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Healthy Garza 'excited' to pitch, prove himself
By Carrie Muskat / MLB.com
MESA, Ariz. -- Cubs pitcher Matt Garza feels he's right on schedule for Spring Training, but what happens beyond this season for the right-hander, who will be a free agent, is not something he's worried about.
"My goal is to go out there and pitch and prove I'm healthy enough and make them want me to stay," Garza said Monday. "That's my job. Right now, there's all the questions -- 'How's he going to come back? How's he going to rebound?' Those are legitimate questions.
"I haven't thrown off the mound competitively for two months," he said. "Now I'm feeling great and having great bullpens and excited to be pitching. All I know is April 1 is Game 1, and I want to be there."
Garza hasn't pitched since July 21. He was shut down after that because of problems with his right elbow, the cause of which is still baffling. Doctors told him he needed rest. Garza then spent the offseason getting everything in shape, and that, he said, includes "every little muscle, every toe muscle, every finger muscle."
"That was my goal this offseason, to make sure everything was in shape," Garza said. "I feel great, and I feel I'm in a great state of mind, great shape, and I'm glad to be back on the mound."
He was a hot topic at the Trade Deadline last July, but his injury ended interest. The Cubs did deal Ryan Dempster and Paul Maholm at that time. Could Garza be going this year?
"My name's popped up every year of my professional career," he said. "It's all the same to me. I'm just happy to be back pitching. Yeah, it's a big year. It doesn't matter -- if we win, we all win. We get to October, that's the only thing I play for is to get to October."
The rules have changed for free agents, which has left a pitcher like Kyle Lohse looking for work as camps open. He declined a qualifying offer from the Cardinals, and any team that signs him would have to give up a Draft pick.
"That's out of my hands," Garza said when asked to compare his situation to Lohse's. "I have to pitch. If I pitch well enough and good enough, that's not going to matter. If I go out there and do what I'm capable of doing and toe the rubber every five days and be as consistent as I need to be and as durable as I have my entire career, it's not going to matter. I'm just going to go out there and pitch and make people want me."
Garza, who was 5-7 with a 3.91 ERA in 18 starts last season with the Cubs, has had no problems in early workouts at Fitch Park. He's already throwing 45 pitches in bullpen sessions, working on fastball command. Cubs manager Dale Sveum mentioned Garza as one of the options for Opening Day.
"Right now, I'm just glad to be in the mix to start," Garza said. "There could've been a lot of setbacks. I'm just glad to be able to start."
Carrie Muskat is a reporter for MLB.com. She writes a blog, Muskat Ramblings, and you can follow her on Twitter @CarrieMuskat. This story was not subject to the approval of Major League Baseball or its clubs.


