Microvascular smooth muscle cells exhibit divergent phenotypic switching responses to platelet-derived growth factor and insulin-like growth factor 1.

OBJECTIVE: Vascular smooth muscle cell (VSMC) phenotypic switching is critical for normal vessel formation, vascular stability, and healthy brain aging. Phenotypic switching is regulated by mediators including platelet derived growth factor (PDGF)-BB, insulin-like growth factor (IGF-1), as well as transforming growth factor-ß (TGF-ß) and endothelin-1 (ET-1), but much about the role of these factors in microvascular VSMCs remains unclear. METHODS: We used primary rat microvascular VSMCs to explore PDGF-BB- and IGF-1-induced phenotypic switching. RESULTS: PDGF-BB induced an early proliferative response, followed by formation of polarized leader cells and rapid, directionally coordinated migration. In contrast, IGF-1 induced cell hypertrophy, and only a small degree of migration by unpolarized cells. TGF-ß and ET-1 selectively inhibit PDGF-BB-induced VSMC migration primarily by repressing migratory polarization and formation of leader cells. Contractile genes were downregulated by both growth factors, while other genes were differentially regulated by PDGF-BB and IGF-1. CONCLUSIONS: These studies indicate that PDGF-BB and IGF-1 stimulate different types of microvascular VSMC phenotypic switching characterized by different modes of cell migration. Our studies are consistent with a chronic vasoprotective role for IGF-1 in VSMCs in the microvasculature while PDGF is more involved in VSMC proliferation and migration in response to acute activities such as neovascularization. Better understanding of the nuances of the phenotypic switching induced by these growth factors is important for our understanding of a variety of microvascular diseases.

Digestive Manifestations in Patients Hospitalized With Coronavirus Disease 2019.

BACKGROUND & AIMS: The prevalence and significance of digestive manifestations in coronavirus disease 2019 (COVID-19) remain uncertain. We aimed to assess the prevalence, spectrum, severity, and significance of digestive manifestations in patients hospitalized with COVID-19. METHODS: Consecutive patients hospitalized with COVID-19 were identified across a geographically diverse alliance of medical centers in North America. Data pertaining to baseline characteristics, symptomatology, laboratory assessment, imaging, and endoscopic findings from the time of symptom onset until discharge or death were abstracted manually from electronic health records to characterize the prevalence, spectrum, and severity of digestive manifestations. Regression analyses were performed to evaluate the association between digestive manifestations and severe outcomes related to COVID-19. RESULTS: A total of 1992 patients across 36 centers met eligibility criteria and were included. Overall, 53% of patients experienced at least 1 gastrointestinal symptom at any time during their illness, most commonly diarrhea (34%), nausea (27%), vomiting (16%), and abdominal pain (11%). In 74% of cases, gastrointestinal symptoms were judged to be mild. In total, 35% of patients developed an abnormal alanine aminotransferase or total bilirubin level; these were increased to less than 5 times the upper limit of normal in 77% of cases. After adjusting for potential confounders, the presence of gastrointestinal symptoms at any time (odds ratio, 0.93; 95% CI, 0.76-1.15) or liver test abnormalities on admission (odds ratio, 1.31; 95% CI, 0.80-2.12) were not associated independently with mechanical ventilation or death. CONCLUSIONS: Among patients hospitalized with COVID-19, gastrointestinal symptoms and liver test abnormalities were common, but the majority were mild and their presence was not associated with a more severe clinical course.

Constraining Cosmological Phase Transitions with the Parkes Pulsar Timing Array.

A cosmological first-order phase transition is expected to produce a stochastic gravitational wave background. If the phase transition temperature is on the MeV scale, the power spectrum of the induced stochastic gravitational waves peaks around nanohertz frequencies, and can thus be probed with high-precision pulsar timing observations. We search for such a stochastic gravitational wave background with the latest data set of the Parkes Pulsar Timing Array. We find no evidence for a Hellings-Downs spatial correlation as expected for a stochastic gravitational wave background. Therefore, we present constraints on first-order phase transition model parameters. Our analysis shows that pulsar timing is particularly sensitive to the low-temperature (T∼1-100 MeV) phase transition with a duration (ß/H_{*})^{-1}∼10^{-2}-10^{-1} and therefore can be used to constrain the dark and QCD phase transitions.

