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Supplemental Table 3: Differential gene expression analysis between B16 KO and WT tumors in vivo at day 6. Showing genes with q < 0.05 and log2FC > 1 or < -1.

Supplementary Figure 1: Impact of PAK4 deletion on the expression of chemokines.

Supplementary Figure 8: PAK4 KO in human melanoma cells impairs B-catenin/WNT signalling.

Supplementary Figure 4: PAK4 deletion does not affect MHC-I and II surface expression.

Supplemental Table 6: List of extracellular matrix genes enriched in PAK4 KO anti-PD-1 treated samples.

Supplementary Figure 2: PAK4 deletion does not affect CCL21 secretion.

Supplementary Figure 3: No difference in signalling through different stimuli between PAK4 KO and WT cells.

Supplemental Table 4: Differential gene expression analysis between B16 PAK4 KO anti-PD-1 vs PAK4 KO isotype tumors in vivo at day 10. Showing genes with q < 0.05 and log2FC > 2 or < -2.

Supplemental Table 2: Differential gene expression analysis between B16 KO and WT cells in vitro. Showing genes with q < 0.05 and log2FC > 1 or < -1.

Supplementary Table 1: Panel of immune markers for dendritic cell characterization.

Supplementary Figure 6: Spatial colocalization of CD8 and CD31 positive cells.

Supplemental Table 5: Differential gene expression analysis between B16 PAK4 KO anti-PD-1 vs WT anti-PD-1 tumors in vivo at day 10. Showing genes with q < 0.05 and log2FC > 1 or < -1.

Supplementary Figure 9: PAK4 inhibitor, A0317859, also synergizes with PD-1 blockade in YUMM2.1 melanoma cells.

Supplementary Figure 7: PAK4 protein expression levels in the different cell lines.

Supplementary Figure 5: PAK4 deletion increases CXCL10 expression in vivo.

PAK4 inhibition can sensitize tumors to immune checkpoint blockade (ICB) therapy; however, the underlying mechanisms remain unclear. We report that PAK4 inhibition reverses immune cell exclusion by increasing the infiltration of CD8 T cells and CD103+ dendritic cells (DC), a specific type of DCs that excel at cross-presenting tumor antigens and constitute a source of CXCL10. Interestingly, in melanoma clinical datasets, PAK4 expression levels negatively correlate with the presence of CCL21, the ligand for CCR7 expressed in CD103+ DCs. Furthermore, we extensively characterized the transcriptome of PAK4 knockout (KO) tumors, in vitro and in vivo, and established the importance of PAK4 expression in the regulation of the extracellular matrix, which can facilitate immune cell infiltration. Comparison between PAK4 wild type and KO anti-PD-1 treated tumors revealed how PAK4 deletion sensitizes tumors to ICB from a transcriptomic perspective. In addition, we validated genetically and pharmacologically that inhibition of PAK4 kinase activity is sufficient to improve antitumor efficacy of anti-PD-1 blockade in multiple melanoma mouse models. Therefore, this study provides novel insights into the mechanism of action of PAK4 inhibition and provides the foundation for a new treatment strategy that aims to overcome resistance to PD-1 blockade by combining anti-PD-1 with a small-molecule PAK4 kinase inhibitor. Significance: Our findings provide new insights into PAK4 inhibition mechanism of action as well as the scientific foundation for specifically blocking PAK4 kinase activity using a novel and specific PAK4 kinase inhibitor to overcome resistance to PD-1 blockade.

We describe a case of mild lower urinary tract symptoms and microscopic hematuria in a 53-year-old-male with hypertension found to have urethral stricture disease suspicious for urothelial carcinoma. During the investigation, cystoscopy and biopsy demonstrated eosinophilic amyloid proteins consistent with primary localized urethral amyloidosis. No systemic evidence of amyloidosis was demonstrated. Following a trial of conservative management with serial dilatations, the patient elected to proceed with surgical management by anterior urethroplasty using an excision and primary anastomosis technique. The patient has done well with resolution of his symptoms and no further recurrence of urethral amyloid disease at ongoing follow up.

