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Spatiotemporal regulation of the cellular transcriptome is crucial for proper protein expression and cellular function. However, the intricate subcellular dynamics of RNA remain obscured due to the limitations of existing transcriptomics methods. Here, we report TEMPOmap—a method that uncovers subcellular RNA profiles across time and space at the single-cell level. TEMPOmap integrates pulse-chase metabolic labeling with highly multiplexed three-dimensional in situ sequencing to simultaneously profile the age and location of individual RNA molecules. Using TEMPOmap, we constructed the subcellular RNA kinetic landscape in various human cells from transcription and translocation to degradation. Clustering analysis of RNA kinetic parameters across single cells revealed ‘kinetic gene clusters’ whose expression patterns were shaped by multistep kinetic sculpting. Importantly, these kinetic gene clusters are functionally segregated, suggesting that subcellular RNA kinetics are differentially regulated in a cell-state- and cell-type-dependent manner. Spatiotemporally resolved transcriptomics provides a gateway to uncovering new spatiotemporal gene regulation principles.

Pathways associated with G9a copy number in the Leeds melanoma cohort

Supplementary Figure Legend

BRAF-targeted therapy results in objective responses in the majority of patients; however, the responses are short lived (∼6 months). In contrast, treatment with immune checkpoint inhibitors results in a lower response rate, but the responses tend to be more durable. BRAF inhibition results in a more favorable tumor microenvironment in patients, with an increase in CD8+ T-cell infiltrate and a decrease in immunosuppressive cytokines. There is also increased expression of the immunomodulatory molecule PDL1, which may contribute to the resistance. On the basis of these findings, we hypothesized that BRAF-targeted therapy may synergize with the PD1 pathway blockade to enhance antitumor immunity. To test this hypothesis, we developed a BRAF(V600E)/Pten−/− syngeneic tumor graft immunocompetent mouse model in which BRAF inhibition leads to a significant increase in the intratumoral CD8+ T-cell density and cytokine production, similar to the effects of BRAF inhibition in patients. In this model, CD8+ T cells were found to play a critical role in the therapeutic effect of BRAF inhibition. Administration of anti-PD1 or anti-PDL1 together with a BRAF inhibitor led to an enhanced response, significantly prolonging survival and slowing tumor growth, as well as significantly increasing the number and activity of tumor-infiltrating lymphocytes. These results demonstrate synergy between combined BRAF-targeted therapy and immune checkpoint blockade. Although clinical trials combining these two strategies are ongoing, important questions still remain unanswered. Further studies using this new melanoma mouse model may provide therapeutic insights, including optimal timing and sequence of therapy. Cancer Immunol Res; 2(7); 643–54. ©2014 AACR.

Epigenetic regulators, when genomically altered, may become driver oncogenes that mediate otherwise unexplained pro-oncogenic changes lacking a clear genetic stimulus, such as activation of the WNT/β-catenin pathway in melanoma. This study identifies previously unrecognized recurrent activating mutations in the G9a histone methyltransferase gene, as well as G9a genomic copy gains in approximately 26% of human melanomas, which collectively drive tumor growth and an immunologically sterile microenvironment beyond melanoma. Furthermore, the WNT pathway is identified as a key tumorigenic target of G9a gain-of-function, via suppression of the WNT antagonist DKK1. Importantly, genetic or pharmacologic suppression of mutated or amplified G9a using multiple in vitro and in vivo models demonstrates that G9a is a druggable target for therapeutic intervention in melanoma and other cancers harboring G9a genomic aberrations.Significance:Oncogenic G9a abnormalities drive tumorigenesis and the “cold” immune microenvironment by activating WNT signaling through DKK1 repression. These results reveal a key druggable mechanism for tumor development and identify strategies to restore “hot” tumor immune microenvironments.This article is highlighted in the In This Issue feature, p. 890

Supplementary Figure 1. BP cells behave like classic BRAFV600E melanomas after BRAF inhibition. Supplementary Figure 2. Subcutaneously implanted BP cells develop into tumors. Supplementary Figure 3. BRAF inhibition is associated with an increase in density of tumor-infiltrating T cells. Supplementary Figure 4. Kinetics of functional CD8 T cell markers. Supplementary Figure 5. BRAF inhibition is associated with an increase in overall PD-L1 and PD-L2 expression in the tumor. Supplementary Figure 6. Cell type specific changes in PD-L1 expression after BRAF inhibitors. Supplementary Figure 7. PD-1 or PD-L1 blockade synergy with BRAF inhibitors is associated with increased density of tumor-infiltrating T cells.

