An investigation of the clinical impact and therapeutic relevance of a DNA damage immune response (DDIR) signature in patients with advanced gastroesophageal adenocarcinoma.

BACKGROUND: An improved understanding of which gastroesophageal adenocarcinoma (GOA) patients respond to both chemotherapy and immune checkpoint inhibitors (ICI) is needed. We investigated the predictive role and underlying biology of a 44-gene DNA damage immune response (DDIR) signature in patients with advanced GOA. MATERIALS AND METHODS: Transcriptional profiling was carried out on pretreatment tissue from 252 GOA patients treated with platinum-based chemotherapy (three dose levels) within the randomized phase III GO2 trial. Cross-validation was carried out in two independent GOA cohorts with transcriptional profiling, immune cell immunohistochemistry and epidermal growth factor receptor (EGFR) fluorescent in situ hybridization (FISH) (n = 430). RESULTS: In the GO2 trial, DDIR-positive tumours had a greater radiological response (51.7% versus 28.5%, P = 0.022) and improved overall survival in a dose-dependent manner (P = 0.028). DDIR positivity was associated with a pretreatment inflamed tumour microenvironment (TME) and increased expression of biomarkers associated with ICI response such as CD274 (programmed death-ligand 1, PD-L1) and a microsatellite instability RNA signature. Consensus pathway analysis identified EGFR as a potential key determinant of the DDIR signature. EGFR amplification was associated with DDIR negativity and an immune cold TME. CONCLUSIONS: Our results indicate the importance of the GOA TME in chemotherapy response, its relationship to DNA damage repair and EGFR as a targetable driver of an immune cold TME. Chemotherapy-sensitive inflamed GOAs could benefit from ICI delivered in combination with standard chemotherapy. Combining EGFR inhibitors and ICIs warrants further investigation in patients with EGFR-amplified tumours.

Smoking among female daily smokers in Surabaya, Indonesia.

OBJECTIVES: Nationally representative studies suggest 1-2% of Indonesian women (2.3 million) smoke various tobacco products daily; however, in recent years, there has been concern that the tobacco industry has successfully increased female smoking. Our objective was to describe current cigarette smoking behaviors, past quit attempts, and intention to quit of female daily smokers in Surabaya, Indonesia. STUDY DESIGN: Survey. METHODS: Female daily smokers (n = 112) in Surabaya, Indonesia, the country's second largest city, were recruited to participate in a survey during 2018. Convenience sampling was utilized in two malls. Potential participants were intercepted in or near designated smoking areas and invited to the nearby data collection site. Survey items from Global Adult Tobacco Survey and the International Tobacco Control Policy Evaluation Project were utilized. RESULTS: Participants self-reported smoking 13.8 cigarettes per day (7.3 white machine-rolled cigarettes per day, 4.2 kreteks per day, and 2.4 roll-your-own cigarettes per day). Over 75% smoked their first cigarette within 30 min of waking. Over 53% had a heaviness of smoking index score suggesting moderate or high addiction. Approximately half (51%) did not attempt to quit smoking in the previous 12 months, and 55% planned to quit beyond 6 months or not at all. CONCLUSIONS: Our sample smoked five to six more cigarettes per day than female daily smokers in previous national surveys. Relative to previous studies, our data suggest an unexpected preference for white machine-rolled cigarettes and that there could be, at a minimum, pockets of increased smoking and addiction among female daily smokers in Indonesia.

Validation of a Metastatic Assay using biopsies to improve risk stratification in patients with prostate cancer treated with radical radiation therapy.

Background: Radiotherapy is an effective treatment of intermediate/high-risk locally advanced prostate cancer, however, >30% of patients relapse within 5 years. Clinicopathological parameters currently fail to identify patients prone to systemic relapse and those whom treatment intensification may be beneficial. The purpose of this study was to independently validate the performance of a 70-gene Metastatic Assay in a cohort of diagnostic biopsies from patients treated with radical radiotherapy and androgen deprivation therapy. Patients and methods: A bridging cohort of prostate cancer diagnostic biopsy specimens was profiled to enable optimization of the Metastatic Assay threshold before further independent clinical validation in a cohort of diagnostic biopsies from patients treated with radical radiotherapy and androgen deprivation therapy. Multivariable Cox proportional hazard regression analysis was used to assess assay performance in predicting biochemical failure-free survival (BFFS) and metastasis-free survival (MFS). Results: Gene expression analysis was carried out in 248 patients from the independent validation cohort and the Metastatic Assay applied. Ten-year MFS was 72% for Metastatic Assay positive patients and 94% for Metastatic Assay negative patients [HR = 3.21 (1.35-7.67); P = 0.003]. On multivariable analysis the Metastatic Assay remained predictive for development of distant metastases [HR = 2.71 (1.11-6.63); P = 0.030]. The assay retained independent prognostic performance for MFS when assessed with the Cancer of the Prostate Assessment Score (CAPRA) [HR = 3.23 (1.22-8.59); P = 0.019] whilst CAPRA itself was not significant [HR = 1.88, (0.52-6.77); P = 0.332]. A high concordance [100% (61.5-100)] for the assay result was noted between two separate foci taken from 11 tumours, whilst Gleason score had low concordance. Conclusions: The Metastatic Assay demonstrated significant prognostic performance in patients treated with radical radiotherapy both alone and independent of standard clinical and pathological variables. The Metastatic Assay could have clinical utility when deciding upon treatment intensification in high-risk patients. Genomic and clinical data are available as a public resource.