A Theoretical Iteration for Predicting the Feasibility for Immediate Functional Dental Implant Loading.

When planning an implant-supported restoration, the dentist is faced with surgical and prosthetic technical issues as well as the patient's expectations. Many patients wish an immediate solution to an edentulous condition. This may be especially true in the esthetic zone, and that zone is determined by the patient. The dentist may consider when it is feasible to load the supporting implants with definitive or provisional prosthetics. In this work, many parameters were theoretically assessed for inclusion: bone density, cortical thickness, insertion torque, parafunction, bite load capacity, number of implants under load, implant/crown ratio, implant diameter, and length. After assessment, the most influential parameters were selected. An iteration, using patient age, implant diameter, bite load capacity, and cortical thickness, is now presented to aid the implant dentist in determining the feasibility for immediate functional loading of a just-placed dental implant in a healed site. Extensive testing is required to develop this concept. According to this iteration, most immediate functional loaded implants would fail. A future refined and definitive formula may enable the clinician to safely and immediately functionally load an implant with a definitive prosthesis. For access to the applet, please go to https://implantloading.shinyapps.io/shiny_app/.

Building the foundations for sustainable development: a case for global investment in the capabilities of adolescents.

Investment in the capabilities of the world's 1·2 billion adolescents is vital to the UN's Sustainable Development Agenda. We examined investments in countries of low income, lower-middle income, and upper-middle income covering the majority of these adolescents globally to derive estimates of investment returns given existing knowledge. The costs and effects of the interventions were estimated by adapting existing models and by extending methods to create new modelling tools. Benefits were valued in terms of increased gross domestic product and averted social costs. The initial analysis showed high returns for the modelled interventions, with substantial variation between countries and with returns generally higher in low-income countries than in countries of lower-middle and upper-middle income. For interventions targeting physical, mental, and sexual health (including a human papilloma virus programme), an investment of US$4·6 per capita each year from 2015 to 2030 had an unweighted mean benefit to cost ratio (BCR) of more than 10·0, whereas, for interventions targeting road traffic injuries, a BCR of 5·9 (95% CI 5·8-6·0) was achieved on investment of $0·6 per capita each year. Interventions to reduce child marriage ($3·8 per capita each year) had a mean BCR of 5·7 (95% CI 5·3-6·1), with the effect high in low-income countries. Investment to increase the extent and quality of secondary schooling is vital but will be more expensive than other interventions-investment of $22·6 per capita each year from 2015 to 2030 generated a mean BCR of 11·8 (95% CI 11·6-12·0). Investments in health and education will not only transform the lives of adolescents in resource-poor settings, but will also generate high economic and social returns. These returns were robust to substantial variation in assumptions. Although the knowledge base on the impacts of interventions is limited in many areas, and a major research effort is needed to build a more complete investment framework, these analyses suggest that comprehensive investments in adolescent health and wellbeing should be given high priority in national and international policy.

Evolution of a holistic systems approach to planning and managing road safety: the Victorian case study, 1970-2015.

BACKGROUND: The Victorian Safe System approach to road safety slowly evolved from a combination of the Swedish Vision Zero philosophy and the Sustainable Safety model developed by the Dutch. The Safe System approach reframes the way in which road safety is viewed and managed. METHODS: This paper presents a case study of the institutional change required to underpin the transformation to a holistic approach to planning and managing road safety in Victoria, Australia. RESULTS: The adoption and implementation of a Safe System approach require strong institutional leadership and close cooperation among all the key agencies involved, and Victoria was fortunate in that it had a long history of strong interagency mechanisms in place. However, the challenges in the implementation of the Safe System strategy in Victoria are generally neither technical nor scientific; they are predominantly social and political. While many governments purport to develop strategies based on Safe System thinking, on-the-ground action still very much depends on what politicians perceive to be publicly acceptable, and Victoria is no exception. CONCLUSIONS: This is a case study of the complexity of institutional change and is presented in the hope that the lessons may prove useful for others seeking to adopt more holistic planning and management of road safety. There is still much work to be done in Victoria, but the institutional cultural shift has taken root. Ongoing efforts must be continued to achieve alert and compliant road users; however, major underpinning benefits will be achieved through focusing on road network safety improvements (achieving forgiving infrastructure, such as wire rope barriers) in conjunction with reviews of posted speed limits (to be set in response to the level of protection offered by the road infrastructure) and by the progressive introduction into the fleet of modern vehicle safety features.

