The deposition of unconjugated bilirubin (UCB) in selected regions of the brain results in irreversible neuronal damage, or Bilirubin Encephalopathy (BE). Although UCB impairs a large number of cellular functions in other tissues, the basic mechanisms of neurotoxicity have not yet been fully clarified. While cells can accumulate UCB by passive diffusion, cell protection may involve multiple mechanisms including the extrusion of the pigment as well as pro-survival homeostatic responses that are still unknown.
Transcriptome changes induced by UCB exposure in SH-SY5Y neuroblastoma cell line were examined by high density oligonucleotide microarrays. Two-hundred and thirty genes were induced after 24 hours. A Gene Ontology (GO) analysis showed that at least 50 genes were directly involved in the endoplasmic reticulum (ER) stress response. Validation of selected ER stress genes is shown by quantitative RT-PCR. Analysis of XBP1 splicing and DDIT3/CHOP subcellular localization is presented.
These results show for the first time that UCB exposure induces ER stress response as major intracellular homeostasis in surviving neuroblastoma cells in vitro.
The toxic effects of bilirubin, a bile pigment produced during the catabolism of heme-containing compounds , have been documented in various biological systems. Nearly all newborn infants develop increased levels of unconjugated bilirubin (UCB) in the blood stream, clinically evident as neonatal jaundice . Although this is a benign and transient phenomenon, abnormal accumulation of bilirubin may cause bilirubin encephalopathy ranging from minimal neurological injury to severe and permanent condition leading to neurodevelopmental dysfunctions . Furthermore, infants affected by the genetic pathology of Crigler-Najjar I present high UCB plasma levels and are particularly exposed to bilirubin encephalopathy (BE) .
The basic mechanisms of hyperbilirubinemia neurotoxicity have not been fully clarified, although it has been shown that UCB may impair a large number of cellular functions, including energy metabolism , cell proliferation , DNA and protein synthesis , receptor functionality , and neurotransmitter uptake and release . Furthermore, in the brain UCB seems more injurious to neurons than to glial cells, with effects that depend on the cellular subtype .
The endoplasmic reticulum (ER) plays a crucial role in the synthesis and folding of newly secretory and membrane proteins . Any perturbation that compromises protein folding and functionality of the ER is referred to as ER stress. The ER stress response can promote cellular repair and survival by reducing the load of unfolded proteins through global attenuation of protein synthesis and by upregulating chaperones . This response is collectively termed as the unfolded protein response (UPR) and is mediated by three ER transmembrane receptors: pancreatic ER kinase PERK (also known as eukaryotic translation initiation factor 2α (eIF2α) kinase 3 (EIF2AK3)), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6).
In resting cells, all of these ER stress receptors are maintained in an active state through their association with the ER chaperone GRP78 (also called BiP). An accumulation of unfolded proteins causes dissociation of GRP78 from PERK, IRE1 and ATF6, thereby initiating the UPR. Thus UPR is a pro-survival response to reduce the accumulation of unfolded proteins and restore normal function . However, when misfolded-protein aggregation persists and ER stress cannot be resolved, signalling switches from a pro-survival to a pro-apoptotic pathway.
Recently, the ER has attracted attention as a key subcellular compartment in which the effects of several cellular stresses may contribute to pathological processes culminating in neuronal injury and degeneration. A central role of ER dysfunction is evident in various pathologies of the brain, including acute injuries (transient ischemia, trauma) and neurodegenerative disorders (Alzheimer's, Parkinson's and Huntington's diseases) .
The present study was designed to analyze the consequences of bilirubin exposure to better understand the molecular processes underlying neuronal damage and homeostasis. We used high density oligonucleotide microarrays to analyze the gene expression profile of human neuroblastoma SH-SY5Y cells upon UCB treatment. Gene expression data and experimental validation point to ER stress as the major intracellular homeostatic response in surviving cells.
The human neuroblastoma SH-SY5Y cell line is a well-established in vitro cellular model to study the biological consequences of neuroactive molecules, drugs and toxins. To assess UCB effects, we have taken advantage of previous studies where the outcome of clinically relevant UCB concentrations was tested and the cytotoxic effects revealed [15, 16]. The sensitivity of SH-SY5Y cells to UCB treatment was thus evaluated by measuring cell viability after exposing cells to a concentration of free bilirubin of 140 nM (Bf 140 nM) for 1, 4 and 24 h. As shown in Figure 1, cell viability was reduced to 60% already after 1 h of UCB treatment and the extent of reduction in cell viability never exceeded the 40% of the cell population, even with longer treatments.
Cell viability in SH-SY5Y cells treated with UCB. SHSY5Y were exposed to UCB treatment at Bf 140 nM during a time course of 1, 4 and 24 hours and cell viability was evaluated by MTT assay. Cell viability of UCB-treated samples was expressed as a percentage of the respective samples treated with DMSO 0.6%. Data are expressed as the mean ± SD of 3 independent experiments performed in triplicate.
To examine the molecular events associated with the homeostatic response to UCB treatment, high density oligonucleotide microarrays (Affymetrix GeneChip Human U133A 2.0) were used to interrogate the expression of over 14,500 transcripts. RNA was isolated from SH-SY5Y neuroblastoma cells treated with Bf 140 nM for 1, 4 and 24 h. Experiments were performed in three biological replicates with control samples monitoring the effects of DMSO used as Bf solvent.
Analysis of the microarray hybridization data, as reported in Methods, produced a list of 258 probe sets with a false discovery rate (FDR) of 10%. This list corresponded to 230 genes upregulated after 24 h of UCB treatment ("late response"). No down-regulated genes were detected. The fold change of transcript levels ranged from 1.2 to 6.9, the majority of which being toward the lower end. A list of all differentially expressed genes is provided in the Additional file 1. No significant changes of gene expression were observed after 1 and 4 h of UCB treatment ("early response"). We obtained a list of 58 probe sets when a FDR of 0% was applied and a list of 196 probe sets with a FDR of 5%. Furthermore, 400 probe sets were induced with a FDR of 20% with no down-regulated genes (data not shown).
Differentially expressed genes were then examined for their Gene Ontology (GO) annotations concerning their cellular localization. As shown in Table 1 and as detailed in the Additional file 2, the analysis revealed that these genes were mainly distributed in the endoplasmic reticulum (ER), the Golgi apparatus (Golgi) and in the nucleus. To obtain insights into the pathways in which they were mainly involved, genes were sorted into distinct functional subgroups. UCB treatment induced genes belonging to functional categories related to the UPR in the protein secretory pathway (Table 2) as well as to regulation of transcription and DNA-dependent processes (Table 3). A set of up-regulated genes included plasma membrane transporters of the family of the solute carrier transporters (SLCs) and other genes involved in amino acid metabolism (Table 4). In the remaining part of the manuscript the attention will be focused on the UPR and ER stress responses, since the majority of up-regulated genes are well known members of these homeostatic pathways.
Identification of the cellular localization of differentially expressed genes in response to 24 h of UCB treatment at Bf 140 nM in SH-SY5Y cells.
The table shows the number and percentage (%) of the 217 total genes that were recognized as valid gene names in the gene_association.goa_human_hgnc file according to GO slim terms from the cellular ontology in regard to Endoplasmic reticulum, Golgi apparatus and nucleus localization. All details are reported in Table S2 in the Additional file 2.
Differentially expressed genes involved in ER stress signalling and UPR in response to UCB treatment in SH-SY5Y cells.
Genes are sub-categorized according to their GO Biological Process annotation. Fold changes (FC) are fold differences for cells treated with Bf 140 nM versus cells grown in DMSO 0.6% (controls) for 24 h. Affymetrix Probe Set-IDs, gene names and symbol, fold change (FC), and cellular location are indicated.
Differential expression of genes mapping to "regulation of transcription, DNA-dependent" (GO Biological Process) in response to UCB treatment in SH-SY5Y cells.
Fold changes (FC) are fold differences for cells treated with Bf 140 nM versus cells grown in DMSO 0.6% (controls) for 24 h. Affymetrix Probe Set-IDs, gene names and symbol, fold change (FC), and cellular location are indicated.
Differential expression of genes involved in protein synthesis/translation, amino acid metabolism/transport and autophagy in response to UCB treatment in SH-SY5Y cells.
Genes are sub-categorized according to their GO Biological Process annotation. Fold changes (FC) are fold differences for cells treated with Bf 140 nM versus cells grown in DMSO 0.6% (controls) for 24 h. Affymetrix Probe Set-IDs, gene names and symbol, fold change (FC), cellular location are indicated.
UPR serves many aspects of the secretory pathway and is predominantly involved in the response to stress conditions when proteins are misfolded. GO categories of UPR components induced by UCB are shown in Table 2. These include stress sensing and signalling, protein folding, translocation, vesicular transport and ER-associated degradation. Genes known or predicted by sequence homology to play a physiological role in secretion or in the biogenesis of organelles were also increased.
The largest group of up-regulated genes was represented by ER-resident molecular chaperone and oxido-reductase proteins. Among the molecular chaperones, we found established UPR target genes such as HSPA5 (heat shock 70 kDa protein 5) (upregulated 2.3 times), known also as GRP78 (glucose-regulated protein, 78 kDa) or BiP , HSP90B (heat shock protein 90 kDa beta (Grp94) (1.5), member 1), DNAJB9 (4.4) and DNAJC10 (1.8). Additional genes included PDIA5 (1.7), PDIA6 (1.5) and ERO1LB (2.1) that are involved in oxidative protein folding .
A second major group was composed by genes involved in co-translational translocation of proteins across the ER membrane, to the Golgi and in the anterograde/retrograde transport back into the cytosol. The group included the signal sequence receptor SSR1 (1.6), the translocating chain-associating membrane protein TRAM1 (1.8), members of the SEC61 complex, such as SEC61A1 (1.6), SEC61A2 (1.4) and SEC63 (1.5), and SERP1 (Stress-associated Endoplasmic Reticulum Protein 1) (1.9), also known as RAMP4 (Ribosome-Associated Membrane Protein 4), that is a Sec61-associated polypeptide specifically induced by ER stress . Up-regulated genes were also represented by the coated vesicle membrane proteins, such as SEC23B (1.8), SEC24D (2.1), SEC31A (1.6) and SCFD1 (1.5), as well as by the transmembrane trafficking proteins TMED2 (1.4), TMED9 (1.6) and TMED10 (1.4).
The ER-associated degradation (ERAD) system eliminates misfolded proteins by their retrotranslocation across the ER membrane into the cytosol, where ubiquitin-conjugating enzymes target them for proteosomal degradation. ERAD requires a number of dedicated ER-resident factors. Indeed, HERPUD1 or HERP (Homocysteine-induced ER Protein) (4.1)  was significantly induced as well as EDEM1 (ER Degradation Enhancer Mannosidase alpha-like 1) (2.1) , SEL1L (2.1), a transmembrane cofactor of the E3 ubiquitin ligase HRD1 , and DERL2 (Derlin membrane-like domain family member 2) (1.6) whose product associates with EDEM1 and is a specific target of the IRE1 branch of the UPR response .
As shown in Table 3, several differentially expressed genes were classified into the GO category of "regulation of transcription, DNA-dependent". Up-regulated transcription factors included two established UPR target genes: DDIT3 (6.7), also known as CHOP or C/EBP homologous protein, and XBP1, X box-binding protein 1 (2.6). CHOP is a 29-kDa leucine zipper transcription factor that is ubiquitously expressed at a low level and robustly up-regulated in response to various stress conditions . CHOP is proapoptotic and a key mediator of ER stress-induced cell death [26, 27]. XBP1 gene, after a nonconventional splicing event , codes for a transcription activator that regulates a subset of ER-resident chaperones that are essential for protein folding, maturation and degradation in the ER . Additional stress-responsive transcriptional regulators induced by UCB included ATF3 (4.9), ATF4 (1.7), ATF5 (1.6), CEBPB (4.9), CEBPG (2.4), JUN (3.5) and JUND (1.6). Interestingly, although ATF4 showed a moderate induction, several of its downstream targets were strongly up-regulated, e.g. DDIT3, ATF3, CEBPB, CEBPG, CREB3L2 (1.7) and TRIB3 (6.7). TRIB3 (also known as NIPK, SKIP3, TRB3 and SINK) is an ER stress-inducible gene that is involved in CHOP-dependent cell death as a second messenger during ER stress .
The translational machinery for a new boost of protein synthesis has been suggested to be amplified during UPR-associated translational attenuation [13, 31]. As shown in Table 4, we observed an up-regulation of 6 tRNA synthetase genes, such as AARS (1.7), CARS (1.8), GARS (1.8), MARS (1.5), SARS (2.2) and WARS (2.9), which are involved in tRNA processing. We also observed the induction of 2 eukaryotic translation initiation factors, that are EIF1 (1.4) and EIF5 (1.6). One of the stronger up-regulated genes was FAM129A (family with sequence similarity 129, member A) (6.9), also known as NIBAN, which has been recently demonstrated to be involved in the ER stress response and to positively affect the protein translation machinery by regulating the mTOR pathway . Interestingly, the list of genes induced during UCB treatment also includes DDIT4 (DNA-Damage-Inducible Transcript 4) (4.4), also known as REDD1/RTP801/Dig2, that is another well-known regulator of the mTOR pathway . The mTOR-dependent pathway is strongly connected to the autophagic machinery, and indeed autophagy-related genes such as GABARAPL1 (GABA(A) receptor-associated protein like 1 or ATG8) (1.9), GABARAPL3 (GABA(A) receptors-associated protein like 3 (pseudogene)) (2.3) and WIPI1 (WD repeat domain, phosphoinositide interacting 1 gene or Atg18) (2.8) were also positively differentially expressed, suggesting that surviving neuroblastoma cells may present an induction of autophagy [34, 35].
UCB exposure also activated genes involved in amino acid metabolism (Table 4). Among the strongest up-regulated genes, SLC7A11 (6.8) and SLC3A2 (3.8) were identified. These two genes encode respectively for xCT, the light chain subunit, and for 4F2hC, the glycosylated heavy-chain subunit, that are components of the system xCT, anionic amino acid transport system specific for cystine and glutamate . Recently, the UCB-induced SLC7A5 (3.2) has been shown to physically and functionally interact with SLC3A2, forming a bidirectional amino acid transporter and representing a key regulator of both the mTOR pathway and autophagy .
For validation, we selected genes considered of interest based on their involvement in ER stress response. Gene expression levels of DDIT3, HSPA5, XBP1, HERPUD1, TRIB3, FAM129A and SLC7A11 were verified by qRT-PCR. Results are reported in Figure 2. The fold differences determined by qRT-PCR were very similar to those obtained by the high density oligonucleotide microarray gene expression analysis.