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ATP-binding cassette A3 (ABCA3) is a lipid transport protein required for synthesis and storage of pulmonary surfactant in type II cells in the alveoli. Abca3 was conditionally deleted in respiratory epithelial cells (Abca3Δ/Δ) in vivo. The majority of mice in which Abca3 was deleted in alveolar type II cells died shortly after birth from respiratory distress related to surfactant deficiency. Approximately 30% of the Abca3Δ/Δ mice survived after birth. Surviving Abca3Δ/Δ mice developed emphysema in the absence of significant pulmonary inflammation. Staining of lung tissue and mRNA isolated from alveolar type II cells demonstrated that ∼50% of alveolar type II cells lacked ABCA3. Phospholipid content and composition were altered in lung tissue, lamellar bodies, and bronchoalveolar lavage fluid from adult Abca3Δ/Δ mice. In adult Abca3Δ/Δ mice, cells lacking ABCA3 had decreased expression of mRNAs associated with lipid synthesis and transport. FOXA2 and CCAAT enhancer-binding protein-α, transcription factors known to regulate genes regulating lung lipid metabolism, were markedly decreased in cells lacking ABCA3. Deletion of Abca3 disrupted surfactant lipid synthesis in a cell-autonomous manner. Compensatory surfactant synthesis was initiated in ABCA3-sufficient type II cells, indicating that surfactant homeostasis is a highly regulated process that includes sensing and coregulation among alveolar type II cells.
- gene regulation
- lipids
- lung
- compensation
pulmonary surfactant reduces surface tension at the air-liquid interface in the alveolus, thereby maintaining lung volumes during the respiratory cycle. Surfactant consists of lipids and associated proteins that are required for surfactant function. Notably, surfactant proteins (SP) B and C (encoded by SFTPB and SFTPC) and ATP-binding cassette (ABC) A3 (ABCA3) play critical roles in surfactant synthesis and function. Various autosomal-recessive mutations in SFTPB and ABCA3 cause fatal respiratory distress in human newborns due to the lack of surfactant (20, 42). In some cases, ABCA3 mutations are associated with interstitial lung diseases in older children (12, 17, 54). Histological analysis of lungs of such patients showed interstitial fibrosis. Heterozygosity for ABCA3 mutations increased the severity of interstitial lung disease associated with mutations in SFTPC, indicating that alterations in ABCA3 may influence the pathogenesis of lung diseases (10).
Coordination of the synthesis and packaging of surfactant components is essential for the transition to air breathing at birth and thereafter. Surfactant content and function are regulated at multiple levels, including synthesis, processing, intracellular transport, assembly, and storage of surfactant components, surfactant secretion, and catabolism of lipids and proteins. Expression of surfactant proteins and regulation of lipid metabolism during development are dependent on a number of transcription factors that are expressed in respiratory epithelial cells (6, 14, 15, 29, 30, 47). Less is known regarding control of surfactant lipid homeostasis in the postnatal lung. Maintenance of surfactant lipid content during lung injury may involve intra- and extracellular sensing modules capable of detecting and regulating lipid content and trafficking.
ABC proteins are members of a large family of transporters associated with ATP-dependent translocation of various substrates across cellular membranes. ABCA3 is expressed in various organs, including lung, brain, and kidney (32, 44). In the lung, Abca3 mRNA increases dramatically prior to birth (44). Abca3 gene expression is regulated by glucocorticoids (53) and cis-acting cassettes that mediate pulmonary cell- and lipid-sensitive pathways (7), allowing surfactant homeostasis at birth and thereafter. ABCA3 is a 1,704-amino acid and 190-kDa protein, highly expressed in alveolar type II cells, where it is present in the limiting membrane of lysosomal-derived intracellular inclusions termed lamellar bodies (LBs) (32, 52, 55). Abnormalities in lipid content and function were observed in surfactant from patients with ABCA3-related pulmonary disease (20). Deletion of the Abca3 gene in mouse models resulted in respiratory failure after birth, which was caused by the absence of surfactant in the alveoli. Loss of mature LBs in Abca3 gene-deleted mice was also observed and was consistent with findings in human infants with mutations in ABCA3 (2, 13, 19, 23). Taken together, ABCA3 is required for LB formation and pulmonary surfactant function.
While deletion of the Abca3 gene in mice demonstrated its requirement at birth, little is known about the effects of Abca3 deficiency in adult lung function. In this study, the Abca3 gene was conditionally deleted in respiratory epithelial cells. Deletion of Abca3 altered lung lipid content and synthesis. Maintenance of surfactant function in Abca3-deleted mice after birth was associated with compensatory lipid synthesis in nontargeted type II cells, revealing a novel compensatory system that senses surfactant deficiency caused by cell-selective deletion of Abca3.
Gene construction for Abca3 floxed mouse.
To generate a conditional Abca3 floxed mutant allele, a 14.4-kb region of the mouse gene was subcloned from a positively identified bacterial artificial chromosome (BAC) clone from inGenious Targeting Lab (Stony Brook, NY) and used to construct the targeting vector. The construct was designed such that the short-homology arm extended 1.9 kb 3′ of the loxP-floxed neomycin (Neo) cassette, in intron 7/8. The long-homology arm extended 12.5 kb 5′ of the Neo cassette, and a single loxP site was inserted in intron 3/4, 5′ of exon 4. The target region was 4.6 kb and included exons 4, 5, 6, and 7 (Fig. 1A). A targeting construct was generated with Neo-resistance (neoR) gene as a selective marker. The linearized targeting construct was electroporated into eukaryotic stem cells and grown in the antibiotic G418. Surviving clones were screened for homologous recombination by Southern and PCR analysis. Ten positive clones were detected in 288 samples analyzed. Eukaryotic stem cells from three positively identified loxP-floxed Abca3 clones (Abca3flx) were injected into blastocysts to generate chimeric mice, and germ-line transmission of the Abca3flx allele was achieved from all clones injected and backcrossed with C57BL6 for at least three generations.
SP-C-rtTA (line 2)/(tetO)7CMV-Cre/Abca3flx/flx triple-transgenic mice were generated as described previously (34, 35, 39, 49). In the presence of doxycycline, exons 4, 5, 6, and 7 of the Abca3 gene were permanently deleted from respiratory epithelial cells prior to birth (Abca3Δ/Δ mice; Fig. 1A). For genotyping, DNA was purified from the tail of experimental mice, and PCR was performed for Abca3flx, SP-C-rtTA, and (tetO)7CMV-Cre genes using the following primers: 5′-AGC ACT TTT CCC TCT CTG GCC TTG AG-3′ and 5′-TGC CCA CCC AGC ACC ATG CT-3′ for Abca3flx, 5′-GAC ACA TAT AAG ACC CTG GCTA-3′ and 5′-AAA ATC TTG CCA GCT TTC CC-3′ for SP-C-rtTA, and 5′-TGC CAC CAA GTG ACA GCA ATG-3′ and 5′-AGA GAC GGA AAT CCA TCG CTCG-3′ for (tetO)7CMV-Cre. Abca3-deleted transgenic (Abca3Δ/Δ) mice and nondeleted (SP-C-rtTAtg/wt, Abca3flx/flx, control) littermates were used for the experiments. FVB/N-TgN(EIIa-Cre)C5379Lmgd mice carrying a Cre transgene, under the control of the adenovirus EIIa promoter, which targets expression of Cre recombinase to the early mouse embryo, were purchased from the Jackson Laboratory (Bar Harbor, ME). Double-transgenic mice were generated by mating EIIa-Crewt/tg/Abca3flx/wt to Abca3flx/flx mice. Abca3flx/flx littermates lacking the Cre gene served as controls.
Animal husbandry and doxycycline administration.
Mice were maintained in a pathogen-free environment in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Cincinnati Children's Hospital Research Foundation. All animals were housed in humidity- and temperature-controlled rooms on a 14:10-h light-dark cycle. Mice were allowed food and water ad libitum. There was no serological evidence of pulmonary pathogens or bacterial infections in sentinel mice maintained within the colony. Gestation was dated by detection of the vaginal plug [as embryonic (E) day 0.5 (E0.5)] and correlated with the weight of each pup at the time of death. Doxycycline (625 mg/kg; Harlan Teklad, Madison, WI) was administered to the dams in the food from E6.5 to E14.5, resulting in extensive deletion of Abca3 in respiratory epithelial cells. Mice were then fed normal food.
Tissue preparation, histology, and immunohistochemistry.
Lung tissue preparation and immunohistochemistry were performed essentially as described previously (28, 46). Tissue sections were stained with hematoxylin and eosin. Primary antibodies were used at the following dilutions: surfactant protein (SP)-B (1:2,000, rabbit polyclonal; Chemicon, Temecula, CA), proSP-C (1:4,000, rabbit polyclonal; AB3428, Chemicon), ABCA3 (1:1,000, rabbit polyclonal; Seven Hills Bioreagents), FOXA2 (1:4,000 and 1:8,000, rabbit polyclonal; Seven Hills Bioreagents), and cleaved caspase-3 (1:1,000, rabbit polyclonal; R & D Systems, Minneapolis, MN). The secondary antibody was goat anti-rabbit IgG (1:200; Vector, Burlingame, CA). All experiments are representative of findings from at least four Abca3Δ/Δ and control mice. Lung sections were stained with orcein to detect elastic fibers (Poly Scientific, Bay Shore, NY).
Transmission electron microscopy.
Lung tissue from postnatal day 0 (PND0) and 9-mo-old Abca3Δ/Δ mice and littermate controls was fixed in modified Karnovsky's fixative consisting of 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer and 0.1% calcium chloride (pH 7.3). Adult tissue was stained en bloc with 4% uracyl acetate, postfixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer, dehydrated, and embedded in epoxy resin (EMbed 812, Electron Microscopy Sciences, Fort Washington, PA), as previously described (56). Ultrathin sections were postfixed with uracyl acetate and lead citrate and viewed in Morgagni 268 and Hitachi 7600 transmission electron microscopes (TEM), and digitized images were collected with an Advantage Plus 2K × 2K TEM equipped with a charge-coupled device digital camera (Advanced Microscopy Techniques, Danvers, MA).
Morphometric measurements were performed on 9 mo-old mice of both genotypes. The overall proportion [%fractional (%Fx) area] of respiratory parenchyma and air space was determined by a point-counting method (50). Measurements were performed on two sections taken at intervals throughout the left, right upper, and right lower lobes. Slides were viewed with an ×20 objective. Images (fields) were acquired randomly with a digitalized camera and quantified using MetaMorph imaging software (version 7.5, Molecular Devices, Downingtown, PA). A computer-generated, 121-point lattice grid was superimposed on each field, and the number of intersections (points) falling over respiratory parenchyma (alveoli and alveolar ducts) or air space was counted. Points falling over bronchioles, large vessels, and smaller arterioles and venules were excluded from the study. %Fx area was calculated by dividing the number of points for each compartment (n) by the total number of points contained within the field (N) and then multiplying by 100: %Fx area = n/N × 100. Fifteen random fields per section were analyzed to gather the data. The x- and y-coordinates for each field measured were selected by a random-number generator (Microsoft Excel 2003, Redmond, WA).
The number of ABCA3-stained cells was determined by a point-counting method. Five mice of both genotypes were studied. Measurements were performed throughout the left, right upper, and right lower lobes, and the number of ABCA3-stained cells was counted manually. The density of ABCA3-stained cells was calculated by dividing the number of ABCA3-stained cells by alveolar area (mm2). Fifteen fields per section were analyzed. The x- and y-coordinates for each field measured were selected by a random-number generator. Bronchioles, large vessels, and smaller arterioles and venules were excluded.
The cross-sectional area of LBs in alveolar type II cells from 9-mo-old control and Abca3Δ/Δ mice was determined from ultrathin (90-nm-thick) sections cut from one to two Epon blocks from each group (n = 1 for control and n = 3 for Abca3Δ/Δ mice) and transferred to 200-mesh copper grids. Ten electron micrographs were randomly selected from one to two sections per block. Each electron micrograph was coded according to genotype and digitally acquired at a final magnification of ×10,000. The cross-sectional areas of all LBs were determined using the region measurement function in MetaMorph imaging software (version 7.5).
The percentage of type II cells with or without LBs was assessed by TEM of tissue from 9-mo-old control and Abca3Δ/Δ mice. A total of 292 type II cells found in 1–2 grids from 3 different Abca3Δ/Δ mice were analyzed.
Isolation of alveolar type II epithelial cells.
Alveolar type II cells were isolated from 9-mo-old control and Abca3Δ/Δ mouse lung with use of collagenase and differential plating, as described previously (38). Type II cells were used 2 h after isolation for RNA analysis.
Flow cytometry analysis and sorting.
After isolation, alveolar type II cells were stained for 10 min in the presence of 1 μM Nile red (NR). Cells were then washed three times in PBS and brought to a concentration of 4 × 106 cells/ml in sorting buffer [1× PBS (Ca2+/Mg2+ free), 1 mM EDTA, 25 mM HEPES (pH 7.0), 1% fetal bovine serum, 50 U of penicillin/ml, and 50 μg of streptomycin/ml]. Cells were sorted and analyzed by a FACSAria II sorter (Becton Dickinson, San Jose, CA) within 2 h of staining. Excitation was at 533 nm using a yellow-green laser, and emission was collected at 628 nm. A more intense red fluorescence of the phospholipid-enriched cells is obtained, because the emission maxima wavelength of phospholipids stained with NR is longer than that observed for neutral lipids (9, 21). NR-positive cells were initially gated on the basis of side scatter and mean fluorescence intensity of the NR signal. Data were exported and analyzed with FACSDiVa software (Becton Dickinson).
Bronchoalveolar lavage fluid.
Nine-mo-old mice (n = 5/group) were anesthetized and killed by exsanguination. Tracheas were cannulated, and five 1-ml aliquots of 0.9% NaCl were flushed into the lungs and withdrawn by syringe three times for each aliquot. The volumes of recovered bronchoalveolar lavage (BAL) fluid (BALF) from all the groups were similar. After centrifugation, BAL cells were counted using a hemocytometer to determine total BAL cell concentration. BAL cells were used for RNA extraction, or, after cytospin, cells were stained to determine differential cell counts (Diff-Quik, Dade Behring, Miami, FL).
RNA extraction and RT-PCR.
RNA was extracted from whole lung homogenate of PND0 Abca3Δ/Δ and control mice or isolated lung alveolar type II cells or BAL cells of adult mice with use of RNeasy Protect Mini kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Samples were obtained from five control or Abca3Δ/Δ mice. Quantitative PCRs using Taqman probes (Applied BioSystems, Foster City, CA) were performed with primer sets specific for Abca3 (Mm00550501_m1), surfactant protein (Sftp) Sftpa1 (Mm00499170_m1), Sftpb (Mm00455681_m1), Sftpc (Mm00488144_m1), fatty acid synthase (Fasn, Mm01253292_m1), stearoyl-CoA desaturase (Scd1, Mm01197142_m1), lysophosphatidylcholine acyltransferase 1 (Lpcat1, Mm00461015_m1), fatty acid-binding protein 5 (Fabp5, Mm00783731_s1), choline phosphate cytidylyltransferase 1 (Pcyt1a, Mm00447774_m1), napsin A aspartic peptidase (Napsa, Mm00492829_m1), steroidogenic acute regulatory domain protein (Stard) 2 (Stard2, Mm00476629_m1), Stard7 (Mm00460536_m1), Stard9 (Mm00626196_m1), Stard10 (Mm00502345_m1), Abca1 (Mm00442646_m1), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (Hmgcs1, 00524111_m1), nuclear factor of activated T cells (Nfatc3, Mm01249200_m1), NK2 homeobox 1 Nkx2.1 (Mm00447558_m1), forkhead box A2 (Foxa2, Mm01976556_s1), CCAAT enhancer-binding protein-α (Cebpa, Mm01265914_s1), macrophage inflammatory protein 1α (Mip1a, Mm00441258), transforming growth factor-β1 (Tgfb1, Mm03024053), TNFα (Tnfa, Mm00443258_m1), IL-1β (Il-1b, Mm01336189_m1), and matrix metallopeptidase 12 (Mmp12, Mm00838401_g1). A probe set for 18S rRNA was used as the normalization standard. The PCR and relative quantifications were performed using 25 ng of cDNA per reaction in a real-time PCR system (model 7300, Applied BioSystems). Relative quantification data from quantitative PCR analysis were statistically analyzed using Student's t-tests.
