A Dedicated Outpatient Pharmacy Improves Access to Discharge Medications in a Pediatric Emergency Department: A Quality Improvement Study.

STUDY OBJECTIVE: Following discharge from a pediatric emergency department (ED) or urgent care, many families do not pick up their prescribed medications. The aim of this quality improvement study was to increase the percentage of patients discharged home with medications in-hand from 6% to 30% within 6 months. METHODS: Due to the planned construction of a new ED, urgent care, and dedicated pharmacy, a multidisciplinary team was formed to increase access to discharge medications. We performed a pilot study in the urgent care to improve the discharge prescription process and expanded its scope to the ED. We evaluated the effect of our interventions on the percentage of patients discharged with medications in-hand through statistical process control charts. Process measures included the percentage of prescriptions electronically prescribed and directed to an on-site pharmacy. RESULTS: Between June 21, 2021 and March 27, 2022, 7,678 patients were discharged with at least 1 medication in-hand. The percentage of patients discharged with medications in-hand increased from 6.2% to 60.6%. The percentage of prescriptions e-prescribed and directed to an on-site pharmacy increased to 94.6% and 65.6% respectively. CONCLUSIONS: In this study, the availability of a 24-hour on-site pharmacy appears to be the most impactful intervention increasing access to discharge medications for families. Other interventions, such as a pilot study in the urgent care and implementing default electronic prescribing, may have potentiated the effect of the new pharmacy.

Bilateral Acute Macular Neuroretinopathy (AMN) after COVID-19 and its Clinical Course. / Bilaterale akute makuläre Neuroretinopathie (AMN) nach COVID-19 und Verlauf.

Acute macular neuroretinopathy (AMN) is a rare disease entity. It is mainly observed in young women with a history of influenza-like infection or who have been taking oral contraceptives for several years. Patients typically describe subjective visual deterioration and mono- or bilateral paracentral relative scotomas. In some cases, funduscopic ophthalmic examination may reveal subtle sharply demarcated flat lesions of reddish-brown or orange colour in the macular region. Diagnosis is usually made by near-infrared fundus imaging which shows hyporeflective areas, and SD-OCT imaging which manifests changes in the outer retinal layers. In the following, three patient cases with bilateral AMN are described which occurred in direct temporal relationship to a recent SARS-CoV-2 infection.

High-coverage plasma lipidomics reveals novel sex-specific lipidomic fingerprints of age and BMI: Evidence from two large population cohort studies.

Obesity and related metabolic diseases show clear sex-related differences. The growing burden of these diseases calls for better understanding of the age- and sex-related metabolic consequences. High-throughput lipidomic analyses of population-based cohorts offer an opportunity to identify disease-risk-associated biomarkers and to improve our understanding of lipid metabolism and biology at a population level. Here, we comprehensively examined the relationship between lipid classes/subclasses and molecular species with age, sex, and body mass index (BMI). Furthermore, we evaluated sex specificity in the association of the plasma lipidome with age and BMI. Some 747 targeted lipid measures, representing 706 molecular lipid species across 36 classes/subclasses, were measured using a high-performance liquid chromatography coupled mass spectrometer on a total of 10,339 participants from the Australian Diabetes, Obesity and Lifestyle Study (AusDiab), with 563 lipid species being validated externally on 4,207 participants of the Busselton Health Study (BHS). Heat maps were constructed to visualise the relative differences in lipidomic profile between men and women. Multivariable linear regression analyses, including sex-interaction terms, were performed to assess the associations of lipid species with cardiometabolic phenotypes. Associations with age and sex were found for 472 (66.9%) and 583 (82.6%) lipid species, respectively. We further demonstrated that age-associated lipidomic fingerprints differed by sex. Specific classes of ether-phospholipids and lysophospholipids (calculated as the sum composition of the species within the class) were inversely associated with age in men only. In analyses with women alone, higher triacylglycerol and lower lysoalkylphosphatidylcholine species were observed among postmenopausal women compared with premenopausal women. We also identified sex-specific associations of lipid species with obesity. Lysophospholipids were negatively associated with BMI in both sexes (with a larger effect size in men), whilst acylcarnitine species showed opposing associations based on sex (positive association in women and negative association in men). Finally, by utilising specific lipid ratios as a proxy for enzymatic activity, we identified stearoyl CoA desaturase (SCD-1), fatty acid desaturase 3 (FADS3), and plasmanylethanolamine Δ1-desaturase activities, as well as the sphingolipid metabolic pathway, as constituent perturbations of cardiometabolic phenotypes. Our analyses elucidate the effect of age and sex on lipid metabolism by offering a comprehensive view of the lipidomic profiles associated with common cardiometabolic risk factors. These findings have implications for age- and sex-dependent lipid metabolism in health and disease and suggest the need for sex stratification during lipid biomarker discovery, establishing biological reference intervals for assessment of disease risk.

