overexpression. Knockdown or overexpression of PHB1 in Huh-7 cells did not affect proliferation
significantly or sensitize cells to sorafenib-induced apoptosis.
Conclusions—Hepatocyte-specific PHB1 deficiency results in marked liver injury, oxidative
stress and fibrosis with development of HCC by 8 months. These results support PHB1 as a tumor
suppressor in hepatocytes.
Keywords Methionine adenosyltransferase 1A; mitochondria; liver-specific knockout; microarray; cyclin D1INTRODUCTION Prohibitin (PHB) proteins are highly conserved and ubiquitously expressed proteins that have diverse cellular functions (1,2). There are two PHB proteins, PHB1 and PHB2, which are encoded by genes located on different chromosomes and form a large multimeric complex (PHB complex) that is found largely in the inner mitochondrial membrane where it exerts a chaperone-like function to stabilize newly synthesized mitochondrial proteins (3).They are essential for mitochondrial function and biogenesis in yeast (4). PHB1 is also found in the nucleus, where it has been shown to interact with Rb and p53 among other proteins to bring about a change in transcriptional activities of E2F (5) and p53 (6). These nuclear events have been associated with inhibition of cell cycle progression (5) and induction of apoptosis (6). In addition, PHB1 is also localized to the plasma membrane of certain cell types and may function as surface receptor although the ligand(s) remains to be identified, found in circulation, and found in the gastrointestinal tract (muscularis,muscularis mucosa and epithelial layers) where it has been implicated to protect against infection and inflammation (7,8). PHB1 was originally cloned in 1989 and identified as having antiproliferative activity and thought to be a tumor suppressor (hence its name) (9).However, this was later found to be associated with the 3′-UTR of PHB1 mRNA and not
due to the protein itself (3). The tumor suppressor activity of PHB1 is highly controversial
as PHB1 expression is actually higher in many transformed cells and tumors (1). The most
well characterized function of PHB1 is its role as a mitochondrial chaperone. While many
studies have examined the effects of PHB1 in cell lines and yeast, little is known about the
in vivo function of these proteins in mammals and nothing has been reported about the
function of PHB1 in mammalian liver. The reason for this is that deletion of PHB1 leads to
embryonic lethality (Lexicon Knockout Mice Phenotype Data Summary NIH-1165,
https://www.doczj.com/doc/7a12306634.html,/external/ko/lexicon/2210.html).
We identified PHB1 to be down-regulated at the protein level from the time of birth in mice
lacking methionine adenosyltransferase 1A (MAT1A) and in mice and patients at risk for
development of non-alcoholic steatohepatitis (10). Mat1a knockout (KO) mice have
increased hepatic oxidative stress, impaired mitochondrial function and develop
steatohepatitis and hepatocellular carcinoma (HCC) spontaneously (11,12). MAT1A is
expressed largely by mature hepatocytes where it encodes for the enzyme MAT that is
responsible for the biosynthesis of S-adenosylmethionine (SAMe) (13). We found that
PHB1 was down-regulated at the protein level when intracellular SAMe level fell (10).
Since PHB1 was down-regulated in the livers of the Mat1a KO mice at birth and persisted
up to the development of steatohepatitis (10), the purpose of the current work was to see if
liver-specific Phb1 KO mice can recapitulate some of the phenotype of the Mat1a KO mice,
specifically impaired mitochondrial function, oxidative stress, steatohepatitis and malignant
transformation. Our findings show the importance of PHB1 in maintaining normal liver
function and support its role as a tumor suppressor in hepatocytes.
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MATERIALS AND METHODS
Materials
α-32P-dCTP (3,000 Ci/mmol) was purchased from PerkinElmer (Boston, MA). All other reagents were of analytical grade and obtained from commercial sources.Generation of the liver-specific Phb1 KO mice Liver-specific Phb1 KO mouse (C57BL/6J) was developed by serial breeding of Phb1loxP/loxP and Albumin-Cre +/+(Alb-Cre +/+) mice as shown in supplemental Figure 1 and described in detail in Supplemental Methods.All experiments were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Southern California. Ages between 3 and 46 weeks mice were used for the experiments. Please see Supplemental Methods for details of specimen handling. Isolated hepatocytes were obtained by the Cell Culture Core of the USC Research Center for Liver Diseases as described (14).Cell Cultures, PHB1 knockdown or overexpression A normal mouse hepatocyte cell line, AML12, was purchased from American Type Culture Collection (ATCC, Manassas, VA), while HepG2 and Huh-7 cells were provided by the Cell Culture Core and cultured in recommended media in a humidified incubator with 37°C temperature and 5% of CO 2. Cells with passage number less than 18 were used for the experiments. Primary human hepatocytes were obtained from CellzDirect (Pittsboro, NC).Cells were washed with phosphate-buffered saline three times and protein was extracted for Western blot analysis as described below.The pre-designed small interfering RNA (siRNA) targeting mouse Phb1 (sense sequence:AGAGCGAGCGGCAACAUUUtt or AGAAACCAAUUAUCUUUGAtt) and negative
control siRNA were purchased from Ambion (Austin, TX). AML12 and Huh-7 cells in 6
well plates (0.2×106 cells/well) were transfected using RNAiMax (5μL/well) from
Invitrogen (Carlsbad, CA) with PHB1 siRNA (12nM) or negative control siRNA for 18h
(AML12) and 48h (Huh-7) for mRNA or protein expression, or 24h (AML12) and 48h
(Huh-7) for proliferation or apoptosis assays, following the manufacturer’s manual.
Phb1 overexpression vector (PHB1-pcDNA3.1) and negative control empty vector were
kindly provided by Dr Mehta (Illinois Institute of Technology Research Institute, Chicago,
IL). Transient transfection was done using 3μL of Lipofectamine 2000 (Invitrogen,
Carlsbad, CA) and 1.4μg of target plasmid per well of 6 well plates. After 4h, transfection
medium was changed to normal medium and cells were cultured for an additional 44h for
mRNA, protein expression, proliferation or apoptosis assays.
Nucleic acid isolations
Genomic DNA for genotyping was isolated from hepatocytes and various organs by the
method of Strauss (15). Total RNA was isolated from livers, AML12 and Huh-7 cells using
Trizol reagent (Invitrogen, Carlsbad, CA) and then purified by total RNA isolation kit
(Bioland Scientific LLC., Cerritos, CA) following the manufacturer’s manuals.
Genotyping
Genotyping was determined by PCR and described in detail in Supplemental Methods.
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Gene and protein expression
Northern blot analysis, autoradiography and densitometry were done as previous described
(12). The specific probes for mouse Phb1 exon 2 and Gapdh were designed to correspond to
published mRNA sequences from +52 to +154 (Phb1, NM_008831.3) and +221 to +810
(Gapdh , NM_008084), respectively. Both were labeled with α-32P-dCTP using a random
primed DNA labeling kit (DECAprime? II, Ambion Inc., Austin, TX) as described (12).
Results of Northern blot analysis were normalized to Gapdh .
See Supplemental Methods for details of the microarray analysis, which examined
differential mRNA expression profiles, and quantitative real-time PCR for confirmation.
Protein expression was examined using Western blot analysis performed as described (16)
using anti-PHB1, PHB2 and β-actin antibodies (Abcam, Cambridge, MA).
Chromatin immunoprecipitation (ChIP) assay
Please see Supplemental Methods for details.
Histology, immunohistochemistry, immunocytofluorescence and EM
Histologic exam was done in blinded fashion as to the genotype or age of the animal. Please
see Supplemental Methods for details of these procedures.Plasma alkaline phosphatase (ALP), bilirubin and alanine transaminase (ALT) levels Plasma bilirubin, ALT and ALP were determined by total bilirubin kit (Thermo Electron Corporation, Waltham, MA), ALT reagent (Raichem, Cliniqa Corporation, San Marcos,CA) and ALP assay kit (Biovision Incorporated, Mountain View, CA), respectively,following manufacture’s protocols.Plasma and hepatic triglyceride and cholesterol levels
Lipid portion of liver was extracted as method of Folch (17) using chloroform/methanol
(2/1, v/v) as solvent matrix. Evaporated and reconstituted lipid from liver and frozen and
thawed plasma were subjected to the assays for cholesterol and triglyceride using
commercial kits (Thermo DMA, Louisville, CO) following manufacturer’s manuals.
