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Prion protein (PrP) concentration controls the kinetics of prion replication and is a genetically and pharmacologically validated therapeutic target for prion disease. In order to evaluate PrP concentration as a pharmacodynamic biomarker and assess its contribution to known prion disease risk factors, we developed and validated a plate-based immunoassay reactive for PrP across 6 species of interest and applicable to brain and cerebrospinal fluid (CSF). PrP concentration varied dramatically across different brain regions in mice, cynomolgus macaques, and humans. PrP expression did not appear to contribute to the known risk factors of age, sex, or common PRNP genetic variants. CSF PrP was lowered in the presence of rare pathogenic PRNP variants, with heterozygous carriers of P102L displaying 55%, and D178N just 31%, of the CSF PrP concentration of mutation-negative controls. In rodents, pharmacologic reduction of brain Prnp RNA was reflected in brain parenchyma PrP and, in turn in CSF PrP, validating CSF as a sampling compartment for the effect of PrP-lowering therapy. Our findings support the use of CSF PrP as a pharmacodynamic biomarker for PrP-lowering drugs and suggest that relative reduction from individual baseline CSF PrP concentration may be an appropriate marker for target engagement.

Prion protein (PrP) concentration controls the kinetics of prion replication and is a genetically and pharmacologically validated therapeutic target for prion disease. In order to evaluate PrP concentration as a pharmacodynamic biomarker and assess its contribution to known prion disease risk factors, we developed and validated a plate-based immunoassay reactive for PrP across six species of interest and applicable to brain and cerebrospinal fluid (CSF). PrP concentration varies dramatically between different brain regions in mice, cynomolgus macaques, and humans. PrP expression does not appear to contribute to the known risk factors of age, sex, or common PRNP genetic variants. CSF PrP is lowered in the presence of rare pathogenic PRNP variants, with heterozygous carriers of P102L displaying 55% and of D178N just 31% the CSF PrP concentration of mutation-negative controls. In rodents, pharmacologic reduction of brain Prnp RNA is reflected in brain parenchyma PrP, and in turn in CSF PrP, validating CSF as a sampling compartment for the effect of PrP-lowering therapy. Our findings support the use of CSF PrP as a pharmacodynamic biomarker for PrP-lowering drugs, and suggest that relative reduction from individual baseline CSF PrP concentration may be an appropriate marker for target engagement.

Retinitis pigmentosa 1 (RP1) is a common inherited retinopathy with variable onset and severity. TheRP1gene encodes a photoreceptor-specific, microtubule-associated ciliary protein containing the doublecortin (DCX) domain. Here we show that another photoreceptor-specific Rp1-like protein (Rp1L1) in mice is also localized to the axoneme of outer segments (OSs) and connecting cilia in rod photoreceptors, overlapping with Rp1.Rp1L1−/− mice display scattered OS disorganization, reduced electroretinogram amplitudes, and progressive photoreceptor degeneration, less severe and slower than inRp1−/− mice. In single rods ofRp1L1−/−, photosensitivity is reduced, similar to that ofRp1−/−. While individual heterozygotes are normal, double heterozygotes ofRp1andRp1L1exhibit abnormal OS morphology and reduced single rod photosensitivity and dark currents. The electroretinogram amplitudes of double heterozygotes are more reduced than those of individual heterozygotes combined. In support, Rp1L1 interacts with Rp1 in transfected cells and in retina pull-down experiments. Interestingly, phototransduction kinetics are normal in single rods and whole retinas of individual or doubleRp1andRp1L1mutant mice. Together, Rp1 and Rp1L1 play essential and synergistic roles in affecting photosensitivity and OS morphogenesis of rod photoreceptors. Our findings suggest that mutations inRP1L1could underlie retinopathy or modify RP1 disease expression in humans.