Western blot analysis for ABCA3.
ABCA3 expression was quantified by Western blot analysis in whole lung homogenate. Samples were obtained from PND0 control and Abca3Δ/Δ mice (n = 3 for each genotype). Proteins were extracted using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL), diluted in Laemmli buffer, and subjected to SDS-PAGE under reducing condition. After transfer, the membrane was blocked for 2 h at room temperature in PBS [20 mM Tris·HCl (pH 7.6) and 137 mM NaCl] + 0.2% Tween 20 containing 5% skim milk and then incubated with primary antibody overnight at 4°C. The following dilutions of antibody were used: anti-ABCA3 (COOH-terminal region, 1:1,000, rabbit polyclonal; a kind gift from Dr. Inagaki, Kyoto University, Kyoto, Japan) (52) and actin (1:1,000, goat polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA). Peroxidase-conjugated secondary antibodies (Calbiochem, EMD Biosciences, San Diego, CA) were used at 1:5,000 dilutions. Immunoreactive bands were detected with enhanced chemiluminescence reagents (Amersham Bioscience, Buckinghamshire, UK).
LB preparation.
Nine-month-old mice (n = 8/group) were anesthetized and killed by exsanguination. After BAL, the lungs were rapidly removed and chilled in 0.25 M sucrose in Tris buffer (0.01 M Tris·HCl, 0.001 M CaCl2, 0.002 M MgSO4, and 0.15 M NaCl, pH 7.4). The right lobes were minced finely with scissors and homogenized with 0.25 M sucrose by use of a loosely fitting Teflon plunger (clearance of ∼0.09 mm) with a Pyrex Potter homogenizer. Debris and intact cells were spun down at 2,000 rpm (600 g) for 5 min at 4°C. The supernatant was strained through lens paper. The crude lung homogenate was brought to 1.2 M sucrose by the addition of 2.5 M sucrose. Four-milliliter aliquots were placed in polyallomer tubes and overlaid by a sucrose gradient consisting of 3 ml of 0.7 M sucrose buffer, 7 ml of 0.5 M sucrose buffer, and 3 ml of 0.3 M sucrose. The tubes were centrifuged for 3 h at 104,000 g at 4°C in a Beckman L-70 ultracentrifuge with an SW28 swinging-bucket rotor. After centrifugation, the LB fraction was collected from the top and stored at −80°C.
Analysis of surfactant components.
Saturated phosphatidylcholine (Sat PC) was measured in homogenized whole lung tissue from PND0 mice (n = 4–6 mice for each genotype) as previously described (3). The phospholipids in chloroform-methanol extracts of homogenized lung were separated by two-dimensional thin-layer chromatography for compositional analyses as previously described (26). The spots were visualized with iodine vapor, scraped, and assayed for phosphorus content as previously described (26).
Electrospray ionization-mass spectrometry of phospholipid molecular species and phosphatidylcholine synthesis.
Mice were injected intraperitoneally with 1 mg of methyl-D9-choline chloride (Cambridge Isotope Laboratories, Andover, MA) 3 h before they were killed. Phospholipids were extracted from BALF, lavaged lung tissue, and LBs with chloroform and methanol according to Bligh and Dyer (8) after addition of the following internal standards: phosphatidylcholine (PC14:0/14:0, 10 pmol), phosphatidylethanolamine (PE14:0/14:0, 4 pmol), phosphatidylglycerol (PG14:0/14:0, 2 pmol), and phosphatidylserine (PS14:0/14:0, 2 pmol). Electrospray ionization-mass spectrometry of phospholipids was performed as previously described (36). In addition, incorporation of methyl-D9-choline as a measure of PC synthesis in the various tissue fractions was determined by precursor scan of mass-to charge ratio (m/z) 193 (P193) expressed relative to total PC, determined as the sum of precursor scan of m/z 184 (P184) and P193 (5). The volumes of recovered BALF from all the groups were similar. Protein concentration was determined using a Bradford assay (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard.
RNA microarray analysis.
For Affymetrix MicroArray, E18.5 lung RNAs from Abca3Δ/Δ mice and littermate controls were prepared using the RNeasy Protect Mini kit according to the manufacturer's protocol. RNA was treated with DNase at room temperature for 15 min. Lung cRNA was hybridized to the Murine Genome 430 2.0 Array (consisting of 45,000 probe sets representing >34,000 mouse genes; Affymetrix) using the manufacturer's protocol. Affymetrix MicroArray Suite version 5.0 was used to scan and quantitate the gene chips using default scan settings. Normalization was performed using the Robust Multichip Average model, which consists of three steps: background adjustment, quartile normalization, and summarization (24, 25). Microarray analysis was performed with the software package BRB Array Tools, developed by the Biometric Research Branch of the National Cancer Institute (http://linus.nci.nih.gov/BRB-ArrayTools.html). Differentially expressed genes were identified using a univariate F test and permutation test (n = 100) with a significance level of 0.05. Data were prefiltered by excluding probe sets as follows: 1) those whose expression differed ≤1.2 from the median in 80% of the samples, 2) those whose expression data were missing in >50% of the samples, or 3) those with >70% of absent calls by the Affimetrix algorithm in six samples. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analysis were performed using the publicly available web-based tool DAVID (Database for Annotation, Visualization, and Integrated Discovery) (16). Gene frequency and probability in each functional category were calculated by Fisher's Exact test using Mouse Genome 430 2.0 as background control. Biological association networks were built using Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, CA).
Statistical analysis.
Student's t-test was used to determine the levels of difference between groups. Values for all measurements are expressed as means ± SE, and P values for significance are indicated for each experiment.
Abca3 is required for postnatal adaptation to air breathing.
Abca3 mRNA was previously detected in alveolar type II cells in the murine lung, as well as in other organs, including brain and kidney (32, 44). To identify the role of Abca3 in alveolar type II cells, triple-transgenic SP-C-rtTAtg/wt, TetO-Cretg/wt, and Abca3flx/flx (Abca3Δ/Δ) mice were produced; in these mice, Abca3 was selectively and permanently deleted in the respiratory epithelium following administration of doxycycline to the dam from E6.5 to E14.5 (Fig. 1A). Analysis of Abca3 gene expression demonstrated a dramatic reduction of Abca3 mRNA and ABCA3 protein in newborn (PND0) Abca3Δ/Δ mice (Fig. 1, B–E); 67% of PND0 Abca3Δ/Δ mice died at birth. The lungs of the Abca3Δ/Δ mice that died at PND0 failed to inflate. Atelectasis was observed (Fig. 1, F–G). Immunostaining for mature SP-B and proSP-C were similar in Abca3Δ/Δ and control mice (Fig. 1, H–K). To confirm the targeting of the conditional Abca3 floxed mutant allele, EIIa-Cretg/wt, Abca3flx/flx mice wherein Abca3 is deleted prior to implantation in the uterine wall, died soon after birth from respiratory failure, confirming the recombination of the Abca3flx allele and supporting its requirement for the transition to air breathing at birth (data not shown).
Adult Abca3Δ/Δ mice develop emphysema.
Approximately 33% of the Abca3Δ/Δ mice survived postnatally and to 9 mo of age. Postnatal survival was better in females than males (Fig. 2A). The efficiency of Abca3 deletion in alveolar type II cells in surviving mice at 9 mo was assessed by quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Both Abca3 mRNA and the number of cells stained for ABCA3 were decreased by ∼50% in type II cells isolated from adult Abca3Δ/Δ mice compared with control littermates (Fig. 2, B and D), indicating the heterogeneity of Abca3 targeting. Deletion of Abca3 in adult mice altered surfactant-associated protein mRNA expression. While a marked decrease in Sftpb was observed in Abca3Δ/Δ lungs, Sftpc and Napsa were similar in both groups of mice (Fig. 2C). In contrast, Sftpa1 was significantly increased in Abca3Δ/Δ mice compared with control littermates.
Surviving Abca3Δ/Δ mice developed normally after birth. While alveolarization was unchanged at 4 wk of age, air space enlargement was observed in 9-mo-old Abca3Δ/Δ mice (Fig. 3, A–D). The relative proportion of respiratory parenchyma (Fig. 3E) and air space (Fig. 3F; fractional area) were altered in 9-mo-old Abca3Δ/Δ mice compared with control littermates, with a significant increase in the fractional area of air space (P < 0.002) in Abca3Δ/Δ mice. The enlargement of alveoli was associated with alterations in the distribution and structure of elastic fibers. Thickened elastin fibers were observed surrounding enlarged air spaces in Abca3Δ/Δ mice, in contrast to fine elastin fibers in septae of normal lung (data not shown). Inflammation was not readily apparent in the Abca3Δ/Δ mice, and total cell counts were similar in lung lavage fluid (Fig. 3G), wherein monocytes/macrophages were the main cell population. Inflammatory cytokine mRNA levels in Abca3Δ/Δ mice were not statistically different from those in control littermates (Fig. 3H). There was no histological or serological evidence of infection, and sentinel mice did not harbor viral or bacterial pathogens.
Deletion of Abca3 alters lamellar body formation and surfactant composition.
In newborn mice, in contrast to control littermates, LBs were not observed in type II cells from PND0 Abca3Δ/Δ mice that died at birth from respiratory distress (Fig. 4, A and B). Alveolar type II cells from PND0 Abca3Δ/Δ mice contained more glycogen, and some contained atypical lipid structures. Secreted surfactant and tubular myelin were absent from the alveoli of the newborn Abca3Δ/Δ mice, while evidence of type I cell injury and early hyaline membrane formation, consistent with respiratory distress syndrome, was also detected by TEM (data not shown).
Numerous LBs were observed in 9-mo-old control mice (Fig. 4C). Although the average LB cross-sectional area was similar between control and Abca3Δ/Δ mice, variations in size and number of LBs were seen in the Abca3Δ/Δ mice (see supplemental Fig. 1 in the online version of this article). In Abca3Δ/Δ mice, two distinct subsets of type II cells were altered. One subset of type II cells contained no LBs (68%), but liposome-like vesicular structures of intermediate electron density with electron-dense inclusions were observed (Fig. 4, E and F). In addition, this subset contained variable numbers of lipid inclusions that were absent from the controls (Fig. 4, G and H). The second subset of type II cells contained normal (26%) and "giant" (6%) LBs, either single lamellar-like bodies or larger structures containing several LBs (Fig. 4, D, I, J; see supplemental Fig. 1). These data support the concept that changes in surfactant lipid homeostasis in nondeleted type II cells may compensate for the absence of LBs in cells in which Abca3 was deleted.
Effect of deletion of Abca3 on phospholipid composition and synthesis.
Consistent with the ultrastructure alterations observed in newborns, Sat PC (Fig. 5A), PC, and, to a lesser extent, PG content were decreased in lungs from PND0 Abca3Δ/Δ mice (Fig. 5, B and C); lung lysophosphatidylcholine and phosphatidylethanolamine contents were increased.
Despite the altered morphology of type II cells in the adult Abca3Δ/Δ mice, the concentration of total PC in lavaged lungs and LBs, calculated as the sum of individual molecular species determined by electrospray ionization-mass spectrometry, was not significantly different in adult Abca3Δ/Δ and control mice (Fig. 6). The concentration of total PC was, however, decreased by 50% in BALF from adult Abca3Δ/Δ mice. Both groups of mice showed a comparable sequential enrichment pattern of surfactant-specific PC species from lavaged lungs, through LBs to BALF. Comparison of profiles of PC synthesis and concentration indicated that acyl remodeling mechanisms were also unaltered in Abca3Δ/Δ compared with control mice (data not shown). PC synthesis, calculated as the sum of methyl-D9-choline incorporation into PC molecular species after 3 h of labeling, was decreased by 20% in lavaged lung in Abca3Δ/Δ mice, from 1.28 ± 0.06 to 1.00 ± 0.12% total PC (mean ± SE, P < 0.05; Fig. 6). Incorporation of stable isotope into PC in isolated LBs was 1.73 ± 0.10% total PC in controls and was significantly decreased in lungs of Abca3Δ/Δ mice to 1.11 ± 0.10% total PC (mean ± SE, P < 0.001). In contrast, methyl-D9-choline enrichment in BALF PC was essentially identical in Abca3Δ/Δ mice and controls, although newly synthesized PC was considerably less abundant in BALF than in lavaged tissue or LB at 0.34 ± 0.07 vs. 0.31 ± 0.06% total PC. PG content was markedly decreased in LBs and BALF fractions, but not in lung parenchyma (Fig. 6). In agreement with previous analyses (37), this detailed lipidomic characterization showed that disaturated PG species consistently represented <20% of PG in tissue, LB, and BALF of all mice. The decrease in PG16:0/16:0 content was accounted for by increased longer-chain PG species (see supplemental Fig. 2, A and B). Analysis of the "minor components" of surfactant showed alterations in phosphatidylinositol (PI). PI was increased twofold in lung tissue and LB fractions. Notably, content of PI16:0/22:6 increased in the progression from tissue, through LB to BALF. The content of PI16:1/18:1, PI16:0/18:1, PI18:1/18:2, and PI18:1/18:1 was increased in Abca3Δ/Δ compared with control mice at the expense of longer-chain PI species (see supplemental Fig. 2, C–E).
Deletion of Abca3 influences genes regulating lipid synthesis and transport in type II epithelial cells.
RNA was extracted from whole lung homogenate from E18.5 Abca3Δ/Δ and control mice. RNAs were hybridized to the murine genome MOE430 chip. Data were analyzed using the publically available web-based tool DAVID. RNA microarray analysis of E18.5 Abca3Δ/Δ mice revealed that deletion of Abca3 influenced a number of mRNAs related to lipid metabolism (Table 1). To determine whether deletion of Abca3 in respiratory epithelial cells altered expression of genes regulating lipid homeostasis, expression of mRNAs of genes associated with lipid synthesis and transport was assessed by qRT-PCR (Fig. 7A). A marked decrease in mRNAs influencing fatty acid (Scd1 and Lpcat1) and cholesterol (Hmgcs1 and Abca1) metabolism was observed in the lungs of newborn Abca3Δ/Δ mice, perhaps indicating response to altered lipid handling in the absence of ABCA3. In contrast, Pcyt1a mRNA coding for the choline cytidylyltransferase-α (CCTα) protein was upregulated in lungs from PND0 Abca3Δ/Δ mice.
Expression of mRNAs of genes associated with lipid synthesis, transport, and secretion in alveolar type II cells isolated from 9-mo-old mice was assessed by qRT-PCR (Fig. 7). A marked decrease in mRNAs influencing fatty acid metabolism (Scd1, Fasn, Fabp5, Lpcat1, and Pcyt1a; Fig. 7B) and cholesterol metabolism (Hmgcs1 and Abca1; Fig. 7C) was observed in total type II cells of 9-mo-old Abca3Δ/Δ mice. Levels of mRNAs encoding a family of lipid transfer proteins, Stard2, Stard7, Stard9, and Stard10, were assessed by qRT-PCR (Fig. 7D). Stard2 was significantly upregulated in total type II cells from Abca3Δ/Δ mice compared with control, whereas Stard9 and Stard10 were slightly reduced.
