The Natural Product Discovery Institute contains a set of more than 30 000 fungal and actinomycete organisms that can be used for natural product discovery.The natural product library contains purified compounds, partially purified column fractions, and crude extracts from natural product‐producing organisms. The collection is available for screening.This site describes a roadmap for analysing the genomes of Actinobacteria for natural product biosynthetic pathways.In this study, data from the large‐scale sequencing of actinomycetes were mined to uncover new metabolic pathways for making phosphonic acid natural products. Biosynthetic phosphonic acids are often metabolite mimics that have interesting biological properties.Microbes in the human gut are known to produce a large array of natural products and mining the genomes provides a pathway to new discovery and delineating differences between microbial populations in different people.Recent papers relating to genomics and natural products are discussed in this article.This paper deals with the discovery of a new class of di‐ and trichlorinated acyl amides with cannabinomimetic activity.This announcement of an industrial cooperation illustrates the current interest in commercial genome mining for natural product discovery surrounding agricultural applications.This review article deals with the mining of metagenomic data for natural products that are generated by microbes from marine environments.This website provides a resource for submitting genome sequences to identify secondary‐metabolite biosynthetic gene clusters.This database contains the structures, properties and commercial sources for over 300,000 natural products.This database contains structures and information on over 225,000 natural products to be used for virtual screening.This multi‐functional database is composed of sub‐databases. One of those deals with small molecules important in Chinese medicine for virtual screening. Another database is valuable for docking and screening for drug development.This database allows for the identification of molecules based on LC‐MS/MS chromatograms.This review details the value of genomic data for natural product identification.This paper describes PRISM, a computational resource for identifying natural product biosynthetic clusters, particularly polyketide clusters.Warp Drive Bio formed to mine the genomes of actinomycetes for novel natural product clusters and identify a new generation of pharmaceuticals.


Computational fluid simulation can support design of bioreactor systems for tissue engineering application.A three-dimensional computational fluid dynamics- (CFD-) model based on a differential pressure laminar flow bioreactor prototype was developed to further examine performance under changing culture conditions. Cell growth inside scaffolds was simulated by decreasing intrinsic permeability values and led to pressure build-up in the upper culture chamber. Pressure release by an integrated bypass system allowed continuation of culture. The specific shape of the bioreactor culture vessel supported a homogenous flow profile and mass flux at the scaffold level at various scaffold permeabilities. Experimental data showed an increase in oxygen concentration measured inside a collagen scaffold seeded with human mesenchymal stem cells when cultured in the perfusion bioreactor after 24 h compared to static culture in a Petri dish (dynamic: 11% O Bioreactor designs for bone tissue engineering applications often use perfusion models where fluid shear stresses through porous scaffolds activate biological processes such as cell proliferation and differentiation –5. The s in vitro, a reduced oxygen tension has been accompanied by an increased proliferation rate and a decreased differentiation capacity of stem and osteoprogenitor cells compared to normoxic culture conditions  was multiplied by the viscosity of the fluid  to get the unit [kg∗m−3∗s−1]. The porous inertial resistance [m−1] was divided by 2 and multiplied with the fluid density  to convert to the unit [kg∗m−4].For unit conversion, the porous viscous resistance  and is given byρ is the density of the fluid, a is the area, and v velocity of the flow (as a component of magnitude).The mass flux is the rate of mass flow per unit area [kg2 and expanded in standard culture medium ), supplemented with 5% fetal calf serum, antibiotics, sodium pyruvate, and nonessential amino acids . Stem cells were continuously tested for multipotency and stem cell surface markers [For biological experiments, human adipose-derived mesenchymal stem cells were derived from fat tissue donors undergoing abdominoplasty after informed consent and in accordance with the guidelines of the Ethical Committee of Hannover Medical School. The full procedure followed standard protocols and has been described elsewhere . Mesench markers .