Analysis of gene expression related to the ER stress response. Gene expression levels of selected genes (X axis) were analyzed by qRT-PCR. Y axis represents the gene expression level normalized to housekeeping genes (GAPDH and β-actin) of SH-SY5Y cells not treated (untreated) or treated with vehicle (DMSO 0.6%) or UCB (Bf 140 nM) for 24 hours. These results represent at least three RNA samples per experimental condition run in triplicate.
We investigated two independent events that are known activators of the ER stress transcriptional program: unconventional splicing of XBP1 and cytoplasm/nuclear shuttling of DDIT3/CHOP. The splicing of XBP1 transcript changes its open reading frame to code for a transcription factor (spliced XBP1 or sXBP1) that activates the expression of a subset of ER stress/UPR related genes [28, 29]. Here we show that UCB treatment is indeed able to trigger this unconventional splicing (Figure 3A). The nuclear localization of the transcription factor DDIT3/CHOP  is one of the most commonly used indicators of ER stress. As presented in Figure 3B, we observed a diffuse cytoplasmic localization in untreated and DMSO 0.6% control cells, but a major nuclear staining in cells treated with Bf 140 nM. This indicates activation of a CHOP-dependent transcriptional program. These data represent an independent proof of the induction of a vigorous ER stress.
Analysis of XBP1 splicing and DDIT3/CHOP subcellular localization in SH-SY5Y cells treated with UCB. (A) mRNA levels of the spliced form of XBP1 (s - spliced, u - unspliced), indicator of ER stress, were examined by RT-PCR after 24 h of treatment. The amplified products were separated on a 2% agarose gel and visualized with ethidium bromide staining. Data are representative of n ≥ 2 separate experiments per condition point. (B) The expression of CHOP (shown in red) was examined with immunocytochemistry as described in Methods. Representative images are shown for SH-SY5Y cells not treated (untreated) or treated with vehicle (DMSO 0.6%) or UCB (Bf 140 nM). The nuclei (shown in blue) were visualized with DAPI staining. Bar = 10 μm.
Bilirubin encephalopathy is one of the consequences of severe hyperbilirubinemia and is characterized by multifocal deposition of UCB in selected regions of the brain, resulting in temporary or permanent impairment of auditory, motor or mental functions . Cellular UCB level is closely related to the free concentration (Bf) of the pigment in plasma and to the mechanisms regulating the traffic of UCB among plasma, cerebral spinal fluid and cells. Although UCB is formed in virtually all cells and may enter by passive diffusion , intracellular concentration is determined by cellular export and metabolic transformation of the pigment. Interestingly, only certain neuronal cell types present a UCB susceptibility to undergo necrosis and apoptosis , whereas astrocytes are relatively spared. This has been linked to a higher content of MRP1, a transporter with high affinity to UCB  able to keep the intracellular UCB concentration low. Importantly, the level of MRP1 expression in human neuroblastoma SH-SY5Y cells is inversely and linearly correlated with UCB toxic effects .
The molecular mechanisms of intracellular UCB toxicity are still unclear . UCB has been initially proved to be a pro-apoptotic agent, suppressing cell growth by inducing DNA fragmentation, mitochondrial release of cytochrome c, activation of caspase-3 and cleavage of poly(ADP)ribose polymerase . More recently, oxidative stress has emerged as a potential crucial event, since its generation mirrors UCB-mediated apoptosis [43, 44]. In various cellular systems, UCB causes reactive oxygen species (ROS) production, protein oxidation, lipid peroxidation and disruption of glutathione metabolism [43, 45–47]. Furthermore, UCB-mediated oxidative stress is also in part responsible for inhibition of cell growth [48, 49]. Inhibition of cell proliferation has been indeed observed in primary vascular smooth muscle cells in vitro as well as inferred by gene expression data on the repression of cell cycle-related genes in the liver of a mouse model of Crigler-Najjar type I disease . To study the effects of intracellular UCB on neurons, we took advantage of the human neuroblastoma SH-SY5Y cell line. These cells have been widely used in neurobiology as the in vitro system of choice to dissect molecular pathways leading to neurodegeneration. Previous studies have already shown that neuroblastoma cells are a good in vitro model to address the consequences of clinically relevant UCB concentrations . Although we are aware of the limitations in the use of a neuroblastoma cell line to recapitulate dysfunctions that occur in neonatal human brain, this experimental model allowed us to dissect in details the molecular events elicited by UCB.
Treatment with Bf 140 nM was highly toxic, triggering death in 40% of the cells within one hour. This is expected since the accepted threshold for bilirubin toxicity approximately occurs at 70 nM . 63% reduction in cell viability at 4 h after bilirubin exposure were previously demonstrated in the rat neuroblastoma N-115 cell line . Furthermore, Silva RF et al.  showed 85 nM Bf triggered deleterious effects on mitochondrial function.
A full understanding of molecular mechanisms of cell death will require additional investigation. UCB has been previously proved to induce apoptosis in neuroblastoma cells, although necrosis could not be excluded .
Furthermore, the criteria to distinguish sensitive from insensitive neuroblastoma cells remain unclear. One may speculate that subtle heterogeneity in the level of differentiation may play a role in susceptibility to insults. Alternatively, cell death induction may depend on cell cycle position.
We then focused on the 60% of surviving cells to test the hypothesis that these neurons may express a homeostatic response.
A gene expression approach was carried out to identify gene expression patterns and signalling pathways induced upon UCB treatment. No significant changes of gene expression were observed after 1 and 4 h of UCB treatment. After 24 h, 230 genes were induced while no down-regulated genes were observed even when an FDR of 20% was applied.
The lack of an "early response" is surprising since it fails to provide a potential mechanism for cell survival within the first hour. Therefore, present data suggest that it is very unlikely that the molecular pathways induced at 24 hours play a role in the survival of cells after one hour of treatment.
GO analysis of the "late response" genes proved for the first time that UCB treatment induces an ER stress response in neuroblastoma cells in vitro. Evidences for ER stress involvement are overwhelming. Among others, genes induced by UCB treatment include several molecular chaperones like BiP, a well-known marker of ER overload, as well as molecular components of the ERAD system, that has been recently positioned in a central stage among the molecular mechanisms of neurodegeneration [53, 54]. Furthermore, two well-known events in ER stress, such as XBP1 unconventional splicing and DDIT3/CHOP nuclear relocation, were proved to be triggered by UCB treatment, therefore providing additional experimental evidences.
UCB also altered several components of the translational machinery including tRNA synthesis genes and translation initiation factors. The translation machinery is considered one of the major targets of the ER stress response to regulate protein synthesis and decrease protein overload. Since no data are available in literature about the relationship between UCB intracellular concentration and translation rate, there is a compelling need for an accurate proteomic analysis of UCB effects in SH-SY5Y neuroblastoma cells. A complementary cellular strategy in ER stress conditions includes an increase in protein degradation. In this context some UCB-induced genes suggested a role for autophagy. It is of notice that autophagy has been recently associated with neurodegenerative diseases as well as with inhibition of cell growth and entering into a quiescent state [55, 56]. This may correlate to previous reports showing a severe inhibition of cell proliferation mediated by UCB . Further experimental validation is needed to prove an increase of autophagy.
Finally, the induction of both subunits of the transport system X(C)(-) was also observed: SLC3A2 (4F2hc) and SLC7A11 (xCT) are able to heterodimerize to mediate cystine-glutamate exchange and regulate intracellular glutathione levels. Since the maintenance of a high glutathione concentration may be protective , we speculate that induction of the X(C)(-) transport system may represent a major pro-survival response, as has been recently shown in neurons . Interestingly, we could not observe any significant differential gene expression of MRP1 at any point of the time course suggesting that this transporter plays a negligible role in UCB-induced adaptation in this in vitro model.
In summary, the present study shows for the first time that intracellular accumulation of UCB in surviving neuroblastoma SH-SY5Y cell line provokes ER stress.
UCB was purchased from Sigma (St. Louis, MO, USA) and purified as described by McDonagh and Assisi . UCB was dissolved in 0.6% v/v of DMSO and diluted in 100 μL of complete medium supplemented with 15% Fetal Bovine Serum containing 54 μM albumin. Bf concentration was 140 nM, measured as described previously . 3(4,5-dimethylthiazolyl-2)-2,5 diphenyl tetrazolium (MTT), HPLC-grade dimethyl sulfoxide (DMSO), and all other chemicals were purchased from Sigma, unless otherwise specified.
SH-SY5Y human neuroblastoma cells (ATCC CRL-2266) were cultured in a humidified incubator at 37°C and 5% CO2 in F12-EMEM (1:1) containing 15% foetal bovine serum (FBS), 2 mM L-glutamine, 25 μg/mL gentamicin, 100 U/ml penicillin, and 100 μg/mL streptomycin at 37°C with 5% CO2. Cells were seeded at 6 × 106 cells in 75 cm2 flasks and grown for 24 h (cells reached 70-80% confluence). The medium was then replaced with experimental medium, which was either free medium (untreated samples) or 140 nM Bf medium (Bf 140 nM samples), or 0.6% DMSO medium (DMSO 0.6% samples) and cells were allowed to grow. After 1, 4 and 24 h cells were collected and processed for cell viability (MTT cell viability assay), microarray, quantitative RT-PCR and immunofluorescence analyses. Separate flasks of cells were used for each of the treatments and assays. Experiments were performed at least in three independent biological replicates at all time points.
Cells were treated as described above and the MTT cell viability assay was performed as previously described by Denizot and Lang . Absorbance was measured at 562 nm using a microtiter plate reader (LC 400, Beckman Coulter, Milan, Italy). Cell viability of UCB treated samples was expressed as a percentage of the respective samples treated with DMSO alone.
Total RNA was isolated using the TRIzol reagent (Invitrogen) following the manufacturer's instructions. Single strand cDNA was obtained from 1 μg of purified RNA using the iSCRIPT™ cDNA Synthesis Kit (Bio-Rad) according to manufacturer's instructions. Quantitative RT-PCR was performed using SYBR-Green PCR Master Mix (Applied Biosystem) and an iCycler IQ Real Time PCR System (Bio-Rad). Sequence of gene specific primers used for qRT-PCR is provided in Table 5. Expression of the gene of interest was normalized to house keeping genes (glyceraldehyde-3-phosphate dehydrogenase or GAPDH and β-actin) and the initial amount of the template of each sample was determined as relative expression versus one of the samples chosen as reference. The relative expression of each sample was calculated by the formula 2 exp-ΔΔCt (User Bulletin 2 of the ABI Prism 7700 Sequence Detection System). GAPDH and β-actin expressions are not modified under the present experimental conditions (data not shown). XBP1 splicing was studied using reverse transcriptase-polymerase chain reaction. cDNA was synthesized as herein reported. Human XBP1 primers (forward: 5'-AAACAGAGTAGCAGCTCAGACTGC-3'; reverse: 5'-TCCTTCTGGGTAGACCTCTGGG AG-3') were designed to generate cDNA products encompassing the IRE1 cleavage site as previously described . The unspliced (uXBP1) and spliced (sXBP1) mRNAs generate 473- and 447-bp cDNA products, respectively. The amplified products were separated on a 2% agarose gel and visualized with ethidium bromide staining.
Total RNA was extracted as described above and purified using the RNeasy mini kit (Qiagen). The quality of total RNA was assessed using a bioanalyzer (Agilent 2100; Agilent Technologies) and RNA was quantified by using a ND-1000 Nanodrop spectrophotometer. Ten μg of each total RNA sample was labelled according to the standard one-cycle amplification and labelling protocol developed by Affymetrix (Santa Clara, CA). Labelled cRNA was hybridized on Affymetrix GeneChip Human U133A 2.0 Arrays containing over 14,500 transcripts. Hybridized GeneChips were stained, washed (GeneChip Fluidic Station 450) and scanned (GeneChip Scanner 3000 7G). Cell intensity values and probe detection calls were computed from the raw array data using the Affymetrix GeneChip Operating Software (GCOS). Further data processing was performed in the R computing environment http://www.r-project.org/, version R 2.5.0 for Windows) using packages from the BioConductor software project http://www.bioconductor.org/. Variance-stabilizing normalization was applied , using the "justvsn" function from the "vsn" library. Normalized data were then filtered based on the Affymetrix detection call, so that only probes that had a Present call in at least one of the arrays were retained . Data were then imported in the MultiExperiment Viewer (MeV) software  (version 4.0.01 for Windows XP), and statistical analysis was performed with the SAM (Significance Analysis of Microarrays) module [65, 66]. A False Discovery Rate (FDR) of about 10% (i.e. 9.317%) was applied to detect significantly differentially expressed genes. Microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) with Accession Number GSE16768. Differentially expressed genes were then specifically examined, based on their Gene Ontology annotation , for their cellular localization and for their involvement in some biological processes of interest. GO slim annotations were based on the Generic GO Term Mapper http://go.princeton.edu/cgi-bin/GOTermMapper using the Gene Association File goa_human_hgnc (Generic GO slim).
For immunofluorescence experiments, SH-SY5Y cells were cultured on glass slides overnight, fixed in 4% paraformaldehyde for 10 minutes, washed with PBS two times, treated with 0.1 M glycine for 5 minutes in PBS and permeabilized with 0.1% Triton X-100 in PBS for additional 5 minutes. After washing with PBS and blocking with 0.2% BSA in PBS, cells were incubated with the anti-CHOP 1:100 (Santa Cruz Biotechnology) diluted in 0.2% BSA in PBS for 90 minutes at room temperature. After washing, cells were incubated with AlexaFluor 594 (Dako Cytomation)-labelled anti-mouse secondary antibody for 1 h. For nuclear staining, cells were incubated with 1 μg/mL DAPI for 5 minutes. Cells were washed and mounted with Vectashield mounting medium (Vector). Images were collected using a confocal microscope (LEICA TCS SP2).
We thank Dr S Vilotti for kindly providing the immunofluorescence images and Dr M Biagioli for her invaluable qRT-PCR guidance. We would also like to thank Prof. G Tell for critically reading the manuscript and all members of Prof. G Tell, CT and SG laboratories for discussions. This work was supported by the Telethon Grant GGP05062, by the Regional Grant DL26 (Regione FVG, Italy) and by The Giovanni Armenise-Harvard Foundation career development award to SG.
RC performed data analysis, assisted with the generation of microarray data, validated targets by qRT-PCR, wrote and revised the manuscript. CB and PG performed the cell culture and the MTT assays. PR performed the bioinformatic and statistical analysis of the microarray experiments. RF performed some qRT-PCR experiments. HK produced the microarray data at the CBM Genomics unit (Trieste, Italy). SG conceived, designed and wrote the paper. CT and SG provided support, direction and oversight of the experiments and revised the final manuscript. All authors read and approved the final version of the manuscript.


