text:
Studies of the Hepatitis C virus (HCV) replication cycle have been made possible with the development of subgenomic selectable RNAs that replicate autonomously in cultured cells. In these replicons the region encoding the HCV structural proteins was replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replication of these RNAs. Subsequent analyses revealed that, within selected cells, HCV RNAs had acquired adaptive mutations that increased the efficiency of colony formation by an unknown mechanism. Using a panel of replicons that differed in their degrees of cell culture adaptation, in this study we show that adaptive mutations enhance RNA replication. Transient-transfection assays that did not require selection of transfected cells demonstrated a clear correlation between the level of adaptation and RNA replication. The highest replication level was found with an adapted replicon carrying two amino acid substitutions located in NS3 and one in NS5A that acted synergistically. In contrast, the nonadapted RNA replicated only transiently and at a low level. The correlation between the efficiency of colony formation and RNA replication was corroborated with replicons in which the selectable marker gene was replaced by the gene encoding firefly luciferase. Upon transfection of naive Huh-7 cells, the levels of luciferase activity directly reflected the replication efficiencies of the various replicon RNAs. These results show that cell culture-adaptive mutations enhance HCV RNA replication.
The Hepatitis C virus(HCV) is one of the most common etiologic agents of chronic liver diseases (reviewed in reference 37). Acute infections are usually subclinical or associated with mild symptoms, but the virus persists in more than 70% of infected individuals. The long-term outcomes of these persistent infections are varied, and they can range from an apparently healthy carrier state to chronic active hepatitis, liver fibrosis, cirrhosis, and eventually hepatocellular carcinoma.
HCV was classified in the genus Hepacivirus of the familyFlaviviridae, to which the genera Flavivirus(with the prototype member Yellow fever virus) andPestivirus (with the representative member Bovine viral diarrhea virus) belong (33). These viruses have enveloped particles that harbor an RNA genome of positive polarity. The HCV genome has a length of about 9,600 nucleotides, and it carries a single long open reading frame (ORF) that is flanked at both termini by nontranslated regions (NTRs). Viral proteins are generated as a polyprotein precursor that is translated via the internal ribosome entry site (IRES) located in the 5′ NTR (45, 47). It permits the direct binding of the 40S ribosome subunit in the absence of additional translation factors (35). The polyprotein precursor is cleaved by viral and host cell enzymes into at least 10 different products (for recent reviews, see references 2 and39). The structural proteins that are located in the amino-terminal region of the polyprotein are the core protein and the envelope glycoproteins E1 and E2 (22). The nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B are separated from the structural proteins by the short hydrophobic polypeptide p7, which has an unknown function (27, 32). NS2 and the amino-terminal region of NS3 constitute the NS2-3 proteinase responsible for cleavage at the NS2/3 site (19, 21). Three enzymatic activities reside in NS3: a serine-type proteinase in the ∼180 amino-terminal residues and nucleoside triphosphatase- helicase activities that are located in the remainder of the protein (3, 20, 26, 41, 44). NS4A is an essential cofactor of the NS3 proteinase that forms a stable heterodimeric NS3-NS4A complex that mediates cleavages at the NS3/4A, NS4A/B, NS4B/5A, and NS5A/B sites (4, 14, 28, 42). The function of NS4B is not known. NS5A is a highly phosphorylated polypeptide that may be involved in resistance to the antiviral activity of alpha interferon (12, 13, 16, 17). Phosphorylation is mediated by an as yet unknown cellular kinase (23, 40, 43). A major phosphoacceptor site has been mapped for a genotype 1a isolate, but this site is not conserved with NS5A proteins of other HCV genotypes (38). Most HCV isolates contain two phosphoprotein variants with apparent molecular masses of 56 and 58 kDa corresponding to the basal and the hyperphosphorylated forms, respectively (25, 40, 43). It is not known whether NS5A has a direct role in RNA replication and whether phosphorylation is important for its function(s). NS5B is the RNA-dependent RNA polymerase (RdRp) (5, 31, 48).
The development of selectable subgenomic HCV replicons for the first time enabled the study of viral RNA replication in cell culture (30). These replicons are composed of the 5′ NTR, which directs translation of the gene encoding the neomycin phosphotransferase; the IRES of the Encephalomyocarditis virus (EMCV), which directs translation of the HCV NS3-NS5B region; and the 3′ NTR (Fig. 1). Upon transfection of cells of the human hepatoma cell line Huh-7 and selection with G418, cell lines that carried high levels of self-replicating HCV RNAs could be established. Subsequent analyses of the sequences of HCV RNAs isolated from selected cell lines revealed that these replicons harbored cell culture-adaptive mutations (6, 29). For instance, a single amino acid substitution in the NS5B RdRp increased the efficiency of colony formation (ECF) ∼500-fold compared with that of the unaltered replicon RNA (29). However, the mechanism by which this cell culture adaptation was achieved remained unknown. In this study we show that cell culture-adaptive mutations enhance RNA replication. Using a transient-transfection assay without subsequent selection of cells, we demonstrate a clear correlation between the level of cell culture adaptation and RNA replication. However, within selected cell lines that were obtained after transfection of replicons with different levels of adaptation, the differences in the replicon copy numbers per cell varied only slightly. These results suggest that the host cell plays an additional important role in determining the replication level.
Cell cultures.Cell monolayers of the human hepatoma cell line Huh-7 (34) were routinely grown in Dulbecco's modified minimal essential medium (DMEM; Life Technologies, Karlsruhe, Germany) supplemented with 2 mM l-glutamine, nonessential amino acids, 100 U of penicillin, 100 μg of streptomycin, and 10% fetal calf serum. G418 (Geneticin; Life Technologies) was added at a final concentration of 500 to 1,000 μg/ml to cell lines carrying HCV replicons.
Plasmid constructions.All nucleotide numbers refer to a complete HCV genome cloned by our group (EMBL database accession numberAJ238799 ). Construction of the vector pFK and the parental replicon construct pFK-I389neo/NS3-3′/wt (EMBL database accession number AJ242654 ) has been described recently (29, 30). To analyze adapted replicon variants from cell line 5-15-9-2-3 for functionality, SfiI-restricted DNA fragments (nucleotides [nt] 3622 to 8499) that were generated by long-distance reverse transcription PCR (RT-PCR) were transferred into the parental replicon pFK-I389neo/NS3-3′/wt. These constructs were designated 5.1 to 5.16 and 1 to 66. Of the four clones with the highest ECFs (5.1, 5.2, 5.16, and 19), the HCV sequence between the two SfiI sites was determined, and with clone 5.1, each mutation leading to an amino acid substitution was introduced individually into the parental replicon. Plasmids pFK2109 Asn → Asp and pFK2197 Ser → Pro were generated by transferring MluI-EcoRI (nt 6529 to 6699) and EcoRI-XhoI (nt 6699 to 7186) fragments, respectively, from pFK5.1 into pFK-I389neo/NS3-3′/wt. To obtain plasmid pFK1202 Glu → Gly/1280 Thr → Ile, which harbors both NS3 mutations, a PmeI-BstXI fragment (5′ end of the EMCV IRES up to nt 4319) from pFK5.1 was transferred into the parental replicon construct. These two mutations were separated by transferring a PmeI-EagI (nt 3991) or anEagI-EcoRI fragment that was obtained from this construct into the parental replicon, resulting in plasmid pFK1202 Glu → Gly or pFK1280 Thr → Ile, respectively. Plasmid pFK1757 Leu → Ile was generated by transferring a SalI-MluI fragment (nt 4725 to 6529) into pFK-I389neo/NS3-3′/wt. To obtain plasmids pFK2327 Pro → Ser and pFK2350 Lys → Glu,XhoI-PshAI (nt 7186 to 7338) andMluI-SpeI fragments (nt 6529 up to the 3′ end of the HCV sequence), respectively, were inserted into the parental replicon construct. Combinations of individual NS3 mutations with the major adaptive mutation in NS5A (2197 Ser → Pro) were obtained by transferring the EcoRI-XhoI fragment from pFK2197 Ser → Pro into pFK1202 Glu → Gly or pFK1280 Thr → Ile. The presence of each mutation was confirmed by sequence analysis. To generate the corresponding luciferase plasmids for the transient-replication assay, the neo gene was replaced by the gene encoding the luciferase of the firefly Photinus pyralis by using the AscI and PmeI restriction sites. These sites were introduced at the 5′ and 3′ ends of the luciferase gene by PCR.
Preparation of total RNA and quantification of HCV RNA by Northern blotting.The methods to prepare total RNA and quantify HCV RNA by Northern blotting have been described recently (29). In brief, total RNA was prepared by a single-step isolation method (8), denatured by treatment with 5.9% glyoxal in a solution containing 50% dimethyl sulfoxide and 10 mM sodium phosphate buffer (pH 7.0), and analyzed after denaturing agarose gel electrophoresis by Northern blotting. Prior to hybridization, the membrane was stained with methylene blue and cut ∼1 cm below the 28S rRNA band and the upper strip containing the HCV replicon RNA was hybridized with a 32P-labeled negative-sense riboprobe complementary to the 3′ end of the NS5B region and part of the 3′ NTR (nt 8362 to 9408). The lower strip that was hybridized with a β-actin-specific antisense riboprobe was used to correct for total RNA amounts loaded in each lane of the gel. Specific bands were quantitated by phosphorimaging with a BAS 2500 scanner (Fuji), and the number of replicon molecules was determined by comparison with a serial dilution of in vitro transcripts loaded in parallel onto the gel.
Determination of the copy number of replicon RNAs.Owing to the dependence of RNA replication on host cell proliferation (36), copy numbers could not be determined from single measurements. Therefore, 5 × 105 cells that had been regularly passaged three times a week at a dilution of 1:3 to 1:5 in the presence of 500 μg of G418 per ml were seeded in a 6-cm-diameter cell culture dish and harvested at daily intervals 3 to 6 days after seeding. The amounts of replicon RNA were determined by Northern blotting analysis as described above. For each replicon, at least three independent cell lines were examined.
Amplification of replicon RNA by RT-PCR and cloning of amplified DNA fragments.Amplification of replicon RNAs was done by long-distance RT-PCR using a mixture of polymerases (7). One microgram of total RNA and 50 pmol of primer A9413 (CAG GAT GGC CTA TTG GCC TGG AG) were mixed in a total volume of 10.5 μl and denatured for 10 min at 65°C. Reverse transcription was performed with Expand-RT (Roche Biochemicals, Mannheim, Germany) in a total volume of 20 μl, and after 1 h at 42°C, different amounts of the reaction mixture were used for PCR with the Expand Long Template PCR system (Roche Biochemicals) and primers S59 (TGT CTT CAC GCA GAA AGC GTC TAG) and A9386 (TTA GCT CCC CGT TCA TCG GTT GG). Cycle conditions were two min of initial denaturation at 94°C and 40 cycles of 10 s at 94°C, 90 s at 54°C, and 540 s at 68°C. After 10 cycles, the extension time was increased 10 s for each additional cycle. After a final 10-min incubation at 68°C, PCR products were purified by preparative agarose gel electrophoresis, restricted with SfiI, and inserted into the parental construct pFK-I389neo/NS3-3′/wt.
Sequence analysis.Sequences were verified using the Thermo Sequenase Fluorescently Labeled Primer Cycle Sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech, Freiburg, Germany) and IRD-41-labeled primers (MWG-Biotech, Ebersberg, Germany) according to the instructions of the manufacturer. Reaction mixtures were analyzed on a model 4000 Licor DNA sequencer (MWG-Biotech).
In vitro transcription, electroporation, and selection of G418-resistant cell lines.In vitro transcripts were generated using the protocol described recently (29). In brief, plasmid DNA was restricted with AseI and ScaI (New England Biolabs, Bad Schwalbach/Taunus, Germany) and after extraction with phenol and chloroform and precipitation with ethanol dissolved in RNase-free water. In vitro transcription reaction mixtures contained 80 mM HEPES (pH 7.5), 12 mM MgCl2, 2 mM spermidine, 40 mM dithiothreitol (DTT), 3.125 mM each nucleoside triphosphate, 1 U of RNasin (Promega, Mannheim, Germany) per μl, 0.1 μg of restricted plasmid DNA per μl, and 0.6 U of T7 RNA polymerase (Promega) per μl. After 2 h at 37°C, an additional 0.3 U of T7 RNA polymerase per μl was added and the reaction mixture was incubated for another 2 h. Transcription was terminated by the addition of 1.2 U of RNase-free DNase (Promega) per μg of plasmid DNA and 30 min of incubation at 37°C. After one extraction with acidic phenol and chloroform, DNA was precipitated with isopropanol and dissolved in RNase-free water. The concentration was determined by measurement of the optical density at 260 nm, and RNA integrity was checked by denaturing agarose gel electrophoresis. The conditions for electroporation and selection of G418-resistant cells have been described in detail elsewhere (29). Briefly, 0.25 to 500 ng of in vitro transcripts adjusted with total RNA from naı̈ve Huh-7 cells to a final amount of 10 μg was mixed with 400 μl of a suspension of 107 Huh-7 cells per ml. Electroporation conditions were 960 μF and 270 V using a Gene pulser system (Bio-Rad, Munich, Germany) and a cuvette with a gap width of 0.4 cm (Bio-Rad). Cells were immediately transferred to 8 ml of complete DMEM containing 1.25% dimethyl sulfoxide and seeded in a 10-cm-diameter cell culture dish. After 24 h, medium was replaced by complete DMEM supplemented with 500 μg of G418 per ml. Medium was changed weekly, and 3 to 4 weeks after electroporation, colonies were stained with Coomassie brillant blue (0.6 g/liter in 50% methanol–10% acetic acid). To determine the number of CFU of a given RNA, serial dilutions from 300 to 0.25 ng of RNA were transfected into Huh-7 cells and processed further as described above. For each replicon, 5 to 20 independent transfections were performed.
