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Abstract Background Flux-balance analysis based on linear optimization is widely used to compute metabolic fluxes in large metabolic networks and gains increasingly importance in network curation and structural analysis. Thus, a computational tool flexible enough to realize a wide variety of FBA algorithms and able to handle batch series of flux-balance optimizations is of great benefit. Results We present FASIMU, a command line oriented software for the computation of flux distributions using a variety of the most common FBA algorithms, including the first available implementation of (i) weighted flux minimization, (ii) fitness maximization for partially inhibited enzymes, and (iii) of the concentration-based thermodynamic feasibility constraint. It allows batch computation with varying objectives and constraints suited for network pruning, leak analysis, flux-variability analysis, and systematic probing of metabolic objectives for network curation. Input and output supports SBML. FASIMU can work with free (lp_solve and GLPK) or commercial solvers (CPLEX, LINDO). A new plugin (faBiNA) for BiNA allows to conveniently visualize calculated flux distributions. The platform-independent program is an open-source project, freely available under GNU public license at http://www.bioinformatics.org/fasimu including manual, tutorial, and plugins. Conclusions We present a flux-balance optimization program whose main merits are the implementation of thermodynamics as a constraint, batch series of computations, free availability of sources, choice on various external solvers, and the flexibility on metabolic objectives and constraints.

Abstract Due to an increasing awareness of the potential hazardousness of air pollutants, new laws, rules and guidelines have recently been implemented globally. In this respect, numerous studies have addressed traffic-related exposure to particulate matter using stationary technology so far. By contrast, only few studies used the advanced technology of mobile exposure analysis. The Mobile Air Quality Study (MAQS) addresses the issue of air pollutant exposure by combining advanced high-granularity spatial-temporal analysis with vehicle-mounted, person-mounted and roadside sensors. The MAQS-platform will be used by international collaborators in order 1) to assess air pollutant exposure in relation to road structure, 2) to assess air pollutant exposure in relation to traffic density, 3) to assess air pollutant exposure in relation to weather conditions, 4) to compare exposure within vehicles between front and back seat (children) positions, and 5) to evaluate "traffic zone"-exposure in relation to non-"traffic zone"-exposure. Primarily, the MAQS-platform will focus on particulate matter. With the establishment of advanced mobile analysis tools, it is planed to extend the analysis to other pollutants including NO2, SO2, nanoparticles and ozone.

Abstract Background Grape powdery mildew is caused by the North American native pathogen Erysiphe necator. Eurasian Vitis vinifera varieties were all believed to be susceptible. REN1 is the first resistance gene naturally found in cultivated plants of Vitis vinifera. Results REN1 is present in 'Kishmish vatkana' and 'Dzhandzhal kara', two grapevines documented in Central Asia since the 1920's. These cultivars have a second-degree relationship (half sibs, grandparent-grandchild, or avuncular), and share by descent the chromosome on which the resistance allele REN1 is located. The REN1 interval was restricted to 1.4 cM using 38 SSR markers distributed across the locus and the segregation of the resistance phenotype in two progenies of collectively 461 offspring, derived from either resistant parent. The boundary markers delimit a 1.4-Mbp sequence in the PN40024 reference genome, which contains 27 genes with known functions, 2 full-length coiled-coil NBS-LRR genes, and 9 NBS-LRR pseudogenes. In the REN1 locus of PN40024, NBS genes have proliferated through a mixture of segmental duplications, tandem gene duplications, and intragenic recombination between paralogues, indicating that the REN1 locus has been inherently prone to producing genetic variation. Three SSR markers co-segregate with REN1, the outer ones confining the 908-kb array of NBS-LRR genes. Kinship and clustering analyses based on genetic distances with susceptible cultivars representative of Central Asian Vitis vinifera indicated that 'Kishmish vatkana' and 'Dzhandzhal kara' fit well into local germplasm. 'Kishmish vatkana' also has a parent-offspring relationship with the seedless table grape 'Sultanina'. In addition, the distant genetic relatedness to rootstocks, some of which are derived from North American species resistant to powdery mildew and have been used worldwide to guard against phylloxera since the late 1800's, argues against REN1 being infused into Vitis vinifera from a recent interspecific hybridisation. Conclusion The REN1 gene resides in an NBS-LRR gene cluster tightly delimited by two flanking SSR markers, which can assist in the selection of this DNA block in breeding between Vitis vinifera cultivars. The REN1 locus has multiple layers of structural complexity compared with its two closely related paralogous NBS clusters, which are located some 5 Mbp upstream and 4 Mbp downstream of the REN1 interval on the same chromosome.