Mouse retina undergoes crucial changes during early postnatal development. By using Affymetrix microarrays, we analyzed gene expression profiles of wild-type 129SvEv/C57BL/6 mouse retinas at postnatal days (P) 7, 10, 14, 18, and 21 and found significantly altered expression of 355 genes. Characterization of these 355 genes provided insight into physiologic and pathologic processes of mouse retinal development during the second and third postnatal weeks, a period that corresponds to human embryogenesis between weeks 12 and 28. These genes formed 6 groups with similar change patterns. Among the genes, sixteen cause retinal diseases when mutated; most of these 16 genes were upregulated in retina during this period. Using the PathArt program, we identified the biological processes in which many of the 355 gene products function. Among the most active processes in the P7-P21 retina are those involved in neurogenesis, obesity, diabetes type II, apoptosis, growth and differentiation, and protein kinase activity. We examined the expression patterns of 58 genes in P7 and adult retinas by searching the Brain Gene Expression Map database. Although most genes were present in various cell types in retinas, many displayed high levels of expression specifically in the outer nuclear, inner nuclear, and/or ganglion cell layers. By combining our 3 analyses, we demonstrated that during this period of mouse retinal development, many genes play important roles in various cell types, multiple pathways are involved, and some genes in a pathway are expressed in coordinated patterns. Our results thus provide foundation for future detailed studies of specific genes and pathways in various genetic and environmental conditions during retinal development.

To understand the mechanisms underlying autosomal dominant progressive retinitis pigmentosa (RP) caused by the mutations of the RP1 gene and to identify molecules that play roles in the early disease process, we used Affymetrix U74Av2 microarrays to compare the gene expression profiles of retinas from Rp1-/- and Rp1+/+ mice at postnatal days (P) 7, 10, 14, 18 and 21. These profiles were independently verified by comparison with results of retinal serial analysis of gene expression, U74Av2 array studies of mouse retinas, real-time PCR and in situ hybridization. We found that the disruption of Rp1 significantly affected the expression of multiple clusters of genes whose products were involved in diverse biological pathways. The molecular responses to the disruption of Rp1 changed dramatically during development and were distinct from responses to the disruption of photoreceptor transcription factors (Crx-/- or Nrl-/-) and a phototransduction molecule (Pde6brd1). We found specific alterations of gene expression in the c-Jun N-terminal kinase (JNK) signaling cascades. Western analysis confirmed that the phosphorylation of key members in the JNK signaling cascades (i.e. JNK1, JNK2, MAP2, MKK4 and c-Jun) is reduced, whereas phospho-ERK and phospho-p38 are unchanged, in Rp1-/- retinas at P18-21. Immunostaining demonstrated that, like Rp1, phospho-JNKs and phospho-MAP2 are present in outer segments of photoreceptors. Our studies reveal unique molecular phenotypes in multiple biological pathways and the specific reduction of JNK signaling cascades in RP1 diseases, and suggest that RP1, a doublecortin-containing microtubule associated protein, and JNK signaling cascades play integral roles in photoreceptor development and maintenance. Our studies further suggest JNK-related therapeutic strategies for RP1 diseases.

POE (Post-Occupancy Evaluation) Methodology is an effective tool to find users' evaluation to built projects and help improve design. In this paper three ALSA prized projects in China as cases are explored, with some common characters of Minimum-Intervention-Design (MID) and have built as urban waterfront park, designed by the same office. The present study was performed to evaluate users' satisfaction and understanding rates to the three parks and their concepts basing on the POE method. Interesting results are: 1) Users' satisfaction rates are high in all of three parks. 2) MID creates diverse familiar spaces and opportunities for people to think of wildness and learn native plants resulting in high recognition. 3) Users' understanding and acceptation of the park have positive correlation with explanation system. 4) Post built maintenance influences the users' altitudes and turns to be a very important factor for more application of MID.

