Motion correlation analysis of two neighbor nucleosomes shows that nucleosomes form physically condensed domains with ~150-nm diameters, even in active chromatin regions. The mean-square displacement analysis between two neighbor nucleosomes demonstrates that nucleosomes behave like a liquid in the condensed domain on the ~150 nm/~0.5 s spatiotemporal scale, which facilitates chromatin accessibility. Beyond the micrometers/minutes scale, chromatin seems solid-like, which may contribute to maintaining genome integrity. Our study reveals the viscoelastic principle of the chromatin polymer; chromatin is locally dynamic and reactive but globally stable.

The conserved DNMT1-dependent methylation regions in human cells are vulnerable to neurotoxicant rotenone exposure

BackgroundAllele-specific DNA methylation (ASM) describes genomic loci that maintain CpG methylation at only one inherited allele rather than having coordinated methylation across both alleles. The most prominent of these regions are germline ASMs (gASMs) that control the expression of imprinted genes in a parent of origin-dependent manner and are associated with disease. However, our recent report reveals numerous ASMs at non-imprinted genes. These non-germline ASMs are dependent on DNA methyltransferase 1 (DNMT1) and strikingly show the feature of random, switchable monoallelic methylation patterns in the mouse genome. The significance of these ASMs to human health has not been explored. Due to their shared allelicity with gASMs, herein, we propose that non-traditional ASMs are sensitive to exposures in association with human disease.ResultsWe first explore their conservancy in the human genome. Our data show that our putative non-germline ASMs were in conserved regions of the human genome and located adjacent to genes vital for neuronal development and maturation. We next tested the hypothesized vulnerability of these regions by exposing human embryonic kidney cell HEK293 with the neurotoxicant rotenone for 24 h. Indeed,14 genes adjacent to our identified regions were differentially expressed from RNA-sequencing. We analyzed the base-resolution methylation patterns of the predicted non-germline ASMs at two neurological genes, HCN2 and NEFM, with potential to increase the risk of neurodegeneration. Both regions were significantly hypomethylated in response to rotenone.ConclusionsOur data indicate that non-germline ASMs seem conserved between mouse and human genomes, overlap important regulatory factor binding motifs, and regulate the expression of genes vital to neuronal function. These results support the notion that ASMs are sensitive to environmental factors such as rotenone and may alter the risk of neurological disease later in life by disrupting neuronal development.

Aurora B kinase is recruited to multiple discrete kinetochore and centromere regions in human cells.

Aurora B kinase has a critical role in regulating attachments between kinetochores and spindle microtubules during mitosis. Early in mitosis, kinase activity at kinetochores is high to promote attachment turnover, and in later mitosis, activity decreases to ensure attachment stabilization. Aurora B localizes prominently to inner centromeres, and a population of the kinase is also detected at kinetochores. How Aurora B is recruited to and evicted from these regions to regulate kinetochore-microtubule attachments remains unclear. Here, we identified and investigated discrete populations of Aurora B at the centromere/kinetochore region. An inner centromere pool is recruited by Haspin phosphorylation of histone H3, and a kinetochore-proximal outer centromere pool is recruited by Bub1 phosphorylation of histone H2A. Finally, a third pool resides ~20 nm outside of the inner kinetochore protein CENP-C in early mitosis and does not require either the Bub1/pH2A/Sgo1 or Haspin/pH3 pathway for localization or activity. Our results suggest that distinct molecular pathways are responsible for Aurora B recruitment to centromeres and kinetochores.

Origin Recognition Complex (ORC) Evolution Is Influenced by Global Gene Duplication/Loss Patterns in Eukaryotic Genomes.