Genetic code expansion has emerged as a powerful tool in enzyme design and engineering, providing new insights into sophisticated catalytic mechanisms and enabling the development of enzymes with new catalytic functions. In this regard, the non-canonical histidine analogue N δ -methylhistidine (MeHis) has proven especially versatile due to its ability to serve as a metal coordinating ligand or a catalytic nucleophile with a similar mode of reactivity to small molecule catalysts such as 4-dimethylaminopyridine (DMAP). Here we report the development of a highly efficient aminoacyl tRNA synthetase (G1PylRS MIFAF ) for encoding MeHis into proteins, by transplanting five known active site mutations from Methanomethylophilus alvus ( Ma PylRS) into the single domain PylRS from Methanogenic archaeon ISO4-G1. In contrast to the high concentrations of MeHis (5-10 mM) needed with the Ma system, G1PylRS MIFAF can operate efficiently using MeHis concentrations of ∼0.1 mM, allowing more economical production of a range of MeHis-containing enzymes in high titres. Interestingly G1PylRS MIFAF is also a 'polyspecific' aminoacyl tRNA synthetase (aaRS), enabling incorporation of five different non-canonical amino acids (ncAAs) including 3-pyridylalanine and 2-fluorophenylalanine. This study provides an important step towards scalable production of engineered enzymes that contain non-canonical amino acids such as MeHis as key catalytic elements.

YeastFab is a Golden Gate-based cloning standard and parts repository. It is designed for modular, hierarchical assembly of transcription units and multi-gene assemblies for expression in Saccharomyces cerevisiae. This makes it a suitable toolbox to optimize the expression strength of heterologous genes in yeast. When cloning heterologous coding sequences into YeastFab vectors, in several cases we have observed toxicity to the cloning host Escherichia coli. The provided protocol details how to clone such toxic genes from multiple synthetic DNA fragments while adhering to the YeastFab standard. The presented cloning strategy includes a C-terminal FLAG tag that allows screening for constructs with a desired protein expression in yeast by western blot. The design allows scarlessly removing the tag through a Golden Gate reaction to facilitate cloning of expression constructs with the native, untagged transgene., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Synthetic biology holds immense promise to tackle key problems in resource use, environmental remediation, and human health care. However, comprehensive safety measures are lacking to employ engineered microorganisms in open-environment applications. Genetically encoded biocontainment systems may solve this issue. Here, we describe such a system based on conditional stability of essential proteins. We used a destabilizing domain degron stabilized by estradiol addition (ERdd). We ERdd-tagged 775 essential genes and screened for strains with estradiol dependent growth. Three genes, SPC110, DIS3 and RRP46, were found to be particularly suitable targets. Respective strains showed no growth defect in the presence of estradiol and strong growth inhibition in its absence. SPC110-ERdd offered the most stringent containment, with an escape frequency of <5×10 -7 . Removal of its C-terminal domain decreased the escape frequency further to <10 -8 . Being based on conditional protein stability, the presented approach is mechanistically orthogonal to previously reported genetic biocontainment systems., (© 2024. The Author(s).)

