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This study was conducted to investigate decreased susceptibility (minimum inhibitory concentrations [MICs] 0.25-4 mg/L) and resistance (MICs > 4 mg/L) to aztreonam-avibactam (ATM-AVI). Contemporary non-replicate clinical isolates of carbapenemase-producing Escherichia coli (CP-EC) (n=90) and ESBL-producing E. coli (EP-EC) (n=12) were used. CP-EC belonged to 25 distinct sequence types (STs) and all EP-EC belonged to ST405. All strains were isolated from 2019 to 2022 at the Karolinska University Laboratory, Stockholm, Sweden. ATM-AVI MICs were determined using broth microdilution. The EUCAST epidemiological cut-off value of 0.125 mg/L was used to define the wild type (WT). Whole-genome sequences (Illumina) were analysed for detecting resistance determinants among WT vs. non-WT isolates. Among 102 isolates, 40 (39%) and 62 (61%) were WT and non-WT, respectively. Among non-WT isolates, resistance was noted for 20 and decreased susceptibility for 42. Resistance was observed among 14/47 New Delhi metallo-β-lactamase (NDM)-producers, 5/43 OXA-48 group producers, and 1/12 EP-EC. Decreased susceptibility was observed among 29/47 NDM, 13/43 OXA-48 group, and 3/12 EP-EC. Resistant isolates predominantly belonged to ST405, followed by STs 410, 361, 167, 617, and 1284. Penicillin-binding protein 3 (PBP3) inserts (YRIK/YRIN) were observed in 20/20 and CMY-42 in 5/20 resistant isolates. Several mutations in the ftsI (encoding PBP3) and regulatory genes of outer membrane proteins (OmpC and OmpF) and efflux pumps (AcrAB-TolC) were detected. A ≥ 2-fold reduction in MICs was observed among 20/20 vs. 7/20 isolates tested in the presence of the membrane permeabiliser, polymyxin B nanopeptide (PMBN) and efflux inhibitor, phenylalanine arginine β-naphthylamide (PAβN), respectively. In conclusion, resistance to ATM-AVI is a result of interplay of various determinants, including target alterations, deactivating enzymes, and decreased permeability., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The properties of MXene flakes, a new class of two-dimensional materials, are strictly determined by their surface termination. The most common termination groups are oxygen-containing (=O or -OH) and fluorine (-F), and their relative ratio is closely related to flake stability and catalytic activity. The surface termination can vary significantly among MXene flakes depending on the preparation route and is commonly determined after flake preparation by using X-ray photoelectron spectroscopy (XPS). In this paper, as an alternative approach, we propose the combination of surface-enhanced Raman spectroscopy (SERS) and artificial neural networks (ANN) for the precise and reliable determination of MXene flakes' (Ti 3 C 2 T x ) surface chemistry. Ti 3 C 2 T x flakes were independently prepared by three scientific groups and subsequently measured using three different Raman spectrometers, employing resonant excitation wavelengths. Manual analysis of the SERS spectra did not enable accurate determination of the flake surface termination. However, the combined SERS-ANN approach allowed us to determine the surface termination with a high accuracy. The reliability of the method was verified by using a series of independently prepared samples. We also paid special attention to how the results of the SERS-ANN method are affected by the flake stability and differences in the conditions of flake preparation and Raman measurements. This way, we have developed a universal technique that is independent of the above-mentioned parameters, providing the results with accuracy similar to XPS, but enhanced in terms of analysis time and simplicity., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Background: Measurements of pleural fluid biomarkers for rapid identification of complicated parapneumonic effusion (CPPE) are crucial for optimal management. Previous studies for biomarker evaluation were however based on pleura culture, not modern DNA technique. Lactate has not been thoroughly studied earlier as a potential biomarker in this regard., Objectives: To evaluate whether the routine biomarkers pH, glucose, lactate dehydrogenase (LDH) measured in pleural fluid in a microbiological well characterised cohort could differentiate simple parapneumonic effusion (SPPE) from CPPE and if pleural fluid lactate could be of additional use in this discrimination., Methods: Pleural fluid prospectively collected from adult patients ( n  = 112) with PPE admitted to the Departments of Infectious Diseases (DIDs) at four Stockholm County hospitals were characterised microbiologically with bacterial culture and 16S rDNA sequencing, and biochemically with pH, glucose, LDH and lactate., Results: Forty and seventy two patients were categorised as SPPE/CPPE. The median values between SPPE/CPPE differed significantly for all biomarkers with varying overlap. Receiver operating characteristics (ROC) curves showed the area under the curve (AUC) for pH 0.905 (CI 0.847-0.963), glucose 0.861 (CI 0.79-0.932), LDH 0.917 (CI 0.860-0.974) and lactate 0.927 (CI 0.877-0.977), corresponding to best cut-off levels and sensitivity/specificity for pH of 7.255, 0.819/0.9, glucose 5.35 mmol/L, 0.847/0.775, LDH 9.8 µcat/L, 0.905/0.825 and lactate 4.9 mmol/L, 0.875/0.85., Conclusions: To distinguish between SPPE/CPPE, pH and LDH performed well, but optimal cut-off values differed from earlier established recommendations. Pleura lactate had the largest AUC of the investigated biomarkers and may be used in the analyses of PPE-staging.