A major challenge to end the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is to develop a broadly protective vaccine that elicits long-term immunity. As the key immunogen, the viral surface spike (S) protein is frequently mutated, and conserved epitopes are shielded by glycans. Here, we revealed that S protein glycosylation has site-differential effects on viral infectivity. We found that S protein generated by lung epithelial cells has glycoforms associated with increased infectivity. Compared to the fully glycosylated S protein, immunization of S protein with N-glycans trimmed to the mono-GlcNAc–decorated state (S MG ) elicited stronger immune responses and better protection for human angiotensin-converting enzyme 2 (hACE2) transgenic mice against variants of concern (VOCs). In addition, a broadly neutralizing monoclonal antibody was identified from S MG -immunized mice that could neutralize wild-type SARS-CoV-2 and VOCs with subpicomolar potency. Together, these results demonstrate that removal of glycan shields to better expose the conserved sequences has the potential to be an effective and simple approach for developing a broadly protective SARS-CoV-2 vaccine.

Cutaneous manifestations have been associated with COVID-19 infection and their significance in hospitalized patients remains unclear. This study catalogues the exanthems observed in hospitalized COVID-19 patients to determine prevalence and inform clinicians in devising diagnostic and management strategies.

Loss-of-function mutations of JAK1/2 impair cancer cell responsiveness to IFNγ and immunogenicity. Therefore, an understanding of compensatory pathways to activate IFNγ signaling in cancer cells is clinically important for the success of immunotherapy. Here we demonstrate that the transcription factor SOX10 hinders immunogenicity of melanoma cells through the IRF4–IRF1 axis. Genetic and pharmacologic approaches revealed that SOX10 repressed IRF1 transcription via direct induction of a negative regulator, IRF4. The SOX10–IRF4–IRF1 axis regulated PD-L1 expression independently of JAK–STAT pathway activity, and suppression of SOX10 increased the efficacy of combination therapy with an anti-PD-1 antibody and histone deacetylase inhibitor against a clinically relevant melanoma model. Thus, the SOX10–IRF4–IRF1 axis serves as a potential target that can bypass JAK-STAT signaling to immunologically warm up melanoma with a "cold" tumor immune microenvironment. Significance: This study identifies a novel SOX10/IRF4 pathway that regulates noncanonical induction of IRF1 independent of the JAK–STAT pathway and can be targeted to improve the efficacy of anti-PD-1 therapy in melanoma.

Addiction to UV light and opioids is increased by VitD deficiency, suggesting that a feedback loop promotes its synthesis., The current opioid epidemic warrants a better understanding of genetic and environmental factors that contribute to opioid addiction. Here we report an increased prevalence of vitamin D (VitD) deficiency in patients diagnosed with opioid use disorder and an inverse and dose-dependent association of VitD levels with self-reported opioid use. We used multiple pharmacologic approaches and genetic mouse models and found that deficiencies in VitD signaling amplify exogenous opioid responses that are normalized upon restoration of VitD signaling. Similarly, physiologic endogenous opioid analgesia and reward responses triggered by ultraviolet (UV) radiation are repressed by VitD signaling, suggesting that a feedback loop exists whereby VitD deficiency produces increased UV/endorphin-seeking behavior until VitD levels are restored by cutaneous VitD synthesis. This feedback may carry the evolutionary advantage of maximizing VitD synthesis. However, unlike UV exposure, exogenous opioid use is not followed by VitD synthesis (and its opioid suppressive effects), contributing to maladaptive addictive behavior.

A major challenge to end the pandemic caused by SARS-CoV-2 is to develop a broadly protective vaccine. As the key immunogen, the spike protein is frequently mutated with conserved epitopes shielded by glycans. Here, we reveal that spike glycosylation has site-differential effects on viral infectivity and lung epithelial cells generate spike with more infective glycoforms. Compared to the fully glycosylated spike, immunization of spike protein with N-glycans trimmed to the monoglycosylated state (Smg) elicits stronger immune responses and better protection for hACE2 transgenic mice against variants of concern. In addition, a broadly neutralizing monoclonal antibody was identified from the Smg immunized mice, demonstrating that removal of glycan shields to better expose the conserved sequences is an effective and simple approach to broad-spectrum vaccine development.One-Sentence SummaryRemoving glycan shields to expose conserved epitopes is an effective approach to develop a broad-spectrum SARS-CoV-2 vaccine.

Although immune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein 1 (PD-1), can deliver durable anti-tumor effects, most patients with cancer fail to respond. Recent studies suggest that ICI efficacy correlates with a higher load of tumor-specific neoantigens and development of vitiligo in patients with melanoma. Here, we report that patients with low melanoma neoantigen burdens who responded to ICIs had tumors with higher expression of pigmentation-related genes. Moreover, expansion of peripheral blood CD8(+) T cell populations specific for melanocyte antigens was observed only in patients who responded to anti-PD-1 therapy, suggesting that ICIs can promote breakdown of tolerance toward tumor-lineage self-antigens. In a mouse model of poorly immunogenic melanomas, skewing of epitope recognition toward wild type melanocyte antigens was associated with markedly improved anti-PD-1 efficacy in two independent approaches: introduction of neoantigens by ultraviolet (UV) B radiation mutagenesis, or the therapeutic combination of ablative fractional photothermolysis plus imiquimod. Complete responses against UV mutation-bearing tumors after anti-PD-1 resulted in protection from subsequent engraftment of melanomas lacking any shared neoantigens, as well as pancreatic adenocarcinomas forcibly overexpressing melanocyte-lineage antigens. Our data demonstrate that somatic mutations are sufficient to provoke strong anti-tumor responses after checkpoint blockade, but long-term responses are not restricted to these putative neoantigens. Epitope skewing toward T cell recognition of wild type tumor-lineage self-antigens represents a common pathway for successful response to ICIs, which can be evoked in neoantigen-deficient tumors by combination therapy with ablative fractional photothermolysis and imiquimod.