To determine whether the alterations in mRNA expression of genes associated with lipid metabolism are triggered by cell death in response to Abca3 deletion in the respiratory epithelium, apoptosis was assessed by immunohistochemistry using cleaved caspase-3 as a marker. No differences in cleaved caspase-3 staining were observed in lungs of 4-wk-old and 9-mo-old Abca3Δ/Δ mice compared with control littermates (data not shown), indicating that alveolar type II cell survival was not affected by the deletion of Abca3.
To assess the potential compensatory regulation of gene expression between the two populations of alveolar type II cells in adult Abca3Δ/Δ mice, type II cells were isolated from the lung and then distinguished by flow cytofluorometry using NR. Highly enriched populations of NR-positive (NR++) and NR weakly (NR+) stained cells were obtained by excluding from the sort cells in the region of overlap between the two peaks. NR is a fluorescent hydrophobic probe that stains both neutral lipid and phospholipid inclusions using specific filter packages. When cells were subjected to flow cytofluorometric analysis under red fluorescence conditions (>630 nm), two cell populations stained with NR were observed in Abca3Δ/Δ mice; only one cell population (NR++) was present in control mice. In Abca3Δ/Δ mice, the phospholipid-loaded (NR++) cells (undeleted Abca3 cells) were differentiated by stronger fluorescence intensity, which was similar to findings in Abca3flx/flx cells in control mice. Abca3Δ/Δ cells displayed weaker fluorescence (NR+; Fig. 8A), because these cells lack LBs and NR is taken up by cell membrane lipids as well as by intracellular lipid droplets. Expression of mRNAs of genes associated with surfactant, lipid synthesis, transport, and secretion was assessed by qRT-PCR in NR++ and NR+ type II cells. Sftpa1 was significantly increased in NR+ compared with NR++ and control type II cells. A marked decrease in mRNAs influencing fatty acid (Scd1, Fasn, and Lpcat1) and cholesterol (Hmgcs1; Fig. 8C) metabolism was observed in NR+ type II cells isolated from 9-mo-old Abca3Δ/Δ mice compared with NR++ type II cells, supporting a decrease in the activity of surfactant synthesis pathways in the targeted cells. In contrast, Fasn, Scd1, and Hmgcs1 mRNA expression was increased in NR++ cells compared with control type II cells, perhaps indicating a selective compensatory lipid synthesis pathway in ABCA3-sufficient cells. On the other hand, mRNA expression of other genes (Sftpb, Pcyt1a, Fabp5, and Abca1) was similarly decreased in NR+ and NR++ type II cells, indicating that compensatory mechanisms were partial in response to deletion of Abca3. Finally, Stard2 was significantly increased in Abca3-deficient cells compared with Abca3-sufficient cells, whereas Stard9 was slightly reduced (Fig. 8D).
Deletion of Abca3 alters the transcriptional network associated with lipid homeostasis in alveolar type II cells.
Nfatc3, Cebpa, and Foxa2 mRNAs, genes known to influence expression of genes regulating surfactant lipid homeostasis, were significantly reduced in NR+ type II cells from Abca3Δ/Δ mice (Fig. 9A). Nkx2.1 mRNA was similar in NR++ and NR+ cells. Immunostaining for FOXA2 was readily detected in type II cells in control mice (Fig. 9, B and C). In contrast, numbers and intensity of FOXA2 staining of type II cells were decreased in Abca3Δ/Δ mice (Fig. 9, D and E).
Abca3, a lipid transporter protein that is highly expressed in the lung alveolar type II cells (32, 52, 55), is critical for surfactant lipid homeostasis and for adaptation to air breathing. Germ-line deletion of the mouse Abca3 gene caused respiratory distress immediately followed by death at birth (2, 13, 19, 23), and severe mutations in ABCA3 caused respiratory failure in newborn infants (42). Since chronic lung disease was reported in older patients (11, 17), we used a conditional system to selectively delete Abca3 in alveolar epithelial cells of the mouse lung to assess the long-term effect of Abca3 deficiency in the lung.
In the present work, respiratory epithelium-specific, conditional deletion of Abca3 caused respiratory distress and death, confirming previous findings that Abca3 was critical for adaptation for air breathing (2, 13, 19, 23). In mice that died at birth, Abca3 mRNA was reduced by 85–90% and was associated with the absence of LBs in type II epithelial cells. Thickened mesenchyme and glycogen accumulation in Abca3Δ/Δ type II cells were consistent with immaturity of the alveolar epithelium, indicating that Abca3 participates in lung maturation. Levels of SP-B and proSP-C, which are important for surfactant homeostasis, were not altered in newborn Abca3Δ/Δ mice. Surviving Abca3Δ/Δ mice developed emphysema, in contrast to previous models, in which lungs of Abca3+/− mice were normal in adulthood (13, 19). ABCA3-deficient and -sufficient cells were identified in the present model. While ABCA3 was decreased by 50% in each type II cell in Abca3+/− mice, lungs of Abca3Δ/Δ mice contained two subpopulations of type II cells: those expressing ABCA3 and those not expressing ABCA3. Thus Abca3Δ/Δ mice represent a model wherein the alveolar epithelium is variably affected, a model that is perhaps consistent with lung injury or infection, in which Abca3 gene expression is altered regionally. The present model indicates that ABCA3-sufficient type II cells compensate for the loss of ABCA3 in other cells to maintain surfactant production. Abca3Δ/Δ mice developed emphysema, perhaps indicating that ABCA3 deficiency resulted in cell injury and lung remodeling. Although the majority of mutations in ABCA3 are associated with neonatal lethality, a few ABCA3 mutations are associated with interstitial lung disease in older children who survive beyond the perinatal period (12, 17, 54). The heterozygosity for an ABCA3 mutation was associated with a more severe clinical presentation in the case of a SFTPC mutation, indicating the importance of ABCA3 in alveolar homeostasis that may influence the pathogenesis of lung disease (10).
Expression of genes involved in lipid metabolism was decreased during the perinatal period as well as in adult Abca3Δ/Δ mice. Expression of genes dedicated to the biosynthesis of fatty acids and phospholipids was decreased, consistent with altered lipid content in the lungs of newborn Abca3Δ/Δ mice. Decreased expression of mRNAs related to lipid metabolism [acyl-CoA oxidase 1 (Acoxl), Lpcat1, Sftpa1, lipase (Lipg), glycerophosphodiester phosphodiesterase domain-containing protein 2 (Gdpd2), pancreatic lipase-related protein 1 (Pnliprp1), delta-like 1 (Dlk1), and patatin-like phospholipase domain-containing protein 2 (Pnpla2)] was observed at E18.5. Such changes in gene expression may represent an indirect effect of ABCA3 deficiency and alteration in cellular lipids or, alternatively, a generalized delay in type II cell maturation. Gene Ontology and network analysis on mRNAs that were altered >1.5-fold suggested "lipid/sterol metabolism" as a major group of associated genes influenced by Abca3 deletion. Expression of genes regulating lipid synthesis (Lpcat1, Scd1, Abca1, and Hmgcs1) was decreased at PND0 in Abca3Δ/Δ mice, consistent with the RNA microarray data. In contrast, Pcyt1a was increased in PND0 Abca3Δ/Δ lungs, perhaps indicating a compensatory role for CCTα in PC synthesis after birth. Lung epithelial cell-specific deletion of Pcyt1a resulted in severe respiratory failure at birth due to a major defect in phospholipid synthesis and LB formation (45). In the adult Abca3Δ/Δ type II cells, decreased expression of several genes, including Scd1, Fasn, Lpcat1, and Pcyt1a, as well as Hmgcs1 (a gene critical for cholesterol synthesis), also was observed, demonstrating that chronic deletion of Abca3 altered lipid biosynthesis in type II epithelial cells after birth. Expression of a number of lipid transporters, including the fatty acid-binding protein 5 (Fabp5) and cholesterol transporter ATP-binding cassette A1 (Abca1), was decreased in type II cells of Abca3Δ/Δ mice. For most of these genes, levels of mRNA were reduced by 40–70%, consistent with the loss of ABCA3 in 50% of type II cells in Abca3Δ/Δ mice. This inhibition of lipid biosynthesis and transport may serve to counterregulate surfactant production, since it can no longer be packaged and secreted properly in the alveolar space. Alternatively, cell injury related to the lack of ABCA3 may initiate an inhibitory effect on lipid biosynthesis. While Sftpa1 mRNA was decreased in newborn Abca3Δ/Δ lungs, it was increased in ABCA3-deficient type II cells in adult Abca3Δ/Δ mice, perhaps indicating a compensatory role for SFTPA1 in the formation of tubular myelin in the ABCA3-deficient cells.
Lungs of surviving Abca3Δ/Δ mice contained two distinct populations of alveolar type II cells: one lacking LBs and the other containing LBs of various size. Absence of LBs related to the deletion of Abca3 in type II cells was consistent with previous mouse and human studies (2, 13, 19, 20, 23, 42). The presence of large atypical LBs and ABCA3 protein in a subset of type II cells indicates both targeting and compensatory responses to the lack of surfactant synthesis in coexisting Abca3 targeted cells, a concept supported by analysis of Abca3 mRNA levels and ABCA3 expression by immunohistochemistry. Mouse Abca3Δ/Δ cells lacking LBs contained structures of intermediate electron density with electron-dense inclusions scattered throughout the cytoplasm of type II cells, similar to findings in patients with ABCA3 mutations (11, 18, 40, 42, 43). Abca3Δ/Δ cells lacking LBs were enriched in atypical lipid vesicles that appear to contain neutral lipids based on their staining characteristics. In contrast, alveolar type II cells wherein deletion of Abca3 did not occur contained heterogeneous populations of LBs, some of which were enlarged. The abnormal accumulation of LBs may indicate the existence of a compensatory pathway that serves to enhance surfactant production. Fasn, Scd1, and Hmgcs1 mRNAs were increased in type II cells from Abca3Δ/Δ mice, wherein deletion of Abca3 did not occur, providing further support for the concept of a compensatory pathway maintaining surfactant synthesis. It is unclear whether the lack of ABCA3 in a subset of cells or compensatory changes in ABCA3-sufficient cells influences cell injury and leads to emphysema in the Abca3Δ/Δ mice. Nonuniform exogenous expression of SFTPB in distal respiratory epithelial type II cells of Sftpb−/− mice, resulting in the presence of both SFTPB-deficient and -sufficient cells, was associated with air space enlargement and alterations in LB structure, phenotype similar to the adult Abca3Δ/Δ mice (33). These data suggest that focal variations of surfactant production and secretion may be responsible for alterations in lung structure and function and that they may be partially corrected by SFTPB- or ABCA3-sufficient cells.
Deletion of Abca3 altered Sat PC levels and phospholipid composition in newborn and adult Abca3Δ/Δ mice. The decreased PC and PG content in newborn Abca3Δ/Δ lungs is consistent with previous reports in Abca3−/− mice (2, 13, 19). PC and PG contribute to surfactant function, and thus their dramatic reduction in newborn Abca3−/− mice is likely directly correlated with respiratory distress and death at birth. Our results and other studies demonstrate that deletion of Abca3 in alveolar type II cells from newborn mice reduces surfactant PC, especially short acyl chain species, which are important for maintaining normal lung function (4, 36). In the surviving Abca3Δ/Δ mice, while PC was significantly reduced in BALF, no differences were observed in the lung tissue (after lavage) and LB fractions, perhaps indicating differences in secretion or recycling. In agreement with the morphological analysis, two metabolically distinct LB populations could be distinguished in the adult Abca3Δ/Δ mouse lung. The methyl-D9-choline incorporation data suggest that one subset of Abca3Δ/Δ mouse lung LBs accumulates and secretes PC at a rate similar to that of control mice. The second subset of LBs contains a more metabolically inert pool of PC that is not secreted. The lower PC content of BALF in adult Abca3Δ/Δ mice would then be a direct consequence of fewer type II cells containing functional LBs. While methyl-D9-choline incorporation data might suggest a defect in surfactant secretion in Abca3Δ/Δ mice, the similarity of fractional incorporation of label into BALF PC in the two groups of animals indicates little change in the kinetics of surfactant PC secretion from functional type II cells in Abca3Δ/Δ mice. PG was markedly reduced in LB and BALF fractions, but not in the lung tissue, indicating that PG transport and packaging in LBs and secretion are altered after deletion of Abca3. Analysis of the minor surfactant phospholipids demonstrated significant increases in PI (lung tissue after lavage and LBs) in Abca3Δ/Δ mice. Similar alterations in PC, PG, and PI were shown in "at-risk" or acute respiratory distress syndrome patients (22). Whether ABCA3 deficiency enhances susceptibility to lung injury is unclear. Adult Stat3Δ/Δ mice, in which levels of Abca3 mRNA and protein and lung Sat PC are reduced, are susceptible to lung injury induced by hyperoxia (31).
Stard2 mRNA was increased in ABCA3-deficient type II cells. The steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain proteins bind lipids (1). The START domain is thought to be a lipid-exchange and/or a lipid sensing domain that mediates lipid transfer between intracellular compartments. PC and PE are known ligands for STARD2 and STARD10. STARD2 may play a role in the maintenance of lipid homeostasis in Abca3Δ/Δ mice. Since START domain proteins frequently contain homeodomains, they may function as lipid-responsive transcription factors or coactivators (41). Kanno et al. (27) showed that STARD2 coactivates transcriptional activity of paired box protein (PAX3) in vitro, providing a potential mechanism by which lipid metabolism is linked to transcriptional programs.
Previous studies established that NKX2.1 (15), FOXA2 (47, 48), C/EBPα (30), and NFATc3 (14) are critical for perinatal lung function, directly regulating surfactant protein, and influencing surfactant lipid synthesis. Comparison of lung mRNAs from CebpaΔ/Δ (30), Cnb1Δ/Δ (14), Foxa2Δ/Δ (47), and Titf1PM/PM mice (15) demonstrated that a number of mRNAs were similarly influenced in each model. Genes involved in the regulation of lung lipid homeostasis were decreased after deletion or mutation of each of these transcription factors. In the present study, Foxa2 and Cebpa mRNAs were markedly reduced in Abca3Δ/Δ type II cells, suggesting their potential role in the downregulation of genes regulating lung lipid homeostasis. Susceptibility of adult CebpaΔ/Δ mice to lung injury induced by hyperoxia was associated with the decrease of genes regulating surfactant lipid homeostasis and surfactant protein biosynthesis, processing, and transport (51). Mechanisms by which ABCA3 deficiency influences transcriptional pathways to modulate lipid homeostasis in type II cells remain unknown.
The present study demonstrates that Abca3 is required not only for respiration at birth, but also for maintaining lung phospholipid homeostasis after birth, influencing lipid biosynthesis and trafficking. Present findings support the presence of a sensing pathway within and between ABCA3-sufficient and -deficient alveolar type II cells that initiates compensation at transcriptional and metabolic levels. Maintenance of respiratory function, despite alterations in lipid synthesis and secretion by a subset of Abca3 gene-deleted type II epithelial cells, indicates compensatory mechanisms by which type II cells maintain surfactant lipid homeostasis in the alveolar epithelium. Alterations of ABCA3 expression may render individuals, newborns, infants, and older individuals more susceptible to acute and chronic lung diseases.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-38859 (J. A. Whitsett).
No conflicts of interest, financial or otherwise, are declared by the authors.
- Copyright © 2010 the American Physiological Society