μm diameter), and uses a two-frequency modulation technique to eliminate interference by background fluorescence [The bioreactor was equipped with a laser-based oxygen measurement system called “OPAL,” which measures oxygen-dependent phosphorescence lifetime of microbeads  to the culture medium in order to maintain a stable pH. At the end of experiments, scaffolds were fixed in formalin-based solution and processed for microscopy analysis .For experiments, cylindrical collagen scaffolds were prepared with phosphorescent microbeads which were placed centrally inside the scaffolds and fixated with 20 The laminar flow bioreactor and the oxygen sensor system are being presented in ∗ 10−13 and 5 ∗ 10−12 when the bypasses in the bioreactor were closed. All other configuration variants showed a solution which converged.CFD simulations were performed for closed and open bypass systems and scaffolds with different permeability values representing different states of cell growth inside the scaffold as stated in ∗ 10−11 and 63 Pa for a permeability of 5 ∗ 10−12 and increases up to 360 Pa when permeability decreases to 5 ∗ 10−13. Opening the bypass system results in pressure release, and pressure values in the upper and lower part of the culture chamber adjust to a pressure difference of about 3.3 Pa, as seen here in ∗ 10−13.Results of the CFD simulation are presented in Figures −10 down to 10−14 m/s for closed or open bypass configuration and represents global values and not velocities in single pores of the scaffold.Results of flow velocity analysis are shown in −2 s−1) simulation in the bioreactor for closed (a) and open (b) bypass configuration. The results show a homogenous distribution and were in the range between 0.05 and 0.15 kg m−2 s−1 for the various permeability values used for the porous media in the model.Oxygen measurement centrally inside the cell-seeded collagen scaffolds demonstrated a significant difference between statically cultured scaffolds in a Petri dish compared to dynamically cultured scaffolds in the bioreactor as demonstrated in In recent years, computational fluid dynamics has gained more and more interest in the development and optimization of systems for biotechnology such as bioreactors , 40, 41.Results from our simulation underline the importance of pressure control in the system during cell growth inside the porous scaffold causing decrease in permeability. In our current operating prototype, the pressure control system is set up that a change in differential pressure of 1 mbar = 100 Pa activates the opening of the bypasses . The speOur oxygen measurement data from the interior of the cell-seeded scaffold suggest an improvement of oxygen supply towards the cells during perfusion culture in the bioreactor. Further work is necessary in order to study oxygen distribution inside scaffolds during long-term perfusion cultures and determine its influence on cell growth and differentiation inside tissue-engineered constructs.A three-dimensional computational model is presented to characterize flow and pressure distribution inside a perfusion bioreactor prototype. Simulation results underscore the importance of pressure control during changes in porous media permeability caused during cell growth and changes in internal scaffold architecture, which was solved by an internal bypass system for pressure release. Oxygen measurement data suggest an improved oxygen supply for the tissue-engineered constructs in the bioreactor during perfusion culture compared to static cultivation in a Petri dish.


V. cholerae O1 strains of the Hikojima serotype that co–express the Inaba and Ogawa antigens of O1 lipopolysaccharide (LPS). Mutation of the wbeT gene reduced LPS perosamine methylation and thereby gave only partial transformation into Ogawa LPS on the cell surface. The strains express approximately equal amounts of Inaba– and Ogawa–LPS antigens which are preserved after formalin–inactivation of the bacteria. Oral immunizations of both inbred and outbred mice with formalin–inactivated whole–cell vaccine preparations of these strains elicited strong intestinal IgA anti–LPS as well as serum vibriocidal antibody responses against both Inaba and Ogawa that were fully comparable to the responses induced by the licensed Dukoral vaccine. Passive protection studies in infant mice showed that immune sera raised against either of the novel Hikojima vaccine strains protected baby mice against infection with virulent strains of both serotypes. This study illustrates the power of using genetic manipulation to improve the properties of bacteria strains for use in killed whole–cell vaccines.We describe here the development of stable classical and El Tor  Vibrio cholerae bacteria of a single serogroup, O1. Cholera displays a wide spectrum of symptoms from asymptomatic infection to severe dehydrating diarrhea in which patients can purge up to 2 liter of fluid per hour. Despite highly effective fluid rehydration therapies, cholera remains an important global health problem with 3–5 million cases worldwide resulting in approximately 120,000 deaths per year Cholera is an acute, often severe diarrheal disease which can be fatal to both children and adults V. cholerae O1 whole cell vaccines comprising a mixture of strains of Inaba and Ogawa serotypes and El Tor and classical biotypes: one  also contains cholera toxin