Objectives: Clinicians worldwide struggle to identify the bacterial aetiology of bone and joint infections. Failure to unequivocally identify the pathogen is linked to poor clinical outcomes. We explored the added value of analysing multiple samples per patient with 16S ribosomal DNA (16S rDNA) sequencing in diagnosing postoperative bone and joint infections. All patients had received antimicrobials prior to sampling, and false-negative cultures could be suspected. Bone biopsies obtained from patients with postoperative bone and joint infections for cultures were also subjected to 16S rDNA sequencing., Results: In 5/28 infectious episodes, sequencing identified the causative organism of the infection when cultures failed. In 8/28 episodes, the methods led to different results, potentially leading to different antimicrobial choices. The analysis of multiple samples per patient helped rule out potential contaminating pathogens. We conclude that 16S rDNA sequencing has diagnostic value for patients receiving antibiotic treatment. We regard the method as a complement to culturing when the cultures are negative. Multiple samples per patient should be analysed to determine the clinical significance of positive findings., (© 2022. The Author(s).)

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.

The aim of this study was to use a 16S rDNA sequencing method in combination with conventional culture in patients with parapneumonic effusions (PPE) to evaluate the methods, study the microbiological spectrum, and examine the presence of bacteria within the different stages of PPE. Adults with community-acquired pneumonia (CAP) and PPE (n = 197) admitted to the Departments of Infectious Diseases at four hospitals in Stockholm County during 2011-2014 were prospectively studied. All patients underwent thoracentesis. Twenty-seven non-infectious pleural effusions were used as controls. The pleural samples were analyzed with culture, 16S rDNA sequencing, pH, glucose, and lactate dehydrogenase. Microbiological etiology was found in 99/197 (50%) of the patients with mixed infections in 20 cases. The most common pathogens were viridans streptococci (n = 37) and anaerobic bacteria (n = 40). Among the 152 patients with both methods performed, 26/152 (17%) and 94/152 (62%) had bacteria identified with culture and 16S rDNA sequencing respectively (p < 0.001). In 24/26 (92%) culture-positive cases, the same organism was identified by 16S rDNA. All controls were negative in both methods. Among the patients with complicated PPE and complete sampling, bacteria were found in 69/74 patients (93%), all detected with 16S rDNA sequencing, compared to 23/74 (31%) culture-positive samples (p < 0.001). Compared with culture, 16S rDNA sequencing substantially improved the microbiological yield, a microbiological diagnosis was achieved in almost all patients with complicated PPE, and the specificity seemed to be high. 16S rDNA sequencing should be used together with culture in patients with PPE to guide antibiotic therapy.