subset: commoncrawl


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- typical airport hotel, had everything we needed for one night, friendly stuff, compfy bed, clean
Published on: 07 June 2016
Situated in a tranquil residential area and just 15 minutes from Birmingham Airport, Coventry Hill Hotel serves as an ideal location for both the business and leisure travellers.
Guests will find comfortable and well-featured accommodation consisting of 192 en-suite bedrooms with direct dial telephone, colour TV, trouser press and hairdryer. Smoking rooms are available on request.
Enjoy fine dining in a relaxed atmosphere at The Seasons Restaurant, with its a la Carte menu and a Sunday carvery. Guests can also enjoy a drink at the Windmill Bar while watching sporting events on the big screen TV.
For those with a little time to spare, why not explore nearby Stratford Upon Avon, Warwick Castle, Coventry Cathedral or the National Exhibition Centre.
|Star rating
Book on arrival at hotel (24 hour operation)£5 return per adults, £2.50 return per child
|Distance to airport
9 miles/15 minutes
|Check in / check out
Earliest Check In: 14:00
|Parking options
|Packaged with Meteor Meet and Greet parking. Drive to hotel on night of stay (parking at the hotel is complimentary) then drive to the Airport on your day of departure where you will be met by the Meteor driver.
Packaged With Long stay 5 on airport parking. Drive to hotel on night of stay ( parking at the hotel is complimentary) then drive to the Airport following the signs for Long stay 5 car park. Transfers take 5 minutes and are complimentary.
|Room types available
Single, Double, Family Room
|Room Facilities
192 En-Suite Bedrooms
|Hotel Amenities
Seasons Restaurant: Breakfast - 07:00-09:30 (Mon-Fri) 08:00-10:00 (Weekends) Lunch - 12:30-14:00 Dinner 18:00-21:00
|Breakfast Options
Breakfast is NOT included.
Full English £10.50, Continental £8.50
|Disabled Facilities
Level access to reception via ramp.
|Internet Available
Wireless Internet Access in all public areas
|Local Information
Nearest Motorway: M6 12 miles
Need help deciding which Birmingham Airport Hotel is right for you? Call us on 01342 859442 and one of our team will be happy to help you.


