Incomplete Specificity Research Articles

Maintenance of specificity in sympatric host-specific fig/wasp pollination mutualisms.

BackgroundFig/wasp pollination mutualisms are extreme examples of species-specific plant-insect symbioses, but incomplete specificity occurs, with potentially important evolutionary consequences. Why pollinators enter alternative hosts, and the fates of pollinators and the figs they enter, are unknown.MethodsWe studied the pollinating fig wasp, Ceratosolen emarginatus, which concurrently interacts with its typical host Ficus auriculata and the locally sympatric alternative host F. hainanensis, recording frequencies of the wasp in figs of the alternative hosts. We measured ovipositor lengths of pollinators and style lengths in female and male figs in the two host species. Volatile organic compounds (VOCs) emitted by receptive figs of each species were identified using GC-MS. We tested the attraction of wasps to floral scents in choice experiments, and detected electrophysiologically active compounds by GC-EAD. We introduced C. emarginatus foundresses into figs of both species to reveal the consequences of entry into the alternative host.ResultsC. emarginatus entered a low proportion of figs of the alternative host, and produced offspring in a small proportion of them. Despite differences in the VOC profiles of the two fig species, they included shared semiochemicals. Although C. emarginatus females prefer receptive figs of F. auriculata, they are also attracted to those of F. hainanensis. C. emarginatus that entered male figs of F. hainanensis produced offspring, as their ovipositors were long enough to reach the bottom of the style; however, broods were larger and offspring smaller than in the typical host. Female figs of F. hainanensis failed to produce seeds when visited by C. emarginatus. These findings advance our current understanding of how these species-specific mutualisms usually remain stable and the conditions that allow their diversification.

Z-DNA as a Tool for Nuclease-Free DNA Methyltransferase Assay.

Methylcytosines in mammalian genomes are the main epigenetic molecular codes that switch off the repertoire of genes in cell-type and cell-stage dependent manners. DNA methyltransferases (DMT) are dedicated to managing the status of cytosine methylation. DNA methylation is not only critical in normal development, but it is also implicated in cancers, degeneration, and senescence. Thus, the chemicals to control DMT have been suggested as anticancer drugs by reprogramming the gene expression profile in malignant cells. Here, we report a new optical technique to characterize the activity of DMT and the effect of inhibitors, utilizing the methylation-sensitive B-Z transition of DNA without bisulfite conversion, methylation-sensing proteins, and polymerase chain reaction amplification. With the high sensitivity of single-molecule FRET, this method detects the event of DNA methylation in a single DNA molecule and circumvents the need for amplification steps, permitting direct interpretation. This method also responds to hemi-methylated DNA. Dispensing with methylation-sensitive nucleases, this method preserves the molecular integrity and methylation state of target molecules. Sparing methylation-sensing nucleases and antibodies helps to avoid errors introduced by the antibody’s incomplete specificity or variable activity of nucleases. With this new method, we demonstrated the inhibitory effect of several natural bio-active compounds on DMT. All taken together, our method offers quantitative assays for DMT and DMT-related anticancer drugs.

Electrodiagnosis of Lumbar Spine Disorders and Cauda Equina Syndrome

Magnetic resonance imaging (MRI) is widely employed in the diagnosis of lumbar spine disorders, such as lumbar spinal stenosis or lumbar disc herniation. However, incomplete specificity is a definite drawback, given that MRI abnormalities are frequently identified in aged control subjects. Relying solely on MRI without considering clinical symptoms/signs or electrodiagnosis may lead to misdiagnosis. In contrast, electrodiagnostic tests are generally considered to have higher specificity than MRI, making them more useful for preventing unnecessary surgeries. Needle EMG can clarify the distribution of the involvement through fibrillation potentials and positive sharp waves, and can complement manual muscle testing. Denervation at the tensor fascia latae muscle can confirm an L5 lesion. Regarding nerve conduction studies (NCS), sensory nerve action potentials are usually normal in lumbar disorders. Somatosensory evoked potentials are useful for localizing the lesion. Amyotrophic lateral sclerosis (ALS) is an important differential diagnosis because spine surgery will promote the disease progression of ALS. Documenting upper extremity and thoracic paraspinal involvements are key to diagnose ALS, in combination with profuse fasciculation potentials in EMG. NCS plays a key role in diagnosing entrapment and demyelinating neuropathies. Electrodiagnostic tests are also useful for confirming functional neurological disorders.

