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Drinking water treatment plants (DWTPs) are designed to remove physical, chemical, and biological contaminants. However, until recently, the role of DWTPs in minimizing the cycling of antibiotic resistance determinants has got limited attention. In particular, the risk of selecting antibiotic-resistant bacteria (ARB) is largely overlooked in chlorine-free DWTPs where biological processes are applied. Here, we combined high-throughput quantitative PCR and metagenomics to analyze the abundance and dynamics of microbial communities, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) across the treatment trains of two chlorine-free DWTPs involving dune-based and reservoir-based systems. The microbial diversity of the water increased after all biological unit operations, namely rapid and slow sand filtration (SSF), and granular activated carbon filtration. Both DWTPs reduced the concentration of ARGs and MGEs in the water by circa 2.5 log gene copies mL -1 , despite their relative increase in the disinfection sub-units (SSF in dune-based and UV treatment in reservoir-based DWTPs). The total microbial concentration was also reduced (2.5 log units), and none of the DWTPs enriched for bacteria containing genes linked to antibiotic resistance. Our findings highlight the effectiveness of chlorine-free DWTPs in supplying safe drinking water while reducing the concentration of antibiotic resistance determinants. To the best of our knowledge, this is the first study that monitors the presence and dynamics of antibiotic resistance determinants in chlorine-free DWTPs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Francesc Corbera-Rubio reports financial support was provided by Dunea Duin & Water. Brent Pieterse reports a relationship with Dunea Duin & Water that includes: employment. David de Ridder reports a relationship with Evides NV that includes: employment., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Despite the frequent co-occurrence of posttraumatic stress disorder and substance use disorder, screening for trauma exposure and posttraumatic stress disorder symptoms is not a routine practice in substance use disorder clinics. The aims of this study were to examine the prevalence of exposure to traumatic events, posttraumatic stress disorder symptoms, and subjective sleep quality in substance use disorder inpatients after detoxification. In addition, we analyzed associations of sociodemographics, direct and indirect exposure to traumatic events, and sleep quality with posttraumatic stress disorder symptom severity., Methods: Adults diagnosed with substance use disorder (n = 188; 25% women, mean age 46.6 ± 12.3 years) from 2 inpatient addiction clinics were assessed at approximately 4 days post-admission for age, gender, educational level, self-reported substance use, trauma exposure, general and posttraumatic stress disorder-specific subjective sleep quality, and posttraumatic stress disorder symptom severity. Correlates of posttraumatic stress disorder symptom severity were identified with linear regression analyses., Results: The prevalence of direct trauma exposure was high (89%), 51% of participants screened positive for posttraumatic stress disorder and 87% reported clinically significant poor sleep quality. Younger age, female gender, direct and indirect exposure to more traumatic events, and poor subjective sleep quality were associated with more severe posttraumatic stress disorder symptoms., Conclusion: Nearly all substance use disorder patients admitted for detoxification in our study had been directly or indirectly exposed to 1 or more traumatic events, and many reported posttraumatic stress disorder symptoms and poor sleep quality. Younger and female substance use disorder patients were at higher risk of posttraumatic stress disorder symptoms. Our results emphasize the need for systematic screening for direct and indirect trauma exposure, posttraumatic stress disorder symptoms, and poor sleep quality in patients admitted for clinical substance use disorder treatment., (2022 authors.)

Background: To conduct a systematic review of early intervention programs (0-5 years) utilising coaching practice characteristics, to identify (i) implementation fidelity; (ii) parent training processes, and (iii) outcome measures of capacity building in parents. The coaching practice characteristics of (1) joint planning, (2) observation, (3) action/practice, (4) reflection and (5) feedback identified by Rush and Shelden were utilised. Method: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement was followed. A comprehensive search of 6 electronic databases was undertaken in March 2016 and updated in February 2018. Results: Of 2397 articles, 18 papers met full inclusion criteria. Of these, 5 were randomised controlled trials. Only one specifically evaluated the impact of parent coaching versus therapist only delivered interventions. Risk of bias and study quality using Downs and Black checklist for clinical trial quality yielded the following descriptive ratings: Seven studies: "Poor" (scores 1-13); Six studies: "Fair" (scores 15-17); and five "Good" (scores 20-24). Conclusion: Coaching in early intervention is well accepted. Nevertheless, this review identified a continued lack of operationalised definitions; inconsistency in the reporting of therapist training and adherence to active ingredients/coaching principles; and an absence of outcome measures focused on parent capacity.Implications for RehabilitationContemporary early intervention services recognise the importance of engaging parents as active participators in their child's development. This is evident by the increase in interventions that utilise parent coaching practices. The findings of this systematic review indicate the need for professionals to:•Describe and document fidelity of coaching practices in the delivery of intervention.•Objectively measure changes in parent capacity and self-efficacy as a result of the coaching based intervention.The reporting of parent capacity measures will allow us to truly examine the effectiveness of coaching practices in empowering families to support their child to realise their full potential.

