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Adenovirus early region 4 open reading frame 4 (E4orf4) protein has been reported to induce p53-independent, protein phosphatase 2A (PP2A)–dependent apoptosis in transformed mammalian cells. In this report, we show that E4orf4 induces an irreversible growth arrest in Saccharomyces cerevisiae at the G2/M phase of the cell cycle. Growth inhibition requires the presence of yeast PP2A-Cdc55, and is accompanied by accumulation of reactive oxygen species. E4orf4 expression is synthetically lethal with mutants defective in mitosis, including Cdc28/Cdk1 and anaphase-promoting complex/cyclosome (APC/C) mutants. Although APC/C activity is inhibited in the presence of E4orf4, Cdc28/Cdk1 is activated and partially counteracts the E4orf4-induced cell cycle arrest. The E4orf4–PP2A complex physically interacts with the APC/C, suggesting that E4orf4 functions by directly targeting PP2A to the APC/C, thereby leading to its inactivation. Finally, we show that E4orf4 can induce G2/M arrest in mammalian cells before apoptosis, indicating that E4orf4-induced events in yeast and mammalian cells are highly conserved.
Protein phosphatase 2A (PP2A),* one of the major protein serine/threonine phosphatases in the cell, plays a role in several cellular processes, including metabolism, transcription, RNA splicing, translation, cell cycle progression, morphogenesis, signal transduction, development, and transformation (Mumby and Walter, 1993; Wera and Hemmings, 1995). The predominant form of PP2A in cells is a heterotrimer consisting of three subunits. The 36-kD catalytic C subunit and the 63-kD regulatory A subunit (PR65) form the core enzyme, and the B subunit binds the core enzyme to form the holoenzyme. The A and C subunits both exist as two isoforms (α and β), which are closely related, whereas the B subunit is variable and its multiple isoforms belong to at least three unrelated gene families, B/B55/PR55, B′/B56/PR61, and B″/PR72/PR130 (Kamibayashi et al., 1994; McCright and Virshup, 1995; Csortos et al., 1996). The cellular B subunits can also be replaced by viral proteins, such as the SV40 small t antigen and the polyomavirus small and middle T antigens (Mumby, 1995).
In Saccharomyces cerevisiae, two closely related genes, PPH21 and PPH22, redundantly encode the major PP2A catalytic subunit (Sneddon et al., 1990). TPD3 encodes the only A subunit, and two distinct B subunits, encoded by CDC55 and RTS1, are homologous to mammalian B and B′, respectively (Healy et al., 1991; van Zyl et al., 1992; Shu et al., 1997). Mutation of both PPH21 and PPH22 eliminates most of the PP2A activity in the cell and drastically reduces growth. Strains lacking PPH21, PPH22, and a third related gene, PPH3, are completely inviable (Ronne et al., 1991). Two other PP2A-like phosphatase catalytic subunits exist, encoded by SIT4 (Sutton et al., 1991) and PPG1 (Posas et al., 1993), which perform nonredundant functions in the cells.
Mutations of CDC55 yield defects in cytokinesis and result in abnormal cell morphology at low temperature, whereas mutation of RTS1 results in growth defects at high temperature (Healy et al., 1991; Shu et al., 1997). PP2A was proposed to play a role in activation of Clb–Cdc28 kinase complexes for progression from G2 to mitosis (Lin and Arndt, 1995). The effect of Cdc55 on cellular morphogenesis is also mediated through Cdc28, and it was proposed that PP2A, regulated by Cdc55, affects the activity of the Cdc28 regulators Mih1 and Swe1 (Minshull et al., 1996; Wang and Burke, 1997; Yang et al., 2000). CDC55 was also implicated as a component of the spindle checkpoint pathway: cdc55Δ mutants are nocodazole sensitive (Wang and Burke, 1997), and in the presence of a defective spindle, Cdc55-deficient cells are still able to separate their sister chromatids and to leave mitosis even though cyclin B destruction is prevented, possibly by promoting inactivation of Cdc28 through inhibitory phosphorylation (Minshull et al., 1996).
Adenovirus early region 4 open reading frame 4 (E4orf4) protein is a multifunctional viral regulator. Our work, as well as work from other laboratories, has shown that the E4orf4 protein downregulates expression of genes that have been activated by E1A and cAMP (Müller et al., 1992; Kleinberger and Shenk, 1993), induces hypophosphorylation of various viral and cellular proteins (Müller et al., 1992; Kanopka et al., 1998), regulates alternative splicing of adenovirus mRNAs (Kanopka et al., 1998), and induces p53-independent apoptosis in transformed cells (Lavoie et al., 1998; Marcellus et al., 1998; Shtrichman and Kleinberger, 1998). Induction of apoptosis has been reported to be caspase independent in CHO cells (Lavoie et al., 1998), but it is caspase dependent in 293T cells (Livne et al., 2001). We have previously shown that E4orf4 interacts with the PP2A holoenzyme through a direct association with the Bα/B55 subunit (Kleinberger and Shenk, 1993). Interaction with PP2A complexes that include the Bα/B55 subunit is required for induction of apoptosis by E4orf4 (Shtrichman et al., 1999). The presence of an active PP2A also contributes to other functions performed by the E4orf4 protein, including down regulation of transcription (Kleinberger and Shenk, 1993; Bondesson et al., 1996; Whalen et al., 1997) and alternative splicing (Kanopka et al., 1998). Recently, we demonstrated that E4orf4 associates not only with PP2A-Bα, but also with several isoforms of PP2A-B′. However, only the interaction of E4orf4 with a PP2A holoenzyme population that includes the Bα subunit is required for induction of apoptosis (Shtrichman et al., 2000).
A key unresolved question is the identity of the targets of the PP2A-Bα–E4orf4 complex involved in induction of apoptosis. To gain insight into the cellular pathway induced by the PP2A-Bα–E4orf4 complex, we expressed E4orf4 in the yeast S. cerevisiae. Although yeast do not possess some of the cellular machinery required for the apoptotic process, such as caspases and Bcl-2 family members, this organism can serve as a powerful tool for apoptosis research (Matsuyama et al., 1999). Here, we report that in yeast, expression of E4orf4 induces irreversible growth arrest through a mechanism that requires Cdc55 and Tpd3, the yeast homologues of the B and A subunits of PP2A. The cells arrested in mitosis and exhibited enhanced accumulation of reactive oxygen species (ROS), a feature of apoptosis in metazoan cells. Our data suggest that E4orf4 acts via modulation of the activity of the anaphase-promoting complex/cyclosome (APC/C) ubiquitination complex. Furthermore, we show that E4orf4 can induce G2/M arrest before apoptosis in mammalian cells, suggesting that E4orf4-induced events are conserved in yeast and mammals.
E4orf4 induces irreversible growth arrest in yeast
Since E4orf4 binds the PP2A-B subunit, and yeast and mammalian PP2A-B subunits share an extensive homology (53% identity and 67% similarity) (Healy et al., 1991), we tested whether expression of E4orf4 may affect yeast cell growth in a PP2A-dependent manner. Wild-type E4orf4 and the A3 mutant, which did not bind an active PP2A and did not induce apoptosis in mammalian cells (Shtrichman et al., 1999), were cloned in a yeast vector under the control of the GAL1,10 promoter. On galactose plates, expression of E4orf4, but not of mutant A3, prevented yeast growth, whereas no growth defect was apparent on GAL1,10-repressing glucose plates (Fig. 1 A). This growth inhibition depended on Cdc55, because in a cdc55Δ strain, growth was unaffected by E4orf4 (Fig. 1 B). Reintroduction of the CDC55 gene into the cdc55Δ strain reinstated E4orf4-induced toxicity in these cells (Fig. 1 B). A yeast strain lacking the RTS1 gene did not lose the ability to respond to E4orf4 (Fig. 1 C), indicating that, as in mammalian cells, Cdc55/B but not Rts1/B′, is required for E4orf4-induced toxicity. Deletion of the TPD3 gene, homologous to mammalian PP2A-A, also resulted in loss of the cellular response to E4orf4 (Fig. 1 D). The altered response to E4orf4 did not result from changes in levels of E4orf4 expression (Fig. 1 E). The E4orf4-expressing plasmid was introduced into yeast strains lacking each of the PP2A-like catalytic subunits: Pph21, Pph22, Pph3, Sit4, and Ppg1. Each of these deletion strains maintained the response to E4orf4 expression (Table I), suggesting a redundancy in the catalytic subunit required for the response to E4orf4.
We next investigated whether the E4orf4-induced growth arrest was reversible. E4orf4 expression was induced by galactose in liquid medium, and growth was monitored by a general count using a hemacytometer, and by a viable count. Fig. 2 A shows that until 6 h after induction there was no change in the apparent growth rate of cells expressing E4orf4 versus control cells. However, at later times, E4orf4-expressing cells showed a slower growth and stopped growing by 10 h after induction. If the cells were diluted to 3 × 106 cells/ml in fresh galactose-containing medium at that time, the E4orf4-containing cells did not regain exponential growth, whereas control cells did (Fig. 2 B). Residual cell growth seen at late time after E4orf4 induction may reflect selection in liquid medium of cells carrying fewer copies of the multicopy 2μ plasmid and expressing less E4orf4. Indeed, Western blot analysis indicated that at 24 and 48 h after induction, E4orf4 levels in the population were lower, compared with 3 and 6 h after induction (results not shown). When E4orf4-expressing cells were transferred to glucose medium, the GAL promoter was shut off and E4orf4 protein levels decreased (results not shown). Nonetheless, cell viability, measured as the ability to produce colonies on glucose plates, dropped rapidly within a few hours of growth in the galactose-containing medium (Fig. 2 C). These results indicate that E4orf4-induced growth arrest is irreversible.
It has been reported that cell death induced in yeast by mammalian proapoptotic genes, such as Bax, is accompanied by changes in cell membranes and DNA degradation, typical of mammalian apoptosis (Zha et al., 1996). We tested whether E4orf4 induced similar changes. However, no alterations in trypan blue exclusion were observed at several time points after induction of E4orf4 expression, and no DNA degradation was detected on agarose gels as late as 48 h after induction (results not shown). Furthermore, it has been previously reported that Bax induction in yeast inhibited cell growth in all cells, but it did not lead to death in cells lacking functional mitochondria (petite) (Greenhalf et al., 1996). We investigated whether E4orf4 expression in petite (rho-0) cells resulted in cell death, measured by colony formation. E4orf4 was expressed from a tetracyclin-regulatable promoter (Gari et al., 1997), since petite cells did not grow well on galactose. E4orf4 induction by doxycycline removal was slower compared with its induction by galactose, and 6 h after induction, its expression reached levels comparable to those seen upon galactose induction 3 h after induction (results not shown). Cells were replated on plates containing doxycycline at various times after induction. Fig. 2 D shows that loss of viability upon induction of E4orf4 was similar in wild type and in petite yeast cells. The toxic effect appeared later than with galactose-induced E4orf4, probably due to the slower rate of E4orf4 induction by doxycycline removal. Thus, this experiment demonstrates that E4orf4-induced toxicity is independent of functions encoded by mitochondrial DNA. The cellular events induced by E4orf4 appear to differ from those induced by Bax expression.
E4orf4 enhances cellular ROS levels
It has been recently reported that generation and accumulation of oxygen radicals is a key event in apoptotic pathways both in yeast and in mammalian cells (Hockenbery et al., 1993; Kane et al., 1993; Slater et al., 1995; Madeo et al., 1999). We tested whether E4orf4 induced enhanced ROS levels in yeast cells. E4orf4 expression was induced with galactose for 6 h, and cells were then stained with 1 μg/ml dihydroethidium for 10 min. Dihydroethidium is oxidized specifically by superoxide ions to fluorescent ethidium (Budd et al., 1997). Fig. 3 (left) shows an example of E4orf4-expressing cells, which accumulated ROS, as compared with control cells. Fluorescence levels in the cells were measured by FACS® analysis, and Fig. 3 (right) demonstrates that 26% of E4orf4-expressing cells were stained above background levels, whereas only 3.3% of vector-containing cells fluoresced above background. Staining with dihydrorhodamine 123, a compound that is oxidized to the fluorescent chromophore rhodamine 123 by ROS (Schultz et al., 1996), led to similar results (data not shown).
E4orf4 induces G2/M arrest
Microscopic examination of cells expressing E4orf4 showed striking morphological changes: 8 h after induction, cells were greatly enlarged, and many displayed abnormal buds. To determine whether E4orf4 affects the cell cycle, DNA content of cells expressing E4orf4 from the GAL promoter was analyzed by FACS®. Fig. 4 A shows that within 4.5 h of galactose induction, E4orf4-expressing cells started to accumulate in G2/M. To determine at what stage of mitosis the cells arrested, we visualized the spindles with green fluorescent protein (GFP)–tubulin. Fig. 4 B shows that cells were arrested at several mitotic stages. Most cells were arrested either with short preanaphase spindles, or with extended telophase spindles (49 and 40%, respectively; n = 100). Some of these cells had undergone a second round of budding. After longer induction times, chains of unseparated buds became visible, and anucleated cells started to accumulate (not shown).
E4orf4 expression is synthetically lethal with reduction in Cdc28 activity
