Peroxisomes perform diverse and vital functions in eukaryotes, and abnormalities in peroxisomal function lead to severe developmental disorders in humans. Peroxisomes are also involved in a wide array of physiological and metabolic functions unique to plants, yet many aspects of this important organelle are poorly understood. In yeast and mammals, various steps in peroxisome biogenesis require the function of peroxin (PEX) proteins, among which PEX12 is a RING finger peroxisomal membrane protein involved in the import of matrix proteins. To investigate the role of PEX12 in plants, we identified a T-DNA knockout allele of PEX12 and generated partial loss-of-function pex12 mutants using RNA interference. We show that pex12 null mutants are developmentally arrested during early embryogenesis, and that the embryo-lethal phenotype can be rescued by overexpression of the PEX12-cyan fluorescent protein fusion protein, which targets to the peroxisome. Using virus-induced gene-silencing techniques, we demonstrate that peroxisomal number and fluorescence of the yellow fluorescent protein-peroxisome targeting signal type 1 protein are greatly reduced when PEX12 is silenced. RNA interference plants with partial reduction of the PEX12 transcript exhibit impaired peroxisome biogenesis and function, inhibition of plant growth, and reduced fertility. Our work provides evidence that the Arabidopsis (Arabidopsis thaliana) PEX12 protein is required for peroxisome biogenesis and plays an essential role throughout plant development.
Peroxisomes perform diverse and crucial functions in eukaryotes, and peroxisomal deficiencies cause developmental disorders in humans and disrupt many physiological and developmental processes in fungi (Powers and Moser, 1998; Gould and Valle, 2000; Parsons et al., 2001; Titorenko and Rachubinski, 2004). Plant peroxisomes, which include glyoxysomes, leaf-type peroxisomes, and unspecialized peroxisomes, contain different sets of enzymes depending on the cell type and developmental stage. They are involved in a wide range of functions, including lipid metabolism, photorespiration, nitrogen metabolism, stress response, and synthesis of some plant hormones (Beevers, 1979; Olsen and Harada, 1995; Hayashi and Nishimura, 2003).
More than 30 yeast genes have been identified that encode components for various aspects of peroxisome biogenesis, including peroxisome assembly, matrix protein import, and peroxisome proliferation (Purdue and Lazarow, 2001; Charlton and Lopez-Huertas, 2002; Brown and Baker, 2003). Proteins in this category are named peroxins, or PEX proteins. The peroxisome protein import machinery is composed of a number of proteins facilitating the import process, although the architecture of this machinery is still elusive. PEX2, PEX10, and PEX12 are RING finger proteins hypothesized to function in close proximity to facilitate the import of cargo proteins and the recycling of receptors in yeast and mammals, and possibly in other eukaryotes. These three peroxins are integral membrane proteins whose N- and C-terminal domains are both predicted to be exposed to the cytosol, yet their precise biochemical function is still unclear (Purdue and Lazarow, 2001; Brown and Baker, 2003). A complicating factor is that some PEX proteins appear to play additional roles outside of the peroxisome. For instance, in the yeast Yarrowia lipolytica, PEX2 is also involved in facilitating the exit of plasma membrane- and cell wall-associated proteins from the endoplasmic reticulum before they enter the secretory pathway (Titorenko et al., 1997). In the filamentous yeast Podospora anserina, PEX2 is also required for nuclear fusion during sexual sporulation (Berteaux-Lecellier et al., 1995). Our current knowledge still cannot explain the functional complexity of these PEX proteins.
The Arabidopsis (Arabidopsis thaliana) genome is predicted to encode about 15 proteins homologous to the yeast peroxins (Mullen et al., 2001; Charlton and Lopez-Huertas, 2002); a few of the genes have been cloned and partially characterized (Lin et al., 1999, 2004; Hayashi et al., 2000, 2005; Zolman et al., 2000; Hu et al., 2002; Schumann et al., 2003; Sparkes et al., 2003; Zolman and Bartel, 2004; Woodward and Bartel, 2005). PEX2 and PEX10 encode essential peroxins because null mutations of either gene cause embryonic lethality (Hu et al., 2002; Schumann et al., 2003; Sparkes et al., 2003). PEX2 was also found to be involved in developmental pathways regulated by the DET1 protein (Hu et al., 2002). Electron microscopy of pex10 null embryos revealed defects in the formation of lipid bodies, protein bodies, and the endoplasmic reticulum, in addition to the lack of peroxisomes (Schumann et al., 2003), although it is hard to distinguish whether these deficiencies are direct or indirect results from the loss of PEX10 function. Owing to embryo lethality of the knockout alleles, clear evidence showing the involvement of these RING peroxins in Arabidopsis peroxisome biogenesis and their roles in later stages of development has yet to be presented.
Mutations in PEX12 lead to failure of matrix protein import in yeast and mammals and result in the Zellweger syndrome, a lethal neurological disorder in humans (Gould and Valle, 2000). To determine the role of PEX12 in plant cellular functions and to address its role in plant development, we characterized Arabidopsis PEX12 and identified mutants deficient in this gene. Here, we report the subcellular localization of the Arabidopsis PEX12 protein and analysis of pex12 mutants generated by T-DNA insertion and two independent strategies of RNA interference (RNAi). This work establishes an essential role for Arabidopsis PEX12 in peroxisome biogenesis and in plant growth and development.
AtPEX12 (At3g04460) is a single-copy gene encoding a putative protein of 44 kD. It shares approximately 27% protein sequence identity with its yeast and mammalian orthologs and contains a C5-type RING finger motif with five conserved Cys (Fig. 1), which is different from the C3HC4-type RING found in AtPEX2 and AtPEX10. To study the function of PEX12 in plant development, we first identified from the Salk Arabidopsis T-DNA knockout collection (Alonso et al., 2003) a line containing a T-DNA insertion in the sixth exon of PEX12 (SALK_13612; Fig. 2A). We were unable to identify plants homozygous for the insertion after screening more than 200 T3 progeny from T2 heterozygous plants. Immature siliques of wild-type plants contained green seeds (Fig. 2B) that turned brown at maturity and, occasionally, one or two aborted seeds. However, immature siliques from heterozygous plants had approximately 22% white seeds (Fig. 2C), which became purplish and shriveled as the siliques matured. These data suggest that the pex12 insertion allele is segregating as a single recessive mutation, which may confer embryonic lethality in homozygotes.
Amino acid sequence alignment of PEX12 from Arabidopsis and other species. Arabidopsis, AtPEX12 (Q9M841); Homo sapiens, HsPEX12 (O00623); Pichia pastoris, PpPEX12 (Q01961). Sequences were aligned using Megalign from DNASTAR. Underlined are putative transmembrane domains. Boxes indicate the conserved Cys residues in the C-terminal RING finger motif.
PEX12 gene structure and knockout phenotypes. A, Schematic representation of PEX12, showing exons as white boxes and introns as single lines. The triangle indicates the position of the T-DNA insertion. Primers shown were used for genotyping. B and C, Immature siliques of wild-type and heterozygous plants. Arrows point to mutant seeds. Bars = 0.5 mm. D, PCR genotyping of embryo DNA. Lanes 1 to 2, Normal embryos; lanes 3 to 4, abnormal embryos. Primers used for 1 and 3, P12F/P12R; for 2 and 4, LB/P12R.
To confirm that the abnormal embryos indeed were homozygous for the T-DNA insertion allele, we dissected both abnormal and normal embryos (torpedo stage) from the seeds and pooled approximately 10 embryos of each type for DNA extraction. PCR analysis demonstrated that normal embryos contained both the insertion and the wild-type alleles (Fig. 2D, lanes 1 and 2), suggesting that they were composed of both wild-type and heterozygous embryos. However, only the T-DNA insertion-specific band was amplified from the abnormal embryos (Fig. 2D, lanes 3 and 4), indicating that these embryos were homozygous knockouts.
To determine which step of embryo development was disrupted by the pex12 null mutation, we analyzed developing seeds in heterozygous plants using Nomarski optics. Knockout embryos of pex12 showed retarded growth and in most cases were able to develop to the heart stage, but failed to grow further into torpedo and mature embryos (Fig. 3, C and D). Occasionally, the mutant embryos ceased to grow at globular or early torpedo stage (data not shown). Our data suggest that homozygous pex12 null embryos were delayed in growth and eventually stopped developing during early stages of embryogenesis.
Characterization of pex12 knockout embryos. A to D, Nomarski optics of developing seeds. A and C are from the same silique, as are B and D. E and F, Electron micrographs of wild-type (E) and mutant (F) embryos. Bars in A to D = 0.1 mm; in E and F = 1 μm. lb, Lipid body; pl, plastid; nu, nucleus; WT, wild type. White arrows in E point to peroxisomes.
We performed electron microscopic analysis with the knockout embryos to determine the effect of PEX12 deficiency at the ultrastructural level. Embryos subjected to examination were from heterozygous plants 6 d after fertilization, when wild-type embryos are usually at the torpedo stage of embryogenesis. Wild-type embryos contained well-developed peroxisomes (also called glyoxysomes in seeds), lipid bodies, and plastids (Fig. 3E). The pex12 null embryos, however, lacked peroxisomes, contained small and underdeveloped plastids, and were missing characteristic lipid body structures (Fig. 3F). Based on these observations, we conclude that PEX12 is required at least for the formation of the peroxisome. The impairment of other subcellular structures, such as lipid bodies and plastids, could be an indirect consequence of the loss of PEX12. For example, lipid bodies are physically associated with peroxisomes during seed germination, providing them with fatty acid substrates. It is thus conceivable that the function and presence of lipid bodies can be affected by a feedback mechanism when no functional peroxisomes are present to carry out lipid metabolism; however, a direct role of PEX12 in lipid body formation cannot be completely ruled out.
To determine the subcellular localization of AtPEX12, we analyzed wild-type Arabidopsis plants coexpressing yellow fluorescent protein (YFP)-peroxisome targeting signal type 1 (PTS1) and PEX12-cyan fluorescent protein (CFP) by fluorescence microscopy. PTS1, which is composed of Ser, Lys, and Leu, is widely used as a tag to localize proteins to the peroxisome in diverse systems. It is the targeting sequence for the majority of known peroxisomal matrix proteins and is recognized by the PTS1 receptor PEX5 (Subramani et al., 2000). PEX12-CFP displayed a punctate pattern of fluorescence, which is typical for peroxisomes, and was colocalized with YFP-PTS1 (Fig. 4, A–C), suggesting that PEX12 is also peroxisomal in Arabidopsis.
Subcellular localization of PEX12-CFP and its functional complementation of the pex12 null mutant. A to C, Localization of PEX12-CFP to the peroxisome. Cells shown are from leaf tissue of a plant coexpressing YFP-PTS1 and PEX12-CFP. Bars = 10 μm. D, Rescue of the pex12 knockout plant by PEX12-CFP. Top seedling, Wild type; bottom seedlings, T3 progeny from three independently rescued pex12 knockout lines.
We also transformed plants heterozygous for the knockout allele with the PEX12-CFP fusion construct driven by the 35S constitutive promoter. T2 plants were screened for homozygosity for the pex12 knockout allele and overexpression of the PEX12-CFP protein. The embryo-lethal phenotype of the knockout plants was rescued by the transgene (Fig. 4D), providing further evidence that the lack of a functional PEX12 gene was responsible for the lethal phenotype and suggesting that the PEX12-CFP fusion protein functions properly.