Biological information can be encoded in the dynamics of signaling components which has been implicated in a broad range of physiological processes including stress response, oncogenesis, and stem cell differentiation. To study the complexity of information transfer across the eukaryotic promoter, we screened 119 dynamic conditions—modulating the frequency, intensity, and pulse width of light—regulating the binding of an epigenome editor to a fluorescent reporter. This system revealed highly tunable gene expression and filtering behaviors and provided the most comprehensive quantification to date of the maximum amount of information that can be reliably transferred across a promoter as ∼1.7 bits. Using a library of over 100 orthogonal epigenome editors, we further determined that chromatin state could be used to tune mutual information and expression levels, as well as completely alter the input-output transfer function of the promoter. This system unlocks the information-rich content of eukaryotic epigenome editing.

Epigenetic regulators, when genomically altered, may become driver oncogenes that mediate otherwise unexplained pro-oncogenic changes lacking a clear genetic stimulus, such as activation of the WNT/β-catenin pathway in melanoma. This study identifies previously unrecognized recurrent activating mutations in the G9a histone methyltransferase gene, as well as G9a genomic copy gains in approximately 26% of human melanomas, which collectively drive tumor growth and an immunologically sterile microenvironment beyond melanoma. Furthermore, the WNT pathway is identified as a key tumorigenic target of G9a gain-of-function, via suppression of the WNT antagonist DKK1. Importantly, genetic or pharmacologic suppression of mutated or amplified G9a using multiple in vitro and in vivo models demonstrates that G9a is a druggable target for therapeutic intervention in melanoma and other cancers harboring G9a genomic aberrations. Significance: Oncogenic G9a abnormalities drive tumorigenesis and the “cold” immune microenvironment by activating WNT signaling through DKK1 repression. These results reveal a key druggable mechanism for tumor development and identify strategies to restore “hot” tumor immune microenvironments. This article is highlighted in the In This Issue feature, p. 890

Objective The aim of the study was to evaluate outcomes after pediatric out-of-hospital cardiopulmonary interventions (CPIs) by emergency medical services (EMS). Methods Children (age, ≤18 years) who received CPI by EMS from 2001 to 2008 were identified from the Utah Department of Health. Cardiopulmonary intervention was defined as oxygenation, ventilation or CPR, and transport to a hospital by EMS. Univariate and multivariable regression analyses evaluated associations between potential predictors and outcomes (death and new neurologic dysfunction). Results A total of 464 patients (58% male) received EMS attention. For the 71% patients (327) who were alive on EMS arrival, 63% (205) received CPI without CPR. Of note, 6% (12) of these patients died after arrival to the hospital and new neurologic dysfunction was diagnosed in 6% (13). Among the 12 patients who died, 50% (6) were younger than 1 year.On multivariable regression analysis, factors associated with increased risk of death before and in-hospital are the following: age younger than 1 year (odds ratio [OR], 0.26; 95% confidence interval [CI], 0.17-0.39), shorter EMS transport time (OR, 0.94; 95% CI, 0.89-0.99), and longer EMS dispatch time (OR, 1.23; 95% CI, 1.08-1.40). Factors associated with increased risk of new neurologic dysfunction are the following: lack of pulse (OR, 0.14; 95% CI, 0.04-0.53), requiring CPR (OR, 6.15; 95% CI, 1.48-25.6), and CPR duration (OR, 1.20; 95% CI, 1.05-1.37). Conclusions Age younger than 1 year, shorter transport time, and longer dispatch time were associated with increased risk of death. Being pulseless upon discovery and receiving CPR were associated with new neurologic dysfunction. Maximizing EMS transport interventions for patients younger than 1 year requiring CPI may improve patient outcomes.

Summary Biological information can be encoded within the dynamics of signaling components, which has been implicated in a broad range of physiological processes including stress response, oncogenesis, and stem cell differentiation. To study the complexity of information transfer across the eukaryotic promoter, we screened 119 dynamic conditions—modulating the pulse frequency, amplitude, and pulse width of light—regulating the binding of an epigenome editor to a fluorescent reporter. This system revealed tunable gene expression and filtering behaviors and provided a quantification of the limit to the amount of information that can be reliably transferred across a single promoter as ∼1.7 bits. Using a library of over 100 orthogonal chromatin regulators, we further determined that chromatin state could be used to tune mutual information and expression levels, as well as completely alter the input-output transfer function of the promoter. This system unlocks the information-rich content of eukaryotic gene regulation.

