Instability of transgene expression is a major challenge for the biopharmaceutical industry, which can impact yields and regulatory approval. Some tRNA genes (tDNAs) can resist epigenetic silencing, the principal mechanism of expression instability, and protect adjacent genes against the spread of repressive heterochromatin. We have taken two naturally occurring clusters of human tDNAs and tested their ability to reduce epigenetic silencing of transgenes integrated into the genome of Chinese hamster ovary (CHO) cells. We find sustained improvements in productivity both in adherent CHO-K1 cells and in an industrially relevant CHO-DG44 expression system (Apollo X, FUJIFILM Diosynth Biotechnologies). We conclude that specific tDNA clusters offer potential to mitigate the widespread problem of production instability.

England's 10 national parks are renowned for their landscapes, wildlife, and recreational value. However, surface waters in the national parks may be vulnerable to pollution from human‐use chemicals, such as active pharmaceutical ingredients (APIs), because of factors like ineffective wastewater treatment, seasonal tourism, a high proportion of elderly residents, and the presence of low‐flow water bodies that limit dilution. The present study determined the extent of API contamination in the English national parks by monitoring 54 APIs in 37 rivers across all national parks over two seasons. Results were compared to existing data sets for UK cities and to concentration thresholds for ecological impacts and antimicrobial resistance selection. Results revealed widespread contamination of the national parks, with APIs detected at 52 out of 54 sites and in both seasons. Thirty‐one APIs were detected, with metformin, caffeine, and paracetamol showing the highest mean concentrations and cetirizine, metformin, and fexofenadine being the most frequently detected. While total API concentrations were generally lower than seen previously in UK cities, locations in the Peak District and Exmoor had higher concentrations than most city rivers. Fourteen locations had concentrations of either amitriptyline, carbamazepine, clarithromycin, diltiazem, metformin, paracetamol, or propranolol above levels of concern for fish, invertebrates, and algae or for selection for antimicrobial resistance. Therefore, API pollution of the English national parks appears to pose risks to ecological health and potentially human health through recreational water use. Given that these parks are biodiversity hotspots with protected ecosystems, there is an urgent need for improved monitoring and management of pharmaceutical pollution and pollution more generally not only in national parks in England but also in similar environments across the world. Environ Toxicol Chem 2024;00:1–14. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

RNA polymerase (pol) III occurs in two forms, containing either the POLR3G subunit or the related paralogue POLR3GL. Whereas POLR3GL is ubiquitous, POLR3G is enriched in undifferentiated cells. Depletion of POLR3G selectively triggers proliferative arrest and differentiation of prostate cancer cells, responses not elicited when POLR3GL is depleted. A small molecule pol III inhibitor can cause POLR3G depletion, induce similar differentiation and suppress proliferation and viability of cancer cells. This response involves control of the fate-determining factor NANOG by small RNAs derived from Alu short interspersed nuclear elements. Tumour initiating activity in vivo can be reduced by transient exposure to the pol III inhibitor. Untransformed prostate cells appear less sensitive than cancer cells to pol III depletion or inhibition, raising the possibility of a therapeutic window.

Chloride channels are known to play critical physiological roles in many cell types. Here, we describe the expression of anion channels using RNA Seq in primary cultures of human bronchial epithelial cells (hBECs). Chloride intracellular channel (CLIC) family members were the most abundant chloride channel transcripts, and CLIC1 showed the highest level of expression. In addition, we characterize the chloride currents in hBECs and determine how inhibition of CLIC1 via pharmacological and molecular approaches impacts these. We demonstrate that CLIC1 is able to modulate cyclic AMP-induced chloride currents and suggest that CLIC1 modulation could be important for chloride homeostasis in this cell type.

Introduction Genome-Wide Association Studies have identified associations between lung function measures and Chronic Obstructive Pulmonary Disease (COPD) and chromosome region 6p21 containing the gene for the Advanced Glycation End Product Receptor (AGER, encoding RAGE). We aimed to (i) characterise RAGE expression in the lung, (ii) identify AGER transcripts, (iii) ascertain if SNP rs2070600 (Gly82Ser C/T) is associated with lung function and serum sRAGE levels and (iv) identify whether the Gly82Ser variant is functionally important in altering sRAGE levels in an airway epithelial cell model. Methods Immunohistochemistry was used to identify RAGE protein expression in 26 human tissues and qPCR was used to quantify AGER mRNA in lung cells. Gene expression array data was used to identify AGER expression during lung development in 38 fetal lung samples. RNA-Seq was used to identify AGER transcripts in lung cells. sRAGE levels were assessed in cells and patient serum by ELISA. BEAS2B-R1 cells were transfected to overexpress RAGE protein with either the Gly82 or Ser82 variant and sRAGE levels identified. Results Immunohistochemical assessment of 6 adult lung samples identified high RAGE expression in the alveoli of healthy adults and individuals with COPD. AGER/RAGE expression increased across developmental stages in human fetal lung at both the mRNA (38 samples) and protein levels (20 samples). Extensive AGER splicing was identified. The rs2070600T (Ser82) allele is associated with higher FEV1, FEV1/FVC and lower serum sRAGE levels in UK smokers. Using an airway epithelium model overexpressing the Gly82 or Ser82 variants we found that HMGB1 activation of the RAGE-Ser82 receptor results in lower sRAGE production. Conclusions This study provides new information regarding the expression profile and potential role of RAGE in the human lung and shows a functional role of the Gly82Ser variant. These findings advance our understanding of the potential mechanisms underlying COPD particularly for carriers of this AGER polymorphism.