Transient-replication assays.Huh-7 cells were transfected by electroporation as described above using 7.5 μg of aneo replicon or 5 μg of a luciferase replicon. After addition of 10 ml of complete DMEM, 1- to 2-ml aliquots of the cell suspension were seeded in a 3-cm-diameter culture dish and harvested at time points given in Results. For Northern blot analysis, total RNA was analyzed as described above. With luciferasereplicons, cells were washed three times with phosphate-buffered saline (PBS) and scraped off the plate into 350 μl of ice-cold lysis buffer (1% Triton X-100, 25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT). One hundred microliters of lysate was mixed with 360 μl of assay buffer (25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT, 2 mM ATP, 15 mM K2PO4[pH 7.8]) and, after addition of 200 μl of a 200 μM luciferin stock solution, measured in a luminometer (Lumat LB9507 from Berthold, Freiburg, Germany) for 20 s. Values obtained with cells harvested 4 h after electroporation were used to determine the transfection efficiency.
Immunofluorescence.Transfected cells (0.7 × 105) were seeded on glass coverslips and, after 4, 15.5, 24, 48, 72, and 96 h, washed three times with PBS. Cell were fixed in an ice-cold mixture of acetone and methanol (90 and 10%) for about 10 min at −20°C and washed thereafter three times with PBS, followed by a 1-h incubation in IF buffer (PBS, 3% bovine serum albumin, 0.1% Triton X-100) at 4°C. An NS3B-specific rabbit polyclonal antibody (3) was added at a dilution of 1:500 in IF buffer, and after 1 h, cells were washed three times with PBS, followed by incubation with a 1:100 dilution of an anti-rabbit antibody conjugated with fluorescein isothiocyanate (Sigma, Deisenhofen, Germany) in IF buffer. Coverslips were washed with PBS and mounted on glass slides with Permafluor (Immunotech, Marseille, France), and cells were examined under a fluorescence microscope (Zeiss, Jena, Germany).
Construction of a cell culture-adapted HCV replicon RNA.To study the mechanism of cell culture adaptation, a highly efficient replicon RNA was required. Although we had recently identified a single amino acid substitution in NS5B that increased the ECF ∼500-fold (29), we attempted to develop an even more efficient replicon. On the assumption that adaptive mutations might accumulate during serial passage of replicon RNAs, total RNA was isolated from a cell line that harbored an NS3-5B replicon (cell line 5-15 [30]) and transfected into naı̈ve Huh-7 cells. After stringent selection with 1 mg of G418 per ml, a fast-growing cell clone was isolated and total RNA was prepared and transfected into parental Huh-7 cells. After three successive passages, cell line 5-15-9-2-3 was obtained. To identify cell culture-adaptive mutations, nearly full-length replicon RNAs were amplified by long-distance RT-PCR using primers S59 and A9386 and, after restriction of the amplified DNA fragments with SfiI, almost the complete HCV ORF was inserted into the parental replicon construct (Fig. 1). In vitro transcripts from 82 different clones were prepared and transfected into naı̈ve Huh-7 cells to determine their ECFs. As summarized in Table 1, 69 clones were replication defective, because no G418-resistant colonies were obtained. Nine of the tested clones were comparable to the wild type (20 to 50 colonies), whereas four clones were more efficient. The sequence analysis of theSfiI fragments of these clones revealed several amino acid substitutions (Fig. 1). Six of them were found in all four clones derived from the cell line 5-15-9-2-3, whereas the number of nonconserved amino acid substitutions was variable. A careful titration of in vitro transcripts derived from the two most efficient clones, which were designated 5.1 and 19, revealed that replicon 5.1 had the highest ECF (∼500,000 CFU per μg of RNA) and that replicon 19 was about sevenfold less efficient (∼70,000 CFU/μg).
Determination of adaptive mutations in the HCV coding sequence.Since adaptive mutations should be generated early during selection and become fixed in all RNA progeny, we focused our further analyses on the conserved amino acid substitutions. Three of these six substitutions were located in NS5A, and two were located in NS3. Only one conserved mutation was found in NS4B, and none were found in NS4A and NS5B (see below). Interestingly, the conserved mutation in the center of NS5A at position 2197 of the HCV polyprotein was also present in two clones that were isolated from the founder cell line 5–15 (Fig. 1). In contrast, a second conserved substitution in NS5A at position 2350 was found in only one of these two clones (5–15/4B). To identify which of the six conserved mutations were responsible for cell culture adaptation, they were introduced individually into the parental replicon construct and serial dilutions of in vitro transcripts were transfected into naı̈ve Huh-7 cells. After selection with G418, the ECF was determined. The results shown in Fig.2 demonstrate that the most adaptive mutation was the one located in the center of NS5A at amino acid position 2197 of the polyprotein. In addition, the glycine substitution for glutamic acid and the isoleucine substitution for threonine in NS3 at positions 1202 and 1280, respectively, increased the ECF too, albeit to a much lesser extent. All other mutations did not affect the number of G418-resistant cell colonies. Interestingly, the highly adaptive NS5A substitution, but not the NS3 mutations, was already found in the replicons isolated from the founder cell line 5–15. Thus, the initial adaptive mutation was the one found in NS5A, and this mutation remained conserved during cell culture passage of the replicon RNAs. The additional adaptive mutations in NS3 must have been acquired at a later stage or were present only in a minor fraction of replicons in the founder cell line and accumulated during successive cell culture passage. Owing to the high selective pressure we used, the presence of these three mutations in the replicon RNA appeared to confer a selection advantage. In agreement with this assumption, we found that these substitutions were synergistic. When the two NS3 mutations were combined, the ECF of the replicon was ∼4-fold higher than that of the RNA harboring only the adaptive substitution at position 1280. However, when both NS3 mutations were combined with the adaptive NS5A substitution at position 2197, the ECF of the triple mutant was dramatically increased compared with the replicon that harbored only this NS5A substitution (Fig. 2). In fact, the G418 transduction efficiency of the triple mutant was as high as the one obtained with replicon 5.1. Since the analysis of sequences of the HCV ORF flanking the SfiI fragment did not reveal additional amino acid substitutions in NS3 and NS5B that were conserved between the four replicons recloned from cell line 5-15-9-2-3, we had identified the major adaptive mutations.
Transient replication of HCV RNAs in nonselected cells.In all experiments performed so far we had measured RNA replication by determining the G418 transduction efficiency. Since this approach did not allow the direct determination of replication kinetics, transient-replication assays in nonselected cells were performed. For this approach we chose the most adapted RNAs, 5.1 and 19, and replicon 9-13F, which had an ECF that was ∼100-fold lower than that of RNA 5.1 (Table 2). Replicon 9-13F carried one essential adaptive mutation in NS5A at position 2163 of the polyprotein (29). A derivative of replicon 5.1 that carried a 10-amino-acid residue deletion spanning the active site of the NS5B RdRp served as a negative control (rep5.1/Δ5B) (Fig.3A). Huh-7 cells were transfected with these neo replicons, and total RNA that was prepared after 3.5, 15, 24, 48, 72, and 96 h was analyzed by Northern blotting to determine the copy numbers of the replicons. The representative result in Fig. 3A shows that input RNA was rapidly degraded and barely detectable 15 h after transfection (lane 28). In the low-level adaptation replicon 9-13F, a much slower gradual reduction of HCV RNA could be observed, and it became undetectable at 96 h posttransfection (lanes 21 to 26). In contrast, an increase of the copy numbers of HCV RNAs was found with the two highly adapted replicons. A careful quantification of the Northern blot by phosphorimaging revealed that the copy numbers of both RNAs increased 24 h after transfection (Fig. 3B), which is the time when transfected cells started growing. In replicon 5.1, at 72 h posttransfection the level of replicating RNA reached a maximum and declined within the next 24 h, when cells became confluent. A similar kinetic was found with replicon 19, but the copy number at the time of the peak was ∼3-fold lower. As described above, the decline in the amounts of replicon RNAs in confluent cells (96 h) was most likely due to the tight linkage between RNA replication and host cell proliferation (36).
The different efficiencies of RNA replication were reflected by the levels of HCV protein expression. As shown by immunofluorescence, 4 h after electroporation all transfected cells were positive for NS3 (Fig. 4). In contrast, at 96 h posttransfection the antigen was found only in cells transfected with the highly adapted RNAs 5.1 and 19, whereas the signal found in cells with the poorly adapted replicon 9-13F was hardly above the background. Overall, the replication kinetics determined by Northern blotting and immunofluorescence in this transient-replication assay were in good agreement with the ECF of each replicon RNA, suggesting that cell culture adaptation is mediated by a direct enhancement of RNA replication.
Transient replication of HCV replicons carrying a luciferase reporter gene.To further substantiate this assumption and to simplify the subsequent analyses, we replaced the neo genes in the various cell culture-adapted replicons by the gene encoding the luciferase of the firefly P. pyralis (Fig.5A). Since the copy number of this reporter gene should be determined by the replication levels of the corresponding replicon RNAs, the luciferase activity in a lysate of transfected cells could be used to directly monitor the replication of an RNA. Huh-7 cells were transfected by electroporation, and each 1/10 of the cells was seeded into a cell culture dish. Cells were harvested at various time points, and luciferase activities were determined. As a negative control, a derivative of the wild-type replicon that carried a single amino acid substitution changing the GDD motif of the RdRp active site to GND was transfected in parallel. Luciferase activities measured 4 h after transfection were used to determine the transfection efficiencies. A representative result is shown in Fig. 5. In this experiment we also analyzed the replication of the HCV RNAs in cells after passage, because we wanted to avoid the reduction of replicon RNA levels when the cells reached confluence (36). Therefore, cells in one culture dish were passaged at a dilution of 1:3 at 72 h posttransfection and passaged a second time 96 h later (Fig. 5B). Cells were harvested at given time points to determine the luciferase activities. Overall, the replication kinetics observed with these luciferase replicons were very similar to the ones obtained with the neo replicons that were analyzed by Northern blot analysis up to 96 h after transfection (Fig. 5C). At every determined time point (except 4 h posttransfection), luciferase activities obtained with the most highly adapted RNA, 5.1, were consistently the highest whereas those found in 9-13F-transfected cells were much lower. Interestingly, in the cells passaged at 72 h posttransfection, an increase in luciferase activity was clearly visible with replicons 5.1, 19, and 9-13F (compare the values obtained 127 and 144 h posttransfection). When these cells were passaged a second time 96 h after the first passage, only in cells transfected with the highly adapted replicons 5.1 and 19 could a second increase of luciferase activity be observed (compare the values at 216 and 244 h posttransfection). These results show that, in unselected cells, the replicons were gradually lost, with the level of decline being determined by the level of cell culture adaptation.