Introduction: The glutathione-S-transferase P1 (GSTP1) protein is highly expressed in melanoma and other human cancers, and is associated with resistance to chemotherapy, aggressive tumor growth, and poor overall patient survival. In addition to its function in phase II metabolism, GSTP1 is a major regulator of downstream signaling pathways that regulate cell survival and apoptosis. In this study, we investigated the role of GSTP1 and of altered MAP kinase signaling in apoptotic signaling and survival of melanoma cells. Methods: Human melanoma cell lines, A375 and DM6, were infected with a lentiviral vector carrying short hairpin RNA (shRNA) targeting GSTP1 or eGFP (vector controls). The infected and uninfected (control) cells were subjected to puromycin selection pressure, and GSTP1 expression in the heterogeneous surviving cells monitored by PCR and western blotting. Population growth kinetics was performed in real time by automated electronic cell sensing. Apoptosis was measured by flow cytometry (FCM) of propidium iodide stained cells and by assaying caspase 3/7 activation levels. Alterations in MAP kinase (P38, ERK, and JNK) levels and phosphorylation state were determined by western blotting, and apoptosis and MAP kinase related gene expression by focused array PCR. Results: GSTP1 transcripts and protein were significantly (up to 90%) suppressed in both cell lines, following GSTP1-shRNA-lentiviral infection and antibiotic selection, with the effect greater in A375 than in DM6. In both cell lines, however, GSTP1 suppression induced a significant decrease in growth rate, R (from 0.058 Hr−1 to 0.007 Hr−1 in A375 and 0.0395 Hr−1 to 0.021 Hr−1 in DM6). Similarly, population doubling times, td, increased from 12.0 hrs to 100.2 hrs in A375 and 17.6 hrs to 33.2 hrs in DM6, and baseline apoptosis increased significantly in both GSTP1-ve cell lines. While MAP kinase (ERK) phosphorylation increased, absolute levels remained unchanged. P38 was unaffected. Focused PCR analysis showed increased transcript levels of ERK regulated cell-cycle genes, including, CDKN2A and CDKN2D in GSTP1-ve A375 cells and CDKN1A, CDKN2C, and CDKN2D in GSTP1-ve DM6 cells. Levels of transcripts of apoptotic signaling genes were also significantly altered by GSTP1 suppression, albeit, with differences between the cell lines; caspase 1 and 10 increased ∼3-fold in A375 while BCL2 and BCL2L1 decreased >2-fold in DM6. Conclusions: Our data indicate that GSTP1 is required for growth of GSTP1-expressing melanoma cells and that in these cells; GSTP1 suppression slows growth and cell-cycle progression, and increases spontaneous apoptosis through altered MAP kinase and pro-apoptotic signaling. These observations provide a rationale for therapeutic targeting of GSTP1 in these tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1635. doi:10.1158/1538-7445.AM2011-1635

Overexpression of glutathione S-transferase p1 (GSTP1), a protein involved in Phase II metabolism and cell signaling, is a hallmark of diverse human malignancies, including gliomas, and is associated with resistance to therapy, aggressive tumor growth and poor patient survival. Although, the function of GSTP1 in drug metabolism and detoxification is well documented, its role in regulating tumor cell growth remains unclear. Here, we report that in human glioma characterized by high GSTP1 expression, GSTP1 is obligately required for the growth and survival of the tumor cells. For this, we stably infected cells of the MGR3 glioblastoma multiforme (GBM) cell line that expresses high de novo levels of GSTP1, with a lentiviral vector that either expresses a short hairpin RNA (shRNA) specifically targeting to GSTP1 or a control shRNA. We isolated two clones, 1 and 2 and showed by western blotting that the GSTP1 specific shRNA mediated decreases in GSTP1 expression of 92% and 100%, respectively, relative to controls while infected with eGFP shRNA. Growth kinetic analysis showed the in vitro growth rates of both clones 1 and 2 to be significantly lower than that of the parental or control clone, with population doubling times of 108.9 ± 17.6 hr for clone 1 and 94.8 ± 4.3 hr for clone 2, compared with 40.6 ± 2.6 hr for the parental cells and 44.7 ± 2.0 hr for the control transfected cells. Cell cycle analysis showed that the GSTP1 knockdown induced a significant almost 2-fold increase in the sub-G1 (apoptotic) population that correlated with an increase in caspase 3/7 activation. Western blotting showed no significant changes in total protein levels of ERK, JNK and p38 MAPK following GSTP1 knockdown, however, phosphorylation levels of both ERK1/2 and JNK1/2 increased significantly, while phospho- p38 MAPK levels did not change. JNK downstream signaling, viz., phosphorylation of c-Jun, increased dramatically in cells of the two clones. These alterations in MAPK signaling in the tumor cells associated with GSTP1 silencing correlated with the decreased survival and growth kinetics and corresponding changes in apoptosis/survival signals, including, decreased bcl2 and increased bax levels in the tumors. Our results show that GSTP1 is required for cell survival and growth of human GBM cells that naturally over-express high GSTP1 levels. This is consistent with the aggressive tumor growth and poor survival of patients whose tumors contain high levels of GSTP1 and provide a rationale for targeting GSTP1 as a potential therapeutic strategy for malignant gliomas. Supported by grants RO1CA112519, RO1CA127872, RO1CA91438, P30CA114236 and P5OCA108786 from the National Institutes of Health (USA). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1061.

On the basis of the treatment of 12 cases of severely burned patients with complicated residual wounds and 20 successive biopsies of the wounds at different stages in two cases, we have observed the recurrences and the healing process of the wounds, their histologic changes, and an appropriate treatment has been suggested in treating these recurrences of wounds.