National joint replacement registries outside North America have been effective in reducing revision risk. However, there is little information on the role of smaller regional registries similar to those found in Canada or the United States. We sought to understand trends in total hip (THA) and knee (TKA) arthroplasty revision patterns after implementation of a regional registry.We reviewed our regional joint replacement registry containing all 30 252 cases of primary and revision THA and TKA performed between Jan. 1, 2005, and Dec. 31, 2013. Each revision case was stratified into early (2 yr), mid (2-10 yr) or late (10 yr), and we determined the primary reason for revision.The early revision rate for TKA dropped from 3.0% in 2005 to 1.3% in 2011 (R(2) = 0.84, p = 0.003). Similarly, the early revision rate for THA dropped from 4.2% to 2.1% (R(2) = 0.78, p = 0.008). Despite primary TKA and THA volumes increasing by 35.5% and 39.5%, respectively, there was no concomitant rise in revision volumes. The leading reasons for TKA revision were infection, instability, aseptic loosening and stiffness. The leading reasons for THA revision were infection, instability, aseptic loosening and periprosthetic fracture. There were no discernible trends over time in reasons for early, mid-term or late revision for either TKA or THA.After implementation of a regional joint replacement registry we observed a significant reduction in early revision rates. Further work investigating the mechanism by which registry reporting reduces early revision risk is warranted.Ailleurs qu'en Amérique du Nord, les registres nationaux des remplacements articulaires ont été efficaces pour réduire le risque de révision. Cependant, il y a peu d'information sur le rôle des plus petits registres régionaux comme ceux qu'on trouve au Canada et aux États-Unis. Nous avons donc cherché à comprendre les tendances en matière de révision des arthroplasties totales de la hanche (ATH) et du genou (ATG) après la création d'un registre régional.Nous avons passé en revue notre registre régional des remplacements articulaires, qui contient les 30 252 ATH et ATG primaires et de révision effectuées entre le 1er janvier 2005 et le 31 décembre 2013. Chaque cas de révision a été classé précoce (2 ans), moyen (de 2 à 10 ans) ou tardif (10 ans), et nous avons déterminé la raison principale de la révision.Le taux de révision précoce pour l'ATG a diminué de 3,0 % en 2005 à 1,3 % en 2011 (Nous avons observé une baisse significative des taux de révision précoce après la mise en œuvre d'un registre régional des remplacements articulaires. Il serait pertinent d'étudier plus en profondeur le mécanisme par lequel le signalement dans un registre réduit le risque de révision précoce.

PurposeThe purpose of this paper is to provide details on implementation of inexpensive and accurate automation solution for performing brisket cutting while the sheep/lamb is hanging by forelegs.Design/methodology/approachThe system developed uses 6 Degree of freedom (DOF) industrial manipulator, custom build tool, sterilizer, and other off the shelf products to create a machine, all the parameters in the system are designed to ensure that the highest levels of safety and hygiene standards are met. The primary system has been developed for sheep/lamb.FindingsIt is shown that the system developed enhanced both the productivity and quality of the process, while adhering to the hygiene standard in the meat processing plants.Research limitations/implicationsThe process of precise brisket can be very challenging; the system designed uses customised tools to provide a good model for the trajectory planning.Originality/valueThe system developed uses statistical tools to calculate the correct trajectories.

Although energy efficiency implementation is increasingly being recognized by policymakers worldwide as one of the most effective means to mitigating rising energy prices, tackling potential environmental risks, and enhancing energy security, mainstreaming its financing in developing country markets continues to be a challenge. Experience shows that converting cost-effective energy savings potential, particularly the demand-side improvement opportunities across sectors, into investments face many barriers and unforeseen transaction costs. This paper draws upon selected experiences with financing energy efficiency in developing countries to explore the key factors of various programmatic approaches and financing instruments that have been applied successfully for delivering energy efficiency solutions. Through case studies, a diverse range of institutional issues are examined related to the identification, packaging, designing, and monitoring approaches that have been used to catalyze traditional and innovative financing of energy efficiency projects. With adequate liquidity in major developing country markets and availability of modern energy savings technologies, it is often the institutional issues that become a key challenge to address in order to finance and implement robust programs. As further operational experience is gained, increased knowledge sharing can lead to scaling-up of such energy efficiency investments. The paper concludes with some ideas for accelerating implementation.

The maize Golden2-like or GLK genes encode proteins belonging to a class of GARP domain transcriptional activators. GLKs have been observed to be involved in chloroplast development and stress adaptation. In spite of the importance of GLKs in plant processes, the in planta targets of GLK regulated transcription, however, have not been identified. We use GLK1 overexpression (OE) to study reprogramming of gene expression networks by GLK1 in Arabidopsis and to identify an associated phenotype. GLK1 OE in Arabidopsis confers resistance to Fusarium graminearum, a broad host pathogen responsible for major losses in cereal crops. Affymetrix Gene Chip and RT-PCR analyses indicated that GLK1 OE in Arabidopsis reprogrammed gene expression networks to enhance a high constitutive expression of genes encoding proteins involved in basal defences but downregulated genes that are associated with programmed cell death and hypersensitive responses. The nature of the role of GLK1 overexpression in selectively reprogramming defence gene networks could explain its effectiveness in enhancing the development of resistance to the necrotrophic phase of F. graminearum pathogenesis.