Second-hand smoke and indoor air quality in public places in Gaza City.

Second-hand smoke is a major cause of preventable disease and death in the Eastern Mediterranean Region. To assess how second-hand smoke impacts air quality, respirable suspended particles (PM2.5) which are emitted during cigarette and waterpipe smoking, were measured inside and outside of 18 smoking and 5 nonsmoking public venues in Gaza city. Median PM2.5 level inside the smoking venues was 117 microg/m3, which exceeds the WHO guidelines for daily PM2.5 exposure (25 microg/m3) by more than 4-fold. The median level inside the smoking venues (117 microg/m3) was significantly higher than outside the venues (43 microg/m3), and significantly higher than the median level inside non-smoking venues (40 microg/m3). By contrast particulate levels outside non-smoking venues (35 microg/m3) did not differ from the corresponding levels inside (40 microg/m3). To protect employees and the public from second-hand smoke exposure, policies prohibiting sinoking in public niaces are needed in Gaza city.

Second-hand smoke and indoor air quality in public places in Gaza city

Second-hand smoke is a major cause of preventable disease and death in the Eastern Mediterranean Region. To assess how second-hand smoke impacts air quality, respirable suspended particles [PM[2.5]], which are emitted during cigarette and waterpipe smoking, were measured inside and outside of 18 smoking and 5 nonsmoking public venues in Gaza city. Median PM[2.5] level inside the smoking venues was 117 microg/m[3], which exceeds the WHO guidelines for daily PM[2.5] exposure [25 microg/m[3]] by more than 4-fold. The median level inside the smoking venues [117 microg/m[3]] was significantly higher than outside the venues [43 microg/m[3]], and significantly higher than the median level inside non-smoking venues [40 microg/m[3]]. By contrast, particulate levels outside non-smoking venues [35 microg/m[3]] did not differ from the corresponding levels inside [40 microg/m[3]]. To protect employees and the public from second-hand smoke exposure, policies prohibiting smoking in public places are needed in Gaza city

BRCA1 and GATA3 corepress FOXC1 to inhibit the pathogenesis of basal-like breast cancers.

In this study we describe a novel interaction between the breast/ovarian tumor suppressor gene BRCA1 and the transcription factor GATA3, an interaction, which is important for normal breast differentiation. We show that the BRCA1-GATA3 interaction is important for the repression of genes associated with triple-negative and basal-like breast cancer (BLBCs) including FOXC1, and that GATA3 interacts with a C-terminal region of BRCA1. We demonstrate that FOXC1 is an essential survival factor maintaining the proliferation of BLBCs cell lines. We define the mechanistic basis of this corepression and identify the GATA3-binding site within the FOXC1 distal promoter region. We show that BRCA1 and GATA3 interact on the FOXC1 promoter and that BRCA1 requires GATA3 for recruitment to this region. This interaction requires fully functional BRCA1 as a mutant BRCA1 protein is unable to localize to the FOXC1 promoter or repress FOXC1 expression. We demonstrate that this BRCA1-GATA3 repression complex is not a FOXC1-specific phenomenon as a number of other genes associated with BLBCs such as FOXC2, CXCL1 and p-cadherin were also repressed in a similar manner. Finally, we demonstrate the importance of our findings by showing that loss of GATA3 expression or aberrant FOXC1 expression contributes to the drug resistance and epithelial-to-mesenchymal transition-like phenotypes associated with aggressive BLBCs.

T-box 2 represses NDRG1 through an EGR1-dependent mechanism to drive the proliferation of breast cancer cells.