The embryo-lethal phenotype of the pex12 knockout plants prevented us from further elucidating the potential roles of PEX12 in peroxisome biogenesis and in later stages of development; thus, mutants with reduced levels of PEX12 were needed. To this end, two RNAi strategies were employed to knock down the expression of PEX12: (1) infecting plants with viruses containing a fragment of the PEX12 coding sequence and (2) making transgenic plants stably expressing a PEX12 double-stranded RNAi (dsRNAi) construct.
A gene-silencing system based on the bipartite geminivirus cabbage leaf curl virus (CbLCV) was recently developed that can efficiently induce diffusible, homology-based systemic silencing of endogenous genes in Arabidopsis (Turnage et al., 2002; Robertson, 2004). This system is composed of two small circular viral DNA genomes: CbLCV A and CbLCV B. To attenuate the viral symptom, the coat protein-encoding AR1 gene was deleted from the A genome and replaced by a fragment of the gene to be silenced. The B genome carries the movement protein for systemic infection (Turnage et al., 2002). To silence the PEX12 gene, a 247-bp cDNA fragment of PEX12 was cloned into the CbLCV A vector in sense or antisense orientation. Viruses containing the silencing constructs were bombarded into Arabidopsis plants in the YFP-PTS1 background. Leaf tissue from infected plants was observed under the fluorescent microscope 3 to 4 weeks after bombardment, when genes encoded by the viruses are expressed at high levels in new leaves. As a control, we also bombarded some plants with viruses containing the CHLORATA42 (CH42) gene. CH42 encodes the small subunit of the chloroplast magnesium-chelatase (Koncz et al., 1990) and confers an albino phenotype when silenced, owing to the lack of chlorophyll production (Supplemental Fig. 1). This control is used as an indicator for massive viral replication and systemic movement and therefore serves as a guide to determine the time for RNA and fluorescent microscopic analyses.
Plants infected by both the sense and antisense PEX12-silencing constructs exhibited a strong reduction in the number of peroxisomes as well as peroxisomal fluorescence of the YFP-PTS1 protein in new leaves (Fig. 5, C and E) compared to old leaves (Fig. 5, D and F), whereas plants infected by the empty vector control did not show a significant difference between old and new leaf tissue (Fig. 5, A and B). Reverse transcriptase (RT)-PCR analysis was subsequently performed to determine the expression level of PEX12 in these tissues. Figure 5G shows that, in plants bombarded with the PEX12-silencing constructs, the transcript level of PEX12 in the new tissue was significantly lower than in the old tissue, suggesting that PEX12 is required for peroxisome biogenesis in leaves. Despite the fact that the CbLCV virus used in this work was attenuated by removal of the AR1 gene, plants still displayed mild viral symptoms after infection, such as wrinkled leaves, stunted growth, and lack of inflorescence. As such, the mutant phenotypes caused by PEX12 silencing in adult plants could not be unambiguously determined by this approach.
Virus-induced gene silencing of PEX12. A to F, YFP-PTS1 fluorescence in plants infected by virus containing the CbLCV empty vector (A and B), vector containing a fragment of PEX12 in the sense orientation (C and D), and vector containing the antisense fragment of PEX12 (E and F). A, C, and E, Leaves from new growth; B, D, and F, old leaves of the same plants. Bars = 10 μm. G, RT-PCR analysis of PEX12 and UBIQUITIN10 (UBQ10) transcripts. Lanes 1 to 6 are PCR products amplified from RNA from A to F.
To elucidate more clearly the impact of PEX12 on plant development, a second RNAi approach was performed simultaneously. Given the lethal phenotype of the null mutants, we aimed to generate weaker and partial loss-of-function mutants of PEX12 by using a smaller fragment of the gene, rather than the entire coding region, in the RNAi construct. The same 247-bp cDNA fragment of PEX12 was cloned as inverted repeats into the dsRNAi vector pFGC5941, obtained from the Arabidopsis Biological Resource Center (ABRC). Plants containing YFP-PTS1 were transformed with the pFGC5941-derived PEX12-silencing construct under the control of the 35S promoter.
Approximately 20% of the 50 T1 transgenic plants showed fairly strong reduction of the PEX12 mRNA. The level of PEX12 suppression correlated with the severity of the mutant phenotypes in both T1 plants and their progeny. Compared with wild-type plants and RNAi plants with a subtle reduction of PEX12 expression (such as RNAi no. 3), RNAi plants with stronger reduction of PEX12 gene expression (such as RNAi nos. 1 and 2) were smaller and paler green, and developed more slowly (Fig. 6, A and B). The mutants were also less fertile with smaller gynoecia and shorter stamens in many mutant flowers (Fig. 6, C and D), and contained reduced numbers of peroxisomes and significantly weakened YFP-PTS1 fluorescence in peroxisomes (Fig. 6, E–H).
Phenotypes of PEX12 RNAi plants. A and B, Two-week-old (A) and 8-week-old (B) plants. Inset in B, RT-PCR (35 cycles) of RNA from leaves of (lanes 1–4) wild-type and PEX12 RNAi number 1, number 2, and number 3 plants. Bands shown are PCR products from PEX12 (top) and UBIQUITIN10 (bottom). C and D, Flower comparison of wild-type and RNAi number 1 plants. E to H, YFP-PTS1 fluorescence in wild-type and PEX12 RNAi plant leaves. Bars in C and D = 1 mm. Bars in E to H = 20 μm. I, Root length measurements of 7-d-old light-grown RNAi seedlings on 0% (black bars) or 1% (white bars) Suc, or on plates containing 1% Suc plus 20 μm IBA (gray bars). Each error bar represents sd from approximately 25 samples.
We also tested sugar dependence and indole-3-butyric acid (IBA) response of the RNAi plants. Through β-oxidation and the glyoxylate cycle, oilseed peroxisomes participate in lipid mobilization, which provides seedlings with energy to grow. As such, peroxisomal mutant seedlings of oilseed species such as Arabidopsis usually grow poorly in the absence of exogenous sugar. Mutants defective in β-oxidation are also resistant to the inhibitory effect of IBA on root elongation because mutant peroxisomes cannot efficiently convert IBA into IAA inside this organelle (Zolman et al., 2000). T3 seeds were plated on medium with or without 1% Suc and on medium containing 20 μm IBA combined with 1% Suc; root length was measured after 7 d of growth in the light. RNAi plants numbers 1 and 2 exhibited defects in root elongation without supplemental sugar, a phenotype that was mostly rescued by Suc (Fig. 6I). In addition, these mutants were also less responsive to IBA (Fig. 6I), which is indicative of a deficiency in β-oxidation.
Taken together, our data support the role of PEX12 as a critical player in peroxisome formation and function, and in plant vegetative and reproductive growth.
The essential role of PEX12 throughout Arabidopsis development led us to examine its expression pattern in the plant. An RT-PCR analysis of the PEX12 transcript suggested that this gene was ubiquitously expressed in young seedlings, leaves, roots, and flowers (data not shown). To assess its expression more completely, we used GENEVESTIGATOR, an Arabidopsis microarray gene expression database and analysis toolbox (Zimmermann et al., 2004; https://www.genevestigator.ethz.ch), to search for expression of the Arabidopsis PEX12 gene in various organs and at several developmental stages. The microarray data, based on experiments with the Arabidopsis full-genome chip (ATH1) arrays, showed that AtPEX12 was ubiquitously expressed and that its expression pattern correlated well with several other genes known to be required for peroxisome biogenesis, including the other two RING peroxin genes, PEX2 and PEX10 (Fig. 7). Consistent with their essential role in embryogenesis, all the three RING peroxins were most highly expressed in seeds (Fig. 7A). The transcript levels of these genes were also high during germination (Fig. 7B), when peroxisomes are needed for lipid metabolism, and in senescent plants (Fig. 7, A and B), in which leaf peroxisomes are transformed into glyoxysomes to convert membrane lipids into carbohydrates. All three PEX genes were also abundant in floral structures, namely, inflorescences, carpels, and pedicels (Fig. 7A), and at the stage when flowering is complete and siliques are formed (Fig. 7B, stage 8). These data are also in agreement with results from our study, which showed that flower formation and fertility were impaired when the expression of PEX12 was strongly reduced in Arabidopsis (Fig. 6). Interestingly, these three PEX genes were also strongly expressed in the shoot apex (Fig. 7A), where a specific function for peroxisomes has not been established, underlying their possible fundamental roles in this tissue.
Expression patterns of the RING PEX genes in Arabidopsis. A, Expression in various plant organs. B, Expression at successive developmental stages, as defined by Boyes et al. (2001). Stage 1, Hypocotyls and cotyledons emerged from seed coat; stage 2, cotyledons completely open; stage 3, seedlings with two rosette leaves; stage 4, 10 rosette leaves; stage 5, first flower buds seen; stage 6, early flowering; stage 7, midflowering; stage 8, flowering completed; stage 9, senescent and ready for seed harvest. The y axis in both A and B indicates the level of gene expression. Data used for the analysis were retrieved from GENEVESTIGATOR (https://www.genevestigator.ethz.ch; Zimmermann et al., 2004).
We have presented evidence that PEX12 is a peroxisomal protein in Arabidopsis and is essential for peroxisome biogenesis and plant development. First, null pex12 embryos (SALK_13612) were slow growing and eventually aborted during early embryogenesis. Second, PEX12-CFP was localized to the peroxisome and complemented the lethal phenotype of the pex12 knockout mutants. In addition, YFP-PTS1 plants infected by the CbLCV virus carrying part of the PEX12 coding sequence displayed strong reduction of the number of peroxisomes and import of PTS1-containing matrix proteins. Furthermore, transgenic plants, in which PEX12 gene expression was partially reduced by a dsRNAi construct, showed partial deficiency in peroxisome biogenesis and function, a smaller stature, and reduced fertility. Finally, a search of the GENEVESTIGATOR microarray database revealed similar expression patterns of the three RING peroxins in some tissues, supporting the essential roles of these PEX genes in seed development, germination, and flower formation.
The two classic and best-known functions for plant peroxisomes are (1) lipid mobilization through β-oxidation and glyoxylate cycle during oilseed germination and senescence, and (2) photorespiration in photosynthetic leaves (Beevers, 1979). Recent data have shown that peroxisomes also play an essential role in seed development, a complicated process that requires the coordinated action of hundreds of genes with diverse functions (Tzafrir et al., 2003). One example of the essential role for peroxisomes in embryogenesis came from the study of acyl-CoA oxidases (ACX), a family of enzymes catalyzing the first step of β-oxidation in the peroxisome with various substrate specificities. The acx3 acx4 double mutant, defective in both ACX3 (specific for medium-chain acyl-CoAs) and ACX4 (specific for short-chain acyl-CoAs), was found to abort during embryo development (Rylott et al., 2003). Here, we show that disruption of AtPEX12 caused developmental arrest of embryos mostly at the heart stage. This result is consistent with previous findings that PEX2 and PEX10 are essential for embryogenesis of Arabidopsis (Hu et al., 2002; Schumann et al., 2003; Sparkes et al., 2003). Interestingly, developmental arrest in the knockouts of PEX2 (J. Fan and J. Hu, unpublished data) and PEX10 (Schumann et al., 2003; Sparkes et al., 2003) also takes place at around the heart stage of embryogenesis. It is conceivable that peroxisome function is fundamental to post-heart stage embryo development, which requires a high level of fatty acid metabolism to provide energy for rapid embryo elongation and differentiation. Alternatively, peroxisomes may be a source of yet-to-be determined signaling molecules that promote further embryogenesis beyond the heart stage. Finally, loss of peroxisome function may also result in the accumulation of toxic levels of lipids or reactive oxygen species detrimental to the embryo because peroxisomes contain catalase, peroxisome membrane-bound ascorbate peroxidase, superoxide dismutase, and other antioxidative enzymes (del Rio et al., 2002). Irrespective of the mechanism underlying the role of peroxisomes in embryogenesis, the three RING finger peroxins appear to comprise basic components of peroxisomes, whereas the role for some other PEX genes may not be so important in Arabidopsis. For example, a null mutation of AtPEX14 caused plants to be short and pale but still fertile (Hayashi et al., 2000).