In this study we investigate opportunities for reducing arsenic (As) to low levels, below 1 μg/L in produced drinking water from artificially infiltrated groundwater. We observe that rapid sand filtration is the most important treatment step for the oxidation and removal of As at water treatment plants which use artificially recharged groundwater as source. Removal of As is mainly due to As co-precipitation with Fe(III)(oxyhydr)oxides, which shows higher efficiency in rapid sand filter beds compared to aeration and supernatant storage. This is due to an accelerated oxidation of As(III) to As(V) in the filter bed which may be caused by the manganese oxides and/or As(III) oxidizing bacteria, as both are found in the coating of rapid sand filter media grains by chemical analysis and taxonomic profiling of the bacterial communities. Arsenic removal does not take place in treatment steps such as granular activated carbon filtration, ultrafiltration or slow sand filtration, due to a lack of hydrolyzing iron in their influent and a lack of adsorption affinity between As and the filtration surfaces. Further, we found that As reduction to below 1 μg/L can be effectively achieved at water treatment plants either by treating the influent of rapid sand filters by dosing potassium permanganate in combination with ferric chloride or by treating the effluent of rapid sand filters with ferric chloride dosing only. Finally, we observe that reducing the pH is an effective measure for increasing As co-precipitation with Fe(III)(oxyhydr)oxides, but only when the oxidized arsenic, As(V), is the predominant species in water., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: The co-occurrence of PTSD and alcohol use disorder (AUD) is common. Therefore, it is important to know which treatments are effective for the group of patients suffering from both disorders.
AIM: To explore the evidence of medical treatment options for PTSD and AUD.
METHOD: We systematically searched the literature using MEDLINE, Embase and psycINFO (PRISMA guideline).
RESULTS: Ten studies were included of which 9 were randomised controlled trials (RCT). Only one or a few RCTs examined several drugs. The combination of sertraline, naltrexone and disulfiram showed the biggest effect, although the results were limited and partly contradictory.
CONCLUSION: At the moment, there is little evidence for a clear pharmacological preference for the treatment of both PTSD and AUD. Based on the current studies there is, although limited, most evidence for the combination of naltrexone and sertraline or monotherapy with disulfiram. Further research is necessary in order to adequately treat this double diagnosis.

A combined chemical and biological analysis of samples from a major obsolete pesticide and persistent organic pollutant (POP) dumpsite in Northern Tajikistan was carried out. The chemical analytical screening focused on a range of prioritized compounds and compounds known to be present locally. Since chemical analytics does not allow measurements of hazards in complex mixtures, we tested the use of a novel effect-based approach using a panel of quantitative high-throughput CALUX reporter assays measuring distinct biological effects relevant in hazard assessment. Assays were included for assessing effects related to estrogen, androgen, and progestin signaling, aryl hydrocarbon receptor-mediated signaling, AP1 signaling, genotoxicity, oxidative stress, chemical hypoxia, and ER stress. With this panel of assays, we first quantified the biological activities of the individual chemicals measured in chemical analytics. Next, we calculated the expected sum activity by these chemicals in the samples of the pesticide dump site and compared the results with the measured CALUX bioactivity of the total extracts of these samples. The results showed that particularly endocrine disruption-related effects were common among the samples. This was consistent with the toxicological profiles of the individual chemicals that dominated these samples. However, large discrepancies between chemical and biological analysis were found in a sample from a burn place present in this site, with biological activities that could not be explained by chemical analysis. This is likely to be caused by toxic combustion products or by spills of compounds that were not targeted in the chemical analysis.