T-box 2 (TBX2) is a transcription factor involved in mammary development and is known to be overexpressed in a subset of aggressive breast cancers. TBX2 has previously been shown to repress growth control genes such as p14(ARF) and p21(WAF1/cip1). In this study we show that TBX2 drives proliferation in breast cancer cells and this is abrogated after TBX2 small interfering RNA (siRNA) knockdown or after the expression of a dominant-negative TBX2 protein. Using microarray analysis we identified a large cohort of novel TBX2-repressed target genes including the breast tumour suppressor NDRG1 (N-myc downregulated gene 1). We show that TBX2 targets NDRG1 through a previously undescribed mechanism involving the recruitment of early growth response 1 (EGR1). We show EGR1 is required for the ability of TBX2 to repress NDRG1 and drive cell proliferation. We show that TBX2 interacts with EGR1 and that TBX2 requires EGR1 to target the NDRG1 proximal promoter. Abrogation of either TBX2 or EGR1 expression is accompanied by the upregulation of cell senescence and apoptotic markers. NDRG1 can recapitulate these effects when transfected into TBX2-expressing cells. Together, these data identify a novel mechanism for TBX2-driven oncogenesis and highlight the importance of NDRG1 as a growth control gene in breast tissue.

Characterization of the metabolic burden on Escherichia coli DH1 cells imposed by the presence of a plasmid containing a gene therapy sequence.

The presence of a plasmid, containing gene sequences for DNA immunotherapy that are not expressed in microbial culture, imposed a degradation in bioreactor performance in cultures of the host E. coli strain. Significant decreases in growth rate (24%) and biomass yield (7%) and a corresponding increase in overflow metabolism were observed in a strain containing a therapeutic sequence (a hepatitis B antigen under the control of a CMV promotor). The observed increase in overflow metabolism was incorporated into a Metabolic Flux Analysis (MFA) model (as acetate secretion). Metabolic flux analysis revealed an increase in TCA cycle flux, consistent with an increased respiration rate observed in plasmid-containing cells. These effects are thought to result from increased ATP synthesis requirements (24%) arising from the expression of the Kanr plasmid marker gene whose product accounted for 18% of the cell protein of the plasmid-containing strain. These factors will necessitate significantly higher aeration and agitation rates or lower nutrient feed rates in high-density cultures than would be expected for plasmid-free cultures.

Role played by BRCA1 in regulating the cellular response to stress.

BRCA1 (breast-cancer susceptibility gene 1) is a tumour suppressor gene that is mutated in the germline of women with a genetic predisposition to breast and ovarian cancer. In this review, we examine the role played by BRCA1 in mediating the cellular response to stress. We review the role played by BRCA1 in detecting and signalling the presence of DNA damage, particularly double-strand DNA breaks, and look at the evidence to support a role for BRCA1 in regulating stress response pathways such as the c-Jun N-terminal kinase/stress-activated protein kinase pathway. In addition, we examine the role played by BRCA1 in mediating both cell-cycle arrest and apoptosis following different types of cellular insult, and how this may be modulated by the presence or absence of associated proteins such as p53. Finally, we explore the possibility that many of the functions associated with BRCA1 may be based on transcriptional regulation of key downstream genes that have been implicated in the regulation of these specific cellular pathways.

The formation of plasmid DNA loaded pharmaceutical powders using supercritical fluid technology.

The invention of novel drugs based on biological macromolecules requires the development of specialized formulation methods. Supercritical fluid technology offers the possibility to produce dry powder formulations suitable for inhalation or needle-free injection. In this article we describe the first application of a process involving supercritical carbon dioxide for the production of plasmid DNA-loaded particles. The technique of solution enhanced dispersion by supercritical fluids (SEDS) is used to coformulate the 6.9 kb plasmid pSV beta with mannitol as excipient. After initial experiments showed a high degradation of the plasmid during powder formation, a systematic investigation of the process revealed pH effects to be crucial for the recovery of intact DNA. The application of high-buffer concentration led to an increase of the recovered supercoiled proportion from 7% to 80%.

Radiation recall dermatitis in a patient treated with dacarbazine.

Radiation recall describes an inflammatory reaction at a previously irradiated site associated with the use of chemotherapeutic agents. Dacarbazine, a tetrazine cytotoxic drug, has not been noted to cause this phenomenon. We report the case history of a 44-year-old female patient who developed a recall dermatitis due to dacarbazine in a site previously irradiated for the treatment of malignant melanoma. The skin erythema responded quickly to oral corticosteroid treatment. Further cycles of dacarbazine were facilitated with oral corticosteroid premedication. We conclude that dacarbazine should be considered as a potential cause of radiation recall dermatitis and that this can be managed and prevented with oral corticosteroids.

Biochemical engineering approaches to the challenges of producing pure plasmid DNA.