Many countries using sensitive screening methods for detection of carriage of methicillin-resistant Staphylococcus aureus (MRSA) have a sustained low incidence of MRSA infections. For diagnostic laboratories with high sample volumes, MRSA screening requires stability, low maintenance and high performance at a low cost. Herein we designed oligonucleotides for a new nuc targeted hydrolysis probe PCR to replace the standard in-house nuc SybrGreen PCR assay. This new, more time-efficient, PCR assay resulted in a 40% increase in daily sample capacity, with maintained high specificity and sensitivity. The assay was also able to detect Staphylococcus aureus clonal cluster 75 (CC75) lineage strains, recently re-classified as Staphylococcus argenteus, with a sensitivity considerably increased compared to our previous assay. While awaiting consensus if the CC75 lineage of S. aureus should be considered as S. argenteus, and whether methicillin-resistant S. argenteus should be included in the MRSA definition, many diagnostic laboratories need to update their MRSA assay sensitivity/specificity towards this lineage/species. The MRSA screening assay presented in this manuscript is comprised of nuc oligonucleotides separately targeting S. aureus and CC75 lineage strains/S. argenteus, thus providing high user flexibility for the detection of CC75 lineage strains/S. argenteus.

Objectives: The need for rapid antibiotic susceptibility testing increases with escalating levels of antimicrobial resistance in Enterobacteriaceae. Our objective was to evaluate the accuracy of reading EUCAST disc diffusion, ROSCO ESBL and carbapenemase detection kits and the Mast Carbapenemase Activity Test (CAT-ID) disc, after 6 h of incubation., Methods: We used a collection of 128 isolates of Escherichia coli and Klebsiella pneumoniae with a wide variety of resistance mechanisms. Inhibition zones read from digital photo images with the BD Kiestra™ Total Lab Automation System after 6 h of incubation were compared with standard reading, after 18 h, of the same Mueller-Hinton agar plates., Results: For WT isolates, zones were generally smaller at 6 h than at 18 h. Cefotaxime had excellent categorical agreement of 99%, despite the high number of challenge isolates. However, for some other antimicrobials, hetero-resistant subpopulations were commonly invisible at 6 h, which resulted in an unacceptable number of errors when using standard EUCAST breakpoints. Accurate ESBL detection was possible at 6 h for isolates lacking other β-lactamases. Carbapenemase detection was not reliable after 6 h., Conclusions: Inhibition zone reading at 6 h is an accurate method for susceptibility testing of extended-spectrum cephalosporins for Enterobacteriaceae. For other antimicrobials, 6 h reading can be used for preliminary reports of clearly resistant or susceptible isolates, preferably with application of adjusted breakpoints including an area of uncertainty between susceptible and resistant values., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Sodium niobate (NaNbO3, or NNO) is known to be antiferroelectric at temperatures between 45 and 753 K. Here we show experimentally the presence of the ferroelectric phase at temperatures between 100 and 830 K in the NNO crystals obtained by top-seeded solution growth. The ferroelectric phase and new phase transitions are evidenced using a combination of thermo-optical studies by variable angle spectroscopic ellipsometry, Raman spectroscopy analysis, and photoelectron emission microscopy. The possibility for strain-induced ferroelectricity in NNO is suggested.

Objectives: The aim was to evaluate 16S rDNA sequencing in heart valves in patients with infective endocarditis undergoing surgery., Methods: Fifty-seven patients with infective endocarditis were examined in this prospective study by analysing heart valves with 16S rDNA sequencing and culturing methods and comparing the results to blood cultures. As controls, heart valves from 61 patients without any signs of endocarditis were examined., Results: All together 77% of the endocarditis patients were positive for 16S rDNA, 84% had positive blood cultures and 23% had positive cultures from heart valves, whereas only 16% of the cultures from heart valves were concordant with results from blood cultures or 16S rDNA. Concordant results between 16S rDNA sequencing and blood cultures were found in 75% patients. All controls were negative for 16S rDNA. In 4 out of 9 patients with negative blood cultures, the aetiology was established by 16S rDNA alone, i.e. viridans group streptococci., Conclusion: In this Swedish study, 16S rDNA sequencing of valve material was shown to be a valuable addition in blood culture-negative cases. The value of heart valve culture was low. Molecular diagnosis using 16S rDNA sequencing should be recommended in patients undergoing valve replacement for infective endocarditis., (Copyright © 2011 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

