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Cholangiocarcinoma (CCA) is a rare biliary tract tumor with poor prognosis that often is challenging to diagnose and the majority of patients present with advanced stage. Squamous cell carcinoma antigen 1 (SCCA1) overexpression has been found in different tumors associated with poor prognosis and chemoresistance.To assess the presence and possible prognostic role of SCCA1/2 isoforms in bile and serum of patients with CCA.Forty seven surgical patients (36 with CCA and 11 with benign diseases) were prospectively included in the study. Serum and bile specimens were collected at the time of surgery and free and IgM-complexed SCCA was quantified by ELISA (Xeptagen, srl).Free or IgM linked SCCA was rarely found in serum, while SCCA was detectable in bile samples of patients with CCA, especially in those with extrahepatic form (43% vs 17%, p = 0.008), but not in controls. Despite similar tumor stage, these positive patients presented a trend toward a higher percentage of portal invasion (27% vs 15%) and of tumor recurrence than negative cases (62% vs 40%), although the difference was not statistically significant.These preliminary results indicate that bile testing for SCCA is a specific marker of extrahepatic CCA, with potential prognostic value.

Purpose: We aimed to investigate a cohort of female and male patients with idiopathic central precocious puberty (CPP), negative for Makorin Ring Finger Protein 3 (MKRN3) defect, by molecular screening for Delta-like 1 homolog (DLK1) defects. DLK1 is an imprinted gene, whose mutations have been described as a rare cause of CPP in girls and adult women with precocious menarche, obesity and metabolic derangement. Methods: We enrolled 14 girls with familial CPP and 13 boys with familial or sporadic CPP from multiple academic hospital centers. Gene sequencing of DLK1 gene was performed. Circulating levels of DLK1 were measured and clinical and biochemical characteristics were described in those with DLK1 defects. Results: A novel heterozygous mutation in DLK1, c.288_289insC (p.Cys97Leufs*16), was identified in a male proband, his sister and their father. Age at onset of puberty was in line with previous reports in the girl and 8years in the boy. The father with untreated CPP showed short stature. No metabolic derangement was present in the father except hypercholesterolemia. Undetectable Dlk1 serum levels indicated the complete lack of protein production in the three affected patients. Conclusion: A DLK1 defect has been identified for the first time in a boy, underscoring the importance of genetic testing in males with idiopathic or sporadic CPP. The short stature reported by his untreated father suggests the need for timely diagnosis and treatment of subjects with DLK1 defects.

Context Hypogonadism in Prader-Willi syndrome (PWS) is generally attributed to hypothalamic dysfunction or to primary gonadal defect. MKRN3, a maternal imprinted gene located on 15q11.2-q13 region, encodes makorin ring finger protein 3, whose deficiency causes precocious puberty, an extremely rare symptom in PWS. Objective This study aimed to evaluate MKRN3 levels in patients with PWS and to analyze its correlation with sexual hormone levels, insulin resistance and Body Mass Index (BMI). Methods We performed an observational cross-sectional study and enrolled 80 patients with genetically confirmed diagnosis of PWS with median age of 9.6 years. Results MKRN3 levels were measurable in 49 PWS patients with a geometric mean of 34.9 +/- 22 pg/ml (median: 28.4). Unmeasurable levels of MKRN3 were found in 31 patients. No statistically significant differences were found between patients with and without measurable MKRN3 levels for any clinical, biochemical, or genetic characteristics. However, MKRN3 levels were inversely correlated with HOMA-IR index (p: 0.005) and HbA1c (p: 0.046) values. No statistically significant correlations were found between MKRN3 and LH, estradiol and testosterone concentrations, pubertal development and genetic defect, whereas a direct correlation with FSH was found (p: 0.007). Conclusions The typical genetic defect of PWS should lead to unmeasurable levels of the MKRN3 protein due to the inactivation of the paternal allele. Measurable circulating MKRN3 could suggest the possible involvement of tissue-specific imprinting mechanisms and other regulatory factors in gene expression. Correlations with HOMA-IR index, HbA1c, and FSH suggest peripheral actions of MKRN3, but future studies are warranted to investigate this topic.

Dexmedetomidine is the sedative agent administered in combination with remifentanil and low dose of sevoflurane in the interventional arm of the ongoing TREX trial (Trial Remifentanil DExmedetomidine). The TREX pilot study (published in Paediatr Anaesth 2019;29:59-67) established infusion rates higher than those initially proposed. This could be attributed to an inappropriate target concentration for sedation or incorrect initial pharmacokinetic parameter estimates.The TREX study is a Phase III, randomized, active controlled, parallel group, blinded evaluator, multicentre, superiority trial comparing neurological outcome after standard sevoflurane anaesthesia with dexmedetomidine/remifentanil and low dose sevoflurane anaesthesia in children aged less than 2 years undergoing anaesthesia of 2 hours or longer. In this report, dexmedetomidine pharmacokinetics were analysed in the interventional arm of the Italian population.There were 162 blood samples from 32 infants (22 male and 10 female). The median (IQR) age was 12 (5.2-15.5) months, weight 9.9 (7.3-10.8) kg. Duration of anaesthesia ranged from 2-6 hours. None of the children were born premature (median postnatal age 39 weeks, IQR 38-40 weeks). A 3-compartment PK model that incorporated allometric scaling and a maturation function demonstrated plasma concentration observations from the current Italian arm of the TREX study were consistent with those predicted by a "universal" model using pooled data obtained from neonates to adults.This current PK analysis from the Italian arm of the TREX study confirms that plasma concentration of dexmedetomidine is predictable using known covariates such as age and size. The initial target concentration (0.6 μg.L