The conservation of orthologs of most subunits of the origin recognition complex (ORC) has served to propose that the whole complex is common to all eukaryotes. However, various uncertainties have arisen concerning ORC subunit composition in a variety of lineages. Also, it is unclear whether the ancestral diversification of ORC in eukaryotes was accompanied by the neofunctionalization of some subunits, for example, role of ORC1 in centriole homeostasis. We have addressed these questions by reconstructing the distribution and evolutionary history of ORC1-5/CDC6 in a taxon-rich eukaryotic data set. First, we identified ORC subunits previously undetected in divergent lineages, which allowed us to propose a series of parsimonious scenarios for the origin of this multiprotein complex. Contrary to previous expectations, we found a global tendency in eukaryotes to increase or decrease the number of subunits as a consequence of genome duplications or streamlining, respectively. Interestingly, parasites show significantly lower number of subunits than free-living eukaryotes, especially those with the lowest genome size and gene content metrics. We also investigated the evolutionary origin of the ORC1 role in centriole homeostasis mediated by the PACT region in human cells. In particular, we tested the consequences of reducing ORC1 levels in the centriole-containing green alga Chlamydomonas reinhardtii. We found that the proportion of centrioles to flagella and nuclei was not dramatically affected. This, together with the PACT region not being significantly more conserved in centriole-bearing eukaryotes, supports the notion that this neofunctionalization of ORC1 would be a recent acquisition rather than an ancestral eukaryotic feature.

X chromosome dosage and presence of SRY shape sex-specific differences in DNA methylation at an autosomal region in human cells

BackgroundSexual dimorphism in DNA methylation levels is a recurrent epigenetic feature in different human cell types and has been implicated in predisposition to disease, such as psychiatric and autoimmune disorders. To elucidate the genetic origins of sex-specific DNA methylation, we examined DNA methylation levels in fibroblast cell lines and blood cells from individuals with different combinations of sex chromosome complements and sex phenotypes focusing on a single autosomal region––the differentially methylated region (DMR) in the promoter of the zona pellucida binding protein 2 (ZPBP2) as a reporter.ResultsOur data show that the presence of the sex determining region Y (SRY) was associated with lower methylation levels, whereas higher X chromosome dosage in the absence of SRY led to an increase in DNA methylation levels at the ZPBP2 DMR. We mapped the X-linked modifier of DNA methylation to the long arm of chromosome X (Xq13-q21) and tested the impact of mutations in the ATRX and RLIM genes, located in this region, on methylation levels. Neither ATRX nor RLIM mutations influenced ZPBP2 methylation in female carriers.ConclusionsWe conclude that sex-specific methylation differences at the autosomal locus result from interaction between a Y-linked factor SRY and at least one X-linked factor that acts in a dose-dependent manner.

DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells

Sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures in the human genome have been implicated in stimulating genomic instability. Previously, we found that a naturally occurring intra-molecular triplex (H-DNA) caused genetic instability in mammals largely in the form of DNA double-strand breaks. Thus, it is of interest to determine the mechanism(s) involved in processing H-DNA. Recently, we demonstrated that human DHX9 helicase preferentially unwinds inter-molecular triplex DNA in vitro. Herein, we used a mutation-reporter system containing H-DNA to examine the relevance of DHX9 activity on naturally occurring H-DNA structures in human cells. We found that H-DNA significantly increased mutagenesis in small-interfering siRNA-treated, DHX9-depleted cells, affecting mostly deletions. Moreover, DHX9 associated with H-DNA in the context of supercoiled plasmids. To further investigate the role of DHX9 in the recognition/processing of H-DNA, we performed binding assays in vitro and chromatin immunoprecipitation assays in U2OS cells. DHX9 recognized H-DNA, as evidenced by its binding to the H-DNA structure and enrichment at the H-DNA region compared with a control region in human cells. These composite data implicate DHX9 in processing H-DNA structures in vivo and support its role in the overall maintenance of genomic stability at sites of alternatively structured DNA.