The Relationship Between Objectively Measured Walking and Risk of Pedestrian-Motor Vehicle Collision.

Safe urban walking environments may improve health by encouraging physical activity, but the relationship between an individual's location and walking pattern and the risk of pedestrian-motor vehicle collision is unknown. We examined associations between individuals' walking bouts and walking risk, measured as mean exposure to the risk of pedestrian-vehicle collision. Walking bouts were ascertained through integrated accelerometry and global positioning system data and from individual travel-diary data obtained from adults in the Travel Assessment and Community Study (King County, Washington) in 2008-2009. Walking patterns were superimposed onto maps of the historical probabilities of pedestrian-vehicle collisions for intersections and midblock segments within Seattle, Washington. Mean risk of pedestrian-vehicle collision in specific walking locations was assessed according to walking exposure (duration, distance, and intensity) and participant demographic characteristics in linear mixed models. Participants typically walked in areas with low pedestrian collision risk when walking for recreation, walking at a faster pace, or taking longer-duration walks. Mean daily walking duration and distance were not associated with collision risk. Males walked in areas with higher collision risk compared with females, while vehicle owners, residents of single-family homes, and parents of young children walked in areas with lower collision risk. These findings may suggest that pedestrians moderate collision risk by using lower-risk routes.

Preference of endoscopic ablation over medical prevention of esophageal adenocarcinoma by patients with Barrett's esophagus.

BACKGROUND & AIMS: Endoscopic intervention or pharmacologic inhibition of cyclooxygenase might be used to prevent progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC). We investigated whether patients with BE prefer endoscopic therapy or chemoprevention of EAC. METHODS: Eighty-one subjects with nondysplastic BE were given a survey that described 2 scenarios. The survey explained that treatment A (ablation), endoscopy, reduced lifetime risk of EAC by 50%, with 5% risk for esophageal stricture, whereas treatment B (aspirin) reduced lifetime risk of EAC by 50% and the risk of heart attack by 30%, yet increased the risk for ulcer by 75%. Subjects indicated their willingness to undergo either treatment A and/or treatment B if endoscopic surveillance were required every 3-5 years, every 10 years, or were not required. Visual aids were included to represent risk and benefit percentages. RESULTS: When surveillance was required every 3-5 years, more subjects were willing to undergo treatment A than treatment B (78%, 63 of 81 vs 53%, 43 of 81; P < .01). There were no differences in age, sex, education level, or history of cancer, heart disease, or ulcer between patients willing to undergo treatment A and those willing to undergo treatment B. Altering the frequency of surveillance did not affect patients' willingness to undergo either treatment. CONCLUSIONS: In a simulated scenario, patients with BE preferred endoscopic intervention over chemoprevention for EAC. Further investigation of the shared decision-making process regarding preventive strategies for patients with BE may be warranted.

IGF1R deficiency in vascular smooth muscle cells impairs myogenic autoregulation and cognition in mice.

Introduction: Cerebrovascular pathologies contribute to cognitive decline during aging, leading to vascular cognitive impairment and dementia (VCID). Levels of circulating insulin-like growth factor 1 (IGF-1), a vasoprotective hormone, decrease during aging. Decreased circulating IGF-1 in animal models leads to the development of VCID-like symptoms, but the cellular mechanisms underlying IGF-1-deficiency associated pathologies in the aged cerebrovasculature remain poorly understood. Here, we test the hypothesis that vascular smooth muscle cells (VSMCs) play an integral part in mediating the vasoprotective effects of IGF-1. Methods: We used a hypertension-based model of cerebrovascular dysfunction in mice with VSMC-specific IGF-1 receptor (Igf1r) deficiency and evaluated the development of cerebrovascular pathologies and cognitive dysfunction. Results: VSMC-specific Igf1r deficiency led to impaired cerebral myogenic autoregulation, independent of blood pressure changes, which was also associated with impaired spatial learning and memory function as measured by radial arm water maze and impaired motor learning measured by rotarod. In contrast, VSMC-specific IGF-1 receptor knockdown did not lead to cerebral microvascular rarefaction. Discussion: These studies suggest that VSMCs are key targets for IGF-1 in the context of cerebrovascular health, playing a role in vessel stability alongside other cells in the neurovascular unit, and that VSMC dysfunction in aging likely contributes to VCID.

