DNA methylation mutants in Physcomitrella patens elucidate individual roles of CG and non-CG methylation in genome regulation.

Cytosine (DNA) methylation in plants regulates the expression of genes and transposons. While methylation in plant genomes occurs at CG, CHG, and CHH sequence contexts, the comparative roles of the individual methylation contexts remain elusive. Here, we present Physcomitrella patens as the second plant system, besides Arabidopsis thaliana, with viable mutants with an essentially complete loss of methylation in the CG and non-CG contexts. In contrast to A. thaliana, P. patens has more robust CHH methylation, similar CG and CHG methylation levels, and minimal cross-talk between CG and non-CG methylation, making it possible to study context-specific effects independently. Our data found CHH methylation to act in redundancy with symmetric methylation in silencing transposons and to regulate the expression of CG/CHG-depleted transposons. Specific elimination of CG methylation did not dysregulate transposons or genes. In contrast, exclusive removal of non-CG methylation massively up-regulated transposons and genes. In addition, comparing two exclusively but equally CG- or CHG-methylated genomes, we show that CHG methylation acts as a greater transcriptional regulator than CG methylation. These results disentangle the transcriptional roles of CG and non-CG, as well as symmetric and asymmetric methylation in a plant genome, and point to the crucial role of non-CG methylation in genome regulation.

A novel ultraviolet B home phototherapy system: Efficacy, tolerability, adherence, and satisfaction.

BACKGROUND: Phototherapy is effective in treating psoriasis and other skin conditions. However, clinic-based phototherapy can be time-consuming, expensive, and inconvenient. Conventional home phototherapy addresses many hurdles, but has other limitations. OBJECTIVE: Assess the treatment efficacy, adherence, and satisfaction of a novel ultraviolet B home phototherapy system. METHODS: Eight patients with stable plaque psoriasis completed a multicenter, prospective, open label, interventional study using a home phototherapy device designed to improve treatment control and adherence. Matched control and study lesions were assessed on each subject. A dosing protocol based on American Academy of Dermatology guidelines for narrowband UVB phototherapy was managed by the phototherapy system. Responsiveness to the treatment was measured using the Psoriasis Severity Index (PSI) at 10 weeks versus control. Patient satisfaction was graded on a five-star Likert scale. RESULTS: At 10 weeks, all patients experienced improvement in the treated lesions, with a mean improvement of 57% in PSI (P<0.0001 compared to baseline and P<0.0002 compared to the control lesions). Patient treatment adherence was 96% and treatment satisfaction was 100% five-star rated. Control lesions did not significantly change in PSI over the 10-week period (P=0.1411). CONCLUSIONS: The home phototherapy system provided a safe and effective means to manage plaque psoriasis.

Corrigendum: A novel ultraviolet B home phototherapy system: Efficacy, tolerability, adherence, and satisfaction.

The original article was published on February 15, 2019 and corrected on April 15, 2019. The third pair of panes of Figure 2 were reversed, such that the pane previously depicted on the left was after phototherapy. The corrected sequence is now before phototherapy, on the left, and after, on the right. This change appears in the revised online PDF copy of this article.

A draft genome of the striped catfish, Pangasianodon hypophthalmus, for comparative analysis of genes relevant to development and a resource for aquaculture improvement.