B–subunit (CTB) and one (Shanchol) lacks CTB but contains a V. cholerae strain of the O139 serogroup . Both vaccines have been shown in several field trials to be well tolerated and confer up to 85% protection against cholera V. cholerae O1 strain co–expressing the Inaba and Ogawa serotype antigens and inactivated by one rather than two methods.In many cholera–endemic countries and in cholera outbreaks following natural or political disasters clean water and basic sanitation measures are lacking and vaccines are increasingly recognized as an important preventive intervention wbeT gene and mutations that inactivate this enzyme such as insertions, deletions or truncation of this gene result in the Inaba serotype The serogroup is determined by the cell wall lipopolysaccharide (LPS), and both Ogawa and Inaba bacteria have a common O1 antigen, referred to as the A–antigen. The difference between the Ogawa and Inaba serotypes lies in the terminal perosamine of the LPS which is methylated in the Ogawa LPS forming the B–antigen, whereas the Inaba LPS is not methylated resulting in a serotype specific C–antigen A third serotype called Hikojima has been described that expresses all three antigens , but this serotype is rare and evidence indicates that it is an unstable transitional serotype observed when a strain undergoes serotype switching from Ogawa to Inaba and does not occur in nature V. cholerae that combines the essential components of the multiple strains used in the currently licensed vaccines. An important aspect of this work has been to generate a stable Hikojima type strain that co–expresses the Ogawa (AB) and Inaba (AC) LPS antigens. We have already reported the generation of one such strain (MS1342) achieved by the insertion into the chromosome of a parent Inaba strain of a wild–type wbeT gene that was stably expressed at low levels Recently we have worked to develop a new generation of killed OCVs based on a single, genetically modified, non–virulent strain of O1 wbeT gene in the constructed strain with a risk for instability due to recombination. In the current paper we describe an alternative approach in which the activity of the endogenous wbeT gene is instead reduced by site–directed mutagenesis. Two further candidate Hikojima strains were constructed: one El Tor and one classical. We demonstrate the genetic and phenotypic stability of these novel strains as well as their ability after formalin inactivation, to elicit strong serum as well as intestinal antibody responses reacting with both Ogawa and Inaba LPS following oral route immunizations in mice.A disadvantage of this approach was the presence of two copies of the Furthermore, passive protection studies showed that immune sera raised against either of the novel Hikojima type whole–cell vaccines protected baby mice against infection with virulent Ogawa as well as Inaba strains.wbeT creating the mutation S158P in the gene product completely destroyed its LPS methylation activity thus resulting in the Inaba serotype. We hypothesized that this site is important for the activity of the wbeT–encoded protein and therefore could be used as a target site for directed mutagenesis in order to generate mutants with reduced activity of the wbeT gene product. A mutation library was constructed in the classical Inaba strain JS1569 as described in the Material and Methods. Fifty different colonies from the mutation library were tested using antibody–based colony blotting. It was clear that the colonies expressed different amounts of Ogawa LPS reflected in different staining intensity from nil to strong. Selected clones from the plate were further analyzed by sequencing the wbeT gene in their plasmids. A typical blot is shown in We determined that a point mutation in s158f mutant was found on the basis of comparison in dot blots with clones expressing the wild type wbeT gene and clones expressing inactive derivatives of wbeT and agglutination assays to result in activity giving the closest balance of Inaba/Ogawa expression this mutation was selected for further experiments. The wbeT gene carrying the s158f encoding mutation together with its native promoter was inserted into the pMT–suicide1–SacB plasmid and conjugated into the classical strain JS1569 and El Tor strain Phil6973. Colonies were selected for chloramphenicol and kanamycin resistance and positive clones were screened for the Hikojima phenotype using agglutination with serotype–specific antisera. Growth on sucrose was used to select for loss of the plasmid and kanamycin resistant strains were screened for expression of the Ogawa antigen. Finally, the kanamycin resistance gene was removed using FLP driven recombination as described in Methods. Three different strains, MS1568 Hikojima and MS1571 Ogawa from Phil6973 and MS1580 Hikojima from JS1569 were generated. The Ogawa strain had the wild type codon TCA (serine) at the position 158 whereas the Hikojima strains had TTT . Both strains from Phil6973 had a wild type codon at position 252 and the strain from JS1569 at position 219, where the respective native strains have stop codons. Both strains