Copper is a trace element essential to cellular function with elevated levels implicated in cancer progression. Clinical trials using copper chelators are associated with improved patient survival, however, the molecular mechanisms by which copper depletion inhibits tumor progression are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Epithelial-mesenchymal transition (EMT) is often exploited by malignant cells to promote growth and metastasis. Transforming growth factor (TGF)-β is a master regulator of EMT and facilitates cancer progression through changes in the tumor and its microenvironment. Herein, we report that a reduction of copper with the chelating agent tetraethylenepentamine (TEPA) inhibited EMT in vitro in three diverse cancer cell types; human triple-negative breast cancer (TNBC), neuroblastoma (NB), and diffuse intrinsic pontine glioma (DIPG) cell lines. Single-molecule imaging demonstrated EMT markers including Vimentin, β-catenin, ZEB1, and p-SMAD2 had increased expression with copper treatment and this pro-mesenchymal shift was rescued by the addition of TEPA. Moreover, SNAI1, ZEB1, and p-SMAD2 demonstrated increased accumulation in the cytoplasm after treating with TEPA. Transcriptomic analyses revealed a significant downregulation of the EMT pathway, including canonical (TGF-β/SMAD2&3) and non-canonical (TGF-β/PI3K/AKT and TGF-β/RAS/RAF/MEK/ERK) TGF signaling pathways. Matrix metalloproteinases MMP-9 and MMP-14 proteins which activate latent TGF-β complexes were also downregulated by TEPA treatment. These molecular changes are consistent with reduced plasma levels of TGF-β we observed in cancer models treated with TEPA. Importantly, copper chelation reduced metastasis to the lung in a TNBC orthotopic syngeneic mouse model. Our studies suggest copper chelation therapy can be used to inhibit EMT-induced metastasis by targeting TGF-β signalling. Because on-target anti-TGF-β therapies are failing in the clinic, copper chelation presents itself as a potential therapy for targeting TGF-β in cancer.

BackgroundLegionella can cause Legionnaires’ disease, a potentially fatal form of pneumonia that occurs as sporadic epidemics. Not all strains display the same propensity to cause disease in humans. Because Legionella pneumophila serogroup 1 is responsible for >85% of infections, the majority of studies have examined this serogroup, but there are 3 commonly used laboratory strains: L pneumophila serogroup 1 Philadelphia (Phil-1)-derived strains JR32 and Lp01 and 130b-derived strain AA100.MethodsWe evaluated the ability of Phil-1, JR32, Lp01, and AA100 to cause disease in guinea pigs.ResultsWe found that, although Phil-1, JR32, and AA100 cause an acute pneumonia and death by 4 days postinfection (100%), strain Lp01 does not cause mortality (0%). We also noted that Lp01 lacks a mobile element, designated p45, whose presence correlates with virulence. Transfer of p45 into Lp01 results in recovery of the ability of this strain to cause mortality, leads to more pronounced disease, and correlates with increased interferon-γ levels in the lungs and spleens before death.ConclusionsThese observations suggest a mechanism of Legionnaires’ disease pathogenesis due to the presence of type IVA secretion systems that cause higher mortality due to overinduction of a proinflammatory response in the host.

The 2-pentadecyl-2-oxazoline (PEA-OXA) is a natural compound with protective action in neuro-inflammation. We have previously shown that PEA-OXA behaves as an α2 adrenergic receptor (α2AR) antagonist and a putative protean agonist on histamine H3 receptors. Recently, neuroinflammation and monoaminergic neurotransmission dysfunction has drawn particular attention in Alzheimer Disease (AD) pathophysiology. In this context, the objective of this study was to investigate the effects of the dual-acting PEA-OXA in an AD-like model in mice. A combined computational and experimental approach was used to evaluate the ability of PEA-OXA to bind α2A-AR subtype, and to investigate the effects of PEA-OXA treatment on neuropathological (behavioural and functional) effects induced by soluble Amyloid β 1-42 (sAβ1-42) intracerebroventricular injection. Computational analysis revealed the PEA-OXA ability to bind the α2A-AR, a pharmacological target for AD, in two alternative poses, one overlapping the Na+ binding site. In vivo studies indicated that chronic treatment with PEA-OXA (10mg/kg, os) restored the cognitive (discriminative and spatial memory) deficits and social impairments induced by sAβ injection. Consistently, electrophysiological analysis showed a recovery of the long-term potentiation in the hippocampus (Lateral Entorhinal Cortex-Dentate Gyrus pathway), while neuroinflammation, i.e., increased pro-inflammatory cytokines levels and microglia cells density were reduced. These data provide the basis for further investigation of the pro-cognitive aptitude of PEA-OXA by proposing it as an adjuvant in the treatment in AD, for which the available pharmacological approaches remain unsatisfactory. Moreover, this study offers new future direction in research investigating the role of α2AR in neuropsychiatric illness and therapies.

Il contributo analizza il rapporto poliedrico della provincia storica di terra di lavoro con la Nazione dall'unificazione al fascismo, momento di dissoluzione della provincia storica .