Pulcherrimin is an iron (III) chelate of pulcherriminic acid that plays a role in antagonistic microbial interactions, iron metabolism, and stress responses. Some bacteria and yeasts produce pulcherriminic acid, but so far, pulcherrimin could not be produced in Saccharomyces cerevisiae. Here, multiple integrations of the Metschnikowia pulcherrima PUL1 and PUL2 genes in the S. cerevisiae genome resulted in red colonies, which indicated pulcherrimin formation. The coloration correlated positively and significantly with the number of PUL1 and PUL2 genes. The presence of pulcherriminic acid was confirmed by mass spectrometry. In vitro competition assays with the plant pathogenic fungus Botrytis caroliana revealed inhibitory activity on conidiation by an engineered, strong pulcherrimin-producing S. cerevisiae strain. We demonstrate that the PUL1 and PUL2 genes from M. pulcherrima, in multiple copies, are sufficient to transfer pulcherrimin production to S. cerevisiae and represent the starting point for engineering and optimizing this biosynthetic pathway in the future., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Technologies to profoundly engineer biology are becoming increasingly affordable, powerful, and accessible to a widening group of actors. While offering tremendous potential to fuel biological research and the bioeconomy, this development also increases the risk of inadvertent or deliberate creation and dissemination of pathogens. Effective regulatory and technological frameworks need to be developed and deployed to manage these emerging biosafety and biosecurity risks. Here, we review digital and biological approaches of a range of technology readiness levels suited to address these challenges. Digital sequence screening technologies already are used to control access to synthetic DNA of concern. We examine the current state of the art of sequence screening, challenges and future directions, and environmental surveillance for the presence of engineered organisms. As biosafety layer on the organism level, we discuss genetic biocontainment systems that can be used to created host organisms with an intrinsic barrier against unchecked environmental proliferation., Competing Interests: D.D. is a co-founder of Lattice Automation, Inc. and Asimov Inc. Both companies create engineered biological systems using software and automation. J.D.B. is a Founder and Director of CDI Labs, Inc., a Founder of and consultant to Neochromosome, Inc, a Founder, SAB member of and consultant to Re-Open Diagnostics, LLC, and serves or served on the Scientific Advisory Board of the following: Sangamo Therapeutics, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc. and the Wyss Institute. T.K. is co-founder of Ginkgo Bioworks, Inc. EL is CEO and co-founder of Twist Bioscience, Inc., and J.D. is an employee of Twist Bioscience, Inc., (© 2023 The Author(s).)

The synthetic yeast, Sc2.0, is nearing completion as consolidation of all 17 synthetic chromosomes into a single cell advances. This organism will be the first synthetic eukaryote and provides a highly plastic biological chassis built from the bottom-up using principles of biological design. This synthetic approach to genome construction has allowed the genetic code to be re-wired in this background to liberate the amber stop codon as a dedicated triplet for encoding non-canonical amino acids. The availability of an expanded set of amino acid building blocks allows precise control of protein structure and function, providing new opportunities to develop protein-based therapeutics, materials and catalysts. In this article, we review the challenges facing genetic code expansion research in yeast and highlight how the development of Sc2.0 provides new and exciting opportunities to address existing limitations., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Computer-aided design (CAD) for synthetic biology promises to accelerate the rational and robust engineering of biological systems. It requires both detailed and quantitative mathematical and experimental models of the processes to (re)design biology, and software and tools for genetic engineering and DNA assembly. Ultimately, the increased precision in the design phase will have a dramatic impact on the production of designer cells and organisms with bespoke functions and increased modularity. CAD strategies require quantitative models of cells that can capture multiscale processes and link genotypes to phenotypes. Here, we present a perspective on how whole-cell, multiscale models could transform design-build-test-learn cycles in synthetic biology. We show how these models could significantly aid in the design and learn phases while reducing experimental testing by presenting case studies spanning from genome minimization to cell-free systems. We also discuss several challenges for the realization of our vision. The possibility to describe and build whole-cells in silico offers an opportunity to develop increasingly automatized, precise and accessible CAD tools and strategies., (Copyright © 2020 Marucci, Barberis, Karr, Ray, Race, de Souza Andrade, Grierson, Hoffmann, Landon, Rech, Rees-Garbutt, Seabrook, Shaw and Woods.)

The ability to redesign and reconstruct a cell at whole-genome level provides new platforms for biological study. The international synthetic yeast genome project-Sc2.0, designed by interrogating knowledge amassed by the yeast community to date, exemplifies how a classical synthetic biology "design-build-test-learn" engineering cycle can effectively test hypotheses about various genome fundamentals. The genome reshuffling SCRaMbLE system implemented in synthetic yeast strains also provides unprecedented diversified resources for genotype-phenotype study and yeast metabolic engineering. Further development of genome synthesis technology will shed new lights on complex biological processes in higher eukaryotes., Competing Interests: Declaration of competing interest The authors declare no potential conflicts of interest., (Copyright © 2020. Published by Elsevier Inc.)

Antisense transcription is common in naturally occurring genomes and is increasingly being used in synthetic genetic circuitry as a tool for gene expression control. Mutual influence on the expression of convergent genes can be mediated by antisense RNA effects and by transcriptional interference (TI). We aimed to quantitatively characterize long-range TI between convergent genes with untranslated intergenic spacers of increasing length. After controlling for antisense RNA-mediated effects, which contributed about half of the observed total expression inhibition, the TI effect was modeled. To achieve model convergence, RNA polymerase processivity and collision resistance were assumed to be modulated by ribosome trailing. The spontaneous transcription termination rate in regions of untranslated DNA was experimentally determined. Our modeling suggests that an elongating RNA polymerase with a trailing ribosome is about 13 times more likely to resume transcription than an opposing RNA polymerase without a trailing ribosome, upon head-on collision of the two.