proved to have a stable phenotype over more than 100 generations of cultivation. The position of the PCR primers of the wbe region before and after gene manipulation is shown in Since the All vaccine candidates were tested for agglutination using serotype–specific antisera before and after formalin–inactivation as described However, the ca 2–fold variation of this assay did not allow for more precise estimates and in order to obtain a better quantification of the relative amounts of each antigen an ELISA–based method was developed testing the reactivity of purified LPS preparations from the Hikojima strains with Inaba– and Ogawa–specific antisera in comparison with the reactivity of the same antisera with defined mixtures of Inaba and Ogawa LPS in different proportions. Using this assay the strains MS1568 and MS1580 were estimated to have 56±4% and 42±4% (mean ± SEM of 3 tests) of their total LPS being of the Ogawa type, respectively.In order to quantify the relative amount of Ogawa antigen using a non–immunological method, LPS preparations from different Hikojima strains and from reference Inaba and Ogawa strains were analyzed using mass spectrometry. It is known that each Ogawa LPS molecule consists of a chain of perosamine residues in which the terminal perosamine is methylated. In contrast, Inaba LPS lacks any methylated perosamine V. cholerae O1 Ogawa and Inaba target bacteria and by ELISA for IgG+IgM antibodies against Ogawa and Inaba LPS; similar analyses were also performed with serum samples that had been absorbed with formalin–inactivated Inaba or Ogawa bacteria to determine serotype–specific antibodies. In addition, after oral immunizations fecal pellets extracts and small intestinal tissue extracts were tested by ELISA for specific IgA antibody levels.To evaluate the immunogenicity of inactivated whole–cell vaccine preparations of the different Hikojima–type candidate vaccine strains in comparison with the widely licensed Dukoral vaccine, both inbred Balb/c and outbred CD1 mice were immunized orally/intragastrically with equivalent amounts (based on the LPS content) of formalin–killed MS1568 or MS1580 formalin– inactivated whole–cell vaccines or with Dukoral. The formalin inactivated MS1568 vaccine and similarly prepared vaccine from its ancestor strain Phil6973, were also used for intraperitoneal immunizations. Following immunizations, serum samples were analyzed for vibriocidal antibodies against The intended use of a whole–cell cholera vaccine is that by immunization through the oral route it should stimulate local IgA anti–LPS antibody production in the small intestine, the immune response known to mediate most or all of the protective effect Closely similar fecal and intestinal IgA anti–LPS responses were obtained in outbred CD1 mice immunized orally/intragastrically by either two or three rounds with formalin–killed MS1568 vaccine as compared with Dukoral. The fecal and intestinal tissue extract IgA anti–LPS responses to the MS1568 vaccine were fully comparable to those against the Dukoral vaccine after either two or three rounds of immunization, which latter resulted in ca 2– to 20–fold further increased locally produced IgA levels compared to those after two immunization rounds .The oral immunizations with MS1568, MS1580, or Dukoral vaccines also gave rise to strong vibriocidal antibody responses . Again tThese results show that both the El Tor (MS1568) and the classical (MS1580) Hikojima formalin–killed whole cell vaccines were strongly immunogenic when given orally and induce intestinal IgA and serum vibriocidal and IgG+IgM anti–LPS responses that were fully comparable to those induced by the Dukoral vaccine.The protective activity of sera from CD1 mice after oral immunizations with formalin–killed MS1568 or Dukoral vaccines was tested against challenge with virulent isogenic V. cholerae O1 Inaba and Ogawa bacteria, using the infant mouse cholera model. Pooled sera from unimmunized mice served as control. The results are shown in Titrations of vibriocidal antibodies showed that three intraperitoneal immunizations with formalin–killed preparations of MS1568 and Phil6973 gave rise to high antibody titers against both Inaba and Ogawa target strains. In the case of Phil6973 most of these antibodies were cross reactive, since absorption of the immune sera with bacteria of the heterologous serotype reduced the antibody titers against the homologous serotype more then 10–fold. The MS1568 vaccine, however, gave rise to high levels of Ogawa (anti–B) and lower levels of Inaba (anti–C) specific antibodies, the latter being produced at similar low levels by the parental Phil6973 Inaba strain, In developing a new generation of killed whole cell OCVs a central aim is to make it affordable for the populations in greatest need. This can be achieved by simplifying manufacture whilst maintaining the levels of protection afforded by currently available OCVs. In current OCVs the use of several strains and two inactivation methods makes their manufacture complex and relatively expensive. Using a single vaccine strain and one inactivation method would