Owing to the high sensitivity of the luciferase reporter, we also determined the replication kinetic of the parental, nonadapted replicon RNA. As shown in Fig. 5C, only at 24, 48, and 72 h posttransfection was a difference found between this replicon and the replication-defective negative control (GND). However, compared to that of even the least-adapted RNA, 9-13F, a dramatic difference in the replication level was found. Therefore, we can conclude that cell culture-adaptive mutations strongly increase RNA replication and that they were generated during a low-level replication of the nonadapted parental replicon. It should be noted that, in more than 10 independent experiments, the absolute values of luciferase activities varied significantly but that, in all cases, the relative differences between the individual replicons remained the same.
Having developed a highly sensitive and fast replication assay for HCV RNAs, we next wanted to confirm the usability of this approach for reverse-genetic studies. To this end, we generated a panel ofluciferase replicon RNAs harboring the single amino acid substitutions in NS3 and NS5A we had already analyzed with respect to colony formation (Fig. 2). These replicons were transfected into naı̈ve Huh-7 cells, and luciferase activities were measured at daily intervals up to 96 h posttransfection. The values obtained 4 h after transfection were used to correct for different transfection efficiencies (not shown). For each replicon, a clear correlation was found between the luciferase activities in the transient-transfection assay and the ECF of the correspondingneo construct (Fig. 6). For instance, the replicon harboring the adaptive mutation in NS3 at position 1202 replicated at a rather low level. In contrast, for each time point, the luciferase activities in lysates of cells transfected with the replicon with the adaptive mutation in NS5A (nt 2197, S → P) were higher. When these two mutations were combined, the resulting RNA replicated at a level that was clearly higher than the sum of the replication levels of the RNAs carrying the substitutions individually. A further increase was found with the replicon harboring in addition the substitution in NS3 at position 1280. Although this mutation, when analyzed individually, only slightly increased replication compared with that of the nonadapted replicon, it did so synergistically both in combination with the other more adaptive NS3 substitution at position 1202 and with the adaptive NS5A mutation at position 2197. In fact, the replication level of this triple mutant was as high as the one observed with replicon 5.1, confirming that these NS3 and NS5A substitutions were responsible for cell culture adaptation and enhanced replication levels synergistically. In summary, the results that were based onluciferase HCV replicons correlated well with the results obtained with the neo replicons, both in transient-replication assays and after selection of stable cell lines. Thus, HCV RNAs that harbor a reporter gene can be used to accurately determine replication of the replicon RNA.
Lack of correlation between the ECF and replicon RNA copy number in G418-selected cell lines.To analyze whether an increase in the ECF and the RNA replication level would directly correlate with the copy number of an HCV RNA in a selected cell line, we took advantage of several HCV replicons that differed in their levels of cell culture adaptation. Replicons 5.1, 19, and 9-13F were the ones described above. Replicon 5B2884Gly harbored a single glycine substitution in NS5B at position 2884 that led to an ∼500-fold increase in the ECF compared with that of the wild type (29) (Table 2). In vitro transcripts of these constructs were transfected into naı̈ve Huh-7 cells, and for each replicon at least three cell clones were isolated and expanded. To determine the copy numbers of these RNAs in selected cells, we had to consider that the replication levels of the replicons depend on host cell metabolism (36). Therefore, copy numbers were determined from serial measurements using cells that had been harvested 3, 4, 5, and 6 days after seeding. A representative example of this analysis is shown in Fig.7 for three different cell lines that were obtained after transfection with the most highly adapted replicon, 5.1, and a summary for all cell lines is given in Table 2. In agreement with the ECFs, the highest copy number was found in cell lines harboring replicon 5.1. However, in spite of the dramatic differences in the numbers of colonies obtained with a given replicon RNA, the replication levels within a selected cell line differed much less. Even between the replicons with the highest and the lowest levels of adaptation (5.1 and 9-13F, respectively), which varied in their ECFs by a factor of ∼100, the differences in the copy numbers were only ∼2-fold. With the other replicons, the replication levels were comparable. This result suggests that the ECF is primarily determined by the initial level of RNA replication and that, during selection, either particular host cells that support high-level replication of the HCV RNA are enriched or further adaptive mutations are acquired.
In this study we developed a novel highly adapted HCV replicon that harbors two synergistic mutations in NS3 and one in NS5A. The ECF of this RNA was ∼5 × 105 CFU per μg of RNA, which is ∼20-fold higher than that of the best-adapted replicon we described recently (29). By analyzing this and several other HCV RNAs that differed with respect to their levels of cell culture adaptation, we found a clear correlation between the ECF and RNA replication as determined by two different transient-transfection assays. These results demonstrate that cell culture-adaptive mutations increase RNA replication. They also provide an explanation of how adaptive replicons are generated. As shown with the parental replicon carrying the luciferase gene, this nonadapted RNA replicated only transiently and at a low level in transfected cells. During this time, mutations must have been generated by the viral RNA polymerase that in a few instances were adaptive. By increasing the level of RNA replication, cells harboring such a replicon developed G418 resistance. Since most mutations did not increase but rather reduced replication or were inactivating, development of an adaptive replicon was a rare event, explaining why the numbers of G418-resistant colonies that were obtained with a nonadapted RNA were very low. Alternatively, the adaptive mutations may have been introduced by the T7 RNA polymerase used for in vitro transcription. However, given the infrequent generation of adaptive mutations, the number of replication-competent RNA molecules in the transcription reaction probably would have been too low to allow detection in the luciferase assay. The fact that we consistently observed a low level of replication with the parental replicon suggests that the adaptive mutations were generated during the initial low-level replication of this RNA.
While a clear correlation was found between replication in the transient-transfection assays and the ECF, this was not the case with respect to the copy number of replicon RNA molecules per cell after selection. For instance, the most highly adapted replicon, 5.1, had an ECF of ∼5 × 105 CFU/μg of RNA and showed the highest initial replication whereas the ECF of the least-adapted replicon was ∼100-fold lower, and this RNA replicated very inefficiently. However, in selected cell lines obtained after transfection with these replicon RNAs, the copy numbers differed only by a factor of ∼2. This result indicates that during selection of cells harboring a poorly adapted replicon, further mutations that increase the replication level are introduced into the RNA. Alternatively, during prolonged passage, we may have selected for particular host cells that support high-level replication. Currently, we cannot distinguish between the two possibilities, but in agreement with the latter assumption, we found that the copy number of replicon RNA was highest in cell line 9-13 (2.5 × 108 per μg of total RNA), which harbored a moderately adapted replicon (rep5B2884Gly with a substitution in NS5B that increased the ECF ∼500-fold over the wild-type level) (29). In contrast, in cell line 5-15-9-2-3 carrying the most highly adapted replicon (rep5.1 with an ECF that is ∼10,000-fold higher than that of the wild type), the replicon copy number was even lower (1.6 × 108 per μg of total RNA). Therefore, the level of cell culture adaptation is determined by the initial replication level, which also determines the level of G418 transduction efficiency. However, during prolonged cell passage and selection, particular host cells that support a higher level of replicon RNA replication may be selected.
By serial cell culture passage of replicon RNAs, we selected for three mutations that enhanced replication synergistically. In the founder cell line, only the highly adaptive substitution in NS5A was found, and it was the only conserved mutation in the two analyzed clones. Thus, the initial adaptation was due to this particular substitution whereas the adaptive mutations in NS3 were generated at a later stage during cell culture passage. Alternatively, these NS3 substitutions might have already been present at a low frequency in the replicons of the founder cell line but were enriched by the stringent selection procedure.
Currently, we can only speculate about the molecular mechanism of cell culture adaptation. There are certainly several ways how it can be achieved. In a previous study we had shown that a single amino acid substitution in NS5B increased the ECF ∼500-fold compared to that of the parental RNA (29). In this report, we identified two substitutions in NS3 and one in the center of NS5A. Three-dimensional structure modeling experiments suggest that the substitution at the carboxy terminus of NS3 (position 1202) is located on the surface of the molecule and probably has no effect on its various enzymatic activities (Neera Borkakoti, personal communication). The same is probably true for the mutation in domain 1 of the NS3 helicase at position 1280. We therefore favor the hypothesis that these amino acid changes affect an interaction site with a cellular or viral protein important for RNA replication. With NS5A, the substitution affected serine 2197, and this residue was shown to be important for hyperphosphorylation (43). The loss of this serine residue in the adapted replicon RNA may suggest that phosphorylation at this position is inhibitory for RNA replication. However, it is not known whether serine 2197 is a phosphoacceptor site or plays some other role in hyperphosphorylation. As deduced from the formation of pp58, the NS5A in cells harboring replicon 5.1 is still hyperphosphorylated (N. Krieger and R. Bartenschlager, unpublished results). However, the appearance of the pp56/58 double band in conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis may not be sufficient to reveal subtle differences in the phosphorylation patterns between the parental NS5A and the adaptive mutation. Therefore, further genetic and biochemical studies will be required to clarify this important point.
While this paper was in preparation, Blight and coworkers (6) identified several cell culture-adaptive mutations too. Focusing their analysis on NS5A, they identified a total of 10 different mutations that were all located in the center of the molecule. Among these was the proline substitution for serine at position 2197 that we identified here as the most adaptive one. While this mutation reduced only NS5A hyperphosphorylation, it was completely blocked with an isoleucine substitution for serine at position 2204, and this mutation conferred the highest level of adaptation (6). Thus, NS5A hyperphosphorylation appears to be nonessential for HCV RNA replication in cell culture.
Cell culture adaptation by specific mutations in the genome has been described for several other viruses, too. In many cases, adaptation was used for the attenuation of a virus. For instance, with the Sindbis virus a selection procedure was used to isolate a noncytopathic replicon (15). This RNA harbored either one of two single amino acid substitutions in nonstructural protein 2 that reduced the level of RNA replication to 1% of that of the wild-type replicon. This reduction led to a loss of cytopathogenicity concomitant with a cell type restriction (1). Mutations conferring cell culture adaptation by enhancing virus growth have been described for HAV. While the parental virus has a slow-growth property, several cell culture-adapted isolates that replicate more efficiently and to higher titers have been described (e.g., see reference 46). The mutations primarily responsible for adaptation were identified in viral proteins 2B and 2C and in the 5′ NTR (9, 18, 24), although other mutations acquired during adaptation could also contribute to efficient virus growth in cell culture (10, 11). However, the molecular basis for cell culture adaptation is not known.
The development of highly cell culture-adapted HCV replicons opens new avenues for studies of RNA replication, in particular with the help of reverse genetics. These analyses can now be performed in transient-transfection assays, circumventing the problem that, during selection of cells, the effect of a mutation to be studied is masked by second-site reversions or compensations. However, the use of selectable HCV replicons is still a valuable tool, in particular for mutants that replicate only poorly. Therefore, the combination of both assays should help to clarify the mechanism of HCV replication.
We are grateful to Ulrike Herian for excellent technical assistance and to Neera Borkakoti for help with three-dimensional modelling of NS3.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB490, Teilprojekt A2) and the European Community (QLK2-1999-00356).
- Received 21 December 2000.
- Accepted 21 February 2001.
- Copyright © 2001 American Society for Microbiology