Assessment of the long-term transcriptional activity of a 550-bp-long human β-actin promoter region

β-actin (ACTB) is one of the genes expressed most abundantly and ubiquitously in human non-muscular tissues. Here, we investigated the long-term activity of a 550-bp-long human ACTB promoter region in human cells in comparison with other commonly used constitutive promoters. We first constructed plasmid vectors expressing enhanced green fluorescent protein (GFP) driven by one of the 5 promoters, human ACTB, human elongation factor-1α (EF1α), cytomegalovirus early enhancer/chicken β-actin (CAG), cytomegalovirus (CMV), and herpes simplex virus thymidine kinase, and transfected them into multiple human somatic cell lines. Stable transfectants were maintained for 45days, and GFP signals from the cells were quantified by fluorescence flow cytometry. GFP signals driven by the human ACTB and the CMV promoters were also compared over time for up to 60days following transfection. We observed robust, prolonged transcriptional activity with the human ACTB promoter that is comparable to the human EF1α and the CAG promoters and significantly more stable than the CMV promoter.

Human telomeres replicate using chromosome-specific, rather than universal, replication programs

Telomeric and adjacent subtelomeric heterochromatin pose significant challenges to the DNA replication machinery. Little is known about how replication progresses through these regions in human cells. Using single molecule analysis of replicated DNA (SMARD), we delineate the replication programs-i.e., origin distribution, termination site location, and fork rate and direction-of specific telomeres/subtelomeres of individual human chromosomes in two embryonic stem (ES) cell lines and two primary somatic cell types. We observe that replication can initiate within human telomere repeats but was most frequently accomplished by replisomes originating in the subtelomere. No major delay or pausing in fork progression was detected that might lead to telomere/subtelomere fragility. In addition, telomeres from different chromosomes from the same cell type displayed chromosome-specific replication programs rather than a universal program. Importantly, although there was some variation in the replication program of the same telomere in different cell types, the basic features of the program of a specific chromosome end appear to be conserved.

RNA Synthesis by the Brome Mosaic Virus RNA-Dependent RNA Polymerase in Human Cells Reveals Requirements for De Novo Initiation and Protein-Protein Interaction

Brome mosaic virus (BMV) is a model positive-strand RNA virus whose replication has been studied in a number of surrogate hosts. In transiently transfected human cells, the BMV polymerase 2a activated signaling by the innate immune receptor RIG-I, which recognizes de novo-initiated non-self-RNAs. Active-site mutations in 2a abolished RIG-I activation, and coexpression of the BMV 1a protein stimulated 2a activity. Mutations previously shown to abolish 1a and 2a interaction prevented the 1a-dependent enhancement of 2a activity. New insights into 1a-2a interaction include the findings that helicase active site of 1a is required to enhance 2a polymerase activity and that negatively charged amino acid residues between positions 110 and 120 of 2a contribute to interaction with the 1a helicase-like domain but not to the intrinsic polymerase activity. Confocal fluorescence microscopy revealed that the BMV 1a and 2a colocalized to perinuclear region in human cells. However, no perinuclear spherule-like structures were detected in human cells by immunoelectron microscopy. Sequencing of the RNAs coimmunoprecipitated with RIG-I revealed that the 2a-synthesized short RNAs are derived from the message used to translate 2a. That is, 2a exhibits a strong cis preference for BMV RNA2. Strikingly, the 2a RNA products had initiation sequences (5'-GUAAA-3') identical to those from the 5' sequence of the BMV genomic RNA2 and RNA3. These results show that the BMV 2a polymerase does not require other BMV proteins to initiate RNA synthesis but that the 1a helicase domain, and likely helicase activity, can affect RNA synthesis by 2a.

SProtP: A Web Server to Recognize Those Short-Lived Proteins Based on Sequence-Derived Features in Human Cells

Protein turnover metabolism plays important roles in cell cycle progression, signal transduction, and differentiation. Those proteins with short half-lives are involved in various regulatory processes. To better understand the regulation of cell process, it is important to study the key sequence-derived factors affecting short-lived protein degradation. Until now, most of protein half-lives are still unknown due to the difficulties of traditional experimental methods in measuring protein half-lives in human cells. To investigate the molecular determinants that affect short-lived proteins, a computational method was proposed in this work to recognize short-lived proteins based on sequence-derived features in human cells. In this study, we have systematically analyzed many features that perhaps correlated with short-lived protein degradation. It is found that a large fraction of proteins with signal peptides and transmembrane regions in human cells are of short half-lives. We have constructed an SVM-based classifier to recognize short-lived proteins, due to the fact that short-lived proteins play pivotal roles in the control of various cellular processes. By employing the SVM model on human dataset, we achieved 80.8% average sensitivity and 79.8% average specificity, respectively, on ten testing dataset (TE1-TE10). We also obtained 89.9%, 99% and 83.9% of average accuracy on an independent validation datasets iTE1, iTE2 and iTE3 respectively. The approach proposed in this paper provides a valuable alternative for recognizing the short-lived proteins in human cells, and is more accurate than the traditional N-end rule. Furthermore, the web server SProtP (http://reprod.njmu.edu.cn/sprotp) has been developed and is freely available for users.