The prehistoric (< 7 ka) Zaro eruption at Ischia island (Southern Italy) produced a lava complex overlaying a pyroclastic deposit. Although being of low energy, the Zaro eruption might have caused casualties among the neolithic population that inhabited that area of Ischia, and damages to their settlements. A similar eruption at Ischia with its present-day population would turn into a disaster. Therefore, understanding the magmatic processes that triggered the Zaro eruption would be important for volcanic hazard assessment and risk mitigation, so as to improve a knowledge that can be applied to other active volcanic areas worldwide. The main Zaro lava body is trachyte and hosts abundant mafic and felsic enclaves. Here all juvenile facies have been fully characterized from petrographic, geochemical and isotopic viewpoints. The whole dataset (major and trace element contents; Sr-Nd isotopic composition) leads to rule out a genetic link by fractional crystallization among the variable facies. Thus, we suggest that the Zaro mafic enclaves could represent a deep-origin mafic magma that mingled/mixed with the main trachytic one residing in the Ischia shallow magmatic system. The intrusion of such a mafic magma into a shallow reservoir filled by partly crystallized, evolved magma could have destabilized the magmatic system presumably acting as a rapid eruption trigger. The resulting processes of convection, mixing and rejuvenation have possibly played an important role in pre- and syn-eruptive phases also in several eruptions of different sizes in the Neapolitan area and elsewhere in the world., The authors are grateful to Roberto de Gennaro for his precious help in obtaining SEM-EDS-WDS data. This study was carried out in the framework of the GEOPAM Research Group (AGAUR, Generalitat de Catalunya, Ref. 2017 SGR 1494). We acknowledge the technical support of M. Rejas of ICTJA-CSIC Laboratory of Geochemistry labGEOTOP, infrastructure co-funded with resources from the ERDF, European Union, (CSIC08-4E-001, and partner of Project EPOS Implementation Phase, EPOS IP, Grant Agreement no. 676564-EPOS IP Call H2020-IFRADEV-12015-1). The INGV, OV laboratories have been financially supported by the EPOS Research Infrastructure through the contribution of the Italian Ministry of University and Research (MUR). Reviews by Prof. Dr. Gerhard Franz, as well as comments and suggestions by the Editor, greatly contributed to the improvement of the original manuscript and are kindly acknowledged. Funding Open access funding provided by Università degli Studi di Napoli Federico II within the CRUI-CARE Agreement

Mitogen‐activated protein kinases (MAPK) are critical mediators of cellular responses to pathogens and are activated in response to infection, but investigation is difficult in multi‐cell hosts due to developmental lethality of mutations. Mycobacterium marinum (Mm) is an established model for tuberculosis, a disease afflicting nearly one‐third of the world's population. We found that Mm‐infected Caenorhabditis elegans display >80% mortality, but nonpathogenic M. smegmatis cause

Aims: To demonstrate the efficacy of five small molecule compounds for inhibiting the growth of Mycobacterium tuberculosis. To present evidence that these compounds will penetrate into the central nervous system. Study Design: Five small molecule compounds bearing a hydrazide group were synthesized utilizing microwave excitation. These compounds were then placed into tissue culture with Mycobacterium tuberculosis at various concentrations for evaluation of bacterial growth inhibition. Place and Duration of Study: The compounds to be tested were prepared at the University of Nebraska Chemistry Department August 2013. The evaluation of antibacterial activity was determined at the Texas A&M Health Science Center during October to December of 2013. Methodology: Applying microwave excitation for generation of hydrazide groups within the structure of small molecule carboxylic acids, five agents were prepared for evaluation Original Research Article British Journal of Pharmaceutical Research, 4(12): 1535-1551, 2014 1536 of bacterial growth inhibition. These agents were dissolved into tissue culture media at various concentrations. Having various levels of tuberculostatic agents, then tuberculosis bacteria were added to determine level of growth inhibition. Growth inhibition of the bacteria was achieved and measured by compound concentration for comparison and evaluation. Results: Five compounds having a hydrazide functional group greatly inhibited the growth of Mycobacterium tuberculosis. All five agents had molecular weight less than 215 grams/mole and polar surface area of less than 70 Angstroms. Values of Log P ranged from -0.226 to 0.998. Values of Log BB (Log [Cbrain/Cblood]) ranged from -0.711 to 0.525, with a range in central nervous system penetration Cbrain/Cblood of 0.195 to 0.299. All compounds showed zero violations of the Rule of 5. Substantial inhibition of bacterial growth was observed at concentrations as low as 30 micrograms/mL, as measured by optical density and colony forming units. Conclusion: These five hydrazide compounds substantially decreased the proliferation of tuberculosis bacteria at concentrations as low as 30 micrograms/mL. In addition, their physicochemical properties are shown to allow high levels of penetration into the central nervous system.

Infection of the zebrafish with Mycobacterium marinum is regarded as a well-established experimental model to study the pathogenicity of Mycobacterium tuberculosis. Herein, a M. marinum transposon mutant library was screened for attenuated M. marinum phenotypes using a Dictyostelium discoideum assay. In one attenuated mutant, the transposon was located within tesA, encoding a putative type II thioesterase. Thin-layer chromatography analyses indicated that the tesA::Tn mutant failed to produce two major cell wall-associated lipids. Mass spectrometry and nuclear magnetic resonance clearly established the nature of missing lipids as phthioglycol diphthioceranates and phenolic glycolipids, respectively, indicating that TesA is required for the synthesis of both lipids. When injected into the zebrafish embryo bloodstream, the mutant was found to be highly attenuated, thus validating the performance and relevance of the Dictyostelium screen. Consistent with these in vivo findings, tesA::Tn exhibited increased permeability defects in vitro, which may explain its failure to survive in host macrophages. Unexpectedly, virulence was retained when bacteria were injected into the notochord. Histological and ultrastructural studies of the infected notochord revealed the presence of actively proliferating mycobacteria, leading to larval death. This work presents for the first time the notochord as a compartment highly susceptible to mycobacterial infection.