She was born a creative soul but was also born in a place that left her feeling on the outskirts of the norm. Knowing she needed more she packed her bags and headed towards London. That's where we pick up the story as Caitlyn shares about the move, happiness and her attachment to red.
Kendra: Were there any reservations about leaving home at 17 to live in London?
Caitlyn Scarlett: Yeah I think the normal amount of fear was there for a teenager leaving the nest, I was essentially starting life from scratch and I heavily relied on my friends at the time. But I also knew the nerves would pass and I was meant to be in the city. Most of the doubt actually came from other people who worried I might not be ready for that level of independence, a lot of them had words with my mother encouraging her to make me wait but she was always very supportive and knew I'd handle myself.
Kendra: Was the move entirely music based?
Caitlyn Scarlett: Primarily yes, but I also craved a more exciting lifestyle than the type you'd find in my hometown. Plus I never felt like I fit in there, I had a few good friends but I was not popular by any means. I knew I'd find my kind of people in London, creative, LGBTQ, outgoing, ambitious etc.
Kendra: Thinking back, do you remember what you were doing leading up to the moment "Happy When" came to mind? Did those events shape the lyrics?
Caitlyn Scarlett: My state of mind in the years before and my journey through changing that were at the forefront of my mind when writing it. I've felt the sentiments in the lyrics personally in times of lower self-esteem and really grown past that. The song's purpose is to subtly point out the destructive nature of that mindset and the Hollywood image of romance.
Kendra: How'd you come to work with Miko on the remix?
Caitlyn Scarlett: He just made it and submitted it I think. As soon as I heard it I emailed my management saying I loved it and that he'd really found a fresh take on the track. This is actually his first release so I'm happy I could support another artist like that.
Kendra: What kinds of themes are you exploring on your upcoming release, Red Tape?
Caitlyn Scarlett: The next tape further explores my experiences with love, anxiety, politics, gender identity and success. Just my inner narratives expressed, hopefully in a way that can entertain and help others.
Kendra: Red seems to be a constant in your world. The EP and you've said you want to leave a red mark on the world. What's that about? And why red?
Kendra: A new EP, does that mean some shows perhaps too?
Caitlyn Scarlett: Yes, I can't say exactly when but I have now started the process of preparing the live set and assembling the band. Anyone who is interested in knowing more should follow me on Instagram or Twitter because I'm sure I'll be announcing something over the next few months!
Kendra: We listen to music when we're in all types of moods, but if you had to make a mixtape of songs you listen to when you are indeed happy, what five would be on it?
Right around the time Christina Holmes and I crossed paths she was coming off the road with Nahko and Medicine for the People. A "dream come true" for the acoustic singer, she nestled back into home life after what her fiance has called her "post-tour coma." A period of one to three days of well-deserved sleep. Now she's wide awake and ready to get back to working on a new album. We talked about what to expect from the follow up to her very personal Stand Up, discovering a new side of herself and more as she sat in her home in the smallest state known to the US.
Taking the life and work of another and interpreting it isn't an easy feat, but it's a challenge that Clara Engel took on for their latest Songs for Leonora Carrington. An interesting concept, we talked about that as well as where they're at in their head when they step on stage, and their plans as far as 2018 is concerned.