Episomal High Copy Number Maintenance of Hairpin-capped DNA Bearing a Replication Initiation Region in Human Cells

We previously found that a plasmid bearing a replication initiation region efficiently initiates gene amplification in mammalian cells and that it generates extrachromosomal double minutes and/or chromosomal homogeneously staining regions. During analysis of the underlying mechanism, we serendipitously found that hairpin-capped linear DNA was stably maintained as numerous extrachromosomal tiny episomes for more than a few months in a human cancer cell line. Generation of such episomes depended on the presence of the replication initiation region in the original plasmid. Despite extrachromosomal maintenance, episomal gene expression was epigenetically suppressed. The Southern blot analysis of the DNA of cloned cells revealed that the region around the hairpin end was diversified between the clones. Furthermore, the bisulfite-modified PCR and the sequencing analyses revealed that the palindrome sequence that derived from the original hairpin end or its end-resected structure were well preserved during clonal long term growth. From these data, we propose a model that explains the formation and maintenance of these episomes, in which replication of the hairpin-capped DNA and cruciform formation and its resolution play central roles. Our findings may be relevant for the dissection of mammalian replicator sequences.

The long and short of RNAs

The known world of RNA is expanding faster than that of any other cellular building block. The latest additions are types of long and short non-coding RNAs formed by bidirectional transcription and unusual processing. The transcriptomes of eukaryotic cells are unexpectedly complex, with virtually the entire non-repeat portions of many genomes being transcribed. This paper from the Affymetrix/Cold Spring Harbor Laboratory ENCODE Transcriptome Project reports a deep sequencing study revealing that a remarkable breadth of RNA species is produced both from within annotated genes and from unannotated intergenic regions in human cells. Importantly, many of these small RNAs possess cap structures and appear to be processed from mature mRNAs resulting in populations of long and short RNAs with capped 5 ends that coincide. Transfecting synthetic PASRs (promoter-associated short RNAs) corresponding to the c-MYC transcriptional start site reduced MYC mRNA abundance, demonstrating a biological impact for at least one class of these small RNAs.

Human immunodeficiency virus type 1 incorporated with fusion proteins consisting of integrase and the designed polydactyl zinc finger protein E2C can bias integration of viral DNA into a predetermined chromosomal region in human cells.

In vitro studies using fusion proteins consisting of human immunodeficiency virus type 1 integrase (IN) and a synthetic polydactyl zinc finger protein E2C, a sequence-specific DNA-binding protein, showed that integration of retroviral DNA can be biased towards a contiguous 18-bp E2C-recognition site. To determine whether the fusion protein strategy can achieve site-specific integration in vivo, viruses were prepared by cotransfection and various IN-E2C fusion proteins were packaged in trans into virions. The resulting viruses incorporated with the IN-E2C fusion proteins were functional and capable of performing integration at a level ranging from 1 to 24% of that of viruses containing wild-type (WT) IN. Two of the more infectious viruses, which contained E2C fused to either the N (E2C/IN) or to the C (IN/E2C) terminus of IN, were tested for their ability to direct integration into a unique E2C-binding site present within the 5' untranslated region of erbB-2 gene on human chromosome 17. The copy number of proviral DNA was measured using a quantitative real-time nested-PCR assay, and the specificity of directed integration was determined by comparing the number of proviruses within the vicinity of the E2C-binding site to that in the whole genome. Viruses containing IN/E2C fusion proteins had sevenfold higher preference for integrating near the E2C-binding site than those viruses containing WT IN, whereas viruses containing E2C/IN had 10-fold higher preference. The results indicated that the IN-E2C fusion protein strategy is capable of directing integration of retroviral DNA into a predetermined chromosomal region in the human genome.