A robust phenotypic plasticity to ward off adverse environmental conditions determines performance and productivity in crop plants. Flax (linseed), is an important cash crop produced for natural textile fiber (linen) or oilseed with many health promoting products. This crop is prone to drought stress and yield losses in many parts of the world. Despite recent advances in drought research in a number of important crops, related progress in flax is very limited. Since, response of this plant to drought stress has not been addressed at the molecular level; we conducted microarray analysis to capture transcriptome associated with induced drought in flax. This study identified 183 differentially expressed genes (DEGs) associated with diverse cellular, biophysical and metabolic programs in flax. The analysis also revealed especially the altered regulation of cellular and metabolic pathways governing photosynthesis. Additionally, comparative transcriptome analysis identified a plethora of genes that displayed differential regulation both spatially and temporally. These results revealed co-regulated expression of 26 genes in both shoot and root tissues with implications for drought stress response. Furthermore, the data also showed that more genes are upregulated in roots compared to shoots, suggesting that roots may play important and additional roles in response to drought in flax. With prolonged drought treatment, the number of DEGs increased in both tissue types. Differential expression of selected genes was confirmed by qRT-PCR, thus supporting the suggested functional association of these intrinsic genes in maintaining growth and homeostasis in response to imminent drought stress in flax. Together the present study has developed foundational and new transcriptome data sets for drought stress in flax.

Arabidopsis thaliana GOLDEN2‐LIKE (GLK1 and 2) transcription factors regulate chloroplast development in a redundant manner. Overexpression of AtGLK1 (35S:AtGLK1) in Arabidopsis also confers resistance to the cereal pathogen Fusarium graminearum. To further elucidate the role of GLK transcription factors in plant defence, the Arabidopsis glk1 glk2 double‐mutant and 35S:AtGLK1 plants were challenged with the virulent oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) Noco2. Compared with Col‐0, glk1 glk2 plants were highly resistant to Hpa Noco2, whereas 35S:AtGLK1 plants showed enhanced susceptibility to this pathogen. Genetic studies suggested that AtGLK‐mediated plant defence to Hpa Noco2 was partially dependent on salicylic acid (SA) accumulation, but independent of the SA signalling protein NONEXPRESSOR OF PATHOGENESIS‐RELATED 1 (NPR1). Pretreatment with jasmonic acid (JA) dramatically reversed Hpa Noco2 resistance in the glk1 glk2 double mutant, but only marginally affected the 35S:AtGLK1 plants. In addition, overexpression of AtGLK1 in the JA signalling mutant coi1‐16 did not increase susceptibility to Hpa Noco2. Together, our GLK gain‐of‐function and loss‐of‐function experiments suggest that GLK acts upstream of JA signalling in disease susceptibility to Hpa Noco2. In contrast, glk1 glk2 plants were more susceptible to the necrotrophic fungal pathogen Botrytis cinerea, whereas 35S:AtGLK1 plants exhibited heightened resistance which could be maintained in the absence of JA signalling. Together, the data reveal that AtGLK1 is involved in JA‐dependent susceptibility to the biotrophic pathogen Hpa Noco2 and in JA‐independent resistance to the necrotrophic pathogen B. cinerea.

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The effects of cold acclimation and long-term elevated CO(2) on photosynthetic performance of wild-type (WT) and BnCBF17-over-expressing line of Brassica napus cv. Westar (BnCBF17-OE) grown at either 20/16 °C (non-acclimated) or 5/5 °C (cold acclimated) and at either ambient (380 μmol C mol(-1)) or elevated (700 μmol C mol(-1)) CO(2) were studied. Compared with non-acclimated WT, the BnCBF17-OE grown at 20 °C mimicked the effects of cold acclimation on WT B. napus with respect to compact dwarf phenotype and increased rates of light-saturated CO(2) assimilation and photosynthetic electron transport. This was associated with enhanced energy conversion efficiency into biomass as assessed by decreased excitation pressure coupled to decreased dependence on non-photochemical energy dissipation for a given irradiance. Growth at elevated CO(2) decreased the light and CO(2)-saturated rates of photosynthesis by 30 % for non-acclimated WT relative to growth at ambient CO(2). This was associated with inhibition in electron transport rates (20 %), decrease in amount of rbcL (35 %) and cytosolic FBPase (70 %) and increased excitation pressure and non-photochemical quenching in elevated versus ambient CO(2)-grown non-acclimated WT. In contrast, light and CO(2)-saturated rates of photosynthesis, electron transport, excitation pressure, non-photochemical quenching and levels of rbcL, cytosolic FBPase and Lhcb1 were insensitive to growth under elevated CO(2) in BnCBF17-OE and cold-acclimated WT. Thus, BnCBF17-over-expression and cold acclimation maintain enhanced energy conversion efficiency and reduced sensitivity to feedback-limited photosynthesis during long-term growth of B. napus under elevated CO(2). Our results indicated that CBFs transcription factors regulate not only freezing tolerance but also has major whole plant effects.