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I.M. Pei papers, 1920-2016
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I. M. Pei
1917, Apr. 26 Born Ieoh Ming Pei, Canton (Guangzhou), China
1935 Traveled to the United States to study architecture
1940 B.S. in architecture, Massachusetts Institute of Technology, Cambridge, Mass.
1942 Married Eileen Loo (died 2014)
1943-1945 Operation analyst, National Defense Research Committee
1945-1948 Assistant professor, Harvard University Graduate School of Design, Cambridge, Mass.
1946 M.S. in architecture, Harvard University Graduate School of Design, Cambridge, Mass.
1948-1955 Director of architecture, Webb & Knapp, Inc.
1954 Became naturalized citizen of the United States
1955-1990 Founded I. M. Pei & Associates (later I. M. Pei & Partners in 1966, and Pei Cobb Freed & Partners in 1989)
1964 Elected fellow, American Institute of Architects
1966-1970 Served on the National Council on the Humanities, appointed by Lyndon B. Johnson
1967 Elected member, American Academy of Arts and Sciences
1975 Elected member, American Academy of Arts and Letters
1975 Elected member, Royal Institute of British Architects
1979 Awarded Gold Medal, American Institute of Architects
1980 Appointed member, National Council on the Arts by Jimmy Carter
1981 Elected member, American Philosophical Society
1981-1984 Member, National Council on the Arts
1983 Awarded Pritzker Architecture Prize
1986 Awarded Medal of Liberty by Ronald Reagan
1988 Awarded National Medal of Art
1990 Retired from Pei Freed Cobb & Partners
1993 Awarded Medal of Freedom by George H. W. Bush
2000 Received Living Legend Award from the Library of Congress
2003 Awarded National Design Award for Lifetime Achievement, Smithsonian Institution, Cooper-Hewitt, National Design Museum
2019, May 16 Died, New York, N.Y.
Chronology of the Work of I. M. Pei
1952-1956 Mile High Center, Denver, Colo.
1954-1963 Luce Memorial Chapel, T'ai-chung shih, Taiwain
1957-1962 Kips Bay Plaza, New York, N.Y.
1957-1964 Society Hill, Philadelphia, Pa.
1959-1964 Cecil and Ida Green Center for Earth Sciences, Massachusetts Institute of Technology, Cambridge, Mass.
1960-1961 Government Center Urban Renewal Plan, Boston, Mass.
1960-1964 Southwest Washington urban renewal plan, Washington, D.C.
1960-1970 National Airlines Terminal, John F. Kennedy International Airport, New York, N.Y.
1961-1964 Everson Museum of Art, Syracuse, N.Y.
1961-1964 Syracuse University S. I. Newhouse Communications Center, Syracuse, N.Y.
1961-1966 University Plaza, New York University, New York, N.Y.
1961-1967 National Center for Atmospheric Research, Boulder, Colo.
1962-1970 Air traffic control towers, various U.S. airports
1963-1967 New College of Florida dormitories, student union, and academic center, Sarasota, Fla.
1963-1971 Wilmington Tower, Wilmington, Del.
1965-1979 John F. Kennedy Library, Boston, Mass.
1966-1968 Des Moines Art Center addition, Des Moines, Iowa
1966-1969 Bedford-Stuyvesant Superblock, Brooklyn, N.Y.
1966-1971 Cleo Rogers Memorial Library, Columbus, Ind.
1966-1977 Dallas City Hall, Dallas, Tex.
1967-1973 Commerce Court, Canadian Imperial Bank of Commerce, Toronto, Canada
1968-1970 Columbia University Comprehensive Master Plan, New York, N.Y.
1968-1972 Paul Mellon Center for the Arts, Choate Rosemary Hall, Wallingford, Conn.
1968-1973 Herbert F. Johnson Museum of Art, Cornell University, Ithaca, N.Y.
1968-1978 East Building of the National Gallery of Art, Washington, D.C.
1970-1976 Oversea-Chinese Banking Corp. Centre, Singapore
1972-1976 Ralph Landau Chemical Engineering Building, Massachusetts Institute of Technology, Cambridge, Mass.
1973-1986 Raffles City, Singapore
1974-1983 Johnson & Johnson World Headquarters, New Brunswick, N.J.
1977-1981 Museum of Fine Arts West Wing, Boston, Mass.
1977-1982 Sunning Plaza, Hong Kong
1977-1984 International Business Machines Corp. Corporate Office Building, Purchase, N.Y.
1978-1982 Texas Commerce Tower and United Energy Plaza, Houston, Tex.
1978-1984 Wiesner Building/Center for Arts and Media Technology, Massachusetts Institute of Technology, Cambridge, Mass.
1979-1982 Fragrant Hill Hotel, Beijing, China
1979-1986 Jacob K. Javits Convention Center and Plaza, New York, N.Y.
1980-1987 Bank of America Tower at International Plaza, Miami, Fla.
1981-1990 Gateway Towers, Singapore
1981-1992 Guggenheim Pavilion, Mount Sinai Medical Center expansion and modernization, New York, N.Y.
1982-1989 Morton H. Meyerson Symphony Center, Dallas, Tex.
1982-1989 Bank of China Tower, Hong Kong
1983-1989 International Business Machines Corp. Office Building Complex, Somers, N.Y.
1983-1993 Musée du Louvre (Grand Louvre), Paris, France
1986-1989 Choate Rosemary Hall Science Center, Wallingford, Conn.
1987-1995 Rock and Roll Hall of Fame and Museum, Cleveland, Ohio
1988-1990 Shinji Shumeikai Bell Tower, Shiga-ken, Japan
1989-1993 Four Seasons Hotel, New York, N.Y.
1988-1992 Kirklin Clinic, Birmingham, Ala.
1990-2007 Musée d'Art Moderne Grand-Duc Jean, Luxembourg, Luxembourg
1991-1997 Miho Museum, Shiga-ken, Japan
1993-1999 Buck Institute for Age Research, Marin County, Calif.
1994-2001 Bank of China Head Office, Beijing, China
1995-2000 Republic of Korea Permanent Mission to the United Nations, New York, N.Y.
1995-2003 Deutsches Historisches Museum, Berlin, Germany
1996-2001 Ronald Reagan UCLA Medical Center, Los Angeles, Calif.
1999-2003 Oare Pavilion, Wiltshire, England
2000-2008 Museum of Islamic Art, Dawḥah, Qatar
2002-2005 Macau Science Center, Macau, China
2006-2009 Miho Institute of Aesthetics chapel, Shiga-ken, Japan