considerably simplify and reduce the cost of manufacture.V. cholerae O1 derivatives co–expressing the Inaba and Ogawa O1 LPS antigens by introducing a weakly expressed wild type wbeT gene into an Inaba strain wbeT genes in Inaba strains of either El Tor or classical biotype V. cholerae, with a mutated wbeT gene giving a single amino acid substitution that reduces the activity of the gene product resulting in only partial methylation of the surface LPS.We have previously shown that it is possible to generate stable and immunogenic Hikojima wbeT gene and carry no genes conferring antibiotic resistance. Apart from the introduced specific mutation in the wbeT gene, the only change from the parental strains is the presence of a 213 base pair insert in the intergenic region between wbeT and wbeV. Both strains are potentially more stable since they carry only a single copy wbeT gene rather than the tandem repeat present in MS1342 and therefore the potential for recombination resulting in either Inaba or Ogawa strains is dramatically reduced.The resulting strains MS1568 (El Tor) and MS1580  have advantages over the previously described wbeT gene would yield the desired level of reduction in LPS methylation activity. We had identified a locus in the wbeT gene at which mutations had a profound effect on the activity of the wbeT encoded protein and employed site–directed mutagenesis in order to generate a library of mutants modifying the amino acid at position 158 of the wbeT gene product. Interestingly, although the mutagenesis worked well, no mutants were isolated in which highly charged residues  occurred at position 158. With the exception of arginine, when mutants with highly charged residues were found, they had additional mutations that introduced stop codons resulting in a truncated inactive product. This suggests that there was selection against these mutants, at least in the E. coli strain into which the library was transformed perhaps due to a harmful methylation activity.In constructing the new strains we had to determine which mutations in the wbeT genes and shown that they conferred differing levels of Ogawa expression, one mutation (S158F) resulting in intermediate methylation activity of O1 LPS was selected for the further work. Using a modified allelic replacement procedure the S158F mutant was used to replace the inactive mutant wbeT genes in two Inaba strains of O1 V. cholerae; one of El Tor biotype  and one of classical biotype (JS1569).Having screened the mutant The generated strains, MS1568 and MS1580 respectively, both stably expressed approximately 50% each of Ogawa and Inaba LPS as tested over more than 100 generations.wbeT gene listed in GenBank from a Hikojima strain (Accession number FJ619106.1). This has a point mutation converting the amino acid at position 158 of the gene product from serine to proline. The same mutation is present in a number of other entries (e.g. Accession number JX565662) where the phenotype is reported as Inaba. We have found the same mutation in additional Inaba strains in our strain collection wbeT mutation has been reported also by other groups as associated with the Inaba serotype V. cholerae stably displaying the Hikojima serotype over several generations has been found.While the serotype Hikojima was originally described for a limited number of clinical isolates, recent evidence indicates that in nature Hikojima is not a stable serotype but rather represents a transition state for Ogawa to Inaba serotype switching To investigate whether the generated MS1568 and MS1580 strains were immunogenic with respect to both Inaba and Ogawa antigens we studied immune responses to these antigens in mice immunized with formalin–killed whole cell MS1568 and MS1580 vaccines and compared the responses to those elicited by immunizations with the Dukoral vaccine . When orally immunized with formalin–killed whole–cell vaccine preparations of the generated Hikojima strains MS1568 or MS1580, all mice gave strong vibriocidal antibody responses in serum effective against Inaba as well as Ogawa test organisms as well as strong serum anti–LPS responses measured by ELISA. Both the vibriocidal and ELISA anti–LPS antibody levels induced by the Hikojima vaccines were fully comparable to those elicited by the Dukoral vaccine. Further, protection studies in infant mice showed that immune serum against the MS1568 strain after oral immunization protected to the same extent against both Ogawa and Inaba challenge and as effectively as serum obtained after similar immunization with Dukoral.Absorption studies were used to further define the epitope specificity of the anti–LPS and vibriocidal antibodies elicited by the different vaccines. Following parenteral immunization with formalin–killed MS1568 and MS1580 bacteria both inbred Balb/c and outbred CD1 mice elicited mainly cross–reactive anti–LPS antibodies as determined by ELISA, but also significant levels of Ogawa–specific and Inaba–specific antibodies most clearly seen in the vibriocidal responses. This distribution of anti–LPS antibody specificities demonstrated that both antigens were present