Cell Proliferation and apoptosis assays
Cell proliferation in PHB1 silenced cells for 24h or 48h was measured by the incorporation
rate of bromodeoxyuridine (BrDU) into DNA using a BrDU assay kit (CalBiochem, San
Diego, CA) as described (18) with 3,000 cells per well in 96 well plates and 4h or 1h of
BrDU incorporation time for AML12 or Huh-7 cells, respectively. Apoptosis was measured
by Hoechst staining as previously described (18) in Huh-7 cells treated with sorafenib
(10μM, last 24h of knockdown or overexpression).
Statistical analysis
Data are given as mean±SEM. Statistical analysis for the microarray data is described in that
section separately. For the rest of the results, statistical analysis was performed using
unpaired Student’s t-test. For changes in mRNA and protein levels, ratios of various genes
and proteins to housekeeping gene or protein densitometric values were compared.
Significance was defined by p<0.05.
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RESULTS
Generation of the liver-specific Phb1 KO mice
Following the scheme shown in supplemental Figure 1, liver-specific Phb1 KO was generated. Of the 120 mice genotyped from 18 litters of heterozygous mating (Phb1loxP/+;Alb-Cre +/?), 10% were liver-specific KOs (Phb1loxP/loxP ; Alb-Cre +/+ or Alb-Cre +/?), 73%were heterozygotes (Phb1loxP/+), and 17% were WT (Phb1+/+).Figure 1A shows deletion of Phb1 occurred only in the liver of 3-week old KO mice.However, deletion was not complete at this age, as there remains a faint band that corresponds to the wild type gene in both liver and isolated hepatocytes. Consistent with this, Northern blot analysis using Phb1 exon 2 as the cDNA probe shows 80% reduction in Phb1 mRNA level (Fig. 1B) and Western blot analyses show a comparable reduction in the PHB1 protein level in the liver and hepatocytes but not in the kidney (Fig. 1C). PHB2protein level was also reduced, but to a lesser degree to 30-40% of controls in the liver and hepatocytes (Fig. 1C).Physical and biological changes in the liver-specific Phb1 KO mice From the time of birth, there is variability in the weight and health of the KO mice. 15% of the pubs (115 of 768) died before weaning (3 weeks old). Although most were not genotyped, of the ones that died before weaning and examined, they were all liver-specific KOs. KOs that survived passed 3 weeks weighed less than WT control littermates and this difference persisted up to 14 weeks of age (supplemental Figures 2 and 3). The relative liver to body weight was higher in the KO mice (Table 1). At 3-weeks of age, many KO mice appeared ill and liver injury is evident biochemically (Table 1). Liver injury is confirmed histologically by marked necrosis and inflammation seen throughout the liver (Fig. 2B, C).There is also bile duct metaplasia (Fig. 2D), anisocytosis of hepatic nuclei (Fig. 2E) and positive staining for OV-6, an oval cell marker (Fig. 3B) and glutathione S-transferase Pi
(GSTP) (Fig. 3D), a preneoplastic marker in the 3-week old KO liver.
Mat1a KO mice have higher hepatic triglyceride levels (11) and develop steatohepatitis (12).
This prompted us to measure lipid levels in the liver-specific Phb1 KO mice. Liver-specific
Phb1 KO mice have elevated plasma cholesterol levels but hepatic cholesterol levels and
both plasma and hepatic triglyceride levels were unchanged from WT controls (Table 1).
As the mice grew older, by 14 weeks hepatic nodule can be seen in some liver sections but
not on gross examination (Fig. 2F). By 38 weeks, many KO livers stain positive for AFP
(Fig. 3F). Since PHB1 is a mitochondrial chaperone protein, we examined mitochondrial
morphology by EM. Supplemental Figure 4 (A and B) shows that mitochondria in the 3-
week old KO liver appear swollen and many have no discernible cristae. Consistent with
impaired mitochondrial function is positive 4-hydroxynonenal (4-HNE) staining from
increased lipid peroxidation in the KO liver as compared to WT control liver (Supplemental
Fig. 4C and D). As the KO mice grew older, there is progressive apoptosis as shown by
activated caspase 3 staining (Fig. 4 top row), persistent proliferation as indicated by
proliferating cellular nuclear antigen (PCNA) staining (Fig. 4 middle row), and progressive
fibrosis on reticulin staining (Fig. 4 bottom row). No frank cancer was noted in 8 KO mice
by 14 weeks on a normal diet based on histologic examination. However, by 20 weeks, all
mice have multiple liver nodules on gross exam of the liver (Fig. 5B) and between the ages
of 35 to 46 weeks, 38% (5 of 13, 1 of 5 male and 4 of 8 female mice) have multi-focal HCC
(Fig. 5C and D).
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Since Phb1 KO mice develop HCC, we next compared PHB1 protein expression in normal
primary human hepatocytes to that of human HCC cell lines Huh-7 and HepG2. Figure 5E
shows that the protein level of PHB1 is markedly decreased in the two liver cancer cell lines.
Differential gene expression in the 3-week old liver-specific Phb1 KO mice livers
Differential gene expression was examined using microarray. Supplemental figure 5 shows
the heatmap generated from the microarray data demonstrating the striking difference in
gene expression between KO and controls. Note the controls were very similar regardless of
age. Microarray analysis (25,000 genes) revealed that 402 genes were up-regulated and 182
genes were down-regulated (fold-change > 2.0; p < 0.05) (see Supplemental Table 2 and 3
for complete list). Quantitative real-time PCR confirmed these changes in more than 15
genes (Table 2). Many of the genes differentially expressed in Phb1 KO mice liver are
involved in growth such as H19, CDC20, PRC1, IGF2B, cyclin D1 (CCND1), EGFR1,
RASAL1, and SRC (Table 2). Several genes involved in fibrogenesis are also markedly up-
regulated including many collagen genes and tissue inhibitor of metalloproteinase 1
(TIMP1). Interestingly, many enzymes are markedly down-regulated, including several
cytochrome P450 (CYP450) family members and UDP glycosyltransferase (Table 2). Genes
differentially expressed fall into many different pathways including angiogenesis,
cytoskeletal regulation, signaling pathways involved in epidermal growth factor receptor,
heterotrimeric G-protein, inflammation, integrin, interleukin, p53, phosphoinositide 3-kinase
(PI3K), platelet-derived growth factor (PDGF), Ras, and vascular endothelial growth factor
(VEGF). Supplemental Tables 6 to 19 describe changes in mRNA level based on different
signaling pathways and biological functions.
Localization of PHB1 in hepatocytes
PHB1 subcellular localization in hepatocytes has not been examined. Using confocal
microscopy, Supplemental figure 6A shows that the bulk of PHB1 is localized in the
mitochondria but there is also staining in the nuclei of normal mouse hepatocytes. PHB1
staining is diminished in both compartments in the hepatocytes isolated from the KO mouse
(Supplemental Fig. 6B). Both mitochondrial and nuclear staining can also be seen in
AML12 cells (Supplemental Fig. 6C).
Effects of acute PHB1 knockdown in non-transformed AML12 cells
To better assess whether the changes observed in the KO mice are due to direct or indirect
effects (compensatory proliferation in response to injury) of PHB1 deficiency, we employed
acute knockdown with siRNA against Phb1 in non-transformed AML12 cells. Figure 6A
shows that after 18 hours of siRNA treatment, the efficiency of PHB1 knockdown is about
90% at the mRNA level while PHB1 protein level fell by only 30% (Fig. 6B). After 18
hours of siRNA treatment, a number of the genes picked up on in vivo microarray analysis
also exhibited a similar change, but with much smaller magnitude, for instance, cyclin D1
(CCND1) was increased 64% instead of 4-fold. Some of the genes exhibited similar
magnitude of change as in vivo microarray, such as KRT18, which increased by 69% and
p53, which increased by 48%. Some of the genes actually showed the opposite change as in
vivo microarray, such as CYP4A12A, which more than doubled instead of being 20-fold
reduced and CYP2F2, which increased by 31% instead of a 10-fold reduction (Fig. 6C and
Supplemental Table 3). Acute reduction of PHB1 for 24 hours resulted in a 50% increase in
cell proliferation (Fig. 6D).