Peroxisomal matrix proteins are mislocalized in the cytoplasm in yeast and animal cells with reduced function of PEX12, indicating that this protein is particularly required for protein import into peroxisomes (Chang et al., 1997; Okumoto et al., 2000). Mammalian cells lacking PEX12 also showed accumulation of the PTS1 receptor PEX5 at the cytosolic side of the peroxisome membrane, suggesting that PEX12 may mediate recycling of PEX5 (Dodt and Gould, 1996). PEX12 was found to interact with PEX5 and PEX10 in yeast and mammals (Chang et al., 1999; Okumoto et al., 2000; Agne et al., 2003; Eckert and Johnsson, 2003). PEX10 was found to interact with PEX4, an ubiquitin-conjugating enzyme-like protein associated with the peroxisomal membrane (Eckert and Johnsson, 2003), yet the biological consequence of this interaction is unknown. Although conflicting results have been shown as to whether PEX2 is physically associated with PEX10 and PEX12 (Okumoto et al., 2000; Agne et al., 2003; Eckert and Johnsson, 2003), it has been postulated that these three RING peroxins may form a subcomplex on peroxisome membranes active in mediating protein import (Purdue and Lazarow, 2001). The similar phenotypes caused by loss of function of each of these three genes in Arabidopsis support the notion that AtPEX2, AtPEX12, and AtPEX10 act closely during peroxisome biogenesis. However, it will be necessary to test for interactions among the three RING peroxins and between these proteins and other peroxins in Arabidopsis to elucidate the biochemical mechanism underlying the action of the RING-type PEX proteins in plants.
Electron microscopic analysis of an Arabidopsis pex10 null mutant demonstrated that, in addition to the lack of characteristic peroxisomes, the lethal embryos also contained small lipid bodies with a half-unit membrane studded with ribosomes and flat lipid body discs (Schumann et al., 2003). However, these abnormal lipid body structures were not apparent in the pex12 null embryos; rather, no characteristic lipid bodies were observed in the lethal embryos examined in this study. This discrepancy may be due to the fact that there is some difference between the functions of PEX12 and PEX10. Alternatively, the mutant embryos analyzed in our study might be at a more degenerating stage. More direct evidence is needed to firmly establish a role of PEX10 or PEX12 in the formation of organelles other than the peroxisome in plants.
The mechanism for peroxisome biogenesis in various organisms shares a significant degree of conservation. All of the Arabidopsis PEX genes that have been cloned and characterized show some level of functional similarity with their yeast counterparts. For example, the PEX2, PEX10, PEX14, and PEX16 proteins all target to peroxisomes (Hayashi et al., 2000; Hu et al., 2002; Schumann et al., 2003; Sparkes et al., 2003; Lin et al., 2004). PEX16 could partially complement a yeast pex16 mutant (Lin et al., 1999), and mutants of PEX5, PEX6, PEX7, and PEX14 exhibit defects in peroxisomal functions (Hayashi et al., 2000; Zolman et al., 2000; Zolman and Bartel, 2004; Woodward and Bartel, 2005). On the other hand, some level of divergence in peroxisome biogenesis has also been observed in different species. For example, the number of import pathways for matrix proteins and the capacity of the receptors seem to vary among species (Otera et al., 1998; Motley et al., 2000; Hayashi et al., 2005). In addition, a number of the yeast and mammalian PEX genes do not have sequence homologs in Arabidopsis; thus, some aspects of peroxisome biogenesis in plants may be unique. It is possible that, in addition to the orthologs of yeast and animal PEX proteins, plants also carry unique peroxins that function in plant-specific aspects of peroxisome biogenesis. Further genetic, bioinformatic, and proteomic analyses are needed to identify novel PEX proteins from plants.
Arabidopsis (Arabidopsis thaliana) plants used in this study are of the Columbia background. Seeds were germinated on 1× Murashige and Skoog medium (Gibco), with or without 1% Suc, 20 μm IBA, and appropriate antibiotics, when necessary. Plants used for most experiments were grown with 16/8-h photoperiod under 80 to 100 μmol m−2 s−1 light conditions at 22°C. Plants used for bombardment in virus-induced gene silencing were grown in the same light intensity and temperature in short-day conditions with an 8/16-h photoperiod.
PCR was performed with genomic DNA using Taq DNA polymerase (Promega) and conditions suggested by the manufacturer. The primers were as follows: left border (LB) from the T-DNA vector, 5′-GCGTGGACCGCTTGCTGCAACT-3′; P12F from intron 5 and exon 6, 5′-ATGCCAAGATAGATGGATACATCCTCAAGG-3′; and P12R from exon 8, 5′-GGAGGGTACACTGTTGGAGCTGATAATCTC-3′. P12F and P12R amplify a 620-bp product from the wild-type allele. LB and P12R amplify an insertion-specific product of 730 bp. An approximately 110-bp piece of DNA of unknown origin was also found at the insertion site, between the T-DNA and the PEX12 genomic DNA.
A Leica MZ125 dissecting microscope (W. Nuhsbaum) was used to observe siliques and floral structures, and a Zeiss Axiophot microscope (Carl Zeiss) was used for Nomarski optics of seeds. For Nomarski optics, fresh siliques harvested 6 to 9 d after pollination were dissected. Developing seeds were treated with clearing solution containing chloral hydrate:water:glycerol (8:2:1, v/v/v) and cleared for 1 to 4 h at room temperature or overnight at 4°C.
A Zeiss Axiophot microscope was used to visualize fluorescent proteins. For in vivo detection of YFP and CFP, leaf tissue was mounted in water and viewed with a YFP filter (excitation 500 ± 12.5 nm, emission 540 ± 20 nm) or a CFP filter (excitation 440 ± 10 nm, emission 480 ± 15 nm).
Abnormal and normal seeds from the same silique (6–7 d after flowering) were fixed separately in 2.5% glutaraldehyde, 0.1 m phosphate buffer, pH 7.2, at room temperature for 2 h, followed by a secondary fixation in 1% (w/v) OsO4 in the same buffer. Samples were dehydrated in a graded series of acetone and embedded in Spurr's epoxy resin. Ultrathin sections (70–90 nm) were cut by a MT-X ultramicrotome, stained with 2% uranyl acetate and lead citrate, and observed under a JEM-100CX II transmission electron microscope (JEOL).
To make YFP-PTS1, a YFP-Ser-Lys-Leu fragment was amplified by PCR from the vector pEYFP-Peroxi (CLONTECH) and cloned into a vector derived from pPZP212 (Hajdukiewicz et al., 1994) and containing the 35S promoter. To clone PEX12-CFP, the coding region of AtPEX12 (At3g04460) was first amplified by RT-PCR from first-strand cDNA made from wild-type Col seedling mRNA, using primers At3g04460Fw (5′-AAGGATCCATCACTAACTAGAAGAAGAGA-3′) and At3g04460Rv (5′-ATGTCGACAGTGTCCTGAAACAACCTCC-3′). The resulting PCR fragment was then cloned into BamHI and SalI sites at the amino terminus of CFP (CLONTECH) in a binary vector derived from pPZP211 (Hajdukiewicz et al., 1994) and containing the 35S promoter.
To clone the PEX12 RNAi construct, the Arabidopsis vector pFGC5941 for dsRNA production was obtained from ABRC (stock no. CD3-447). A 247-bp fragment of PEX12 cDNA (position 364–611) was amplified by PCR using primers hpsipex12L (5′-GCTCTAGAGGCGCGCCGTTGTGTTACCGTATTTC-3′) and hpsipex12R (5′-CGGGATCCATTTAAATTCACTTGATGCATGTATC-3′). The PCR product was digested with AscI and SwaI, and ligated into pFGC5941 to generate plasmid pFGC5941-PEX12a. The same PCR product was digested again with BamHI and XbaI, and ligated into pFGC5941-PEX12a to generate plasmid pFGC5941-PEX12. As a result, the pFGC5941-PEX12 plasmid has a copy of the 247-bp fragment inserted into AscI to SwaI sites, and an inverted repeat of the fragment inserted into BamHI to XbaI sites.
All PCR amplifications were carried out using Pfu DNA polymerase (Stratagene) and protocols suggested by the manufacturer. Agrobacterium-mediated transformation of Arabidopsis plants was performed using the floral dip method (Clough and Bent, 1998). Transgenic plants were selected on Murashige and Skoog plates containing 50 ng/μL kanamycin for YFP-PTS1, 90 ng/μL gentamycin for PEX12-CFP, or 10 ng/μL BASTA for PEX12 RNAi.
A 247-bp fragment from PEX12 cDNA (position 364–611) was amplified by PCR using primers husi2L (5′-GCTCTAGAGTTGTGTTACCGTATTTC-3′) and husi2R (5′-GAAGATCTTCACTTGATGCATGTATC-3′). The PCR product was digested with XbaI and BglII and cloned into the pCPCbLCV.007 vector (Turnage et al., 2002) to generate plasmid p007-husi2.
A reverse copy of the 247-bp fragment was also generated by PCR using primers husi2aL (5′-GAAGATCTGTTGTGTTACCGTATTTC-3′) and husi2aR (5′-GCTCTAGACTTCACTTGATGCATGTATC-3′). The PCR product was digested with XbaI and BglII and cloned into the pCPCbLCV.007 vector (Turnage et al., 2002) to generate plasmid p007-husi2a.
Arabidopsis plants in the YFP-PTS1 background were grown in individual pots in short-day conditions and bombarded with an equal amount of each silencing construct DNA (in CbLCV A) and the pCPCbLCV.008 DNA (CbLCV B) as described in a previous study (Turnage et al., 2002). Each plant was bombarded at the age of 3 to 4 weeks old, according to the protocol provided by Turnage et al. (2002). Two to three plants were bombarded with each construct. The experiment was repeated three times. "Old" and "new" leaf tissue was collected separately from infected plants approximately 4 weeks after bombardment. "New leaves" were those from around the center of the rosette that emerged after bombardment, and the "old leaves" were older rosette leaves that were present at the time of the bombardment. Because of the distinct colors of silenced and nonsilenced leaves, the CH42-infected plants served as a guide to distinguish "new" from "old" tissue for microscopic and RT-PCR characterizations.
Total RNA was extracted with TRIzol reagent (Invitrogen) and subjected to Reverse Transcription reaction (Gibco). The PEX12-specific primers PEX12F2 (5′-GCGAGATTGAGATTGAGGAAAGACAGTGCC-3′) from exon 3 and PEX12R (5′-GGAGGGTACACTGTTGGAGCTGATAATCTC-3′) from exon 8 amplify a 684-bp product from PEX12 cDNA. The ubiquitin-specific primers UBQ10-1 (5′-TCAATTCTCTCTACCGTGATCAAGATGCA-3′) and UBQ10-2 (5′-GGTGTCAGAACTCTCCACCTCAAGAGTA-3′) from the UBIQUITIN10 gene (At4g05320) amplify a cDNA product of approximately 320 bp. PCR conditions were as follows: 94°C for 3 min, followed by cycles (27 for Fig. 5 and 35 for Fig. 6) of 94°C for 45 s, 57°C for 45 s, 72°C for 1 min, and a final extension of 72°C for 7 min.
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers Q9M841, O00623, and Q01961.
We thank Drs. Andreas Weber and Gregg Howe for their comments on the manuscript, and Ms. Karen Bird for editing. We are also grateful to Renato Perez and Deb Lin for technical assistance, Dr. Dominique Robertson for the CbLCV vectors, Dr. Pablo Cerdan for the UBQ10 primers, and the Salk Institute and ABRC for the PEX12 T-DNA insertion lines and the dsRNA vector pFGC5941.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.066811.
↵1 This work was supported by the U.S. Department of Energy and Michigan State University start-up funds to J.H., and by the National Science Foundation and the Howard Hughes Medical Institute (funding to J.C.).