Sodium-dependent myo-inositol transporter 1 is a cellular receptor for Mus cervicolor M813 murine leukemia virus.

Retrovirus infection is initiated by binding of the surface (SU) portion of the viral envelope glycoprotein (Env) to specific receptors on cells. This binding triggers conformational changes in the transmembrane portion of Env, leading to membrane fusion and cell entry, and is thus a major determinant of retrovirus tissue and species tropism. The M813 murine leukemia virus (MuLV) is a highly fusogenic gammaretrovirus, isolated from Mus cervicolor, whose host range is limited to mouse cells. To delineate the molecular mechanisms of its restricted host range and its high fusogenic potential, we initiated studies to characterize the cell surface protein that mediates M813 infection. Screening of the T31 mouse-hamster radiation hybrid panel for M813 infectivity localized the receptor gene to the distal end of mouse chromosome 16. Expression of one of the likely candidate genes (slc5a3) within this region in human cells conferred susceptibility to both M813 infection and M813-induced fusogenicity. slc5a3 encodes sodium myo-inositol transporter 1 (SMIT1), thus adding another sodium-dependent transporter to the growing list of proteins used by MuLVs for cell entry. Characterization of SMIT1 orthologues in different species identified several amino acid variations within two extracellular loops that may restrict susceptibility to M813 infection.

Characterization of transcriptional activity of taurine transporter using luciferase reporter constructs.

Although taurine transporter (TAUT) activity has been known to be regulated by diverse intracellular and extracellular factors involved in the signal transduction pathway, such as protein kinase C, intracellular Ca concentration, and glucocorticoids, little is known concerning the underlying mechanisms. Evidence suggests that such stimulation-mediated changes in TAUT activity in mammalian cells are partly achieved through the modulation of TAUT transcription activity. In order to better understand the regulation of TAUT transcription activity and subsequently the role of taurine in the signal transduction pathway, we have cloned and sequenced the 5' flanking region of the human TAUT gene, and characterized the TAUT promoter region in human cells. For these reasons, the TAUT luciferase reporter vector was constructed using the 5' flanking region of the TAUT gene (1800 bp). The TAUT luciferase reporter vector was then transfected into SiHa cells, and luciferase activity was measured. The construct containing its own promoter of TAUT (pGL3 b TAU31) showed a 10 fold higher luciferase activity compared to the value found in the empty vector (pGL3 b). This implies a functional transcription of the homologous TAUT promoter. Similar results were obtained with the exon deleted construct [pGL3 b TAU31(-e)]. We also constructed the TAUT luciferase reporter gene (pGL3 pro TAU13) using a heterologous promoter. About 2.5 fold higher luciferase activity was observed in cells transfected with this construct containing the heterologous promoter compared to the value found in the control vector (PGL3 pro).

The 55 kDa regulatory subunit of protein phosphatase 2A plays a role in the activation of the HPV16 long control region in human cells with a deletion in the short arm of chromosome 11.

Previous results indicated that SV40 small t is essential for SV40-induced transformation of diploid cells but dispensable for the transformation of cells with a deletion on the short arm of chromosome 11 (del-11 cells). From these results we concluded that del-11 cells contain a cellular 'SV40 small t-like' factor, which is able to transactivate the HPV16 long control region (LCR) and to complement SV40 large T in transformation. Since SV40 small t and the regulatory 55 kDa subunit (PR55) of protein phosphatase 2A (PP2A), have been shown to inhibit the enzyme activity of PP2A, the PR55 beta subunit could be the putative 'small t-like' factor. In accordance with this hypothesis, we show that the PR55 beta subunit is highly expressed in del-11 but not in diploid cells and is able to trans-activate the HPV16 LCR in diploid cells. Moreover, inhibition of PP2A by okadaic acid resulted in trans-activation of the HPV16 LCR in diploid cells. Alignment of PR55 and SV40 small t showed a common four amino acid motif DKGG. We present evidence that the integrity of this motif is necessary for the PP2A-mediated ability of SV40 small t to trans-activate the HPV16 LCR.