To see if the effect of PHB1 knockdown on cyclin D1 expression may be exerted at the level
of E2F binding to its consensus sites on the cyclin D1 promoter, we performed ChIP
analysis comparing E2F binding to different regions of the promoter that contain E2F
binding sites. Figure 6E shows that E2F binding increased particularly in region ?513 to
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?697 (500±12% of scrambled control from three experiments, p<0.05) of the cyclin D1
promoter in cells where PHB1 expression was reduced. E2F binding to the other regions
also increased significantly but to a much lesser degree (150 to 200%).
Effects of PHB1 overexpression
Overexpression of PHB1 in AML12 cells reduced proliferation (Fig. 7B and D). However,
while overexpression in Huh-7 cells tended to lower proliferation, it was not statistically
significant (Fig. 7A and C).
PHB1 expression and sensitivity to sorafenib
To see if PHB1 expression in liver cancer cells can affect sensitivity to sorafenib, Huh-7
cells were treated with siRNA against PHB1 or overexpression vector to raise PHB1. This
was then followed by sorafenib treatment. Apoptosis and proliferation were measured
thereafter. Figure 8 shows that PHB1 knockdown did not sensitize Huh-7 cells to sorafenib-
induced apoptosis or inhibition in proliferation. Overexpression of PHB1 also had no
influence on sorafenib-induced apoptosis or inhibition of proliferation (data not shown).DISCUSSION MAT is an essential enzyme for survival as it is responsible for the biosynthesis of SAMe,the principal biological methyl donor and in mammalian liver, a precursor of GSH (13).MAT1A is one of two MAT genes that encode for the catalytic subunit of MAT that is largely expressed in normal differentiated mammalian liver (13). The expression and activity of hepatic MAT falls in patients with liver disease due to lower MAT1A mRNA level and inactivation of the MAT1A-encoded isoenzymes (13). This work was originally prompted by our observation that Mat1a KO mice have reduced PHB1 protein level from birth and persisted up to 8 months of age (10). Since PHB1 is known to stabilize mitochondrial proteins, we speculated that reduced PHB1 might have led to impaired
mitochondrial function, oxidative stress and susceptibility to many liver injuries in Mat1a
KO mice (10-12). Mat1a KO mice also develop HCC spontaneously (11). Whether reduced
PHB1 could have contributed to this was unclear since there is tremendous controversy with
regards to PHB1’s role as a tumor suppressor (1). Although the functional role of PHB
complex as mitochondrial chaperone is well characterized, particularly in yeast (1,3),
whether it plays a similar role in mammals in vivo has been unclear since Phb1 and Phb2
knockout mice are lethal embryonically
(https://www.doczj.com/doc/7a12306634.html,/external/ko/lexicon/2210.html,19). Development of the
liver-specific Phb1 KO mice has allowed us to address whether reduced PHB1 contributes
to the Mat1a KO phenotype and more importantly, to study the role of PHB1 in normal liver
physiology.
Using the albumin promoter to drive Cre expression has resulted in hepatocytes-specific
deletion of Phb1. However, at 3-weeks of age the deletion was not complete. This is
consistent with known efficiency of the albumin-Cre transgene as reported by Postic and
Magnuson (20), which was 40% immediately after birth, 60% at one week and 75% at 3
weeks. Deletion of liver-specific Phb1 resulted in striking liver injury very early on. Since
the proportion of homozygotes (KO) is much lower than the expected 18.8% (25% chance
from Phb1lox/lox and 75% from Alb-Cre +, or 0.25×0.75=0.188) based on Mendelian
genetics, we suspect there is fetal wastage. This is plausible as albumin can be expressed
very early during mouse development, stage 7 to 8 somites (20). In yeast, it is known that
PHB1 and PHB2 are interdependent (3). Thus, loss of one results in the loss of the other.
Whether this is also true in higher organisms was unclear. Our results show that this is also
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true in mammalian liver as PHB2 was also reduced (although to a lesser degree) when PHB1was markedly reduced.Many of the liver-specific Phb1 KO mice died before weaning and at only 3 weeks of age,there is biochemical and histological evidence of marked liver injury. Histologically the liver is characterized by necrosis and inflammation at 3 weeks. There is also increased apoptosis, which progressed as the mice grew to 14 weeks. Consistent with its known role as a mitochondrial chaperone, marked reduction in PHB1 resulted in abnormal mitochondrial morphology and oxidative stress. There is also increased proliferation, as indicated by PCNA staining. Interestingly as early as 3-weeks of age, there is already increased staining for OV-6 and GSTP, oval cell and preneoplastic markers, respectively. By 14-weeks,dysplastic hepatic nodules were evident microscopically and by 20-weeks, all mice have multiple hepatic nodules on gross examination. By 35 to 46 weeks, more than 1/3 of the mice developed multi-focal HCC. Since increased proliferation and stem cell expansion observed in the livers of the KO mice may be due to a compensatory response to injury, we examined the effect of acute reduction in PHB1 on cell proliferation in non-transformed AML12 cells. Acute loss of PHB1 in a non-transformed hepatocyte resulted in increased proliferation whereas overexpression of PHB1 resulted in the opposite. Although PHB1expression also tended to have a similar effect in Huh-7 cells, changes were not statistically significant. Taken together, these observations would support a role for PHB1 as a tumor suppressor, at least in normal hepatocytes. It is possible that the effect of PHB1 on growth is different in normal versus malignant hepatocytes as many signaling pathways are altered in cancer. This is an area that will require further investigation.There is compelling data from several laboratories using a variety of techniques that support presence of PHB1 in the nucleus in steroid-responsive cells (breast and prostate cancer cell lines) and physical interaction with Rb, E2F, and p53 as well as other proteins to repress the transcriptional activity of E2F (5,21,22) and activate the transcriptional activity of p53 (6).
Our results show that in hepatocytes, a small amount of PHB1 can be found in the nucleus.
Furthermore, reduced PHB1 expression in AML12 cells resulted in increased E2F binding to
the cyclin D1 promoter and cyclin D1 expression. However, if PHB1 inhibits cell cycle
progression and induces apoptosis, as these studies suggest, why would it be highly
expressed in many human cancers? Although it is highly expressed in many types of cancer
(23), it has not been well studied in HCC and whether it is functionally normal has not been
examined. Indeed, we found much higher PHB1 protein level in normal human hepatocytes
when compared to two human liver cancer cell lines. PHB1 expression level also doesn’t
equate function. A recent study found liver cells and transgenic mice that express hepatitis C
virus core protein have increased mitochondrial PHB1 protein level but despite higher level,
it is functionally impaired (24). Thus, it would be of interest to see if there is a difference in
subcellular localization of PHB1 in various cancers and whether it is functionally intact.
Consistent with this notion, a recent study found lung cancer cells that have increased
membrane associated PHB1 (cell surface and microsomal membrane fractions) were more
resistant to paclitaxel (23) and interestingly, total whole cell levels of PHB1 were
unchanged. We did not observe any difference in sensitivity to sorafenib in Huh-7 cells
when PHB1 expression was varied. Thus, the effect of PHB1 may be cancer and cell line
specific.
The microarray data revealed many pathways are affected by reduced PHB1 expression.
Many are growth related and oncogenes. Interestingly, cyclin D1 and PCNA, which are up-
regulated in Mat1a KO (25) and Phb1 KO livers, are known E2F targets (26). These
findings, along with our current observations, would support PHB1’s repressive role on E2F
transcriptional activity in mouse liver. We also examined whether these genes are similarly
affected after acute knockdown of PHB1 in AML12 cells. Although many of the growth-
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related genes are similarly affected, the magnitude of change is much smaller than in vivo.
This may be partly due to the fact that PHB1 protein level fell only by 30% after 18 hours of
siRNA treatment even though the mRNA level fell by 90%. PHB1 protein appears to be
quite stable with a half-life that exceeds 10 hours (27). Interestingly, CYP4A12A and
CYP2F2 are both increased in the AML12 cells following PHB1 knockdown, which is the
opposite of what happened in the KO livers. This likely reflects some adaptive changes that
occurred in vivo and influence of the in vivo microenvironment.