The answer to these simple questions ultimately is defined not by the product or services these companies offer, but the feelings and emotion you have when using or interacting with their products. These companies know this and even market to it, and you may not even realize it.
Have you looked for your name or the name of someone close to you? And if you found it, did you take a picture of it and share it with someone?
Coca-Cola found a way for consumers to interact, talk about and share their brand without a drop of their product needing to be consumed. Regardless if you prefer the taste of Pepsi over Coke, there is a very good chance that you have talked about this brand experience campaign or Coca-Cola in general which only reinforces this brand as an experience than a product.
Bud Light has been running the 'Up for Whatever' Campaign for almost a year now. If you're not familiar, unsuspecting Bud Light customers are selected for 'random' dream day adventures where they may meet their favorite UFC fighters and line dance with them and attend a concert. Or they may even press a button and a flash party erupts with Lil' John flying in on a helicopter to perform. One customer even came home to have his favorite football player in his living room and his backyard was transformed into a pirate ship with cannons that shot off when his favorite football team scored a touchdown. I don't think you need to ask what any of this has to do with beer, because the answer… is nothing!
Instead of telling you how great their products and services are, brands are recognizing and taking advantage of positive experience as a tool for brand exposure. These experiences are then shared through social media platforms by the end users, completely unsolicited by the brand and the reach is staggering.
In the future when you are thinking of an awareness campaign, try not to think about the bells and whistles your product or service is capable of providing, but instead, think about an experience that the end user could have and capitalize on it. Think about your target demographic and make sure you keep them and their interests in mind. Be creative and take risks. This is also an experience for you!