To pinpoint the cell cycle target of E4orf4, specific cell cycle mutants were assayed for synthetic effects with E4orf4 expression. To that end, an alternative expression vector was used, expressing lower levels of E4orf4 upon galactose induction (pDAD-E4orf4). Comparison between levels of E4orf4 expression from the pDAD and p426-GalL vectors is shown in Fig. 5 A. When pDAD-E4orf4 was introduced into wild-type yeast cells, only a weak effect on yeast growth was detected, manifested by a slightly smaller colony size, compared with control cells (Fig. 5 B). However, mutants of the main yeast cell cycle kinase, Cdc28, were significantly more sensitive to E4orf4 expression. Temperature-sensitive CDC28 mutants, such as cdc28-4 and cdc28-1N (Surana et al., 1991), as well as a mitotic cyclin–defective strain (clb1Δ clb2ts clb3Δ clb4Δ), were unable to grow even in the presence of the low levels of E4orf4 expressed from the pDAD vector (Table I). In contrast, cell cycle mutants specifically affected in the G1/S transition, such as cdc34 or cdc53, or mutants affected in mitotic exit, such as cdc15 or sic1Δ, were not supersensitive to E4orf4 (Table I), confirming that E4orf4 acts specifically at mitosis. The kinase Swe1 (the S. cerevisiae homologue of mammalian Wee1) and the phosphatase Mih1 (the S. cerevisiae homologue of Cdc25), respectively, inhibit and activate Cdc28 activity (Russell et al., 1989; Booher et al., 1993). mih1Δ cells were more sensitive to E4orf4 expression (Fig. 5 B), and overexpression of Swe1 was synthetically lethal with E4orf4 expression (Table I), suggesting that E4orf4 may act via activation of the inhibitory kinase Swe1. However, deletion of SWE1 did not suppress the toxicity of E4orf4 (Table I). Furthermore, although Swe1 and Mih1 both act on Tyr19 of Cdc28, expression of the constitutively active Cdc28-F19 mutant in wild-type cells did not suppress the toxicity of E4orf4 (Table I), suggesting that in these cells the mechanism of action of E4orf4 does not involve modulation of phosphorylation of Cdc28 on Tyr19.
To further investigate the E4orf4 effect on Cdc28 activity, Cdc28-Clb2 kinase activity was compared in cells containing p426-GalL-E4orf4 or the empty vector 6 h after induction with galactose. As seen in Fig. 5 C, enhanced kinase activity was observed in wild-type cells in the presence of E4orf4, whereas no differences in kinase activity were seen in an mih1Δ mutant. The levels of Clb2 protein were similar in control cells and in cells expressing E4orf4 (Fig. 5 D). These results indicate that E4orf4 expression leads to an Mih1-dependent increase in specific Cdc28-Clb2 kinase activity. Thus, the hyperactivation of Cdc28 by E4orf4 may be a secondary E4orf4 effect that partially counteracts the main E4orf4 inhibitory effect on cell cycle progression. The inability of the mih1Δ strain to hyperactivate Cdc28 by Tyr19 dephosphorylation might explain its supersensitivity to E4orf4. To test this hypothesis, the nonphosphorylatable Cdc28-A18F19 mutant was introduced in the mih1Δ strain. The resulting strain reverted to a level of E4orf4 sensitivity similar to the wild-type strain (Fig. 5 E), indicating that inability to dephosphorylate Cdc28 is the reason for the hypersensitivity of the mih1Δ mutant to E4orf4.
E4orf4-expressing cells are supersensitive to benomyl
Cdc55 has been identified as a component of the spindle assembly checkpoint (Wang and Burke, 1997). Mutations in genes that affect the kinetochore/spindle checkpoint result in cells that are more sensitive to the microtubule-depolymerizing drug benomyl than wild-type cells, and deletion of CDC55 causes enhanced benomyl sensitivity (Minshull et al., 1996; Wang and Burke, 1997). We tested whether E4orf4 affects benomyl sensitivity of cells. Yeast cells expressing E4orf4, mutant A3, or the empty vector were serially diluted and plated on galactose plates containing sublethal concentrations of benomyl. Fig. 6 shows that cells expressing low E4orf4 levels are highly sensitive to benomyl, compared with control cells. Cells that express mutant E4orf4 (A3), even at high levels, are no more sensitive to benomyl than are the control cells. Thus, E4orf4, through its interaction with PP2A, may functionally interact with the spindle assembly checkpoint, or it may directly inhibit the target of this checkpoint. Several yeast strains containing mutations in genes involved in spindle checkpoint control were tested for a synthetic effect with E4orf4. However, these genes, including MAD1, -2, -3 and Bub1 and -2 did not manifest such an effect in the absence of benomyl (Table I).
The APC/C is a target of the E4orf4 pathway
The APC/C ubiquitination complex is required, with its Cdc20 subunit, for the metaphase-to-anaphase transition, and with another subunit, Hct1/Cdh1, it is involved in mitotic exit. We found that APC/C mutants, as well as the cdc20-1 and cdc20-3 mutants, and the cdh1/hct1 deletion, are supersensitive to E4orf4 (Table I). It was reported that the exit from mitosis involves a biphasic inactivation of the Cdc28-Clb2 kinase and degradation of Clb2. The first phase is dependent on Cdc20 and starts at the metaphase-to-anaphase transition. The second phase occurs in telophase and requires activation of the Cdc20 homologue, Hct1/Cdh1 (Visintin et al., 1997; Baumer et al., 2000; Yeong et al., 2000). To characterize the effect of E4orf4 on APC/C activity, we followed accumulation and degradation of substrates of both APC/C complexes. To measure degradation rates by APC/CCdh1, the substrate Ase1 was used, since APCCdh1 activity is highest in G1, and ectopic Clb2 expression was incompatible with maintenance of G1 arrest (Juang et al., 1997; Visintin et al., 1997). As shown in Fig. 7 A, Ase1 was significantly stabilized in the presence of E4orf4, its half-life increasing from 2.5 to 7 min. Pds1, an inhibitor of the metaphase-to-anaphase transition, is degraded by APCCdc20 (Visintin et al., 1997). In E4orf4-expressing cells, Pds1 accumulated to significantly higher levels than in control cells, suggesting that Pds1 degradation is also inhibited by E4orf4 (Fig. 7 B). To directly assay the effect of E4orf4 on Pds1 degradation by pulse–chase, we artificially arrested the cells with α-factor in G1, a cell cycle phase where Pds1 had been shown to be unstable (Cohen-Fix et al., 1996). Under these conditions, however, no difference was seen in Pds1 degradation between control and E4orf4-expressing cells—in both cases, Pds1 was degraded with a half-life of 3 min (results not shown). Since Pds1 degradation normally occurs at the metaphase-to-anaphase transition, its ectopic degradation in G1-arrested cells may reflect an alternative mechanism that is different from its normal mechanism of degradation. Therefore, an attempt was made to arrest the cells in metaphase by expressing nondegradable Pds1 (Cohen-Fix and Koshland, 1999). However, cell cycle arrest could not be maintained during the relatively long period of time required for E4orf4 accumulation. Thus, we could not reliably determine directly the effect of E4orf4 on Pds1 stability.
E4orf4 physically interacts with the APC/C and recruits PP2A to the complex
One possibility for explaining the effect of E4orf4 on APC/C activity is that it directly targets PP2A to the APC/C. We tested this hypothesis by assaying whether E4orf4 physically interacts with the APC/C. HA-tagged Cdc16 was expressed in E4orf4 (wild type or the A3 mutant)–expressing versus control cells, and cell extracts were prepared 5 h after induction with galactose. E4orf4 was immunoprecipitated from the various samples, and the presence of the HA-tagged APC/C subunit Cdc16 in the immune complexes was detected by Western blot analysis. Fig. 8 A demonstrates that wild-type E4orf4 coimmunoprecipitated with Cdc16, whereas the A3 mutant associated with HA-Cdc16 more weakly. The A3 mutant did not bind Tpd3 at all, similar to our findings in mammalian cells (Shtrichman et al., 2000). No HA-Cdc16 was immunoprecipitated in the absence of E4orf4 proteins. These results indicate that E4orf4 specifically associates with APC/C in yeast cells. We next tested whether E4orf4 expression leads to enhanced recruitment of PP2A to the APC/C. HA-Cdc16 was expressed in wild-type yeast cells or in tpd3Δ cells, with wild-type E4orf4 or with the empty vector. Immunoprecipitation reactions were carried out with antibodies to Tpd3, and the presence of HA-Cdc16 in the immune complexes was detected by a Western blot. Fig. 8 B demonstrates that Cdc16 was present in a complex with PP2A in cell extracts expressing E4orf4. No interaction above background levels, determined by a similar immunoprecipitation from tpd3Δ cell extracts, was detected in the absence of E4orf4. These results could suggest that in the absence of E4orf4, PP2A and the APC/C do not interact; alternatively, PP2A and the APC/C may normally interact, but this interaction may be too weak to be detected by immunoprecipitation in the absence of E4orf4.
To test for functional interactions between PP2A and the APC/C, the pADH-CDC55 plasmid, overexpressing Cdc55, was transformed into wild-type yeast cells or into cdc20-1 cells. As seen in Fig. 8 C, although wild-type cells were efficiently transformed with the pADH-CDC55 plasmid, no transformants were obtained in the cdc20-1 background, suggesting that increased PP2A-Cdc55 activity is lethal in this background. Conversely, it has been previously reported that deletion of CDC55 partially suppresses the temperature sensitivity of the cdc20-1 mutant (Wang and Burke, 1997). Together, these data suggest that even in the absence of E4orf4, PP2A-Cdc55 acts as a negative regulator of the APC/CCdc20. Thus, the results presented in Fig. 8 suggest that E4orf4 may be merely stabilizing an existing interaction between these two protein complexes.
E4orf4 can induce G2/M arrest in mammalian cells
The work presented here demonstrated that yeast cells expressing E4orf4 accumulated in G2/M, whereas previous work in mammalian cells indicated that E4orf4 expression led to apoptosis. Upon transient transfection of E4orf4 into various mammalian cell lines, no cell cycle effects were detected. However, in a stably transfected 293-derived cell line, expressing E4orf4 from a tetracycline-regulated promoter, G2/M arrest was observed 24 h after induction with doxycycline (Fig. 9). No such arrest was observed upon treatment of a parallel control cell line. 48 h after induction, the G2/M arrest was released, and cells with sub-G1 DNA content undergoing apoptosis started to accumulate. These results indicate that, under certain physiological conditions, E4orf4 can initially induce cell cycle arrest, from which the cells eventually escape and undergo apoptosis.
In this work, we showed that adenovirus E4orf4, which induces apoptosis in transformed mammalian cells through a mechanism involving its interaction with PP2A complexes containing the B, but not the B′ subunit, causes irreversible growth arrest in S. cerevisiae. This growth arrest requires yeast PP2A complexes containing Cdc55, but not Rts1, and occurs at a specific cell cycle stage. These observations, and our recent findings indicating that nontoxic E4orf4 mutants selected in yeast could not induce apoptosis in mammalian cells (Afifi et al., 2001), underscore the high conservation of the E4orf4 targets between yeast and mammalian cells. Thus, as a first step towards elucidating the E4orf4-induced apoptotic pathway in mammalian cells, we took advantage of the wealth of available yeast cell cycle mutants to map the interaction of E4orf4 with the yeast cell cycle machinery.
Genetic interactions between E4orf4 and Cdc28
Genetic interactions suggested that reducing the activity of the Cdc28 kinase resulted in hypersensitivity to E4orf4. Thus, the growth arrest was more severe in cdc28 or mitotic cyclin mutants, and in mutants lacking the activating Mih1 phosphatase or overexpressing the inhibitory Swe1 kinase (Fig. 5; Table I). Absence of the Swe1 kinase, or of the Swe1 target on Cdc28 (Cdc28-F19) did not affect sensitivity to E4orf4. Strikingly however, in the presence of E4orf4, Clb2-associated kinase was hyperactivated in an Mih1-dependent manner (Fig. 5). Thus, E4orf4 may partially counteract its own cell cycle inhibitory effect by stimulating dephosphorylation of Cdc28-Y19 by Mih1. The hypersensitivity of the mih1 mutant would be due to its inability to respond to this stimulation. Indeed, the hypersensitivity was overcome by expression of a constitutively active Cdc28-A18F19 mutant in the mih1Δ cells (Fig. 5 E).
E4orf4 inhibits APC/C activity
Morphologically, E4orf4-arrested cells appear to be in preanaphase and telophase stages (Fig. 4) in wild type, as well as in mih1Δ cells, however the proportion of short preanaphase spindles was higher in the mih1Δ mutant (data not shown). Biochemical examination of the E4orf4-expressing cells indicates that they are defective in Ase1 degradation and accumulate high levels of Pds1 as well (Fig. 7). These results suggest that E4orf4 specifically inhibits APCCdh1 and may also inhibit APCCdc20. This is supported by the hypersensitivity of both cdc20ts mutants and cdh1Δ mutants to E4orf4, as well as by the dual morphology of the arrested cells. The lack of an effect of E4orf4 on Pds1 degradation in G1-arrested cells does not support an inhibition of APCCdc20; however, whereas Pds1 degradation normally occurs at the metaphase-to-anaphase transition, it could for technical reasons only be assayed in G1-arrested cells (Visintin et al., 1997). Thus, it is possible that under these artificial conditions, the effect of E4orf4 on APCCdc20 activity is lost. An arrest of the cells in metaphase by expression of a nondegradable Pds1 is transient (Cohen-Fix and Koshland, 1999) and does not provide enough time for optimal E4orf4 expression. In any case, it should also be noted that since pds1Δ cells are still sensitive to E4orf4 (Table I), there must be another substrate of APCCdc20 that is required for E4orf4-induced arrest. The supersensitivity of E4orf4-expressing cells to benomyl (Fig. 6) is also best explained by inhibition of APCCdc20. Benomyl, which transiently triggers the spindle assembly checkpoint by inhibiting APCCdc20 (Fang et al., 1998; Hwang et al., 1998), might cause permanent arrest when the APC is already partially inhibited.
E4orf4 targets PP2A to the APC/C
The observations that E4orf4 coimmunoprecipitates with APC/C subunits and that PP2A associates with the APC/C in E4orf4-expressing cells (Fig. 8) suggest that E4orf4 functions by directly targeting PP2A to the APC/C. Several yeast APC/C subunits were shown to be phosphorylated (Peters et al., 1996). Furthermore, phosphorylation of the APC/C by Cdk1 was shown to be required for its activation both in metazoans (Lahav-Baratz et al., 1995; Shteinberg et al., 1999) and in yeast (Rudner and Murray, 2000), either directly or indirectly via activation of the Cdc5/Polo kinase (Charles et al., 1998). Thus, E4orf4 may inhibit the APC by inducing its dephosphorylation by PP2A. We suggest that APC/C activity in E4orf4-expressing cells results from the net effect of phosphorylation by Cdc28 and dephosphorylation by E4orf4/PP2A. Therefore, mutants having reduced Cdc28 activity are hypersensitive to E4orf4. This model is summarized in Fig. 10.