Genome-Wide Association Study (GWAS) meta-analyses have identified a strong association signal for lung function, which maps to a region on 4q24 containing two oppositely transcribed genes: glutathione S-transferase, C-terminal domain containing (GSTCD) and integrator complex subunit 12 (INTS12). Both genes were found to be expressed in a range of human airway cell types. The promoter regions and transcription start sites were determined in mRNA from human lung and a novel splice variant was identified for each gene. We obtained the following evidence for GSTCD and INTS12 co-regulation and expression: (i) correlated mRNA expression was observed both via Q-PCR and in a lung expression quantitative trait loci (eQTL) study, (ii) induction of both GSTCD and INTS12 mRNA expression in human airway smooth muscle cells was seen in response to TGFβ1, (iii) a lung eQTL study revealed that both GSTCD and INTS12 mRNA levels positively correlate with percent predicted FEV1, and (iv) FEV1 GWAS associated SNPs in 4q24 were found to act as an eQTL for INTS12 in a number of tissues. In fixed sections of human lung tissue, GSTCD protein expression was ubiquitous, whereas INTS12 expression was predominantly in epithelial cells and pneumocytes. During human fetal lung development, GSTCD protein expression was observed to be highest at the earlier pseudoglandular stage (10-12 weeks) compared with the later canalicular stage (17-19 weeks), whereas INTS12 expression levels did not alter throughout these stages. Knowledge of the transcriptional and translational regulation and expression of GSTCD and INTS12 provides important insights into the potential role of these genes in determining lung function. Future work is warranted to fully define the functions of INTS12 and GSTCD.

Meta-analyses of genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) spanning the 5-hydroxytryptamine receptor 4 (5-HT4R) gene (HTR4) associated with lung function. The aims of this study were to i) investigate the expression profile of HTR4 in adult and fetal lung tissue and cultured airway cells, ii) further define HTR4 gene structure and iii) explore the potential functional implications of key SNPs using a bioinformatic approach.

OBJECTIVES:The postinfectious irritable bowel syndrome (PI-IBS) suggests that impaired resolution of inflammation could cause IBS symptoms. The authors hypothesised that polymorphisms in genes whose expression were altered by gastroenteritis might be linked to IBS with diarrhoea (IBS-D) which closely resembles PI-IBS. DESIGN:Part 1: 25 healthy volunteers (HVs), 21 patients 6 months after Campylobacter jejuni infection, 37 IBS-D and 19 IBS with constipation (IBS-C) underwent rectal biopsy for gene expression analysis and peripheral blood mononuclear cell cytokine production assessment. Part 2: Polymorphisms in genes whose expression was altered in Part 1 were assessed in 179 HV, 179 IBS-D, 122 IBS-C and 41 PI-IBS. RESULTS:Part 1: Mucosal expression of seven genes was altered in IBS: CCL11, CCL13, Calpain 8 and TNFSF15 increased while NR1D1, GPR161 and GABRE decreased with similar patterns after infection with C jejuni. Part 2: The authors assessed 21 known single nucleotide polymorphisms (SNPs) in these seven genes and one SNP in each of the TNFα and IL-10 genes. Three out of five TNFSF15 SNPs (rs6478108, rs6478109 and rs7848647) showed reduced minor allele frequency (MAF) (0.28, 0.27 and 0.27) in subjects with IBS-D compared with HV (0.38, 0.36 and 0.37; p=0.007, 0.015 and 0.007, respectively) confirming others recent findings. The authors also replicated the previously reported association of the TNFα SNP rs1800629 with PI-IBS which showed an increase in the MAF at 0.30 versus 0.19 for HV (p=0.04). CONCLUSION:IBS-D and PI-IBS patients are associated with TNFSF15 and TNFα genetic polymorphisms which also predispose to Crohn's disease suggesting possible common underlying pathogenesis.