text:
With closure of sporting goods store, Marquis shopping center in York loses an original tenant
Parkland shooter pleads guilty to 17 murders, 17 attempted murders in school massacre
Not a pop star, but Christina Petty's still a singer | Back to Reality
MAN GETS 17 1/2-YEAR TERM IN 1992 AMTRAK DERAILMENT
Hampton University alum awarded $250K in Pharrell Williams' minority entrepreneurship program
ALLEN AGREES TO ACCEPT GOALS 2000 MONEY
Virginia Government & Politics
Youngkin taps Fauquier County Sheriff as Secretary of Public Safety, says first goal is to fire parole board
By Jane Harper
Getting rid of Virginia's current parole board members has been a top priority for Gov.-elect Glenn Youngkin. (Eva Russo/AP)
Governor-elect Glenn Youngkin on Monday selected Fauquier County Sheriff Robert "Bob" Mosier to serve as secretary of Public Safety and Homeland Security, and said among his first goals will be to raise pay for law enforcement officers and fire the current parole board.
"Sheriff Mosier will play an important role in keeping our communities safe," Youngkin said in a statement provided to The Virginian-Pilot. "We will get to work on this key priority by fully funding and raising pay for our law enforcement officers. Together, we will protect qualified immunity, and on Day One fire the Parole Board."
Getting rid of the current parole board members has been a top priority for Youngkin and Attorney General Elect Jason Miyares after the board granted parole in several controversial cases last year. Among the most contentious was board's decision to release Vincent Lamont Martin, who was sentenced to life in prison for the 1979 murder of Richmond patrol officer Michael Connors. In many of the cases, victims and family members were not asked for input, nor were they even told about the board's decision.
Mosier, a Republican, has been in law enforcement for more than 30 years, with experience in local, county, federal, and international law enforcement, the statement said. He began as a police dispatcher at the age of 19.
Historic Triangle legislators push to expand protection of senior citizens from financial abuse
In Fauquier County, Mosier has served as commander of the special operations, patrol and criminal investigations divisions. He also developed and implemented community policing programs, managed security for large public events, and earned numerous awards and commendations over the years. He was elected sheriff in 2015.
In 1996, Mosier was selected by the U.S. Department of State to serve in Bosnia and Herzegovina with the International Police Task Force as a station commander, where he represented the Fauquier County Sheriff's Office.
He later served as director of investigations for the human rights organization International Justice Mission, as a senior representative for a U.S. Department of Defense law enforcement program, and as a GS-15 deputy senior executive police advisor for the Iraq Bureau of International Narcotics and Law Enforcement Affairs with the U.S. Department of State, according to the Youngkin statement.
All of the governor-elect's cabinet nominations must be approved by the Virginia General Assembly. Youngkin will be sworn in Jan. 15.
Jane Harper, 757-408-3714, jane.harper@pilotonline.com
robert mosier
public safety and homeland security
youngkin names
Jane Harper
Jane Harper covers state and federal courts for The Pilot. A native of Southeast Texas, she earned her journalism degree from The University of Texas at Austin and worked at newspapers in Texas and Maryland before moving to Norfolk in 1994.
Latest Virginia Government & Politics
Crittenden Middle School honor roll
TIPS LEAD TO ARREST