Host range analysis of a chimeric simian virus 40 genome containing the BKV capsid genes

Simian virus 40 (SV40) propagates poorly in cells from human embryonic kidney (HEK) and human fetal fibroblasts (HFF) while BK virus grows well in many human cell types. It has been suggested that sequences within the SV40 late region but not within the BKV late region may act to inhibit growth of virus in HEK and HFF cells. In order to test this and to identify a late region host range function, we have replaced the late region of wtSV40 DNA with the late region of RFV (a variant of BKV) to produce an intermolecular hybrid or chimera. The constructed SV40/RFV chimeric genome contained approx. 5900 base pairs, more than 650 base pairs greater than wtSV40. Nevertheless, when introduced by transfection the chimera appeared to be infectious. Three chimeric genomes were recovered from infected cells and all contained deletions of nearly 600 base pairs, exclusively at the region of the 3' terminal junction. Since all three chimeras propagated in human HFF and HEK cells, the RFV late region and not the RFV regulatory region possesses a host range function required for growth in human cells. Analysis of T-antigen gene expression suggests that the replacement of the SV40 late region with the BKV late region leads to full expression of the SV40 early region in human cells. Two chimeras exhibited a BKV-like host range and the third exhibited both a BKV and an SV40-like host range. We determined precisely which sequences were deleted in each chimera and we exchanged 3' terminal junction fragments containing these deletions, between two chimeras with different host ranges. From these experiments we demonstrated that: (1) The 3' terminus of the SV40 large T-antigen gene is required for growth of SV40/RFV in TC-7 and CV-1 simian cells but not for growth in human cells; (2) while the SV40 late region is refractory for growth in human cells, the RFV late region is not refractory for growth in simian cells; (3) the 3' terminus of the RFV T-antigen gene is not required for growth in human cells. The results of the 3' terminal junction exchanges and studies of early gene expression also demonstrate that BKV and SV40 can penetrate human and simian cells, even when they failed to grow in one cell type.

Expression of SV40 large T antigen, but not small t antigen, is required for the induction of chromosomal aberrations in transformed human cells

Expression of the Simian virus 40 (SV40) early region in human cells results in the induction of chromosomal aberrations and polyploidy, and in transformation. To understand how genetic damage occurs and what role it plays in transformation, human diploid fibroblasts and embryonic kidney cells were transfected with plasmids encoding wild type or mutant forms of the viral early region, and the neo gene. Clones selected for G418 resistance and expressing viral genes were initially analyzed within 20 cell divisions. Our results demonstrate that expression of the SV40 large T antigen is sufficient for the induction of chromosomal damage and ploidy changes, and that small t does not contribute to these processes. Mutant plasmids lacking the SV40 origin of DNA replication were as proficient as wild type plasmids, indicating that viral DNA replication is not required for cytogenetic damage. We have also shown that chromosome aberrations, but not necessarily polyploidy, increase in frequency and complexity upon subculturing of the clones regardless of whether such populations arrest at crisis or yield immortal lines. Our results are compatible with the hypothesis that large T antigen destabilizes the cellular genome, and that specific mutations arising from this process may contribute to cell immortalization.

Sequential silver staining and hybridization in situ on nucleolus organizing regions in human cells.

Chromosome preparations from eight individuals were first stained with silver nitrate to reveal the nucleolus organizing regions (NORs) and then hybridized in situ with ribosomal RNA. In six individuals the size of the silver-staining regions was positively correlated with the amount of label present after hybridization in situ. Thus the variation in silver-staining intensity among chromosomes was largely explained by variation in the number of rDNA gene copies per NOR. However, in two individuals this correlation was absent, suggesting that other factors can also influence the size of the silver-staining region.