in an immunogenic form in the novel vaccine strains.E. coli (ETEC) We attached particular importance to the ability of the candidate vaccines strains to elicit locally produced intestinal IgA antibodies to LPS, since such antibodies are regarded to be essential for immune protection after oral immunization Our results suggest that either of the Hikojima strains MS1568 and MS1580 could replace the mixture of strains in currently licensed OCVs. However, MS1568 may be preferable since it tended to give slightly higher antibody responses than MS1580 and is of the El Tor biotype which accounts for all cases of cholera today. It also has the advantage that it is derived from Phil6973, which is used as a formalin killed strain in both the Dukoral and Shanchol OCVs and hence has been extensively tested in humans As mentioned, a single strain and a single inactivation method should significantly simplify future manufacture of killed OCVs. Work is now in progress to develop a practical and thermo–stable OCV formulation based on formalin–inactivated MS1568 bacteria combined with a stomach acid protected recombinant cholera toxin B subunit (rCTB) that is significantly cheaper than currently licensed OCVs.From a broader perspective this study demonstrates the value of genetically manipulated bacteria in generating killed whole cell vaccines. Unlike live attenuated vaccine strains the demands on the properties of the living bacteria are not as stringent. Although one of the generated strains (MS1580) is non–toxigenic, the second strain (MS1568) retains the toxin genes which are however not expressed under the growth conditions used for cultivation for use in the vaccine formulation.In conclusion, the current work takes advantage of the advances made in elucidating the immunological basis of protective immunity against cholera to construct novel vaccines strains that can contribute to a novel effective and affordable cholera vaccine that is accessible to all who need it.c. Liquid cultures were grown in LB broth at 37°c as previously described The bacterial strains and plasmids used in the current study are shown in wbeT gene are shown in Primers and oligonucleotides used in the cloning and analysis of the wbeT gene was generated by synthesizing the oligonucleotide, wbeT m1, containing the random codon (NNB) at amino acid position 158. The oligonucleotide was mixed with the primer wbeT m3 for annealing at room temperature for 1 h. A double stranded product was generated by filling in the missing bases using T4 DNA polymerase for 5 min at room temperature in the presence of dNTPs. The enzyme was then inactivated by heating the reaction at 72°C for 10 min. The double stranded product was digested with BstBI and PflMI and ligated into the previously described V. cholerae strain JS1569 to create a mutation library which was used subsequently for the analysis of wbeT expression. The spread plates demonstrated the transformation frequency but also acted as a source of individual transformants that were subsequently analyzed by sequencing to determine the success of the mutagenesis.A library of mutations of the V. cholerae strain JS1569 were isolated and checked for wbeT expression using an antibody based colony blot as previously described Single colonies from the mutation library created in wbeT gene.Colonies expressing different levels of Ogawa antigen were isolated and the inserts in the pML–wbeTtac–S158x plasmids were sequenced in order to determine the amino acid present at position 158 of the cloned V. cholerae strains is shown in wbeT gene was amplified from the appropriate pML–wbeTtac–S158x plasmid with the primers wbeT 1 and wbeT 2. Upstream DNA was amplified from chromosomal DNA from V. cholerae strain vx44945 with the primers wbeT 5 and wbeT 7 (wbeT gene (part of the wbeV gene) from strain vx44945 amplified with primers wbeV litmus 2 and wbeV f. The two resulting fragments were joined to the first generated fragment by primerless pcr essentially as described above (t4 DNA polymerase and ligated into pMT–Suicide1–SacB (GenBank: KF188719.1) plasmid digested with EcoRV. Ligated DNA was transformed into E. coli strain S17–1 and transformants were selected on the basis of their resistance to both kanamycin and chloramphenicol. The insert in plasmids carried by clones with the correct antibiotic resistance were isolated and analyzed first by restriction analysis and then by sequencing to confirm that the sequence was correct. The resulting plasmid was pMT–sucide1–SacB–wbeT(S158x)–Km.A scheme of the steps in the construction of the suicide plasmid used for site–directed mutagenesis of O1 d wbeT 7 . Templated above . The fined above . The casV. cholerae strains Phil6973 and JS1569 by transconjugation achieved by patch mating on LB plates. Cells from these matings were grown on selective plates containing Km/PB for Phil6973 and Km/Ra for JS1569. Single colonies were isolated and checked for a Hikojima phenotype by agglutination with Inaba and Ogawa–specific antisera as described before R) and chloramphenicol sensitivity (CmS) as well as for a Hikojima phenotype. CmS