The aim of this study was the molecular characterization of primary drug-resistant Mycobacterium tuberculosis strains in Thailand. We examined a group of M. tuberculosis isolates from newly registered tuberculosis (TB) cases, collected at the largest university hospital, the Siriraj Hospital, in Thailand. Of 76 selected drug-resistant M. tuberculosis strains recovered from previously untreated pulmonary TB patients whose sputum samples were sent to this hospital, 29 (38%) were single-drug resistant, 26(34%) multidrug resistant and two (2.6%) extensively drug resistant. Fifty (66%) strains belonged to Beijing genotype. The study demonstrated a severe problem of drug resistance among recently detected TB patients, and two large clusters of genetically similar strains indicated ongoing transmission of drug-resistant TB.

Recently we reported noncovalent functionalization of nanotubes in an aqueous medium with ionic liquid-based surfactants, 1-dodecyl-3-methylimidazolium bromide (1) and 1-(12-mercaptododecyl)-3-methylimidazolium bromide (2), resulting in positively charged single-wall carbon nanotube (SWNT)-1,2 composites. Thiolation of SWNTs with 2 provides their self-assembly on gold as well as templating gold nanoparticles on SWNT sidewalls via a covalent -S-Au bond. In this investigation, we studied the electronic structure, intermolecular interactions, and packing within noncovalently thiolated SWNTs and also nanotube alignment in the bulk of SWNT-2 dried droplets and self-assembled submonolayers (SAMs) on gold by high-resolution X-ray photoemission spectroscopy (HRXPS), C K-edge X-ray absorption fine structure (NEXAFS) spectroscopy, and polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS). HRXPS data confirmed the noncovalent nature of interactions within the nanocomposite of thiolated nanotubes. In PM-IRRAS spectra of SWNT SAMs on gold, the IR-active vibrational SWNT modes have been observed and identified. According to PM-IRRAS data, the hydrocarbon chains of 2 are oriented with less tilt angle to the bare gold normal in a SAM deposited from an SWNT-2 dispersion than those of 1 deposited from an SWNT-1 dispersion on the mercaptoethanesulfonic acid-primed gold. For both the dried SWNT-2 bulk and the SWNT-2 SAM on gold, the C K-edge NEXAFS spectra revealed the presence of CH-pi interactions between hydrocarbon chains of 2 and the pi electronic nanotube structure due to the highly resolved vibronic fine structure of carbon 1s --> R*/sigma*C-H series of states in the alkyl chain of 2. For the SWNT-2 bulk, the observed splitting and upshift of the SWNT pi* orbitals in the NEXAFS spectrum indicated the presence of pi-pi interactions. In the NEXAFS spectrum of the SWNT-2 SAM on gold, the upshifted values of the photon energy for R*/sigma*C-H transitions indicated close contact of 2 with nanotubes and with a gold surface. The angle-dependent NEXAFS for the SWNT-2 bulk showed that most of the molecules of 2 are aligned along the nanotubes, which are self-organized with orientation parallel to the substrate plane, whereas the NEXAFS for the SWNT-2 SAM revealed a more normal orientation of functionality 2 on gold compared with that in the SWNT-2 bulk.