For various experimental applications, microbial cultures at defined, constant densities are highly advantageous over simple batch cultures. Due to high costs, however, devices for continuous culture at freely defined densities still experience limited use. We have developed a small-scale turbidostat for research purposes, which is manufactured from inexpensive components and 3D printed parts. A high degree of spatial system integration and a graphical user interface provide user-friendly operability. The used optical density feedback control allows for constant continuous culture at a wide range of densities and offers to vary culture volume and dilution rates without additional parametrization. Further, a recursive algorithm for on-line growth rate estimation has been implemented. The employed Kalman filtering approach based on a very general state model retains the flexibility of the used control type and can be easily adapted to other bioreactor designs. Within several minutes it can converge to robust, accurate growth rate estimates. This is particularly useful for directed evolution experiments or studies on metabolic challenges, as it allows direct monitoring of the population fitness.

The interest in quantitative single photon emission computer tomography (SPECT) shows potential in a number of clinical applications and now several vendors are providing software and hardware solutions to allow 'SUV-SPECT' to mirror metrics used in PET imaging. This brief technical report assesses the accuracy of activity concentration measurements using a new algorithm 'xSPECT' from Siemens Healthcare. SPECT/CT data were acquired from a uniform cylinder with 5, 10, 15 and 20 s/projection and NEMA image quality phantom with 25 s/projection. The NEMA phantom had hot spheres filled with an 8 : 1 activity concentration relative to the background compartment. Reconstructions were performed using parameters defined by manufacturer presets available with the algorithm. The accuracy of activity concentration measurements was assessed. A dose calibrator-camera cross-calibration factor (CCF) was derived from the uniform phantom data. In uniform phantom images, a positive bias was observed, ranging from ∼6% in the lower count images to ∼4% in the higher-count images. On the basis of the higher-count data, a CCF of 0.96 was derived. As expected, considerable negative bias was measured in the NEMA spheres using region mean values whereas positive bias was measured in the four largest NEMA spheres. Nonmonotonically increasing recovery curves for the hot spheres suggested the presence of Gibbs edge enhancement from resolution modelling. Sufficiently accurate activity concentration measurements can easily be measured on images reconstructed with the xSPECT algorithm without a CCF. However, the use of a CCF is likely to improve accuracy further. A manual conversion of voxel values into SUV should be possible, provided that the patient weight, injected activity and time between injection and imaging are all known accurately.

Induced pluripotent stem cells (iPSCs) hold great promise to model diseases, where the disease affected cell type is difficult to access. A major obstacle for the development of disease models is the lack of well characterized control iPSCs from old people not affected by such a disease. Furthermore, gene-editing approaches often require iPSCs from healthy donors, where pathogenic mutations can be inserted if patient material is not available. Here, we report the generation of an iPSC line (16423 #6) from a 77-year-old woman, who did not display any disease symptoms at the time, when the skin biopsy was taken., (Copyright © 2016 Michael Boutros, German Cancer Research Center, Heidelberg, Germany. Published by Elsevier B.V. All rights reserved.)

We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong σ(70) dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a 'forward' gene interferes with the expression of a 'reverse' gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The rat pancreatic progenitor cell line B-13 is of interest for research on drug metabolism and toxicity since the cells trans-differentiate into functional hepatocyte-like cells (B-13/H) when treated with glucocorticoids. In this study we investigated the trans-differentiation and liver-specific functions of B-13/H cells in a three-dimensional (3D) multi-compartment bioreactor, which has already been successfully used for primary liver cell culture. Undifferentiated B-13 cells were inoculated into the bioreactor system and exposed to dexamethasone to promote hepatic trans-differentiation (B-13/HT). In a second approach, pre-differentiated B-13 cells were cultured in bioreactors for 15 days to evaluate the maintenance of liver-typical functions (B-13/HP). During trans-differentiation of B-13 cells into hepatocyte-like cells in the 3D bioreactor system (approach B-13/HT), an increase in glucose metabolism and in liver-specific functions (urea and albumin synthesis; cytochrome P450 [CYP] enzyme activity) was observed, whereas amylase - characteristic for exocrine pancreas and undifferentiated B-13 cells - decreased over time. In bioreactors with pre-differentiated cells (approach B-13/HP), the above liver-specific functions were maintained over the whole culture period. Results were confirmed by gene expression and protein analysis showing increased expression of carbamoyl-phosphate synthase 1 (CPS-1), albumin, CYP2E1, CYP2C11 and CYP3A1 with simultaneous loss of amylase. Immunohistochemical studies showed the formation of 3D structures with expression of liver-specific markers, including albumin, cytokeratin (CK) 18, CCAAT/enhancer-binding protein beta (CEBP-β), CYP2E1 and multidrug resistance protein 2 (MRP2). In conclusion, successful culture and trans-differentiation of B-13 cells in the 3D bioreactor was demonstrated. The requirement for only one hormone and simple culture conditions to generate liver-like cells makes this cell type useful for in vitro research using 3D high-density culture systems.