Correction: High-coverage plasma lipidomics reveals novel sex-specific lipidomic fingerprints of age and BMI: Evidence from two large population cohort studies.

[This corrects the article DOI: 10.1371/journal.pbio.3000870.].

Population-based plasma lipidomics reveals developmental changes in metabolism and signatures of obesity risk: a mother-offspring cohort study.

BACKGROUND: Lipids play a vital role in health and disease, but changes to their circulating levels and the link with obesity remain poorly characterized in expecting mothers and their offspring in early childhood. METHODS: LC-MS/MS-based quantitation of 480 lipid species was performed on 2491 plasma samples collected at 4 time points in the mother-offspring Asian cohort GUSTO (Growing Up in Singapore Towards healthy Outcomes). These 4 time points constituted samples collected from mothers at 26-28 weeks of gestation (n=752) and 4-5 years postpartum (n=650), and their offspring at birth (n=751) and 6 years of age (n=338). Linear regression models were used to identify the pregnancy and developmental age-specific variations in the plasma lipidomic profiles, and their association with obesity risk. An independent birth cohort (n=1935), the Barwon Infant Study (BIS), comprising mother-offspring dyads of Caucasian origin was used for validation. RESULTS: Levels of 36% of the profiled lipids were significantly higher (absolute fold change > 1.5 and Padj < 0.05) in antenatal maternal circulation as compared to the postnatal phase, with phosphatidylethanolamine levels changing the most. Compared to antenatal maternal lipids, cord blood showed lower concentrations of most lipid species (79%) except lysophospholipids and acylcarnitines. Changes in lipid concentrations from birth to 6 years of age were much higher in magnitude (log2FC=-2.10 to 6.25) than the changes observed between a 6-year-old child and an adult (postnatal mother) (log2FC=-0.68 to 1.18). Associations of cord blood lipidomic profiles with birth weight displayed distinct trends compared to the lipidomic profiles associated with child BMI at 6 years. Comparison of the results between the child and adult BMI identified similarities in association with consistent trends (R2=0.75). However, large number of lipids were associated with BMI in adults (67%) compared to the children (29%). Pre-pregnancy BMI was specifically associated with decrease in the levels of phospholipids, sphingomyelin, and several triacylglycerol species in pregnancy. CONCLUSIONS: In summary, our study provides a detailed landscape of the in utero lipid environment provided by the gestating mother to the growing fetus, and the magnitude of changes in plasma lipidomic profiles from birth to early childhood. We identified the effects of adiposity on the circulating lipid levels in pregnant and non-pregnant women as well as offspring at birth and at 6 years of age. Additionally, the pediatric vs maternal overlap of the circulating lipid phenotype of obesity risk provides intergenerational insights and early opportunities to track and intervene the onset of metabolic adversities. CLINICAL TRIAL REGISTRATION: This birth cohort is a prospective observational study, which was registered on 1 July 2010 under the identifier NCT01174875 .

Shark liver oil supplementation enriches endogenous plasmalogens and reduces markers of dyslipidemia and inflammation.