text:
By Mary Ellen Flannery
Hispanic students of all ages are more likely than ever to be something really marvelous: a college student.
Last year, more than 2 million Hispanic young adults were enrolled in college, according to the Pew Hispanic Center, reaching a record 16.5 percent share of all college enrollments. And it's not simply because the United States is home to more Hispanic students—although it is. It's not simply because President Barack Obama has created new opportunities for poor and middle-class students to pay for college—although he certainly has done that too.
It's also thanks to the hard work of NEA educators at all levels, said NEA Vice President Lily Eskelsen, who serves on the White House Commission on Hispanic Education. "Educators, teachers, education support professionals are working like crazy to build bridges between schools and homes," she said. "When you see something good like this happening, it's not an accident. It's planned. We have put at the top of our agenda to close the achievement gaps."
Creating opportunities for all students to attend college, if they choose, starts early—really early. In small-town Crete, Nebraska, for example a gateway town for Central and South Americans working in the Midwest's meat-packing industry, fully certified school district teachers keep more than 150 pre-kindergartners busily hopping between interactive learning centers and pre-literacy centers.
"(Early education) isn't just a good remedy for English Language Learners. It's not just a good remedy for Hispanic students. It's not just a good remedy for low-SES (socioeconomic status) students. It's a good investment for all children," writes Ellen Frede, co-director of the National Institute for Early Education Research, in ETS Policy Notes.
Creating college-bound students continues in elementary school, in places like Reading, Pennsylvania, where Gladys Méndez, the parent coordinator at Thirteenth and Green Elementary School, delivers resources to needy families. Her work can be as simple as handing out a warm winter coat or as ambitious as hosting a bilingual night meeting, attended by nearly 100 parents, to discuss the school's improvement plan. "Once you get the parents involved, the students actually do succeed," Méndez said.
And, of course, these efforts continue in middle and high school. Even in community colleges, programs like the Puente Program at California's Rio Hondo College help Hispanic students to transfer to universities. Earlier this year, Rio Hondo professor Mary Ann Pacheco won NEA's César Chávez Acción y Compromiso Human and Civil Rights Award for a lifetime of advocacy.
"She taught me that I belong here in college. That I have just as much as right as anybody else does to be here," said Monique Lopez, a Puente Program student and mother of three.
Studies have shown that the problem of Hispanic college attendance has never been a problem of will. A survey by the Pew Hispanic Center in 2010 found that 89 percent of Hispanic students said they knew college was important to be successful in life, just 48 percent said they actually saw that for themselves. The biggest obstacle was money.
College is expensive—and the price tag still puts it out of reach of many students, Hispanic and otherwise. That's why it's critically important for states and the federal government to invest in public higher education. At the state levels, budget cuts have forced tuition hikes. At the federal level, President Obama has made great strides in broadening the reach of Pell Grants, work-study programs, and direct access student loans. "President Obama has put his money where his mouth is to make sure minority students have the ability to go college," Eskelsen noted.
Related posts:
- Must Reads – College Enrollment
- NEA's Eskelsen to Serve on White House Commission on Hispanic Education
- Duncan Calls for More Black and Hispanic Male Teachers
via NEA Today http://neatoday.org/2012/08/28/whats-behind-the-surge-in-hispanic-college-enrollment/