Carriers of a novel frame-shift insertion in WNT16a possess elevated pancreatic expression of TCF7L2.

BACKGROUND: The discovery of TCF7L2 as a global type 2 diabetes (T2D) gene has sparked investigations to explore the clinical utility of its variants for guiding the development of new diagnostic and therapeutic strategies. However, interpreting the resulting associations into function still remains unclear. Canonical Wnt signaling regulates ß-catenin and its binding with TCF7L2, which in turn is critical for the production of glucagon-like peptide-1 (GLP-1). This study examines the role of a novel frame-shift insertion discovered in a conserved region of WNT16a, and it is proposed that this mutation affects T2D susceptibility in conjunction with gene variants in TCF7L2. RESULTS: Our results predicted that the insertion would convert the upstream open reading frame in the Wnt16a mRNA to an alternative, in-frame translation initiation site, resulting in the prevention of nonsense-mediated decay, leading to a consequent stabilization of the mutated WNT16a message. To examine the role of Wnt16a in the Wnt signaling pathway, DNA and serum samples from 2,034 individuals (48% with T2D) from the Sikh Diabetes Study were used in this investigation. Prevalence of Wnt16a insertion did not differ among T2D cases (33%) and controls (32%). However, there was a 3.2 fold increase in Wnt16a mRNA levels in pancreatic tissues from the insertion carriers and a significant increase (70%, p < 0.0001) in luciferase activity in the constructs carrying the insertion. The expression of TCF7L2 mRNA in pancreas was also elevated (~23-fold) among the insertion carriers (p=0.003). CONCLUSIONS: Our results suggest synergistic effects of WNT16a insertion and the at-risk 'T' allele of TCF7L2 (rs7903146) for elevating the expression of TCF7L2 in human pancreas which may affect the regulation of downstream target genes involved in the development of T2D through Wnt/ß-catenin/TCF7L2 signaling pathway. However, further studies would be needed to mechanistically link the two definitively.

Whole animal knockout of smooth muscle alpha-actin does not alter excisional wound healing or the fibroblast-to-myofibroblast transition.

The contractile phenotype and function of myofibroblasts have been proposed to play a critical role in wound closure. It has been hypothesized that smooth muscle α-actin expressed in myofibroblasts is critical for its formation and function. We have used smooth muscle α-actin-null mice to test this hypothesis. Full-thickness excisional wounds closed at a similar rate in smooth muscle α-actin-null and wild-type mice. In addition, fibroblasts in smooth muscle α-actin-null granulation tissue when immunostained with a monoclonal antibody that recognizes all muscle actin isoforms exhibited a myofibroblast-like distribution and a stress fiber-like pattern, showing that these cells acquired the myofibroblast phenotype. Dermal fibroblasts from smooth muscle α-actin-null and wild-type mice formed stress fibers and supermature focal adhesions, and generated similar amounts of contractile force in response to transforming growth factor-ß1. Smooth muscle γ-actin and skeletal muscle α-actin were expressed in smooth muscle α-actin-null myofibroblasts, as shown by immunostaining, real-time polymerase chain reaction, and mass spectrometry. These results show that smooth muscle α-actin is not necessary for myofibroblast formation and function and for wound closure, and that smooth muscle γ-actin and skeletal muscle α-actin may be able to functionally compensate for the lack of smooth muscle α-actin in myofibroblasts.

MMP-2 expression by fibroblasts is suppressed by the myofibroblast phenotype.