The slow growth rate and genetic intractability of tubercle bacilli has hindered progress toward understanding tuberculosis, one of the most frequent causes of death worldwide. We overcame this roadblock through development of near-infrared (NIR) fluorogenic substrates for β-lactamase, an enzyme expressed by tubercle bacilli, but not by their eukaryotic hosts, to allow real-time imaging of pulmonary infections and rapid quantification of bacteria in living animals by a strategy called reporter enzyme fluorescence (REF). This strategy has a detection limit of 6 ± 2 × 102colony-forming units (CFU) of bacteria with the NIR substrate CNIR5 in only 24 h of incubation in vitro, and as few as 104CFU in the lungs of live mice. REF can also be used to differentiate infected from uninfected macrophages by using confocal microscopy and fluorescence activated cell sorting.Mycobacterium tuberculosisand the bacillus Calmette–Guérin can be tracked directly in the lungs of living mice without sacrificing the animals. Therapeutic efficacy can also be evaluated through loss of REF signal within 24 h posttreatment by using in vitro whole-bacteria assays directly in living mice. We expect that rapid quantification of bacteria within tissues of a living host and in the laboratory is potentially transformative for tuberculosis virulence studies, evaluation of therapeutics, and efficacy of vaccine candidates. This is a unique use of an endogenous bacterial enzyme probe to detect and image tubercle bacilli that demonstrates REF is likely to be useful for the study of many bacterial infections.

Persistence ofMycobacterium tuberculosisin humans represents a major roadblock to elimination of tuberculosis. We describe identification of a locus inM. tuberculosis, mel2, that displays similarity to bacterial bioluminescent loci and plays an important role during persistence in mice. We constructed a deletion of themel2locus and found that the mutant displays increased susceptibility to reactive oxygen species (ROS). Upon infection of mice by aerosol the mutant grows normally until the persistent stage, where it does not persist as well as wild type. Histopathological analyses show that infection with themel2mutant results in reduced pathology and both CFU and histopathology indicate that dissemination of themel2mutant to the spleen is delayed. These data along with growth in activated macrophages and infection of Phox−/−and iNOS−/−mice and bone marrow-derived macrophages suggest that the primary mechanism by whichmel2affects pathogenesis is through its ability to confer resistance to ROS. These studies provide the first insight into the mechanism of action for this novel class of genes that are related to bioluminescence genes. The role ofmel2in resistance to ROS is important for persistence and dissemination ofM. tuberculosisand suggests that homologues in other bacterial species are likely to play a role in pathogenesis.

Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7-hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)-regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI-opmD multidrug efflux pump and genes involved in the synthesis of QS-regulated virulence factors including pyocyanin (phz operon), 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole-related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa.

We recently identified two loci, mel1 and mel2 , that affect macrophage infection by Mycobacterium marinum . The ability of these loci to confer enhanced infection in trans is presumably due to gene dosage effects since their presence on plasmids increases expression from five- to eightfold. Reasoning that this phenomenon would allow identification of other mycobacterial genes involved in macrophage infection, we conducted a screen of an M. marinum DNA library that provides 2.6-fold coverage of the entire genome for clones that affect macrophage infection. Our preliminary screen identified 76 plasmids that carry loci affecting macrophage infection. We eliminated plasmids that do not confer the expected phenotype when retransformed (70%), that have identical physical maps (5%), or that carry either of the mel1 or mel2 loci (14%) from further consideration. Four loci that confer enhanced infection ( mel ) and four that confer repressed infection ( mrl ) of macrophages were identified, and two of each group were chosen for detailed analysis. Saturating transposon mutagenesis was used to identify the loci responsible, and M. marinum mutants were constructed in the genes involved. We expect these genes to provide insight into how mycobacteria parasitize macrophages, an important component of innate immunity.

Primary autosomal recessive microcephaly (MCPH) is a developmental disorder characterized by prenatal onset of abnormal brain growth. MCPH occurs both alone and as part of a broad range of neurodevelopmental syndromes with or without cortical malformations and growth retardation. Here we report a consanguineous Moroccan family with two siblings affected by severe primary microcephaly, failure to thrive, congenital dermatitis and severe developmental delay. Brain magnetic resonance imaging showed lissencephaly of frontal lobes and periventricular heterotopia of the gray matter. We performed both Comparative Genomic Hybridization array and whole exome sequencing (WES) analyses of the kindred. No quantitative defects were detected. However, WES identified a new homozygous missense variation in the penultimate nucleotide of exon 23 of RTTN gene (c.2953AG;pArg985Gly). cDNA sequencing revealed two abnormal spliced products, one lacking only exon 23 and the other lacking exons 22 and 23 (out-of-frame). RTTN is a protein involved in cilia structure and function. Homozygous mutations in RTTN gene have been described in bilateral diffuse isolated polymicrogyria and, more recently, in microcephalic primordial dwarfism (PD). We found a novel homozygous mutation in RTTN associated with microcephalic PD as well as complex brain malformations and congenital dermatitis, thus expanding the phenotypic spectrum of both RTTN-associated diseases and ciliary dysfunction.

Forensic odontology is the branch of dentistry that applies dental science to the law. Legal investigation involving dentistry may include human identification, oro-facial trauma, and dental malpractice. In many countries identification of the deceased is both a legal requirement to progress estate management and a social obligation to allow closure for relatives. In single and multiple fatality incidents odontology can assist authorities assigning identity to deceased persons. This chapter outlines the basis for dental involvement in identifying the dead and presents key concepts for each stage of the dental identification process.