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.

[Image: see text] Spleen tyrosine kinase (SYK) is a critical regulator of signaling in a variety of immune cell types such as B-cells, monocytes, and macrophages. Accordingly, there have been numerous efforts to identify compounds that selectively inhibit SYK as a means to treat autoimmune and inflammatory diseases. We previously disclosed GS-9973 (entospletinib) as a selective SYK inhibitor that is under clinical evaluation in hematological malignancies. However, a BID dosing regimen and drug interaction with proton pump inhibitors (PPI) prevented development of entospletinib in inflammatory diseases. Herein, we report the discovery of a second-generation SYK inhibitor, GS-9876 (lanraplenib), which has human pharmacokinetic properties suitable for once-daily administration and is devoid of any interactions with PPI. Lanraplenib is currently under clinical evaluation in multiple autoimmune indications.

SUMMARY Immune checkpoint inhibitors have improved patient survival in melanoma, but the innate resistance of many patients necessitates the investigation of alternative immune targets. Many immune checkpoint proteins lack proper characterization, including V-domain Ig suppressor of T cell activation (VISTA). VISTA expression on immune cells can suppress T cell activity; however, few studies have investigated its expression and regulation in cancer cells. In this study, we observe that VISTA is expressed in melanoma patient samples and cell lines. Tumor cell-specific expression of VISTA promotes tumor onset in vivo, associated with increased intratumoral T regulatory cells, and enhanced PDL-1 expression on tumor-infiltrating macrophages. VISTA transcript levels are regulated by the stemness factor Forkhead box D3 (FOXD3). BRAF inhibition upregulates FOXD3 and reduces VISTA expression. Overall, this study demonstrates melanoma cell expression of VISTA and its regulation by FOXD3, contributing to the rationale for therapeutic strategies that combine targeted inhibitors with immune checkpoint blockade., In Brief VISTA is an understudied immune checkpoint protein. Through the analysis of patient samples and studies in mouse models, Rosenbaum et al. investigate the functional consequences of VISTA expression on melanoma cells. Furthermore, they demonstrate that the BRAF-regulated transcription factor FOXD3 negatively regulates VISTA expression., Graphical Abstract

Background: Cutaneous manifestations have been associated with COVID-19 infection and their clinical significance in hospitalized patients remains unclear Methods: A retrospective chart review of 1216 patients older than 18 years of age hospitalized with laboratory-confirmed SARS-CoV-2 infection from March 12, 2020 to May 31, 2020 at a large urban academic medical center A keyword search query of patient records combined with manual chart review by at least two dermatologists identified a study group having cutaneous manifestations concurrent with COVID-19 infection, specifically between 14 days prior to admission and up to discharge Results: 122 patients with 195 skin lesions concurrent with COVID-19 hospitalization were identified Dermatology reviewers evaluated clinical photographs for 116 lesions (59 5%) and inpatient dermatology consultations for 42 lesions (21 5%) The most common cutaneous findings in patients with COVID-19 hospitalization were pressure injuries (n=118;60 5%) and morbilliform eruptions (n=33;16 9%) A very small number of patients (0 6%;n=7/1216) had exanthems occurring within 2 weeks of COVID-19 symptom onset The majority of exanthems developed within 14 days of exposure to possible culprit drugs and beyond the 14-day window of COVID-19 symptom onset, making viral association unlikely Conclusion: Skin lesions concurrent with COVID-19 hospitalization were most frequently linked to hospitalization-related factors, such as pressure injuries or drug-related exanthems, rather than due to novel pathologies related to SARS-CoV-2 itself

Fatty liver, or hepatic steatosis, is an alarmingly common pathology in western societies, in large part because if left unheeded, it can lead to life-threatening forms of nonalcoholic fatty liver disease, including nonalcoholic steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. As such, it is essential that we attain a greater understanding of the pathways that control energy partitioning in the liver and ultimately how they are impacted by environmental factors. Here, we define the essential requirement for a member of the Ras-related protein in the brain (RAB)-like (RABL) clade of small GTPases, RABL2, in fatty acid metabolism including in microtubule-associated mitochondrial movement within the liver. RABL2 dysfunction, even in mice fed a low-fat chow diet, leads to retarded hepatic mitochondria movement associated with and a cascading phenotype of interrelated metabolic defects reminiscent of a type 2 diabetic state: hepatic steatosis, insulin resistance, glucose intolerance, and adult onset obesity. RABL2 dysfunction does not, however, alter mitochondrial content, or the inherent respiratory capacity of individual mitochondria per se. Rather, it is associated with a decreased capacity for fatty oxidation in the context of the intact cell, suggesting a complex, and important, role for mitochondrial movement in metabolic health. Our data highlight the importance of RABL2 and mitochondrial dynamics in hepatic fatty acid oxidation and in the achievement of metabolic balance.