redpajama_set_name: RedPajamaCommonCrawl


text:
Vinyl Record 3
Attack on Titan Season 2 soundtrack comes to vinyl
Following the release of the Season 1 soundtrack vinyl earlier this year, we're delighted to announce that our upcoming Attack on Titan Season 2 Soundtrack vinyl releases will be arriving in February 2021! There will be Deluxe (pictured below) and standard edition vinyl versions available, plus a CD version too! Pre-orders for these will launch […]
1 month ago	https://blog.alltheanime.com/attack-on-titan-season-2-soundtrack-comes-to-vinyl/
My Hero Academia: Heroes Rising vinyl soundtrack coming to All The Anime Shop
Following the recent news of our upcoming Attack on Titan Season 1 sound track vinyl release; us taking pre-orders for a Cowboy Bebop soundtrack vinyl produced by Milan Records; and the addition of a number of vinyl soundtracks (including Death Note) produced by Tiger Lab Vinyl, we've got more vinyl soundtrack news to share with […]
4 months ago	https://blog.alltheanime.com/my-hero-academia-heroes-rising-vinyl/
Back in October of last year, we revealed that our next big vinyl record release would be the soundtrack of the juggernaut that is Attack on Titan, from the famed composer Hiroyuki Sawano! Ever since that announcement you've been hungry for more news, and today we're excited to reveal everything you need to know about our upcoming Attack on […]
5 months ago	https://blog.alltheanime.com/attack-on-titan-season-1-soundtrack-comes-to-vinyl/


Business Continuity Planning (BCP) is best described as the planning and preparation that will keep your business afloat during any operational emergency. Business owners need to plan for the threats and risks their company faces, and implement safeguards to protect the business in case of disaster.
Registered members have access to a free Business Continuity Planning (BCP) kit, ebooks and webinars created with our partner Inoni and, for more complicated cases, we provide a BCP service at a special discounted rate.
Although you know the risks (flooding, cyber-attack, terrorism to name a few), the effects these have on you are often unique to your business. There is no standard response. Your business is complex with many highly interconnected and synchronised components known only to you, so rebuilding it (whilst continuing to operate) is rarely easy or straightforward, even with help.
Any business could face an unexpected, significant event that has the potential to put them out of business. By putting together a plan you are ensuring that should the worst happen, you know exactly who is responsible for what and there is no panic as to how you will cope.
Putting together a plan can seem daunting, but this kit helps to break it into manageable chunks and, if you are feeling overwhelmed, Inoni are offering exclusive discounts on BCP software and services to guide you through the process and make it even simpler.
Read about how we implemented our Business Continuity Plan when our offices flooded.


Just today we got two reports that indicate manufacturing activity is slowing. If you buy into the idea that the U.S. is now sliding into a recession, it is not a stretch to assume that industrial stocks will decline. The question is, are we closer to the beginning of the decline or closer to the end?
Let's take a look at today's two reports.
"Industrial production fell 0.5 percent in February after having increased 0.1 percent in January. Much of the decrease in February resulted from a weather-related drop of 3.7 percent in the output of utilities. In the manufacturing sector, output decreased 0.2 percent in February, and declines were fairly widespread across industries. The output of mines moved up 0.4 percent. At 113.7 percent of its 2002 average, total industrial production was 1.0 percent above its year-earlier level. The capacity utilization rate for total industry in February fell 0.6 percentage point, to 80.9 percent, the lowest rate since November 2005."
"The factory operating rate fell 0.3 percentage point in February, to 79.3 percent, a level 0.5 percentage point below its 1972-2007 average. The production of durable goods moved down 0.4 percent. Large production declines were recorded in wood products, primary metals, motor vehicles and parts, furniture and related products, and miscellaneous products."
All told, production of both non-durables and durable goods declined. In addition, capacity utilization was seen to be slightly less. It can't be said that the wheels are coming off manufacturing but on the other hand the numbers are not particularly encouraging either.
The Federal Reserve Bank of New York released their Empire State Manufacturing Index. It fell to a negative 22.2 in March from a negative 11.7 in February. A number below zero indicates contraction. The report showed somewhat of a mixed picture as new orders edged up slightly but stayed in negative territory. Inventories and shipments both fell further below previous unexpectedly negative values.
As readers may recall, the Philadelphia Fed also had a surprisingly bad read on manufacturing in their report last month. That was followed by a bad number from the Institute for Supply Management whose factory index dropped below 50 and pointed to a more widespread contraction in manufacturing.
After today's market action, one could argue that I might have been premature in determining the financials had bottomed. It appears from the latest batch of reports, however, that industrials are just building up some momentum on the downside.
Many U.S. manufacturers have been benefiting from the weak dollar and bolstering their income statements via strong exports. There is discussion that, with financial and economic problems spreading, other countries may begin to lower their interest rates, making the dollar look stronger in comparison. Overseas demand may also begin to flag if the malaise in the U.S. continues to spread globally.
Are we on the verge of an acceleration in the decline of the manufacturing stocks? Industrials, as represented by the SPDR Industrials ETF (XLI) are off about 16% from their peak in October 2007. XLI, which closed today $35.90, is currently well above the January intraday low of $32.40.
If you believe that the industrials have room to fall further, you may be interested in the ProShares UltraShort Industrials ETF (SIJ). At about $67, SIJ is riding along above its 50-day moving average which at the beginning of this year crossed above its 200-day moving average. The Aroon indicators show the strong up-trend that has been in place may be weakening but the Chaikin Money Flow indicates there is still plenty of buying pressure. The RSI measurement looks like the ETF has a way to go before it could be considered over-bought. This ETF is somewhat thinly traded but it could be the right investment to play a recession-driven slowdown in manufacturing.


text:
package org.mini2Dx.core.engine.geom;

import org.junit.Before;
import org.junit.Test;
import org.mini2Dx.core.engine.PositionChangeListener;
import org.mini2Dx.core.engine.SizeChangeListener;

import com.badlogic.gdx.math.Vector2;

import junit.framework.Assert;