There is a great need for alternative testing methods for reproductive toxicants that are practical, fast, cost-effective and easy to interpret. Previously we followed a pragmatic approach using readily available tests, which was successful in predicting reproductive toxicity of chemicals [13]. This initial battery still contained apical tests and is fairly complex and low in its throughput. The current study aimed to simplify this screening battery using a mechanistic approach and a panel of high throughput CALUX reporter gene assays. A mechanistic approach was taken to validate this high throughput test battery. To this end it was challenged with two preselected sets of chemicals addressing two major apical effect classes relevant in reproductive toxicity. We found selectivity in this battery in that 82% of the compounds inducing reproductive organ deformities were predicted correctly, while for compounds inducing neural tube defects this was the case in 47% only. This is consistent with the mechanisms of toxicity covered in the battery. The most informative assays in the battery were ERalpha CALUX to measure estrogenicity and the AR-anti CALUX assay to measure androgen receptor antagonism., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Due to increasing energy demand and limited fossil fuels, renewable energy sources have gained in importance. Particulate matter (PM) in general, but also PM from the combustion of wood is known to exert adverse health effects in human. These are often related to specific toxic compounds adsorbed to the PM surface, such as polycyclic aromatic hydrocarbons (PAH), of which some are known human carcinogens. This study focused on the bioavailability of PAHs and on the tumor initiation potential of wood combustion PM, using the PAH CALUX® reporter gene assay and the BALB/c 3T3 cell transformation assay, respectively. For this, both cell assays were exposed to PM and their respective organic extracts from varying degrees of combustion. The PAH CALUX® experiments demonstrated a concentration-response relationship matching the PAHs detected in the samples. Contrary to expectations, PM samples from complete (CC) and incomplete combustion (IC) provided for a stronger and weaker response, respectively, suggesting that PAH were more readily bioavailable in PM from CC. These findings were corroborated via PAH spiking experiments indicating that IC PM contains organic components that strongly adsorb PAH thereby reducing their bioavailability. The results obtained with organic extracts in the cell transformation assay presented the highest potential for carcinogenicity in samples with high PAH contents, albeit PM from CC also demonstrated a carcinogenic potential. In conclusion, the in vitro assays employed emphasize that CC produces PM with low PAH content however with a general higher bioavailability and thus with a nearly similar carcinogenic potential than IC PM., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The river Meuse serves as a drinking-water source for more than 6 million people in France, Belgium, and The Netherlands. Pharmaceuticals and pesticides, both designed to be biologically active, are important classes of contaminants present in this river. The variation in the presence of pharmaceuticals in time and space in the Dutch part of the Meuse was studied using a multicomponent analytical method for pharmaceuticals combined with univariate and multivariate statistical analyses of the results. Trends and variation in time in the presence of pharmaceuticals were investigated in a dead-end side stream of the Meuse that serves as an intake point for the production of drinking water, and 93% of the selected compounds were detected. Highest concentrations were found for the antidiabetic metformin. Furthermore, a spatial snapshot of the presence of pharmaceuticals and pesticides was made along the river Meuse. Principal component analysis was successfully applied to reveal that wastewater-treatment plant effluent and water composition at the Belgian border were the main factors determining which compounds are found at different locations. The Dutch part of the river basin appeared responsible for approximately one-half of the loads of pharmaceuticals and pesticides discharged by the Meuse into the North Sea. The present study showed that multicomponent monitoring in combination with principal component analysis is a powerful tool to provide insight into contamination patterns in surface waters., (© 2013 SETAC.)