Objectives: Viridans group streptococci (VGS) cause severe diseases such as infective endocarditis and septicaemia. Genetically, VGS species are very close to each other and it is difficult to identify them to species level with conventional methods. The aims of the present study were to use sequence analysis of the RNase P RNA gene (rnpB) to identify VGS species in clinical blood culture isolates, and to compare the results with the API 20 Strep system that is based on phenotypical characteristics., Methods: Strains from patients with septicaemia or endocarditis were analysed with PCR amplification and sequence analysis of the rnpB gene. Clinical data were registered as well., Results: One hundred and thirty two VGS clinical blood culture isolates from patients with septicaemia (n=95) or infective endocarditis (n=36) were analysed; all but one were identified by rnpB. Streptococcus oralis, Streptococcus sanguinis and Streptococcus gordonii strains were most common in the patients with infective endocarditis. In the isolates from patients with haematological diseases, Streptococcus mitis and S. oralis dominated. In addition in 76 of the isolates it was possible to compare the results from rnpB analysis and the API 20 Strep system. In 39/76 (51%) of the isolates the results were concordant to species level; in 55 isolates there were no results from API 20 Strep., Conclusion: Sequence analysis of the RNase P RNA gene (rnpB) showed that almost all isolates could be identified. This could be of importance for evaluation of the portal of entry in patients with septicaemia or infective endocarditis.

We report a case of infective endocarditis caused by Actinobacillus actinomycetemcomitans (Aggregatibacter actinomycetemcomitans) from the HACEK group diagnosed by PCR/16S from the heart valve. Multiple blood cultures and cultures from heart valve were negative and cardiac surgery was performed due to therapeutic and cardiac failures. Molecular biological methods are useful in such a patient, to choose an optimal antibiotic treatment post-surgery.

Normal oral keratinocytes (NOKs), and a Simian virus 40 T-antigen-immortalised oral keratinocyte line termed SVpgC2a, were cultured in an effort to model the human oral epithelium in vitro, including normal and dysplastic tissue. Monolayer and organotypic cultures of NOKs and SVpgC2a were successfully established in a standardised serum-free medium with high levels of amino acids, by using regular tissue culture plastic for monolayers and collagen gels containing oral fibroblasts as the base for generating tissue equivalents. NOKs express many characteristics of normal tissue, including those associated with terminal squamous differentiation. After > 150 passages, SVpgC2a cells retained an immortal, nontumourigenic phenotype that, relative to NOKs, was associated with aberrant morphology, enhanced proliferation, deficiency in terminal differentiation, proneness to apoptosis, and variably altered expression of structural epithelial markers. Transcript and protein profiling, as well as activity assays, demonstrated the expression of multiple xenobiotic-metabolising enzymes in SVpgC2a cells, some of which were higher in comparison to NOKs. A generally preserved, or even activated, ability for xenobiotic metabolism in long-term cultures of SVpgC2a cells indicated that this cell line could be useful in safety testing protocols--for example, in the development of consumer products in the oral health care field. However, SVpgC2a cells displayed some features reminiscent of a severe oral dysplasia, implying that this cell line could also to some extent serve as a model of a premalignant oral epithelium.

Gene expression underlying cellular growth and differentiation is only partly understood. This study analyzed transcript levels of the formaldehyde-metabolizing enzyme alcohol dehydrogenase 3 (ADH3) and various growth and differentiation-related genes in human oral keratinocytes. Culture of confluent cells both with and without fetal bovine serum inhibited colony-forming efficiency and induced a squamous morphology. Confluency alone decreased the transcript levels of ADH3, the proliferation markers cell division cycle 2 (CDC2) and proliferating cell nuclear antigen (PCNA), and the basal cell marker cytokeratin 5 (K5), but increased transcripts for the suprabasal differentiation markers involucrin (INV) and small proline-rich protein 1B (SPR1). These changes were variably influenced by serum, i.e., loss of CDC2 and PCNA was inhibited, loss of K5 promoted, increase of SPR1 transcripts inhibited, and increase of INV promoted. The extent and onset of the effects implied that ADH3 transcription serves as a proliferation marker and that confluency with or without serum exposure can serve to selectively analyze proliferative and differentiated cellular states.