The identity of the specific phosphorylation sites on the APC/C targeted by E4orf4 still eludes us. It appears that the phosphorylation sites described previously in Cdc16, Cdc23, and Cdc27 (Rudner and Murray, 2000) are not the substrates of the E4orf4–PP2A complex, since substitution of these sites to alanine residues has only a weak effect on APC/C function (Rudner and Murray, 2000), and do not render the yeast cells resistant to E4orf4 toxicity (Table I). Furthermore, although it was reported that APC/C phosphorylation at these sites affects only the activity of the APC/CCdc20 complex (Rudner et al., 2000), our results suggest that E4orf4, through its association with both PP2A and the APC/C, affects the APC/CCdh1 complex as well. It should be noted that Cdh1 itself is activated, rather than repressed, by dephosphorylation; however, this dephosphorylation is carried out by a distinct phosphatase, Cdc14 (Visintin et al., 1998; Zachariae et al., 1998).
The inhibitory effect of PP2A on APC/C activity in the presence of E4orf4 could represent a novel interaction induced by the viral protein; alternatively, E4orf4 could be merely enhancing a physiological interaction of PP2A with the APC/C. We found that overexpression of Cdc55 was synthetically lethal with the cdc20-1 mutation even in the absence of E4orf4 (Fig. 8 C). Conversely, Wang and Burke (1997) reported that deletion of CDC55 partially suppressed the cdc20-1 temperature sensitivity. Therefore, we suggest that one of the normal functions of PP2A is to inhibit the APC/C. Deletion of the PP2A regulator CDC55 would result in derepression of APC/C activity, counterbalancing the reduction of Cdc20 activity of the cdc20-1 mutant, thereby resulting in suppression of the mutant phenotype. This may also provide an additional explanation for the loss of spindle assembly checkpoint activity detected in the cdc55Δ mutant by Minshull et al. (1996). These authors attributed the loss of the checkpoint to increased inhibitory phosphorylation of Cdc28 on Tyr19. However, even in the presence of the Cdc28-V18F19 mutant, a reduction in Clb2 levels, and a concomitant reduction in Clb2-associated kinase activity, was detectable in the cdc55Δ cells (Minshull et al., 1996). Therefore, we suggest that derepression of APC/C activity in the cdc55Δ mutant may additionally make inactivation of the APC/C by the checkpoint pathway less effective, contributing to loss of checkpoint activity.
The similarity between E4orf4-initiated pathways in yeast and mammalian cells
In a 293-derived cell line, E4orf4 induces G2/M arrest before induction of apoptosis (Fig. 9). In a previous report, Wersto et al. (1998) demonstrated that, in the absence of the E1 region of adenovirus, the E4 region of the virus (excluding E4orf6) can induce G2 growth arrest and elevation in cyclins A and B and p34cdc2 kinase protein levels. The finding of elevated levels of mitotic cyclins in E4-arrested mammalian cells could result from interference with the degradation of mitotic cyclins (Wersto et al., 1998). Thus, the similar effect of E4orf4 on mammalian and yeast cells could reflect a conservation not only of the PP2A–E4orf4 interaction, but also of the interaction of PP2A–E4orf4 with its target, the APC/C. This possibility is currently being investigated. Many viral proteins cause cell cycle dysregulation, either as their primary function to create optimal conditions for viral replication, or as a by-product of their primary function. However, whether the effect of E4orf4 on the cell cycle reflects one of its natural functions or whether it is a consequence of its ectopic expression outside the viral life cycle is not clear yet.
Our initial motivation in expressing E4orf4 in yeast was to gain more information on the pathway of apoptosis induction by E4orf4 in transformed mammalian cells. Although apoptosis has mainly been described in multicellular organisms, in recent years there have been several observations of apoptosis-like phenomena in unicellular eukaryotes, including S. cerevisiae (Madeo et al., 1997). Despite the fact that none of the genes involved in the basic apoptotic machinery in metazoans, such as Bcl-2 or caspases, were identified in yeast, introduction of proapoptotic genes, such as BAX or BAK into yeast, resulted in cytotoxicity with apoptotic-like phenotypes (Greenhalf et al., 1996; Zha et al., 1996; Ink et al., 1997; Tao et al., 1997). Furthermore, the generation of ROS plays a role in the apoptotic process both in metazoans and in yeast (Hockenbery et al., 1993; Kane et al., 1993; Slater et al., 1995; Madeo et al., 1999). Thus, yeast has become a useful model system for the study of parts of the apoptotic pathway (Matsuyama et al., 1999).
We found that yeast cells expressing E4orf4 irreversibly stopped proliferating and exhibited one of the hallmarks of apoptosis, namely, accumulation of ROS. E4orf4 expression also leads to accumulation of ROS in mammalian cells. However, this similarity in phenotype does not necessarily reflect a similarity in mechanism, especially since yeast cells lack the death receptor pathway that is recruited by E4orf4 in mammalian cells to induce ROS accumulation (Livne et al., 2001). Other regulators shown to be involved in E4orf4-induced apoptosis in mammalian cells are also lacking in yeast, such as the Src family of kinases (Lavoie et al., 2000). However, E4orf4 induces both caspase-dependent and caspase-independent cell death pathways in mammalian cells (Lavoie et al., 1998; Livne et al., 2001). The cell cycle arrest induced by E4orf4 in yeast may be mechanistically relevant to at least one of these two apoptotic pathways.
Perturbations in cell cycle regulation and induction of apoptosis
We found that in a 293-derived cell line, the E4orf4-induced G2/M arrest is followed by apoptosis (Fig. 9). Several compounds—PKC inhibitors (Begemann et al., 1998), taxol (Jordan et al., 1996), and viral genes such as HIV-1 Vpr (Stewart et al., 1997)—have been reported to induce both G2/M arrest and apoptosis in mammalian cells. Conversely, several reports demonstrated that induction of apoptosis by various stimuli required CDC2 or CDK2 activity (Shi et al., 1994; Meikrantz and Schlegel, 1996; Yao et al., 1996). Thus, perturbations in cell cycle control can lead to apoptosis. Furthermore, a specific link between APC/C inhibition and apoptosis has been previously established in a report showing that the APC/C Cdc27 subunit was cleaved by caspases in Jurkat cells induced to undergo apoptosis by Fas, resulting in APC/C inhibition and cyclin A and B stabilization (Zhou et al., 1998). CDC2 kinase activity was further induced in these cells due to caspase cleavage and inactivation of Wee1, resulting in reduced tyrosine phosphorylation at the inhibitory site. In these cells, similar to our findings in yeast expressing E4orf4, the APC/C is inhibited and Cdks are stimulated. It is possible that this combination of APC/C inhibition and Cdk stimulation contributes to induction of the apoptotic process in mammalian cells, and that this is one of the pathways through which E4orf4 selectively kills transformed cells. This notion, which can now be tested directly, may eventually result in novel therapeutic strategies.
Materials And Methods
Yeast strains, media, and mammalian cell lines
Yeast were grown either in YPD (1% yeast extract, 2% bacto peptone, 0.015% l-tryptophan, 2% glucose) or in synthetic complete medium (Sherman et al., 1982). For induction with galactose, cells were grown in synthetic medium with 2% raffinose overnight. They were then diluted to A600 0.3 and allowed to grow another 2 h before addition of galactose to 2%. Alternatively, cells were grown in 2% glucose to midlog phase, washed once, and resuspended in 2% galactose. For induction by doxycycline removal, cells were grown overnight in medium containing 2% glucose and 5 μg/ml doxycycline. They were then diluted to A600 0.3 and allowed to grow another 2 h in the same medium before being washed twice in water and transferred to medium without doxycycline.
All yeast strains used in this study are listed in Table II, along with their sources. All the strains are isogenic with strain W303. Derivation of a rho-0 strain was carried out as described (Sherman et al., 1982). Strain KY520 was built by crossing strain CY5580 (a, leu2, ura3, cdc55::LEU2) (Wang and Burke, 1997) with W303. Similarly, KY679 was obtained by crossing Y1366 (a, his3Δ1, lys2-1, leu2-3, 112 ura3-52, tpd3Δ::LEU2) (van Zyl et al., 1992) with W303. Strains KY596 and KY600 were obtained by transformation with mih1::LEU2 (Russell et al., 1989) and swe1::LEU2 (Booher et al., 1993) deletion plasmids, respectively. Strain KY620 was obtained by crossing a swe1Δ::LEU2 with a mih1Δ::LEU2 strain. Presence of the two markers was deduced from tetrad analysis and confirmed by backcross. Strains KY630, KY631 were obtained by integrating the YiP CLB2-3xHA plasmid (L. Johnston, National Institute for Medical Research, London, UK) into the W303 and KY620 strains at CLB2.
A 293-derived cell line expressing E4orf4 from a tetracycline-inducible promoter was prepared by introducing E4orf4 cDNA cloned in the pUHG10-3 vector (Gossen and Bujard, 1992) and pBabe-Puro (Morgenstern and Land, 1990) into the 293 Tet-On cell line (CLONTECH Laboratories) and selection for puromycine-resistant colonies. A cell line selected in the same way but not expressing E4orf4 is used as a negative control.
E4orf4 and mutant A3 were subcloned into the BamHI-ClaI sites of pCM190 (Gari et al., 1997), into the BamHI-EcoRI sites of p414 GALL (Mumberg et al., 1994), or into the BglII-EcoRI sites of pDAD2, a yeast 2μ plasmid containing the GAL1,10 promoter (Kornitzer et al., 1994). PDS1 was cloned by PCR under the CUP1 promoter of plasmid KB354 (Meimoun et al., 2000) and fused to the triple Myc epitope tag. Other plasmids have been previously described: YCp50-CDC55 (Nickels and Broach, 1996), pDB20(HA-CDC55) (Zhao et al., 1997), GAL10:SweI (Booher et al., 1993), HA-Cdc16 (Hwang and Murray, 1997), GAL-Ase1-Mycx3 (Juang et al., 1997).
Detection of ROS accumulation
Free intracellular radicals were detected with dihydroethidium or with dihydrorhodamine 123 (Sigma-Aldrich). Dihydroethidium was added at 1 μg/ml of cell culture, and cells were viewed through a rhodamine optical filter after a 10-min incubation. Dihydrorhodamine 123 was added at 5 μg/ml of cell culture, and cells were viewed through a rhodamine filter after a 2-hr incubation. For FACS® analysis, cells were incubated with dihydroethidium as described and analyzed using FACSCalibur® (Becton Dickinson).
Histone H1 kinase assay
∼3 × 108 cells treated as indicated were pelleted, washed once, resuspended in 0.1 ml extraction buffer (20 mM Tris-Cl, pH 7.5, 100 mM NaCl, 10 mM EDTA, 1% Triton X-100, 5% glycerol, 1 mM each of NaF, Na-pyrophosphate, NaVO3, β-glycerolphosphate, EGTA, and the protease inhibitors PMSF, TPCK, TLCK, leupeptin, pepstatin, and chymostatin at 20–50 μg/ml), and broken by vortexing in the presence of glass beads. Extract containing 0.15 mg protein was incubated 1 h on ice with 1 μg of α-HA antibody, followed by incubation 1 h with protein A–sepharose beads (Amersham Pharmacia Biotech). The beads were washed three times in kinase assay buffer (25 mM MOPS, pH 7.2, 15 mM MgCl2, 5 mM EGTA, 1 mM dithiothreitol, 60 mM β-glycerolphosphate, 0.1 mM NaVO3). Reactions were performed at 30°C for 30 min in 10 μM kinase assay buffer containing 0.5 mM cold ATP, 2 μCi γ[32P]ATP, and 5 μg histone HI. The reaction products were separated by polyacrylamide electrophoresis and histone HI phosphorylation was quantitated with a phosphorimager.
Protein degradation assay
Ase1 and Pds1 degradation were measured in bar1Δ cells arrested in G1 with α-factor. Overnight cultures grown in raffinose were diluted in raffinose medium with 1 μM α-factor. When more than 90% of the cells had reached G1 arrest, typically within 3–4 h, E4orf4 expression was induced by the addition of 3% galactose for 7 h. Pulse–chase analysis was then performed as described (Meimoun et al., 2000). Maintenance of G1 arrest was confirmed by subjecting an aliquot of the culture taken at the time of the chase to FACS® analysis.
Immunoblot analysis and coimmunoprecipitations
For immunoblots, yeast cells were collected at various times after induction and lysed in the presence of 1.85 M NaOH and 7.4% β-mercaptoethanol. Proteins were TCA precipitated and resuspended in protein loading buffer (125 mM Tris-HCl, pH 6.8, 1% SDS, 10% glycerol). For visualization of E4orf4, proteins were chromatographed on 15% SDS polyacrylamide gels and subjected to Western blotting and detection by specific antibodies as previously described (Shtrichman et al., 1999).
For coimmunoprecipitations, yeast extracts were prepared by bead beating cells for 4 min at 4°C in lysis buffer (250 mM NaCl, 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.5% Nonidet P-40, 0.1% Triton X-100, 50 mM NaF, 100 μM sodium vanadate, 100 nM okadaic acid [Calbiochem], complete protease inhibitor cocktail [Roche Molecular Biochemicals]). The lysates were spun to separate beads and debris from the clear lysate. The beads were washed twice more in the lysis buffer, and immunoprecipitations were carried out in the same lysis buffer. Antibodies to E4orf4 were covalently coupled to protein sepharose beads (Shtrichman et al., 1999). Antibodies to the HA tag were from Babco, and were used at 1:600. Lysates, antibodies, and protein A beads were rotated for 3 h at 4°C, and then washed three times in lysis buffer.
Antibodies used in this work were anti-HA (Babco), anti-E4orf4 (Shtrichman and Kleinberger, 1998), anti-Tpd3 (from J.R. Broach, Princeton University, Princeton, NJ), and anti-Myc monoclonal (9E10).
We are grateful to the following scientists for gifts of plasmids and yeast strains: D.J. Burke (University of Virginia, Charlottesville, VA), R. Hallberg (Syracuse University, Syracuse, NY), J.R. Broach, H. Ronne (Ludwig Institute for Cancer Research, Uppsala, Sweden), J. Arino (Universitat Autonoma de Barcelona, Barcelona, Spain), G. Fink (Whitehead Institute, Cambridge, MA), A. Amon (Massachusetts Institute of Technology), A.W. Murray (Harvard University, Cambridge, MA), K. Nasmyth (Research Institute of Molecular Pathology, Vienna, Austria), W. Zacchariae (Max Planck Institute, Dresden, Germany), A.A. DePaoli-Roach (Indiana University, Indianapolis, IN), S. Kron (University of Chicago, Chicago, IL), D. Pellman (Dana Farber Cancer Institute, Boston, MA), and L. Johnston. We thank Shavit Gonen-Fein for technical assistance and S. Selig for critical reading of the manuscript.
This work was supported by grants from the Israel Science Foundation, founded by the Israel Academy of Sciences and Humanities, the Israeli Ministry of Sciences, and from the Fund for the Promotion of Research at the Technion (to D. Kornitzer and T. Kleinberger).
Abbreviations used in this paper: APC/C, anaphase-promoting complex/cyclosome; E4orf4, early region 4 open reading frame 4; GFP, green fluorescent protein; PP2A, protein phosphatase 2A; ROS, reactive oxygen species.