Quantitative evaluation of candidate genes and development of a multiplex RT-PCR assay for the forensic identification of vaginal fluid

The identification of vaginal fluid from a mixed sample containing semen provides important probative evidence of vaginal intercourse in sexual assaults. In studies on the mRNA-based identification of body fluids, vaginal characteristic genes, such as MUC4 and HBD1, have been used to identify vaginal fluid; however, these are insufficient to discriminate vaginal fluid from other body fluids because of incomplete specificity and detectability. The aim of this study was to develop a more specific procedure for identifying vaginal fluid. First, previously reported and newly selected candidate genes were evaluated quantitatively using real-time reverse transcription-polymerase chain reaction (RT-PCR). Then, we developed a multiplex RT-PCR assay to detect probable candidates simultaneously. Each amplicon was detected and quantified by chip electrophoresis. Furthermore, we examined the specificity and robustness of the developed multiplex RT-PCR assay using various body fluids and aged vaginal fluid stains. As a result of this real-time RT-PCR assay, we selected five candidate genes—MUC4, CYP2B7P1, KLK13, ESR1, and SERPINB13—on the basis of their specificity and sensitivity. Then, the simultaneous amplification of these genes was performed successfully, and each fragment could be separated and quantified automatically by chip electrophoresis. The specificity and detectability of the multiplex detection of vaginal characteristic genes were almost comparable to those of real-time RT-PCR and it could be applied to vaginal fluid samples stored at room temperature for 1.5 years. Although discrimination criteria should be set, the multiplex RT-PCR assay developed in this study could be an effective tool for the identification of vaginal fluid.

Concept of uncertainty in relation to the foresight research

Abstract Uncertainty is one of the most important features of many areas of social and economic life, especially in the forward-looking context. On the one hand, the degree of uncertainty is associated with the objective essence of randomness of the phenomenon, and on the other, with the subjective perspective of a man. Future-oriented perception of human activities is laden with an incomplete specificity of the analysed phenomena, their volatility, and lack of continuity. A man is unable to determine, with complete certainty, the further course of these phenomena. According to the author of this article, in order to significantly reduce the uncertainty while making strategic decisions in a complex environment, we should focus our actions on the future through systemic research of foresight. This article attempts to answer the following research questions: 1) What is the relationship between foresight studies in the system perspective to studies of the uncertainty? 2) What classes of foresight methods enable the research of uncertainty in the process of system inquiry of the future? This study conducted deductive reasoning based on the results of the analysis methods and criticism of literature.

Evaluation of single reactor bovine tuberculosis breakdowns based on analysis of reactors slaughtered at an Irish export meat plant

The ‘Singleton Protocol’ was adopted by the Irish Department of Agriculture Fisheries and Food (DAFF) in 1996 to address the incomplete specificity of the single intra-dermal comparative tuberculin test (SICTT)...

Spatial distributions of cone inputs to cells of the parvocellular pathway investigated with cone-isolating gratings.

Receptive fields of midget ganglion cells and parvocellular lateral geniculate nucleus (LGN) neurons show color-opponent responses because they receive antagonistic input from the middle- and long-wavelength sensitive cones. It has been controversial as to whether this opponency can derive from random connectivity; if receptive field centers of cells near the fovea are cone-specific due to midget morphology, this would confer some degree of color opponency even with random cone input to the surround. A simple test of this mixed surround hypothesis is to compare spatial frequency tuning curves for luminance gratings and gratings isolating cone input to the receptive field center. If tuning curves for luminance gratings were bandpass, then with the mixed surround hypothesis tuning curves for gratings isolating the receptive field center cone class should also be bandpass, but to a lesser extent than for luminance. Tuning curves for luminance, chromatic, and cone-isolating gratings were measured in macaque retinal ganglion cells and LGN cells. We defined and measured a bandpass index to compare luminance and center cone-isolating tuning curves. Midget retinal ganglion cells and parvocellular LGN cells had bandpass indices between 0.1 and 1 with luminance gratings, but the index was usually near 1 (meaning low-pass tuning) when the receptive field center cone class alone was modulated. This is strong evidence for a considerable degree of cone-specific input to the surround. A fraction of midget and parvocellular cells showed evidence of incomplete specificity. Fitting the data with receptive field models revealed considerable intercell variability, with indications in some cells of a more complex receptive structure than a simple difference of Gaussians model.