Summary Whole-transcriptome analysis was used here for the first time in the rhizosphere to discern the genes involved in the pathogenic response of Pseudomonas aeruginosa PAO1 as well as to discern the response of the poplar tree. Differential gene expression shows that 185 genes of the bacterium and 753 genes of the poplar tree were induced in the rhizosphere. Using the P. aeruginosa transcriptome analysis, isogenic knockout mutants, and two novel plant assays (poplar and barley), seven novel PAO1 virulence genes were identified (PA1385, PA2146, PA2462, PA2463, PA2663, PA4150 and PA4295). The uncharac- terized putative haemolysin repressor, PA2463, upon inactivation, resulted in greater poplar virulence and elevated haemolysis while this mutant remained competitive in the rhizosphere. In addition, disrup- tion of the haemolysin gene itself (PA2462) reduced the haemolytic activity of P. aeruginosa, caused less cytotoxicity and reduced barley virulence, as ex- pected. Inactivating PA1385, a putative glycosyl transferase, reduced both poplar and barley viru- lence. Furthermore, disrupting PA2663, a putative membrane protein, reduced biofilm formation by 20-fold. Inactivation of PA3476 (rhlI) increased viru- lence with barley as well as haemolytic activity and cytotoxicity, so quorum sensing is important in plant pathogenesis. Hence, this strategy is capable of elu- cidating virulence genes for an important pathogen.

The ability to infect macrophages is a common characteristic shared among many mycobacterial species.Mycobacterium avium,Mycobacterium tuberculosis, andMycobacterium kansasiienter macrophages, using the complement receptors CR1, CR3, CR4, and the mannose receptor. To identifyM. aviumgenes and host cell pathways involved in the bacterial uptake by macrophages, we screened aM. aviumtransposon mutant library for the inability to enter macrophages. Uptake-impaired clones were selected. Sequence of sixM. aviumclones identified one gene involved in glycopeptidolipid biosynthesis, one gene encoding the conserved membrane protein homologue to theM. aviumsubsp.paratuberculosis MAP2446cgene and four others belonging to the same region of the chromosome. Analysis of the chromosome region revealed a pathogenicity island inserted between two tRNA sequences with 58% of G+C content versus 69% in theM. aviumgenome. The region is unique forM. aviumand is not present inM. tuberculosisorM. paratuberculosis. Although the mutants did not differ from the WT bacterium regarding the binding to macrophage cell membrane, analysis of macrophage proteins after 1 h infection revealed a deficiency in the mutant to phosphorylate certain proteins on uptake. To understandM. aviuminteraction with two evolutionarily distinct hosts, the mutants were evaluated forAcanthamoeba castellaniiinvasion. The defect in the ability of the mutants to invade both cells was highly similar, suggesting thatM. aviummight have evolved mechanisms that are used to enter amoebas and human macrophages.

Two dipeptide drugs are synthesized utilizing an acetylsalicylic acid or nicotinic acid molecule for the framework. A D-alanine-D-alanine dipeptide moiety is attached to the carbonyl carbon of acetylsalicylic acid (I) and nicotinic acid (II). Dipeptide derivatives (I) and (II) showed significant reduction of Escherichia coli (E. coli) bacterial growth and colony-forming units. A mixture of (I) and (II) induced growth inhibition of 8%, 17.5%, 28%, and 42.5% at concentrations of 100, 200, 300, and 400 microg/mL, respectively. Ampicillin demonstrated much less growth inhibition of this penicillin-resistant E. coli bacteria. Derivatives (I) and (II) showed significant reduction of colony-forming units at concentrations higher than 200 microg/mL, whereas ampicillin showed no significant affect on colony-forming units. Both (I) and (II) produced no violations of the Rule of 5, indicating favorable characteristics for bioavailability. Molecular properties were determined and showed (I) to have a Log Kow of -0.22 with aqueous solubility of 683.8 mg/L, whereas (II) had a Log Kow of -1.00 and aqueous solubility of 6859 mg/L. A mixture of the parent compounds acetyl salicylic acid and nicotinic acid demonstrated some antibacterial activity.

Two derivatives of cephalothin, compound I and II, were synthesized and demonstrated strong growth inhibition of ampicillin resistant Escherichia coli (E. coli). Compound I is the propyl ester of the parent cephalothin antibiotic, while compound II is the butyl ester derivative. The ester substituent replaces the former carboxyl group of cephalothin. Compounds I and II are stable at room temperature and have increased lipophilicity compared to cephalothin due to the presence of the ester substituent. The MIC50 of I and II were determined to be 55 microg/mL and 30 microg/mL, respectively. Cephalothin showed less than 20% growth inhibition of E. coli at all concentrations based on assay of colony forming units. Compounds I and II showed greater than 50% growth inhibition of E. coli at all concentrations greater than 50 microg/mL (more than 65% at 400 microg/mL). Pharmacological properties such as octanol/water partition Log P and polar surface area were determined. Values for polar surface area suggested 35% of I or II present in the intestinal system would be absorbed. The Log P values for I and II are 2.061 and 2.62, respectively, which indicate I and II will penetrate the blood-brain barrier more effectively than cephalothin. The lipophilic substituent constant (pi) for I and II are 1.592 and 1.95, respectively, which indicates the ester substituents contribute a strong lipophilic trait. Properties of molar refractivity, parachor, and molar volume, which describe van-der-Waals interactions, are also determined.