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Introduction The heritability of irritable bowel syndrome (IBS) has been estimated at approximately 50–60%. An important subgroup (18%) of all IBS patients develop symptoms after gastrointestinal infection (postinfective IBS, PI-IBS). PI-IBS develops in 10% of Campylobacter jejuni infection, 2/3 of which are of the IBS with diarrhoea subtype (IBS-D). Aims To use changes in rectal mucosal gene expression following C jejuni infection which were also seen in irritable bowel syndrome as a guide to identify and test candidate genes in IBS. Methods Study 1: 31 patients with documented C. jejuni infection, 37 IBS with diarrhoea and 25 healthy volunteers provided blood and rectal biopsies for mRNA assessment using an Affymetrix array. Genes showing >1.5-fold change in both Group 1 and 2 compared with HV were validated by qPCR. Polymorphisms of these candidate gene were assessed in 168 IBS-D, 132 IBS with constipation (IBS-C) and 153 healthy volunteers (HV). Results As previously reported1 the following proinflammatory genes were increased in rectal biopsies following C jejuni infections and in IBS-D: CCL11, CCL13, TNFSF15, Calpain and GABRE while NR1D1 and GPR161 were depressed. We found no difference in the distribution of genetic polymorphisms of the CCL11, CCL13, genes between HV and IBS. We found an increased frequency of the T allele in the TNFSF15 SNP rs6478108 in IBS-D (72% versus 62% in HV, p=0.007) but not IBS-C, in keeping with the T allele being associated with an increased risk of Crohn9s disease. Similarly we found increased frequency of the proinflammatory C allele from the TNFSF15 SNP rs7848647 and the G allele from TNFSF15 SNP rs6478109 in D-IBS (p=0.007 and p=0.015 respectively). Although owing to small numbers we found a non-significant difference in TNFSF15 gene expression associated with the risk alleles (p=0.1) there was a linear test for trend for the CC genotype of rs6478108 to have a lower TNFSF15 expression. Conclusion We have identified a proinflammatory genetic tendency for increased TNFSF15 expression which may predispose to the development of IBS with diarrhoea.

BACKGROUND & AIMS Patients with irritable bowel syndrome with diarrhea (IBS-D) have increased mucosal serotonin (5-hydroxytryptamine [5-HT]) availability, possibly because immune activation reduces activity of the 5-HT transporter (SERT). We investigated the relationship between mucosal and platelet SERT and immune activation of the duodenal mucosa in patients with IBS-D. METHODS We quantified mucosal intraepithelial lymphocytes (IELs), mast cells, and enterochromaffin cells in blood samples, measured levels of SERT messenger RNA (mRNA) in mucosal samples, and assessed platelet uptake of 5-HT and platelet membrane binding of (3)H-paroxetine in samples from 29 healthy volunteers (HVs), 20 patients with IBS-D, and 20 untreated patients with celiac disease. RESULTS Patients with IBS-D or celiac disease had increased numbers of IELs and mast cells compared with HVs (both P < .001). Levels of SERT mRNA were reduced in the mucosa of patients with IBS-D or celiac disease and were inversely correlated with numbers of IELs (r = -0.72, P < .0001). Uptake of 5-HT by platelets from patients with IBS-D or celiac disease was reduced (mean, 17.1 ± 3.5 and 28.3 ± 4.1 nmol·min(-1)·mg(-1), respectively) compared with HVs (50.8 ± 8.0 nmol·min(-1)·mg(-1), P < .01 and P = .05, respectively). Binding of paroxetine to membranes of platelets from patients with IBS-D (median [interquartile range], 226 [92-405] fmol/mg protein) was significantly greater than that from HVs (109 [69-175] fmol/mg protein) and correlated inversely with platelet uptake of 5-HT (r = -0.62, P = .03). Tryptase release from incubated biopsy samples was significantly increased in patients with IBS-D (2.2 [0.42-3.5] vs 0.50 [0.25-0.86] ng·mL(-1)·mg(-1) for HVs; P = .03). CONCLUSIONS Platelet SERT is reduced in IBS-D and associated with reduced levels of SERT mRNA and duodenal immune activation.

Airway hyper-responsiveness (AHR) is a key feature of asthma and a causal relationship between airway inflammation and AHR has been identified. The aim of the current study was to clarify the effect of proinflammatory cytokines and asthma medication on primary human airway smooth muscle (ASM) inositol phosphate (IPx) signalling and define the regulatory loci involved.

Cysteinyl leukotrienes play an important role in the pathophysiology of many inflammatory disorders, including asthma. The aim of this study was to characterize the mechanisms underlying transcriptional regulation of the human cysteinyl leukotriene receptor 1 (hCYSLTR1) gene. 5′RACE was performed on human airway smooth muscle (HASM) and peripheral blood mononuclear cells. A 1128-bp region of the hCYSLTR1 main putative promoter was screened for polymorphisms by sequencing of 48 individuals. Luciferase reporter gene assays were performed using fragments of the core promoter (232 bp to 1128 bp) in HASM and THP1 cells. Three hCYSLTR1 transcripts were found, one representing 90% of all messenger RNA identified. The genomic location of the transcription start sites suggested there are two putative hCYSLTR1 promoters. The majority of the transcriptional activity of the main putative promoter was detected between −232 and −679 bp. Four single-nucleotide polymorphisms in strong linkage disequilibrium were found in the region studied: −561 (rs7066737), −642 (rs2806489), −781 (rs2637204), and −940 (rs321029), with three haplotypes observed. In THP1 cells, the G allele (−642) caused a twofold decrease in luciferase expression compared to the A allele. These data suggest that the majority of hCYSLTR1 transcripts in HASM and monocytes arise from a single promoter located immediately upstream of the 5′ untranslated region, although rarer transcripts can also occur. This study also raises the possibility that cell-type-dependent differences in transcriptional activity caused by the presence of specific haplotypes within the main CYSLTR1 promoter may be a predictor of disease risk or treatment response.