subset: commoncrawl


text: Sophisticated Lake Shore Drive Co-up with Private Living Quarters The sophisticated living area of this Lake Shore Drive Co-Up, designed by Gibbons, Fortman, & Associates, features a charming open space with grand columns. A long gallery leads to the private quarters. The master bath mirrors frame a skyline view of the city. Cove lighting illuminates the space. This charming library is flanked by two columns, giving the room an added sense of privacy and comfort. Custom millwork wraps around two adjacent walls while a marble fireplace is tucked away in a custom mantle designed by Gibbons, Fortman & Associates. The entry to the home office is framed by two columns opposite of each other, inviting you in. The beautiful swinging doors reside behind two classic columns, giving the home office space elegance and grandeur. Custom cabinetry lines the wall adjacent to the stainless steel appliances, completing this stunning kitchen.


text:
I recently spent the time going through my roster of credit cards to see which ones I should be using for various purchases. I believe I've come up with a pretty universal strategy, so I figured I'd share it since I've already done the research.
To preface, if you are ever paying late fees or interest on credit cards, you are better off without them. Points and cashback are fantastic, but they aren't worth paying fees for. But if you are discplined about paying your credit cards off in full every month, you can take advantage of cashback programs and earn a significant amount of money.
Here are the cards I use currently:
Uber Visa
This card has no annual fee and gives you 4% cash back on all restaurants. If you go to Chipotle every day and spend $10.23 each time, that's $150 in cash back every year.
The card also gives back 3% on travel, which is pretty good, and 2% on all online purchases (including Paypal). For a no-fee card, this one really gives you some great cashback.
Amazon Synchrony Store Visa
Amazon has two credit cards which have no annual fees. Both give 5% cash back if you have a Prime account, which I do. Considering the huge percentage of my purchases that I make each year that come from Amazon, this saves me a ton of money.
The Visa one also gives you 5% back at Whole Foods. I didn't get it because I already have a lot of cards issued by Chase (their issuer), but it's very valuable if you shop at Whole Foods.
Chase Ink Cash
Chase Ink Cash also has no annual fees and gives a 5% cashback on phone and internet.
Chase Sapphire Reserve
This card does have an annual fee, but all of the benefits including travel credit and lounge access make it worth it even without the points you can earn. It gets 3 points back on travel and dining. I use the Uber card over this one for food since I don't often make the best use of my Chase points, but I always use it for travel.
You can go even further down the rabbithole, but these four cards will cover the basics other than gas. I don't buy a lot of gas, so I don't optimize for that. If you don't want to pay any fees, don't get the Reserve card and use Uber for travel as well. Three percent back on travel is respectable.
Photo is my friend Brian and I drinking tea on the island. We finally got furniture! That reminds me... Reserve and Uber Visa don't have foreign exchange fees, so you can use them anywhere.
There are three items I own which I'll always upgrade when a significant upgrade exists: my computer, my camera, and my Kindle. Yesterday I got my new Kindle, the fourth generation one that was just released. Before I talk about this specific Kindle, I want to address some general points about the Kindle.
Some people balk at the $189 price tag of the newest 3G Kindle (which is the only one to buy, by the way). It's expensive, but only if you consider it a drop in replacement for books. I consider it $200 to ensure that I read at least 10X more than I used to.
Key Points:
A credit card is secondary to your checking account. I suggest getting your primary finances in order first.
There are advanced tricks to getting the most out of your credit cards, but before you can do that, you should make sure the one you have is right for you.
First, check your credit score on Credit Karma.


text:
July 1, 1959, Page 0Buy Reprints
IT has been bruited about by Walt Disney's press corps that the producer of "Darby O'Gill and the Little People," which moved into the Normandie Theatre yesterday, is of Irish descent. A view of this overpoweringly charming concoction of standard Gaelic tall stories, fantasy and romance should leave no doubt in an observer's mind that there is no blarney in this bit of information. Only a man less than 6 years of age who has never quaffed a dollop of poteen in a shebeen would argue the point.The fact is that a large, restless contingent of small fry at yesterday's initial showing of the picture, who obviously were not members of one of Dublin's nursery schools, seemed more entranced by the cavortings of Donald Duck and Pluto in an old short titled "Beach Picnic" than in the adventures of "Darby" and his ilk. Perhaps a dram or two of matured Irish spirits taken in the friendly company of the boyos in a local shebeen would go a long way toward making the strange adventures of the Kerry folk in the hamlet of Rathcullen less exotic and more palatable.Mr. Disney, Lawrence E. Watkin, his scenarist, and Robert Stevenson, his director, have given us in "Darby" more fairy folk, legends and brogue than are heard on any soft summer's night at the Abbey Theatre or a Third Avenue saloon. They are spinning the tale of Darby, the aging rapscallion caretaker of Lord Fitzpatrick's manor house in Rathcullen, who is about to be retired by his benign master. Since he is the only man in the Rathcullen Arms who has "seen" leprechauns, it follows that he will capture Brian Connors, the 5,000-year-old, twenty-one inch king of the "little people" and force him to grant him three wishes.Our hero, says this impish but redoubtable monarch, is a "knowledgeable" man. So Darby saves his wishes for the time he'll need them. And eventually he uses them to fix it so his daughter marries a broth of a Dublin man who has come to replace him as caretaker. Darby, it should go without saying, not only outwits the leprechauns in their mountain lair of Knocknasheega, but also carries their protesting king around in a sack like the rabbit he appears to be to the villagers of Rathcullen.Mr. Disney and his Hollywood folklorists have dressed their fable up in lovely colors, and they have the services of Albert Sharpe in the title role. As Darby, Mr. Sharpe is a man who has seen lots of service in the name of Irish folklore both in films and in the theatre, notably in "Finian's Rainbow," where he gave real value to the role of a man in search of that pot of gold at the foot of a rainbow. Although he has his chance at a similar reward in "Darby," he forsakes it but is, nevertheless, a truly lovable, glinty'-eyed, unshaven darlin' of a rascal who charms a viewer as much as a leprechaun.Janet Munro is pretty and pert as his poor but proud colleen whom he marries off, finally, to Sean Connery, who is merely tall, dark and handsome as the caretaker. Jimmy O'Dea, in red chin whiskers, ermine cape and crown, and his "little people," are, thanks to trick photography, a cute enough sight to make a man swear off legends and whisky.Kieron Moore, as the scoffing village bully who contests Darby for his job and his daughter, makes the film's most trenchant remark by angrily observing: "I've heard enough silly blather about the little people to last me a lifetime." Obviously, he's a knowledgeable man, too.
DARBY O'GILL AND THE LITTLE PEOPLE; written by Lawrence Edward Watkin; suggested by H. T. Kavanagh's "Darby O'Gill" stories; directed by Robert Stevenson; presented by Walt Disney and distributed by the Buena Vista Film Distribution Company. At the Normandie Theatre, Fifty-seventh Street west of Sixth Avenue. Running time: ninety-three minutes.Darby O'Gill . . . . . Albert SharpeKatle O'Gill . . . . . Janet MunroMichael McBride . . . . . Sean ConneryKing Brian Connors . . . . . Jimmy O'DeaPony Sugrue . . . . . Kieron MooreSheelah . . . . . Estelle WinwoodLord Fitzpatrick . . . . . Walter FitzgeraldFather Murphy . . . . . Dennis O'DeaTom Kerrigan . . . . . J. G. DevlinPhadrig Oge . . . . . Jack MacGowranPaddy Scanlon . . . . . Farrell PellyMolly Malloy . . . . . Nora O'Mahony


text:
By: Frank Armstrong, CFP, AIF
Place one hand on a hot stove, and the other on a slab of dry ice. Any statistician will tell you that on average, you are comfortable–but, of course, you know it's not so. The markets' average returns hide very long periods of both very weak and very strong performance. What's more, averages are derived from past returns, but future returns are unlikely to exactly mirror the past. Naively relying on "average" numbers without considering the market's wide variations can lead to disaster.
The models we use to describe market behavior become critical to our retirement planning. We often say that stock prices follow a random walk, in which each price change is independent of the one that preceded it. But studies have shown that there is more serial correlation than a random distribution would predict. In other words, both winners and losers repeat more often than they should. We also typically say that stock-market returns are normally distributed, but actually there are far more very good and very bad periods than there should be if market returns mapped out as a true bell-shaped curve; the "tails," as they are called, are too fat.
For instance, based on the average daily market volatility of the preceding 50 years, the crash of 1987 should happen just once in 55 million years. (A statistician would say that the crash was a 16 sigma event, or outside of 16 standard deviations from the average.) So, investors can all go back to sleep, right? Well, not quite. It would be foolish to build a plan that didn't take into account at least one more good crash during our lifetimes.
Getting more and bigger winners than you should is not catastrophic. But what if the losers are bigger and more often than we expect? How might that affect the plan? And if some of those big losses show up during the first few years of retirement, will you be able to recover?
Imagine that you retired in 1972. You had $500,000 invested in equities and planned to withdraw 8% ($40,000) each year for income. From 1973 to 1974, the market dropped 50%, and you made your withdrawals as expected. In just two short years, you were down more than 60%. Your remaining capital was about $200,000, so depleted that it could never recover under the weight of your planned annual $40,000 withdrawals–which, after the disaster, amounted to 20% of the remaining capital!
Investors must assess both the probabilities and the potential consequences of their strategies. For example, we know that if we play Russian roulette with the typical revolver, we have but a one in six chance of losing. Unfortunately, while the probability of losing is low, the consequences of a loss could ruin your whole day. The consequences of failure for a retiree, while not quite so absolute, are still unacceptable. Nothing can turn those golden years into a nightmare faster than being broke.
Given the possibility of market crashes, the wide variation of investment returns in the short term, and the consequences of failure, retirees must adopt strategies with the highest probability of success. They will want to build in a little extra pad for safety. The market neither knows nor cares whether you have retired. It can go down at any time, and stay there for several years.
Armstrong's Laws
This situation leads me to Armstrong's Law of Averages: Don't expect to ever see an average year. From this profound axiom, we can derive the corollary: Don't expect retirement to be a long string of average years.
Conceptually, at least, it's easy to structure an accumulation program. But the game changes at retirement. Both economically and psychologically, the stakes get higher. Time is no longer on your side. With no more salary coming in, there is no way to make up for investment mistakes. Risk takes on an entirely new meaning. The pucker factor is up.
The retiree looks at whatever he has accumulated and somewhat fearfully asks two questions: How much can I safely withdraw? How can I structure my account to maximize the probability that it will last forever?
What's Amortization?
In this instance, amortization refers to withdrawing a certain percentage of your nest egg each year in the expectation that it will reach zero after a predetermined number of years.
We often hear people say that the best plan is to die happy having just spent their last dollar. But amortization is out of the question. The time horizon of one's retirement is long and not easily predicted. After all, if we plan to spend our last dollar at our average projected life expectancy, what happens if we screw up and live another five years? Remember that by definition half of all people will live longer than the average. In any event, over long periods of time, amortization doesn't add much to income. Besides, drawing down your nest egg over a long period of time doesn't really add much to your income. Even if we don't care whether there is anything left over for the kids, there's no alternative to keeping the principal intact.
As if the problem weren't already tricky enough, we must expect at least modest amounts of inflation. So, our income requirement will grow throughout life. The longer our time horizon, the more inflation will eat into the buying power.
Retirees will soon discover that they are unlikely to meet any reasonable income needs using fixed-income investments alone. They must usually endure some equity risk to meet their objectives. The trick is to generate at least the required minimum return at the lowest possible risk level. Too little risk, and the portfolio may not generate enough return, while too much risk may "blow up" the portfolio during a string of bad years.
As objectives and risk tolerance are now quite different, the asset-allocation plan we put together during the accumulation phase must change. An appropriate plan must generate at least the minimum required total return at the lowest possible risk level. And it should have a good pad for safety. As a matter of policy, investors will want to complete the transition to a more conservative posture well in advance of their expected retirement dates.
The old schoolbook "low risk" income solution for retirees, widows, and orphans was to load up on money-market funds, municipal bonds, convertible bonds, utilities, real estate, preferred stocks, and other high-dividend-paying or interest-bearing securities. But this income-only approach generates an inefficient portfolio with far higher risk per unit of return than is necessary.
A far more efficient (lower risk) solution is to invest for total return rather than income. Risk can be more effectively controlled by varying the ratio of stocks to bonds in the portfolio than by purchasing the "low risk" income portfolio.
A variation of a "balanced" portfolio, including a diversified global-equity portion and a short-term bond portion, will generally come closest to meeting all the conflicting criteria. However, if available capital falls short of what is required, something will have to give. Either retirement must be delayed, or one's lifestyle must be curtailed.
Coming Up: How much can I withdraw?
One big risk in retirement planning is directly related to one's withdrawal rate. It is therefore under the direct control of the retiree. In the next installment, we will consider how much investors should reasonably expect to safely withdraw from their nest eggs.
Retirement Planning: Not Relying On The Averages
By: Frank Armstrong, CFP, AIF