Macrophages produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) in response to bacterial infections. Mycobacteria are relatively resistant to ROS, but RNS inhibit growth of, and possibly even kill, mycobacteria in activated macrophages. We recently constructed a Mycobacterium marinum mel2 locus mutant, which is known to affect macrophage infection. We found previously that the mel2 locus confers resistance to ROS and RNS in laboratory medium, suggesting that this locus might play a similar role during growth in macrophages. Since J774A.1 murine macrophages produce high levels of ROS and RNS upon activation with gamma interferon (IFN-γ), we examined the effects of IFN-γ on ROS and RNS production by these cells as well as the effects on growth of M. marinum in these cells. We found that an M. marinum mutant with mutation of the first gene in the mel2 locus, melF , is defective for growth in IFN-γ-plus-lipopolysaccharide-treated J774A.1 cells and that this defect is abrogated by the presence of either inhibitors of nitric oxide synthase or ROS scavengers. Furthermore, the M. marinum melF mutant displays a defect at late stages in the mouse footpad model of infection. These phenotypic characteristics could be complemented fully by the entire mel2 locus but only partially by the presence of melF alone, supporting data suggesting that this insertion mutation has polar effects on downstream genes in the mel2 locus. These observations demonstrate that the M. marinum mel2 locus plays a role in resistance to ROS and RNS produced by activated macrophages.

The slow growth of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP) expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: tdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-tdTomato displayed the highest fluorescence. We used the tdTomato-labeled M. bovis BCG to obtain real-time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-tdTomato and Hsp60-tdTomato revealed that L5-tdTomato carried four-fold more tdTomato gene copies than Hsp60-tdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-tdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.

Floods are becoming a very huge problem in most developed and developing countries, due to natural and human-induced risks and threats, which occur at river areas. In many cases, catastrophic events are caused by some inadequate human behaviour connected to, inter alia, reduction of the natural water retention by land due to industrial purposes or exploitation of meadows along river basins; no maintenance of levees; not respecting hydrogeological equilibrium; or not considering technical prescriptions or provisions to build along river basins. The adoption of flood risk management and planning in flood-prone areas are priorities to be dealt with in many river areas, as the United Nations and European Union stated. In this context, law plays a fundamental role to identify protection structures and liabilities in river areas, to define regulations and provisions in all stages of risk management. This research is aimed at showing how law intervenes in flood risk management and which subjects are covered; the importance to assess legal provisions which influence flood management or more generally water and river basin management and the interconnection at international, national and local level.

Mycobacterium marinum is an important pathogen of humans, amphibians and fish. Most pathogenic mycobacteria, including M. marinum, infect, survive and replicate primarily intracellularly within macrophages. We constructed a transposon mutant library in M. marinum using Tn5367 delivered by phage transduction in the shuttle phasmid phAE94. We screened 529 clones from the transposon library directly in macrophage infection assays. All clones were screened for their ability to initially infect macrophages as well as survive and replicate intracellularly. We identified 19 mutants that fit within three classes: class I) defective for growth in association with macrophages (42%), class II) defective for macrophage infection (21%) and class III) defective for infection of and growth in association with macrophages (37%). Although 14 of the macrophage infection mutants (Mim) carry insertions in genes that have not been previously identified, five are associated with virulence of mycobacteria in animal models. These observations confirm the utility of mutant screens directly in association with macrophages to identify new virulence determinants in mycobacteria. We complemented four of the Mim mutants with their M. tuberculosis homologue, demonstrating that secondary mutations are not responsible for the observed defect in macrophage infection. The genes we identified provide insight into the molecular mechanisms of macrophage infection by M. marinum.