text:
Pokémon Go is always striving to improve and make its gameplay more inviting for players. The game has consistently adapted content from the mainline games, including the ability to Mega Evolve your Pokémon. Now, Niantic is officially changing how Mega Evolutions are going to work in the game.
Mega Evolutions has undergone a few changes, you will can now Mega Evolve from the raid and battle prep screens. You will also notice an additional animation on the page of Pokémon that you already have Mega Evolved, and there is a new Mega Mastery system.
What is Mega Mastery?
Mega Mastery is a new way to level up your Mega Evolutions quicker. Here are the changes being made to help you do this.
- When you catch a Pokémon of the same type, you now have a chance to earn Candy XL.
- You get extra experience when catching a Pokémon of the same type.
- As a Pokémon’s rest level decreases, its Mega Level will increase.
The more you use a Mega Evolution, the quicker that Pokémon’s Mega Level will be increased. Each Pokémon has a chance to earn various bonuses when leveled up, like a higher chance to earn candy.
For more on Pokémon Go, check out How to catch Klefki – Pokémon Go on GameTips.PRO!

domain: entertainment


text:
Whether you're on your way to the studio or on your way to brunch, the Tonic Selene Jacket will keep you stylish and comfortable anywhere you go. Made of soft fleece fabric, in a Black color, this top offers comfort and understated style. An elegant wide collar, single button colsure, and a rounded front hem, give this jacket a style boost that looks good with leggings or denim. Wear for studio-to-street transitions and everyday style!
Why we love: Love the elegant design and super soft fabric!
Fabric: French Terry fleece.

domain: design


text:
Affordable Housing Trust Fund (AHTF) Program Overview
In May 2003, the City Council combined previously separate multifamily affordable housing funding programs into the Affordable Housing Trust Fund (AHTF) to assist in the financing of the production and preservation/stabilization of affordable and mixed-income rental housing projects in Minneapolis. The AHTF Program assists with providing decent, safe and affordable housing opportunities, assists with community revitalization and blight removal, and supports tax base enhancement, job creation, and increasing density along critical corridors. The AHTF Program administration is guided by the Minneapolis Plan, the Unified Housing Policy, and the HUD Consolidated Plan for Housing and Community Development.
The AHTF awards are typically awarded pursuant to an annual Request for Proposals (“RFP”) and structured as a loan with a note and mortgage.
Funds not awarded through the RFP may be available in a pipeline round based on the following priority:
- An application scored well in the current round and met thresholds but didn’t receive funding or received a prior award but did not maximize the funding request.
- Projects that preserve Naturally Occurring Affordable Housing (NOAH): Rental housing projects provided by the private market without government subsidy that have at least 20% of the units with rents affordable to households with incomes at or below 50% of the area median income, and/or rental housing projects that previously received local subsidy or low income housing tax credits, and will no longer be subject to income and/or rent restrictions.
- Projects that are a priority in accordance with the Interagency Stabilization Group (ISG).
- Projects that further achieve the goal of the Senior Housing Policy of a minimum of 35 units in each Ward by 2025. The remaining eligible Wards are: 1, 4, 5, 7, 10, 11, 12, and 13. The Wards that have already achieved this goal are: Ward 2 Minnehaha Commons, 41 units; Ward 3 Mill City Quarter, 150 units; Ward 6 Snelling Apartments, 60 units; Ward 8 Sabathani Senior Housing, 50 units; and Ward 9 Hi Lake Triangle Apartments, 64 units.
The AHTF has three funding sources: Local funds (City General Fund and Tax Increment), federal Community Development Block Grant (CDBG) funds, and federal HOME Investment Partnership Program funds. CDBG and HOME have differing policy objectives and differing restrictions on where and how those funds can be used in housing projects.
The AHTF Program has established selection criteria used to review and score the proposals. There are two thresholds that a project must meet to be evaluated for funding. First, a project must meet the Minimum Point Threshold of at least 20 points combined in four selection criteria: 1. Financial Soundness and Management; 2. Capacity of Property Manager and Quality of Property Management Plan; 3. Capacity of Owner and Developer’s Comparable Project Experience; and 4. Economic Integration. A project must also meet the Total Point Threshold, which means a project must receive a minimum of 86 points in all selection criteria. In addition, a project must have complete site control.
On July 18, 2017, the Development Finance Committee (DFC) reviewed the AHTF funding recommendation for both the Maya Commons and Green on Fourth Apartment project’s proposed financing structure and the overall capacity of the developer and recommended approval. See attached action.
AHTF Funding Recommendations
Below is a summary of the projects:
1. Maya Commons – Ward 2 – Como – Score 126
937 13th Ave SE
- 50 units (42 @ 50% AMI or lower) of new construction
- Financing structure: 9% LIHTC, grants, and public deferred loans
- Policy alignment and public benefit summary: workforce housing; LTH
- The Total Development Cost of the project is $14,328,758 which is a TDC/unit of $243,435
2. Green on Fourth Apartments – Ward 2 – Prospect Park – Score 112
2901 4th Street SE
- 243 units (49 @ 50% AMI or lower) of new construction
- Financing structure: HRB 4%, grants, TIF, and public deferred loans
- Policy alignment and public benefit summary: affordable housing; family and mixed income
- The Total Development Cost of the project is $56,048,406 which is a TDC/unit of $230,652
Timely Completion Conditions and Rescission of Awards
The AHTF policies in the RFP require timely completion of projects within the context of the established funding cycles of the other funders such as Minnesota Housing. The AHTF project awards are reserved for 15 months from the City Council approval date. The developer must submit monthly progress reports describing incremental project financing and development achievements. At the end of 15 months, if the developer can demonstrate that at least one-third of the total development funds have been raised; and can provide evidence that the balance of the development money is likely to be raised; and can provide evidence that a closing will occur within the next twelve months, the funding reservation may be administratively extended for an additional 12 month period. Projects unable to meet the timelines may have their awards rescinded. Projects receiving a supplemental AHTF award are held to the timely completion conditions from the year of the first AHTF award for that project, which means that receiving a supplemental AHTF award does not extend the 27 month timeframe in which the project should be fully financed.
These projects had both received prior awards. Those funds are being relinquished and recommitted to these projects which will establish a timeframe that begins upon Council approval on August 18, 2017.
Maya Commons is seeking the same AHTF award as was originally applied for in 2016, $1,250,000. This award is being renewed due to the structure of the project changing. This project originally consisted of two buildings with a total of 150 units being developed simultaneously. The project has since been broken out into two phases with two separate developers. The end project will remain largely the same; two buildings with a total of 145 units. This AHTF award is for the first phase consisting of one building with 50 units. No AHTF award is being sought for the second phase which consists of 95 units.
Green on Fourth Apartments original AHTF award was in 2014 in the amount of $1,025,000. The project is seeking an additional $200,000 in trust funds as the number of units for this project increased allowing it to qualify for additional funds. This project will thus have a total AHTF award of $1,225,000.
AHTF Repayment and Recapture Terms
The AHTF funds are typically provided as a deferred payment loan with a 30 to 40 year term with 0% to 1% simple interest. Additionally, the City seeks to recapture a percentage of net cash flow in mixed income projects, where possible, pursuant to CPED’s approved policy guidelines. In this round, there are no mixed income projects subject to this requirement.
AHTF Application and Loan Origination Fees
In May 2011, the City Council approved the collection of a $1,000 application fee for each AHTF proposal and a one percent (1%) loan origination fee for each AHTF project. The loan origination fee for the recommended projects will be collected at project closing, except for projects with HOME funding because HUD prohibits collecting an origination fee with HOME funds.

domain: finance


text:
CHFFF curriculum files are available to download and use for the limited purpose of education and training by non-profit organizations. To use the curriculum, please email Wendy Wolfe, PhD, Division of Nutritional Sciences, Cornell University, with your name, agency/organization, state and intended use.
We highly recommend that CHFFF be used in its entirety and as written. If you only use parts of it, please be sure that Cornell copyright is acknowledged, adding “From Choose Health: Food, Fun, and Fitness, Copyright 2011-15, Cornell University” to documents as needed. See complete CHFFF Copyright Information.

domain: library


text:
I'm a big fan of bodyweight training and if you stick to the bodyweight training, you probably don't need any of those until you can do 10 pistols.
With that in mind, I would get pull-up bar or gymnastics rings first.
If you have them or easily access them, I think either kettlebell or vest is okay.
BTW, weight loss is more about diet than exercise, and the major role of exercise on weight loss is increasing metabolism by building muscle rather than burning calories directly.
Please keep in mind that the strength (or muscle) you can build with only one kettlebell is very limited especially if you are a guy.
If you are going to add more kettlebells later, kettlebells can be a good choice, but if you are a minimalist, vest would be better.
Last edited by vincebae; 07-02-2013 at 10:35 AM.
what is your current workout routine? i'm voting for the weight vest. its 1000x more functional than the kettlebell. you can add the weight vest to every single bodyweight exercise that you can do. you can sprint with it. you can hike with it.
a kettlebell is decent for a few things, but is probably the single most over-hyped piece of fitness equipment on the market today. its a neat addition to a solid home gym, but 1 kettlebell isn't going to get you that far. definitely the vest.