redpajama_set_name: RedPajamaCommonCrawl


text:
using System;
using System.Collections;
using System.Text;
using System.IO;

public class FreestyleScriptPrecompiler
{
    static public bool Compile(ref string content, string scriptFile, bool isPrimaryScript, Hashtable context)
    {
        if (!isPrimaryScript)
            return false;

        var code = new StringBuilder(4096);
        code.AppendLine(@"
//Auto-generated file

using System;
using System.IO;
using System.Linq;
using System.Text;
");

        bool headerProcessed = false;

        string line;

        using (var sr = new StringReader(content))
            while ((line = sr.ReadLine()) != null)
            {
                if (!headerProcessed && (!line.StartsWith("//") && line.Trim() != "" && (!line.TrimStart().StartsWith("using "))))
                {
                    headerProcessed = true;
                    code.AppendLine(
@"

namespace ScriptNamespace
{
    public class ScriptClass
    {
        public static int Main(string[] args)
        {
            // args = new[] { ""-service"" };

            try
            {
                if (args.Length == 0)
                {
                    Console.WriteLine(ScriptClass.ScriptLogic(args));
                }
                else if (args.Length == 1 && args[0].StartsWith(""-service""))
                {
                    using (var host = StartScriptLogicAsRest(args[0].Substring(""-service"".Length), () => ScriptLogic(args.Skip(1).ToArray())))
                    {
                        Console.ReadLine();
                        host.Close();
                    }
                }
                return 0;
            }
            catch (Exception ex)
            {
                return 1;
            }
        }

        public static string ScriptLogic(string[] args)
        {
                    // <CODE from repo>
                    ");
                        }

                code.AppendLine(line);
            }

        code.AppendLine(
@"
             // </CODE from repo>

            return null;
        }

        public static System.Net.HttpListener StartScriptLogicAsRest(string serviceParameter, Func<object> func)
        {
            var listener = new System.Net.HttpListener();
            listener.Prefixes.Add(string.Format(""http://localhost:4567/{0}/"", System.Diagnostics.Process.GetCurrentProcess().ProcessName));
            listener.Start();

            while (true)
            {
                System.Net.HttpListenerContext context = listener.GetContext();
                System.Net.HttpListenerRequest request = context.Request;
                System.Net.HttpListenerResponse response = context.Response;

                object returnValue = func();
                if(returnValue == null)
                {
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");

        content = code.ToString();
        return true;
    }
}

redpajama_set_name: RedPajamaGithub


Ah! My delivery from Mayberry Medical Supply! It's so convenient for people with limited mobility.
Just hurry up and sign. Your house smells like sour milk.
I'll only take a minute more of your time. I have a something to send back to them.
This stool sample needs a spectral analysis.
Just sticking a stamp on it isn't enough. You need to actually wrap it.
8 +1 for "Your house smells like sour milk"
7 Deeply disturbing. Good pace.
5 ok. typo not good.
4 have a something ?


text:
Q: Data Uri for each intent in android Android defines various of intent actions, How can I know what uri-data I can use with each intent?

A:
How can I know what uri-data I can use with each intent?

In some cases, it may be described in the documentation of the action. In other cases, the action is generic (e.g., ACTION_EDIT), and so there may be any number of possible MIME types that will be supported, depending on what software is installed on the device.

And Each action (such as Intent.ACTION_VIEW, Intent.ACTION_EDIT,...) I want to see a full reference of all Data URI which it can receive.

That is not knowable in advance. It depends on what software is installed on your device.

I just want to see a full list of actions which were defined by Android

There is no such list.

redpajama_set_name: RedPajamaStackExchange


The interface is very clean and well organized so that one can access all functions with minimum effort. You can find more details about how USB Guardian protects your PC against worms and viruses on this page. It is possible to give a USB flash drive / USB memory stick a behaviour that is very similar to autostarting CD's and DVD's.


The Zoning Board of Appeals as it is presently constituted is hereby reaffirmed as having been established in accordance with the provisions of the General City Law. The Board shall consist of 7 members appointed by the Mayor subject to confirmation by the Common Council. All appointments shall be made for a term of three years.
Vacancies for the unexpired term of any member shall be filled for such unexpired period only.
No person shall be appointed to more than 3 consecutive full 3-year terms.
The Mayor shall have the power to remove, after public hearing, any member of the Zoning Board of Appeals for cause. A Board member may be removed for failure to attend 3 consecutive meetings or for noncompliance with any minimum training requirement established by this chapter.
No more than 4 members of the Board may be enrolled in any 1 political party.
Each member of the Board shall complete a basic qualification program for Zoning Board of Appeals persons within 12 months of appointment and thereafter shall participate annually in a continuing education course to be approved by the Common Council upon the recommendation of the Board.
The Board shall hold an annual meeting in January of each year to elect, from its members, a Chairperson and a Vice Chairperson to assume the duties of the Chairperson in his absence. Chairpersons and Vice Chairpersons shall be elected for a 2-year term. No person shall be elected to more than 2 consecutive terms as Chairperson.
Under General City Law § 35, it may grant a building permit to a property owner aggrieved by an Official Map of the City adopted under General City Law § 26 and § 29. In this case, a notice of a public hearing must be published in a newspaper of general circulation in the City 15 days prior to the public hearing. The property owner must show that the property is not yielding a fair return on its value and demonstrate that his request is causing a minimum impact upon the intent of the Official Map.
It may grant reasonable exceptions under special circumstances to the prohibitions of General City Law § 36 concerning property without access to public streets.
Under General Municipal Law § 239-j, it may grant a building permit to a property owner aggrieved by an Official Map of the county adopted under General Municipal Law § 239-k. Editor's Note: General Municipal Law §§ 239-j, relating to permits for buildings in rights-of-way or sites preserved by the Official Map, and 239-k, relating to regulations for control of development, were repealed by L.1997, c. 451, § 2, effective 7-1-1998. See now General Municipal Law §§ 239-e and 239-f. In this case, a majority vote of 2/3 is required. The property owner must show that the property is not yielding a fair return on its value and that the building will have a minor impact upon the intent of the Official Map.
It may decide any question involving the intent of any provision of this chapter, including the determination of the exact location of any district boundary if there is uncertainty with respect thereto.
It may review any decision or order made by the Zoning Officer.
It may grant area variances subject to the applicable provisions of Article XV of this chapter.
It may grant use variances subject to the applicable provisions of Article XV of this chapter.