Evaluation of current cancer immunotherapy: hemato-oncology.

Hematologic malignancies were the first diseases in clinical oncology for which the potential of harnessing the immune system as targeted therapy was unequivocally demonstrated. Unfortunately, the use of this highly efficacious modality has been limited to only a subset of patients and diseases because of immune-mediated toxicities resulting from incomplete specificity, and disease-specific determinants of sensitivity versus resistance to immune effector mechanisms. Recent studies, however, have begun to elucidate the molecular basis of the observed clinical effects allowing the rational development of next generation of immunotherapeutic combinations. We discuss here cancer antigen targets in hematologic malignancies and the specific approaches to induce immunity being pursued, the importance of modulating the host immunoregulatory environment, and the special features of immunological monitoring in clinical investigation. The hematologic malignancies represent an ideal setting for the development of immunotherapy due to logistical, clinical monitoring, and disease biology factors and may represent an exemplar for immune-based treatment in other cancer types.

High sensitivity assay using serum sample for IL28B genotyping to predict treatment response in chronic hepatitis C patients

Recent human genome-wide association studies (GWAS) revealed a strong association between IL28B gene variation and the pegylated interferon-α with ribavirin (PEG-IFN-α/RBV) treatment response in chronic hepatitis C patients. Two single nucleotide polymorphisms (SNP), rs8103142 and rs11881222 located in the IL28B gene, were found in significant association with the viral clearance. The present study employed these SNPs to develop a new accessible screening method allowing identification of potential non-responders before starting the therapy. Primer sets were designed to amplify rs8103142 and rs11881222 fragments from genomic DNA extracted from serum samples. This method was validated using microarray typing (GWAS) and applied for genotyping of 68 hepatitis C virus-infected patients with PEG-IFN-α/RBV treatment at baseline. In comparison with GWAS, the screening method showed 100% and 95.6% accuracy in typing of rs8103142 and rs11881222, respectively, indicating incomplete specificity but 100% of sensitivity in both. Genotyping by both SNP showed that 53 (77.9%), 14 (20.6%) and one (1.5%) of the patients were of major homozygous, heterozygous and minor homozygous type, respectively. The majority (85%) of homozygous patients exhibited response to therapy in contrast to heterozygous patients (29%). Among all genotyped only one case was found with the minor homozygous genotype which had late virological response to therapy before relapsing. This study described a highly sensitive assay that can be useful in determining SNP genotypes as well as in predicting the response to IFN-based treatment.

Diagnostic Challenges in Celiac Disease and the Role of the Tissue Transglutaminase–Neo-Epitope

The diagnosis of celiac disease (CD) remains a clinical challenge based on the incomplete specificity and sensitivity rates of current non-invasive tests. Furthermore, histological assessments fail to identify all overt cases and, in particular, do not manifest pathognomonic alterations in silent cases. Accordingly, the majority of CD cases are diagnosed with great delay. Recent research into the pathogenesis of CD, allowed us to identify a neo-antigen that appears to be the most promising serological tool for the detection of anti-tissue transglutaminase as well as anti-gliadin antibodies.

KIT (CD117): A Review on Expression in Normal and Neoplastic Tissues, and Mutations and Their Clinicopathologic Correlation