Plasmalogens are membrane glycerophospholipids with diverse biological functions. Reduced plasmalogen levels have been observed in metabolic diseases; hence, increasing their levels might be beneficial in ameliorating these conditions. Shark liver oil (SLO) is a rich source of alkylglycerols that can be metabolized into plasmalogens. This study was designed to evaluate the impact of SLO supplementation on endogenous plasmalogen levels in individuals with features of metabolic disease. In this randomized, double-blind, placebo-controlled cross-over study, the participants (10 overweight or obese males) received 4-g Alkyrol® (purified SLO) or placebo (methylcellulose) per day for 3 weeks followed by a 3-week washout phase and were then crossed over to 3 weeks of the alternate placebo/Alkyrol® treatment. SLO supplementation led to significant changes in plasma and circulatory white blood cell lipidomes, notably increased levels of plasmalogens and other ether lipids. In addition, SLO supplementation significantly decreased the plasma levels of total free cholesterol, triglycerides, and C-reactive protein. These findings suggest that SLO supplementation can enrich plasma and cellular plasmalogens and this enrichment may provide protection against obesity-related dyslipidemia and inflammation.

Evaluating tank acclimation and trial length for dynamic shuttle box temperature preference assays in aquatic animals.

Characterizing the thermal preference of fish is important in conservation, environmental and evolutionary physiology and can be determined using a shuttle box system. Initial tank acclimation and trial lengths are important considerations in experimental design, yet systematic studies of these factors are missing. Three different behavioral assay experimental designs were tested to determine the effect of tank acclimation and trial length (hours of tank acclimation:behavioral trial: 12:12, 0:12, 2:2) on the temperature preference of juvenile lake whitefish (Coregonus clupeaformis), using a shuttle box. Average temperature preferences for the 12 h:12 h, 0 h:12 h, 2 h:2 h experimental designs were 16.10±1.07°C, 16.02±1.56°C and 16.12±1.59°C respectively, with no significant differences between experimental designs (P=0.9337). Ultimately, length of acclimation time and trial length had no significant effect on thermal preference.

Time to implement a national referral pathway for suspected cauda equina syndrome: review and outcome of 250 referrals.

INTRODUCTION: Cauda equina syndrome (CES) is a condition with significant implications and medico-legal profile. The literature still lacks large primary studies to provide strong evidence for a robust management pathway. Statements from Neurosurgical and Spinal societies support early diagnosis and imaging but this has not resulted in any noticeable shift in referral pattern. We strongly feel the need for a nationally agreed, evidence-based referral pathway in practice. We present our large series and in-depth analysis of the referral pathway to provide strong evidence for more robust referrals and management. METHODS: We reviewed 250 referrals of suspected CES (sCES) to the regional neurosurgical unit, evaluating the importance of clinical findings and the imaging pathway. RESULTS: After clinico-radiological evaluation only 32 (13%) had confirmed CES requiring urgent surgery. There was no significant difference in terms of clinical presentation between these true cases of CES (tCES) and false cases (fCES). Imaging was therefore the key rate-limiting step. MRI was the most common investigation used. 73 patients presented without imaging out of hours (OOH). In this group, investigation was delayed to the next day in 60/73 (82%) patients while only 13 (18%) patients underwent OOH MRI. Only 2 (3%) were able to have this at their local hospital. CONCLUSIONS:  As with previous studies we conclude that signs/symptoms are insufficient to identify tCES. Taking into consideration the improved outcome with early diagnosis, the importance of early scanning in diagnosing tCES, and the poor availability of OOH MRI scanning outside of neurosurgical units, we recommend a national policy of 24/7 MRI availability for cases of sCES at all hospitals with MRI scanners. This would remove the 87% of patients not requiring urgent surgery from an unnecessary and distracting referral process.

Protein Activity of the Fusarium fujikuroi Rhodopsins CarO and OpsA and Their Relation to Fungus-Plant Interaction.

Fungi possess diverse photosensory proteins that allow them to perceive different light wavelengths and to adapt to changing light conditions in their environment. The biological and physiological roles of the green light-sensing rhodopsins in fungi are not yet resolved. The rice plant pathogen Fusarium fujikuroi exhibits two different rhodopsins, CarO and OpsA. CarO was previously characterized as a light-driven proton pump. We further analyzed the pumping behavior of CarO by patch-clamp experiments. Our data show that CarO pumping activity is strongly augmented in the presence of the plant hormone indole-3-acetic acid and in sodium acetate, in a dose-dependent manner under slightly acidic conditions. By contrast, under these and other tested conditions, the Neurospora rhodopsin (NR)-like rhodopsin OpsA did not exhibit any pump activity. Basic local alignment search tool (BLAST) searches in the genomes of ascomycetes revealed the occurrence of rhodopsin-encoding genes mainly in phyto-associated or phytopathogenic fungi, suggesting a possible correlation of the presence of rhodopsins with fungal ecology. In accordance, rice plants infected with a CarO-deficient F. fujikuroi strain showed more severe bakanae symptoms than the reference strain, indicating a potential role of the CarO rhodopsin in the regulation of plant infection by this fungus.