text:
photo © 2005 Michael Pereckas | more info (via: Wylio)
"What, me worry?" – Alfred E. Neuman
I'm a child of the 50's. I remember the atomic bomb drills in school and people putting fallout shelters at their home. In. their. front. yard! Remember those?
So now I find myself thinking, am I sensitized or de-sensitized to the current nuclear power plant disaster in Japan? Should I have iodine pills on hand? Result – I'm sensitized as as far as having knowledge of the situation and an awareness of what's going on. I'm de-sensitized as far as not being worried about it.
In the 50's I was concerned about how to save myself from the bomb and nuclear fallout. In the 60's I was of the age where I was certain I was indestructible and would not be affected. In the 70's I was sitting on top of the bomb in my F-4 knowing full-well that if the horn went off and I dropped it, there would be no place to land by the time I got there. In the 80's – détente. 90's – cold war over!
So, Exactly How Close Are We To One of Those Nuclear Power Plants?
Oklahoma does not have a nuclear power plant. We still have coal-fired plants. Comes from Coal County I guess. Our neighbors, Kansas and Texas do have them. CNNMoney.com has a nifty map where you just put in your zip code and it'll tell you how far you are from the nearest nuclear power plants. Our humble abode and office are 210 miles from the Wolf Creek 1 plant in Kansas.
So here I sit in the good ole 73013 – fat, dumb and happy! 210 miles from the nearest nuclear power plant. I don't have duct tape. I don't have iodine pills.


text:
Fred Garnett is running an enquiry into creativity and heutagogy, and he's asked me to write 50 words on this topic. Heutagogy, in a nutshell, is the study of learning that is determined by the learner. Self-determined learners can in turn become heutagogics themselves by developing critical awareness of their own learning preferences, goals and practices. Being a self-determined sort of learning person, I've resisted Fred's limit of 50 words ('I didn't have time to write a shorter letter…') but you can take what 50 word bits you want from this.
My thesis is this: All effective or meaningful learning depends on a good balance between Creative and Methodical modes.
Creative modes can flourish even in areas of learning that are not expressive, performative, or making-based, such as Science. On the other hand, some delivery of the more 'creative' subjects does not allow creativity to flourish because it can be too Methodical. For example, drama education can be driven too much by set methods for performing established repertoire and how to 'make it' in a stage career, leaving out Creative modes such as the interpretation of scripts, improvisation, personal emotion and imaginative writing.
The Methodical modes are not bad per se, but they can be too dominant. When they are, this is like walking with one leg, or breathing in without ever breathing out. The Double Diamond of the design process demonstrates how generation of new ideas flows between open ideation to closing down, and then repeats. Unsurprising then that a discipline such as Design, while becoming so vital across industries, is becoming devalued in our schools. It is too balanced!
It may be that when we bemoan the lack of creativity in a school, or curriculum, or in our work, we should more precisely be criticising the lack of self-determined and balanced learning. Where self-determined learning is allowed and nurtured, whether through the design of learning settings or deliberate 'deschooling', this balance is more likely to occur because self-motivated learners naturally draw from each mode in an integrated flow. They respond to the context, for example, their peers doing activities they'd like to try, or to problems that clearly need solving, and they are pushed on by their feelings of joy, absorption and curiosity about these tasks.
Self-determined learning approaches are repressed in conventional schooling in England and in many other countries, where the Methodical mode dominates along with high stakes testing. In England, the subjects that favour balanced learning are being dropped, from schools' own offer (e.g. numbers taking Design and Technology are falling) and from exam syllabi (e.g. History of Art will no longer be offered as an A level) because they are 'discounted' in data regimes. They require too much imaginative enquiry or original inventiveness to be objectively examined.
The dominance of the Methodical mode (combined with high stakes tests) can be worse than ineffective, leading to daily demotivation, mental stress from a sense of failure and ultimately to a society that lacks imagination of the future and empathy for others. At this time, a context of global ecological collapse and, in the UK, of impending economic collapse via Brexit, we have never had such an urgent need for self-care to ensure individual wellbeing, imagination about the future and empathy for others.
Schools (at least in England) have so many formal requirements to measure children's progress and attainment that they lack the capacity to reflect on progress in areas of learning that grow self-motivated and balanced people who can do well in life and work.
Schools and informal learning programmes (e.g. run by cultural organisations) need to explore alternative systems to fill this gap. They need to help young people develop as heutagogics, i.e. to be aware of their own preferences and motivations, and of what skills are needed for any context they find themselves in. Such a system would help learners identify and describe skills across their formal and informal learning experiences. Off the top of my head, these seem to be the skills that are developed through balanced self-determined learning and which are also increasingly needed in future work:
Critical skills: Questioning, challenging, comparing, analysing, evaluating
Ethical skills: Being able to think fairly about the needs of others when working, including people with particular needs, different cultural values, or the environment.
Interpersonal and social skills: Co-operation, empathy, listening, putting others at ease
Communication skills: Presenting, writing, speaking, visual communication, using social media effectively. (Applied and multimodal literacy, beyond the emphasis on skills measured in English Language GCSE.)
Technical skills: Using equipment and materials to make things and solve problems, following instructions, refining processes, keeping on top of digital technology as it changes.
Aesthetic skills: Understanding and using elements such as colour, sound effects, shape and movement when creating or appreciating art (e.g. in music, dance, digital art, craft etc).
Imaginative skills: Visualising images or inventing stories, seeing new ways of doing something, inventing a new game, creating social media content, creating original films, artworks or designs.
Physical, spatial and manual skills: Creating maps of places, making 3D drawings, building a structure, choreographing a dance, kinaesthetic prowess in sport.
Project management: Organising or leading a creative project, managing a work schedule and other people, including numeracy and financial skills.
Self-management: Awareness of your own learning goals and preferences, caring for your mental and physical health, timekeeping, maintaining intrinsic motivation and discipline.


text:
KBzine: the original kitchen and bathroom industry e-news - since 2002
25th May 2020
We strongly recommend viewing KBzine full size in your web browser. Click our masthead above to visit our website version.
KBB Preview: Exploring the true potential of Mistral
Stand: M100
Visitors to the Karonia stand can expect to be stunned by the ground-breaking displays of Mistral 25mm solid surfacing and by three new additions to the range. The stand has been specifically designed to explore the true potential of Mistral and fuel the imagination of visitors.
Since kbb Birmingham 2012, Karonia has seen Mistral grow steadily, inspiring designers and homeowners to create stylish and unique home environments. Now Karonia will use kbb Birmingham 2104 to demonstrate further the undoubted design versatility of Mistral with concept pieces and creative features.
The practicality of working with Mistral and the expansion of Karonia's customer support programme have been key to the growth in its popularity. Installers now have a product that is straightforward and efficient to install but which also allows them to show their precision skills and attention to detail. In visiting the stand they will be able to meet our support team to discuss ideas as well as view Karonia's dedicated tooling.
The company has traditionally used this show as the launch pad for its new products and kbb Birmingham 2014 will be no exception. Three new colours, available from April this year, will be previewed at the show. The new effects will not only add further diversity to proven colour groups but will also invite designers to create bold colour schemes by using these new finishes as strong statement pieces or indeed as a complement to interesting door and fascia combinations.
This year's exhibition will also see the launch of a brand new Mistral brochure. Karonia firmly believes 'seeing is believing' and the brochure will use real-life installations to demonstrate the tremendous scope for imaginative design that this versatile, completely solid product offers.
Image: Glacier from the Mistral range of 25mm solid surfacing with inset wooden chopping board
W: www.karonia.com
7th February 2014


text:
I really wanted to make a card today that was not a client card. I've been so busy with those that I haven't had time to participate in any challenges for at least two weeks. I decided to use Susan Raihala's OLW14 Challenge which is to make a card using an outline stamp with little or no colouring. Here's my card except for one important detail... it was supposed to be square! This card uses more of the scrumptious stamps from Unity's July KOTM. Since little or no colouring was allowed, I used various colours of ink for the apples, stems and leaves. It was a bit difficult since I don't have markers but I managed to press the corners of my ink pads into the appropriate areas. I rushed to do this before the sun went down so I could photograph it. Therefore, I won't be making a square version. Sorry! One is enough for this tired brain. Have a great night, everyone! My husband should be home with some Italian wine very soon. :)
Sources: GinaK 120lb pure luxury white cardstock; Unity July 2010 Kit of the Month - Happily Ever After; Vivid Red & ButterScotch inks; SU Garden Green ink; Memories Artprint Brown ink