text:
Global Defense & Military
Army & Security Forces
New Zealand Army
Thread starter Whiskyjack
army artilery gunner new zealand sas
RegR
Rob c said:
My current view on electric vehicles is that they are a waste of time , because the extra energy to run them is generated by fossil fuel generation, which runs at a thermal efficiency of 25% and a modern petrol car runs at between 30 to 40%. so not counting the huge amount of co2 required to make the lithium they are still making the situation worse than a petrol engine. All that they are doing is transferring the carbon footprint from the car to the power station. A greenie that I was pointing this out to said " but we are 80% renewable to which I replied that we are already using the 80% and you cannot use it twice. The fact is that the renewable energy in NZ is a finite figure of KW and any extra requirements such as extra electric vehicles is generated with fossil fuels. The use of the 80% figure in this context is a complete wrongful miss use of statistics. The use of electric vehicles is only justified if they are charged from a renewable source and the additional load on the national grid is not renewable. However non plug in Hybrids are good for the environment.
Robbing Peter to pay Paul, "solving" one problem to then only cause another/others. Wait until we start stockpiling the used batteries!
Whilst I agree the govt should be seen to be doing their "part" it needs to be tangible and overall effective otherwise a pointless exercise and making any organisations (such as NZDF, police etc) day to day job harder/costlier/limited with no clear benefits just to prove a point is ironically a waste of resources imo. I'm sure there are certain applications but an overall blanket implementation policy is wishful thinking at best, down right dangerous at worst.
Reactions: recce.k1
ngatimozart
Verified Defense Pro
You are a heretical heathen you are. I shall report you to the PM's office for uttering such malicious fallacies against the holy religious think speak.
That's actually a considerable problem that not much thought has been given too. The next point is that electric car batteries are hideously expensive to replace and at the present point in time are unrecyclable so they end up in land fill. For the amount of money that you have to fork out on purchasing a new battery to replace the stuffed one, and then pay for a qualified person to install it, you might as well buy a new car.
Reactions: Oldman1023, oldsig127 and Rob c
Wrong about the batteries. Lithium-ion batteries can be & some are recycled. The problem is cost, but that's coming down. There's potentially a lot of money to be made from cost-efficient recycling of Li-ion batteries, & people are working hard on it.
Reactions: kiwipatriot69
swerve said:
"Can be", "some are" and "potentially" are not exactly great confidence builders when we are only just beginning this mass migration towards a new global phenomenon? Will do, all and definitely are catchphrases we should be associating with this now/already whilst manageable and not "possibly", "potentially", "maybe" later on when full mass production really kicks in and we have no alternative but EV batteries in large scale quantities. Costs are a universal problem in any scenario and are generally either offset, absorbed or passed on but either way, someone ultimately pays. It is literally too early days to consider the full potential costs as we are still in the honeymoon period (using not used) so to speak ie imagine defence having to pay for a fleet of milspec EV batteries to be disposed of in NZ at the end of their LOT through, no doubt, some stringent self imposed "environmentally concious" process to either recycle/destroy? Potentially huge costs again...time will tell.
htbrst
We are above 80% renewable generation now and aim to be at 100% within 10 years. Your assertion that most of the extra power that goes into the electric fleet is from the thermal proportion takes a very simplistic view of how charging an electric vehicle works - the vast majority of their time on charge is overnight when thermal stations are offline.
Presumably NZDF will start by replacing the garrison Corollas et al ( you can tell they are NZDF operated by the "how's my driving sticker"). Electric LAVs will be decades away but it can only be strategically good for the country to not have to import fuel to provide motive power to our overall vehicle fleet.
If your buying a new car, travel over 20,000kms per year or so and petrol is over $3/litre an electric car is a no brainer in the current environment already.
Reactions: Gracie1234
Gracie1234
Adding to the renewables conversation. NZ does have a plan to move to 100% renewable electricity generations with plans to increase generation at our existing sites without additional dams constructed will be the first phase. Battery technology in use today will be replaced with cheaper to produce, more environmentally friendly and easier to recycle technologies. The goal is to make a battery without using rare elements such as Lithium.
As a country, we are also well on the way to switching our heavy vehicle fleet to hydrogen. A network of refueling stations will be completed in a year or so, that will cover 90% of our transport routes. $5B has been secured to generate green hydrogen which will make us probably the first country that will actually be exporting it to other countries, agreements are already in place with Japan and South Korea.
NZ Hydrogen Projects — New Zealand Hydrogen Council
www.nzhydrogen.org
Meridian's Southern Green Hydrogen Project selects Woodside Energy as preferred partner | Meridian Energy
www.southerngreenhydrogen.co.nz
For me it is not a matter of if but when, there will be first-mover advantages for our economy and a strong economy is needed to provide resources to defend it.
I would expect that we will move to hybrid power for most of our next vehicle fleet.
Reactions: kiwipatriot69 and CJohn
Rob c
The Bunker Group
htbrst said:
From the information I received ( sorry lost the link) the highest the renewable rate at night is 92%. and the electric vehicle is still an extra drain on the system. Even if we were 100% at night the extra drain would cause further drain on hydro, reducing the amount of water available available for power production at other times. Again this is not an unlimited resource and will still cause additional fossil fuel consumption. As for the 100% in 10 years, I have never been able to find out what the plan is on how to achieve this in the required timeframe and at this time I have the feeling that it may be just some political talk to sound good but not commit to anything. I do believe that we need to be fully committed to the 100% renewable energy as quickly as possible, but to achieve this we need a strong fully focused plan of action and financing with cross party support to achieve this and action taking place now. This not happening and until we get a focus on this I think the electric vehicle is only a politically symbolic show at trying to stop climate change and we need to get serious and focus on getting our power supply fully renewable first. I am sorry but empty political statements don't cut it.
While there are a lot for very worth while projects that are underway, I very much doubt that they will come anyway close to being completed in time as the time taken to advance from project status to hardware in this country is miles too long. Even a small project locally for a rail siding to load logs took 4 years to get approval to change the land use from agricultural to industrial and with the caveats that were included, this may not now happen. This is just a small example of how with the best of intentions we continue to shoot ourselves in the foot.
Reactions: RegR
RegR said:
Right now, they're recyclable. That's what I was replying to: "unrecyclable". It's wrong. Not only can they be recycled, but some are.
Of course, a lot of other types of battery have been profitable to recycle for a very long time. At the moment recycling Li-ion batteries is like solar cells some years ago: a niche, not competitive with other ways of generating electricity. Then someone (Germany) provided incentives & money piled in. They're now very competitive. Their price has crashed & efficiency shot up. People in the business are saying they're confident the same will happen to Li-ion recycling. There are ideas a-plenty for how to do it more cheaply. They just need investment - & that's flowing now, because there are getting to be enough of them out there to justify it. It doesn't need new physics or the like. Some of it is just a question of applying existing mass production techniques, which just needs enough throughput to pay for the capital investment.
Lithium's rather expensive, BTW, & the price has been going up. There's a lot of money going into finding new sources & ways to extract it. That's driving investment in recycling technology, right now. This isn't a future airy-fairy thing. Hard-nosed businesspeople are taking it seriously. And there's also money going into promising alternative battery technologies. Given demand, supply follows.
the vast majority of their time on charge is overnight when thermal stations are offline.
This is not quite correct as while the industrial load goers down the domestic load goes up until 10 to 11pm then the low load factor comes in to play. Most electric vehicle commuters would plug in when they got home and would have done the majority of their charging before this happens. Also the majority of use of public chargers would be used during the day.
OldTex
The use by military forces of electric vehicles would suggest that the first application will be in garrison only type vehicles. My reasoning for that is that they would utilise the civilian power generation and distribution infrastructure. The use of EV in the tactical space becomes more problematic as the power generation and distribution network will be limited or non-existent, and this is before considering use in a conflict area. It is reasonable therefore to suppose that tactical military EV will be self-charging hybrids. This does not reduce the emissions to zero. The use of other fuels or power sources such as fuel cells or hydrogen introduce challenges for the storage, handling and use of those fuels (which is the basis for many armed forces having moved from petrol to diesel).
John Fedup
How many nations can produce green hydrogen economically, damn few. Sufficient electricity for electrolysis needs carbon free fuel so any talk about a 90-100% electric vehicle future can't happen without fission or hopefully fusion.
Solar & wind can be used for electrolysis. A problem that both share is that they're intermittent. Making hydrogen while electricity output is more than demand would be a partial solution.
In addition both are influenced by geography. North Africa, much of Australia, and the American SW are all prime solar regions. Certain areas have consistent winds making them idea for windmills. Both options are great for those regions. Transmission from remote desert locations will reduce efficiency somewhat but is not a show stopper. One thing I object to in Ontario is using valuable and limited farmland for solar farms (and urban sprawl). Regardless of how much more solar and power generation expands, a huge new nuclear capacity will still be required for a zero carbon emission world.
Reactions: swerve and Nudge
Just to clarify my thoughts on electric vehicles, firstly we are at the beginning of the development cycle of these vehicles and in comparison terms one could say the Model T era of their development and the will improve greatly with time. My objection at this stage is that they are being sold to the public as saving the climate, before we have the renewable power production in place to support them. In other words the pollies are putting the cart before the horse. This is not unusual for NZ pollies as most of them could not organize a good party at a brewery.
Reactions: Teal
recce.k1
Morning, if any kiwi's are online and interested, there's a live feed underway of the "grand opening of the new QAMR Regimental Headquarters and Scots Sqn".
QAMR was live. | By QAMR | Facebook
1.4K views, 70 likes, 19 loves, 16 comments, 3 shares, Facebook Watch Videos from QAMR: QAMR was live.
fb.watch
Reactions: htbrst and RubiconNZ
Exactly right on renewable power and I would add in that most local utilities lack infrastructure for large scale recharging of electric vehicles. This is applicable to most nations. Not sure how many jurisdictions are following BC's plan to phase out all natural gas furnaces and stoves by 2030, that is a significant amount of replacement electricity that will be needed by BC, an anti-nuclear region.
CJohn
recce.k1 said:
Curiously in this video I noticed a couple of servicemen carrying Sterling SMG's, to those in the know, is this just ceremonial use or are they still in active service with the QAMR Regiment ?
CJohn said:
Purely ceremonial. Usually accompanies the regimental banners, in this case QAMR
Reactions: recce.k1 and CJohn
It is the perennial problem for the provision of infrastructure. The justification for the investment in infrastructure must be supported by a sustained level of demand (with an appropriate cost recovery mechanism) and at the same time a sustained level of demand cannot be supported without an adequate amount of infrastructure. One way to reasonably break out of this situation is that infrastructure is funded by the government as a public good even though it will not generate a return on the investment in the short or medium term (or possibly ever).
Reactions: swerve
Nicked this from the US Army thread.
ngatimozart said:
The US Army has finally chosen a new light tank The Army Just Selected Its First Light Tank In Decades | The Drive However its procurement history is littered with a lot of what ifs and failures to proceed so I am not holding my breath. They've been down this road before with a light tank. This one is C-130 transportable, has a 105mm gun and the hull controls and sensors are based on the latest M-1 Abrams controls and sensors. It has modular add-on armour as well. It is thought that the turret is a remote turret.
If this project goes through (noting previous disappointments) does anyone think NZ Army could be taking an interest? Perhaps as (small) part of the LAV3 replacement programme eg perhaps 20-odd vehicles (or if the unbelievable happens, as a seperate project if a future NZG decided to better equip the Army in these worsening times)? Noting that Cadredave previously discussed the M8 Light Tank as being an option to replace the Scorpion FSV's ... pre-LAV3 selection of course).
Appears to be tailored to support light infantry (which also fits the NZ Army approach) by providing them with direct fire support (not obviously for an anti-tank role a la M1A1 Abrams etc), provides deployable lightweight firepower for our Asia-Pacific region (via sealift or allied C-17 - which can apparently fit two), the 105mm gun would give the NZ Army the firepower to destroy fortifications and so on, and according to some of the article comments the 105mm could potentially fire LAHAT semi-active laser homing guided anti-tank guided missiles, and the turret have space for gun launched missiles.
Reg & co any thoughts? Noting previous discussions of having trained crews available being another issue (which could also impact LAV3 crewing).


This course is eligible for Spiritual Education and Enrichment (S.E.E) credits through Unity Worldwide Ministries.
Classes are held at two different time slots- you choose which works best for you and you must sign up for the series in advance.
NOTE: Classes for MARCH/APRIL have all been filled but other SEE credit courses will be offered quarterly and this class will be repeated again in 2019.


text:
Home/Africa/Tunisia detains 30, identifies bomber after attack
Tunisia detains 30, identifies bomber after attack
Tunis – Tunisian authorities have detained 30 people suspected of having extremism links after a suicide bombing targeting presidential guards, and identified the bomber as a local street vendor.
The Interior Ministry said in a statement on Thursday that forensic police identified the attacker as 27-year-old Houssam ben Hedi ben Miled Abdelli by his DNA. It said he was from a working class neighborhood on the edge of Tunis.
ISIS claimed responsibility for Tuesday's attack on a bus in central Tunis, which left 12 dead plus the attacker.
In a later statement, the ministry said it detained 30 people suspected of links to extremist groups and seized several weapons in 526 raids around the country over the past 24 hours. It didn't say whether any of the suspects had ties to the attack.
The blast shook Tunisia and its fragile young democracy after two attacks on tourist sites this year by Islamic radicals that killed 60 people.
A disgruntled Tunisian vendor set himself on fire in 2010, sparking a nationwide uprising that overthrew the president and led to revolts across the Arab world.
Previous Mali arrests 2 suspects over Bamako hotel attack
Next S Sudan peace deal monitors urge action
The peoples of the North Caucasus and the majority of Russians are going in different …


text:
REVIEW album Band Of Gold Band Of Gold
Band Of Gold mine a rich seam!
This act have been likened to Haim, albeit with sifter harmonies, and one reviewer referred to them as the new Fleetwood Mac. Whilst I agree that there is an element of both of those groups in Band Of Gold's sound they have a massive amount of their own original sounds. Frankly I have never liked referring to anyone as the 'new' something or other.
Anyway this eponymously titled début album from a very talented Norwegian band stands up really well without comparisons. The production by Nikolai Hængsle Eilertsen is impeccable as are the harmonies. The musicianship is outstanding too. Singer Nina Elisabeth Mortvedt has the voice of an angel. Overall it has a really chilled feel but it is by no means soporific. I was tapping my feet and swaying in time both times I listened in advance of this review.
I can picture myself listening to this album out in the sunshine with a glass of wine and maybe a joint. But you won't really need any stimulation to enjoy it, trust me. In a perfect world "Gush" would be keeping Justin Bieber from the number one spot in the charts.
Apart from the aforementioned "Gush" the beautifully crafted "But In The Movie Baby" is my current favourite song of the album's nine tracks. But after my first listen "No Way Around" and "Surface Me" were my favourites. The songs exude the confidence of a really established artist which is superb for a first major release.
I mentioned the chilled feel of the album earlier and it would be an excellent tonic after being out clubbing all night. However I think that a number of Band Of Gold's songs would fare well if remixed for use in a club. All round this is a marvellous début!
When are Band Of Gold playing here in the UK? Someone please tell me because I can't wait to see them live.