Extracellular matrix proteins affect the growth and survival of epithelial tissues. Accordingly, surface coating with fibronectin and collagen is a common practice for promoting keratinocyte culture. In this study, the expression of fibronectin and collagen-related factors, including integrins, by normal (NOK), SV40 T-antigen-immortalised (SVpgC2a) and malignant (SqCC/Y1) human oral keratinocytes, under standardised, serum-free conditions, was investigated by using microarray analysis. Cell growth was also studied in the presence and absence of a matrix consisting of human fibronectin and bovine collagen type I (FN-COL). Fibronectin transcripts were abundant in all cells, whereas 16 of 29 collagen chains and 14 of 24 integrin subunits were variably detected. With regard to both the expression level and the number of transcripts, higher collagen and lower integrin expression was observed in SVpgC2a cells than in NOKs and SqCC/Y1 cells. The cell types differed with regard to colony-forming efficiency and the rate and kinetics of growth at high cell density. For all cell types, FN-COL coating consistently stimulated cell migration, without influencing growth in mass culture or clonal density. The results demonstrate the transcription of genes associated with the formation and function of fibronectin and collagen in oral epithelium, and variably altered expression patterns in transformed states, and show that keratinocyte lines can be successfully transferred without the stimulus from extracellular FN-COL.

The metabolic detoxification capacity may critically regulate the susceptibility of human tissues to cancer development. We used standardized and quantitative, reverse transcription-polymerase chain reaction (StaRT-PCR) and microarray chip techniques to analyze transcript levels of multiple detoxification enzymes in cultured normal human oral keratinocytes (NOK) and the Siman virus 40 T antigen-immortalized oral keratinocyte line SVpgC2a, viewing the latter as a model of a benign tumor state. With good agreement between the 2 methodologies, NOK and SVpgC2a were found to express transcripts for cytochrome P450 enzymes (CYPs), factors related to CYP induction and enzymes involved in conjugation reactions or detoxification of reactive oxygen. The cell types expressed similar levels of CYP 2B6/7, CYP 2E1, P450 oxidoreductase, the aryl hydrocarbon receptor nuclear translocator, sulfotransferase 1A1, sulfotransferase 1A3, epoxide hydrolase, glutathione S-transferase M3, glutathione S-transferase pi and catalase, superoxide dismutase 1, glutathione peroxidase 1 and glutathione peroxidase 3. In contrast, SVpgC2a exhibited comparatively higher levels of CYP1A1, 1B1, aryl hydrocarbon receptor, glutathione S-transferase M1, 2, 4, 5, glutathione S-transferase theta 1 and superoxide dismutase 2 and comparatively lower levels of UDP glycosyltransferase 2 and microsomal glutathione S-transferase 1. Some transcripts, e.g., CYP 2A6/7, were not detected by either standard, non quantitative RT-PCR or the above methods, whereas others were barely quantifiable by StaRT-PCR, i.e., were present at 1-10 molecules/10(6) molecules of actin. Overall, the expression analysis demonstrated presence of mRNA for multiple enzymes involved in foreign compound metabolism and detoxification pathways, including several enzymes not previously reported for oral epithelium. Generally, the comparison of NOK from 2 individuals indicated relatively similar transcript levels of these enzymes. In contrast, differences between NOK and SVpgC2a, e.g., for CYP1B1, may reflect alteration caused by immortalization and aid identification of early stage tumor markers in oral epithelium., (Copyright 2002 Wiley-Liss, Inc.)

Background: A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for conducting multi-institutional clinical studies based on expression measurement. Standardized RT (StaRT)-PCR has all these characteristics. In addition, the method must be reproducible. StaRT-PCR has high intralaboratory reproducibility. The purpose of this study is to determine whether StaRT-PCR provides similar interlaboratory reproducibility., Methods and Results: In a blinded interlaboratory study, expression of ten genes was measured by StaRT-PCR in a complementary DNA sample provided to each of four laboratories. The average coefficient of variation for interlaboratory comparison of the nine quantifiable genes was 0.48. In all laboratories, expression of one of the genes was too low to be measured., Conclusion: Because StaRT-PCR data are standardized and numerical and the method is reproducible among multiple laboratories, it will allow development of a meaningful gene expression database.