Primary human hepatocytes represent an important cell source for in vitro investigation of hepatic drug metabolism and disposition. In this study, a multi-compartment capillary membrane-based bioreactor technology for three-dimensional (3D) perfusion culture was further developed and miniaturized to a volume of less than 0.5 ml to reduce demand for cells. The miniaturized bioreactor was composed of two capillary layers, each made of alternately arranged oxygen and medium capillaries serving as a 3D culture for the cells. Metabolic activity and stability of primary human hepatocytes was studied in this bioreactor in the presence of 2.5% fetal calf serum (FCS) under serum-free conditions over a culture period of 10 days. The miniaturized bioreactor showed functions comparable to previously reported data for larger variants. Glucose and lactate metabolism, urea production, albumin synthesis and release of intracellular enzymes (AST, ALT, GLDH) showed no significant differences between serum-free and serum-supplemented bioreactors. Activities of human-relevant cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP3A4/5, CYP2C9, CYP2D6, CYP2B6) analyzed by determination of product formation rates from selective probe substrates were also comparable in both groups. Gene expression analysis showed moderately higher expression in the majority of CYP enzymes, transport proteins and enzymes of Phase II metabolism in the serum-free bioreactors compared to those maintained with FCS. In conclusion, the miniaturized bioreactor maintained stable function over the investigated period and thus provides a suitable system for pharmacological studies on primary human hepatocytes under defined serum-free conditions., (Copyright © 2012 John Wiley & Sons, Ltd.)

Light-switchable proteins offer numerous opportunities as tools for manipulating biological systems with exceptional degrees of spatiotemporal control. Most designed light-switchable proteins currently in use have not been optimised using the randomisation and selection/screening approaches that are widely used in other areas of protein engineering. Here we report an approach for screening light-switchable DNA-binding proteins that relies on light-dependent repression of the transcription of a fluorescent reporter. We demonstrate that the method can be used to recover a known light-switchable DNA-binding protein from a random library., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: This study evaluated the genetic similarity and prevalence of the stx1, stx2, eae, and ehxA genes in Escherichia coli isolated from pasteurized cow milk. Eighty-seven E. coli isolates from pasteurized cow milk from 22 dairies located in northwestern Paraná state, Brazil, were analyzed. Genetic similarity was evaluated using enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) and repetitive extragenic palindromic sequence PCR (REP-PCR). E. coli isolates were also analyzed by PCR to investigate the presence of the stx1, stx2, eae, and ehxA genes. ERIC-PCR and REP-PCR clustered 87 bacterial isolates in 76 and 81 genomic profiles, respectively. Both techniques revealed high genetic diversity among the E. coli isolates, confirming the possibility of their use in epidemiological studies. The stx1, stx2, eae, and ehxA virulence genes were not detected in E. coli isolates, indicating a low prevalence of Shiga toxin-producing E. coli in milk produced in the region studied., Practical Application: Knowledge about the presence of diarrheagenic Escherichia coli in pasteurized milk is important developing and implementing control measures in milk and dairy production., (© 2014 Institute of Food Technologists®)

Neural precursor cells of the ventricular zone give rise to all neurons and glia of the central nervous system and rely for maintenance of their precursor characteristics on the closely related SoxB1 transcription factors Sox1, Sox2 and Sox3. We show in mouse spinal cord that, whereas SoxB1 proteins are usually downregulated upon neuronal specification, they continue to be expressed in glial precursors. In the oligodendrocyte lineage, Sox2 and Sox3 remain present into the early phases of terminal differentiation. Surprisingly, their deletion does not alter precursor characteristics but interferes with proper differentiation. Although a direct influence on myelin gene expression may be part of their function, we provide evidence for another mode of action. SoxB1 proteins promote oligodendrocyte differentiation in part by negatively controlling miR145 and thereby preventing this microRNA from inhibiting several pro-differentiation factors. This study presents one of the few cases in which SoxB1 proteins, including the stem cell factor Sox2, are associated with differentiation rather than precursor functions.