Mycobacterium marinum is closely related to Mycobacterium tuberculosis, the cause of tuberculosis in humans. M. marinum has become an important model system for the study of the molecular mechanisms involved in causing tuberculosis in humans. Through molecular genetic analysis of the differences between pathogenic and nonpathogenic mycobacteria, we identified two loci that affect the ability of M. marinum to infect macrophages, designated mel1 and mel2. In silico analyses of the 11 putative genes in these loci suggest that mel1 encodes secreted proteins that include a putative membrane protein and two putative transglutaminases, whereas mel2 is involved in secondary metabolism or biosynthesis of fatty acids. Interestingly, mel2 is unique to M. marinum and the M. tuberculosis complex and not present in any other sequenced mycobacterial species. M. marinum mutants with mutations in mel1 and mel2, constructed by allelic exchange, are defective in the ability to infect both murine and fish macrophage cell lines. These data suggest that the genes in mel1 and mel2 are important for the ability of M. marinum to infect host cells. Although mycobacteria were among the first organisms associated with disease in humans (19, 23), they remain possibly the most important cause of death due to a single infectious agent throughout the world. Possible reasons for this are the relative difficulty of manipulating mycobacteria in the laboratory and the relatively low growth rate (20-h generation time) of the most important mycobacterial species, Mycobacterium tuberculosis. Investigators have sought appropriate models that are both relevant and easy to manipulate to speed progress in tuberculosis research. Recently, there has been a great deal of interest in Mycobacterium marinum as a model for study of M. tuberculosis pathogenesis because of its ease of genetic manipulation (2, 17, 33, 37), close genetic relationship to M. tuberculosis (36, 42, 46), relatively high growth rate (4-h generation time) (10), and the presence of a number of useful laboratory models for in vitro (16) and in vivo (14, 34, 38, 45) virulence studies. Pathogenic mycobacterial species, such as M. tuberculosis, differ from nonpathogenic species, such as Mycobacterium smegmatis, in that they invade mammalian cells more efficiently (6, 13, 39), block lysosomal fusion (4, 20), and replicate well in eukaryotic cells (29, 40, 47, 48). Investigators have taken advantage of these differences to identify the genes involved in host cell interactions by cloning genomic DNA from pathogenic species into nonpathogenic mycobacterial species (6, 29, 47, 48). Although these studies have resulted in identification of genes that are potentially important, further analyses have been slowed by the fact that they were isolated from slowgrowing pathogenic mycobacterial species. As a result, no strains have been constructed with mutations in these genes, and it remains unclear whether mutants would be defective for host cell interactions. Our group recently found that the relatively rapidly growing pathogenic mycobacterium M. marinum also differs from nonpathogenic mycobacteria in its ability to invade, prevent lysosomal fusion, and replicate in macrophages (16). However, no genetic analyses of these differences in host cell interactions have been carried out. In the present study we further characterized the differences between M. marinum and M. smegmatis at the cellular and molecular level in order to better understand the mechanisms of host cell interaction by pathogenic mycobacteria. We observed significant differences in the abilities of M. marinum to adhere to and invade mammalian monocytic cell lines. Using the ability to infect the human monocytic cell line THP-1 as a selection, we identified two loci that confer enhanced host cell infection and designated them mel1 and mel2 for “mycobacterial enhanced infection loci. ” All of the genes in these loci are present in M. tuberculosis in nearly the same gene order. In addition, mel2 appears to be present only in M. marinum and M. tuberculosis complex. We constructed a specific M. marinum mutation in each of these loci by allelic exchange and demonstrated that the resulting mutants were defective in the ability to infect both fish and murine macrophages. These data indicate that mel1 and mel2 are involved in the ability of M. marinum to infect macrophages and support the use of M. marinum as a model to speed progress in molecular analysis of mycobacterial virulence mechanisms.

Legionella pneumophila infects humans, causing Legionnaires' disease, from aerosols generated by domestic and environmental water sources. In aquatic environments L. pneumophila is thought to replicate primarily in protozoa. A 'repeats in structural toxin' (RTX) gene, rtxA, from L. pneumophila was identified recently that plays a role in entry and replication in human macrophages and also has the ability to infect mice. However, the role of this gene in the interaction of L. pneumophila with environmental protozoa and its distribution in different Legionella species has not been examined. Southern analyses demonstrated that rtxA is present in all L. pneumophila isolates tested and correlates with species that have been shown to cause disease in humans. To evaluate the importance of rtxA in the interaction with protozoa a series of studies was carried out in an environmental host for L. pneumophila, Acanthamoeba castellanii. The L. pneumophila rtxA gene plays a role in both adherence and entry into A. castellanii similar to that observed in human monocytic cells. Furthermore, it was found that rtxA is involved in intracellular survival and trafficking. In addition to demonstrating involvement of rtxA in the interaction of L. pneumophila with host cells, these data support a role for this gene both during disease in humans and in environmental reservoirs.

Legionnaires' disease is a potentially lethal pneumonia that is primarily due to infection by the species Legionella pneumophila , although more than 40 other species are known. Certain L . pneumophila subgroups, particularly serogroup 1, are associated with the majority of the epidemics. The genetic bases for these differences in virulence have not been determined. Three strains, AA100, JR32, and Lp01, have been used in many molecular pathogenesis studies of L . pneumophila . We found genetic differences between these strains by PCR and Southern analyses that may be related to their ability to cause disease. We also examined the distribution of these genetic loci in clinical and environmental isolates of Legionella and found a correlation between the presence of two of these loci, rtxA and lvh , and the ability to cause disease in humans. Examination of the interactions of these strains with host cells suggested that they differ in important phenotypic characteristics including adherence, entry, and intracellular replication. Furthermore, in the mouse model of infection they display differing levels of replication in lungs. These studies emphasize the importance of further investigation into the genetic makeup of these strains, which is likely to lead to the identification of additional factors involved in Legionella pathogenesis.

We have developed Random Inducible Controlled Expression (RICE), a high throughput genetic approach to identify regulated virulence pathways in pathogenic mycobacteria. RICE allows expression of bacterial genes under conditions where they are normally off, e.g. under laboratory growth conditions, via the use of an inducible or constitutive promoter as well as gene dosage effects due to the presence of the gene on a plasmid. Mycobacterial genomic DNA can be digested to yield random fragments for cloning into a suicide expression vector downstream of a mycobacterial promoter or with their own promoter on a replicating plasmid increasing expression by gene dosage effects. The plasmid DNA is normally amplified in Escherichia coli and delivered into mycobacteria to select for recombinants or plasmid transformants. The resulting library is then directly screened for enhanced host cell interactions in functional assays that evaluate the efficiency of adherence, entry and replication inside host cells. This approach has resulted in identification of several virulence factors from pathogenic mycobacteria. Our analysis of one such locus identified by RICE, the mycobacterial enhanced entry locus (mel2), found that the genes present facilitate bacterial persistence inside the host by protecting the pathogen against oxidative damage. Thus, we have developed a genetic strategy that offers several advantages: (i) it allows identification of bacterial genetic elements that have a direct role during host-pathogen interactions (ii) it can be used to identify virulence factors in a broad range of pathogens and (iii) it can reveal genes that are only induced at specific stages of infection.