During wound healing, fibroblasts transition from quiescence to a migratory state, then to a contractile myofibroblast state associated with wound closure. We found that the myofibroblast phenotype, characterized by the expression of high levels of contractile proteins, suppresses the expression of the pro-migratory gene, MMP-2. Fibroblasts cultured in a 3-D collagen lattice and allowed to develop tension showed increased contractile protein expression and decreased MMP-2 levels in comparison to a stress-released lattice. In 2-D cultures, factors that promote fibroblast contractility, including serum or TGF-ß, down-regulated MMP-2. Pharmacologically inducing F-actin disassembly or reduced contractility increased MMP-2 expression, while conditions that promote F-actin assembly suppressed MMP-2 expression. In all cases, changes in MMP-2 levels were inversely related to changes in the contractile marker, smooth muscle α-actin. To determine if the mechanisms involved in contractile protein gene expression play a direct role in MMP-2 regulation, we used RNAi-mediated knock-down of the myocardin-like factors, MRTF-A and MRTF-B, which induced the down-regulation of contractile protein genes by fibroblasts under both serum-containing and serum-free conditions. In the presence of serum or TGF-ß, MRTF-A/B knock-down resulted in the up-regulation of MMP-2; serum-free conditions prevented this increased expression. Together, these results indicate that, while MMP-2 expression is suppressed by F-actin formation, its up-regulation is not simply a consequence of contractile protein down-regulation.

Toxoplasma gondii activates hypoxia-inducible factor (HIF) by stabilizing the HIF-1alpha subunit via type I activin-like receptor kinase receptor signaling.

Toxoplasma gondii is an intracellular protozoan parasite that can cause devastating disease in fetuses and immune-compromised individuals. We previously reported that the alpha subunit of the host cell transcription factor, hypoxia-inducible factor-1 (HIF-1), is up-regulated by infection and necessary for Toxoplasma growth. Under basal conditions, HIF-1alpha is constitutively expressed but rapidly targeted for proteasomal degradation after two proline residues are hydroxylated by a family of prolyl hydroxylases (PHDs). The PHDs are alpha-ketoglutarate-dependent dioxygenases that have low K(m) values for oxygen, making them important cellular oxygen sensors. Thus, when oxygen levels decrease, HIF-1alpha is not hydroxylated, and HIF-1 is activated. How Toxoplasma activates HIF-1 under normoxic conditions remains unknown. Here, we report that Toxoplasma infection increases HIF-1alpha stability by preventing HIF-1alpha prolyl hydroxylation. Infection significantly decreases PHD2 abundance, which is the key prolyl hydroxylase for regulating HIF-1alpha. The effects of Toxoplasma on HIF-1alpha abundance and prolyl hydroxylase activity require activin-like receptor kinase signaling. Finally, parasite growth is severely diminished when signaling from this family of receptors is inhibited. Together, these data indicate that PHD2 is a key host cell factor for T. gondii growth and represent a novel mechanism by which a microbial pathogen subverts host cell signaling and transcription to establish its replicative niche.

TGF-beta suppresses the upregulation of MMP-2 by vascular smooth muscle cells in response to PDGF-BB.

During platelet-derived growth factor (PDGF)-BB-mediated recruitment to neovascular sprouts, vascular smooth muscle cells (VSMCs) dedifferentiate from a contractile to a migratory phenotype. This involves the downregulation of contractile markers such as smooth muscle (SM) alpha-actin and the upregulation of promigration genes such as matrix metalloproteinase (MMP)-2. The regulation of MMP-2 in response to PDGF-BB is complex and involves both stimulatory and inhibitory signaling pathways, resulting in a significant delay in upregulation. Here, we provide evidence that the delay in MMP-2 upregulation may be due to the autocrine expression and activation of transforming growth factor (TGF)-beta, which is known to promote the contractile phenotype in VSMCs. Whereas PDGF-BB could induce the loss of stress fibers and focal adhesions, TGF-beta was able to block or reverse this transition to a noncontractile state. TGF-beta did not, however, suppress early signaling events stimulated by PDGF-BB. Over time, though PDGF-BB induced increased TGF-beta1 levels, it suppressed TGF-beta2 and TGF-beta3 expression, leading to a net decrease in the total TGF-beta pool, resulting in the upregulation of MMP-2. Together, these findings indicate that MMP-2 expression is suppressed by a threshold level of active TGF-beta, which in turn promotes a contractile VSMC phenotype that prevents the upregulation of MMP-2.

Fecal metagenomics and metabolomics reveal gut microbial changes after bariatric surgery.

