Description: Aims NADPH oxidase (Nox) isozymes that generate intracellular reactive oxygen species (ROS) and Toll-like receptor 2 (TLR2), an inflammatory mediator, are both involved in the development of atherosclerotic lesions. To identify the molecular connection between TLR2 and Nox isozymes in vascular remodelling, we analysed generation of ROS and pro-inflammatory cytokines in aortic smooth muscle cells from Nox1-deficient mice in response to the synthetic triacylated lipoprotein Pam3CSK, a TLR2 agonist. Methods and results We showed that TLR2 signalling stimulates progression of the pro-inflammatory phenotype in mouse aortic smooth muscle cells (MASMCs) through activation of Nox1. We demonstrated the interaction of TLR2 with Nox1 using yeast two-hybrid and co-immunoprecipitation assays. MASMCs from Nox1-deficient mice failed to generate of ROS in response to Pam3CSK4, indicating that Nox1 is essential for TLR2-dependent production of ROS. We also found that Pam3CSK4 stimulated migration of MASMCs from wild-type mice in a Transwell system, but MASMCs from Nox1-deficient mice failed to show this response. Wild-type MASMCs produced matrix metalloprotease 2 in response to Pam3CSK4, whereas Nox1-deficient MASMCs failed to generate this protease. Moreover, stimulation of MASMCs with Pam3CSK4 resulted in increased expression of the pro-inflammatory cytokine macrophage inflammatory protein 2 in a Nox1-dependent manner, leading to enhanced monocyte-endothelial cell adhesion and trans-endothelial migration of U937 cells. Conclusion These data suggest that Nox1 plays an important role in TLR2-mediated intracellular H 2 O 2 generation, activation of matrix metalloprotease 2, and secretion of pro-inflammatory cytokines, which in turn stimulate MASMC migration and vascular remodelling.

Description: Changes in cold hardiness, carbohydrate content and β-amylase gene expression were monitored in the shoots of the highbush blueberry ( Vaccinium corymbosum L.) cultivars ‘Sharpblue’ and ‘Jersey’ during cold acclimation (CA) and deacclimation (DA). The seasonal patterns were similar in both cultivars, but the levels of cold hardiness determined by electrolyte leakage analysis were significantly different; ‘Jersey’ was hardier than ‘Sharpblue’. Cold hardiness was closely related to total soluble sugar content ( r = –0.98** and –0.99** for ‘Sharpblue’ and ‘Jersey’, respectively). In ‘Jersey’, more soluble sugars accumulated during CA. Of the detected soluble sugars, glucose, fructose and raffinose contents were significantly associated with cold hardiness in both cultivars. Sucrose was abundant in both cultivars, and stachyose content changed significantly during CA and DA. However, they were not associated with cold hardiness. A sharp decrease in starch contents in the middle of CA coincided with β-amylase gene ( VcBMY ) expression, indicating the conversion of starch into soluble sugars. During CA, VcBMY was expressed up to twofold higher in ‘Jersey’ than in ‘Sharpblue’. These results suggest that intraspecies differences in the cold hardiness of highbush blueberries are associated with total soluble sugar content, which is driven partly by differential expression of VcBMY .

Description: Double-stranded ribonucleic acid-activated protein kinase (PKR) downregulates translation as a defense mechanism against viral infection. In fish species, PKZ, a PKR-like protein kinase containing left-handed deoxyribonucleic acid (Z-DNA) binding domains, performs a similar role in the antiviral response. To understand the role of PKZ in Z-DNA recognition and innate immune response, we performed structural and functional studies of the Z-DNA binding domain (Zα) of PKZ from Carassius auratus (caZα PKZ ). The 1.7-Å resolution crystal structure of caZα PKZ :Z-DNA revealed that caZα PKZ shares the overall fold with other Zα, but has discrete structural features that differentiate its DNA binding mode from others. Functional analyses of caZα PKZ and its mutants revealed that caZα PKZ mediates the fastest B-to-Z transition of DNA among Zα, and the minimal interaction for Z-DNA recognition is mediated by three backbone phosphates and six residues of caZα PKZ . Structure-based mutagenesis and B-to-Z transition assays confirmed that Lys56 located in the β-wing contributes to its fast B-to-Z transition kinetics. Investigation of the DNA binding kinetics of caZα PKZ further revealed that the B-to-Z transition rate is positively correlated with the association rate constant. Taking these results together, we conclude that the positive charge in the β-wing largely affects fast B-to-Z transition activity by enhancing the DNA binding rate.