CD117 (KIT) is a type III receptor tyrosine kinase operating in cell signal transduction in several cell types. Normally KIT is activated (phosphorylated) by binding of its ligand, the stem cell factor. This leads to a phosphorylation cascade ultimately activating various transcription factors in different cell types. Such activation regulates apoptosis, cell differentiation, proliferation, chemotaxis, and cell adhesion. KIT-dependent cell types include mast cells, some hematopoietic stem cells, germ cells, melanocytes, and Cajal cells of the gastrointestinal tract, and neoplasms of these cells are examples of KIT-positive tumors. Other KIT-positive normal cells include epithelial cells in skin adnexa, breast, and subsets of cerebellar neurons. KIT positivity has been variably reported in sarcomas such as angiosarcoma, Ewing sarcoma, synovial sarcoma, leiomyosarcoma, and MFH; results of the last three are controversial. The variations in published data may result from incomplete specificity of some polyclonal antibodies, possibly contributed by too high dilutions. Also, KIT is expressed in pulmonary and other small cell carcinomas, adenoid cystic carcinoma, renal chromophobe carcinoma, thymic, and some ovarian and few breast carcinomas. A good KIT antibody reacts with known KIT positive cells, and smooth muscle cells and fibroblasts are negative. KIT deficiency due to hereditary nonsense/missense mutations leads to disruption of KIT-dependent functions such as erythropoiesis, skin pigmentation, fertility, and gastrointestinal motility. Conversely, pathologic activation of KIT through gain-of-function mutations leads to neoplasia of KIT-dependent and KIT-positive cell types at least in three different systems: mast cells/myeloid cells--mastocytosis/acute myeloid leukemia, germ cells--seminoma, and Cajal cells--gastrointestinal stromal tumors (GISTs). KIT tyrosine kinase inhibitors such as imatinib mesylate are the generally accepted treatment of metastatic GISTs, and their availability has prompted an active search for other treatment targets among KIT-positive tumors such as myeloid leukemias and small cell carcinoma of the lung, with variable and often nonconvincing results.

Psychogenic movement disorders

This review focuses on recent studies assessing clinical features and laboratory findings that may help diagnose psychogenic movement disorders, and the ongoing controversy about the relationship of these disorders with preceding peripheral injury. 'Organic' movement disorders may still be misdiagnosed as psychogenic. Probably more commonly, however, psychogenic movement disorders are underdiagnosed. Most features typically associated with recognized movement disorders, including geste antagoniste or treatment-induced dyskinesias, can be seen in psychogenic movement disorder, and abnormal movements that would not normally be considered psychogenic or produced by psychological factors, such as palatal tremor, may occur on a psychogenic basis. On the other hand, psychiatric features are sometimes seen in neurologically based movement disorders. The diagnostic criteria for psychogenic movement disorders provide a degree of diagnostic certainty based on a combination of clinical and psychiatric features. Laboratory investigations can help exclude specific diagnoses, such as Parkinson's disease with (123I)beta-CIT single photon emission computed tomography, and neurophysiological methods can demonstrate characteristic features of psychogenic movement disorders, such as entrainment or suppression of psychogenic tremor with contralateral hand movements. However, some tests reported to differentiate psychogenic from neurological movement disorders may have incomplete specificity; for example, psychogenic tremor may not always be associated with complete coherence of tremor frequency. An ongoing controversy surrounds movement disorders following peripheral injuries, but recent evidence suggests that such patients should always be screened for the presence of a psychogenic movement disorder. Psychogenic movement disorder continues to be a difficult diagnosis to make and is likely to be underrecognized. Clinical and laboratory features are emerging, however, that support this diagnosis. The controversy regarding posttraumatic movement disorders continues, but a diagnosis of a psychogenic movement disorder should be actively sought in such patients.

Intracellular delivery of drugs to macrophages.

Toxic side effects which often complicate successful therapy in a number of diseases possibly arise due to the fact that at therapeutically effective concentrations the non-target cells in the body are also exposed to the cytotoxic effects of the drugs. Minimization of such adverse reactions might be feasible through drug delivery modalities that would reduce the uptake of the drugs by non-target cells and selectively deliver the drug only to the target cells (and/or intracellular sites) at relatively low extracellular concentrations. The current generic approach to site-specific drug delivery consists of attaching the therapeutic agent to a carrier recognized only by the cells where the pharmacological action is desired. Two types of recognition elements on the surface of target cells are being exploited for this purpose, viz., (i) antigens capable of generating specific, non-cross reactive antibodies, and (ii) receptors on the cell surface capable of efficient transport of the ligands. In general, incomplete specificity for the target cells and poor internalization of antibody-drug conjugates still limit the usefulness of antibodies for site-specific drug delivery applications necessitating exploration of alternatives. The alternate possibility is to exploit the exquisite cell type specificity and high efficiency of endocytosis of macromolecules mediated by specific receptors present on the surface of target cells for delivering drugs. A large number of infectious, metabolic, and neoplastic diseases are associated with macrophages leading to morbidities and mortalities to millions of people worldwide, thus an appropriate design of a drug delivery system to macrophages will be of tremendous help.

Protein-protein interactions in pathogen recognition by plants.