BACKGROUND: Evidence from longitudinal patient studies regarding gut microbial changes after bariatric surgery is limited. OBJECTIVE: To examine intraindividual changes in fecal microbiome and metabolites among patients undergoing Roux-en-Y gastric bypass or vertical sleeve gastrectomy. SETTING: Observational study. METHODS: Twenty patients were enrolled and provided stool samples before and 1 week, 1 month, and/or 3 months after surgery. Shallow shotgun metagenomics and untargeted fecal metabolomics were performed. Zero-inflated generalized additive models and linear mixed models were applied to identify fecal microbiome and metabolites changes, with adjustment for potential confounders and correction for multiple testing. RESULTS: We enrolled 16 women and 4 men, including 16 white and 4 black participants (median age = 45 years; presurgery body mass index = 47.7 kg/m2). Ten patients had Roux-en-Y gastric bypass, 10 had vertical sleeve gastrectomy, and 14 patients provided postsurgery stool samples. Of 47 samples, median sequencing depth was 6.3 million reads and 1073 metabolites were identified. Microbiome alpha-diversity increased after surgery, especially at 3 months. Significant genus-level changes included increases in Odoribacter, Streptococcus, Anaerotruncus, Alistipes, Klebsiella, and Bifidobacterium, while decreases in Bacteroides, Coprocosccus, Dorea, and Faecalibacterium. Large increases in Streptococcus, Akkermansia, and Prevotella were observed at 3 months. Beta-diversity and fecal metabolites were also changed, including reduced caffeine metabolites, indoles, and butyrate. CONCLUSIONS: Despite small sample size and missing repeated samples in some participants, our pilot study showed significant postsurgery changes in fecal microbiome and metabolites among bariatric surgery patients. Future large-scale, longitudinal studies are warranted to investigate gut microbial changes and their associations with metabolic outcomes after bariatric surgery.

Reduced Road Traffic Injuries for Young People: A Preliminary Investment Analysis.

PURPOSE: Deaths and serious injuries from road accidents remain a serious issue in developing countries, including for young people, for whom they are the largest cause of death. This article provides an assessment of interventions to reduce these deaths and injuries for adolescents in 75 developing countries. METHODS: We draw on new data on deaths and injuries by age, gender, and accident type for the 75 countries and on the road safety experience of developed and, more recently, developing countries. Critical tasks are to identify key interventions in road safety and estimate their impact and cost. We incorporate these impact and cost estimates in a modeling framework to calculate the reduction in deaths and serious injuries achieved out to 2030, relative to the base case. Finally, established methods are used to value the economic and social benefits arising from these reductions, and hence to calculate benefit-cost ratios. RESULTS: For the unchanged policy case, we estimate that there will be about 3 million deaths and 7.4 million serious injuries from road accidents for persons aged 10-24 years in the 75 countries to 2030. The preferred interventions avert one million of these deaths and 3 million serious injuries, at a cost of $6.5 billion per annum over 2016-2030, or $1.2 per capita across the total population of these countries. After valuing the benefits of the deaths and serious injuries averted, we find a benefit-cost ratio of 7.6 for 2016-2030, but of 9.9 if the interventions continue to 2050. CONCLUSIONS: Proven methods, suitably adjusted to local conditions, are available to reduce the tragic toll of road accidents in developing countries. These initiatives are likely to have strong economic and social returns, and should be given high priority.

Nanomaterial Applications in Photothermal Therapy for Cancer.

As a result of their unique compositions and properties, nanomaterials have recently seen a tremendous increase in use for novel cancer therapies. By taking advantage of the optical absorption of near-infrared light, researchers have utilized nanostructures such as carbon nanotubes, gold nanorods, and graphene oxide sheets to enhance photothermal therapies and target the effect on the tumor tissue. However, new uses for nanomaterials in targeted cancer therapy are coming to light, and the efficacy of photothermal therapy has increased dramatically. In this work, we review some of the current applications of nanomaterials to enhance photothermal therapy, specifically as photothermal absorbers, drug delivery vehicles, photoimmunological agents, and theranostic tools.

BSA-bioinspired gold nanorods loaded with immunoadjuvant for the treatment of melanoma by combined photothermal therapy and immunotherapy.