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Android 2.2 update now available for Samsung Vibrant via Kies Mini
Update: T-Mobile has posted an official guide here.
If you have a Samsung Vibrant, the wait for Android 2.2 FroYo is now over. We reported yesterday that T-Mobile would be pushing out an over-the-air update, but it's actually available right now via a wired update. All you have to do is download ... Read More »


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62 comments found.
This is awesome. Will be purchasing soon for my own portfolio site.
Another cracking Resume theme – ironically comes at just about the same time I need one so it'll either be this or your other one I buy.
nice work!
Glad you like it
awesome! this is really nice!
Thanks for the nice comments guys
Five stars! love the theme along with the bonuses
Thank you very much! I'm glad you like the item
I was about to buy your other CV template but I think I just changed my mind. Very clean and beautiful design.
You are making the right choice! I've taken all the feedback and request I received on my last template and put them into this one. So from my point of view this is the best bundle you can get at a reasonable price too
This is great work!
As an option, instead of a picture of yourself could you provide some sort of free icon/graphics to replace the photo?
You could try and use http://www.iconfinder.com/ and find a picture that you like or you could just delete the picture and leave just the name and job title.
Is there anyway to provide the download CV link as a Word doc instead of indd?
The first obvious choice would be to reproduce the .indd cv template in Word. I decided to not include a Word version of the cv because not everyone will have it available and it would be pretty hard to create a Word template that would be easy to edit nicely.
A much better alternative to a Word file would be to offer a Pdf version for download – wide spread, smaller file size and people can't edit your cv.
To create a pdf version of the cv you can either export the modified .indd file as a pdf or install a virtual printer like http://www.dopdf.com/ and print the html page (it will print to pdf. If you use firefox make sure to opt out of printing additional info like url, date and time, page title etc for a better looking pdf)
I did think of the PDF route but recruitment agents always seem to want a Word version.
Then just copy the cv part of the page and paste it in a word file.
You might need to fix some spacings but aside from that it gives you a great starter word template on which you could possibly improve by changing some fonts and colors etc.
How I can rate it?
You can rate an item by going to the Downloads section of your profile. There is a star rating next to each item.
The tweet isnt working. I looked in Firebug and I get a 401 Unauthorized. Do I need to do anyhting twitter side?
First of all please don't make such statements as it doesn't work. If you look at the preview of the contact page http://bitpublimedia.ro/themeforest/cv-portfolio/contact.html you will see that it is working and the version included in the preview is the same with the one in the download zip.
In contact.html find this lines:
// display last tweet $(".tweet").tweet({ username: "bitpub", ...
All you have to do to get the tweet script working is to replace the username from mine (which is set to show by default) with yours.
If it still doesn't work for you then most likely the source of the problem could be some of the modifications you have done.
Please contact me through the form on my user page http://themeforest.net/user/bitpub if you still have problems as we might need to exchange files and doing so will provide me with your email address and make communications easier.
Update for anybody following this discussion: The problem was that the buyer had their tweets protected. Needless to say the script couldn't access the tweets hence the 401 Unauthorized error. So to use the tweet script make sure you have your tweets set public!
All this high quality templates for just $10?...and not kidding? Thank you! Now I will be ahead of the competition (a.K.a: candidates)
I'm glad to see that you are happy with the item!
I'd like to think that you would have an advantage over the competition as well – after all this is the most complete package you can buy at the moment addressing both online and offline personal promoting needs.
Good luck on what ever you are trying to achieve with this
Is it possible to have playable video (Youtube or Vimeo) in the portfolio section, rather than image galleries?
Do you want videos to open in the lightbox or do you want to have the videos on the actual portfolio page? Either way, that can be done relatively easily
- just change the lines of code that add a image on the page
  with the embed code for youtube video for example
- you might have to edit the css a little if you want to add a border around the video to resemble the one that is around the images now
If you buy and don't manage just send me an message through the contact form on my user page ( http://themeforest.net/user/bitpub bottom right corner) and I'll be glad to help you make the customization
Yeah, I wanted the video to just pop up in the lightbox. Thanks for your help and quick response. I'll contact you if I can't figure it out.
Is it possible that I don't include stars in the technical skills section?
Yes it is as easy to remove the stars and replace them with text descriptions as deleting a class.
Extensive experience
If you leave the l-6 class there it will show 6 starts but if you delete it, it will show the text "Extensive experience". Quite easy and flexible . The documentation also covers this topic.
Just bought this template. There are so many options with it and it is very easy to customize. Great Job. Thanks for the awesome template!
I'm glad you like it and find it easy to edit!
Great looking template!
I'm having some difficulty in getting the contact form to send out emails to me, and my tweets aren't showing up on the contact.html page.
For the contact form, I sent out a test mail, and received "success!" window. But when I checked the inbox, no email was received.
For the twitter setup: 1) Modified the contact.html for my twitter username, and 2) Modified jquery.tweet.js file for my twitter username
Thank you in advance! Ron
1. If you see a javascript pop-up saying success then something is wrong with the code.
2. Make sure your tweets are not protected. That is the main reason the script won't work.
For both problems, I can't offer you a solution until I get to see the source code. Please send me a message through the contact form on my user page ( http://themeforest.net/user/bitpub ) so I can have your email address and be able to exchange files with you. thank you
I figured out the cause of the bugs. It was an error on my part. I didn't copy the contents of several folders that are responsible for the contact and tweets modules.
Great template. Recommending this to friends!
Very nice template. Do you have RTL version?
I'm not quite sure what a RTL version is. Could you be more specific? Thank you!
RTL style is for anguages written right to left (arabic for example).
The template doesn't come with a RTL version, sorry
I bought this theme and loving it.
I have 2 questions with regards to he Resume section:
1. How can I add spaces (or tabs) before any sentence to align it with something from the previous line? Thing is that, in CV-2, under personal details, the address is 2 lines long and so I want the 2nd line to start from the beginning of the actual address and not start from under the title "Address".
2. How can I have 2nd level bullet-in points? As in dots for the 1st level and maybe indented squares for the 2nd level.
Thanks a lot!
Thank you for the purchase!
These modifications require two changes in the site's css. Could you please contact me using the contact form on the lower right corner of my user page ( http://themeforest.net/user/bitpub ) and i will send you an file with the modifications.
Great work!
The only issue that I've run across is the following error when sending a message via the contact form:
\r\n" ."Reply-To: ".$email."\r\n" ."X-Mailer: PHP/" . phpversion()); if($mail) echo 'OK'; } else echo '
'; } ?>
I did input my email address here:
define("WEBMASTER_EMAIL", 'firstname.lastname@example.com');
Do you have php available? From what I see the php code is not being executed. if you tried to run it locally copy it on a web server and it should be fine.


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Primitive Dogs of the Southeast
When the first primitive humans crossed the Bering land-bridge into North America from Asia, they were accompanied by a primitive form of dog. The animals resulted from the domestication of southwest Asian wolves in the region of Iraq a few thousand years earlier. These small, non-descript dogs moved quickly with their human companions down through the western part of North America. Skeletal remains and mummified bodies of these dogs have been found along with artifacts from primitive Southwest Indians.
From the Southwest, these primitive dogs moved into the eastern United States. Archaeological investigations have indicated their presence in the southeastern forested woodlands. There they were companions of the Indians of the Southeast long before the arrival of the first European explorers of the New World.
Recent studies of free-ranging dogs in parts of South Carolina and Georgia have revealed the continued existence of medium-sized, foxlike primitive dogs. Their appearance, as well as behavior and general ecology, suggest a close ancestry - if not direct descent of type - from the first primitive dogs to enter North America more than 8,000 years ago.
Called the "Carolina Dog," these animals most closely resemble Australia's Dingo, which may indeed be among their closest living relatives. Scientists suggest that the striking resemblance between Carolina Dogs and the Dingo stems from the way in which both animals have filled a free-living, or "pariah," niche, on the fringe of human civilization and culture.


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Heroes of the Heartland from August 9, 2014 with host Master Sargent Eric Martz.
Mike has been a fixture on Fargo radio for almost 20 years. Known by many listeners as "Big Dog", Mike has programmed and hosted morning shows on several different FM stations in Fargo-Moorhead. Now, Mike brings his unique brand of humor and opinions to 970 WDAY each weekday from 9am to 11am! Mike has lived in Fargo-Moorhead his entire life; he attended Fargo South and MSUM.