BACKGROUND: The striped catfish, Pangasianodon hypophthalmus, is a freshwater and benthopelagic fish common in the Mekong River delta. Catfish constitute a valuable source of dietary protein. Therefore, they are cultured worldwide, and P. hypophthalmus is a food staple in the Mekong area. However, genetic information about the culture stock, is unavailable for breeding improvement, although genetics of the channel catfish, Ictalurus punctatus, has been reported. To acquire genome sequence data as a useful resource for marker-assisted breeding, we decoded a draft genome of P. hypophthalmus and performed comparative analyses. RESULTS: Using the Illumina platform, we obtained both nuclear and mitochondrial DNA sequences. Molecular phylogeny using the mitochondrial genome confirmed that P. hypophthalmus is a member of the family Pangasiidae and is nested within a clade including the families Cranoglanididae and Ictaluridae. The nuclear genome was estimated at approximately 700 Mb, assembled into 568 scaffolds with an N50 of 14.29 Mbp, and was estimated to contain ~ 28,600 protein-coding genes, comparable to those of channel catfish and zebrafish. Interestingly, zebrafish produce gadusol, but genes for biosynthesis of this sunscreen compound have been lost from catfish genomes. The differences in gene contents between these two catfishes were found in genes for vitamin D-binding protein and cytosolic phospholipase A2, which have lost only in channel catfish. The Hox cluster in catfish genomes comprised seven paralogous groups, similar to that of zebrafish, and comparative analysis clarified catfish lineage-specific losses of A5a, B10a, and A11a. Genes for insulin-like growth factor (IGF) signaling were conserved between the two catfish genomes. In addition to identification of MHC class I and sex determination-related gene loci, the hypothetical chromosomes by comparison with the channel catfish demonstrated the usefulness of the striped catfish genome as a marker resource. CONCLUSIONS: We developed genomic resources for the striped catfish. Possible conservation of genes for development and marker candidates were confirmed by comparing the assembled genome to that of a model fish, Danio rerio, and to channel catfish. Since the catfish genomic constituent resembles that of zebrafish, it is likely that zebrafish data for gene functions is applicable to striped catfish as well.

Synthesis and in vitro evaluation of tetrahydroisoquinolines with pendent aromatics as sigma-2 (σ2) selective ligands.

5-Bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl)]-2,3-dimethoxybenzamide 1 is a potent and selective σ2 receptor ligand suitable for further development. A series of new analogues, incorporating a variety of isoquinoline and carboxylic acid moieties, linked together with either a linear or cyclic amine spacer have been synthesised and assessed for their σ1/σ2 binding affinity and selectivity. Compounds with a rigid piperidine spacer gave Ki values for the σ2 receptor between 8.7-845 nM. Changing the configuration of the methoxy groups on the isoquinoline moiety resulted in molecules with σ2Ki values of 4.4-133 nM whereas varying the length and flexibility of the carbon spaces gave σ2Ki values 0.88-15.0 nM, some of the most active, selective σ2 ligands to date. Thus, the flexibility and length of the carbon linker and the carboxylic acid moiety are confirmed to be key to the exceptional binding affinity and selectivity for this active series. Additionally, the incorporation of a halogen on selected carboxylic acid moieties provided a convenient strategy for the introduction of a radiohalogen for applications in pharmacological and imaging studies.

Highly stable and immunogenic CMV T cell vaccine candidate developed using a synthetic MVA platform.

Human cytomegalovirus (CMV) is the most common infectious cause of complications post-transplantation, while a CMV vaccine for transplant recipients has yet to be licensed. Triplex, a multiantigen Modified Vaccinia Ankara (MVA)-vectored CMV vaccine candidate based on the immunodominant antigens phosphoprotein 65 (pp65) and immediate-early 1 and 2 (IE1/2), is in an advanced stage of clinical development. However, its limited genetic and expression stability restricts its potential for large-scale production. Using a recently developed fully synthetic MVA (sMVA) platform, we developed a new generation Triplex vaccine candidate, T10-F10, with different sequence modifications for enhanced vaccine stability. T10-F10 demonstrated genetic and expression stability during extensive virus passaging. In addition, we show that T10-F10 confers comparable immunogenicity to the original Triplex vaccine to elicit antigen-specific T cell responses in HLA-transgenic mice. These results demonstrate improvements in translational vaccine properties of an sMVA-based CMV vaccine candidate designed as a therapeutic treatment for transplant recipients.

Targeting fat mass and obesity-associated protein mitigates human colorectal cancer growth in vitro and in a murine model.