/**
 * Unit tests for {@link CollisionPolygon}
 */
public class CollisionPolygonTest implements PositionChangeListener<CollisionPolygon>, SizeChangeListener<CollisionPolygon> {
	private CollisionPolygon polygon1, polygon2;
	private int positionNotificationReceived, sizeNotificationReceived;

	@Before
	public void setup() {
		positionNotificationReceived = 0;
		sizeNotificationReceived = 0;

		polygon1 = new CollisionPolygon(new float [] { 0f, 0f,
				10f, 0f,
				10f, 10f,
				0f, 10f
				});
	}

	@Test
	public void testSet() {
		polygon1.addPostionChangeListener(this);
		polygon1.set(10f, 10f);
		Assert.assertEquals(1, positionNotificationReceived);
	}

	@Test
	public void testAddPoint() {
		polygon1.addPostionChangeListener(this);
		polygon1.addPoint(new Vector2(-5f, 5f));
		Assert.assertEquals(1, positionNotificationReceived);
	}

	@Override
	public void positionChanged(CollisionPolygon moved) {
		positionNotificationReceived++;
	}

	@Override
	public void sizeChanged(CollisionPolygon changed) {
		sizeNotificationReceived++;
	}
}

redpajama_set_name: RedPajamaGithub


text:
Proposal Could Keep Trump Off Connecticut Ballot in 2020
Connecticut is one of 18 states with a proposal to mandate that anyone running for president has to disclose their federal tax returns in order for that person to appear on the state ballot.
By Max Reiss	• Published at 4:58 pm on April 23, 2019
Connecticut could end up ending one of the most heated political debates in Washington, or it could end up leaving President Donald Trump off of the 2020 ballot altogether.
Connecticut is one of 18 states with a proposal to mandate that anyone running for president has to disclose their federal tax returns in order for that person to appear on the state ballot. In Connecticut, the proposal also extends to candidates running for governor.
Sen. Mae Flexer, (D – Killingly), who chairs the Government and Elections Administration Committee in the Connecticut General Assembly, acknowledges that the bill exists because President Donald Trump broke the norm of providing several years of tax returns in the lead up to the 2016 election.
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"I think there's a recognition from my colleagues and from the American people and the people of Connecticut that they need to understand the full breadth of the president's financial interests and to do that we need to see the president's tax returns," she said.
The proposal, which passed the GAE Committee earlier in April, would require gubernatorial and presidential candidates wishing to appear on the ballot to disclose three years of federal tax returns.
The issue is significant, because it could set up a scenario where if the General Assembly passes it, and then Gov. Ned Lamont signs it into law, then it would set up a choice for the president. He could sue to block the law, cooperate and provide his tax returns going back three years, or he could settle with being a write-in candidate only.
On Monday, the president sued to block his financial records from being disclosed to Rep. Elijah Cummings, the Democratic chairman of the House Oversight Committee.
Republicans in Connecticut describe the measure as a political cheap shot aimed at the president.
"It's simply here for political purposes and I think that's unfortunate," said Sen. Rob Sampson, (R – Wolcott), who is the ranking member on the GAE Committee. He said he believes the proposal is unconstitutional.
"There is a very clear requirement in the United States Constitution about what is required to be an eligible candidate for president and I believe that supersedes any Connecticut law that would say because a presidential candidate doesn't disclose their tax returns that they would not be allowed," he said.
Flexer said she thinks it's possible that lawmakers approve the measure this year. She said voters have a right to know what financial interests candidates have.
"Obviously President Trump is a strong case why the American people need to understand the financial interests of their president but we're looking actually at a proposal that would do the same thing for gubernatorial candidates in our state as well," she said.


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I turned 32 this year. That seems weird. However, it doesn't seem weird in the way you think it might. I don't have a problem being in my thirties. In fact, I looked forward to it for years! So many people have told me that this decade will be one of self acceptance, stability, and introspection. So far? They were right. My twenties were a whirlwind. Life changed for me every five minutes it seemed like. I always landed on my feet, but the rug was certainly pulled out from under them more than a few times. I didn't have a great grasp on who I was and I certainly hadn't figure out that I was enough. Over the last few years, I have truly learned what it means to be "fearlessly independent".
I have thought about things that I have learned or realized up until this point in my adult life that I want to share with others. I certainly haven't always known these things, and many of them have been learned more recently. Here are 32 important life lessons from my 32 trips around the sun.
5 | Let go of being a certain size or weight. You are a human being, and not designed to stay the same. Your body will change. Don't fight it.
9 | Don't set unattainable expectations for people. In fact, the lower the expectation- the better. Everyone is human and people have bad phases and deserve grace. I have, and I am thankful for second chances.
10 | Someone else is happy with less than what you have. Someone always has less than you. Always come back to gratitude.
14 | Motherhood is an insanely stressful, emotional, and difficult job and no one can fully respect a mother the way another mother can. Link arms with other mothers.
16 | Hurt people hurt people. Take a breath when you are hurt and understand that it's probably not about you.
24 | You can learn how to do anything on YouTube. Literally, anything. If anyone needs an appendectomy in the apocalypse, I got you.
25 | Other regions of the United States are 100% worth exploring. There is so much more to life than your own back yard. Also, live in another region if you can. There is so much to be learned outside of your comfort zone.
29 | It's ok to be on both sides of an issue. You are not required to be passionately rigid on one side or the other.
31 | There is nothing shameful or less than about renting. It comes with a lot of benefits versus owning a house.
32 | The best changes and life resets never start on a Monday. Make the change when you feel the urge.
I love brands whose corporate values fall in line with my personal ones. New Balance began developing performance products in 1906. They were committed to designing a unique brand without fearing what others might think. New Balance also subscribes to the idea of winning, but not at all costs. Wins must have integrity and purpose. The brand continues its "Fearlessly Independent" nature in creating products that combine both function and fashion. For my active lifestyle, New Balance is the perfect addition.
I got these New Balance sneakers on Zappos.com and was thoroughly impressed with their fast and free shipping. They also have a 365 return policy, a range of styles for all ages, and world class customer service. Zappos has long been a staple for me, as it embodies convenience and quality for the modern parent.
With all that said, how have you learned to be fearlessly independent over the years? What advice would you give?


Hamill, W. (1833). Letter, To: Hayes, Engerer & Co. From: William Hamill, November 2, 1833.
Hamill, William. Letter, To: Hayes, Engerer & Co. From: William Hamill, November 2, 1833. 1833.
"Letter, To: Hayes, Engerer & Co. From: William Hamill, November 2, 1833." Series XII Hayes Engerer and Co. Digital Library @ Villanova University. Date Accessed: 22 April 19, 10:41 AM.