Recently, conditional deletion of Phb2 using mouse embryonic fibroblasts (MEFs) from
homozygous Phb2fl/fl embryos transduced with Cre-recombinase was reported (19). Loss of
PHB2 in MEFs was accompanied by loss of PHB1, confirming their interdependence in
mammalian system. Loss of PHB2 resulted in aberrant mitochondrial cristae morphogenesis
and increased apoptosis, which is similar to Phb1 KO. However, loss of PHB2 in MEFs led
to impaired cellular proliferation (19). Given that these two proteins function as a complex
at least in the mitochondria, it is intriguing that they should have such different effects on
growth. Our findings are consistent with an earlier report during liver regeneration in rats
where the expression of PHB1 is abundant in quiescent hepatocytes and nearly absent during
the 3 to 12 hour period following 2/3 partial hepatectomy and returning to normal levels at
24 hours (28). These changes correlated with entry of hepatocytes into the cell cycle and
support the notion that a fall in PHB1 facilitates cell cycle entry and proliferation.
Based on the findings thus far, reduced PHB1 expression that occurs in the Mat1a KO livers
can contribute to liver injury, increased oxidative stress, impaired mitochondrial function,
expansion of liver progenitor cells and development of HCC in the Mat1a KO mouse model
(10-12,29). However, whether it also contributes to the susceptibility to develop fatty liver
in the Mat1a KO mice (12) is not clear. Although there is no evidence for increased fat
accumulation in Phb1 KO livers up to 14 weeks of age, there is increased plasma cholesterol
level, which may signal impairment in cholesterol uptake by the liver. This possibility will
require further investigation.
In summary, liver-specific deletion of Phb1 results in marked liver injury at an early age that
is characterized by necrosis, apoptosis, swollen mitochondria, oxidative stress, fibrosis and
increased expression of progenitor cell and preneoplastic markers. Multi-focal HCC occurs
by 8 months. Marked reduction of PHB1 alters the expression of genes involved in multiple
cellular pathways, ranging from growth, inflammation, and xenobiotic metabolism. Our
study demonstrates for the first time a vital role for PHB1 in normal liver physiology and
supports PHB1 as a tumor suppressor in liver.Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
Financial support. This work was supported by NIH grants DK51719 (S. C. Lu), AA8116 (S. W. French),
AT1576 (S. C. Lu and J. M. Mato), pilot/feasibility funding from the USC Research Center for Liver Diseases
(P30DK48522) (B. Stiles) and SAF 2008-04800, HEPADIP-EULSHM-CT-205 and ETORTEK-2008 (J. M. Mato).
Ciberehd is funded by the Instituto de Salud Carlos III. Isolated mouse hepatocytes were prepared by the Cell
Culture Core, while liver tissue sectioning and H&E staining were performed by the Cell and Tissue Imaging Core
of the USC Research Center for Liver Diseases (P30DK48522). Immunohistochemistry for 4-HNE, reticulin,
OV-6, GSTP, and AFP were done by the Morphology Core of the Southern California Research Center for
Alcoholic Liver and Pancreatic Diseases and Cirrhosis (P50AA11999). Liver-specific Phb1 KO mouse was
generated with the help of the Transgenic Mouse Core and Microarray was performed by the Microarray Core of
the Norris Cancer Center at the Keck School of Medicine USC.
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List of abbreviations (in alphabetical order)
4-HNE
4-hydroxynonenal AFP
alpha-fetoprotein ALP
alkaline phosphatase ALT
alanine transaminase BrDU
bromodeoxyuridine ChIP
chromatin immunoprecipitation EM
electron microscopy GSTP
glutathione S-transferase Pi HCC
hepatocellular carcinoma KO
knockout MAT
methionine adenosyltransferase MEF
mouse embryonic fibroblasts PCR polymerase chain reaction
PCNA proliferating cellular nuclear antigen
PDGF platelet-derived growth factor
PHB1prohibitin 1
PHB2prohibitin 2
PI3K phosphoinositide 3-kinase
ROS
reactive oxygen species
SAMe
S-adenosylmethionine
TIMP1
tissue inhibitor of metalloproteinase 1
VEGF
vascular endothelial growth factor REFERENCES
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7. Mishra S, Murphy LC, Nyomba BLG, Murphy LJ. Prohibitin: a potential target for new therapeutics. TRENDS Mol. Med. 2005; 11:192–197. [PubMed: 15823758]8. Theiss AL, Vijay-Kumar M, Obertone TS, Jones DP, Hansen JM, Gewirtz AT, et al. Prohibitin is a novel regulator of antioxidant response that attenuates colonic inflammation in mice.Gastroenterology. 2009; 137:199–208. [PubMed: 19327358]9. McClung JK, Danner DB, Stewart DA, Smith JR, Schneider EL, Lumpkin CK, et al. Isolation of a cDNA that hybrid selects antiproliferative mRNA from rat liver. Biochem Biophys Res Comm.1989; 164:1316–1322. [PubMed: 2480116]10. Santamaria E, Avila MA, Latasa MU, Rubio A, Martin-Duce A, Lu SC, et al. Functional proteomics of nonalcoholic steatohepatitis: mitochondrial proteins as targets of S-adenosylmethionine. Proc Nat Acad Sci USA. 2003; 100:3065–3070. [PubMed: 12631701]11. Martínez-Chantar ML, Corrales FJ, Martínez-Cruz LA, García-Trevijano ER, Huang ZZ, Chen L,et al. Spontaneous oxidative stress and liver tumors in mice lacking methionine adenosyltransferase 1A. FASEB J. 2002; 10 1096/fj.02-0078fje.12. Lu SC, Alvarez L, Huang ZZ, Chen L, An W, Corrales FJ, et al. Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation. Proc Natl Acad Sci USA. 2001; 98:5560–5565. [PubMed: 11320206]13. Mato JM, Lu SC. Role of S-adenosyl-L-methionine in liver health and injury. Hepatology. 2007;45:1306–1312. [PubMed: 17464973]14. Moldeus P, Hogberg J, Orrenius S. Isolation and use of liver cells. Methods Enzymol. 1978;51:60–70. [PubMed: 672656]15. Strauss WM. Preparation of genomic DNA from mammalian tissue. Curr Protoc Mol Biol. 2001Chapter 2:Unit 2.2.16. Ko K, Yang H, Noureddin M, Iglesia-Ara A, Xia M, Wagner C, et al. Changes in S-adenosylmethionine and GSH homeostasis during endotoxemia in mice. Lab Invest. 2008;88:1121–1129. [PubMed: 18695670]17. Folch J, Lees M, Stanley GH Sloane. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957; 226:497–509. [PubMed: 13428781]18. Ramani K, Yang H, Kuhlenkamp J, Tomasi ML, Tsukamoto H, Mato JM, et al. Changes in the
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Fig 1.
Confirmation of deletion of Phb1 in DNA, mRNA and protein levels. Genotyping of Phb1
exon 2 region including loxP flanking Phb1 exon 2 sites shows the same size of bands
(predicted 731 bp) in all of the organs tested (A, left) in WT control mice. However, deletion
of loxP flanking Phb1 exon 2 sequence (predicted 334 bp) is detected only in liver and
hepatocytes but not in other organs from liver-specific Phb1 KO mice (A, right), which
indicated that Phb1 exon 2 is deleted only in the liver of liver-specific Phb1 KO mice.
Northern blots of total RNA from the livers of WT control and liver-specific Phb1 KO mice
show that Phb1 exon 2 is significantly removed in the livers of liver-specific Phb1 KO mice
compared to those of WT control littermates (B, left). The graph on the right shows
densitometric changes in % of WT control, which is expressed as mean±SEM from 2 in
each group (B, right), *p<0.01. Protein expression of PHB1 and PHB2 by Western blots
shows only PHB1 protein expression is dramatically decreased in whole livers and
hepatocytes, but not in kidney (C, top). PHB2 expression is also decreased along with PHB1
protein in the whole livers and hepatocytes in liver-specific Phb1 KO mice (C, top). The
graph shows densitometric changes in % of WT control, which is expressed as mean±SEM
from 2 mice each for kidneys and hepatocytes and 8 mice each for livers (C, bottom),
*p<0.01.