Description: Background Patient navigation is a promising intervention to address cancer disparities but requires a multisite controlled trial to assess its effectiveness. Methods The Patient Navigation Research Program compared patient navigation with usual care on time to diagnosis or treatment for participants with breast, cervical, colorectal, or prostate screening abnormalities and/or cancers between 2007 and 2010. Patient navigators developed individualized strategies to address barriers to care, with the focus on preventing delays in care. To assess timeliness of diagnostic resolution, we conducted a meta-analysis of center- and cancer-specific adjusted hazard ratios (aHRs) comparing patient navigation vs usual care. To assess initiation of cancer therapy, we calculated a single aHR, pooling data across all centers and cancer types. We conducted a metaregression to evaluate variability across centers. All statistical tests were two-sided. Results The 10521 participants with abnormal screening tests and 2105 with a cancer or precancer diagnosis were predominantly from racial/ethnic minority groups (73%) and publically insured (40%) or uninsured (31%). There was no benefit during the first 90 days of care, but a benefit of navigation was seen from 91 to 365 days for both diagnostic resolution (aHR = 1.51; 95% confidence interval [CI] = 1.23 to 1.84; P 〈 .001)) and treatment initiation (aHR = 1.43; 95% CI = 1.10 to 1.86; P 〈 .007). Metaregression revealed that navigation had its greatest benefits within centers with the greatest delays in follow-up under usual care. Conclusions Patient navigation demonstrated a moderate benefit in improving timely cancer care. These results support adoption of patient navigation in settings that serve populations at risk of being lost to follow-up.

Description: Background Loss of 18q22.3 is a prognostic marker in pancreatic ductal adenocarcinoma (PDAC). This study investigated genes encoded by this cytoband. Methods We studied mRNA/protein expression in radically resected (n = 130) and metastatic patients (n = 50). The role of CYB5A was tested in 11 PDAC cell lines and five primary cultures through retrovirus-mediated upregulation and small interfering RNA using wound-healing, invasion, annexin-V, electron microscopy, and autophagic assays, as well as autophagy genes and kinases arrays. CYB5A+ orthotopic models (n = 6 mice/group) were monitored by Firefly and Gaussia-luciferase bioluminescence, magnetic resonance imaging, and high-frequency ultrasound. Data were analyzed by t test, Fisher exact-test, log-rank test and Cox proportional hazards models. All statistical tests were two-sided. Results Both resected and metastatic patients with low mRNA or protein expression of CYB5A had statistically significantly shorter survival (eg, median = 16.7 months, 95% confidence interval [CI] = 13.5 to 19.9; vs median = 24.8 months, 95% CI = 12.8 to 36.9; P = .02, two-sided log-rank test; n = 82 radically resected PDACs), and multivariable analyses confirmed prognostic relevance. Moreover, we characterized a novel function to CYB5A, autophagy induction, concomitant with reduced proliferation and migration/invasion of PDAC cells. Network analysis of proautophagic pathways suggested CYB5A interaction with TRAF6, which was confirmed by TRAF6 downregulation after CYB5A reconstitution (–69% in SU.86.86-CYB5A+; P = .005, two-sided t test). CYB5A silencing had opposite effects, restoring TRAF6 expression and wound healing. In vivo studies showed that CYB5A induced autophagy while inhibiting tumor growth/metastasis and increasing survival (median = 57 days, 95% CI = 52 to 61; vs median = 44 days, 95% CI = 21 to 57; P = .03, two-sided log-rank test). Conclusions These results define CYB5A as a novel prognostic factor for PDAC that exerts its tumor-suppressor function through autophagy induction and TRAF6 modulation.