Introduction: Colorectal cancer (CRC) remains a significant cause of cancer related mortality. Fat mass and obesity-associated protein (FTO) is a m6A mRNA demethylase that plays an oncogenic role in various malignancies. In this study we evaluated the role of FTO in CRC tumorigenesis. Methods: Cell proliferation assays were conducted in 6 CRC cell lines with the FTO inhibitor CS1 (50-3200 nM) (± 5-FU 5-80 mM) and after lentivirus mediated FTO knockdown. Cell cycle and apoptosis assays were conducted in HCT116 cells (24 h and 48 h, 290 nM CS1). Western blot and m6A dot plot assays were performed to assess CS1 inhibition of cell cycle proteins and FTO demethylase activity. Migration and invasion assays of shFTO cells and CS1 treated cells were performed. An in vivo heterotopic model of HCT116 cells treated with CS1 or with FTO knockdown cells was performed. RNA-seq was performed on shFTO cells to assess which molecular and metabolic pathways were impacted. RT-PCR was conducted on select genes down-regulated by FTO knockdown. Results: We found that the FTO inhibitor, CS1 suppressed CRC cell proliferation in 6 colorectal cancer cell lines and in the 5-Fluorouracil resistant cell line (HCT116-5FUR). CS1 induced cell cycle arrest in the G2/M phase by down regulation of CDC25C and promoted apoptosis of HCT116 cells. CS1 suppressed in vivo tumor growth in the HCT116 heterotopic model (p< 0.05). Lentivirus knockdown of FTO in HCT116 cells (shFTO) mitigated in vivo tumor proliferation and in vitro demethylase activity, cell growth, migration and invasion compared to shScr controls (p< 0.01). RNA-seq of shFTO cells compared to shScr demonstrated down-regulation of pathways related to oxidative phosphorylation, MYC and Akt/ mTOR signaling pathways. Discussion: Further work exploring the targeted pathways will elucidate precise downstream mechanisms that can potentially translate these findings to clinical trials.

Engineering and visualization of bacteria for targeting infarcted myocardium.

Optimization of the specific affinity of cardiac delivery vector could significantly improve the efficiency of gene/protein delivery, yet no cardiac vectors to date have sufficient target specificity for myocardial infarction (MI). In this study, we explored bacterial tropism for infarcted myocardium based on our previous observations that certain bacteria are capable of targeting the hypoxic regions in solid tumors. Out of several Escherichia coli or Salmonella typhimurium strains, the S. typhimurium defective in the synthesis of ppGpp (ΔppGpp S. typhimurium) revealed accumulation and selective proliferation in the infarcted myocardium without spillover to noncardiac tissue. The Salmonellae that were engineered to express a variant of Renilla luciferase gene (RLuc8), under the control of the E. coli arabinose operon promoter (P(BAD)), selectively targeted and delivered RLuc8 in the infarcted myocardium only upon injection of L-arabinose. An examination of the infarct size before and after infection, and estimations of C-reactive protein (CRP) and procalcitonin indicated that intravenous injection of ΔppGpp S. typhimurium did not induce serious local or systemic immune reactions. This current proof-of-principle study demonstrates for the first time the capacity of Salmonellae to target infarcted myocardium and to serve as a vehicle for the selective delivery of therapeutic agents in MI.

Differential treatment regimen-related effects of HU210 on CB(1) and D(2)-like receptor functionality in the rat basal ganglia.

BACKGROUND/AIMS: Functional linkages between the cannabinoid CB(1) and the dopaminergic systems have been reported although the observations and the mechanisms hypothesizing their interactions at the G protein-coupled receptor (GPCR) functionality level are conflicting. METHODS: Administration of a potent cannabinoid agonist, HU210, at various doses (25-100 µg/kg) and treatment regimens (1- to 14-day treatment) in rats was carried out to investigate the effect of HU210 treatment on the CB(1) and D(2)-like agonist-mediated GPCR activation. RESULTS: The desensitizations (reduced coupling) of both D(2) agonist- and CB(1) agonist-mediated GPCR activation was found to be treatment duration dependent and region specific, suggesting implication of receptor tolerance and adaptation due to the cannabinoid treatment. The effect of HU210 on the CB(1) agonist-mediated GPCR desensitization in all treatment groups was not dose dependent. CONCLUSIONS: The desensitization of D(2)-like receptors found after a cannabinoid treatment in this study strengthens the evidence that the two neurotransmitter systems interact at the intercellular level; this interaction might occur via multiple mechanisms, which also vary according to region.

COH04S1 and beta sequence-modified vaccine protect hamsters from SARS-CoV-2 variants.