Polycyclic aromatic hydrocarbons (PAHs) represent a class of ubiquitously occurring environmental compounds that are implicated in a wide range of toxicological effects. Routine measurement of PAH contamination generally involves chemical analytical analysis of a selected group of representatives, for example, EPA-16, which may result in underestimation of the PAH-related toxicity of a sample. Many high molecular weight PAHs are known ligands of the aryl hydrocarbon receptor (AhR), a nuclear receptor that mediates toxic effects related to these compounds. Making use of this property we developed a PAH CALUX assay, a mammalian, H4IIe- cell-based reporter assay for the hazard identification of total PAH mixtures. The PAH CALUX reporter cell line allows for specific, rapid (4 h exposure time) and reliable quantification of AhR-induced luciferase induction relative to benzo[a]pyrene (BaP), which is used as a positive reference PAH congener. Full dose response relationships with inductions over 100-fold were reached within only 2 h of exposure to BaP. The PAH CALUX is highly sensitive, that is, using a 4 h exposure time, a limit of detection (LOD) of 5.2 × 10(-11) M BaP was achieved, and highly accurate, that is, a repeatability of 5.9% and a reproducibility of 6.6% were established. Screening of a selection of PAHs that were prioritized by the European Union and/or the U.S. Environmental Protection Agency showed that the PAH CALUX bioassay has a high predictability, particularly for carcinogenic PAHs. Experiments with synthetic mixtures and reference materials containing complex PAH mixtures show the suitability of the assay for these types of applications. Moreover, the presented results suggest that application of the PAH CALUX will result in a lower risk of underestimation of the toxicity of a sample than chemical analytical approaches that focus on a limited set of prioritized compounds.

This study examined the developmental toxicity of the polycyclic aromatic hydrocarbons (PAHs) 11H-benzo(b)fluorene (BBF) and 4-azapyrene (AP) in comparison to the known teratogen retene. Developmental toxicity assays were performed in zebrafish embryos exposed for 120 h. BBF and retene induced a similar dioxin-like phenotype, whereas AP showed distinct effects, particularly craniofacial malformations. Microarray analysis revealed that for BBF and retene, drug metabolism pathways were induced, which were confirmed by subsequent studies of cyp1a gene expression. For AP, microarray analysis revealed the regulation of genes involved in retinoid metabolism and hematological functions. Studies with a panel of CALUX(®) bioassays to screen for endocrine disrupting activity of the compounds also revealed novel antagonistic effects of BBF and retene on androgen and progesterone receptors. Classification analysis revealed distinct gene expression profiles for both individual and combined PAH exposure. This study highlights the potential health risk of non priority PAHs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The PR CALUX® cell line is a stably transfected human U2-OS cell line expressing the human PR and a luciferase reporter construct containing three progesterone-responsive elements coupled to a minimal promoter. The validity of this assay has been studied as an alternative to the McPhail assay in rabbits, an in vivo assay to detect progestins. The PR CALUX assay was characterized by its stable expression of PR protein which leads to induction of endogenous PR target genes by progestins. It was found to have a highly selective response to low levels of different progestins, as well as an insignificant response to other nuclear hormone receptor ligands. As an important step in their validation, the PR CALUX bioassay was compared with another earlier described in vitro bioassay, a Chinese Hamster Ovary (CHO) cell-based PR-CHO reporter gene assay as well as with an in vitro PR-binding (PR-BIN) assay, and the in vivo McPhail assay. This was done using 35 (with the most accurate potency determinations in all tests) and 50 (with less reliable potency determinations in some tests) compounds tested in all assays. The correlation scores between PR CALUX and PR-CHO were r(2)=0.77, and 0.93, respectively; between PR CALUX and PR-BIN r(2)=0.69 and 0.80. Comparison between either the PR CALUX or the PR-CHO transactivation assay and the in vivo McPhail assay revealed very good correlations of r(2)=0.68 (n=35), and 0.85 (n=50). The transactivation assays can discriminate very potent, from potent, weak and inactive compounds rather easily. Besides testing the biological activity of pure chemicals and pharmaceuticals in vitro, the PR CALUX and PR-CHO transactivation assays proved to be relatively good predictors of in vivo progestagenic activity, allowing the use of these assays as prescreening methods or in vitro alternatives., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Dithiocarbamates (DTCs) have a wide variety of applications in diverse fields ranging from agriculture to medicine. DTCs are teratogenic to vertebrates but the mechanisms by which they exert these effects are poorly understood. Here, we show that low nanomolar exposure to three DTCs, tetraethylthiuram (thiram), tetramethylthiuram (disulfiram), and sodium metam (metam), leads to craniofacial abnormalities in developing zebrafish embryos that are reminiscent of DTC-induced abnormalities found in higher vertebrates. In order to better understand the molecular events underlying DTC teratogenesis, we exposed embryonic zebrafish (PAC2) cells to thiram and disulfiram and measured changes in gene expression with microarrays. We found differential expression of 166 genes that were specific for exposure to DTCs and identified a network of genes related to connective tissue development and function. Additionally, we found eight downregulated genes related to transforming growth factor beta-1 (TGF-beta1) signaling, including an essential transcription factor for zebrafish craniofacial development, SRY-box-containing gene 9a (sox9a). Finally, we show that sox9a expression is perturbed in the ceratobranchial arches of DTC-exposed zebrafish, suggesting that this is an important event in the development of DTC-induced craniofacial abnormalities. Together, we provide evidence for a novel teratogenic endpoint and a molecular basis for a better understanding of DTC-induced teratogenesis in vertebrates.