Characterization of a splice-site mutation in the tumor suppressor gene FLCN associated with renal cancer.

BACKGROUND: Renal cell carcinoma is among the most prevalent malignancies. It is generally sporadic. However, genetic studies of rare familial forms have led to the identification of mutations in causative genes such as VHL and FLCN. Mutations in the FLCN gene are the cause of Birt-Hogg-Dubé syndrome, a rare tumor syndrome which is characterized by the combination of renal cell carcinoma, pneumothorax and skin tumors. METHODS: Using Sanger sequencing we identify a heterozygous splice-site mutation in FLCN in lymphocyte DNA of a patient suffering from renal cell carcinoma. Furthermore, both tumor DNA and DNA from a metastasis are analyzed regarding this mutation. The pathogenic effect of the sequence alteration is confirmed by minigene assays and the biochemical consequences on the protein are examined using TALEN-mediated transgenesis in cultured cells. RESULTS: Here we describe an FLCN mutation in a 55-year-old patient who presented himself with progressive weight loss, bilateral kidney cysts and renal tumors. He and members of his family had a history of recurrent pneumothorax during the last few decades. Histology after tumor nephrectomy showed a mixed kidney cancer consisting of elements of a chromophobe renal cell carcinoma and dedifferentiated small cell carcinoma component. Subsequent FLCN sequencing identified an intronic c.1177-5_-3delCTC alteration that most likely affected the correct splicing of exon 11 of the FLCN gene. We demonstrate skipping of exon 11 to be the consequence of this mutation leading to a shift in the reading frame and the insertion of a premature stop codon. Interestingly, the truncated protein was still expressed both in cell culture and in tumor tissue, though it was strongly destabilized and its subcellular localization differed from wild-type FLCN. Both, altered protein stability and subcellular localization could be partly reversed by blocking proteasomal and lysosomal degradation. CONCLUSIONS: Identification of disease-causing mutations in BHD syndrome requires the analysis of intronic sequences. However, biochemical validation of the consecutive alterations of the resulting protein is especially important in these cases. Functional characterization of the disease-causing mutations in BHD syndrome may guide further research for the development of novel diagnostic and therapeutic strategies.

Revealing the hidden functional diversity of an enzyme family.

Millions of protein database entries are not assigned reliable functions, preventing the full understanding of chemical diversity in living organisms. Here, we describe an integrated strategy for the discovery of various enzymatic activities catalyzed within protein families of unknown or little known function. This approach relies on the definition of a generic reaction conserved within the family, high-throughput enzymatic screening on representatives, structural and modeling investigations and analysis of genomic and metabolic context. As a proof of principle, we investigated the DUF849 Pfam family and unearthed 14 potential new enzymatic activities, leading to the designation of these proteins as ß-keto acid cleavage enzymes. We propose an in vivo role for four enzymatic activities and suggest key residues for guiding further functional annotation. Our results show that the functional diversity within a family may be largely underestimated. The extension of this strategy to other families will improve our knowledge of the enzymatic landscape.

Bats carry pathogenic hepadnaviruses antigenically related to hepatitis B virus and capable of infecting human hepatocytes.