COVID-19 vaccine efficacy is threatened by emerging SARS-CoV-2 variants of concern (VOC) with the capacity to evade protective neutralizing antibody responses. We recently developed clinical vaccine candidate COH04S1, a synthetic modified vaccinia Ankara vector (sMVA) co-expressing spike and nucleocapsid antigens based on the Wuhan-Hu-1 reference strain that showed potent efficacy to protect against ancestral SARS-CoV-2 in Syrian hamsters and non-human primates and was safe and immunogenic in healthy volunteers. Here, we demonstrate that intramuscular immunization of Syrian hamsters with COH04S1 and an analogous Beta variant-adapted vaccine candidate (COH04S351) elicits potent cross-reactive antibody responses and protects against weight loss, lower respiratory tract infection, and lung pathology following challenge with major SARS-CoV-2 VOC, including Beta and the highly contagious Delta variant. These results demonstrate efficacy of COH04S1 and a variant-adapted vaccine analog to confer cross-protective immunity against SARS-CoV-2 and its emerging VOC, supporting clinical investigation of these sMVA-based COVID-19 vaccine candidates.

Synthetic multiantigen MVA vaccine COH04S1 protects against SARS-CoV-2 in Syrian hamsters and non-human primates.

Second-generation COVID-19 vaccines could contribute to establish protective immunity against SARS-CoV-2 and its emerging variants. We developed COH04S1, a synthetic multiantigen modified vaccinia Ankara-based SARS-CoV-2 vaccine that co-expresses spike and nucleocapsid antigens. Here, we report COH04S1 vaccine efficacy in animal models. We demonstrate that intramuscular or intranasal vaccination of Syrian hamsters with COH04S1 induces robust Th1-biased antigen-specific humoral immunity and cross-neutralizing antibodies (NAb) and protects against weight loss, lower respiratory tract infection, and lung injury following intranasal SARS-CoV-2 challenge. Moreover, we demonstrate that single-dose or two-dose vaccination of non-human primates with COH04S1 induces robust antigen-specific binding antibodies, NAb, and Th1-biased T cells, protects against both upper and lower respiratory tract infection following intranasal/intratracheal SARS-CoV-2 challenge, and triggers potent post-challenge anamnestic antiviral responses. These results demonstrate COH04S1-mediated vaccine protection in animal models through different vaccination routes and dose regimens, complementing ongoing investigation of this multiantigen SARS-CoV-2 vaccine in clinical trials.

Safety and immunogenicity of a synthetic multiantigen modified vaccinia virus Ankara-based COVID-19 vaccine (COH04S1): an open-label and randomised, phase 1 trial.

Background: COH04S1, a synthetic attenuated modified vaccinia virus Ankara vector co-expressing SARS-CoV-2 spike and nucleocapsid antigens, was tested for safety and immunogenicity in healthy adults. Methods: This combined open-label and randomised, phase 1 trial was done at the City of Hope Comprehensive Cancer Center (Duarte, CA, USA). We included participants aged 18-54 years with a negative SARS-CoV-2 antibody and PCR test, normal haematology and chemistry panels, a normal electrocardiogram and troponin concentration, negative pregnancy test if female, body-mass index of 30 kg/m2 or less, and no modified vaccinia virus Ankara or poxvirus vaccine in the past 12 months. In the open-label cohort, 1·0 × 107 plaque-forming units (PFU; low dose), 1·0 × 108 PFU (medium dose), and 2·5 × 108 PFU (high dose) of COH04S1 were administered by intramuscular injection on day 0 and 28 to sentinel participants using a queue-based statistical design to limit risk. In a randomised dose expansion cohort, additional participants were randomly assigned (3:3:1), using block size of seven, to receive two placebo vaccines (placebo group), one low-dose COH04S1 and one placebo vaccine (low-dose COH04S1 plus placebo group), or two low-dose COH04S1 vaccines (low-dose COH04S1 group). The primary outcome was safety and tolerability, with secondary objectives assessing vaccine-specific immunogenicity. The primary immunological outcome was a four times increase (seroconversion) from baseline in spike-specific or nucleocapsid-specific IgG titres within 28 days of the last injection, and seroconversion rates were compared with participants who received placebo using Fisher's exact test. Additional secondary outcomes included assessment of viral neutralisation and cellular responses. This trial is registered with ClinicalTrials.gov, NCT046339466. Findings: Between Dec 13, 2020, and May 24, 2021, 56 participants initiated vaccination. On day 0 and 28, 17 participants received low-dose COH04S1, eight received medium-dose COH04S1, nine received high-dose COH04S1, five received placebo, 13 received low-dose COH04S1 followed by placebo, and four discontinued early. Grade 3 fever was observed in one participant who received low-dose COH04S1 and placebo, and grade 2 anxiety or fatigue was seen in one participant who received medium-dose COH04S1. No severe adverse events were reported. Seroconversion was observed in all 34 participants for spike protein and 32 (94%) for nucleocapsid protein (p<0·0001 vs placebo for each comparison). Four times or more increase in SARS-CoV-2 neutralising antibodies within 56 days was measured in nine of 17 participants in the low-dose COH04S1 group, all eight participants in the medium-dose COH04S1 group, and eight of nine participants in the high-dose COH04S1 group (p=0·0035 combined dose levels vs placebo). Post-prime and post-boost four times increase in spike-specific or nucleocapsid-specific T cells secreting interferon-γ was measured in 48 (98%; 95% CI 89-100) of 49 participants who received at least one dose of COH04S1 and provided a sample for immunological analysis. Interpretation: COH04S1 was well tolerated and induced spike-specific and nucleocapsid-specific antibody and T-cell responses. Future evaluation of this COVID-19 vaccine candidate as a primary or boost vaccination is warranted. Funding: The Carol Moss Foundation and City of Hope Integrated Drug Development Venture programme.