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Fig 2.Histologic changes in liver-specific Phb1 KO livers. A to D (×400), E (×800) and F (×200)show H&E staining of livers from 3-week old WT control (A) and liver-specific Phb1 KO (B to E) mice (except for F, 14-week old KO). Characteristic findings in liver-specific Phb1KO livers are (B) foci of necrosis, (C) increased inflammatory cell infiltrate, (D) bile duct metaplasia as indicated by arrow, and (E) anisocytosis (varying sizes) of hepatic nuclei as indicated by arrows, consistent with hepatocyte dysplasia. Consistent with this, 14-week old KO mouse exhibits dysplastic nodule (F, ×200).
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Fig 3.Liver-specific Phb1 livers exhibit increased staining for oval cell and preneoplastic markers.OV6 staining is negative in 3-week old WT control (A, ×800) but positive in 3-week old KO mice (bright green staining, B, ×800). GSTP staining with DAPI co-staining for nuclei shows negative GSTP in 3-week old WT control (C, ×800) but positive cells in 3-week old KO (D, red staining, ×800). Note these GSTP positive cells are also OV6 positive (B). A and C show the same microscopic field as B and D, double OV6/GSTP immunofluorescent staining. AFP staining was negative in 38-week old WT control (E, ×400) but strongly positive in KO (F, ×400).
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Fig 4.Changes in liver-specific Phb1 KO livers from 3-week to 14-week of age. Apoptosis is detected by immunohistochemical staining for active caspase 3 (top row, ×200). Slightly Increased number of active caspase 3 positive cells (brown staining) was found in the liver of 3-week old liver-specific Phb1 KO mouse, which progressed as the mice aged.Proliferation is detected by immunohistochmical staining for PCNA (middle row, ×400).Liver-specific Phb1 KO mice have more PCNA positive cells (brown staining) regardless of their age than those of their control littermates. Fibrosis is detected by reticulin staining (bottom row, ×200 except for 14-week WT, ×400). Significant amount of reticulin is
accumulated in the liver of 3-week old liver-specific Phb1 KO mouse, which progressed in
the 14-week old KO livers.
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Fig 5.Development of HCC in the liver-specific Phb1 KO mice and PHB1 expression in human liver cancer cell lines. Histologic changes on H&E are shown below the gross picture of
each liver. A shows no significant change in the liver of WT mouse on gross or histologic
exam (×200). B shows multiple nodules (arrows) on the surface of the liver of a 35-week old
KO mouse, one of which shows well differentiated HCC with fat deposit on H&E staining
(×400). C shows different stages of HCC development in the liver of a 46-week KO mouse
(arrows), where neoplastic cells (arrows) are clearly observed on H&E staining (×800), and
D shows multi-focal HCC in a 38-week KO mouse by gross exam (arrows) and on H&E
(×600). PHB1 protein level as determined by Western blot analysis (E) is much lower in
Huh-7 and HepG2 cells as compared to normal primary human hepatocytes (PHH).
Densitometric analyses from three independent experiments are shown below the blot and
are expressed as mean±SEM % of PHH, p<0.05 between Huh-7 or HepG2 and PHH.
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Fig 6.
Effects of Phb1 siRNA treatment in AML12 cell line. AML12 cells were treated with
siRNA of Phb1 for 18 (expression) or 24 (growth and ChIP) hours. PHB1 mRNA level was
reduced by 90% as compared to control and scrambled siRNA (SC) (A, n=6) and PHB1
protein expression decreased by 30% (B, n=3). C (n=4 to 6) shows mRNA levels in Phb1
siRNA treated AML12 cells for the genes that are up- or down-regulated most in the livers
of liver-specific Phb1 KO mice. All of mRNA expression changes in C are statistically
significant against control and/or scrambled siRNA (p<0.05, Supplement Table 3). D shows
Phb1 siRNA treated AML12 cells have increased proliferation rate measured by BrDU
incorporation into DNA for 4 hours (n=4). *p<0.005 vs. control and scrambled siRNA. E
shows the effect of Phb1 siRNA on E2F binding to different regions of the cyclin D1
promoter (numbers are in reference to the transcription start site). E2F binding is increased
in all four regions but particularly to the region ?513~?697. These changes were measured
densitometrically and were significantly different from SC control with p<0.05.
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Fig 7.Effects of PHB1 overexpression in AML12 and Huh-7 cell line. AML12 and Huh-7 cells were treated with the human PHB1-pcDNA3.1 PHB1 overexpression plasmid for 48 hours.A and B show the efficiency of PHB1 overexpression in protein level by Western blot.PHB1 protein level increased by 35 to 60% Huh-7 or AML12 cells treated with the
overexpression vector (oe PHB1) as compared to empty vector (EV) control, respectively.
Densitometric analyses from three independent experiments are shown below the blots and
are expressed as mean±SEM % of control, p<0.05 between oe PHB1 and EV. Proliferation
was measured by BrDU incorporation and are shown in C and D. AML12 cells
overexpressing PHB1 had a significant decrease in proliferation (D) as compared to control
and EV, while no significant change was found in Huh-7 cells (C). *p<0.05 vs. control and
EV.
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Fig 8.
Effects of Phb1 siRNA and sorafenib (SF) treatments in Huh-7 cells. Huh-7 cells were
treated with Phb1 siRNA or SC for 48 hours and sorafenib was added during the last 24
hours. PHB1 siRNA reduced PHB1 protein level by 50% as shown on Western blot (A, n=3,
results are mean±SEM densitometric values expressed as % of control). B shows % over
control apoptosis in Huh-7 cells treated with si PHB1 or SC control, with or without SF.
Randomly selected 10× optic magnification fields were taken and apoptotic cells to normal
cells ratio was calculated in 4 fields in each group. si PHB1 treatment did not affect
apoptosis or sensitize cells to apoptosis induced by SF. C shows proliferation changes in
similarly treated Huh-7 cells. SF inhibited proliferation and this was not affected by si PHB1.
*p<0.05 vs. DMSO control.
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如何写先进个人事迹 篇一：如何写先进事迹材料 如何写先进事迹材料 一般有两种情况：一是先进个人，如先进工作者、优秀党员、劳动模范等；一是先进集体或先进单位，如先进党支部、先进车间或科室，抗洪抢险先进集体等。无论是先进个人还是先进集体，他们的先进事迹，内容各不相同，因此要整理材料，不可能固定一个模式。一般来说，可大体从以下方面进行整理。 (1)要拟定恰当的标题。先进事迹材料的标题，有两部分内容必不可少，一是要写明先进个人姓名和先进集体的名称，使人一眼便看出是哪个人或哪个集体、哪个单位的先进事迹。二是要概括标明先进事迹的主要内容或材料的用途。例如《王鬃同志端正党风的先进事迹》、《关于评选张鬃同志为全国新长征突击手的材料》、《关于评选鬃处党支部为省直机关先进党支部的材料》等。 (2)正文。正文的开头，要写明先进个人的简要情况，包括：姓名、性别、年龄、工作单位、职务、是否党团员等。此外，还要写明有关单位准备授予他(她)什么荣誉称号，或给予哪种形式的奖励。对先进集体、先进单位，要根据其先进事迹的主要内容，寥寥数语即应写明，不须用更多的文字。 然后，要写先进人物或先进集体的主要事迹。这部分内容是全篇材料
的主体，要下功夫写好，关键是要写得既具体，又不繁琐；既概括，又不抽象；既生动形象，又很实在。总之，就是要写得很有说服力，让人一看便可得出够得上先进的结论。比如，写一位端正党风先进人物的事迹材料，就应当着重写这位同志在发扬党的优良传统和作风方面都有哪些突出的先进事迹，在同不正之风作斗争中有哪些突出的表现。又如，写一位搞改革的先进人物的事迹材料，就应当着力写这位同志是从哪些方面进行改革的，已经取得了哪些突出的成果，特别是改革前后的．经济效益或社会效益都有了哪些明显的变化。在写这些先进事迹时，无论是先进个人还是先进集体的，都应选取那些具有代表性的具体事实来说明。必要时还可运用一些数字，以增强先进事迹材料的说服力。 为了使先进事迹的内容眉目清晰、更加条理化，在文字表述上还可分成若干自然段来写，特别是对那些涉及较多方面的先进事迹材料，采取这种写法尤为必要。如果将各方面内容材料都混在一起，是不易写明的。在分段写时，最好在每段之前根据内容标出小标题，或以明确的观点加以概括，使标题或观点与内容浑然一体。 最后，是先进事迹材料的署名。一般说，整理先进个人和先进集体的材料，都是以本级组织或上级组织的名义；是代表组织意见的。因此，材料整理完后，应经有关领导同志审定，以相应一级组织正式署名上报。这类材料不宜以个人名义署名。 写作典型经验材料-般包括以下几部分： (1)标题。有多种写法，通常是把典型经验高度集中地概括出来，一
How to manage time Time treats everyone fairly that we all have 24 hours per day. Some of us are capable to make good use of time while some find it hard to do so. Knowing how to manage them is essential in our life. Take myself as an example. When I was still a senior high student, I was fully occupied with my studies. Therefore, I hardly had spare time to have fun or develop my hobbies. But things were changed after I entered university. I got more free time than ever before. But ironically, I found it difficult to adjust this kind of brand-new school life and there was no such thing called time management on my mind. It was not until the second year that I realized I had wasted my whole year doing nothing. I could have taken up a Spanish course. I could have read ten books about the stories of successful people. I could have applied for a part-time job to earn some working experiences. B ut I didn’t spend my time on any of them. I felt guilty whenever I looked back to the moments that I just sat around doing nothing. It’s said that better late than never. At least I had the consciousness that I should stop wasting my time. Making up my mind is the first step for me to learn to manage my time. Next, I wrote a timetable, setting some targets that I had to finish each day. For instance, on Monday, I must read two pieces of news and review all the lessons that I have learnt on that day. By the way, the daily plan that I made was flexible. If there’s something unexpected that I had to finish first, I would reduce the time for resting or delay my target to the next day. Also, I would try to achieve those targets ahead of time that I planed so that I could reserve some more time to relax or do something out of my plan. At the beginning, it’s kind of difficult to s tick to the plan. But as time went by, having a plan for time in advance became a part of my life. At the same time, I gradually became a well-organized person. Now I’ve grasped the time management skill and I’m able to use my time efficiently.