text:
What is life planning? Life planning is all about making sound decisions and deciding what you want to be when you grow up. It includes assessing your interests, exploring career options, using labor market information and making career decisions. On Cape Cod and the Islands we have many choices but the first place to start is by charting your course or coming up with a plan.
Career decision making is probably one of the most important decisions you will ever make. This decision should not be made lightly. Careful thought, research and consideration should be made before making a decision. There are seven steps to sound decision making. These seven steps can help you decide what to wear in the morning, what car you would like to buy all the way to deciding what career would most interest you.
Before deciding on a career you must complete the following steps:
- Evaluate your skills, interests and abilities
- Decide on your work values
- Use the 7 step process to choose a career
How do you evaluate your skills, interests and abilities?
The following links bring you to several online assessment tools that can help you understand yourself and what you like to do.
- Mass Career Information System career cluster inventory helps you assess what you enjoy doing and the careers that use similar skill interests. Mass Career Information System Career Cluster Inventory
- Career Games is a site that features games, workshops and assessments to help you discover what your skills are, what types of jobs/ occupations interest you, job hunting tips, interview help, etc. Career Games
- My Next Move – an O*NET Interest Profiler can help you find out what your interests are, how they relate to the world of work and what kinds of careers you might want to explore. My Next Move
- The Perfect Career – an online tool that helps you "discover the career that's right for you!" The Perfect Career
What ideals and core beliefs are most important to you? What motivates you to work hard?
You will most likely to be comfortable and thrive in a work environment that is compatible with your own values. Difficulties may arise if you find yourself in conflict with a work situation or environment that conflicts with your values. Here are links to sites that can help you discover your core values:
- Core Values Exercise – a PDF that helps you evaluate 150 key values The Core Values Exercise
- Identify Personal Interests, Skills and Values – a Self-assessment tool that is a foundation for career decision making, planning and action. Assessing and understanding your interests, skills and values is the first step in identifying the career path that is right for you. The Self Assessment Tool
Read the entire article about Choosing a Career at this link.
1. Assess yourself – Each individual has different goals, talents, interests, and values. In other words, there are certain careers each individual should not pursue and others where they'd excel and be satisfied.
2. Make a list of potential occupations– After conducting some self-assessment, it should be apparent the types of careers you should pursue. It is not possible to pursue each one, so the list should be used to determine where to begin your career search.
3. Explore the options – Learn about each potential career after narrowing your list. Be sure to learn about educational or training requirements, job duties, employment outlook, annual earnings, and promotion opportunities. Information can be obtained from the Internet, but try to meet with a professional in each field to obtain in-depth details about each profession.
4. Narrow down your list – Eliminate careers that no longer interest you after thoroughly reviewing each one.
5. Set goals – After your list has been narrowed, establish attainable goals. You should be informed enough to establish short and long term goals. Typically, short-term goals can be met between 1-3 years and long-term between 3-5 years.
6. Create a career action plan – Once you've established career goals, begin developing a career action plan consisting of goals and specific steps to reach them.
7. Obtain training – Obtaining required career training will probably consume the majority of your time and efforts as you pursue a career.
Didn't Find Your Answer? Maybe the Career Center can help
Please don't hesitate to contact us and we will be happy to help you find the answers to your questions!


text:
MIDDLETOWN — Set to play on a chilly Saturday morning, Marlboro's Ryan Cary packed a few essentials for the Section 9 Class B semifinals.
MIDDLETOWN — Set to play on a chilly Saturday morning, Marlboro's Ryan Cary packed a few essentials for the Section 9 Class B semifinals.
Under Armour.
Hand warmers.
Still, it didn't take Cary long to warm up at blustery Faller Field. He answered a quick Red Hook score in the first quarter with a 73-yard kickoff return for a touchdown, and Marlboro rolled from there.
The Iron Dukes are going back to the Class B championship for the second year in a row after a convincing 49-12 win. Marlboro (8-1) plays Spackenkill in the Class B championship at 7 p.m. Saturday at Dietz Stadium in Kingston. Spackenkill defeated Marlboro, 21-6, Sept. 17, but the Iron Dukes have won six games in a row since.
"It was nice, it changed the whole momentum of the game," said Cary, who lost his hand warmers on the kickoff return. "We had the momentum back and finished it. It showed them that we were in the game. Just because they scored, doesn't mean we are out of it. I didn't need the hand warmers, I guess."
Red Hook (6-3) took a 6-0 lead just 3:49 into regulation on a 2-yard run by Kirby Roeder. It was set up by a blocked punt by Brandon Tator and the Raiders seemed poised to give Marlboro all it could handle. The teams played Oct. 14 with the Iron Dukes winning, 45-28, but in that game Marlboro led by only three points with five minutes remaining.
Cary's kickoff return, up the right side, was the start of 28 unanswered points by Marlboro (8-1). Marlboro quarterback Mike Schoonmaker added a 20-yard touchdown score and hit Cary with a 5-yard score. An 80-yard kickoff return by Willie Brown to start off the third quarter made it 28-6 Marlboro.
Schoonmaker rushed for 77 yards and two touchdowns and Cornelius Williams had 107 rushing yards and a score. Matt Tamburri added a 32-yard interception return for a touchdown for the Iron Dukes.
"Ryan (Cary) did everything he could to stop Red Hook with that kickoff return," Schoonmaker said. "Red Hook is a phenomenal teams and we had to hold them back. That is a deflating thing. You score and you expect to have the momentum. Ryan did a great job."
Now, Marlboro tries to do something it's never done in school history. For all of its success, the Iron Dukes have never won a Section 9 title. Marlboro is 0-8 in Section 9 championship games (0-5 in Class B: 1994, 2000, '04, '06, '10; 0-3 in Class C: 1990, '92, '95). The Iron Dukes lost to New Paltz 13-0 in last year's Class B championship.
"We wanted to go to the championship game and bring the first Section 9 title to Marlboro," Schoonmaker said. "It's been our goal since day one. We have talked about it during the regular-season and it will be our goal until we win it."


text:
Our Vision & Mission
At Gerry Dunleavy Construction, we believe the homeowner, architect, designers, and our entire staff should work as an assembled team where every idea has merit. As a building company, we focus on one main goal, which is to deliver the best possible product safely, at a fair price, and in a timely fashion. Our staff are instructed to respect your privacy and ensure that the home remodeling process is as stress-free as possible.