domain: health


text:
Submitted to: Meeting Abstract
Publication Type: Proceedings
Publication Acceptance Date: 3/20/2007
Publication Date: 6/10/2007
Citation: Nelson, S.O., Wen-Chuan, G., Trabelsi, S., Kays, S.J. 2007. Sensing Quality of Watermelons Through Dielectric Permittivity. Meeting Abstract.
Interpretive Summary: Interpretive Summary Sensing Quality of Watermelons through Dielectric Permittivity Techniques for nondestructive determination of quality of agricultural products are helpful to producers, handlers and processors, those marketing the produce, and consumers. Visible and physical characteristics of many fresh fruits and vegetables are available for correlation with quality, and some of these, such as color, size, weight, density, elasticity, and firmness are used in automatic sorting of some produce into different categories for the market. Electrical characteristics of fruit tissue known as dielectric properties can be sensed with electric fields for possible detection of fruit quality. Therefore, four miniature watermelons were grown and harvested with a range of maturities for dielectric spectroscopy measurements of the melon tissue to learn whether there might be differences in the dielectric properties that could be correlated with quality. The best criterion for quality is the content of soluble solids, which are mostly sugars and therefore a measure of sweetness. This requires the extraction of tissue samples from the melons and measurement of expressed juice with a refractometer instrument, which has been calibrated to indicate percentage of soluble solids. The dielectric properties of the watermelon tissues were measured over the frequency range from 10 MHz to 1.8 GHz along with refractometer determinations of soluble solids content (SSC). A high correlation was found between SSC and the dielectric properties as expressed in a complex-plane plot of the dielectric constant and loss factor, each divided by SSC. Through this mathematical relationship, SSC was calculated from measured dielectric properties, but predictions were not as high as desired, and further research is needed to assess the dielectric technique for sensing watermelon quality. The challenge in using this new principle for practical nondestructive sensing of honeydew melon quality is considerable, but development of such a melon quality meter would be helpful to melon growers and handlers in the marketing of high quality produce for American consumers.

domain: chemistry


text:
This is Dr. David
assault courses can be quite challenging
did you train before hand?
what is your most recent TSH levels?
have you had pulmonary function testing and cardiac stress testing?
that could definitely been due to your low iron
have you had your stools checked for blood?
it is normal for iron to cause your stools to be black.
the question should be why has your iron been low.
iron gets recycled in the body and usually most people get enough of it in their diets
through meat, steaks, spinach, etc.
if you are slowing loosing iron for any reason, or loosing blood slowly this can cause low iron.
women are more at risk, because they loose blood on a monthly basis through their period.
but another common area where you can loose blood slowly is through the GI system.
a small hemorrhoid could be slowly loosing blood or a small poly can slowly be bleeding
or a small diverticulosis in the colon could be bleeding slowly as well.
that is when a stool occult blood test is a good idea to try to detect and small slow blood loss.
I am glad you had testing for celiac and pernicious anemia.
it sounds like you don't hold on to B12 and iron very well.
this can be very common.
you might eventually need an upper endoscopy to look in your stomach to check for stomach inflammation or gastritis.
sometimes low stomach acid issues can cause poor absorption of iron and B12 and calcium in the stomach.
so your stomach might not be making enough acid to help with nutrient absorption in the stomach
or if you take excessive stomach acid blockers this can cause poor stomach absorption of iron and B12 as well.
but it doesn't sound like you are taking acid blockers or proton pump inhibitors.
perhaps your hemorrhoids are slowly bleeding into your stool
and that is what is causing the low iron issues.
and the low B12.
B12 low of 200 is good.
Normal values are 200 - 900 pg/mL (picograms per milliliter).
sometimes viral infections can cause severe fatigue.
so that can be checked as well
like ebstein barr virus infections.
good luck to you
let me know if you have other questions.
those can sometimes be caused by low magnesium blood levels as well.
so get that checked as well.
low magnesium can cause the nerves and the muscles to not function correctly sometimes.
and low B vitamins can do this as well
you should probably take a B complex vitamin daily
and get your magnesium levels checked.
if they are low as well, you might need to go on magnesium supplements.
if done for now, please leave positive feedback below the chat box
you might have to scroll down to see the ratings area below
you can always reach me as Dr. David if you have other questions.
is my website
if you need to reach me in the future
we are only given credit for helping clients after positive feedback
thanks for using JustAnswer.com
do you see the ratings area below?
do you have any other questions?
good luck with the visual and sensory evoke potential testing.
you are welcome
don't forget to leave positive feedback below
let me know how it goes
MS would show up plaques in the brain on MRI scan
low mag and low B12 issues would not typically show up on MRI scan
but could show up in evoke potentials.
with slowly nerve conduction velocities.


text:
Department of Labor expands apprenticeship opportunities on public works projects
Over the next year, Delaware’s Department of Labor will roll out a more robust program to provide building trade apprenticeships.
The goal is to help breathe new life into the state’s skilled construction workforce.
A 2019 Delaware law requires contractors bidding on large public works projects to provide apprenticeships – offering on-the-job experience while the state’s Department of Labor manages the apprentice’s education. But that law didn’t include an enforcement mechanism or offer small contractors who couldn’t hire an apprentice any backup options, limiting the number of contractors able to meet the requirement.
The Labor Department is now implementing a new law to remedy those problems. Aside from establishing penalties for contractors who don’t comply, Secretary of Labor Karryl Hubbard says the new rules also allow smaller contractors to pay into a state fund to support training programs in lieu of bringing on an apprentice.
“Smaller employers, or employers who could not run an apprenticeship program would have an opportunity to bid on public works jobs and still further the idea of expanding apprenticeships in Delaware," she said.
Hubbard says her department’s push to promote apprenticeships in the building trades is a response to the growing number of skilled tradespeople in Delaware aging out of the workforce – a trend that could hobble the state’s efforts to maintain or update its infrastructure.
“This country has an infrastructure problem has an infrastructure problem, and we really need an army of people with skilled craft trades," she said. "And we’re getting to the point where there’s a deficit.”
Delaware’s Office of Apprenticeship currently supports more than 1,600 apprentices statewide, though the Department of Labor and organized labor leadership believe that number needs to rise to both meet demand and to diversify the construction workforce – particularly by bringing more women into the building trades.

domain: law


text:
OWENS KIDS SOCKSMen #B200A14OWE
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This product is excluded from promotions, rebates and cash-back offers. See Details.
This item may not be eligible for free shipping. See Details.


Downloads key to Capcom PC future
Speaking with Rock Paper Shotgun, Capcom US boss-man Christian Svensson has said that such is the nature of the PC market that downloads are now more important than full retail releases for the publisher.
When asked whether downloads were more important than boxed releases, Svensson replied: "Absolutely. No question in my mind. Digital distribution on PC ties directly into our strategy. Capcom is trying to lead in digital distribution, and I would go as far as to say that in the console space we are already the leading software publisher."
He continued, comparing the situation with the Xbox Live Arcade download platform, stating: "We've had the highest revenue-generating Xbox Live title, we've had the highest revenue-generating Wii title, we're definitely in the top three or four on the PlayStation network. To that end, on the PC side, I've spent the past year building up a digital distribution channel that has about twenty different partners. We're ready on the console side, and we were the first Japanese publisher to do anything on Steam."
More PC downloadables on the way from Capcom, then? It seems a dead-cert.
- Microsoft announces the feature list for the November Xbox One update
- EA Sports patches in some more missing features for the next-gen versions of NHL 15
- Blizzard begins rolling out a new patch for Starcraft II
- Far Cry 4 Season Pass details announced
- Shinji Mikami: I never use women as objects in my games
- Microsoft releases the first trailer for Halo: Nightfall TV series, first episode to premiere at Halo Fanfest
- Silence – The Whispered World 2 announced for Xbox One
- Titanfall to get its very own Horde mode called Frontier Defense
- John Riccitiello joins Unity as their new CEO