Plasmid-based genes offer promise for a new generation of vaccines and for gene therapy, but the size and character of plasmids pose new challenges to biochemical engineers. By acknowledging these and using bioprocess-design information based on fundamental studies of the system's properties, it will be possible to create efficient and consistent processes for these materials. This review addresses the purity required, the key issue of the sensitivity of the chromosomal DNA contaminant and larger plasmids to hydrodynamic forces, and the impact of this and other characteristics of plasmids on the recovery and purification of DNA for pharmaceutical purposes.

Effects of growth medium selection on plasmid DNA production and initial processing steps.

Cultures of recombinant Escherichia coli containing the plasmid pSVbeta were grown in three medium formulations to assess their effects on the characteristics of supercoiled plasmid DNA production for plasmid-based gene therapy. A semi-defined medium containing casamino acids (SDCAS) was found to support higher cell densities and higher plasmid stability than a similar medium containing soya amino acids (SDSOY) or Luria-Bertani medium (LB). Differences were observed in the cell harvest characteristics, plasmid DNA primary recovery, plasmid DNA yield and quality between cells grown on LB and on SDCAS medium. Cells grown on SDCAS medium were more difficult to resuspend after harvest than those grown in LB medium and were less susceptible to alkaline lysis. The plasmid DNA content from SDCAS was predominantly supercoiled and was less contaminated by chromosomal DNA than plasmid DNA extracts derived from cells grown on LB medium. It was hypothesised that the different carbon:nitrogen ratio (C:N) of the medium may have been responsible for changing the cell wall polysaccharide composition resulting in the change in cell harvest and lysis characteristics. Results indicated that changing the C:N ratio of SDCAS medium between 1.21:1 and 12.08:1 resulted in no alteration in cell wall polysaccharide composition or in cell susceptibility to chemical lysis or physical breakage. Plasmid DNA yields increased ten-fold with ten-fold increase in the C:N ratio of SDCAS medium.

Rapid quantitation and monitoring of plasmid DNA using an ultrasensitive DNA-binding dye.

A sensitive fluorescence-based method for monitoring plasmid DNA during production was investigated. This simple method of assaying for plasmid DNA allows rapid monitoring of plasmid yields from a recombinant Escherichia coli fed-batch fermentation. The assay has several advantages over traditional methods of plasmid DNA measurement. The fluorescent dye is highly specific and can measure total plasmid DNA concentration in about 5 min. The assay is sensitive over a wide range of plasmid concentrations of between 15 and 280 ng/mL, even in the presence of impurities that occur within alkaline lysate preparations. The technique can also be applied to monitoring fermentation and downstream purification steps.

Fatal occupational injuries associated with forklifts, United States, 1980-1994.

BACKGROUND: This paper describes deaths of American workers involving forklifts during the 15-year period from January 1, 1980 to December 31, 1994. METHODS: Death certificate data were obtained from the National Institute for Occupational Safety and Health's (NIOSH's) National Traumatic Occupational Fatality (NTOF) surveillance system. The narrative fields on the death certificate were searched for keywords indicating that a powered industrial vehicle (PIV) or forklift was involved in the death. This study examined the circumstances of the forklift-related deaths, the nature of the injury, and the decedent's age, gender, race, occupation, and industry. Average annual employment data from the Bureau of the Census were used to calculate civilian fatality rates by age, gender, industry, and occupation. RESULTS: A total of 1,021 deaths were identified. The average age of the fatally injured worker was 38 years; the 1,021 forklift-related deaths resulted in a total of 27,505 years of productive life lost. The three most common circumstances of the fatalities were forklift overturns (22%), pedestrian struck by forklifts (20%), and worker crushed by forklift (16%). The greatest proportion of the fatalities (37%) occurred to workers in Manufacturing, followed by Transportation, Communication, and Public Utilities, (TCPU), (17%), Construction (16%), Wholesale Trade (8%), and Agriculture, Forestry, and Fishing (AFF) (7%). The highest forklift-related fatality rates per ten million workers occurred among transport operatives (34.0) and laborers (32.0). CONCLUSIONS: Many of the fatalities resulting from forklift "overturns" might have been prevented if the operator had been restrained with a lap/shoulder belt. Careful consideration should be given to separating pedestrian and forklift traffic, and restricting the use of forklifts near time clocks, exits, and other areas where large numbers of pedestrians pass through an area in a short time. Additionally, systematic traffic control, including rules for pedestrian and forklift traffic, will be necessary to reduce the enormous injury and death toll associated with forklifts. Am. J. Ind. Med. 36:504-512, 1999. Published 1999 Wiley-Liss, Inc.

The isolation of strains of Saccharomyces cerevisiae showing altered plasmid stability characteristics by means of selective continuous culture.