Glassio announces an upcoming EP with a cross-genre mover.
JNTHN STEIN & The Age of L.U.N.A team up for a laid back groover.
James Carter leans on some cultural influence for this impressive mover.
Don't miss this flight: Sparrows swoops well within earshot with latest single.
Okayché lays out a darkened blend of electronic RnB/hip hop (★★★).
Golden Features shows us why he's is at the top of the game at a sold-out Forum Theatre.
Lonnie Liston Smith took Sydney to cosmic places.
Ukiyo delivers an impeccable EP full of ambiance and force.
Strap yourselves in for the ride of your life courtesy of RL Grime.


This aim lamp and more than just a lamp it is an artwork. Lamps are usually hung in the middle of the room. With the aim lamp this doesn't matter, since the grounding occurs via a long cable. Secondly it breaks the rule that the cable is only there to be hidden, the aim lamp presents a forest of cables with impetuous lines and an aesthetic function.


text:
Home > Commentaries > Geneva Study Bible > Romans > Chapter 9
• Barnes' New Testament
• Darby's Synopsis
• People's New Testament
• Robertson's Word Pictures
9:1 I say 1 the truth in Christ, I lie not, my conscience also bearing me witness in the Holy Ghost,
(1) The third part of this epistle, which goes to the twelfth chapter, in which Paul ascends to the higher causes of faith: and first of all, because he purposed to speak much of the casting off of the Jews, he uses a declaration, saying by a double or triple oath, and by witnessing of his great desire towards their salvation, his singular love towards them, and in addition granting to them all their privileges.
9:3 For I could wish that myself were a accursed from Christ for my brethren, my kinsmen according to the b flesh:
(a) The apostle loved his brethren so completely that if it had been possible he would have been ready to have redeemed the castaways of the Israelites with the loss of his own soul forever: for this word "accursed" signifies as much in this place.
(b) Being brethren by flesh, as from one nation and country.
9:4 Who are Israelites; to whom [pertaineth] the adoption, and the c glory, and the d covenants, and the giving of the e law, and the f service [of God], and the g promises;
(c) The ark of the covenant, which was a token of God's presence.
(d) The tables of the covenant, and this is spoken by the figure of speech metonymy.
(e) Of the judicial law.
(f) The ceremonial law.
(g) Which were made to Abraham and to his posterity.
9:5 Whose [are] the fathers, and of whom as concerning the flesh Christ [came], 2 who is over all, God blessed for ever. Amen.
(2) Or, "who is God over all, blessed for ever." A most manifest testimony of the Godhead and divinity of Christ.
9:6 3 Not as though the word of God hath taken none effect. For they [are] not all h Israel, which are of Israel:
(3) He enters into the handling of predestination, by means of presenting an objection: How may it be that Israel is cast off, and that in addition we must also make the covenant which God made with Abraham and his seed, frustrated and void? He answers therefore that God's word is true, although Israel is cast off: for the election of the people of Israel is so general and common, that nonetheless the same God chooses by his secret council those as it pleases him. So then this is the proposition and state of this treatise: the grace of salvation is offered generally in such a way, that in spite of how it is offered, the efficacy of it pertains only to the elect.
(h) Israel in the first place, is taken for Jacob: and in the second, for the Israelites.
9:7 Neither, because they are the seed of Abraham, [are they] all children: 4 but, In i Isaac shall thy seed be called.
(4) The first proof is taken from the example of Abraham's own house, in which Isaac only was considered the son, and that by God's ordinance: although Ishmael also was born of Abraham, and circumcised before Isaac.
(i) Isaac will be your true and natural son, and therefore heir of the blessing.
9:8 5 That is, They which are the children of the k flesh, these [are] not the children of God: but the children of the l promise are counted for the seed.
(5) A general application of the former proof or example.
(k) Who are born of Abraham by the course of nature.
(l) Who are born by virtue of the promise.
9:9 6 For this [is] the word of promise, At this time will I come, and Sara shall have a son.
(6) A reason of that application: because Isaac was born by the power of the promise, and therefore he was not chosen, no, he was not at all, except by the free will of God: by which it follows that the promise is the fountain of predestination, and not the flesh, from which promise the particular election proceeds, that is, that the elect are born elect, and not that they are first born, and then after elected, by God who predestinates.
9:10 7 And not only [this]; but when Rebecca also had conceived by one, [even] by our father Isaac;
(7) Another strong and persuasive proof taken from the example of Esau and Jacob, who were both born of the same Isaac, who was the son of promise of one mother, and were born at the same time, and not at different times as Ishmael and Isaac were: and yet nonetheless, as Esau was cast off, only Jacob was chosen: and that before their birth, that neither any goodness of Jacob's might be thought to be the cause of his election, neither any wickedness of Esau to be the cause of his casting away.
9:11 (For [the children] being not yet born, neither having done any good or evil, that the m purpose of God according to election might 8 stand, not of works, but of him that calleth;)
(m) God's decree which proceeds from only his good will, by which it pleases him to choose one, and refuse the other. (8) Paul does not say, "might be made", but "being made might remain". Therefore they are deceived who make foreseen faith the cause of election, and foreknown infidelity the cause of reprobation.
9:12 9 It was said unto her, The elder shall serve the younger.
(9) He proves the casting away of Esau in that he was made servant to his brother: and proves the choosing of Jacob in that he was made lord of his brother, although his brother was the first begotten. And in order that no man might take what God had said, and refer it to external things, the apostle shows out of Malachi, who is a good interpreter of Moses, that the servitude of Esau was joined with the hatred of God, and the lordship of Jacob with the love of God.
9:14 10 What shall we say then? [Is there] n unrighteousness with God? God forbid.
(10) The first objection: if God loves or hates without any consideration of worthiness or unworthiness, then is he unjust, because he may love those who are unworthy, and hate those who are worthy? The apostle detests this blasphemy, and afterward responds to it in depth, point by point.
(n) Man knows no other causes of love or hatred, but those that are in the persons, and thereupon this objection arises.
9:15 11 For he saith to Moses, I will o have mercy on whom I will have mercy, and I will have p compassion on whom I will have compassion.
(11) He answers first with regard to those who are chosen to salvation, in the choosing of whom he denies that God may seem unjust, although he chooses and predestinates to salvation those that are not yet born, without any respect of worthiness: because he does not bring the chosen to the appointed end except by the means of his mercy, which is a cause discussed under predestination. Now mercy presupposes misery, and again, misery presupposes sin or voluntary corruption of mankind, and corruption presupposes a pure and perfect creation. Moreover, mercy is shown by her degrees: that is, by calling, by faith, by justification and sanctification, so that at length we come to glorification, as the apostle will show afterwards. Now all these things orderly following the purpose of God, do clearly prove that he can by no means seem unjust in loving and saving his.
(o) I will be merciful and favourable to whom I wish to be favourable.
(p) I will have compassion on whoever I wish to have compassion.
9:16 12 So then [it is] not of him that q willeth, nor of him that runneth, but of God that sheweth mercy.
(12) The conclusion of the answer: therefore God is not unjust in choosing and saving from his free goodness, such as it pleases him: as he also answered Moses when he prayed for all of the people.
(q) By "will" he means the thought and endeavour of heart, and by "running", good works, to neither of which he gives the praise, but only to the mercy of God.
9:17 13 For the r scripture saith unto Pharaoh, Even for this same purpose have I s raised thee up, that I might 14 shew my power in thee, and that my name might be declared throughout all the earth.
(13) Now he answers concerning the reprobate, or those whom God hates who are not yet born, and has appointed to destruction, without any respect of unworthiness. And first of all he proves this to be true, by alleging the testimony of God himself concerning Pharaoh, whom he stirred up to this purpose, that he might be glorified in Pharaoh's hardening and just punishing.
(r) God speaks unto Pharaoh in the scripture, or, the scripture in talking about God, in this way talks to Pharaoh.
(s) Brought you into this world.
(14) Secondly, he brings the goal of God's counsel, to show that there is no unrighteousness in him. Now the main goal is not properly and simply the destruction of the wicked, but God's glory which appears in their rightful punishment.
9:18 15 Therefore hath he mercy on whom he t will [have mercy], and whom he will he hardeneth.
(15) A conclusion of the full answer to the first objection: therefore seeing that God does not save those whom he freely chose according to his good will and pleasure, but by justifying and sanctifying them by his grace, his counsels in saving them cannot seem unjust. And again, there is not injustice in the everlasting counsel of God, with regard to the destruction of those whom he lifts to destroy, because he hardens before he destroys: therefore the third answer for the maintenance of God's justice in the everlasting counsel of reprobation, consists in this word "hardening": which nonetheless he concealed in the former verse, because the history of Pharaoh was well known. But the force of the word is great, for hardening, which is set against "mercy", presupposes the same things that mercy did, that is, a voluntary corruption, in which the reprobate are hardened: and again, corruption presupposes a perfect state of creation. Moreover, this hardening also is voluntary, for God hardens in such a way, being offended with corruption, that he uses their own will whom he hardens, for the executing of that judgment. Then follow the fruits of hardening, that is, unbelief and sin, which are the true and proper causes of the condemnation of the reprobate. Why does he then appoint to destruction? Because he wishes: why does he harden? Because they are corrupt: why does he condemn? Because they are sinners. Where then is unrighteousness? Nay, if he would destroy all after this manner, to whom would he do injury?
(t) Whom it pleased him to appoint, to show his favour upon.
9:19 16 Thou wilt say then unto me, Why doth he yet find fault? For who hath resisted his will?
(16) Another objection, but only for the reprobate, rising upon the former answer. If God appoints to everlasting destruction, such as he wishes, and if that which he has decreed cannot be hindered nor withstood, how does he justly condemn those who perish by his will?
9:20 17 Nay but, O man, who art thou that repliest against God? 18 Shall the thing u formed say to him that formed [it], Why hast thou made me thus?
(17) The apostle does not answer that it is not God's will, or that God does not either reject or elect according to his pleasure, which thing the wicked call blasphemy, but he rather grants his adversary both the antecedents, that is, that it is God's will, and that is must of necessity so happen, yet he denies that God is therefore to be thought an unjust avenger of the wicked: for seeing that it appears by manifest proof that this is the will of God, and his doing, what impudency is it for man, who is but dust and ashes, to dispute with God, and as it were to call him into judgment? Now if any man say that the doubt is not so dissolved and answered, I answer, that there is no surer demonstration in any matter, because it is grounded upon this principle, that the will of God is the rule of righteousness.
(18) An amplification of the former answer, taken from a comparison, by which it also appears that God's determinate counsel is set by Paul as the highest of all causes: so that it depends not in any way on the second causes, but rather shapes and directs them.
(u) This similitude agrees very properly to the first creation of mankind.
9:21 19 Hath not the potter power over the clay, of the same lump to make one 20 vessel unto x honour, and another unto 21 dishonour?
(19) Alluding to the creation of Adam, he compares mankind not yet made (but who are in the creators mind) to a lump of clay: who afterwards God made, and daily makes, according as he purposed from everlasting, both such as should be elect, and such as should be reprobate, as also this word "make" declares.
(20) Whereas in the objection propounded, mention was only made of vessels to dishonour, yet he speaks of the others also in this answer, because he proves the Creator to be just in either of them.
(x) To honest uses.
(21) Seeing then, that in the name of dishonour the shame of everlasting death is signified, those agree with Paul, who say that some are made by God for most just destruction: and they that are offended with this kind of speech betray their own folly.
9:22 22 [What] if God, willing to shew [his] wrath, and to make his power known, endured with much longsuffering the y vessels of wrath fitted to 23 destruction:
(22) The second answer is this, that God, moreover and besides that he justly decrees whatever he decrees, uses that moderation in executing his decrees, as is declared his singular mercifulness even in the reprobate, in that he endures them a long time, and permits them to enjoy many and singular benefits, until at length he justly condemns them: and that to good end and purpose, that is, to show himself to be an enemy and avenger of wickedness, that it may appear what power he has by these severe judgments, and finally by comparison of contraries to set forth indeed, how great his mercy is towards the elect.
(y) By vessels, the Hebrews understand all types of instruments.
(23) Therefore again, we may say with Paul, that some men are made by God the creator for destruction.
9:23 And that he might make known the z riches of his glory on the vessels of mercy, which he had afore prepared unto glory,
(z) The unmeasurable and marvellous greatness.
9:24 24 Even us, whom he hath called, not of the a Jews only, but also of the Gentiles?
(24) Having established the doctrine of the eternal predestination of God on both parts, that is, on the part of the reprobate as well as of the elect, he comes now to show its use, teaching us that we ought not to seek its testimony in the secret counsel of God, but by the calling which is made manifest, and set forth in the Church, propounding to us the example of the Jews and Gentiles, that the doctrine may be better perceived.
(a) He does not say that each and every one of the Jews are called, but some of the Jews, and some of the Gentiles.
9:25 25 As he saith also in Osee, I will call them my people, which were not my people; and her beloved, which was not beloved.
(25) Our vocation or calling is free, and of grace, even as our predestination is: and therefore there is no reason why either our own unworthiness, or the unworthiness of our ancestors should cause us to think that we are not the elect and chosen of God, if we are called by him, and so embrace through faith the salvation that is offered us.
9:27 26 Esaias also crieth concerning Israel, Though the number of the children of Israel be as the sand of the sea, a remnant shall be saved:
(26) Contrary to this, neither any outward general calling, neither any worthiness of our ancestors, is a sufficient witness of election, unless by faith and belief we answer God's calling: which thing came to pass in the Jews, as the Lord had foretold.
9:28 For he will finish the work, and cut [it] b short in righteousness: because a short work will the Lord make upon the earth.
(b) God chooses and goes about to reduce the unkind and unthankful people to a very small number.
9:29 And as Esaias said before, Except the Lord of c Sabaoth had left us a d seed, we had been as Sodoma, and been made like unto Gomorrha.
(c) Armies, by which word the greatest power that exists is attributed to God.
(d) Even as very few.
9:30 27 What shall we say then? That the Gentiles, which followed e not after righteousness, have attained to righteousness, even the righteousness which is of faith.
(27) The declaration and manifestation of our election is our calling apprehended by faith, as it came to pass in the Gentiles.
(e) So then, the Gentiles had no works to prepare and procure God's mercy before hand: and that the Gentiles attained to that which they did not seek, the mercy of God is to be thanked for it: and in that the Jews did not attain that which they sought after, they can only thank themselves, because they did not seek for it in the proper way.
9:31 28 But Israel, which followed after the law of righteousness, hath not attained to the law of righteousness.
(28) The pride of men is the reason that they reject their calling, so that the cause of their damnation need not to be sought for in any other place but themselves.
9:32 Wherefore? Because [they sought it] not by faith, but as it were by the s works of the law. For they stumbled at that stumblingstone;
(s) Seeking to attain righteousness, they followed the law of righteousness.
Beza, Theodore. "Commentary on Romans 9". "The 1599 Geneva Study Bible". <http://classic.studylight.org/com/gsb/view.cgi?book=ro&chapter=009>. 1599-1645.