HONEYWELL INTERNATIONAL, INC. and HONEYWELL INTELLECTUAL PROPERTIES, INC., Plaintiffs-Appellants, v. ITT INDUSTRIES, INC. and ITT AUTOMOTIVE, INC., Defendants-Appellees, TG North America Corporation, TG Fluid Systems USA Corporation, and A. Raymond, Inc., Defendants-Appellees.
Honeywell International, Inc. and Honeywell Intellectual Properties, Inc. (collectively “Honeywell”) appeal from the final decision of the United States District Court for the Eastern District of Michigan granting summary judgment of noninfringement of U.S. Patent 5,164,879 in favor of ITT Industries, Inc., ITT Automotive, Inc., TG North America Corporation, TG Fluid Systems USA Corporation, and A. Raymond, Inc. (collectively “ITT/TG”). Honeywell Int'l, Inc. v. ITT Indus., Inc., Civ. No. 02-73948 (E.D. Mich. April 27, 2005). Because the district court correctly construed the claim limitation “fuel system component” and determined that the accused products do not meet that limitation, we affirm its grant of summary judgment of noninfringement. We further conclude that under our modified construction of the claim limitation “electrically conductive fibers,” the accused products do not meet that limitation either, thereby providing a separate ground for affirming the district court's grant of summary judgment of noninfringement.
The '879 patent, entitled “Electrostatically Dissipative Fuel System Component,” discloses a fuel filter that is specially made for use in motor vehicles that have electronic fuel injection (“EFI”) systems. Before motor vehicles began using EFI systems, the housing of a fuel filter was commonly made of metal or a polymer material. '879 Patent, col.1 ll.10-12 (filed July 1, 1991). Once vehicles began using EFI systems, as the '879 patent's written description recognizes, fuel filters with polymer housing began to break down and start leaking. Id., col.1 ll.17-20. It was discovered that the breakdowns were caused by the contact between the fuel, which flows at a high velocity in EFI systems, and the fuel filter's polymer housing. The resultant friction strips electrons from the hydrocarbon fuel and traps them in the non-conductive polymer housing, which leads to an electrostatic charge buildup within the housing of the fuel filter. Id., col.1 ll.26-30. The charge continues to build up until it finally discharges by “arcing” onto the vehicle's metal frame and becomes grounded. “Arcing” forms microscopic holes in the fuel filter's housing. Id., col.2 l.59 to col.3 l.2. When enough microscopic holes are formed, the fuel begins to leak. Fuel filters with metal housing avoid the “arcing” phenomenon because they allow no charge buildup. The conductive nature of metal prevents the electrons from being trapped inside the fuel filter and allows them to pass through to the vehicle's frame. Fuel filters with housing made of polymer material, however, are more desirable than their metal housing counterparts because of their lower cost and weight. Id., col.1 ll.13-14.
The patented invention addresses the “arcing” problem in fuel filters with polymer housing by providing an electrically conductive pathway between the fuel filter and the vehicle's metal frame. Id., col.3 ll.41-43. The electrically conductive pathway prevents the electrostatic charge from building up within the housing of the fuel filter. Id., col.3 ll.3-6. According to the written description, the electrically conductive pathway is created by incorporating small amounts of a “conductive filler material” into the polymer housing. Id., col.3 ll.47-51. The written description further discloses that stainless steel is an ideal “conductive filler material” because it has high conductivity, allowing it to be used in fibers with a high aspect ratio. Id., col.3 l.53 to col.4 l.13. The written description also notes that stainless steel fibers are ductile, which allows them to better maintain their integrity during melt-processing. Id. The benefits of stainless steel fibers are contrasted in the written description to electrically conductive carbon fibers, which are said to have less desirable characteristics, e.g., they must be used in fibers with smaller aspect ratios, are more rigid, and act as stress concentrators. Id., col.3 ll.56-60; col.4 ll.1-5.
Turning to the prosecution history of the patent in suit, the '879 patent issued from a divisional application of U.S. Patent Application 575,260, which issued as U.S. Patent 5,076,920 and was entitled “Electrostatically Dissipative Fuel Filter.” Also issuing from a divisional application of the '260 application was U.S. Patent 5,164,084, also entitled “Electrostatically Dissipative Fuel Filter.” The drawing and written description sections for the '879, '920, and '084 patents appear to be identical. See Honeywell Int'l, Inc. v. ITT Indus., Inc., 330 F.Supp.2d 865, 871 (E.D.Mich.2004). The patent examiner for the '260 application issued a restriction requirement in that application because it claimed three distinct inventions: (1) a method for preventing breakdown of a fuel filter, (2) the fuel filter itself, and (3) a moldable polymeric material. Id. Faced with this restriction requirement, the patentee chose the first invention for immediate prosecution, a method for preventing breakdown of a fuel filter, and that became the claimed subject matter of what issued as the '920 patent. The patentee filed divisional applications for the remaining inventions resulting in two other patents, the '084 patent (for a fuel filter) and the '879 patent (for a moldable polymeric material), the patent at issue in this case.
The '879 application, as initially filed, was entitled “Electrostatically Dissipative Fuel Filter” and contained one independent claim directed to a “moldable material for fuel system components.” 1 The patent examiner rejected the claim on the ground of, inter alia, indefiniteness under 35 U.S.C. § 112, ¶ 2. According to the examiner, it was “not clear what fuel system components [were] intended to be constructed of the electrically conductive moldable material.” In addressing the indefiniteness rejection, the patentee deleted the “moldable material for” language so that the claims were directed to a “fuel system component.” The patentee also argued that the independent claim was not indefinite because, although the specification only referred to fuel filters and fuel lines, “it is Applicant's position that he is entitled to a claim broad enough to cover all fuel system components manufactured of the moldable material disclosed and claimed in the specification.” While the application was pending, the title of the '879 application was changed to “Electrostatically Dissipative Fuel System Component,” because, the patentee contended, it “more accurately reflect[ed] the scope of the claims.” After an interview between the patentee and examiner, which resulted in the “arcing” limitation being added, the claims were allowed and the patent issued. Following a reexamination proceeding, the sole independent claim was further amended to recite “fuel injection system component” instead of “fuel system component” as the subject matter of the claims.
On October 2, 2002, Honeywell filed suit against ITT/TG for infringement of the '879 patent. Claim 1 of the '879 patent, the patent's only independent claim, provides as follows:
Fuel injection system component for communicating fuel to the engine of a motor vehicle, said motor vehicle having an electrical plane maintained at a predetermined electrical potential, said fuel system component being made of a composite material comprising a polymer having electrically conductive fibers distributed randomly throughout the material to provide an electrically conductive path through said component between the fuel communicated through said component and said electrical plane, so that at least a portion of the electrically conductive path extends through the component to thereby prevent build-up of electrostatic charge in the fuel and the resultant arcing which causes the breakdown of the polymer material comprising the fuel injection system component.
The products accused of infringement are “quick connects” manufactured and sold by ITT/TG. Quick connects are nut-like structures that join the various components of a fuel injection system together, such as a fuel line to a fuel filter. The accused quick connects have polymer housing that is interlaced with carbon fiber. ITT/TG denied the charge of infringement, and the district court subsequently held a Markman hearing to construe various limitations of the '879 patent, including “fuel injection system component” and “electrically conductive fiber.”
The district court construed the “fuel injection system component” limitation to mean “a fuel filter.” In arriving at its construction, the court recognized that the ordinary meaning of the term “refers to any constituent part of the fuel injection system of a motor vehicle including, for example, fuel filters, fuel lines, and connectors.” Honeywell, 330 F.Supp.2d at 878. The court also recognized that the patentee made statements during the prosecution of the '879 and '084 applications that could be interpreted to mean that the scope of the “fuel injection system component” limitation was broader than only fuel filters, at least in the patentee's view.
Notwithstanding the ordinary meaning or the prosecution history, the court determined that the written description clearly limited the “fuel injection system component” to a fuel filter, and that the statements in the prosecution history could not be used to enlarge the content of the written description. Id. at 882-83. According to the court, on several occasions in the written description, the “invention” was identified to be only a fuel filter. Id. at 879. Moreover, the court explained, “[t]he entire specification of the '879 patent, as well as the sole drawing, describe the elements and operation of a fuel filter with electrically conductive fibers. No other parts are described.” Id. Given the written description, the court concluded that “the patentee characterized a fuel filter as the only embodiment of his invention, not merely a ‘preferred’ version of all possible embodiments.” Id. at 880.
The district court also construed the claim term “electrically conductive fibers” to mean “fibers of a material that conducts electricity, including, without limitation, metal and carbon.” Id. at 888. According to the court, that was the ordinary meaning of “electrically conductive fibers.” The court gave the term its ordinary meaning despite a detailed disclosure in the written description why metal fibers were preferable to carbon fibers. Id. at 884. In the court's view, the disclosure was “not like the clear specification language defining the ‘invention’ as a fuel filter.” Id. The court determined that the disclosure distinguishing metal and non-metal conductive fibers merely reflected the patentee's preferred embodiment. Id. at 884-85. The court further noted that the written description never stated that carbon fibers could not be used as electrically conductive fibers. Id.
After the court issued its claim construction, ITT/TG filed a motion for summary judgment of noninfringement, which the court granted. In concluding that the accused products did not infringe the '879 patent, the court determined that quick connects are not fuel filters, and thus they do not literally infringe. Honeywell Int'l, Inc. v. ITT Indus., Inc., Civ. No. 02-73948, slip op. at 9 (E.D.Mich. May 17, 2005). Moreover, the court found that quick connects do not infringe under the doctrine of equivalents because they are not interchangeable with a fuel filter, and they do not compete commercially with fuel filters. Id. The also court determined that Honeywell could not invoke the doctrine of equivalents to encompass the accused quick connects in view of the written description's identification of a fuel filter as “the present invention.” According to the court, the patentee knew of fuel system components other than fuel filters, and that because he limited the claims to a fuel filter, all other fuel system components were dedicated to the public, and thus outside the reach of the doctrine of equivalents. Id., slip op. at 13-14.
The district court entered final judgment on April 27, 2005. Honeywell timely appealed, and we have jurisdiction pursuant to 28 U.S.C. § 1295(a)(1).
We review a district court's grant of summary judgment de novo, reapplying the same standard used by the district court. Ethicon Endo-Surgery, Inc. v. U.S. Surgical Corp., 149 F.3d 1309, 1315 (Fed.Cir.1998); Stratienko v. Cordis Corp., 429 F.3d 592, 597 (6th Cir.2005). Summary judgment is appropriate if there is no genuine issue as to any material fact and the moving party is entitled to a judgment as a matter of law. Fed.R.Civ.P. 56(c). “The evidence of the non-movant is to be believed, and all justifiable inferences are to be drawn in his favor.” Anderson v. Liberty Lobby, Inc., 477 U.S. 242, 255, 106 S.Ct. 2505, 91 L.Ed.2d 202 (1986). Claim construction is an issue of law, Markman v. Westview Instruments, Inc., 52 F.3d 967, 970-71 (Fed.Cir.1995) (en banc), that we also review de novo, Cybor Corp. v. FAS Techs., Inc., 138 F.3d 1448, 1456 (Fed.Cir.1998) (en banc).
On appeal, Honeywell argues that the district court erred by limiting the “fuel injection system component” limitation to a fuel filter and including no other component of a fuel injection system. In doing so, Honeywell contends that the court imported a limitation from the specification into the claims and thereby improperly limited the scope of the claims to the specification's preferred embodiment. According to Honeywell, nothing in the specification explicitly limits the claim term to a “fuel filter.” Honeywell relies on a statement contained in the specification referring to “the metallic components used in prior art systems,” '879 Patent, col.1 ll.32-33, to argue that the term “component” was meant to be broad. It also cites the patent's abstract, which summarized the invention using the term “component,” and the title of the patent as amended, to further argue that the specification did not limit the “fuel injection system component” to a fuel filter.
In addition, Honeywell points to the prosecution history in assigning error to the district court's construction of the “fuel injection system component” limitation. According to Honeywell, the patentee stated during prosecution that the intended scope of the claims was to include “all fuel components manufactured of the moldable material disclosed and claimed in the specification.” Honeywell also notes that the patent examiner issued a restriction requirement during prosecution of the '084 application (which also included claims to “fuel filters”) because “the fuel system component [claims] do[ ] not specifically require that the component be a fuel filter.”