Management guidelines for refractory Kawasaki disease (KD) are vague. We sought to assess practice variation and identify factors associated with large/complex coronary artery aneurysms (LCAA) and resource utilization in refractory KD. This retrospective cohort study identified patients aged ≤18 years with KD (2004 to 2014) using the Pediatric Health Information System. Refractory KD was defined as receiving1 dose of intravenous immunoglobulin. Demographics, medications, concomitant infections, length of stay (LOS), and charges were collected. Antithrombotic therapy was a surrogate for LCAA. LOS and hospital charges assessed resource utilization. Multivariate regression identified factors associated with LOS, charges, and LCAA. Of 14,194 patients with KD, 2,974 (21%) had refractory KD and 203 of those 2,974 (7%) had LCAA. Additional intravenous immunoglobulin was the sole medication in 77%. Other medications added were steroids (18%), infliximab (2%), and both (3%). Warfarin, low-molecular-weight heparin, tissue plasminogen activator, and clopidogrel were prescribed with equal frequency (2%). Male gender (adjusted relative risk 1.52, 95% confidence interval [CI] 1.08 to 2.16, p0.01), admission to an intensive care unit (4.79, 95% CI 3.40 to 6.74, p0.001), arrhythmia (3.00, 95% CI 1.94 to 4.65, p0.001), and concomitant viral infection (2.29, 95% CI 1.49 to 3.52, p0.001) were associated with LCAA. Severe illness, race, region, and payer were independently associated with increased charges (p0.05 for all). In conclusion, treatment for refractory KD varies widely. Concomitant viral infection was associated with a greater risk of LCAA in refractory KD. Better understanding of optimal management may improve outcomes and decrease both variability in management and resource utilization for refractory KD.

Summary The influenza virus hemagglutinin (HA) and coronavirus spike (S) protein mediate virus entry. HA and S proteins are heavily glycosylated, making them potential targets for carbohydrate binding agents such as lectins. Here, we show that the lectin FRIL, isolated from hyacinth beans (Lablab purpureus), has anti-influenza and anti-SARS-CoV-2 activity. FRIL can neutralize 11 representative human and avian influenza strains at low nanomolar concentrations, and intranasal administration of FRIL is protective against lethal H1N1 infection in mice. FRIL binds preferentially to complex-type N-glycans and neutralizes viruses that possess complex-type N-glycans on their envelopes. As a homotetramer, FRIL is capable of aggregating influenza particles through multivalent binding and trapping influenza virions in cytoplasmic late endosomes, preventing their nuclear entry. Remarkably, FRIL also effectively neutralizes SARS-CoV-2, preventing viral protein production and cytopathic effect in host cells. These findings suggest a potential application of FRIL for the prevention and/or treatment of influenza and COVID-19., Graphical Abstract, Highlights • FRIL is a plant lectin with potent anti-influenza and anti-SARS-CoV-2 activity • FRIL preferentially binds to complex-type N-glycans on viral glycoproteins • FRIL inhibits influenza virus entry by sequestering virions in late endosomes • Intranasal administration of FRIL protects against lethal H1N1 challenge in mice, Liu et al. demonstrate that FRIL, a plant lectin isolated from the hyacinth bean, has potent antiviral activity against SARS-CoV-2 and diverse influenza virus strains. FRIL is effective in vivo against H1N1. FRIL’s antiviral activity is mediated by binding to complex-type N-glycans on viral glycoproteins, interfering with viral entry.

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

Melanoma, the deadliest form of skin cancer, is an aggressive disease that is rising in incidence. Although melanoma is a historically treatment-resistant malignancy, in recent years unprecedented breakthroughs in targeted therapies and immunotherapies have revolutionized the standard of care for patients with advanced disease. Here, we provide an overview of recent developments in our understanding of melanoma risk factors, genomics, and molecular pathogenesis and how these insights have driven advances in melanoma treatment. In addition, we review benefits and limitations of current therapies and look ahead to continued progress in melanoma prevention and therapy. Remarkable achievements in the field have already produced a paradigm shift in melanoma treatment: Metastatic melanoma, once considered incurable, can now be treated with potentially curative rather than palliative intent.

Summary Despite compelling rates of durable clinical responses to programmed cell death-1 (PD-1) blockade, advances are needed to extend these benefits to resistant tumors. We found that tumor-bearing mice deficient in the chemokine receptor CXCR3 responded poorly to anti-PD-1 treatment. CXCR3 and its ligand CXCL9 were critical for a productive CD8+ T cell response in tumor-bearing mice treated with anti-PD-1 but were not required for the infiltration of CD8+ T cells into tumors. The anti-PD-1-induced anti-tumor response was facilitated by CXCL9 production from intratumoral CD103+ dendritic cells, suggesting that CXCR3 facilitates dendritic cell-T cell interactions within the tumor microenvironment. CXCR3 ligands in murine tumors and in plasma of melanoma patients were an indicator of clinical response to anti-PD-1, and their induction in non-responsive murine tumors promoted responsiveness to anti-PD-1. Our data suggest that the CXCR3 chemokine system is a biomarker for sensitivity to PD-1 blockade and that augmenting the intratumoral function of this chemokine system could improve clinical outcomes.