Based on a hollow fiber perfusion technology with internal oxygenation, a miniaturized bioreactor with a volume of 0.5 mL for in vitro studies was recently developed. Here, the suitability of this novel culture system for pharmacological studies was investigated, focusing on the model drug diclofenac. Primary human liver cells were cultivated in bioreactors and in conventional monolayer cultures in parallel over 10 days. From day 3 on, diclofenac was continuously applied at a therapeutic concentration (6.4 µM) for analysis of its metabolism. In addition, the activity and gene expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 were assessed. Diclofenac was metabolized in bioreactor cultures with an initial conversion rate of 230 ± 57 pmol/h/10(6) cells followed by a period of stable conversion of about 100 pmol/h/10(6) cells. All CYP activities tested were maintained until day 10 of bioreactor culture. The expression of corresponding mRNAs correlated well with the degree of preservation. Immunohistochemical characterization showed the formation of neo-tissue with expression of CYP2C9 and CYP3A4 and the drug transporters breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the bioreactor. In contrast, monolayer cultures showed a rapid decline of diclofenac conversion and cells had largely lost activity and mRNA expression of the assessed CYP isoforms at the end of the culture period. In conclusion, diclofenac metabolism, CYP activities and gene expression levels were considerably more stable in bioreactor cultures, making the novel bioreactor a useful tool for pharmacological or toxicological investigations requiring a highly physiological in vitro representation of the liver., (Copyright © 2012 Wiley Periodicals, Inc.)

Inhalation of asbestos and oxidant-generating pollutants causes injury and compensatory proliferation of lung epithelium, but the signaling mechanisms that lead to these responses are unclear. We hypothesized that a protein kinase (PK)Cdelta-dependent PKD pathway was able to regulate downstream mitogen-activated protein kinases, affecting pro- and anti-apoptotic responses to asbestos. Elevated levels of phosphorylated PKD (p-PKD) were observed in distal bronchiolar epithelial cells of mice inhaling asbestos. In contrast, PKCdelta-/- mice showed significantly lower levels of p-PKD in lung homogenates and in situ after asbestos inhalation. In a murine lung epithelial cell line, asbestos caused significant increases in the phosphorylation of PKCdelta-dependent PKD, ERK1/2, and JNK1/2/c-Jun that occurred with decreases in the BH3-only pro-apoptotic protein, Bim. Silencing of PKCdelta, PKD, and use of small molecule inhibitors linked the ERK1/2 pathway to the prevention of Bim-associated apoptosis as well as the JNK1/2/c-Jun pathway to the induction of apoptosis. Our studies are the first to show that asbestos induces PKD phosphorylation in lung epithelial cells both in vivo and in vitro. PKCdelta-dependent PKD phosphorylation by asbestos is causally linked to a cellular pathway that involves the phosphorylation of both ERK1/2 and JNK1/2, which play opposing roles in the apoptotic response induced by asbestos.

Objective: To assess the utility of the appearances of occipital lobe perfusion on HMPAO SPECT in the diagnosis of dementia with Lewy bodies (DLB) using the 123I-FP-CIT findings as the diagnostic 'gold standard'., Methods: Eighty-four consecutive patients underwent both HMPAO SPECT and 123I-FP-CIT as part of their routine investigations for suspected DLB., Results: Thirty-nine of the 84 FP-CIT scans were abnormal indicating a prevalence of 44% of patients with DLB in this series. In those patients classified as DLB, 28% of HMPAO SPECT scans demonstrated occipital hypoperfusion. In those patients with a dementia other than DLB 31% of patients demonstrated occipital hypoperfusion (P=0.8)., Conclusion: Occipital lobe hypoperfusion as demonstrated by HMPAO SPECT in patients with suspected Lewy body dementia does not appear to be able to either rule in, or rule out, the diagnosis of DLB.