The hepatitis B virus (HBV), family Hepadnaviridae, is one of most relevant human pathogens. HBV origins are enigmatic, and no zoonotic reservoirs are known. Here, we screened 3,080 specimens from 54 bat species representing 11 bat families for hepadnaviral DNA. Ten specimens (0.3%) from Panama and Gabon yielded unique hepadnaviruses in coancestral relation to HBV. Full genome sequencing allowed classification as three putative orthohepadnavirus species based on genome lengths (3,149-3,377 nt), presence of middle HBV surface and X-protein genes, and sequence distance criteria. Hepatic tropism in bats was shown by quantitative PCR and in situ hybridization. Infected livers showed histopathologic changes compatible with hepatitis. Human hepatocytes transfected with all three bat viruses cross-reacted with sera against the HBV core protein, concordant with the phylogenetic relatedness of these hepadnaviruses and HBV. One virus from Uroderma bilobatum, the tent-making bat, cross-reacted with monoclonal antibodies against the HBV antigenicity determining S domain. Up to 18.4% of bat sera contained antibodies against bat hepadnaviruses. Infectious clones were generated to study all three viruses in detail. Hepatitis D virus particles pseudotyped with surface proteins of U. bilobatum HBV, but neither of the other two viruses could infect primary human and Tupaia belangeri hepatocytes. Hepatocyte infection occurred through the human HBV receptor sodium taurocholate cotransporting polypeptide but could not be neutralized by sera from vaccinated humans. Antihepadnaviral treatment using an approved reverse transcriptase inhibitor blocked replication of all bat hepadnaviruses. Our data suggest that bats may have been ancestral sources of primate hepadnaviruses. The observed zoonotic potential might affect concepts aimed at eradicating HBV.

Evidence for novel hepaciviruses in rodents.

Hepatitis C virus (HCV) is among the most relevant causes of liver cirrhosis and hepatocellular carcinoma. Research is complicated by a lack of accessible small animal models. The systematic investigation of viruses of small mammals could guide efforts to establish such models, while providing insight into viral evolutionary biology. We have assembled the so-far largest collection of small-mammal samples from around the world, qualified to be screened for bloodborne viruses, including sera and organs from 4,770 rodents (41 species); and sera from 2,939 bats (51 species). Three highly divergent rodent hepacivirus clades were detected in 27 (1.8%) of 1,465 European bank voles (Myodes glareolus) and 10 (1.9%) of 518 South African four-striped mice (Rhabdomys pumilio). Bats showed anti-HCV immunoblot reactivities but no virus detection, although the genetic relatedness suggested by the serologic results should have enabled RNA detection using the broadly reactive PCR assays developed for this study. 210 horses and 858 cats and dogs were tested, yielding further horse-associated hepaciviruses but none in dogs or cats. The rodent viruses were equidistant to HCV, exceeding by far the diversity of HCV and the canine/equine hepaciviruses taken together. Five full genomes were sequenced, representing all viral lineages. Salient genome features and distance criteria supported classification of all viruses as hepaciviruses. Quantitative RT-PCR, RNA in-situ hybridisation, and histopathology suggested hepatic tropism with liver inflammation resembling hepatitis C. Recombinant serology for two distinct hepacivirus lineages in 97 bank voles identified seroprevalence rates of 8.3 and 12.4%, respectively. Antibodies in bank vole sera neither cross-reacted with HCV, nor the heterologous bank vole hepacivirus. Co-occurrence of RNA and antibodies was found in 3 of 57 PCR-positive bank vole sera (5.3%). Our data enable new hypotheses regarding HCV evolution and encourage efforts to develop rodent surrogate models for HCV.

MicroScope--an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data.

MicroScope is an integrated platform dedicated to both the methodical updating of microbial genome annotation and to comparative analysis. The resource provides data from completed and ongoing genome projects (automatic and expert annotations), together with data sources from post-genomic experiments (i.e. transcriptomics, mutant collections) allowing users to perfect and improve the understanding of gene functions. MicroScope (http://www.genoscope.cns.fr/agc/microscope) combines tools and graphical interfaces to analyse genomes and to perform the manual curation of gene annotations in a comparative context. Since its first publication in January 2006, the system (previously named MaGe for Magnifying Genomes) has been continuously extended both in terms of data content and analysis tools. The last update of MicroScope was published in 2009 in the Database journal. Today, the resource contains data for >1600 microbial genomes, of which ∼300 are manually curated and maintained by biologists (1200 personal accounts today). Expert annotations are continuously gathered in the MicroScope database (∼50 000 a year), contributing to the improvement of the quality of microbial genomes annotations. Improved data browsing and searching tools have been added, original tools useful in the context of expert annotation have been developed and integrated and the website has been significantly redesigned to be more user-friendly. Furthermore, in the context of the European project Microme (Framework Program 7 Collaborative Project), MicroScope is becoming a resource providing for the curation and analysis of both genomic and metabolic data. An increasing number of projects are related to the study of environmental bacterial (meta)genomes that are able to metabolize a large variety of chemical compounds that may be of high industrial interest.