Inhibition of tumor growth and metastasis by a combination of Escherichia coli-mediated cytolytic therapy and radiotherapy.

We have reported that Escherichia coli K-12 colonizes hypoxic and necrotic tumor regions after intravenous injection into tumor-bearing mice. In this study, we established a novel strategy for cancer therapy using engineered bacteria to enhance the therapeutic effects of radiation. E. coli strain K-12 was engineered to produce cytolysin A (ClyA), and its effects on tumor growth in primary and metastatic tumor models were evaluated. A single treatment with E. coli-expressing ClyA significantly decreased tumor growth rates initially (9 days after treatment); however, the tumors tended to grow thereafter. With only radiotherapy (RT; 21 Gy), the tumor growth rates were retarded, but not the tumor sizes. A combination of therapy with E. coli-expressing ClyA and radiation [a total of 5 x 10(7) colony-forming units (CFU) and 21 Gy] resulted in significant tumor shrinkage and even complete disappearance of tumors in mice with tumors derived from murine CT26 colon cancer. Furthermore, treatment with E. coli-expressing ClyA markedly suppressed metastatic tumor growth and prolonged the survival time in mice. The results described here indicate that therapy with engineered E. coli could significantly improve the results of RT, and could exert a striking inhibitory effect on the development of lung metastasis.

School-based nutrition interventions can improve bone health in children and adolescents.

OBJECTIVES: Osteoporosis is a major global health problem, and optimizing bone health during childhood and adolescence with adequate calcium and vitamin D intake is a recommendation to prevent the disease. School milk programs may be a viable approach to help children and adolescents increase calcium and vitamin D intakes while improving their bone health. METHODS: A review of the literature was conducted on school milk programs, including examination of details regarding these studies and their participants, and assessments of their effects on bone health in children and adolescents. RESULTS: Nearly all studies reviewed showed significant increases in the bone health behaviors and outcomes that were measured, such as bone mineral density, milk consumption, and calcium and vitamin D intake. CONCLUSIONS: School milk programs can be effective in improving bone health in children and adolescents, and public health policies should be considered to help support and fund effective programs that promote bone health and prevent osteoporosis in population health.

Regionalization of Acute Myeloid Leukemia Treatment in a Community-Based Population: Implementation and Early Results.

INTRODUCTION: Regionalization of care for acute myeloid leukemia (AML) has not been described for community-based settings. In 2015, we shifted AML induction from 21 local centers to 3 regional centers. METHODS: Using time-specific inception cohorts, we assessed whether regionalization was associated with the frequency of use of induction therapy, receipt of bone marrow transplantation, 60-day mortality (treatment toxicity), and 180-day mortality (treatment effectiveness). Information for all adult patients diagnosed with AML from 2013 to 2017 was obtained from the electronic health record. Multivariable methods were used to estimate the adjusted associations of induction, bone marrow transplantation, and death in relation to year of diagnosis before and after regionalization. RESULTS: Of 661 patients diagnosed during 2013 to 2017, 53% were ≥ 70 years, 22% were ≥ 80 years, and 10% died within the week following diagnosis. Comparing 2017 with 2013, the proportion of patients who received induction therapy increased 2.88 times (95% confidence interval [CI] = 1.55-5.35), and the proportion of non-acute promyelocytic leukemia patients receiving bone marrow transplantation increased 2.00 times (95% CI = 0.89-4.50). Regionalization was associated with lower 180-day mortality (hazard ratio [HR] = 0.64; 95% CI = 0.44-0.92), whereas change in 60-day mortality was not statistically significant (HR = 0.67; 95%CI = 0.43-1.04). CONCLUSION: In this community-based population, many patients were of advanced age yet benefitted from AML induction therapy delivered at a regionally specialized center. These early results suggest the benefit of regionalizing subspecialty leukemia care.