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One of the best garages.
"Good communication, Fast and good quality work. Good price. Next year, I will choose this garage again."
5* as always
"Used Mendem Motors for over 25 years can't fault "
Outstanding service
"No quibbles about the service provided"
Great service
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Very helpful staff
"I booked this online but unfortunately it didn't get to the garage so when I turned up they was unaware of booking, but they obliged and managed to fit it in."
Excellent Service
"Honest and great professional service as always. Kept informed and advised accordingly."
Speedy service
"Easy, informative mot"
Great service.
"Happy with service and good value, I did ask for a problem with brake squeal to be looked at and this was not sorted out, so dissapointed with that aspect, especially because I specifically had it written on the job sheet (otherwise it would have been 5 stars for quality as well). I will however without hesitation use them again."
Easy to book online
"Only 2 points of criticism I have is; Received an email confirmation and telephone call stating car was booked in for 9am. When I arrived it wasn't booked in till 12, half 12 has told by a mechanic. Secondly, no call was received to say car was ready to pick up. However, would use again and recommend."
Great service
"Really pleased with the efficiency of service and ease of booking/car collection. They kept me informed throughout the day with updates and information."
It's a fact of life that cars, like all machines, need care, attention, and repairs from time to time. Whether it's a major service or an interim service, or you just feel that something's not quite right with your motor, use BookMyGarage to search for the best garage in Okehampton for your needs, and book the work at a fixed price online there and then. No need to wait around for quotes from lots of garages, BookMyGarage gives instant transparent pricing so you can choose and book there and then.
Straight out of the Swedish city of Gothenberg, Volvo has become well-known for pushing out safe, reliable cars, making them one of the vehicles of choice for families around the globe. Initially made all the way back in 1924 to withstand the rigours of Sweden's rough roads and cold temperatures, you could say they've come a long way!
The current Volvo range focusses mainly on traditional vehicle platforms, including hatchbacks, saloons, SUVs and estate cars. The 'XC' models cater to drivers who regularly get their cars a bit muddy and dirty off-road, while the 'Cross Country' versions are available for people going for a more traditional combination of on and off-road ability
About the Volvo XC70
The Volvo XC70 isn't one of the most popular Volvos on the road, although it has the ability to go off-road! Its reviews have been reasonable, but not many drivers rave about it - the reviewers out there say it's not enjoyable to drive, and it isn't as comfy on the road as its predecessor. However, if you are a driver of this particular make of Volvo, you may love its rugged looks and spacious interior. We've come across complaints of power failure of the all-wheel drive, transmission troubles, and low-pressure lights staying on. If those have bothered you too, you've come to the right place to find some help!
Problems with Volvo
Of course it's not just the XC70 that has troubles. If you drive a Volvo S80 you may come across problems with a whining noise during acceleration, or early transmission failure. If you're driving the Volvo 240 you might also be hearing a whining noise, or have problems with the fuse blowing on your power windows. And if you are driving the sporty Volvo V40 you may be dealing with windshield problems or those pesky transmission issues again.
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text:
– where good jobs are as hard to find as Mitt Romney's tax returns? A new report by the Center for Economic and Policy Research find that our economy has lost one-third of its ability to create what are defined as "good jobs." A good job is one that pays at least $37,000 and offers health insurance and a retirement plan. And the data shows that since 1979, Americans workers of all education levels have lost these good jobs. That includes workers with less than a high school degree and workers with more than a college degree.
Why is this happening? Well, the report concludes: "[T]he real culprit is the systematic decline in the bargaining power of workers — reflected in a drop in… the minimum wage, a collapse in the unionization rate in the private-sector, the deregulation of previously well-paying industries, the privatization of state and local government jobs, a series of business-biased trade deals, a dysfunctional immigration system, poor enforcement of already weak labor standards, and high unemployment." In other words, Reaganomics is to blame.


text:
Route Grade Votings
Adjacent Routes
1) 6a+, 2) 6b, 3) 6a+. There are three options for the final pitch - all the same grade. The left line splits offering a fine corner crack, or an alternative line further left. The right-hand split joins the belay of Les Trois Mousquetons.
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text: Self Storage North Carolina Self Storage Shelby Self Storage 28152 Self Storage 28152 Self Storage Search Results Refine Search Size 5x5 5x10 5x15 10x10 10x15 10x20 10x30 Features Climate Controlled Drive-up Access Auto/Boat/RV Neighborhood Need North Carolina storage in another city? Need storage in another North Carolina zip code? View additional North Carolina self storage facilities?


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10 Healthy Habits For Better Arthritis Management
In a perfect world, pain wouldn't exist, our weight would be optimal and we'd enjoy daily exercise and have energy to spare. But as arthritis pain and stiffness invade joints, the desire to exercise and the energy for much of anything can wane.
The world is not perfect, but there are ways to manage weight, minimize pain and improve energy levels. By changing habits, anyone can make small changes that will have a big impact over time.
Not sure where to start? We asked experts to help compile this top 10 list of habits to adopt.
Continue reading 10 Healthy Habits For Better Arthritis Management


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At Gemvara, we believe that the jewelry you wear should be one of a kind, like you. That's why we make each piece to order, one at a time, just the way you want it. Choose aquamarine and sterling silver or garnet and yellow gold: it's your choice. Mix and match gems and precious metals for a look that's exactly you. It's jewelry for all life's occasions, from an anniversary or Mother's Day to your own personal milestones and celebrations. Celebrate your wedding day with brilliant jewelry that captures the joy of this moment forever. Gemvara uses only the highest quality materials to craft your customer favorite jewelry design with meticulous attention to detail. Our precious metals are recycled or responsibly mined. Our wide selection of customer favorite jewelry by Just Like You Designs, Jessica Behzad, Tony Rodrigues, and other talented designers means you can find jewelry that's exactly your style. You can also shop by style, choosing our Mothers, Featured Set, or Classic Collections. Gemvara's fine quality customer favorite jewelry is designed to be passed down for generations to come, crafted by hand in the United States.
We can help right away. Chat with a jewelry expert now!


text:
Tony Flanders was saying
Binoculars are very individual things; one person's experience definitely doesn't apply to another.
THIS ^^^^^^^ to a surprising degree.
Jump to content
Posted 06 August 2014 - 06:59 PM
Tony Flanders was saying
Binoculars are very individual things; one person's experience definitely doesn't apply to another.
THIS ^^^^^^^ to a surprising degree.
Posted 08 August 2014 - 12:09 PM
As I get older I am leaning more toward lower mag binoculars. I think my hands are still steady (as I still play classical guitar decently) but any pulses from heartbeats and body movements no doubt magnify the unpleasant viewing. So this question seems like should I use a tripod or not for 10x or 12X? Then yes, 12X is better if it is mountable on a tripod. Otherwise I think it is not worth the extra power. But if you use the binos regularly and not just for astronomy then I think the 10X is more useful. Just my 2 cents...
Posted 08 August 2014 - 04:33 PM
The smallest IS model that is recommended for astronomy is 10x30 and it usually costs around 500 USD. that is as much as my main telescope (127 mm mak on EQ3) or as much as two of my secondary scopes combined (Shorttube 102 and 90mm mak)
I'm just not sure if I'm willing to spend that amount of money on a 30mm bino, even if it has IS-function and great optics. However if I could get them for 200-300 USD ( or if I would have lived in a region with 300+ clear nights per year), maybe I would have thought differently.
For many of us, objective size doesn't correlate to the instrument's significance in our overall observing experience. If you spend a lot of your observing time with binoculars, it may worth it to spend more so you can have the observing experience you want. Of course, you can always hold out for a good deal.
The 30mm objective doesn't bother me at all, what I meant is that this model is the cheapest but it costs 500 USD. The 8x25 did not get really good reviews so I'm not considering it. My 6x30 finderscope is also sometimes used like a primary instrument, (for variable stars), so I'm sure 10x30 would be great. The problem is that, I'm not a big bino-observer (yet), so I'm not sure if Canon-IS is gonna be worth the money.
Clear skies!
Edited by DJastronomer, 08 August 2014 - 04:52 PM.
Posted 10 August 2014 - 09:18 AM
Yes, lower power is easier to handhold and is less fatiguing.
Garrett pistol grip monopod will enable you to bump up power to 15x63 and views are great!
Cloudy Nights LLC
Cloudy Nights Sponsor: Astronomics


text:
- Juan Burgueñoa,
- José Crossa *b,
- José Miguel Cotesc,
- Felix San Vicented and
- Biswanath Dasd
- a Biometrics and Statistics Unit, Crop Research Informatics Laboratory (CRIL), International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal 6-641, 06600, México D.F
b Biometrics and Statistics Unit, CRIL, CIMMYT
c Departamento de Ciencias Agronómicas, Facultad de Ciencias Agropecuarias, Universidad Nacional de Colombia
d Global Maize Program, CIMMYT
Fixed linear models have been used for describing genotype × environment interaction (GE). Previous attempts have been made to assess the predictive ability of some linear mixed models when GE components are treated as random effects and modeled by the factor analytic (FA) model. This study compares the predictive ability of linear mixed models when the GE is modeled by the FA model with that of simple linear mixed models when the GE is not modeled. A cross-validation scheme is used that randomly deletes some genotypes from sites; the values for these genotypes are then predicted by the different models and correlated with their observed values to assess model accuracy. A total of six multienvironment trials (one potato [Solanum tuberosum L.] trial, three maize [Zea mays L.] trials, and two wheat [Triticum aestivum L.] trials) with GE of varying complexity were used in the evaluation. Results show that for data sets with complex GE, modeling GE using the FA model improved the predictability of the model up to 6%. When GE is not complex, most models (with and without FA) gave high predictability, and models with FA did not seem to lose much predictive ability. Therefore, we concluded that modeling GE with the FA model is a good thing.Please view the pdf by using the Full Text (PDF) link under 'View' to the left.
Copyright © 2011.


text:
Communication and Team Building
Discuss the importance of identifying norms within a
team. Why is clear communication considered an essential element during
team development? Provide an example when you were a team member and the
lack of communication caused significant problems for the team.
Tutor Answer
Brown University
1271 Tutors
California Institute of Technology
2131 Tutors
Carnegie Mellon University
982 Tutors
Columbia University
1256 Tutors
Dartmouth University
2113 Tutors
Emory University
2279 Tutors
Harvard University
599 Tutors
Massachusetts Institute of Technology
2319 Tutors
New York University
1645 Tutors
Notre Dam University
1911 Tutors
Oklahoma University
2122 Tutors
Pennsylvania State University
932 Tutors
Princeton University
1211 Tutors
Stanford University
983 Tutors
University of California
1282 Tutors
Oxford University
123 Tutors
Yale University
2325 Tutors