A lipid atlas of human and mouse immune cells provides insights into ferroptosis susceptibility.

The cellular lipidome comprises thousands of unique lipid species. Here, using mass spectrometry-based targeted lipidomics, we characterize the lipid landscape of human and mouse immune cells ( www.cellularlipidatlas.com ). Using this resource, we show that immune cells have unique lipidomic signatures and that processes such as activation, maturation and development impact immune cell lipid composition. To demonstrate the potential of this resource to provide insights into immune cell biology, we determine how a cell-specific lipid trait-differences in the abundance of polyunsaturated fatty acid-containing glycerophospholipids (PUFA-PLs)-influences immune cell biology. First, we show that differences in PUFA-PL content underpin the differential susceptibility of immune cells to ferroptosis. Second, we show that low PUFA-PL content promotes resistance to ferroptosis in activated neutrophils. In summary, we show that the lipid landscape is a defining feature of immune cell identity and that cell-specific lipid phenotypes underpin aspects of immune cell physiology.

Bats worldwide carry hepatitis E virus-related viruses that form a putative novel genus within the family Hepeviridae.

Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis in tropical and temperate climates. Tropical genotypes 1 and 2 are associated with food-borne and waterborne transmission. Zoonotic reservoirs (mainly pigs, wild boar, and deer) are considered for genotypes 3 and 4, which exist in temperate climates. In view of the association of several zoonotic viruses with bats, we analyzed 3,869 bat specimens from 85 different species and from five continents for hepevirus RNA. HEVs were detected in African, Central American, and European bats, forming a novel phylogenetic clade in the family Hepeviridae. Bat hepeviruses were highly diversified and comparable to human HEV in sequence variation. No evidence for the transmission of bat hepeviruses to humans was found in over 90,000 human blood donations and individual patient sera. Full-genome analysis of one representative virus confirmed formal classification within the family Hepeviridae. Sequence- and distance-based taxonomic evaluations suggested that bat hepeviruses constitute a distinct genus within the family Hepeviridae and that at least three other genera comprising human, rodent, and avian hepeviruses can be designated. This may imply that hepeviruses invaded mammalian hosts nonrecently and underwent speciation according to their host restrictions. Human HEV-related viruses in farmed and peridomestic animals might represent secondary acquisitions of human viruses, rather than animal precursors causally involved in the evolution of human HEV.

NLRP10 enhances Shigella-induced pro-inflammatory responses.

Members of the NLR family evolved as intracellular sensors for bacterial and viral infection. However, our knowledge on the implication of most of the human NLR proteins in innate immune responses still remains fragmentary. Here we characterized the role of human NLRP10 in bacterial infection. Our data revealed that NLRP10 is a cytoplasmic localized protein that positively contributes to innate immune responses induced by the invasive bacterial pathogen Shigella flexneri. SiRNA-mediated knock-down studies showed that NLRP10 contributes to pro-inflammatory cytokine release triggered by Shigella in epithelial cells and primary dermal fibroblasts, by influencing p38 and NF-κB activation. This effect is dependent on the ATPase activity of NLRP10 and its PYD domain. Mechanistically, NLRP10 interacts with NOD1, a NLR that is pivotally involved in sensing of invasive microbes, and both proteins are recruited to the bacterial entry point at the plasma membrane. Moreover, NLRP10 physically interacts with downstream components of the NOD1 signalling pathway, such as RIP2, TAK1 and NEMO. Taken together, our data revealed a novel role of NLRP10 in innate immune responses towards bacterial infection and suggest that NLRP10 functions as a scaffold for the formation of the NOD1-Nodosome.

The CanOE strategy: integrating genomic and metabolic contexts across multiple prokaryote genomes to find candidate genes for orphan enzymes.