Inhibition of de novo pyrimidine synthesis augments Gemcitabine induced growth inhibition in an immunocompetent model of pancreatic cancer.

Leflunomide (Lef) is an agent used in autoimmune disorders that interferes with DNA synthesis. De Novo pyrimidine synthesis is a mechanism of Gemcitabine (Gem) resistance in pancreatic cancer. This study aims to assess the efficacy and changes in the tumor microenvironment of Lef monotherapy and in combination with Gem, in a syngeneic mouse model of pancreatic cancer. Methods: MTS proliferation assays were conducted to assess growth inhibition by Gem (0-20 nM), Lef (0-40 uM) and Gem+Lef in KPC (KrasLSL.G12D/+;p53R172H/+; PdxCretg/+) cells in vitro. An in vivo heterotopic KPC model was used and cohorts were treated with: PBS (control), Gem (75 mg/kg/q3d), Lef (40 mg/kg/d), or Gem+Lef. At d28 post-treatment, tumor burden, proliferation index (Ki67), and vascularity (CD31) were measured. Changes in the frequency of peripheral and intratumoral immune cell subsets were evaluated via FACS. Liquid chromatography-mass spectrometry was used for metabolomics profiling. Results: Lef inhibits KPC cell growth and synergizes with Gem in vitro (P<0.05; Combination Index 0.44 (<1 indicates synergy). In vivo, Lef alone and in combination with Gem delays KPC tumor progression (P<0.001). CTLA-4+T cells are also significantly decreased in tumors treated with Lef, Gem or in combination (Gem+Lef) compared to controls (P<0.05). Combination therapy also decreased the Ki67 and vascularity (P<0.01). Leflunomide inhibits de novo pyrimidine synthesis both in vitro (p<0.0001) and in vivo (p<0.05). Conclusions: In this study, we demonstrated that Gem+Lef inhibits pancreatic cancer growth, decrease T cell exhaustion, vascularity and as proof of principle inhibits de novo pyrimidine synthesis. Further characterization of changes in adaptive immunity are necessary to characterize the mechanism of tumor growth inhibition and facilitate translation to a clinical trial.

Synthetic Multiantigen MVA Vaccine COH04S1 Protects Against SARS-CoV-2 in Syrian Hamsters and Non-Human Primates.

Second-generation COVID-19 vaccines could contribute to establish protective immunity against SARS-CoV-2 and its emerging variants. We developed COH04S1, a synthetic multiantigen Modified Vaccinia Ankara-based SARS-CoV-2 vaccine that co-expresses spike and nucleocapsid antigens. Here, we report COH04S1 vaccine efficacy in animal models. We demonstrate that intramuscular or intranasal vaccination of Syrian hamsters with COH04S1 induces robust Th1-biased antigen-specific humoral immunity and cross-neutralizing antibodies (NAb) and protects against weight loss, lower respiratory tract infection, and lung injury following intranasal SARS-CoV-2 challenge. Moreover, we demonstrate that single-dose or two-dose vaccination of non-human primates with COH04S1 induces robust antigen-specific binding antibodies, NAb, and Th1-biased T cells, protects against both upper and lower respiratory tract infection following intranasal/intratracheal SARS-CoV-2 challenge, and triggers potent post-challenge anamnestic antiviral responses. These results demonstrate COH04S1-mediated vaccine protection in animal models through different vaccination routes and dose regimens, complementing ongoing investigation of this multiantigen SARS-CoV-2 vaccine in clinical trials.

Biodiesels Manufactured from Different Feedstock: From Fuel Properties to Fuel Atomization and Evaporation.