It is certainly not the first movie to unite a coming-of-age story with a horror movie, but it might be the one with the most heart and the scariest clown.
After two of the weakest weekends at the box office this year, the release of the highly anticipated adaptation of Stephen King's novel was just what movie audiences desperately needed.
Directed by Andres Muschietti, It is the type of film that wears its heart on its sleeve, delivering both an earnest story about growing up and a genuinely horrifying tale in equal measure. The trailer and marketing for the film promise plenty of scares, but what you might not expect is the charm and poignancy of a story about youth and companionship.
At a length of more than a 1,200 pages, King's novel is by no means an easy story to adapt. The book switches back and forth between telling the story of a group of teens and the same group as adults.
Luckily, Muschietti's film does not try to take on both timelines. Instead, It focuses exclusively on the teens, allowing the film time to develop its characters and the horrors they face.
The first act of the film focuses on introducing the characters to the audience, developing their personalities and, more importantly, their fears.
The characters' fears are essential to how the film combines the coming-of-age story with the horror genre. It uses horror elements to reflect the characters' anxieties about growing up and entering adulthood.
The film's antagonist, Pennywise the Clown, is able to shape-shift into whatever the kids are afraid of. Muschietti centers the first half of the movie on the kids' encounters with Pennywise, ending each one in terrified screams.
The effect of these encounters is two-fold. First, it works to develop the characters. In taking this story from page to screen, the characters end up much flatter than they were in the book. By characterizing them through their fears, they are each giving a uniqueness that helps keep them distinct.
Second, these scenes help the movie to establish a narrative about how useless adults are in protecting the kids against Pennywise. Almost all of the encounters with Pennywise start or end with adults showing up, but offering no meaningful protection or guidance to the kids.
The isolated encounters with Pennywise combined with the apathy of the parents in the story drives the kids closer together. It's from this companionship that the film derives its heart; the universality of the way kids share fears gives the movie a unique and affecting resonance.
But this coming-of-age tale is only part of the movie. It contains two entirely separate yet parallel narratives that Muschietti attempts to conflate as one.
The film attempts to contextualize the coming-of-age story within the framework of a horror film. While the film functions rather well as a heartfelt and impassioned coming-of-age tale, it stumbles in connecting that story to the kids' fight against Pennywise.
There is such a focus in the film on ensuring characters' fears are properly articulated that it stumbles when drawing the connection between those fears into the actual horror story.
Pennywise, as a character and entity, is depicted as largely one-dimensional. This ultimately takes away from the film's ability to build a more sustained tension throughout the story.
Despite the time the film commits to introducing the audience to the different characters, it can't avoid moments that feel rushed and underwritten. This may simply be an unavoidable consequence of adapting such a hefty story, but it's clear that the film loses something by attempting to balance so much.
Photo: Brooke Palmer/Warner Bros. Entertainment Inc.
For example, Eddie's fear of illness is depicted rather shallowly through his encounters with a terrifying, disease-afflicted man. On the other hand, and arguably worse, is Mike's characterization. His entire backstory about his parents' death is tacked on like a footnote and the racial elements of the original story are completely omitted.
Beverly suffers from this as well; the film never really manages to draw a distinct connection between her encounter with Pennywise that ends with an explosion of blood in her bathroom to her fear of sexual abuse at the hands of her father.
This is not to say that It doesn't deliver the kind of scares that audiences crave. The box office numbers alone speak volumes. It broke records last weekend, making a record $117 million — the largest debut for a horror film ever.
There's no denying that It contains a variety of successful scares, but upon leaving the theater, it's increasingly clear how textbook those scares really are. It rarely pushes itself outside the comfort zone it establishes in the first act of the film, repeating the same elements in different settings until it reaches the climax.
But even with these weaknesses, It succeeds through the grace of its portrayal of friendship and the endless empathy it has for youth. The horror elements flourish because the movie makes the audience care about the characters, even those that are underwritten. The moments where the group comes together to stand up and protect one another are quite affecting and help to raise the stakes of the horror elements.
Ultimately, it's okay that some of the characters are underwritten because the film makes a choice to be less about the individual arcs of the characters and more about their union in the face of a universal struggle.
This is why the scenes with the kids bicycling down a street or swimming together in a reservoir feel so moving. The movie is, first and foremost, about companionship. So while it may struggle to develop well-rounded characters, it succeeds in capturing that indeterminate feeling of youth, where it seems the whole world is against you except the friends at your side.
In the movie's final scene, the kids make a blood oath to return to the town if Pennywise ever returns. Like so much of the film, a sort of artificial danger looms heavily over them, but their companionship, empathy for one another, and earnest belief in the threat posed by Pennywise gives the film the kind of heart it needs to balance the demands of a coming-of-age story.