Of all biochemically characterized metabolic reactions formalized by the IUBMB, over one out of four have yet to be associated with a nucleic or protein sequence, i.e. are sequence-orphan enzymatic activities. Few bioinformatics annotation tools are able to propose candidate genes for such activities by exploiting context-dependent rather than sequence-dependent data, and none are readily accessible and propose result integration across multiple genomes. Here, we present CanOE (Candidate genes for Orphan Enzymes), a four-step bioinformatics strategy that proposes ranked candidate genes for sequence-orphan enzymatic activities (or orphan enzymes for short). The first step locates "genomic metabolons", i.e. groups of co-localized genes coding proteins catalyzing reactions linked by shared metabolites, in one genome at a time. These metabolons can be particularly helpful for aiding bioanalysts to visualize relevant metabolic data. In the second step, they are used to generate candidate associations between un-annotated genes and gene-less reactions. The third step integrates these gene-reaction associations over several genomes using gene families, and summarizes the strength of family-reaction associations by several scores. In the final step, these scores are used to rank members of gene families which are proposed for metabolic reactions. These associations are of particular interest when the metabolic reaction is a sequence-orphan enzymatic activity. Our strategy found over 60,000 genomic metabolons in more than 1,000 prokaryote organisms from the MicroScope platform, generating candidate genes for many metabolic reactions, of which more than 70 distinct orphan reactions. A computational validation of the approach is discussed. Finally, we present a case study on the anaerobic allantoin degradation pathway in Escherichia coli K-12.

Deficiency in four and one half LIM domain protein 2 (FHL2) aggravates liver fibrosis in mice.

BACKGROUND: Four and one half LIM domain protein 2 (FHL2) has been reported to be a key regulator in many cellular processes being associated with fibrogenesis such as cell migration and contraction. Moreover, hepatic FHL2 is involved in regulation pathways mediating proliferation and cell death machineries. We here investigated the role of FHL2 in the setting of experimental and clinical liver fibrosis. METHODS: FHL2(-/-) and wild type (wt) mice were challenged with CCl(4). Fibrotic response was assessed by quantitative real time PCR (qRT-PCR) of fibrotic marker genes, measurement of hydroxyproline content and histological methods. Murine FHL2(-/-) and hepatic stellate cells (HSC) were isolated and investigated via immunofluorescence. Human fibrotic and normal liver samples were analysed immunohistochemically using antibodies directed against FHL2. RESULTS: FHL2(-/-) mice displayed aggravated liver fibrosis compared to wt mice. However, immunofluorescence revealed no significant morphological changes in cultured FHL2(-/-) and wt myofibroblasts (MFB). In human liver samples, FHL2 was strongly expressed both in the nucleus and cytoplasm in MFB of fibrotic livers. In contrast, FHL2 expression was absent in normal liver tissue. CONCLUSIONS: Deficiency of FHL2 results in aggravation of murine liver fibrosis. In human liver samples, FHL2 is expressed in activated HSCs and portal fibroblasts in human fibrotic livers, pointing to a central role of FHL2 for human hepatic fibrogenesis as well.

Hepatocarcinogenesis in non-cirrhotic liver is associated with a reduced number of clonal hepatocellular patches in non-tumorous liver parenchyma.

We investigated circumscribed cell proliferations in healthy livers in comparison to non-cirrhotic livers bearing hepatocellular carcinoma. Using histochemical staining for cytochrome c oxidase, the fourth complex of the respiratory chain, we visualized patch-forming descendents of regeneratively active liver cells. The clonal nature of these patches was verified by laser-capture microdissection and Sanger sequencing of the enzyme's core subunits in patches carrying marker mutations on the mtDNA. We demonstrate a highly significant increase of the patch size and also a highly significant increase in the number of patches carrying marker mutations between hepatocellular carcinoma-free and -bearing livers. Thus, the carcinoma-bearing livers accumulated more genetic damage on mtDNA than the control group. Furthermore, for the first time, we present evidence in hepatocellular carcinoma-bearing non-cirrhotic livers of a significantly reduced pool of regeneratively active liver cells that are genetically and functionally altered. The analogy to ageing-related changes is suggestive of premature ageing of stem cells in non-cirrhotic hepatocellular carcinoma-bearing liver as an early step to hepatocarcinogenesis.