The Glu167Lys (E167K) transmembrane 6 superfamily member 2 (TM6SF2) variant has been associated with liver steatosis, high alanine transaminase (ALT) levels and reduced plasma levels of liver-derived triglyceride-rich lipoproteins.The objectives of this study were to investigate in a group of obese children the association among the 167K allele of TM6SF2 gene and ALT, cholesterol and triglycerides levels, and hepatic steatosis, and to evaluate the potential interaction between this variant and the I148M patatin like phospholipase 3 gene (PNPLA3) polymorphism on liver enzymes.We genotyped 1010 obese children for TM6SF2 E167K and PNPLA3 I148M polymorphisms. Anthropometrical and biochemical data were collected. Ultrasound imaging of the liver was performed.The 167K allele showed an association with steatosis (P 0.0001), higher ALT levels (P 0.001) and lower total cholesterol (P 0.00001), low-density lipoprotein cholesterol (P 0.0001), triglycerides (P = 0.02) and non-high-density lipoprotein cholesterol levels (P 0.000001). The subjects homozygous for the PNPLA3 148M allele carrying the rare variant of TM6SF2 showed an odds ratio of 12.2 (confidence interval 3.8-39.6, P = 0.000001) to present hypertransaminasaemia compared with the remaining patients.Although the TMS6SF2 E167K variant predisposes the obese children to non-alcoholic fatty liver disease, there is an association between this variant and lower levels of cardiovascular risk factors. Overall, the data suggest differential effects of TMS6SF2 E167K variant on liver and heart health.

Background This study aimed to assess the early caries experience and the efficacy of a community based dental referral pathway in preschool refugees in Western Australia. Methods Preschool refugee children referred to the Western Australian paediatric hospital Refugee Health Clinic were prospectively screened for caries by a paediatric dentist before being referred to community dental clinics. Dental forms and medical records were audited to assess decayed, missing and filled teeth (dmft), medical data and dental services engagement. Poisson regression analysis determined the contribution of count variables to the final model. Results Among the 105 screened children (54% male, median age 3.2 years, 41% Burmese), community dental clinic engagement was low (46%, n = 48). Of the 62% with caries (n = 65/105, mean dmft 5.2, SD 4.1), 45% were recommended for specialist dental services and 48% were treated. After adjustment for age, gender and total number of teeth, caries incidence was significantly associated with BMI-for-age Z score (p = 0.02). Conclusions Preschool refugee caries burden was high. The community dental referral pathway was ineffective compared to co-located intersectorial dental screening. Specialist dental service needs are high in this cohort and require a targeted approach.

Successful parasitism of host cells by intracellular pathogens involves adherence, entry, survival, intracellular replication, and cell-to-cell spread. Our laboratory has been examining the role of early events, adherence and entry, in the pathogenesis of the facultative intracellular pathogenLegionella pneumophila. Currently, the mechanisms used byL. pneumophilato gain access to the intracellular environment are not well understood. We have recently isolated three loci, designatedenh1,enh2, andenh3, that are involved in the ability ofL. pneumophilato enter host cells. One of the genes present in theenh1locus,rtxA, is homologous to repeats in structural toxin genes (RTX) found in many bacterial pathogens. RTX proteins from other bacterial species are commonly cytotoxic, and some of them have been shown to bind to β2integrin receptors. In the current study, we demonstrate that theL. pneumophila rtxAgene is involved in adherence, cytotoxicity, and pore formation in addition to its role in entry. Furthermore, anrtxAmutant does not replicate as well as wild-typeL. pneumophilain monocytes and is less virulent in mice. Thus, we conclude that the entry genertxAis an important virulence determinant inL. pneumophilaand is likely to be critical for the production of Legionnaires' disease in humans.

The incidence of extrapulmonary tuberculosis is increasing, possibly due to the high frequency of co-infection with HIV. Extrapulmonary infections complicate diagnosis, have higher mortality rates and are more difficult to treat. Insight into the mechanisms involved in extrapulmonary spread of tuberculosis is critical to improving management. We set out to better understand extrapulmonary spread kinetics in mice and guinea pigs as well as the effects of infectious dose. We found that extrapulmonary spread occurs at a discrete time point when infected by low-dose aerosol, but at high-dose aerosol it occurs within the first 24 h. The ability to follow tuberculosis in real-time during infection would allow us to better address the mechanisms involved. We found that mycobacteria can be optically imaged after pulmonary infection in the mouse lung, suggesting that this technology could be applied to study of extrapulmonary spread of tuberculosis.

Since Legionella pneumophila is an intracellular pathogen, entry into and replication within host cells are thought to be critical to its ability to cause disease. L. pneumophila grown in one of its environmental hosts, Acanthamoeba castellanii , is phenotypically different from L. pneumophila grown on standard laboratory medium (BCYE agar). Although amoeba-grown L. pneumophila displays enhanced entry into monocytes compared to BCYE-grown bacteria, the mechanisms of entry used and the effects on virulence have not been examined. To explore whether amoeba-grown L. pneumophila differs from BCYE-grown L. pneumophila in these characteristics, we examined entry into monocytes, replication in activated macrophages, and virulence in mice. Entry of amoeba-grown L. pneumophila into monocytes occurred more frequently by coiling phagocytosis, was less affected by complement opsonization, and was less sensitive to microtubule and microfilament inhibitors than was entry of BCYE-grown bacteria. In addition, amoeba-grown L. pneumophila displays increased replication in monocytes and is more virulent in A/J, C57BL/6 Beige, and C57BL/6 mice. These data demonstrate for the first time that the intra-amoebal growth environment affects the entry mechanisms and virulence of L. pneumophila .