Recently, we reported that melanoma risk in redheads is linked not only to pale skin, but also to the synthesis of the pigment - called pheomelanin - that gives red hair its color. We demonstrated that pheomelanin synthesis is associated with increased oxidative stress in the skin, yet we have not uncovered the chemical pathway between the molecule pheomelanin and the DNA damage that drives melanoma formation. Here, we hypothesize two possible pathways. On one hand, pheomelanin might generate reactive oxygen species (ROS) that directly or indirectly cause oxidative DNA damage. On the other hand, pheomelanin synthesis might consume cellular antioxidant stores and make the cell nucleus more vulnerable to other endogenous ROS. Uncovering the mechanistic pathway between pheomelanin and oxidative DNA damage will be an important step in developing strategies to lower melanoma risk in redheads.

There are established epidemiological links between obesity and the severity of prostate cancer. We directly tested this relationship by assessing tumorigenicity of patient-derived xenografts (PDXs) of moderate-grade localized prostate cancer in lean and obese severe combined immunodeficiency (SCID) mice. Mice were rendered obese and insulin resistant by high-fat feeding for 6 weeks prior to transplantation, and PDXs were assessed 10 weeks thereafter. Histological analysis of PDX grafts showed no differences in tumor pathology, prostate-specific antigen, androgen receptor and homeobox protein Nkx-3.1 expression, or proliferation index in lean versus obese mice. Whilst systemic obesity per se did not promote prostate tumorigenicity, we next asked whether the peri-prostatic adipose tissue (PPAT), which covers the prostate anteriorly, plays a role in prostate tumorigenesis. In vitro studies in a cellularized co-culture model of stromal and epithelial cells demonstrated that factors secreted from human PPAT are pro-tumorigenic. Accordingly, we recapitulated the prostate-PPAT spatial relationship by co-grafting human PPAT with prostate cancer in PDX grafts. PDX tissues were harvested 10 weeks after grafting, and histological analysis revealed no evidence of enhanced tumorigenesis with PPAT compared to prostate cancer grafts alone. Altogether, these data demonstrate that prostate cancer tumorigenicity is not accelerated in the setting of diet-induced obesity or in the presence of human PPAT, prompting the need for further work to define the at-risk populations of obesity-driven tumorigenesis and the biological factors linking obesity, adipose tissue and prostate cancer pathogenesis.

Considering all types of restaurants in Hong Kong, Chinese restaurants have the greatest numerical count, and they represent a significant proportion of local restaurant receipts. Chinese restaurants are also very popular all over the world. However, previous researches showed that Chinese restaurants generally consume more water than other types of restaurant. Specifically, the unique cold-water thawing process in commercial Chinese cooking may increase water consumption in Chinese restaurants. This unique feature has yet been explored in printed literature, thus two case studies and a series of field tests were conducted to examine the cold-water thawing process in Chinese restaurants. The aims were to determine the proportion of water consumed by cold-water thawing, compare the performance and estimate the payback period of various cold-water thawing options. Findings showed that cold-water thawing accounted for around 30% of the water consumption in the studied restaurants. Among various cold-water thawing options, the thawing machine and the microbubble machine showed better overall performance. Their payback periods were around 15.5 months and 10 months respectively. The information and recommendation from this paper can serve as a useful reference for restaurant investors, operators, managers, and consultants in controlling water consumption, and minimizing sewage-related environmental impacts.

127 Background: Disruption of the immune system with immune checkpoint inhibitors can result in a multitude of immune-related adverse effects (irAE). While irAEs have been well-reported in clinical trials, the impact and magnitude of irAE’s in the real-world, particularly inpatient is unclear. Methods: Data was collected on patients with advanced malignancy who experienced a suspected irAE needing admission to an academic hospital (Feb 2011 to June 2017). Each case was reviewed comprehensively by minimum of two reviewers, including one sub-specialist. In addition, oncologists at our institution were surveyed regarding their confidence about managing patients with irAEs. Results: Over a span of 6 years, there were 343 hospitalizations for suspected irAEs and the majority (65%; N = 223) were confirmed irAEs that required treatment with immunosuppression or therapy stopped as result. The mean length of stay was 6.3 days (range 1 to 31 days), readmission rate for another irAE event 25%, total readmission rate 61.7%, and inpatient mortality 8%. The most common irAEs were enterocolitis (43.9%), pulmonary (16%), hepatic (15%), neurological (8.9%), endocrinopathies (7.1%), rheumatological (4%), dermatological (3%), cardiovascular (3%), renal (1.8%), and allergy (1.3%). Over the past 5 years, there was a significant increase in admissions due to irAEs (admissions in 2016 vs 2011, odds ratio = 3.07; p < 0.01). The Cancer Center survey (N = 26) revealed majority of oncologists do not feel very comfortable managing irAEs, and 48% felt that irAE complications should be managed on a different service. Conclusions: irAEs from immune checkpoint inhibitors can result in prolonged hospitalizations, high rate of readmissions, and mortality. The number of patients admitted due to irAEs has significantly increased by more than three-fold in the recent years, but majority of oncologists do not feel very comfortable managing irAEs. Consequently, there is a critical need for coordinated multidisciplinary approach, comprehensive provider education, and translational research program for early detection and intervention.