Description: It is currently difficult to determine the molecular and cellular basis for radioscintigraphic signals obtained during macroscopic in vivo imaging. The field is in need of technology that helps bridge the macroscopic and microscopic regimes. To solve this problem, we developed a fiducial marker (FM) simultaneously compatible with 2-color near-infrared (NIR) fluorescence (700 and 800 nm), autoradiography, and conventional hematoxylin–eosin (HE) histology. Methods: The FM was constructed from an optimized concentration of commercially available human serum albumin, 700- and 800-nm NIR fluorophores, 99m Tc-pertechnetate, dimethyl sulfoxide, and glutaraldehyde. Lymphangioleiomyomatosis cells coexpressing the sodium iodide symporter and green fluorescent protein were labeled with 700-nm fluorophore and 99m Tc-pertechnatate and then administered intratracheally into CD-1 mice. After in vivo SPECT imaging and ex vivo SPECT and NIR fluorescence imaging of the lungs, 30-μm frozen sections were prepared and processed for 800-nm NIR fluorophore costaining, autoradiography, and HE staining on the same slide using the FMs to coregister all datasets. Results: Optimized FMs, composed of 100 μM unlabeled human serum albumin, 1 μM NIR fluorescent human serum albumin, 15% dimethyl sulfoxide, and 3% glutaraldehyde in phosphate-buffered saline (pH 7.4), were prepared within 15 min, displayed homogeneity and stability, and were visible by all imaging modalities, including HE staining. Using these FMs, tissue displaying high signal by SPECT could be dissected and analyzed on the same slide and at the microscopic level for 700-nm NIR fluorescence, 800-nm NIR fluorescence, autoradiography, and HE histopathologic staining. Conclusion: When multimodal FMs are combined with a new technique for simultaneous same-slide NIR fluorescence imaging, autoradiography, and HE staining, macroscopic in vivo images can now be studied unambiguously at the microscopic level.

Description: Two red algal classes, the Florideophyceae (approximately 7,100 spp.) and Bangiophyceae (approximately 193 spp.), comprise 98% of red algal diversity in marine and freshwater habitats. These two classes form well-supported monophyletic groups in most phylogenetic analyses. Nonetheless, the interordinal relationships remain largely unresolved, in particular in the largest subclass Rhodymeniophycidae that includes 70% of all species. To elucidate red algal phylogenetic relationships and study organelle evolution, we determined the sequence of 11 mitochondrial genomes (mtDNA) from 5 florideophycean subclasses. These mtDNAs were combined with existing data, resulting in a database of 25 florideophytes and 12 bangiophytes (including cyanidiophycean species). A concatenated alignment of mt proteins was used to resolve ordinal relationships in the Rhodymeniophycidae. Red algal mtDNA genome comparisons showed 47 instances of gene rearrangement including 12 that distinguish Bangiophyceae from Hildenbrandiophycidae, and 5 that distinguish Hildenbrandiophycidae from Nemaliophycidae. These organelle data support a rapid radiation and surprisingly high conservation of mtDNA gene syntheny among the morphologically divergent multicellular lineages of Rhodymeniophycidae. In contrast, we find extensive mitochondrial gene rearrangements when comparing Bangiophyceae and Florideophyceae and multiple examples of gene loss among the different red algal lineages.