Different from fossil diesel, biodiesels can be manufactured from different sources of biomass or animal fat. Each biodiesel manufactured from a certain feedstock consists of different fatty acid methyl esters (FAMEs). Its FAME types and fractions are unique and are solely controlled by the mother feedstock and not the manufacturing process. One key feature that makes biodiesels different from their fossil counterparts is the oxygen contained in biodiesels. The oxygen content, FAME types, and FAME fractions vary in a wide range among biodiesels made from different feedstock and this in turn affects the fuel properties and physical processes, including atomization and evaporation. An extensive analysis has been successfully carried out in this study to examine the role of oxygen content, carbon chain length, and unsaturation degree in different biodiesels and the influence of FAMEs on key fuel properties (heating value, cetane number, viscosity, and surface tension). Furthermore, some useful information related to (i) the morphology and density of fuel fragments derived close to the nozzle exit and (ii) drop evaporation is reported. The atomization characteristics are experimentally observed using a high-speed imaging technique developed earlier, while the evaporation study is theoretically conducted using the well-known D-square model. It shows that the oxygen in the biodiesel is directly linked to the carbon chain length and the number of double bonds in the fuel molecules as well as to the key fuel properties. The viscosity of biodiesels and their constituents has a certain impact on the morphology and population of fuel fragments derived in the breakup zone, while the thermal properties have a significant effect on biodiesel evaporation. The dependence of fuel properties on atomization at the downstream locations of the spray, where the breakup process has completed, is minimal.

Inhibition of Autophagy Amplifies Baicalein-Induced Apoptosis in Human Colorectal Cancer.

Baicalein is a Chinese herbal compound extracted from Scutellaria baicalensis that has anti-tumor properties. The aim of this study was to elucidate the mechanisms of action of baicalein against human colorectal cancer cell lines and to assess whether the anti-proliferative effects of baicalein may be amplified with autophagy inhibition. Human colon cancer cell lines (HT-29, HCT-116, SW480, and SW620) were treated with baicalein alone and in combination with the autophagy inhibitor chloroquine (CQ). Baicalein reduced cell viability in all four colon cancer lines in a dose-dependent fashion. Combination treatment of baicalein and the autophagy inhibitor CQ significantly decreased cell viability compared with baicalein alone in HT-29 and HCT-116 cell lines. Western blot analysis of the HCT-116 cell line treated with both baicalein and CQ demonstrated increased expression of LC3-II, a component of autophagy. The combination of baicalein with CQ culminated in activation of caspase-3-mediated apoptosis. These findings demonstrate that inhibition of autophagy enhanced apoptotic cell death induced by baicalein treatment in colon cancer cell lines. Future work will assess other targetable apoptotic pathways activated by baicalein and autophagy inhibition.

Salmonella-mediated therapy targeting indoleamine 2, 3-dioxygenase 1 (IDO) activates innate immunity and mitigates colorectal cancer growth.

Patients with colon cancer remain largely refractory to current immunotherapeutic strategies. This is, in part, due to the overexpression of the immune checkpoint protein indoleamine 2,3-dioxygenase 1 (IDO). IDO is an important enzyme contributing to tumor-mediated immunosuppression and also correlates with poor prognosis in colon cancer patients. The aim of this study was to assess the therapeutic efficacy of attenuated Salmonella typhimurium delivering an shRNA plasmid targeting IDO (shIDO-ST) in two mouse models of colorectal cancer. In vitro, the CT26 and MC38 murine colon cancer cell lines were shown to upregulate IDO expression following stimulation with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Transfection of both cell lines with shIDO plasmid reduced IDO protein expression and function. In vivo, shIDO-ST treatment significantly delayed CT26 and MC38 tumor progression compared to mice treated with scrambled shRNA control (shScr-ST) or the clinically tested IDO inhibitor epacadostat. Increased tumor infiltration of neutrophils was found to be the primary immune cell population associated with shIDO-ST treatment, suggesting robust activation of innate immunity. Although increased tumor expression of IDO is associated with resistance to antibody therapy against programed cell death-1 (anti-PD1), co-administration of anti-PD1 with shIDO-ST did not provide additional tumor growth control in either model of colorectal cancer. Altogether, we demonstrate that treatment with shIDO-ST markedly delays tumor growth in two immunocompetent colorectal mouse models and this appears to be a superior therapeutic strategy compared to epacadostat or blocking anti-PD1 antibody therapy in colon cancer.