Protein-protein interactions have emerged as key determinants of whether plant encounters with pathogens result in disease or successful plant defense. Genetic interactions between plant resistance genes and pathogen avirulence genes enable pathogen recognition by plants and activate plant defense. These gene-for-gene interactions in some cases have been shown to involve direct interactions of the products of the genes, and have indicated plant intracellular localization for certain avirulence proteins. Incomplete specificity of some of the interactions in laboratory assays suggests that additional proteins might be required to confer specificity in the plant. In many cases, resistance and avirulence protein interactions have not been demonstrable, and in some cases, other plant components that interact with avirulence proteins have been found. Investigation to date has relied heavily on biochemical and cytological methods including in vitro binding assays and immunoprecipitation, as well as genetic tools such as the yeast two-hybrid system. Observations so far, however, point to the likely requirement for multiple, interdependent protein associations in pathogen recognition, for which these techniques can be insufficient. This article reviews the protein-protein interactions that have been described in pathogen recognition by plants, and provides examples of how rapid future progress will hinge on the adoption of new and developing technologies.

Neutral glycolipids of Schistosoma mansoni as feasible antigens in the detection of schistosomiasis.

The neutral glycolipid fraction from mouse-propagated, Schistosoma mansoni adult worms has been investigated as to its chromatographic and antigenic properties, and whether it fulfills the serodiagnostic antigen requirements of sensitivity and specificity in the detection of schistosomiasis. Serological analyses were performed by thin-layer chromatography immunostaining and ELISA. In the acute-phase form of mouse schistosomiasis, the kinetics of development of neutral glycolipid-specific antibody levels was correlated with the intensity of the initial infection and the response was dominated by IgG, as represented by the subclass IgG1. With the experimental animal helminthiases screened, glycolipid antigenicity fulfilled the fundamental traits for a serodiagnostic reagent. In the chronic-phase form of human schistosomiasis mansoni, neutral glycolipid-specific antibody levels were not correlated with the intensity of infection, as estimated from the faecal content of parasite eggs, whilst the isotypic response was dominated by IgM and IgG, the latter represented primarily by IgG1 and secondarily by IgG3. With other human helminthiases, glycolipid antigenicity was incomplete, in that, the specificity was only partially fulfilled. The reason for this incomplete specificity has been clarified, in part, by the detection of cryptic schistosomiasis infections in the cohorts of African patient sera examined.

Identifying Determinants of Recombination Specificity: Construction and Characterization of Chimeric Bacteriophage Integrases

Bacteriophage integrases are members of a family of structurally related enzymes that promote recombination between DNA molecules that carry specific sites. Phages λ and HK022 encode closely related integrases that recognize different sets of sequences within the core regions of their respective attachments sites. To locate the amino acid residues that determine this difference in specificity, we isolated recombinant phages that produce chimeric integrases and measured the ability of these chimeras to promote recombination of λ and HK022 sites in vivo. A chimera that is of λ origin except for one HK022 residue at position 99 and 12 HK022 residues located between positons 279 and 329 had wild-type HK022 specificity and activity for both integrative and excisive recombination. Chimeras containing certain subsets of these 13 residues had incomplete specificity. The region around position 99 is not well-conserved in other members of the integrase family, but the 279-329 segment includes residues that are highly conserved and believed to be directly involved in catalysis. Many chimeras were inactive in recombining either HK022 or λ sites. Selection for mutants that restored activity to these chimeras revealed sets of residues that are likely to interact with each other.

Highly selective cleavage of furanosyl linkages in liquid hydrogen fluoride

Earlier experiments indicated that anhydrous liquid I-IF at Dry Ice-acetone temperatures was nearly specific for cleavage of arabinofuranosyl linkages in plant cell-walls’. Proof of this indication became necessary because of our need for a method to remove arabinofuranosyl residues from the plant cell-wall protein “ Extensin2 “, in order to render the protein susceptible to proteolysis by trypsin, without cleavage of any other sugar linkages associated with the protein. Early experiments showed incomplete specificity for furanose linkages. However, recently we have found that our norma procedures for deactivating HF, forming a relatively unreactive complex with ether, is highly exothermic3. We now report that, if precautions are taken to ensure that during the reaction the HF is maintained at temperatures below -70°C and that during quenching of the reaction equally low temperatures are maintained, only furanosyl linkages are cleaved. We have tested the lability of three different furanosides in several different compounds by comparing the ‘H and/or 13C NMR spectra of the compounds before and after such an HF treatment. Fructofiranosidtx-Sucrose and related oligomers are readily available examples containing fructofuranosyl linkages. These oligomers contain the cr-p-glucopyranosyl-(1 + 2)+?-D-fructofuranoside linkage, which we presumed would be labile at the very low temperature conditions being tested. The higher oligomers raffinose, stachyose, and melezitose contain additional less-labile linkages which could illustrate the specificity of HF at such a low temperature. Table I summarizes our results with four oligomers of this type. Fig. 1 shows the ‘H NMR spectrum of one of them, raffinose, before and after the HF treatment. In all cases