Aim: To assess the role of 99mTc-hexamethylpropyleneamine oxime single-photon emission computed tomography (99mTc-HMPAO SPECT) imaging of the precuneus and medial temporal lobe in the individual patient with mild Alzheimer's disease and dementia with Lewy bodies (DLB) using statistical parametric mapping and visual image interpretation., Methods: Thirty-four patients with mild late-onset Alzheimer's disease, 20 patients with early-onset Alzheimer's disease, 15 patients with DLB and 31 healthy controls were studied. All patients fulfilled appropriate clinical criteria; the DLB patients also had evidence of dopaminergic presynaptic terminal loss on 123I-N-omega-fluoropropyl-2beta-carbomethoxy-3beta-(4-iodophenyl)-tropane imaging. 99mTc-HMPAO SPECT brain scans were acquired on a multidetector gamma camera and images were assessed separately by visual interpretation and with SPM99., Results: Statistical parametric maps were significantly more accurate than visual image interpretation in all disease categories. In patients with mild late-onset Alzheimer's disease, statistical parametric mapping demonstrated significant hypoperfusion to the precuneus in 59% and to the medial temporal lobe in 53%. Seventy-six per cent of these patients had a defect in either location. No controls had precuneal or medial temporal lobe hypoperfusion (specificity, 100%). Statistical parametric mapping also demonstrated 73% of patients with DLB to have precuneal abnormalities, but only 6% had medial temporal lobe involvement., Conclusion: These findings illustrate the capability of statistical parametric mapping to demonstrate reliable abnormalities in the majority, but not all, patients with either mild Alzheimer's disease or DLB. Precuneal hypoperfusion is not specific to Alzheimer's disease and is equally likely to be found in DLB. In this study, medial temporal hypoperfusion was significantly more common in Alzheimer's disease than in DLB. Statistical parametric maps appear to be considerably more reliable than simple visual interpretation of 99mTc-HMPAO images for these regions.

Activation of extracellular signal-regulated kinases (ERK) has been associated with the advent of asbestos-associated apoptosis and proliferation in mesothelial and alveolar epithelial cells and may be linked to the development of pulmonary fibrosis. The objective of studies here was to characterize the development of inflammation, cellular proliferation, and fibrosis in asbestos-exposed C57Bl/6 mice in relationship to patterns of ERK phosphorylation. Inflammation occurred after 10 and 20 days of asbestos exposure as evidenced by increases in total protein and neutrophils in bronchoalveolar lavage fluid. Increases in cell proliferation were observed at 30 days in bronchiolar epithelia and at 4, 14, and 30 days in the alveolar compartment of the lung. Trichrome-positive focal lesions of pulmonary fibrosis developed at 30 days in the absence of elevations in lung hydroxyproline or procollagen mRNA levels. Striking increases in ERK phosphorylation were observed within pulmonary epithelial cells at sites of developing fibrotic lesions after 14 and 30 days of inhalation. In addition to characterizing a murine inhalation model of asbestosis, we provide the first evidence showing activation of ERK signaling within lung epithelium in vivo, following inhalation of asbestos fibers.

The advent of effective antimicrobial agents has been justifiably hailed as one of the most significant medical advances. However, as experience with older agents has grown and as newer compounds have been added to the clinician's armamentarium, certain disadvantages associated with the use of antimicrobials have become increasingly apparent. Toxicity and cost are among the disadvantages, but drug resistance is probably the major adverse consequence of widespread antimicrobial use. This article reviews the extent, mechanisms, and consequences of antimicrobial resistance--particularly in the hospital environment--and offers strategies for minimizing both its emergence and its spread.

High-level resistance to gentamicin among clinical isolates of enterococci has been found with increasing frequency in recent years. In this issue, Zervos and colleagues report findings from a prospective study in which they assessed the frequency of colonization and infection with such organisms at a university medical center, demonstrating probable person-to-person spread. Their findings suggest that hospitals should conduct systematic screening for enterococci with high-level resistance to gentamicin, that antimicrobial treatment habits be modified to limit the emergence of such organisms, and that rigorous infection control be practiced to minimize their spread. These observations are particularly timely because it has become clear that enterococci are extremely versatile pathogens which are both well suited for survival and capable of causing serious illness, especially in hospitalized patients treated with some of the newer broad-spectrum antibiotic agents. Enterococci with high-level resistance to gentamicin are also of growing concern because their resistance to many antibiotic agents severely limits the clinician's options for treatment.