Polybrominated diphenylethers (PBDEs) are ubiquitous in the environment, with the lower brominated congener 2,2',4,4'-tetrabromodiphenylether (BDE47) among the most prevalent. The phenolic PBDE, 6-hydroxy-BDE47 (6-OH-BDE47) is both an important metabolite formed by in vivo metabolism of BDE47 and a natural product produced by marine organisms such as algae. Although this compound has been detected in humans and wildlife, including fish, virtually nothing is known of its in vivo toxicity. Here we report that 6-OH-BDE47 is acutely toxic in developing and adult zebrafish at concentrations in the nanomolar (nM) range. To identify possible mechanisms of toxicity, we used microarray analysis as a diagnostic tool. Zebrafish embryonic fibroblast (PAC2) cells were exposed to 6-OH-BDE47, BDE47, and the methoxylated metabolite 6-MeO-BDE47. These experiments revealed that 6-OH-BDE47 alters the expression of genes involved in proton transport and carbohydrate metabolism. These findings, combined with the acute toxicity, suggested that 6-OH-BDE47 causes disruption of oxidative phosphorylation (OXPHOS).Therefore, we further investigated the effect of 6-OH-BDE47 on OXPHOS in zebrafish mitochondria. Results show unequivocally that this compound is a potent uncoupler of OXPHOS and is an inhibitor of complex II of the electron transport chain. This study provides the first evidence of the in vivo toxicity and an important potential mechanism of toxicity of an environmentally relevant phenolic PBDE of both anthropogenic and natural origin. The results of this study emphasize the need for further investigation on the presence and toxicity of this class of polybrominated compounds.

Messenger RNA levels change on a minutes scale due to both degradation and de novo transcription. Consequently, alterations in the transcript profiles that are not representative for the condition of interest are easily introduced during sample harvesting and work-up. In order to avoid these unwanted changes we have validated a -45 degrees C methanol-based quenching method for obtaining reliable and reproducible 'snapshot' samples of Lactobacillus plantarum cells for transcriptome analyses. Transcript profiles of cells harvested with the quenching method were compared with transcript profiles of cells that were harvested according to two different commonly applied protocols. Significant differences between the transcript profiles of cells harvested by the different methods from the same steady-state culture were observed. In total, 42 genes or operons were identified from which the transcript levels were altered when the cells were not immediately quenched upon harvesting. Among these, several have previously been associated with cold-shock response. Furthermore, the reproducibility of transcript profiles improved, as indicated by the fact that the variation in the data sets obtained from the quenched cells was smaller than in the data sets obtained from the cells that were harvested under non-quenched conditions.

The value of the multivariate data analysis tools principal component analysis (PCA) and principal component discriminant analysis (PCDA) for prioritizing leads generated by microarrays was evaluated. To this end, Pseudomonas putida S12 was grown in independent triplicate fermentations on four different carbon sources, i.e. fructose, glucose, gluconate and succinate. RNA isolated from these samples was analysed in duplicate on an anonymous clone-based array to avoid bias during data analysis. The relevant transcripts were identified by analysing the loadings of the principal components (PC) and discriminants (D) in PCA and PCDA, respectively. Even more specifically, the relevant transcripts for a specific phenotype could also be ranked from the loadings under an angle (biplot) obtained after PCDA analysis. The leads identified in this way were compared with those identified using the commonly applied fold-difference and hierarchical clustering approaches. The different data analysis methods gave different results. The methods used were complementary and together resulted in a comprehensive picture of the processes important for the different carbon sources studied. For the more subtle, regulatory processes in a cell, the PCDA approach seemed to be the most effective. Except for glucose and gluconate dehydrogenase, all genes involved in the degradation of glucose, gluconate and fructose were identified. Moreover, the transcriptomics approach resulted in potential new insights into the physiology of the degradation of these carbon sources. Indications of iron limitation were observed with cells grown on glucose, gluconate or succinate but not with fructose-grown cells. Moreover, several cytochrome- or quinone-associated genes seemed to be specifically up- or downregulated, indicating that the composition of the electron-transport chain in P. putida S12 might change significantly in fructose-grown cells compared to glucose-, gluconate- or succinate-grown cells.