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Content Marketing can help you reach more customers and generate the leads you need to continue growing your business and reaching your goals. Content marketing is something more than 70% of businesses rely on for their marketing and promotion efforts.
What sets Reliable Writers apart is we have more than 20 years of creating powerful stories, original content, and content marketing strategy experience.
We take the time to understand who YOU are, what YOU do, and WHY you do it. It's not enough to trigger keywords in searches or have the most backlinks. What matters today is original, high quality content that is relevant to your target market, includes keywords that help people looking for what you offer find your website, and a marketing strategy that ties it all together.
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A high-speed chase ended Tuesday in La Plata County with the arrest of three people.
The sequence of events got started at 1:04 p.m. when a deputy with the La Plata County Sheriffs Office attempted to stop a driver in a black van for speeding on Colorado Highway 140, south of Hesperus.
The driver, traveling southbound, tried to avoid contact by hitting speeds in excess of 100 mph, according to a news release Wednesday from the Sheriffs Office.
The deputy stopped the pursuit when it reached 100 mph for safety but continued to follow the vehicle while notifying other area police agencies.
The vans driver drove into the back of a white van in the 14000 block of Highway 140. The deputy stopped to offer assistance to three occupants, but all three declined medical transport.
The motorist in the black van drove away from the scene.
For the next 1½ hours, officers with the Sheriffs Office, Southern Ute Indian Police Department and Southern Ute Rangers searched for the black van.
At 2:45 p.m., someone reported a suspicious vehicle with significant front-end damage on a dirt road beyond the end of County Road 130, west of Kline.
Deputies spotted three people on foot walking away from the van.
Two of them were arrested: Jeffrey Cuthair, 26, of Cortez, and Lakota Rabbit, 24, of Towaoc.
The third person, later identified as Sheldon Begay, 23, of Cortez, fled on foot across an open field.
After a brief standoff, Begay was arrested. Begay admitted to driving the black van during the pursuit and crashing into the white van. An officer with the Southern Ute Police Department was also injured in the melee.
Because all three are Native American and the incident took place on reservation lands, the case was turned over to the Southern Ute Indian Police Department.


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OAKLAND — San Francisco Mayor Gavin Newsom came to Oakland on Tuesday evening to drum up support as he tests the waters for a run in 2010 at the state's top executive position. His critics were not far behind, testing his efforts to talk about how he would handle the state's fate and future as the governor of California.
"I always want to tap into that optimism," he told the crowd of about 200 residents from both sides of the Bay, as well as elected officials, volunteers and community advocates gathered at the Rotunda Building downtown.
Arriving about 30 minutes later than the scheduled 6 p.m. start time, Newsom defended his five-year track record as mayor and laid out a general idea of what voters could expect from the young mayor who has been a lightning rod for conservative critics for his progressive stance on issues such as same-sex marriage. He also has been consistently criticized by sectors of San Francisco's progressive groups, especially about environmental issues in the former industrial shipyards of the Bayview Hunters Point area.
Tuesday was no exception.
"It's a shame we have to come to Oakland to talk to you," one man yelled.
Newsom has been on an early campaign trail for four months, holding such open forums to drum up support and hear community concerns.
Tuesday he championed universal health care, a version of which was implemented in San Francisco in 2007, called the Healthy San Francisco program for uninsured San Francisco residents.
The move, however, requiring small businesses with more than 20 employees to provide health care, has been unpopular among some owners who complain they have had to decrease their staff to avoid the requirement.
Newsom also said he would restore funding for local public transit funding that was cut by Gov. Arnold Schwarzenegger this month to balance the state budget. Public transit is critical to the state, Newsom said.
He promised to champion biotechnology and green technology jobs. This is the growth area for the Bay Area, he said. The next governor has to focus on supporting "green tech jobs" and "the workforce of tomorrow," he said, including a focus on job training for Latino and African-American communities.
"That's the economic development strategy we're promoting in the context of this campaign," Newsom said.
He also defended his record as mayor in protecting undocumented workers, in response to a query from a San Francisco resident who asked if Newsom would expand the city's municipal identification cards for all residents to the rest of the state if he became governor. The San Francisco Board of Supervisors approved the identification card legislation in 2007 for all residents — whether they are in the country legally or not, which was written by San Francisco Supervisor Tom Ammiano.
That Newsom is exploring a run for governor came as a surprise to Jane Goldberg, an Oakland small-business owner. Goldberg, the founder of Plant-It Water, came to hear Newsom address environmental issues. Goldberg said she would support Newsom as governor because he is "newer." Newsom, she said, is not "mired in Washington politics" as much as his potential opponents U.S. Sen. Dianne Feinstein, D-Calif., and state Attorney General Jerry Brown, a former Oakland mayor and governor.
Patricia Lewis arrived early to the forum to hear Newsom. Lewis said she was confident he would do a "pretty good job" as governor because of his general views and the way he seemed to speak his mind about gay rights.
Although she did not agree with his 2004 move to recognize same-sex marriage in San Francisco, Lewis said gay rights was "one of the issues that needed to be addressed."
She also has been following his efforts to tackle a $575 million shortfall facing San Francisco. Newsom recently backed off calling the budgetary chasm a crisis as he looks for ways to cut deeper into the city's $6 billion budget, which he, as the mayor, controls $1.2 billion in discretionary dollars for the city and county. San Francisco is the only city in the state for which the mayor governs the city and the county.
When Newsom took over as mayor in 2004, San Francisco faced a $300 million deficit. As governor he would inherit California's $42 billion deficit.
It is challenging to govern in these difficult economic times, Newsom said.
"But governing the cities of San Francisco and Oakland are probably the most challenging," he said, nodding to Oakland Mayor Ron Dellums.
Reach Angela Woodall at 510-208-6413 or at firstname.lastname@example.com.


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Beyond the fact that Dantoni either refuses or is incapable of adjusting to the players he has, which should be an acknowledged fact by now...
I have a simple test: With the players we had this year, I don't believe Dantoni or his system had any impact on our .500ish record above what any other coach would have. Dantoni as a coach did not make us better. Any coach should have been able to finish about 42-40.
A good coach, who has the adaptability to adjust and play to the strengths and weaknesses of the roster, in my opinion, would have been able to improve on our record.
Obviously, this can not be objectively proved. But ask yourselves two questions: 1) With the players we had this year, shouldn't and couldn't virtually any NBA-caliber head coach achieved a 42-40 record? 2) If so, why not hire one that has the flexibility to adjust to the roster, and theoretically maximize the players' impact?
Dantoni is inflexible in his system and philosophy, his rigid style does not maximize the talent of the roster, and I argue that almost any other NBA-caliber coach could do better.
Any other coach would have been fired a while ago but he had his excuses to get him by and frankly I feel that he is still the Knicks coach because Donnie Walsh didn't want to fire him before his contract ended which would have made him look bad which would have him not come back for the extension. So you have to look at it many ways to realize that Mike D has gotten a pass and it's really not fair, especially when you are in NY.
He has one more shot.
He has a training camp and off season with Amare, Anthony, and Billups...I expect more wins but I think the same result will happen.
"The Knicks are back" - Amare
Fellas this is the true Test:
Could MOA take Boston or Chicago and win as many games? Highly unlikely.
Could Thibs or Rivers do better than MOA did with the Knicks? MOST DEFINITELY.
The Celtics started out like 20-3 in their chip season.
I won't even get into the details about their defense masking any growing pains because like that saying goes "If I score I might win, but if they don't score I can't lose" I'll leave that alone, and I'll just say this:
Your logic = Doc Rivers overrated because KG, Pierce, and Allen were the reason for that team's success.
So by that definition what does that say about Mike?
Guess if I was simple in the mind...everything would be fine.
Regardless of what you might think about Rivers, he clearly outcoached Mike D'Antoni in the series.
Either way it doesn't bode well for D'Antoni. If Rivers is actually a good coach, then D'Antoni isn't good enough to compete in the East against better coaches, which I think we've seen by now. If Rivers is actually a terrible coach, that means we got burned by a bad coach, which means D'Antoni sucks ass...
I'll also add this.
Doc River may be overrated to some, that's a subjective opinion, but his philosophy seems to be spot on. It mirrors that of several other championship teams that we've seen in the past, and it's produced a championship. Of course all of this occurred after his team acquired the players needed to win....but it's also got him to the Finals twice in 3 years.
MDA had "his players" in Phoenix and it produced a "maybe we could have advanced to the Finals if it weren't for the refs".....Avery Johnson has been to the Finals more times than MDA and he's the coach of the NJ Nets. Some people can't understand the fascination with Doc Rivers?????? I can't understand what the **** MDA has done. period.
Guess if I was simple in the mind...everything would be fine.
I dont know how good Doc Rivers is. But he is demanding of his players and he holds them accountable. The players love Doc Rivers. They would run through the wall for him.
I think players like Dantoni, but they dont love him. You know what I'm saying. Players love Phil Jackson, players love Doug Collins. Not Dantoni. He doesnt inspire his players.
Now Doc and Phil Jackson have had players that held themselves and their teammates accountable.
Melo and STAT dont do that. We need a coach with a resume and when he walks into a room he demands respect.
That doesnt happen for Dantoni.
The comparison makes no sense tho..
We got Melo and Amare together w thirty or so games left in the season. No training camp, no time to get used to playing together, just some real, live NBA comp to get out there and make it work against.
The Celts and Doc Rivers got there big three in the offseason. They had time to get everybody on the same page in training camp and preseason, and their collective talent level and chemistry were just off the charts which sorta surprised the league IMO.
Our team needed some time to adjust to losing 4 players. The also needed time together to get used to Melo's rhythm. Billups needed time to get to know where everybody likes the ball, how coach wanted him to play etc.. Just a horrible compare/contrast if your trying to make an argument for one style of coaching over another because you're excluding other pertinent factors.
Last edited by ronoranina; Apr 29, 2011 at 00:00.