Mainly reiterating the points made by the district court in its claim construction decision, ITT/TG responds that the claim term “fuel injection system component” was correctly limited to a fuel filter. ITT/TG also argues, however, that the court erred in its construction of the “electrically conductive fibers” limitation. ITT/TG contends that the court should have construed that term to include only metal fibers with a high aspect ratio, not carbon fibers. According to ITT/TG, the written description compared the properties of metal and carbon fibers, and “disparaged” the use of the latter as an electrically conductive fiber. ITT/TG contends that there was a clear disavowal of carbon fibers from the scope of the claims. Moreover, because the accused quick connects are indisputably made with carbon fibers, ITT/TG asserts that there can be no infringement either literally or under the doctrine of equivalents.
We agree with the district court that the claim term “fuel injection system component” is limited to a fuel filter. In Phillips v. AWH Corp., 415 F.3d 1303, 1315 (Fed.Cir.2005) (en banc), this court recognized that “claims ‘must be read in view of the specification, of which they are a part.’ ” We further stated that “the specification ‘is always highly relevant to the claim construction analysis. Usually, it is dispositive; it is the single best guide to the meaning of a disputed term.’ ” Id. (internal citations omitted). Here, the written description uses language that leads us to the conclusion that a fuel filter is the only “fuel injection system component” that the claims cover, and that a fuel filter was not merely discussed as a preferred embodiment. On at least four occasions, the written description refers to the fuel filter as “this invention” or “the present invention”:
This invention relates to a fuel filter for use in the fuel line that delivers fuel to a motor vehicle engine. '879 Patent, col.1 ll.8-9.
According to the present invention, a fuel filter for a motor vehicle is made from a moldable material which may be safely used in vehicles equipped with electronic fuel injection system. Id., col.1 ll.40-43.
This and other advantages of the present invention will become apparent from the following descriptions, with reference to the accompanying drawing, the sole Figure of which is a cross-sectional view of a fuel filter made pursuant to the teachings of the present invention․ Id., col.1 ll.43-49.
According to the present invention, an electrically conductive path is provided between the fuel within the inlet cavity 42 [of the fuel filter] and the [vehicle] body 38. Id., col.3 ll.41-43.
The public is entitled to take the patentee at his word and the word was that the invention is a fuel filter.
Moreover, the written description does not indicate that a fuel filter is merely a preferred embodiment of the claimed invention. The fuel filter was the only component of an EFI system that the written description disclosed as having a polymer housing with electrically conductive fibers interlaced therein. The only other fuel component specifically mentioned in the written description, the fuel line, was not required by the patentee to be made of an electrically conductive polymer material, as the claims require. See id., col.1 ll.59-60 (stating that the “fuel line may also be made of a non-conductive material”). The written description's detailed discussion of the prior art problem addressed by the patented invention, viz., leakage of non-metal fuel filters in EFI systems, further supports the conclusion that the fuel filter is not a preferred embodiment, but an only embodiment. Id., col.1 ll.10-25. Given the written description's disclosure, we conclude that the patentee has limited the scope of the '879 patent claims to a fuel filter.
Nor are we persuaded by Honeywell's argument that the patentee confirmed a broader scope of his claims during prosecution. Honeywell relies mainly on the patentee's response to the examiner's indefiniteness rejection in which he stated that the claims cover “all fuel components manufactured of the moldable material disclosed and claimed in the specification.” Honeywell places too much weight on that statement, as we find it to be ambiguous and possibly inconsistent with the written description. After all, the only fuel component disclosed and claimed in the patent was a fuel filter. In any event, such a broad and vague statement cannot contradict the clear statements in the specification describing the invention more narrowly.
We also do not assign much weight to the patent examiner's restriction requirement with respect to claims for a “fuel filter” and a “fuel system component” during prosecution of the '084 application. In making the restriction requirement, the examiner did not construe the claim term “fuel system component” or determine its meaning in light of the written description. He merely required that the applicant elect one aspect of his invention for prosecution without applying it to the specification.
Nevertheless, even if we were to agree with Honeywell that the patentee clearly expressed his intention during prosecution to have the “fuel injection system component” limitation include components in addition to a fuel filter, it would not change the result in this case. As we determined above, the written description provides only a fuel filter that is made with polymer housing and electrically conductive fibers interlaced therein. No other fuel injection system component with the claimed limitations is disclosed or suggested. Where, as here, the written description clearly identifies what his invention is, an expression by a patentee during prosecution that he intends his claims to cover more than what his specification discloses is entitled to little weight. See Biogen, Inc. v. Berlex Labs., 318 F.3d 1132, 1140 (Fed.Cir.2003) (stating that “[r]epresentations during prosecution cannot enlarge the content of the specification”).
We disagree, however, with the district court's construction of the claim term “electrically conductive fibers” to the extent that it encompasses carbon fibers. This court has recognized that “[w]here the specification makes clear that the invention does not include a particular feature, that feature is deemed to be outside the reach of the claims of the patent, even though the language of the claims, read without reference to the specification, might be considered broad enough to encompass the feature in question.” SciMed Life Sys. v. Advanced Cardiovascular Sys., 242 F.3d 1337, 1341 (Fed.Cir.2001). It is true, as the district court noted, that the '879 patent's written description did not expressly define “electrically conductive fibers,” as it did for “fuel injection system component.” Nevertheless, based on the disclosure in the written description, which demeaned the properties of carbon fibers, we conclude that the patentee thereby disavowed carbon fibers from the scope of the '879 patent's claims.
The written description contains the following disclosure:
Stainless steel also has the advantage of requiring smaller quantities for providing the required conductivity than other conductive fillers, such as carbon black. '879 Patent, col.3 ll.56-60 (emphasis added).
Other electrically conductive fillers, such as the aforementioned carbon, act as stress concentrators and, at the relatively high filler loadings required to achieve conductivity, restrict the ability of the resin matrix to yield under stress. Id., col.4 ll.1-5 (emphasis added).
Also, stainless steel fibers are ductile and non-rigid unlike straight or metallized carbon fibers․ This allows stainless steel fibers to maintain their integrity better during melt-processing. Id., col.4 ll.5-10 (emphasis added).
Unlike the non-metallic fibers, stainless steel fibers also do not increase mechanical strength or stiffness of the base resin significantly. Other metal fibers with high aspect ratios can be satisfactorily substituted for stainless steel. Id., col.4 ll.10-14 (emphasis added).
In making the above statements, the patentee informed its readers specifically why carbon fibers would not be suitable as “electrically conductive fibers” in the claimed invention. If the written description could talk, it would say, “Do not use carbon fibers.” There is no other way to interpret the written description's listing of all the reasons that metal fibers fare better than carbon fibers for use in the claimed invention, even though both materials are electrically conductive, viz., that metal fibers require smaller quantities to achieve the desired conductivity than carbon fibers, create less stress concentration, are more ductile, are less rigid, and increase the mechanical strength of the polymer housing. Contrary to the district court's understanding, the written description has gone beyond expressing the patentee's preference for one material over another. Its repeated derogatory statements concerning one type of material are the equivalent of disavowal of that subject matter from the scope of the patent's claims.
In reaching this decision, we reject Honeywell's argument in support of the district court's construction of “electrically conductive fibers.” Honeywell argues that the written description did identify carbon fibers as electrically conductive fibers, and that stainless steel fibers were merely preferred embodiments. Honeywell's argument misses the point. It is precisely because the written description has identified carbon fibers as electrically conductive, and yet it denigrated carbon fibers' applicability to the claimed invention, that we find a disavowal of that subject matter. Moreover, it is irrelevant whether stainless steel fibers are a preferred embodiment of the claimed invention. We are not here modifying the district court's claim construction to limit its scope to stainless steel fibers. We only modify it to exclude carbon fibers from the scope of the '879 patent claims.
Because Honeywell does not appeal the district court's judgment of lack of literal infringement, we turn to the question whether the accused quick connects infringe under the doctrine of equivalents. Even if the district court correctly construed the “fuel injection system component” limitation, Honeywell argues that the court erred in finding that the accused quick connects do not infringe under the doctrine of equivalents as a matter of law. According to Honeywell, although quick connects do not filter fuel like a fuel filter, the '879 claims do not require that the “fuel injection system component” perform that function. Honeywell contends that the claims only require that the “fuel injection system component” perform the functions of “communicat[ing] fuel to the engine of a motor vehicle,” and “provid[ing] an electrically conductive path through said component and [the] electrical plane.” Honeywell also asserts that the claims require that the only result needed to be achieved is to “prevent the build-up of electrostatic charge in the fuel and the resultant arcing which causes the breakdown of the polymer material comprising the fuel injection system component.” Because, Honeywell argues, the accused quick connects perform these functions in the same way as the fuel filter to achieve the same result, there is infringement.
We agree with the district court that the fuel filter and quick connects are not equivalent devices. They are substantially different. The accused quick connects do not filter fuel. Once we agree with the district court to construe a fuel system component to be a fuel filter, the fuel filter is not merely a limitation of the '879 patent claims. It is central to the patented invention. If one utilizes the conventional function/way/result analysis, it is beyond question that the accused quick connects do not perform the function of the fuel filter. Any equivalent of a fuel filter must necessarily perform the function of fuel filter-filtering fuel-in order to be an equivalent. As Honeywell recognizes in its own brief, “a fuel filter inherently filters fuel.” Honeywell Op. Br., at 49.
Having established that any structure equivalent to a fuel filter must substantially perform the function of filtering fuel, we affirm the district court's judgment of noninfringement. Honeywell concedes in its brief, as it must, that the accused quick connects do not perform the fuel filtering function. Honeywell Op. Br., at 47. Thus, no reasonable trier of fact could find that these two structures are equivalent. Given that quick connects do not perform substantially the same function as a fuel filter, there is no need for us to conduct a further analysis of the way that the accused products perform and the result of that performance.
We also conclude that the accused quick connects do not meet, either literally or under the doctrine of equivalents, the “electrically conductive fibers” limitation as we have construed it. There is of course no literal infringement because, as we have held and Honeywell has admitted, a quick connect is not a fuel filter. There can also be no infringement under the doctrine of equivalents because the accused quick connects use carbon fibers, and such fibers were disavowed from the scope of the “electrically conductive fibers” limitation, as we have discussed above. See J & M Corp. v. Harley-Davidson, Inc., 269 F.3d 1360, 1366 (Fed.Cir.2001) (“The scope of equivalents may [ ] be limited by statements in the specification that disclaim coverage of certain subject matter.” Dawn Equip. Co. v. Ky. Farms, Inc., 140 F.3d 1009, 1016 (Fed.Cir.1998)).
Because the accused quick connects do not meet, literally or equivalently, the “fuel injection system component” or “electrically conductive fibers” claim limitations, the district court's grant of summary judgment of noninfringement of the '879 patent in favor of ITT/TG is affirmed.
1. The '879 application's independent claim, as originally filed, read as follows:Moldable material for fuel system components for communicating fuel to the engine of a motor vehicle, said motor vehicle having a common electrical plane maintained at a common electrical potential, said material comprising a polymer material having electrically conductive fibers distributed randomly throughout the material to provide an electrically conductive path through said components between the fuel communicated through said components and said common electrical plane.
LOURIE, Circuit Judge.