The development of therapeutic methods that can effectively delay tumor growth, inhibit tumor metastases, and protect the host from tumor recurrence still faces challenges. Nanoparticle-based combination therapy may provide an effective therapeutic strategy. Herein, we show that bovine serum albumin (BSA)-bioinspired gold nanorods (GNRs) were loaded with an immunoadjuvant for combined photothermal therapy (PTT) and immunotherapy for the treatment of melanoma. In this work, cetyltrimethylammonium bromide (CTAB)-coated GNRs were successively decorated with polyethylene glycol (PEG) and BSA, and loaded with an immunoadjuvant imiquimod (R837). The synthesized mPEG-GNRs@BSA/R837 nanocomplexes under near-infrared (NIR) irradiation could effectively kill tumors and trigger strong immune responses in treating metastatic melanoma in mice. Furthermore, the nanocomplex-based PTT prevented lung metastasis and induced a strong long-term antitumor immunity to protect the treated mice from tumor recurrence. The nanocomplex-based PTT in combination with immunotherapy may be potentially employed as an effective strategy for the treatment of melanoma and other metastatic cancers.

Identification of the RLBP1 gene promoter.

PURPOSE: Cellular retinaldehyde-binding protein (CRALBP), transcribed from the RLBP1 gene, is a 36-kDa water-soluble protein with 316 amino acids found in the retinal pigment epithelium (RPE) and in retinal Müller cells. It is thought to play a critical role in the visual cycle by functioning as an acceptor of 11-cis-retinol from the isomerohydrolase reaction. The goal here was to evaluate the functional promoter of this gene. METHODS: 5' RACE analysis, promoter-reporter assays, and semiquantitative PCR with exon-specific primers were performed using human-derived RPE cells (ARPE-19 and D407) in culture to evaluate the 5' sequence flanking the RLBP1 gene. In addition, the murine, bovine, and porcine RLBP1 genes were evaluated in silico to identify likely proximal promoter/exon 1 sequences similar to the human gene. RESULTS: 5' RACE analysis revealed the presence of a previously undescribed exon in the RLBP1 gene. This was confirmed by analysis of the GenBank Human EST database, which revealed the presence of 18 sequences matching exon 1. Exon-specific PCR revealed that most CRALBP transcripts expressed in ARPE-19 cells contain both exon 1 and the final exon, suggesting that the primary promoter of CRALBP exists 5' of the newly identified exon 1. Highly homologous sequences in the murine, bovine, and porcine genes were also identified. Finally, promoter-reporter constructs revealed a minimal sequence necessary for promoter function and indicated significantly greater promoter activity compared with previously described RLBP1 promoters. CONCLUSIONS: The findings presented here suggest that CRALBP transcripts in RPE cells contain a noncoding exon in addition to a newly described promoter and, by definition, an additional intron. This finding sets the stage for a mechanistic understanding of the high degree of cell type-specific expression of RLBP1.

Angiotensin II type 2 receptor gene deficiency attenuates susceptibility to tobacco-specific nitrosamine-induced lung tumorigenesis: involvement of transforming growth factor-beta-dependent cell growth attenuation.

To clarify an involvement of angiotensin II signaling in lung neoplasia, we have examined the effect of angiotensin II receptor deficiency on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis. Male angiotensin II type 2 receptor (AT2)-null mice with an SWR/J genetic background and control wild-type mice were treated with NNK (100 mg/kg, i.p.) or saline vehicle. NNK treatment caused the development of lung tumors in all wild-type control mice (100 % tumor prevalence), but only 85% of AT2-null mice developed tumors. The tumor multiplicity in AT2-null mice (1.9 +/- 0.3) was significantly smaller than that in wild-type mice (4.1 +/- 0.9). Primary cultured lung fibroblasts prepared from both AT2-null and wild-type mice markedly increased the colony counts of A549 lung cancer cells in soft agar, but a consistently higher colony count was observed with the wild-type fibroblasts (fold increase in colony number, 5.6 +/- 0.5) than with the AT2-null fibroblasts (3.5 +/- 0.8). The underlying mechanism by which angiotensin II regulates cancer cell growth is due to the regulation of active transforming growth factor-beta (TGF-beta) production. Although the total level of TGF-beta was significantly stimulated when A549 cells were cocultured with either type of fibroblasts, the level of active TGF-beta in the conditioned medium was consistently higher with AT2-null fibroblasts than with wild-type fibroblasts. These results imply that the AT2 receptor negatively regulates the level of active TGF-beta and thus increases NNK-induced lung tumorigenesis. The AT2 receptor function in lung stromal fibroblasts may be a potential modulator of tumor susceptibility in chemical carcinogen-induced lung tumorigenesis.