Enzymes involved in various protective and metabolic processes of carbonyl compounds were analysed utilising a micro-array method in a three-stage in vitro model for oral carcinogenesis involving cultured normal, immortalised and malignant human oral keratinocytes. A complete transcript profiling of identified carbonyl-metabolising enzymes belonging to the ADH, ALDH, SDR and AKR families is presented. Expression of 17 transcripts was detected in normal, 14 in immortalized and 19 in malignant keratinocytes of a total of 12,500 genes spotted on the micro-array chip. For the detected transcripts, about half were changed by cell transformation, and for the various enzyme families, differences in expression patterns were observed. The detected AKR transcripts displayed a conserved pattern of expression, indicating a requirement for the keratinocyte phenotype, while most of the detected SDRs displayed changed expression at the various stages of malignancy. The importance of multiple experiments in using a microarray technique for reliable results is underlined and, finally, the strength of the method in detecting co-expressed enzymes in metabolic pathways is exemplified by the detection of the formaldehyde-scavenging pathway enzymes and the polyol pathway enzymes.

Constituents in food and fluids, tobacco chemicals and many drugs are candidates for oral absorption and oxidative metabolism. On this basis, the expression of cytochrome P450 isozymes (CYPs) and the conversion of CYP substrates were analysed in reference to buccal mucosa. A RT-PCR based analysis of human buccal tissue from 13 individuals demonstrated consistent expression of mRNA for the CYPs 1A1, 1A2, 2C, 2E1, 3A4/7 and 3A5. CYP 2D6 was expressed in six out of the 13 specimens, whereas all samples were negative for 2A6 and 2B6. Serum-free monolayer cultures of the Siman virus 40 large T-antigen-immortalized SVpgC2a and the carcinoma SqCC/Y1 buccal keratinocyte lines expressed the same CYPs as tissue except 3A4/7 and 3A5 (SVpgC2a), and 2C, 2D6 and 3A4/7 (SqCC/Y1). Dealkylation of ethoxyresorufin and methoxyresorufin in both normal and transformed cells indicated functional 1A1 and 1A2, respectively. SVpgC2a showed similar activity as normal keratinocytes for both substrates, whereas SqCC/Y1 showed about 2-fold lower 7-ethoxyresorufin O-deethylation and 7-methoxyresorufin O-demethylation activities. SVpgC2a showed detectable and many-fold higher activity than the other cell types towards chlorzoxazone, a substrate for 2E1. Absent or minute catalytic activity of 2C9, 2D6 and 3A4 in the various cell types was indicated by lack of detectable diclofenac, dextromethorphan and testosterone metabolism (<0.2-0.5 pmol/min/mg). Metabolic activation of the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the mycotoxin aflatoxin B1 (AFB1) to covalently bound adducts was indicated by autoradiographic analysis of both monolayer and organotypic cultures of SVpgC2a. In contrast, SqCC/Y1 showed lower or absent metabolic activity for these substrates. Finally, measurements of various non-reactive AFB1 metabolites indicated rates of formation <0.1 pmol/min/mg in both normal and transformed cells. The results indicate presence of several CYPs of which some may contribute to significant xenobiotic metabolism in human buccal epithelium. Notably, metabolic activation of AFB1 was not previously implicated for oral mucosa. Further, the results show that CYP-dependent metabolism can be preserved or even activated in immortalized keratinocytes. Metabolic activity in SVpgC2a under both monolayer and organotypic culture conditions suggests that this cell line may be useful to pharmaco-toxicological and carcinogenesis studies.

Nurse scheduling is a difficult and time consuming task. The schedule has to determine the day to day shift assignments of each nurse for a specified period of time in a way that satisfies the given requirements as much as possible, taking into account the wishes of nurses as closely as possible. This paper presents a constraint-based, artificial intelligence approach by describing a prototype implementation developed with the Charme language and the first results of its use in the Rouen University Hospital. Horoplan implements a non-cyclical constraint-based scheduling, using some heuristics. Four levels of constraints were defined to give a maximum of flexibility: French level (e.g. number of worked hours in a year), hospital level (e.g. specific day-off), department level (e.g. specific shift) and care unit level (e.g. specific pattern for week-ends). Some constraints must always be verified and can not be overruled and some constraints can be overruled at a certain cost. Rescheduling is possible at any time specially in case of an unscheduled absence.