text:
Home » 2020 Topics & Objectives » Maternal, Infant, and Child Health
Maternal, Infant, and Child Health
Objectives(active tab)
Interventions & Resources
National Snapshots
Expand All Objectives Collapse All Objectives
MICH-1 Reduce the rate of fetal and infant deaths
MICH-1.1 Reduce the rate of fetal deaths at 20 or more weeks of gestation
Baseline: 6.2 fetal deaths at 20 or more weeks of gestation per 1,000 live births and fetal deaths occurred in 2005
Target: 5.6 fetal deaths per 1,000 live births and fetal deaths
Target-Setting Method: 10 percent improvement
National Vital Statistics System-Fetal Death (NVSS-FD), CDC/NCHS; National Vital Statistics System-Natality (NVSS-N), CDC/NCHS
HP2020 data for this objective
Spotlight on Disparities:
Disparities by geographic location
Disparities by sex
Disparities by fetal and/or live birth weight
Details about the methodology and measurement of this HP2020 objective
The HP2010 objective with the same definition was 16-01a.
Search data for all HP2010 objectives
Related research articles on PubMed
MICH-1.2 Reduce the rate of fetal and infant deaths during perinatal period (28 weeks of gestation to less than 7 days after birth)
Baseline: 6.6 fetal and infant deaths per 1,000 live births and fetal deaths occurred during the perinatal period (28 weeks of gestation to less than 7 days after birth) in 2005
Target: 5.9 perinatal deaths per 1,000 live births and fetal deaths
Linked Birth/Infant Death Data Set, CDC/NCHS; National Vital Statistics System-Fetal Death (NVSS-FD), CDC/NCHS
The HP2010 objective with the same definition was 16-01b.
MICH-1.3 Reduce the rate of all infant deaths (within 1 year)
LHI
Leading Health Indicators are a subset of Healthy People 2020 objectives selected to communicate high-priority health issues.
Baseline: 6.7 infant deaths per 1,000 live births occurred within the first year of life in 2006
Target: 6.0 infant deaths per 1,000 live births
Linked Birth/Infant Death Data Set, CDC/NCHS
The HP2010 objective with the same definition was 16-01c.
Related Leading Health Indicator (LHI) information
MICH-1.4 Reduce the rate of neonatal deaths (within the first 28 days of life)
Baseline: 4.5 neonatal deaths per 1,000 live births occurred within the first 28 days of life in 2006
Target: 4.1 neonatal deaths per 1,000 live births
The HP2010 objective with the same definition was 16-01d.
MICH-1.5 Reduce the rate of postneonatal deaths (between 28 days and 1 year)
Baseline: 2.2 postneonatal deaths per 1,000 live births occurred between 28 days and 1 year of life in 2006
Target: 2.0 postneonatal deaths per 1,000 live births
Disparities by marital status
Disparities by race and ethnicity
The HP2010 objective with the same definition was 16-01e.
MICH-1.6 Reduce the rate of infant deaths related to birth defects (all birth defects)
Baseline: 1.4 infant deaths per 1,000 live births were attributed to birth defects (all birth defects) in 2006
Disparities by country of birth
The HP2010 objective with the same definition was 16-01f.
MICH-1.7 Reduce the rate of infant deaths related to birth defects (congenital heart defects)
Baseline: 0.38 infant deaths per 1,000 live births were attributed to congenital heart and vascular defects in 2006
Target: 0.34 infant deaths per 1,000 live births
The HP2010 objective with the same definition was 16-01g.
MICH-1.8 Reduce the rate of infant deaths from sudden infant death syndrome (SIDS)
Baseline: 0.55 infant deaths per 1,000 live births were attributed to sudden infant death syndrome in 2006
Disparities by age group
The HP2010 objective with the same definition was 16-01h.
MICH-1.9 Reduce the rate of infant deaths from sudden unexpected infant deaths (includes SIDS, Unknown Cause, Accidental Suffocation, and Strangulation in Bed)
Baseline: 0.93 infant deaths per 1,000 live births were attributed to sudden unexpected/unexplained causes in 2006
MICH-2 Reduce the 1-year mortality rate for infants with Down syndrome
Baseline: 48.6 deaths within the first year of life per 1,000 infants diagnosed with Down syndrome occurred in 2005–06
Target: 43.7 deaths within the first year of life per 1,000 infants with Down syndrome
National Birth Defects Prevention Network (NBDPN), CDC/NCBDDD
MICH-3 Reduce the rate of child deaths
MICH-3.1 Reduce the rate of deaths among children aged 1 to 4 years
Revised
This objective was revised. See Revision History for Details.
Baseline: 29.4 deaths among children aged 1 to 4 years per 100,000 population occurred in 2007
Target: 26.5 deaths per 100,000 population
National Vital Statistics System-Mortality (NVSS-M), CDC/NCHS; Bridged-race Population Estimates, CDC/NCHS and Census
Map of state-level data for this objective
Revision History: This objective was revised. Read more about the revision history.
MICH-4 Reduce the rate of adolescent and young adult deaths
MICH-4.1 Reduce the rate of deaths among adolescents aged 10 to 14 years
Baseline: 16.5 deaths among adolescents aged 10 to 14 years per 100,000 population occurred in 2007
MICH-4.3 Reduce the rate of deaths among young adults aged 20 to 24 years
Baseline: 98.1 deaths among young adults aged 20 to 24 years per 100,000 population occurred in 2007
MICH-5 Reduce the rate of maternal mortality
Baseline: 12.7 maternal deaths per 100,000 live births occurred in 2007
Target: 11.4 maternal deaths per 100,000 live births
National Vital Statistics System-Mortality (NVSS-M), CDC/NCHS; National Vital Statistics System-Natality (NVSS-N), CDC/NCHS
The HP2010 objective with the same definition was 16-04.
MICH-6 Reduce maternal illness and complications due to pregnancy (complications during hospitalized labor and delivery)
Baseline: 31.1 percent of pregnant females suffered complications during hospitalized labor and delivery in 2007
Target: 28.0 percent
National Hospital Discharge Survey (NHDS), CDC/NCHS
Disparities by geographic region
Disparities by health insurance status
MICH-7 Reduce cesarean births among low-risk (full-term, singleton, and vertex presentation) women
MICH-7.1 Reduce cesarean births among low-risk women with no prior cesarean births
Baseline: 26.5 percent of low-risk females with no prior cesarean birth had a cesarean birth in 2007
National Vital Statistics System-Natality (NVSS-N), CDC/NCHS
MICH-7.2 Reduce cesarean births among low-risk women giving birth with a prior cesarean birth
Baseline: 90.8 percent of low-risk females giving birth with a prior cesarean birth had a cesarean birth in 2007
MICH-8 Reduce low birth weight (LBW) and very low birth weight (VLBW)
MICH-8.1 Reduce low birth weight (LBW)
Baseline: 8.2 percent of live births were low birth weight in 2007
Target: 7.8 percent
Target-Setting Method: Projection/trend analysis
MICH-8.2 Reduce very low birth weight (VLBW)
Baseline: 1.5 percent of live births were very low birth weight in 2007
MICH-9 Reduce preterm births
MICH-9.1 Reduce total preterm births
Baseline: 10.4 percent of live births were preterm in 2007
MICH-9.2 Reduce late preterm or live births at 34 to 36 weeks of gestation
Baseline: 7.5 percent of live births were late preterm or occurred at 34 to 36 weeks of gestation in 2007
MICH-9.3 Reduce live births at 32 to 33 weeks of gestation
Baseline: 1.2 percent of live births occurred at 32 to 33 weeks of gestation in 2007
MICH-9.4 Reduce very preterm or live births at less than 32 weeks of gestation
Baseline: 1.7 percent of live births occurred at less than 32 weeks of gestation in 2007
Pregnancy Health and Behaviors
MICH-10 Increase the proportion of pregnant women who receive early and adequate prenatal care
MICH-10.1 Increase the proportion of pregnant women who receive prenatal care beginning in the first trimester
Baseline: 70.8 percent of females delivering a live birth received prenatal care beginning in the first trimester in 2007
MICH-10.2 Increase the proportion of pregnant women who receive early and adequate prenatal care
Baseline: 70.5 percent of pregnant females received early and adequate prenatal care in 2007
MICH-11 Increase abstinence from alcohol, cigarettes, and illicit drugs among pregnant women
MICH-11.1 Increase abstinence from alcohol among pregnant women
Baseline: 89.4 percent of pregnant females aged 15 to 44 years reported abstaining from alcohol in the past 30 days in 2007–08
National Survey on Drug Use and Health (NSDUH), SAMHSA
Disparities by educational attainment
Disparities by family income (percent poverty threshold)
MICH-11.2 Increase abstinence from binge drinking among pregnant women
Baseline: 95.0 percent of pregnant females aged 15 to 44 years reported abstaining from binge drinking during the past 30 days in 2007–08
Target: 100 percent
Target-Setting Method: Total coverage
MICH-11.3 Increase abstinence from cigarette smoking among pregnant women
Baseline: 89.6 percent of females delivering a live birth reported abstaining from smoking cigarettes during pregnancy in 2007
MICH-11.4 Increase abstinence from illicit drugs among pregnant women
Baseline: 94.8 percent of pregnant females aged 15 to 44 years reported abstaining from illicit drugs in the past 30 days in 2007–08
MICH-12 (Developmental) Increase the proportion of pregnant women who attend a series of prepared childbirth classes
This objective was archived due to lack of data source, changes in science, or was replaced with other objectives. See Revision History for Details.
Potential Data Sources:
Pregnancy Risk Assessment Monitoring System (PRAMS), CDC/NCCDPHP; California's Maternal and Infant Health Assessment (MIHA), California Department of Public Health (CDPH)
Revision History: This objective was archived due to lack of a viable data source. Read more about the revision history.
MICH-13 (Developmental) Increase the proportion of mothers who achieve a recommended weight gain during their pregnancies
Preconception Health and Behaviors
MICH-14 Increase the proportion of women of childbearing potential with intake of at least 400 µg of folic acid daily from fortified foods or dietary supplements
Baseline: 23.8 percent of nonpregnant females aged 15 to 44 years reported a usual daily total intake of at least 400 µg of folic acid from fortified foods or dietary supplements in 2003–06
National Health and Nutrition Examination Survey (NHANES), CDC/NCHS
Disparities by disability status
MICH-15 Reduce the proportion of women of childbearing potential who have lower (below the 25th percentile) red blood cell folate concentrations
Baseline: 24.9 percent of nonpregnant females aged 15 to 44 years had lower (below the 25th percentile) red blood cell folate concentrations in 2007–10
MICH-16 Increase the proportion of women delivering a live birth who received preconception care services and practiced key recommended preconception health behaviors
MICH-16.1 Increase the proportion of women delivering a live birth who discussed preconception health with a health care worker prior to pregnancy
Baseline: 24.5 percent of women who delivered a live birth in 2012 discussed preconception health with a health care worker prior to pregnancy
MICH-16.2 Increase the proportion of women delivering a live birth who took multivitamins/folic acid prior to pregnancy
Baseline: 30.3 percent of females delivering a recent live birth took multivitamins/folic acid every day in the month prior to pregnancy, as reported in 2007
MICH-16.3 Increase the proportion of women delivering a live birth who did not smoke prior to pregnancy
Baseline: 79.8 percent of females delivering a recent live birth did not smoke in the 3 months prior to pregnancy, as reported in 2011
MICH-16.4 Increase the proportion of women delivering a live birth who did not drink alcohol prior to pregnancy
Baseline: 50.6 percent of females delivering a recent live birth did not drink alcohol in the 3 months prior to pregnancy, as reported in 2007
MICH-16.5 Increase the proportion of women delivering a live birth who had a healthy weight prior to pregnancy
Baseline: 52.5 percent of females delivering a recent live birth had a normal weight (i.e., a BMI of 18.5 to 24.9) prior to pregnancy, as reported in 2007
MICH-16.6 Increase the proportion of women delivering a live birth who used a most effective or moderately effective contraception method postpartum
Baseline: 53.2 percent of women delivering a live birth used a most effective or moderately effective contraception method postpartum in 2012
MICH-17 Reduce the proportion of persons aged 18 to 44 years who have impaired fecundity (i.e., a physical barrier preventing pregnancy or carrying a pregnancy to term)
MICH-17.1 Reduce the proportion of women aged 18 to 44 years who have impaired fecundity
Baseline: 12.7 percent of females aged 18 to 44 years had impaired fecundity in 2002
National Survey of Family Growth (NSFG), CDC/NCHS
Disparities by family type
MICH-17.2 (Developmental) Reduce the proportion of men aged 18 to 44 years who have impaired fecundity
Postpartum Health and Behavior
MICH-18 Reduce postpartum relapse of smoking among women who quit smoking during pregnancy
Baseline: 42.4 percent of postpartum women who quit smoking during pregnancy relapsed in 2011
MICH-19 Increase the proportion of women giving birth who attend a postpartum care visit with a health care worker
Baseline: 90.1 percent of women giving birth in 2012 attended a postpartum care visit with a health care worker
Target-Setting Method: Minimal statistical significance
MICH-34 (Developmental) Decrease the proportion of women delivering a live birth who experience postpartum depressive symptoms
MICH-20 Increase the proportion of infants who are put to sleep on their backs
Baseline: 68.9 percent of infants were put to sleep on their backs in 2007
MICH-21 Increase the proportion of infants who are breastfed
MICH-21.1 Increase the proportion of infants who are ever breastfed
Baseline: 74.0 percent of infants born in 2006 were ever breastfed, as reported in 2007–09
National Immunization Survey (NIS), CDC/NCIRD and CDC/NCHS
MICH-21.2 Increase the proportion of infants who are breastfed at 6 months
Baseline: 43.5 percent of infants born in 2006 were breastfed at 6 months, as reported in 2007–09
MICH-21.3 Increase the proportion of infants who are breastfed at 1 year
Baseline: 22.7 percent of infants born in 2006 were breastfed at 1 year, as reported in 2007–09
MICH-21.4 Increase the proportion of infants who are breastfed exclusively through 3 months
Baseline: 33.6 percent of infants born in 2006 were breastfed exclusively through 3 months, as reported in 2007–09
MICH-22 Increase the proportion of employers that have worksite lactation support programs
Baseline: 25 percent of employers reported providing an onsite lactation/mother's room in 2009
Target: 38 percent
Employee Benefits Survey, Society for Human Resource Management (SHRM)
MICH-23 Reduce the proportion of breastfed newborns who receive formula supplementation within the first 2 days of life
Baseline: 24.2 percent of breastfed newborns born in 2006 received formula supplementation within the first 2 days of life, as reported in 2007–09
MICH-24 Increase the proportion of live births that occur in facilities that provide recommended care for lactating mothers and their babies
Baseline: 2.9 percent of 2007 live births occurred in facilities that provide recommended care for lactating mothers and their babies, as reported in 2009
Breastfeeding Report Card, CDC/NCCDPHP
Disability and Other Impairments
MICH-25 Reduce the occurrence of fetal alcohol syndrome (FAS)
Baseline: 3.6 cases of fetal alcohol syndrome per 10,000 live births in 2006 were suspected or confirmed among children born in 2001–04 (standardized to 2006 U.S. live births)
Target: Not applicable
Target-Setting Method: This measure is being tracked for informational purposes. If warranted, a target will be set during the decade.
Fetal Alcohol Surveillance System Network (FASSNet), CDC/NCBDDD; National Vital Statistics System-Natality (NVSS-N), CDC/NCHS
MICH-26 Reduce the proportion of children diagnosed with a disorder through newborn bloodspot screening who experience developmental delay requiring special education services
Baseline: 15.1 percent of children born from 1988–96 who were diagnosed with a disorder through newborn bloodspot screening were receiving special education services between 3-10 years of age
Metropolitan Atlanta Developmental Disabilities Surveillance Program (MADDSP), CDC/NCBDDD
MICH-27 Reduce the proportion of children with cerebral palsy born low or very low birth weight
MICH-27.1 Reduce the proportion of children aged 8 years with cerebral palsy born low birth weight (less than 2,500 grams)
Baseline: 50.0 percent of children aged 8 years with cerebral palsy were born low birth weight (less than 2,500 grams) as reported in 2006
Autism and Developmental Disabilities Monitoring Network (ADDM), CDC/NCBDDD
MICH-27.2 Reduce the proportion of children aged 8 years with cerebral palsy born very low birth weight (less than 1,500 grams)
Baseline: 32.5 percent of children aged 8 years with cerebral palsy were born very low birth weight (less than 1,500 grams) as reported in 2006
MICH-28 Reduce occurrence of neural tube defects
MICH-28.1 Reduce the occurrence of spina bifida
Baseline: 34.2 cases of spina bifida per 100,000 live births in 2005–06
Target: 30.8 cases of spina bifida per 100,000 live births
National Birth Defects Prevention Network (NBDPN), CDC/NCBDDD; National Vital Statistics System-Natality (NVSS-N), CDC/NCHS
MICH-28.2 Reduce the occurrence of anencephaly
Baseline: 24.6 cases of anencephaly per 100,000 live births in 2005–06
Target: 22.1 cases of anencephaly per 100,000 live births
MICH-29 Increase the proportion of young children with autism spectrum disorder (ASD) and other developmental delays who are screened, evaluated, and enrolled in special services in a timely manner
MICH-29.1 Increase the proportion of children (aged 10-35 months) who have been screened for Autism Spectrum Disorder (ASD) and other developmental delays
Baseline: 22.6 percent of children aged 10 to 35 months were screened for Autism Spectrum Disorder (ASD) and other developmental delays in the past year as reported in 2007
National Survey of Children's Health (NSCH), HRSA/MCHB and CDC/NCHS
Disparities by special health care needs
MICH-29.2 Increase the proportion of children with ASD having a first evaluation by 36 months of age
Baseline: 42.7 percent of children aged 8 years with ASD had a first evaluation by 36 months of age, as reported in 2006
MICH-29.3 Increase the proportion of children with ASD enrolled in special services by 48 months of age
Baseline: 52.4 percent of children aged 8 years with ASD were enrolled in special services by 48 months of age, as reported in 2006
MICH-29.4 (Developmental) Increase the proportion of children with a developmental delay with a first evaluation by 36 months of age
MICH-29.5 (Developmental) Increase the proportion of children with a developmental delay enrolled in special services by 48 months of age
MICH-30 Increase the proportion of children, including those with special health care needs, who have access to a medical home
MICH-30.1 Increase the proportion of children who have access to a medical home
Baseline: 57.5 percent of children under age 18 years had access to a medical home in 2007
MICH-30.2 Increase the proportion of children with special health care needs who have access to a medical home
Baseline: 47.1 percent of children under age 18 years with special health care needs had access to a medical home in 2005–06
National Survey of Children with Special Health Care Needs (NS-CSHCN), HRSA/MCHB and CDC/NCHS
MICH-31 Increase the proportion of children with special health care needs who receive their care in family-centered, comprehensive, and coordinated systems
MICH-31.1 Increase the proportion of children aged 0 to 11 years with special health care needs who receive their care in family-centered, comprehensive, and coordinated systems
Baseline: 20.4 percent of children aged 0 through 11 years with special health care needs received their care in family-centered, comprehensive, and coordinated systems in 2005–06
MICH-31.2 Increase the proportion of children aged 12 to 17 years with special health care needs who receive their care in family-centered, comprehensive, coordinated systems
Baseline: 13.8 percent of children aged 12 through 17 years with special health care needs received their care in family-centered, comprehensive, and coordinated systems in 2005–06
MICH-32 Increase appropriate newborn bloodspot screening and follow-up testing
MICH-32.1 Increase the number of States and the District of Columbia that verify through linkage with vital records that all newborns are screened shortly after birth for conditions mandated by their State-sponsored screening program
Baseline: 21 States verified through linkage with vital records that all newborns were screened shortly after birth for conditions mandated by their State-sponsored screening program in 2010
Target: 45 States (44 States and the District of Columbia)
National Newborn Screen and Global Resource Center (NNSGRC), University of Texas Health Science Center at San Antonio, Department of Pediatrics (UTHSCSA)
MICH-32.2 Increase the proportion of screen-positive children who receive followup testing within the recommended time period
Baseline: 98.3 percent of screen-positive children received followup testing within the recommended time period in 2003–06
Title V Information System (TVIS), HRSA/MCHB
MICH-32.3 (Developmental) Increase the proportion of children with a diagnosed condition identified through newborn screening who have an annual assessment of services needed and received
MICH-33 Increase the proportion of very low birth weight (VLBW) infants born at Level III hospitals or subspecialty perinatal centers
Baseline: 75.0 percent of VLBW infants were born at Level III hospitals or subspecialty perinatal centers in 2003–06