text:
Title: Keep That
Author: Fidomom
Disclaimer: This is a work of fiction. It is only loosely based on characters from the tv series Roswell as I imagine they could be from different points throughout and beyond the three seasons of the show. I am not affiliated with any of the original creations nor creators from the series. I will make no monetary gains from publishing this work of fiction.
Pairings/Couples/Category: Michael/Maria, CC
Rating: Adult
Summary: Michael's Christms gift from Maria is something he gets to keep.
Author's Note: The episode A Roswell Christmas Carol always bothered me. Maria made such a huge deal about EXCHANGING gifts with Michael on Christmas Day. He got her that bumper and she got real pearl earrings but then we never got to see what she got for him for Christmas. I made up a shippy reason why not, lol. This story picks up moments after she gets the earrings.
Also: In all my Candy stories, I treat spaceboy as a term of endearment like honey, so I rarely capitalise it, and I choose to make it all one word like Superman.
Keep That
by Fidomom
Michael looked at Maria expectantly and asked, "So since you made such a big deal about us exchanging gifts, where's mine, then?"
Maria grinned and said conspiratorially, "Bet you think I didn't bring it, right? No worries, spaceboy, you'll get the gifts I got you, that's right, plural, I was feeling very moved by the Christmas spirit. Okay well, actually, one of them is just for you, it's in the car but the other one is more kind of for both of us."
Michael narrowed his eyes and asked, "You went over the agreed limit, didn't you?"
Maria cutely pouted and admitted, "Maybe just a little. Are you mad?"
Michael sighed heavily, shook his head briefly and declared, "Not when you look at me like that. So what did you get us?"
Maria squirmed off his lap, reached for his hand and tugged him to his feet as she excitedly suggested, "We're gonna need to go indoors for that! Come on, come on, Michael!"
Michael grumbled a little but followed her indoors. Maria closed all the curtains and chain locked the front door. Michael watched her suspiciously but didn't say anything until she started taking off her clothes, "Whoa! Hold up! What are you doing, Maria?"
Maria chuckled and declared, "You are surprisingly a bit of a prude when getting my clothes off is not your own idea! Relax, spaceboy; I'm not going full birthday suit."
Michael frowned when she stopped undressing and stood in front of him in a bra and a matching pair of drawstring men's boxers. He was clearly baffled when he asked, "How is this a gift for both of us?"
Maria smirked, undid the drawstring, let the boxers fall in a puddle around her feet, leaving her in very skimpy matching bikini briefs and informed him, "Those are for you. I uhh like you in boxers, and what's left is technically for me but I was definitely thinking of you when I gave into the impulse. Do you like any of this? Did I disappoint you?"
Michael stared intently at her eyes and asked, "Depends on if the rest of that is coming off now, too, or not. And why Aladdin?"
Maria looked momentarily disconcerted and she started to explain, "Well, because of the day you -"
Michael's eyes glinted in mischief and he admitted, "I was just kidding, Maria. I got the reference. I like this part of my gift. Don't know how you're going to top this, though."
Maria stepped out of the boxers, picked them up and handed them to Michael, "I don't know that the other part will necessarily top this, but we'll get to that later. I'm gonna need you to model these for me, and then it seems to me that you and I are long overdue for a massive make out session. Any objections?"
Michael wordlessly shook his head and stripped down to the underwear he was already wearing. He motioned for her to turn around while he changed into the Aladdin boxers and Maria laughed but complied as she teased, "It's not like we haven't seen and are not about to see each others everything, anyway, Michael! Really now!"
Michael argued, "Context matters. Okay, you can look now. I feel more than a little ridiculous."
Maria's eyes slowly roved over his body, she bit her lip and she assured him, "You look about as far from ridiculous as I can imagine. You can go ahead and label those your lucky shorts. Damn, spaceboy."
Michael's brow twitched upward briefly and he nodded as he spoke, "Duly noted. Definitely not disappointed. Come closer, Maria."
Maria stood belly to belly with him, put her arms around his neck and asked, "Is this close enough?"
Michael shrugged and answered, "It'll do. For now. How's it going?"
Maria laughed and teased him, "Oh, so we're doing the exchanging pleasantries thing today, are we? It's umm, it's going. You?"
Michael stopped just a breath shy of kissing her on the lips before answering, "Day just keeps getting better and better.", and then he kissed her slowly and thoroughly on the lips for several minutes.
Maria struggled to come up for air first and she suggested, "Couch or bed, Michael, I don't care which one, but I intend to give you some very pleasant memories to associate with your gift."
Michael kissed her neck and face over and over as he shuffled them to the couch, explaining, "It's closer. What kind of memories?"
Maria slipped her hand inside his boxers and stroked his erection, as she spoke huskily into his ear, "Well, it's nothing to do with talking or kissing but it is to do with something my mouth could be doing."
Michael stilled her hand with his own and asked her in concern, "You know you don't have to go that far with me, right? I'm fine with just making out and touching each other."
Maria kissed him softly on the lips and nodded. answering, "I know but I want to, Michael. Admittedly, I don't really know how yet but I have some ideas. I think we can figure it out together. Unless, you're specifically against it for some reason?"
Michael shook his head and let her continue touching him. He clarified, "It's not that. I just don't want you to think it's a problem for me if you don't. Just, you know, don't do anything you aren't comfortable with or ready for. Okay? Oh, God, Maria, honestly that already feels good enough with just your hand."
Maria looked him in the eye and said sincerely, "I really want to at least try it, Michael. Maybe we're missing out on something really great? But you know, like you said to me, if you aren't into it we can just stick to what we already know."
Michael caressed her cheek with his thumb, kissed her gently on the lips once and then nodded as he said, "Okay. You can go for it Just no faking allowed; if you don't like it, I want you to stop. Deal?"
Maria smiled at him appreciatively, gave him a hug and a quick kiss on the lips, and agreed, "Deal. Thank-you, Michael."
Michael let Maria physically position him exactly how she wanted him and he watched in nervous anticipation as her mouth got closer and closer to his naked erection. He sucked in a hard breath as he saw her tongue make contact in the same moment that he felt it. He closed his eyes, bit his lip and mangled a throw cushion in his clenched fist, as a wet, hot sensation enveloped his erection. He groaned in surprised pleasure when she started to suck, "Oh God, Maria. Maria. Is this really okay for you?"
With her mouth still full she answered, "Mmmmhmmm.", and Michael blindly reached for her hair. He slid his fingers into her hair close to her scalp and rubbed his thumb pad against her hair over and over again.
Despite Maria's inexperience and likely because of Michael's, their little experiment didn't take very long. Michael tried to warn her but he wasn't fast enough. Maria choked a little and seemed about to move away and then she made an odd noise and started sucking and swallowing voraciously until Michael was fully spent. She finally released him, gently returned his family jewels to the confines of his boxers, daintily pinched the corners of her lips between her thumb and forefinger, licked both afterward and then announced, "We're gonna need to do that some more. All that sugar you consume. Apparently it goes somewhere. Can I assume you're fine with this plan?"
Michael's brow twitched in amusement. He nodded and agreed, "As long as you enjoy yourself, yes. Are you interested in reciprocation, Maria?"
Maria blushed and fidgeted, then shook her head, "Interested, yes, ready for that, not so much. I mean you're good with your fingers; as you know full well, I about lose my mind when you touch me. I just don't think I could handle more pleasure than that and from what I've heard, it's far more intense when it's oral. We're intense enough, already, for me. For now, anyway. Are you disappointed?"
Michael shrugged, nodded and admitted, "Maybe a little. I've been curious. I don't mind waiting until you're ready and hey if that is something we never end up trying together I'll be okay with that, too. I feel no urgency to pressure you. Like you said we get plenty intense doing what we've been doing. It's not broke, so we don't need to try to fix it. Mission accomplished by the way."
Maria looked at him curiously and asked, "What do you mean?"
Michael pulled her onto his lap and kissed her, then answered, "Very pleasant memories associated with my lucky Aladdin boxers. Thank-you for the gifts. I love you, Maria. Merry Christmas."
Maria leapt off his lap with a start and exclaimed, "But I didn't give you the one that's only for you, yet! Wait here! I'll just get dressed and go back to the car to get it. But don't you get dressed, Michael! We're not done, okay?"
Michael made a point of lounging on the couch, like he was on vacation and he said, "I'll be right here waiting for you."
Maria returned less than ten minutes later, an apology on her lips, "I'm sorry I didn't wrap it up all Christmas-y. But here.", and she handed him a shadowbox, inside of which was a parchment, with words on it, written in calligraphy.
Michael read it and looked at Maria in surprise, "You found this in a shop? How? I've looked for years and never found anything like this."
Maria shook her head and corrected him, "No, I did not find it in a shop. Oh. Well I mean the shadowbox yes, because we both know how I am with woodworking, but no I wrote that out myself and put it in there. You are looking at attempt number five. The first one I smeared the first line, the second one I missed a word, the third one I heard a loud noise and suddenly there was a huge line across what I'd already done. The fourth one though was the worst, it was almost done, and I knocked my ink bottle over onto it.
Calligraphy is an artform, Michael, I got hand cramps even, going through so many attempts before I finally got it to look like that. So yeah, I definitely put thought into that and I hope it's personal enough. Do you like it?"
Michael was staring at the calligraphy on the parchment and he spoke quietly, "I love it, Maria. Thank-you. I mean it. I can't believe you actually remembered about this quote from Ulysses."
Maria clapped her hands happily and instructed, "Oh good! I'm so glad you like it! Turn it over! I left you a little love note on the back panel, too!"
Michael turned it over and read out loud, "Merry Christmas, Michael. You abducted more than me and the Jetta that day; you abducted my heart. It's okay; you get to keep that. Love Always, Maria. XO Wait, is this your actual kiss print, Maria?"
Maria laughed and nodded, bragging proudly, "Of course it is! And red, of course, you know, to be sort of Christmas-y. Too mushy for you, spaceboy?"
Michael set the shadowbox aside, put his arms around Maria, kissed the side of her head and answered, "No. Not even close. I love you, too. Merry Christmas, Maria."
Maria leaned back in his arms, smiling up at him flirtatiously, as she suggested, "Actions, pal-ly. Please explain why I'm still fully clothed?"
Michael chuckled briefly, kissed her neck and whispered against her ear, "You won't be, for long enough to get through the explanation.", and he made good on his words.
Keep That by Fidomom (CC,M/M,ADULT) Pt 1/1 - 01/02/18
This is the place to post all your General Roswell fanfiction. Any Canon fics, which pick up directly from any episode of the show and that focus on Max/Liz, Michael/Maria, Isabel/Alex or Isabel/Jesse, Kyle/Tess, or all the couples together! Rule of Thumb: If Max healed Liz in the Crashdown in September 1999, then your fic belongs here. If it picks up from the show in any way, it belongs here.
Moderators: Anniepoo98, Rowedog, jbangelo, ISLANDGIRL5, Itzstacie, truelovepooh, FSU/MSW-94, Forum Moderators
Post Reply
3 posts • Page 1 of 1
- Fidomom
- Enthusiastic Roswellian
- Posts: 50
- Joined: Wed Nov 29, 2017 11:35 pm
- Location: Vancouver, BC Canada
- Contact:
Keep That by Fidomom (CC,M/M,ADULT) Pt 1/1 - 01/02/18
I'm also a moderator at Roswell Ardor fan fiction forum.
I'm also a moderator at Roswell Ardor fan fiction forum.
- Roswell Fanatic
- Posts: 2373
- Joined: Thu Jun 28, 2007 9:34 pm
Re: Keep That by Fidomom (CC,M/M,ADULT) Pt 1/1 - 01/02/18
Love the Aladdin boxers!
Michael got quite a Christmas present after all.
Michael got quite a Christmas present after all.
- Fidomom
- Enthusiastic Roswellian
- Posts: 50
- Joined: Wed Nov 29, 2017 11:35 pm
- Location: Vancouver, BC Canada
- Contact:
Re: Keep That by Fidomom (CC,M/M,ADULT) Pt 1/1 - 01/02/18
He deserved something special - that bumper was so thoughtful! Plus he let Maria keep the very expensive earrings knowing he'll have to pay Isabel back for them. Thanks for the feedback, Carolyn.keepsmiling7 wrote:Love the Aladdin boxers!
Michael got quite a Christmas present after all.
I'm also a moderator at Roswell Ardor fan fiction forum.
I'm also a moderator at Roswell Ardor fan fiction forum.
Post Reply
3 posts • Page 1 of 1


text:
Oh baby, it's a wild world! It sure is. So many awesome places to visit. The green elf-laden landscape of Scotland, the turquoise waters of exotic white sand beaches, and the temples and pyramids of Mexico and Egypt. There is so much to see, so much to experience. There are so many doors to be opened by living amidst other ...Read More »
Travel Medicine
A penis fungal infection can affect a man at any point in his life. He doesn't have to be sexually active to contract candida albicans, however if a man is sexually active with a partner who has an active vaginal yeast infection outbreak, he is certainly at risk for getting yeast in his penis (and surrounding genitals). Since these types ...Read More »
Infectious Diseases of Scalp and Hair
There are two main types of hair and scalp diseases: infectious and non-infectious. Infectious diseases are those that they can go contagiously from one man to another. The most common examples of them are: impetigo, head-lice, ringworm, scabies, warts and some more. Severity of these infections and their danger to surrounding people may differ greatly. Therefore, it is of primary ...Read More »
What Is The Cost of Travel Vaccinations?
If you decide to travel to a foreign country like Africa means that you are going to need to get the required vaccinations to be able to travel to the country you desire. Travel vaccinations should be done at least four to six weeks before traveling outside of the United States. There are a number of vaccinations required by the ...Read More »
How To Recognize Fish Disease Symptoms
When my son was small, one of the angelfish that lived in his aquarium suddenly became covered in what looked like a white film and after a few days, his fins started becoming damaged. After a few more days, the fish died and others started getting the same condition. It was not long before the aquarium had no fish in ...Read More »
Scalp Infections – Symptoms, Causes & Natural Options For Fast Relief & Treatment
There are many definitions and symptoms commonly described as "scalp infections". There are in fact quite a few quite different reasons why your scalp may be sore, itchy, inflamed burning red or infected. If you are like many people with chronic scalp issues – it may be driving you completely up the wall and not knowing what it is or ...Read More »
Tropical Disease Dengue Fever Found in Florida
Most Americans have never heard of dengue fever. It is an ugly tropical disease that recently showed up in Key West, Florida. The symptoms include a fever, serious headaches, chills, muscle and joint pain and bloody urine. Although the disease is rarely fatal, it is downright unpleasant. And it is dangerous to those with a compromised immune system or other ...Read More »