Description: Telomerase, a unique ribonucleoprotein complex that contains the telomerase reverse transcriptase (TERT), the telomerase RNA component (TERC) and the TERC-binding protein dyskerin, is required for continued cell proliferation in stem cells and cancer cells. Here we identify SRSF11 as a novel TERC-binding protein that localizes to nuclear speckles, subnuclear structures that are enriched in pre-messenger RNA splicing factors. SRSF11 associates with active telomerase enzyme through an interaction with TERC and directs it to nuclear speckles specifically during S phase of the cell cycle. On the other hand, a subset of telomeres is shown to be constitutively present at nuclear speckles irrespective of cell cycle phase, suggesting that nuclear speckles could be the nuclear sites for telomerase recruitment to telomeres. SRSF11 also associates with telomeres through an interaction with TRF2, which facilitates translocation of telomerase to telomeres. Depletion of SRSF11 prevents telomerase from associating with nuclear speckles and disrupts telomerase recruitment to telomeres, thereby abrogating telomere elongation by telomerase. These findings suggest that SRSF11 acts as a nuclear speckle-targeting factor that is essential for telomerase association with telomeres through the interactions with TERC and TRF2, and provides a potential target for modulating telomerase activity in cancer.

Description: Phosphorylation of the histone variant H2AX forms -H2AX that marks DNA double-strand break (DSB). Here, we generated the sequencing-based maps of H2AX and -H2AX positioning in resting and proliferating cells before and after ionizing irradiation. Genome-wide locations of possible endogenous and exogenous DSBs were identified based on -H2AX distribution in dividing cancer cells without irradiation and that in resting cells upon irradiation, respectively. -H2AX-enriched regions of endogenous origin in replicating cells included sub-telomeres and active transcription start sites, apparently reflecting replication- and transcription-mediated stress during rapid cell division. Surprisingly, H2AX itself, prior to phosphorylation, was specifically located at these endogenous hotspots. This phenomenon was only observed in dividing cancer cells but not in resting cells. Endogenous H2AX was concentrated on the transcription start site of actively transcribed genes but was irrelevant to pausing of RNA polymerase II (pol II), which precisely coincided with -H2AX of endogenous origin. -H2AX enrichment upon irradiation also coincided with actively transcribed regions, but unlike endogenous -H2AX, it extended into the gene body and was not specifically concentrated on the pausing site of pol II. Sub-telomeres were less responsive to external DNA damage than to endogenous stress. Our findings provide insight into DNA repair programs of cancer and may have implications for cancer therapy.

Description: Metabolic tumor volume (MTV) and total lesion glycolysis (TLG) from 18 F-FDG PET/CT are emerging prognostic biomarkers in various human cancers. This study examined the prognostic value of these metabolic tumor parameters measured by pretreatment 18 F-FDG PET/CT in patients with salivary gland carcinomas. Methods: Forty-nine patients with intermediate- or high-grade salivary gland carcinomas who underwent definitive surgery with or without radiotherapy or chemoradiotherapy were evaluated preoperatively by 18 F-FDG PET/CT. Maximum standardized uptake values (SUV max ), MTV, and TLG were measured for each patient. Univariate and multivariate analyses were used to identify clinicopathologic and imaging variables associated with progression-free survival (PFS) and overall survival (OS). Univariate analyses included the following variables: age, sex, pT and pN classifications, overall pTNM stage, histologic grade, resection margin, tumor lymphovascular invasion and perineural invasion, postoperative adjuvant therapy, gross total volume, SUV max , MTV, and TLG. Results: The 3-y PFS and OS rates for all study patients were 66.9% and 81.6%, respectively. The median SUV max , MTV, and TLG were 5.1 (range, 1.7–21.5), 16.2 mL (1.0–115.1 mL), and 24.4 g (2.1–224.4 g), respectively. Univariate analyses showed that there were significant correlations between pT classification, pN classification, MTV, and TLG and both PFS and OS ( P 〈 0.05). However, SUV max was not associated with either PFS ( P = 0.111) or OS ( P = 0.316). Multivariate analyses revealed that MTV ( P = 0.011; hazard ratio, 11.50; 95% confidence interval, 1.45–91.01) and TLG ( P = 0.038; hazard ratio, 3.55; 95% confidence interval, 1.07–11.76) were independent variables for PFS. Conclusion: Pretreatment values of MTV and TLG are independent prognostic factors in patients with intermediate or high-grade salivary gland carcinomas.