Background: There are currently 31 million licensed drivers (or 15% of all licensed drivers) over the age of 65. This number is expected to grow rapidly as the elderly population is predicted to double by 2030. With this impending increase of elderly drivers and their higher susceptibility to physical and mental deterioration, it is important to consider their safety as well as the safety of others on the road. State laws vary significantly as to elderly license renewal periods and renewal requirements, which could be particularly important in assessing elderly drivers’ continued ability to drive. We looked at the duration of license renewal in several states and compared elderly driver crash statistics among states with short and long renewal periods.Methods: 2009 accident data from the NHTSAwas collected and sorted with respect to age and state. Total population estimates, again sorted by age and state, were also collected from the US Census Bureau. The data sets were then compiled to create elderly accident ratios by state and compared to data on license renewal duration (RD). By performing t-tests on data stratified by different RDs, the relationship between state licensing periods and elderly driving characteristics was assessed. Results: When comparing RD to crash rates, only a renewal period of 2 years is associated with lower crash rates (p1⁄4 0.028) among the elderly. The crash rates for these states vary from 55 to 177 crashes per million elderly drivers. The five safest states for elderly drivers (DC, HI,MA, CT, andNY) have variable RDs (2-8 years). The worst 5 states for elderly drivers involved in crashes (MS, AK, MT, KY, and ND) all havemedian license RDs of 4 to 5 years.Conclusions: Frequent renewal (2 years) is associated with a reduction in elderly crashes in our analysis using state level data. However, some of the safest elderly driving states are MA and NY, with 5and 8-year renewal durations respectively. A license renewal period of more than 2 years does not seem to be a efficient predictor of elderly driving crash rates when looking at all 50 states.

A cDNA clone, pBN115, encoding a low-temperature-regulated transcript in winter Brassica napus has been isolated. Northern blot analyses show that levels of transcripts hybridizing to pBN115 increase within 24 h of exposure of B. napus to low temperature, peak at 3 d, and then remain at an elevated level for the duration of the cold treatment (up to 10 weeks). Transferring plants from 2[deg]C to room temperature results in the loss of detectable transcripts hybridizing to pBN115 within 1 d. The transcript was not detected in RNA isolated from roots of cold-acclimated B. napus. Results of in vivo labeling of nascent RNA in leaf discs of B. napus with thiouridine suggest that regulation of expression may be transcriptional, at least at the onset of cold temperature. Although drought stress leads to a slight increase in transcript level at room temperature, neither a brief exposure to elevated temperatures nor exogenous application of abscisic acid resulted in the appearance of the transcript represented by pBN115. Furthermore, transcripts hybridizing to pBN115 were present at the same levels whether the plants were acclimated in the light or dark. Hybridization experiments show that pBN115 hybridizes strongly to cold-regulated transcripts in Arabidopsis thaliana, Descurania sophia, and spring B. napus, all of which are cruciferous plants capable of cold acclimation. No hybridizing transcript could be detected in cold-acclimated Spinacea oleracea, winter Secale cereale, or cold-grown Nicotiana tabacum. DNA sequence analysis of pBN115 reveals a single open reading frame that potentially encodes a protein of 14.8 kD. This size closely approximates that of a polypeptide produced by in vitro transcription/translation experiments. Two additional cDNA clones, pBN19 and pBN26, with divergent 5[prime]- and 3[prime]-untranslated regions, were also isolated and found to encode similar, but not identical, polypeptides.