Identification of nuclear receptor-mediated endocrine activities is important in a variety of fields, ranging from pharmacological and clinical screening, to food and feed safety, toxicological monitoring, and risk assessment. Traditionally animal studies such as the Hershberger and Allen-Doisy tests are used for the assessment of androgenic and estrogenic potencies, respectively. To allow fast analysis of the activities of new chemicals, food additives, and pharmaceutical compounds, high-throughput screening strategies have been developed. Here, a panel of mainly steroidal compounds, screened in different in vitro assays, was compared with two human U2-OS cell line-based CALUX (Chemically Activated LUciferase eXpression) reporter gene assays for androgens (AR CALUX) and estrogens (ERalpha CALUX). Correlations found between the data of these two CALUX reporter gene assays and data obtained with other in vitro screening assays measuring receptor binding or reporter gene activation (CHO cell line-based) were good (correlation coefficients (r2) between 0.54 and 0.76; p < 0.0001). Good correlations were also found between the in vitro and in vivo data (correlation coefficient r2 = 0.46 for the AR CALUX vs. Hershberger assay and r2 = 0.87 for the ERalpha CALUX vs. Allen-Doisy assay). The variations in the results obtained with the reporter gene assays (CALUX vs. CHO cell line based) were relatively small, showing the robustness of these types of assays. Using hierarchical clustering, bioactivity relationships between compounds but also relationships between various bioassays were determined. The in vitro assays were found to be good predictors of in vivo androgenic or estrogenic activity of a range of compounds, allowing prescreen and/or possible reduction of animal studies.

The organic acid lactate is the predominant fermentation product of Lactobacillus plantarum. The undissociated form of this organic acid is a strong growth inhibitor for the organism. Different theories have been postulated to explain the inhibitory effects of lactic acid: (i) toxicity arising from the dissipation of the membrane potential, (ii) acidification of the cytosol, or (iii) intracellular anion accumulation. In general, organic acid stresses are complex to study, since their toxicity is highly dependent on their degree of dissociation and thus on the pH. In this study, transcription profiles of L. plantarum grown in steady-state cultures that varied in lactate/lactic acid concentration, pH, osmolarity and absolute and relative growth rate, were compared by microarray analysis. By doing so, the differential expression of multiple groups of genes could specifically be attributed to the different aspects of lactic acid stress. A highly coherent group of lactic acid-responsive, cell surface protein-encoding genes was identified, to which no function has previously been assigned. Moreover, a group of genes that showed increased expression in response to the combination of lactic acid and a lower growth rate is expected to be involved in the formation of the alternative fermentation end-products malate, acetate and ethanol. One of these pathways is the phosphoketolase by-pass that is typical for bifidobacteria.

Background: Clone-based microarrays, on which each spot represents a random genomic fragment, are a good alternative to open reading frame-based microarrays, especially for microorganisms for which the complete genome sequence is not available. Since the generation of a genomic DNA library is a random process, it is beforehand uncertain which genes are represented. Nevertheless, the genome coverage of such an array, which depends on different variables like the insert size and the number of clones in the library, can be predicted by mathematical approaches. When applying the classical formulas that determine the probability that a certain sequence is represented in a DNA library at the nucleotide level, massive amounts of clones would be necessary to obtain a proper coverage of the genome., Results: This paper describes the development of two complementary equations for determining the genome coverage at the gene level. The first equation predicts the fraction of genes that is represented on the array in a detectable way and cover at least a set part (the minimal insert coverage) of the genomic fragment by which these genes are represented. The higher this minimal insert coverage, the larger the chance that changes in expression of a specific gene can be detected and attributed to that gene. The second equation predicts the fraction of genes that is represented in spots on the array that only represent genes from a single transcription unit, which information can be interpreted in a quantitative way., Conclusion: Validation of these equations shows that they form reliable tools supporting optimal design of prokaryotic clone-based microarrays.