With the continuing strong growth in international arrivals to Hong Kong, more hotels are planning to open in the coming years. The sewage problem stemming from local hotels is expected to be worsened. The current study covers the years 1998 to 2005, plus a projection for the years 2006 to 2009. A survey of water use and conservation in 28 local hotels has been conducted. All established norms and ratios related to water consumption in this study are on a per room basis. The environmental impact of sewage discharged is converted into monetary terms on the basis of how much the hotel industry would need to spend in order to avoid the impacts. It is found that the average volume of water consumed per occupied room decreased from 1.145m3in 1994-1996 to 0.904m3in 2001-2002. In 2005, the environmental costs value was more than HK$187 million and exceeded HK$364 million in 2008. It is also found that flow regulator and submeter are the two most commonly used water-saving devices in local hotels. Some recommendations are made to save water, particularly in the laundry, kitchen, and guestroom.

In recognition of the growing concern about the likely detrimental environmental impacts of hotel activities, an increasing number of tourism research publications highlight the importance of achieving sustainable tourism and accommodation. However, there is still a scarcity of numerical data which reflect the severity of the negative impacts on the environment created by the operations of hotel owners. This study compiles a database for hotels to design and construct an appropriate environmental impact index for Hong Kong as a first significant step towards creating a ‘green’ tourism satellite account for the city. It is estimated that the total environmental cost of the local hotel sector has increased by 400% in 16 years to HK$265 million in 2005. Computable or measurable environmental costs were used to establish a composite environmental index and it was discovered that it reached a peak in 2005. In the final analysis, the growth rate of the composite environmental index is compared with that of the value-added index of the local hotel industry and it is found that they generally have the same trend over the study period.

Magnetic resonance imaging (MRI) studies have revealed fronto-temporal cortical gray matter volume reductions in schizophrenia. However, to date studies have not examined whether age- and sex-matched unmedicated schizotypal personality disorder (SPD) patients share some or all of the structural brain-imaging characteristics of schizophrenia patients. We examined cortical gray/white matter volumes in a large sample of unmedicated schizophrenia-spectrum patients (n = 79 SPD, n = 57 schizophrenia) and 148 healthy controls. MRI images were reoriented to standard position parallel to the anterior–posterior commissure line, segmented into gray and white matter tissue types, and assigned to Brodmann areas (BAs) using a postmortem-histological atlas. Group differences in regional volume of gray and white matter in the BAs were examined with MANOVA. Schizophrenia patients had significantly reduced gray matter volume widely across the cortex but more marked in frontal and temporal lobes. SPD patients had reductions in the same regions but only about half that observed in schizophrenia and sparing in key regions including BA10. In schizophrenia, greater fronto-temporal volume loss was associated with greater negative symptom severity and in SPD, greater interpersonal and cognitive impairment. Overall, our findings suggest that increased prefrontal volume in BA10 and sparing of volume loss in temporal cortex (BAs 22 and 20) may be a protective factor in SPD which reduces vulnerability to psychosis.

The goal of cancer immunotherapy is to harness the immune system to recognize and destroy tumor cells, with the potential to produce durable responses that may translate into curative outcomes in patients with metastatic cancers. Results from multiple randomized clinical trials have established immune checkpoint inhibitors as the most successful class of immunotherapies to date. These include monoclonal antibodies that reinvigorate T cell responses by blocking cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 (PD-1), two coinhibitory receptors that regulate T cell activation.

The global epidemic of obesity is closely linked to the development of serious co-morbidities, including many forms of cancer. Epidemiological evidence consistently shows that obesity is associated with a similar or mildly increased incidence of prostate cancer but, more prominently, an increased risk for aggressive prostate cancer and prostate cancer-specific mortality. Studies in mice demonstrate that obesity induced by high-fat feeding increases prostate cancer progression; however, the mechanisms underpinning this relationship remain incompletely understood. Adipose tissue expansion in obesity leads to local tissue dysfunction and is associated with low-grade inflammation, alterations in endocrine function and changes in lipolysis that result in increased delivery of fatty acids to tissues of the body. The human prostate gland is covered anteriorly by the prominent peri-prostatic adipose tissue and laterally by smaller adipose tissue depots that lie directly adjacent to the prostatic surface. We discuss how the close association between dysfunctional adipose tissue and prostate epithelial cells might result in bi-directional communication to cause increased prostate cancer aggressiveness and progression. However, the literature indicates that several ‘mainstream’ hypotheses regarding obesity-related drivers of prostate cancer progression are not yet supported by a solid evidence base and, in particular, are not supported by experiments using human tissue. Understanding the links between obesity and prostate cancer will have major implications for the health policy for men with prostate cancer and the development of new therapeutic or preventative strategies.