- Filed Under
Determined to keep pushing its message that "gun-free zones don't work," the Asheville Tea Party is promoting the First National Gun Appreciation Day on Saturday.
The party drew heavy criticism in December for holding a raffle of a gun similar to the .223-caliber assault weapon used in the Sandy Hook Elementary School massacre. Twenty students and six school staffers were murdered in the attack.
Now, says Asheville Tea Party Chairwoman Jane Bilello, is not the time to back off their message. ...


text:
SYMPOSIUM. Greater Magadha: Evaluation and Retrospective
Greater Magadha: Evaluation and Retrospective
In his 2007 book Greater Magadha, Johannes Bronkhorst proposed a new theory for the historical formation of Indian culture in the mid-first millennium BCE. The theory proposed that a cultural and geographical area, Greater Magadha, was settled by an early wave of Indo-European speakers. A second wave of Indo-European speakers, carriers of Vedic culture, subsequently encountered the Greater Magadhans to the east and the resultant cultural meeting gave rise to the main features known in later Indian history. The Greater Magadha theory addresses many deep problems about the early formation of Indian culture that have long puzzled historians. It has been widely accepted, but has also given rise to serious criticism. This workshop will bring together some of the principal scholars who have engaged with the theory to discuss and retrospectively evaluate the theory just over a decade after it was proposed.

domain: history