英语演讲稿：未来的工作 这篇《英语演讲稿范文：未来的工作》，是特地，希望对大家有所帮助！ 热门演讲推荐：竞聘演讲稿 | 国旗下演讲稿 | 英语演讲稿 | 师德师风演讲稿 | 年会主持词 | 领导致辞 everybody good afternoon:. first of all thank the teacher gave me a story in my own future ideal job. everyone has a dream job. my dream is to bee a boss, own a pany. in order to achieve my dreams, i need to find a good job, to accumulate some experience and wealth, it is the necessary things of course, in the school good achievement and rich knowledge is also very important. good achievement and rich experience can let me work to make the right choice, have more opportunities and achievements. at the same time, munication is very important, because it determines whether my pany has a good future development. so i need to exercise their municative ability. i need to use all of the free time to learn
小学生个人读书事迹简介怎么写800字 书,是人类进步的阶梯,苏联作家高尔基的一句话道出了书的重要。书可谓是众多名人的“宠儿”。历来,名人说出关于书的名言数不胜数。今天小编在这给大家整理了小学生个人读书事迹,接下来随着小编一起来看看吧! 小学生个人读书事迹1 “万般皆下品,惟有读书高”、“书中自有颜如玉,书中自有黄金屋”,古往今来,读书的好处为人们所重视,有人“学而优则仕”,有人“满腹经纶”走上“传道授业解惑也”的道路……但是,从长远的角度看,笔者认为读书的好处在于增加了我们做事的成功率,改善了生活的质量。 三国时期的大将吕蒙,行伍出身,不重视文化的学习,行文时,常常要他人捉刀。经过主君孙权的劝导,吕蒙懂得了读书的重要性,从此手不释卷,成为了一代儒将,连东吴的智囊鲁肃都对他“刮目相待”。后来的事实证明,荆州之战的胜利,擒获“武圣”关羽,离不开吕蒙的“运筹帷幄,决胜千里”,而他的韬略离不开平时的读书。由此可见,一个人行事的成功率高低,与他的对读书,对知识的重视程度是密切相关的。 的物理学家牛顿曾近说过,“如果我比别人看得更远,那是因为我站在巨人的肩上”,鲜花和掌声面前,一代伟人没有迷失方向,自始至终对读书保持着热枕。牛顿的话语告诉我们,渊博的知识能让我们站在更高、更理性的角度来看问题,从而少犯错误,少走弯路。
读书的好处是显而易见的,但是,在社会发展日新月异的今天,依然不乏对读书,对知识缺乏认知的人,《今日说法》中我们反复看到农民工没有和用人单位签订劳动合同,最终讨薪无果;屠户不知道往牛肉里掺“巴西疯牛肉”是犯法的;某父母坚持“棍棒底下出孝子”,结果伤害了孩子的身心,也将自己送进了班房……对书本,对知识的零解读让他们付出了惨痛的代价,当他们奔波在讨薪的路上,当他们面对高墙电网时,幸福,从何谈起?高质量的生活,从何谈起? 读书,让我们体会到“锄禾日当午,汗滴禾下土”的艰辛;读书,让我们感知到“四海无闲田,农夫犹饿死”的无奈;读书,让我们感悟到“为报倾城随太守,西北望射天狼”的豪情壮志。 读书的好处在于提高了生活的质量,它填补了我们人生中的空白,让我们不至于在大好的年华里无所事事,从书本中,我们学会提炼出有用的信息,汲取成长所需的营养。所以,我们要认真读书,充分认识到读书对改善生活的重要意义,只有这样,才是一种负责任的生活态度。 小学生个人读书事迹2 所谓读一本好书就是交一个良师益友,但我认为读一本好书就是一次大冒险,大探究。一次体会书的过程,真的很有意思,咯咯的笑声,总是从书香里散发;沉思的目光也总是从书本里透露。是书给了我启示,是书填补了我无聊的夜空,也是书带我遨游整个古今中外。所以人活着就不能没有书,只要爱书你就是一个爱生活的人,只要爱书你就是一个大写的人,只要爱书你就是一个懂得珍惜与否的人。可真所谓
关于坚持的英语演讲稿 Results are not important, but they can persist for many years as a commemoration of. Many years ago, as a result of habits and overeating formed one of obesity, as well as indicators of overall physical disorders, so that affects my work and life. In friends to encourage and supervise, the participated in the team Now considered to have been more than three years, neither the fine rain, regardless of winter heat, a day out with 5:00 time. The beginning, have been discouraged, suffering, and disappointment, but in the end of the urging of friends, to re-get up, stand on the playground. 成绩并不重要，但可以作为坚持多年晨跑的一个纪念。多年前，由于庸懒习惯和暴饮暴食，形成了一身的肥胖，以及体检指标的全盘失常，以致于影响到了我的工作和生活。在好友的鼓励和督促下，参加了晨跑队伍。现在算来，已经三年多了，无论天晴下雨，不管寒冬酷暑，每天五点准时起来出门晨跑。开始时，也曾气馁过、痛苦过、失望过，但最后都在好友们的催促下，重新爬起来，站到了操场上。 In fact, I did not build big, nor strong muscles, not a sport-born people. Over the past few years to adhere to it, because I have a team behind, the strength of a strongteam here, very grateful to our team, for a long time, we encourage each other, and with sweat, enjoying common health happy. For example, Friends of the several run in order to maintain order and unable to attend the 10，000 meters race, and they are always concerned about the brothers and promptly inform the place and time, gives us confidence and courage. At the same time, also came on their own inner desire and pursuit for a good health, who wrote many of their own log in order to refuel for their own, and inspiring. 其实我没有高大身材，也没健壮肌肉，天生不属于运动型的人。几年来能够坚持下来，因为我的背后有一个团队，有着强大团队的力量，在这里，非常感谢我们的晨跑队，长期以来，我们相互鼓励着，一起流汗，共同享受着健康带来的快
1.How to build a business that lasts100years 0:11Imagine that you are a product designer.And you've designed a product,a new type of product,called the human immune system.You're pitching this product to a skeptical,strictly no-nonsense manager.Let's call him Bob.I think we all know at least one Bob,right?How would that go? 0:34Bob,I've got this incredible idea for a completely new type of personal health product.It's called the human immune system.I can see from your face that you're having some problems with this.Don't worry.I know it's very complicated.I don't want to take you through the gory details,I just want to tell you about some of the amazing features of this product.First of all,it cleverly uses redundancy by having millions of copies of each component--leukocytes,white blood cells--before they're actually needed,to create a massive buffer against the unexpected.And it cleverly leverages diversity by having not just leukocytes but B cells,T cells,natural killer cells,antibodies.The components don't really matter.The point is that together,this diversity of different approaches can cope with more or less anything that evolution has been able to throw up.And the design is completely modular.You have the surface barrier of the human skin,you have the very rapidly reacting innate immune system and then you have the highly targeted adaptive immune system.The point is,that if one system fails,another can take over,creating a virtually foolproof system. 1:54I can see I'm losing you,Bob,but stay with me,because here is the really killer feature.The product is completely adaptive.It's able to actually develop targeted antibodies to threats that it's never even met before.It actually also does this with incredible prudence,detecting and reacting to every tiny threat,and furthermore, remembering every previous threat,in case they are ever encountered again.What I'm pitching you today is actually not a stand-alone product.The product is embedded in the larger system of the human body,and it works in complete harmony with that system,to create this unprecedented level of biological protection.So Bob,just tell me honestly,what do you think of my product? 2:47And Bob may say something like,I sincerely appreciate the effort and passion that have gone into your presentation,blah blah blah-- 2:56(Laughter) 2:58But honestly,it's total nonsense.You seem to be saying that the key selling points of your product are that it is inefficient and complex.Didn't they teach you 80-20?And furthermore,you're saying that this product is siloed.It overreacts, makes things up as it goes along and is actually designed for somebody else's benefit. I'm sorry to break it to you,but I don't think this one is a winner.