A recombinant strain of Saccharomyces cerevisiae containing a plasmid-encoded lacZ gene from Escherichia coli was grown for 420 generations under selective conditions in glucose-limited continuous culture. A ura3-based auxotrophic system was used to apply selection in favour of plasmid-containing organisms. A similar strategy had previously proved successful at evolving clones of Bacillus subtilis, showing improved plasmid stability characteristics. In this study a series of clones were isolated which exhibited large variation in their ability to retain the recombinant plasmid. Clones showed both significantly increased and reduced capacity to maintain the recombinant plasmid. The probabilities of obtaining clones in either category were essentially equal so that selection was not seen to enrich for more stable clones. Periodic selection events appeared to exert a greater influence on the distribution of stability characteristics amongst clones than did the applied selective pressure. Alterations in plasmid retention characteristics could be associated with host or plasmid. The most stable clone isolated exhibited a approximately 30% improvement of its overall stability (sigma(N+)) and an 80% improvement in productivity, when compared to the parental strain CGpLG. This improved stability was associated with alterations in the plasmid genome.

Use of a reduced-carbohydrate, modified-fat enteral formula for improving metabolic control and clinical outcomes in long-term care residents with type 2 diabetes: results of a pilot trial.

Physiologic responses of 30 enterally-fed long-term care residents with type 2 diabetes receiving total nutrition support via either a disease-specific (reduced-carbohydrate, modified-fat) formula or a standard high-carbohydrate formula for 3 mo were compared. Objectives of the study included evaluating metabolic response (glycemic control and lipids) and clinical outcomes. Thirty-four subjects requiring total enteral nutrition support by tube were enrolled in this prospectively randomized, double-blind, controlled, parallel group 3-mo pilot trial. Thirty were evaluable in that they completed 4 wk. Twenty-seven completed all 12 wk. The groups were well-matched for physiologic and demographic parameters at baseline. Fasting serum glucose and capillary (fingerstick) glucose values demonstrated better control in the disease-specific formula-fed group. Serum lipid profiles of this group were similar to or better than those of the standard formula-fed group. The amount of insulin administered to insulin-using subjects in the disease-specific formula-fed group was consistently less than before initiation of the formula, whereas the amount administered was consistently higher in the group fed the standard formula. Overall, subjects randomized to the disease-specific formula experienced better numerical biochemical control and better clinical outcomes when expressed on a numerical and percentage basis. These included surrogate markers of diabetes control such as serum glucose and glycohemoglobin, as well as clinical outcomes such as incidence of infections and pressure ulcers. These findings confirm that the disease-specific formula provides better glycemic control, poses no risk to lipoprotein metabolism, and provides for better clinical outcomes.

Effects of medium composition and nutrient limitation on loss of the recombinant plasmid pLG669-z and beta-galactosidase expression by Saccharomyces cerevisiae.

The effects of medium composition, nutrient limitation and dilution rate on the loss of the recombinant plasmid pLG669-z and plasmid-borne beta-galactosidase expression were studied in batch and chemostat cultures of Saccharomyces cerevisiae strain CGpLG. The difference in growth rates between plasmid-free and plasmid-containing cells (delta mu) and the rate of segregation (R) were determined and some common factors resulting from the effect of medium composition on plasmid loss were identified. Glucose-limited chemostat cultures of CGpLG grown on defined medium were more stable at higher dilution rates and exhibited delta mu-dominated plasmid loss kinetics. Similar cultures grown on complex medium were more stable at lower dilution rates and exhibited R-dominated plasmid loss kinetics. Overall plasmid stability was greatest in phosphate-limited chemostat cultures grown on defined medium and was least stable in magnesium-limited cultures grown on defined medium. delta mu decreased and R increased with increased dilution rate, irrespective of medium composition. Increased plasmid loss rates at high or low dilution rates would appear to be characteristic of loss kinetics dominated by R or delta mu, respectively. Growth of glucose-limited chemostat cultures on complex medium decreased delta mu values but increased R values, in comparison to those cultures grown on defined medium. Any increased stability that a complex medium-induced reduction of delta mu may have conferred was counteracted by an increased R value. Increased beta-galactosidase productivity was correlated with increased plasmid stability only in glucose-limited chemostat cultures grown on defined medium and not in those grown on complex medium. Previous studies have yielded contrasting responses with regard to the effect of dilution rate on recombinant plasmid loss from S. cerevisiae. Our findings can account for these differences and may be generally valid for the stability of similar yeast plasmid constructs. This information would facilitate the design of bioprocesses, where recombinant plasmid instability results in reduced culture productivity.