This book explores energy savings performance contracts (ESPCs) as a means of overcoming some of the more difficult hurdles in promoting energy efficiency in public facilities. ESPCs represent a very attractive solution to many of the problems that are unique to public agencies, since they involve outsourcing a full project cycle to a service provider. From the detailed audit through implementation and savings verification, ESPCs can relieve public agencies of bureaucratic hassles, while service providers can secure the off-budget project financing and be paid from the actual energy savings, thus internalizing project performance risks. ESPC bidding also allows public agencies to select from a range of technical solutions, maximizing the benefit to the agency. Global experience suggests that ESPCs have been more effective at realizing efficiency gains than many other policy measures and programs, since the service providers have a vested interest in ensuring that a project is actually implemented. Many of the country governments interviewed for the study also saw enormous potential in bundling, financing, and implementing energy efficiency projects on a larger scale in the public sector, a method that increases the rate of efficiency gains and creates further benefits through economies of scale.

Aim: To assess the association of floppy iris behaviour during cataract surgery with use of α-1-antagonists and diabetes mellitus. Methods: 1842 eyes of 1786 patients undergoing phacohoemulsification surgery were prospectively enrolled. The use of commonly prescribed α-1-antagonists and the presence or absence of diabetes mellitus were noted. The occurrence of any of the features of the intraoperative floppy iris syndrome (IFIS) was noted by surgeons blinded to the patient’s history. Results: 57% of patients receiving tamsulosin showed features of IFIS compared with 1% of the non-tamsulosin group (p

The effects of overexpression of two Brassica CBF/DREB1-like transcription factors (BNCBF5 and 17) in Brassica napus cv. Westar were studied. In addition to developing constitutive freezing tolerance and constitutively accumulating COR gene mRNAs, BNCBF5- and 17-overexpressing plants also accumulate moderate transcript levels of genes involved in photosynthesis and chloroplast development as identified by microarray and Northern analyses. These include GLK1- and GLK2-like transcription factors involved in chloroplast photosynthetic development, chloroplast stroma cyclophilin ROC4 (AtCYP20-3), beta-amylase and triose-P/Pi translocator. In parallel with these changes, increases in photosynthetic efficiency and capacity, pigment pool sizes, increased capacities of the Calvin cycle enzymes, and enzymes of starch and sucrose biosynthesis, as well as glycolysis and oxaloacetate/malate exchange are seen, suggesting that BNCBF overexpression has partially mimicked cold-induced photosynthetic acclimation constitutively. Taken together, these results suggest that BNCBF/DREB1 overexpression in Brassica not only resulted in increased constitutive freezing tolerance but also partially regulated chloroplast development to increase photochemical efficiency and photosynthetic capacity.

Mortality from prostate cancer is associated with progression of tumors to androgen-independent growth and metastasis. Eicosanoid products of both the cyclooxygenase (COX) and lipoxygenase (LOX) pathways are important mediators of the proliferation of prostate cancer cells in culture and regulate tumor vascularization and metastasis in animal models. Pharmacologic agents that block either COX or LOX products effectively reduce the size of prostate cancer xenografts. Phospholipase A(2) (PLA(2)) enzymes regulate the provision of arachidonic acid to both COX- and LOX-derived eicosanoids, and a secreted form of the enzyme (sPLA(2)-IIA) is elevated in prostate cancer tissues. Here, we show by immunohistochemistry, in patients receiving androgen ablation therapy, that sPLA(2)-IIA remains elevated in remaining cancer cells relative to benign glands after treatment. Furthermore, sPLA(2)-IIA expression seen in benign glands is substantially decreased after androgen depletion, whereas cytosolic PLA(2)-alpha (cPLA(2)-alpha) levels are unchanged. sPLA(2)-IIA mRNA expression is detectable and inducible by androgen (0.01-10 nmol/L) in the androgen-sensitive cell line LNCaP, and exogenous addition of sPLA(2)-IIA (1-100 nmol/L), but not an inactive sPLA(2)-IIA mutant (H(48)Q), results in a dose-dependent increase in cell numbers or the fraction of cells in G(2)-M phase, which is inhibited by sPLA(2)-IIA-selective inhibitors. The effect of exogenous sPLA(2)-IIA can also be blocked by inhibition of cPLA(2)-alpha, suggesting a role for cPLA(2)-alpha in mediating sPLA(2)-IIAlpha action. sPLA(2)-IIA inhibitors suppressed basal proliferation in LNCaP cells and in the androgen-independent, sPLA(2)-positive cell line PC3 but not in the sPLA(2)-IIA-negative androgen-independent cell line DU145. Established PC3 xenograft tumors grew more slowly in mice treated with sPLA(2)-IIA inhibitors than those treated with saline only. The PLA(2) enzymes, and sPLA(2)-IIA in particular, thus represent important targets for the treatment of sPLA(2)-IIA-positive androgen-independent prostate cancer.