Chromosomal Aberrations in Non-Hodgkin’s Lymphoma: Biologic and Clinical Correlations

Molecular characterization of chromosomal aberrations in non-Hodgkin's lymphoma (NHL) has provided a basic understanding of mechanisms of oncogene deregulation. Cytogenetic aberrations have been correlated with histologic subtype, immunophenotype, and clinical features of NHL. Although the incomplete specificity of some of the observed associations may limit the routine use of these markers in a clinical setting, the utilization of cytogenetic analysis can contribute to the diagnosis and management of patients with NHL.

HEPATITIS C VIRUS INFECTION IN PATIENTS UNDERGOING ALLOGENEIC BONE MARROW TRANSPLANTATION

Antibody to the recently identified hepatitis C virus was investigated in sera of 128 patients treated with allogeneic bone marrow transplantation, to determine the prevalence of HCV infection and its role in post-transplant liver complications. The overall prevalence of anti-HCV positivity was 28.6% (38/128 patients). The presence of pretransplant anti-HCV positivity (in 10/35 tested patients) did not seem to predict a more severe liver disease. In fact 8/10 anti-HCV+ and 15/25 anti-HCV- patients had elevated transaminases at BMT, and posttransplant liver failure (due to VOD or subacute hepatitis), and post-BMT rises in transaminases occurred regardless of anti-HCV serology (P = 0.6 and 0.2, respectively). In patients tested for anti-HCV after BMT (n = 128), only two (one anti-HCV+ and one anti-HCV-) experienced VOD; the number of patients in whom liver failure contributed to death was comparable in anti-HCV-positive and anti-HCV- negative patients (P = 0.4). Among 17 patients with documented posttransplant seroconversion (from anti-HCV- to anti-HCV+) the appearance of anti-HCV was concomitant with hepatitis exacerbation in 9 (53%). Histologic changes demonstrated a more severe liver damage in anti-HCV+ patients: a chronic hepatitis was diagnosed in 9/11 anti-HCV+ versus 1/7 anti-HCV- cases. Based on these observations, we conclude that hepatitis C virus has a role in liver disease in such patients, although its evaluation by the anti-HCV test is still of limited accuracy, due to low sensitivity and incomplete specificity.

Detection of cationic and non-cationic markers in the rat glomerulus by electron probe analysis

Acidic glycans (glomerular polyanion substances) in the rat kidney were visualized ultrastructurally by three cationic markers: colloidal iron, ruthenium red, and polyethylenimine-phosphotungstic acid (PEI-PTA). Heavy metal atoms (Fe, Ru and W) were detected in ultrathin sections by energy-dispersive electron probe microanalysis (EPMA). Characteristic peaks of the locally bound elements were obtained in spectra derived from the dense structures seen by transmission electron microscopy (TEM)--i.e. the glycocalyx of podocytes and/or the polyanion sites in the lamina rara externa of the glomerular basement membrane. Weaker signals were emitted by some extraglomerular structures. This finding may reflect a low concentration of glycans in structures lacking apparent density by TEM, and/or incomplete specificity of the markers, partial dislocation of reactive substances or the presence of an endogenous element (Fe). Experimental argyrosis was elicited by the peroral administration of silver nitrate. Dense Ag precipitates were seen chiefly in the lamina densa and characteristic peaks of silver were displayed in this site by EPMA, and was best demonstrated in non-contrasted sections. A single i.v. injection of Ag proteinate failed to produce glomerular pigmentation. The only dense granular product in tubular cells yielded characteristic peaks of Fe (endogenous siderosomes) but EPMA excluded detectable amounts of silver.