关于工作的优秀英语演讲稿 Different people have various ambitions. Some want to be engineers or doctors in the future. Some want to be scientists or businessmen. Still some wish to be teachers or lawers when they grow up in the days to come. Unlike other people, I prefer to be a farmer. However, it is not easy to be a farmer for Iwill be looked upon by others. Anyway,what I am trying to do is to make great contributions to agriculture. It is well known that farming is the basic of the country. Above all, farming is not only a challenge but also a good opportunity for the young. We can also make a big profit by growing vegetables and food in a scientific way. Besides we can apply what we have learned in school to farming. Thus our countryside will become more and more properous. I believe that any man with knowledge can do whatever they can so long as this job can meet his or her interest. All the working position can provide him with a good chance to become a talent. 1 ————来源网络整理，仅供供参考
个人先进事迹简介 01 在思想政治方面,xxxx同学积极向上,热爱祖国、热爱中国共产党,拥护中国共产党的领导.利用课余时间和党课机会认真学习政治理论,积极向党组织靠拢. 在学习上,xxxx同学认为只有把学习成绩确实提高才能为将来的实践打下扎实的基础,成为社会有用人才.学习努力、成绩优良. 在生活中,善于与人沟通,乐观向上,乐于助人.有健全的人格意识和良好的心理素质和从容、坦诚、乐观、快乐的生活态度,乐于帮助身边的同学,受到师生的好评. 02 xxx同学认真学习政治理论,积极上进,在校期间获得原院级三好生,和校级三好生,优秀团员称号,并获得三等奖学金. 在学习上遇到不理解的地方也常常向老师请教,还勇于向老师提出质疑.在完成自己学业的同时,能主动帮助其他同学解决学习上的难题,和其他同学共同探讨,共同进步. 在社会实践方面,xxxx同学参与了中国儿童文学精品“悦”读书系,插画绘制工作,xxxx同学在班中担任宣传委员,工作积极主动,认真负责,有较强的组织能力.能够在老师、班主任的指导下独立完成学院、班级布置的各项工作. 03 xxx同学在政治思想方面积极进取,严格要求自己.在学习方面刻苦努力,不断钻研,学习成绩优异,连续两年荣获国家励志奖学金；作
为一名学生干部,她总是充满激情的迎接并完成各项工作,荣获优秀团干部称号.在社会实践和志愿者活动中起到模范带头作用. 04 xxxx同学在思想方面,积极要求进步,为人诚实,尊敬师长.严格 要求自己.在大一期间就积极参加了党课初、高级班的学习,拥护中国共产党的领导,并积极向党组织靠拢. 在工作上,作为班中的学习委员,对待工作兢兢业业、尽职尽责 的完成班集体的各项工作任务.并在班级和系里能够起骨干带头作用.热心为同学服务,工作责任心强. 在学习上,学习目的明确、态度端正、刻苦努力,连续两学年在 班级的综合测评排名中获得第1.并荣获院级二等奖学金、三好生、优秀班干部、优秀团员等奖项. 在社会实践方面,积极参加学校和班级组织的各项政治活动,并 在志愿者活动中起到模范带头作用.积极锻炼身体.能够处理好学习与工作的关系,乐于助人,团结班中每一位同学,谦虚好学,受到师生的好评. 05 在思想方面,xxxx同学积极向上,热爱祖国、热爱中国共产党,拥护中国共产党的领导.作为一名共产党员时刻起到积极的带头作用,利用课余时间和党课机会认真学习政治理论. 在工作上,作为班中的团支部书记,xxxx同学积极策划组织各类 团活动,具有良好的组织能力. 在学习上,xxxx同学学习努力、成绩优良、并热心帮助在学习上有困难的同学,连续两年获得二等奖学金. 在生活中,善于与人沟通,乐观向上,乐于助人.有健全的人格意 识和良好的心理素质.
尊敬的领导，老师，亲爱的同学们， 大家好！我是5班的梁浩东。今天早上我坐车来学校的路上，我仔细观察了路上形形色色的人，有开着小车衣着精致的叔叔阿姨，有市场带着倦容的卖各种早点的阿姨，还有偶尔穿梭于人群中衣衫褴褛的乞丐。于是我问自己，十几年后我会成为怎样的自己，想成为社会成功人士还是碌碌无为的人呢，答案肯定是前者。那么十几年后我怎样才能如愿以偿呢，成为一个受人尊重，有价值的人呢？正如我今天演讲的题目是：自主管理。 大家都知道爱玩是我们孩子的天性，学习也是我们的责任和义务。要怎样处理好这些矛盾，提高自主管理呢？ 首先，我们要有小主人翁思想，自己做自己的主人，要认识到我们学习，生活这一切都是我们自己走自己的人生路，并不是为了报答父母，更不是为了敷衍老师。 我认为自主管理又可以理解为自我管理，在学习和生活中无处不在，比如通过老师，小组长来管理约束行为和同学们对自身行为的管理都属于自我管理。比如我们到一个旅游景点，看到一块大石头，有的同学特别兴奋，会想在上面刻上：某某某到此一游话。这时你就需要自我管理，你需要提醒自己，这样做会破坏景点，而且是一种素质低下的表现。你设想一下，如果别人家小孩去你家墙上乱涂乱画，你是何种感受。同样我们把自主管理放到学习上，在我们想偷懒，想逃避，想放弃的时候，我们可以通过自主管理来避免这些，通过他人或者自己的力量来完成。例如我会制定作息时间计划表，里面包括学习，运动，玩耍等内容的完成时间。那些学校学习尖子，他们学习好是智商高于我们吗，其实不然，在我所了解的哪些优秀的学霸传授经验里，就提到要能够自我管理，规范好学习时间的分分秒秒，只有辛勤的付出，才能取得优异成绩。 在现实生活中，无数成功人士告诉我们自主管理的重要性。十几年后我想成为一位优秀的，为国家多做贡献的人。亲爱的同学们，你们们？让我们从现在开始重视和执行自主管理，十几年后成为那个你想成为的人。 谢谢大家！
关于工作的英语演讲稿 【篇一：关于工作的英语演讲稿】 关于工作的英语演讲稿 different people have various ambitions. some want to be engineers or doctors in the future. some want to be scientists or businessmen. still some wish to be teachers or lawers when they grow up in the days to come. unlike other people, i prefer to be a farmer. however, it is not easy to be a farmer for iwill be looked upon by others. anyway,what i am trying to do is to make great contributions to agriculture. it is well known that farming is the basic of the country. above all, farming is not only a challenge but also a good opportunity for the young. we can also make a big profit by growing vegetables and food in a scientific way. besides we can apply what we have learned in school to farming. thus our countryside will become more and more properous. i believe that any man with knowledge can do whatever they can so long as this job can meet his or her interest. all the working position can provide him with a good chance to become a talent. 【篇二：关于责任感的英语演讲稿】 im grateful that ive been given this opportunity to stand here as a spokesman. facing all of you on the stage, i have the exciting feeling of participating in this speech competition. the topic today is what we cannot afford to lose. if you ask me this question, i must tell you that i think the answer is a word---- responsibility. in my elementary years, there was a little girl in the class who worked very hard, however she could never do satisfactorily in her lessons. the teacher asked me to help her, and it was obvious that she expected a lot from me. but as a young boy, i was so restless and thoughtless, i always tried to get more time to play and enjoy myself. so she was always slighted over by me. one day before the final exam, she came up to me and said, could you please explain this to me? i can not understand it. i