BRAF-targeted therapy results in objective responses in the majority of patients; however, the responses are short lived (∼6 months). In contrast, treatment with immune checkpoint inhibitors results in a lower response rate, but the responses tend to be more durable. BRAF inhibition results in a more favorable tumor microenvironment in patients, with an increase in CD8+ T-cell infiltrate and a decrease in immunosuppressive cytokines. There is also increased expression of the immunomodulatory molecule PDL1, which may contribute to the resistance. On the basis of these findings, we hypothesized that BRAF-targeted therapy may synergize with the PD1 pathway blockade to enhance antitumor immunity. To test this hypothesis, we developed a BRAF(V600E)/Pten−/− syngeneic tumor graft immunocompetent mouse model in which BRAF inhibition leads to a significant increase in the intratumoral CD8+ T-cell density and cytokine production, similar to the effects of BRAF inhibition in patients. In this model, CD8+ T cells were found to play a critical role in the therapeutic effect of BRAF inhibition. Administration of anti-PD1 or anti-PDL1 together with a BRAF inhibitor led to an enhanced response, significantly prolonging survival and slowing tumor growth, as well as significantly increasing the number and activity of tumor-infiltrating lymphocytes. These results demonstrate synergy between combined BRAF-targeted therapy and immune checkpoint blockade. Although clinical trials combining these two strategies are ongoing, important questions still remain unanswered. Further studies using this new melanoma mouse model may provide therapeutic insights, including optimal timing and sequence of therapy. Cancer Immunol Res; 2(7); 643–54. ©2014 AACR.

To prime reverse transcription of Moloney murine leukaemia virus, a transfer RNA molecule must bind two regions of the retroviral RNA, the primer binding site (PBS) and primer activation signal within the U5-PBS; here, the NMR structures of the U5-PBS RNA and tRNA primer are solved, with and without the retroviral nucleocapsid protein, which remodels these regions. Retroviruses preferentially use specific host tRNAs as primers for the first step of reverse transcription. Moloney murine leukaemia virus (MLV) uses tRNAPro, which binds to two regions of the retroviral RNA (U5-PBS and PAS). Victoria D'Souza and colleagues have solved NMR structures of the U5-PBS RNA and tRNA primer, with and without retroviral nucleocapsid protein, to understand how it remodels these regions as part of the priming process. In contrast to other ATP-independent nucleocapsid chaperones that bind many RNA sites with low affinity, this retroviral protein uses an entropy-driven capture-and-release remodelling mechanism where few sites are bound with high affinity. This results in local destabilization of the regions specifically required for the intermolecular interaction. To prime reverse transcription, retroviruses require annealing of a transfer RNA molecule to the U5 primer binding site (U5-PBS) region of the viral genome1,2. The residues essential for primer annealing are initially locked in intramolecular interactions3,4,5; hence, annealing requires the chaperone activity of the retroviral nucleocapsid (NC) protein to facilitate structural rearrangements6. Here we show that, unlike classical chaperones, the Moloney murine leukaemia virus NC uses a unique mechanism for remodelling: it specifically targets multiple structured regions in both the U5-PBS and tRNAPro primer that otherwise sequester residues necessary for annealing. This high-specificity and high-affinity binding by NC consequently liberates these sequestered residues—which are exactly complementary—for intermolecular interactions. Furthermore, NC utilizes a step-wise, entropy-driven mechanism to trigger both residue-specific destabilization and residue-specific release. Our structures of NC bound to U5-PBS and tRNAPro reveal the structure-based mechanism for retroviral primer annealing and provide insights as to how ATP-independent chaperones can target specific RNAs amidst the cellular milieu of non-target RNAs.

Due to the increasing attention on green hotels, this article aims to investigate environmental assessment methods (EAMs) and eco-labels in China. In this study, three EAMs for the evaluation of hotel environmental performance are included: Green Globe 21 Certification, Hotel Building Environmental Assessment Scheme, and Green Hotel Certification are chosen for reviewing and benchmarking. General features are compared, then key issues are categorized, dissected, and analyzed. These major issues include energy, water, waste, building systems, design, environmental quality, emissions, health, and safety management. The results show that these assessments lack uniformity. In addition, results indicate that benefits from EAMs are uncertain. Queries on the weight and credit formulation are also raised. The study also implies that there is a need to create comprehensive guidelines for hotel operators to select the most suitable assessments for their hotels.