Dear teacher and colleagues: my topic is on “spare time”. It is a huge blessing that we can work 996. Jack Ma said at an Ali's internal communication activity, That means we should work at 9am to 9pm, 6 days a week .I question the entire premise of this piece. but I'm always interested in hearing what successful and especially rich people come up with time .So I finally found out Jack Ma also had said :”i f you don’t put out more time and energy than others ,how can you achieve the success you want? If you do not do 996 when you are young ,when will you ?”I quite agree with the idea that young people should fight for success .But there are a lot of survival activities to do in a day ,I want to focus on how much time they take from us and what can we do with the rest of the time. As all we known ,There are 168 hours in a week .We sleep roughly seven-and-a-half and eight hours a day .so around 56 hours a week . maybe it is slightly different for someone . We do our personal things like eating and bathing and maybe looking after kids -about three hours a day .so around 21 hours a week .And if you are working a full time job ,so 40 hours a week , Oh! Maybe it is impossible for us at
关于人英语演讲稿(精选多篇) 关于人的优美句子 1、“黑皮小子”是我对在公交车上偶遇两次的一个男孩的称呼代号。一听这个外号，你也定会知道他极黑了。他的脸总是黑黑的；裸露在短袖外的胳膊也是黑黑的；就连两只有厚厚耳垂的耳朵也那么黑黑的，时不时像黑色的猎犬竖起来倾听着什么；黑黑的扁鼻子时不时地深呼吸着，像是在警觉地嗅着什么异样的味道。 2、我不知道，如何诠释我的母亲，因为母亲淡淡的生活中却常常跳动着不一样的间最无私、最伟大、最崇高的爱，莫过于母爱。无私，因为她的爱只有付出，无需回报；伟大，因为她的爱寓于
普通、平凡和简单之中；崇高，是因为她的爱是用生命化作乳汁，哺育着我，使我的生命得以延续，得以蓬勃，得以灿烂。 3、我的左撇子伙伴是用左手写字的，就像我们的右手一样挥洒自如。在日常生活中，曾见过用左手拿筷子的，也有像超级林丹用左手打羽毛球的，但很少碰见用左手写字的。中国汉字笔画笔顺是左起右收，适合用右手写字。但我的左撇子伙伴写字是右起左收的，像鸡爪一样迈出田字格，左看右看，上看下看，每一个字都很难看。平时考试时间终了，他总是做不完试卷。于是老师就跟家长商量，决定让他左改右写。经过老师引导，家长配合，他自己刻苦练字，考试能够提前完成了。现在他的字像他本人一样阳光、帅气。 4、老师，他们是辛勤的园丁，帮助着那些幼苗茁壮成长。他们不怕辛苦地给我们改厚厚一叠的作业，给我们上课。一步一步一点一点地给我们知识。虽然
他们有时也会批评一些人，但是他们的批评是对我们有帮助的，我们也要理解他们。那些学习差的同学，老师会逐一地耐心教导，使他们的学习突飞猛进。使他们的耐心教导培养出了一批批优秀的人才。他们不怕辛苦、不怕劳累地教育着我们这些幼苗，难道不是美吗？ 5、我有一个表妹，还不到十岁，她那圆圆的小脸蛋儿，粉白中透着粉红，她的头发很浓密，而且好像马鬓毛一样的粗硬，但还是保留着孩子一样的蓬乱的美，卷曲的环绕着她那小小的耳朵。说起她，她可是一个古灵精怪的小女孩。 6、黑皮小子是一个善良的人，他要跟所有见过的人成为最好的朋友！这样人人都是他的好朋友，那么人人都是好友一样坦诚、关爱相交，这样人与人自然会和谐起来，少了许多争执了。 7、有人说，老师是土壤，把知识化作养分，传授给祖国的花朵，让他们茁壮成长。亦有人说，老师是一座知识的桥梁，把我们带进奇妙的科学世界，让
优秀党务工作者事迹简介范文 优秀党务工作者事迹简介范文 ***，男，198*年**月出生，200*年加入党组织，现为***支部书记。从事党务工作以来，兢兢业业、恪尽职守、辛勤工作，出色地完成了各项任务，在思想上、政治上同党中央保持高度一致，在业务上不断进取，团结同事，在工作岗位上取得了一定成绩。 一、严于律己，勤于学习 作为一名党务工作者，平时十分注重知识的更新，不断加强党的理论知识的学习，坚持把学习摆在重要位置，学习领会和及时掌握党和国家的路线、方针、政策，特别是党的十九大精神，注重政治理论水平的提高，具有坚定的理论信念;坚持党的基本路线，坚决执行党的各项方针政策，自觉履行党员义务，正确行使党员权利。平时注重加强业务和管理知识的学习，并运用到工作中去，不断提升自身工作能力，具有开拓创新精神，在思想上、政治上和行动上时刻同党中央保持高度一致。 二、求真务实，开拓进取 在工作中任劳任怨，踏实肯干，坚持原则，认真做好学院的党务工作，按照党章的要求，严格发展党员的每一个步骤，认真细致的对待每一份材料。配合党总支书记做好学院的党建工作，完善党总支建设方面的文件、材料和工作制度、管理制度等。
三、生活朴素，乐于助人 平时重视与同事间的关系，主动与同事打成一片，善于发现他人的难处，及时妥善地给予帮助。在其它同志遇到困难时，积极主动伸出援助之手，尽自己最大努力帮助有需要的人。养成了批评与自我批评的优良作风，时常反省自己的工作，学习和生活。不但能够真诚的指出同事的缺点，也能够正确的对待他人的批评和意见。面对误解，总是一笑而过，不会因为误解和批评而耿耿于怀，而是诚恳的接受，从而不断的提高自己。在生活上勤俭节朴，不铺张浪费。 身为一名老党员，我感到责任重大，应该做出表率，挤出更多的时间来投入到**党总支的工作中，不找借口，不讲条件，不畏困难，将总支建设摆在更重要的位置，解开工作中的思想疙瘩，为攻坚克难铺平道路，以支部为纽带，像战友一样团结，像家庭一样维系，像亲人一样关怀，践行入党誓言。把握机遇，迎接挑战，不负初心。
关于时间的英语演讲稿范文_演讲稿 and organizing people to learn from advanced areas to broaden their horizons in order to understand the team of cadres working conditions in schools, the ministry of education has traveled a number of primary and secondary schools to conduct research, listen to the views of the school party and government leaders to make school leadership cadres receive attention and guidance of the ministry of education to carry out a variety of practical activities to actively lead the majority of young teachers work hard to become qualified personnel and for them to put up the cast talent stage, a single sail swaying, after numerous twists and turns arrived in port, if there is wind, a hand, and naturally smooth arrival and guide students to strive to e xcel, need to nazhen “wind” - teacher. teachers should be ideological and moral education, culture education and the needs of students organically combining various activities for the students or students to carry out their own. for example: school quiz competitions, essay contests, ke benju performances and other activities to enable students to give full play to their talents. teachers rush toil, in order to that will enable students to continue to draw nutrients, to help them grow up healthily and become pillars of the country before. for all students in general education, the government departments have also not forget those who cared about the