colonies with desired phenotype were sequenced with primer wbeT 87>. A clone carrying the correct sequence was made electro competent and the flipase (FLP)–expressing plasmid 708–FLPe  was introduced, and bacteria grown at 30°C on LB agar plates supplemented with Cm overnight. Clones that were resistant against both chloramphenicol and kanamycin were patched onto a new LB–Cm plate and grown at 37°C overnight. Colonies with the desired Hikojima phenotype that were also KmS were analyzed by PCR amplification of chromosomal DNA with the primers wbeT 5 and wbeV f2. Amplified DNA was sequenced using primer wbeT 87>.This plasmid was then transferred into LPS was isolated from bacteria using the phenol–water extraction method as described 600 reached 1.2. The cells were then harvested by centrifugation  and re–suspended in PBS to an approximate density of 2.5×109 bacteria/ml. To kill the cells formaldehyde  was added to a final concentration of 0.02 M, and the bacterial suspension was incubated for 4 h at room temperature with shaking at 120 rpm in an airtight Erlenmeyer flask. The treated suspension was centrifuged at 10,000×g for 20 min and the resulting pellet was washed three times in PBS and then re–suspended in PBS to an OD600 of 10.0. In order to test for complete inactivation aliquots of the resulting cell suspension were spread onto horse blood agar plates and incubated at 37°C for 48 h.Three colonies from a freshly grown culture on an LB agar plate (with or without antibiotic supplementation as appropriate for different strains) were used to inoculate a starter culture of 25 ml LB medium grown at 37°C with shaking at 180 rpm overnight. Five ml of this starter culture was then used to inoculate 500 ml fresh pre–warmed LB medium which was grown at 37°C with shaking until the ODA mass spectrometric method and two immunological assays were used to quantify the relative amounts of Ogawa and Inaba antigens in LPS preparations from Hikojima strains and in formalin–killed Hikojima vaccine formulations.A mass spectrometric method (MS) was devised to assess the relative amount of Ogawa antigen by determining the proportions of methylated versus non–methylated perosamine in purified LPS preparations from Hikojima strains as compared to reference Inaba and Ogawa LPS preparations. We first removed the lipid A from the LPS as described in detail elsewhere The resulting polysaccharide  was separated on a 200×0.150 mm column with 5 µm polyamine II particles  packed in–house, and eluted with a water gradient . Samples were analyzed on an LTQ linear quadrupole ion trap mass spectrometer (Thermo Electron) by LC–ESI/MS at –3.5 kV. Full–scan  was performed, followed by data dependent MS2 scans  with normalized collision energy of 35%, an isolation window of 2.5 units, an activation q = 0.25, and an activation time of 30 ms. Unmetylated perosamine (m/z ratio 742.6) and methylated perosamine (m/z ratio 756.6) peaks were localized and the respective areas under the curve for these components were calculated and used to determine the proportion of methylated to total (methylated+unmethylated) perosamine see . This raJS1569) purified LPS. The plates were then washed and incubated with an appropriate dilution of either anti–Ogawa (anti–B) specific polyclonal rabbit antiserum To determine the relative amount of Ogawa LPS antigen in the different vaccine strains purified LPS preparations of different strains were examined with Ogawa–specific (anti–B) and serotype cross–reactive (anti–A) antibodies by ELISA as described before An inhibition ELISA performed as described before 9 bacteria).Oral/intragastric and intraperitoneal immunizations were performed in inbred 6–8 week old female Balb/c  or outbred CD1 mice  and collections and preparations of sera, fecal and intestinal extracts were done as described before Eleven days after the last vaccine administration fecal pellets (5 from each mouse) were collected and extracts prepared Directly after bleeding, the sacrificed mice were extensively intravenously perfused with a heparin–PBS solution to maximally remove blood from tissue, where after a defined part of the small intestine was excised and extracted with saponin as described 8 bacteria per dose. Seven days after the last dose the animals were anesthetized with isofluoran, sacrificed, and serum samples prepared and frozen at –20°C until analyzed.In addition to the orally immunized mice, other groups were immunized intraperitoneally on days 0, 8, and 18 with formalin–killed preparations of strains MS1568 and Phil6973, approximately 1×10V. cholerae O1 as previously described Serum, fecal extracts, and small intestine tissue extracts were analyzed for antibodies to V. cholerae O1 Ogawa and Inaba challenge. For the challenge it was desired to use strains where the only difference was the Ogawa and Inaba serotype.The infant mouse cholera model et alV. cholerae. Briefly, the strain was grown in LB broth at 37°C with shaking (180 rpm) and a log phase suspension of approximately 4×106 bacteria/ml was mixed with heat inactivated absorbed polyclonal rabbit anti–Ogawa serum together with guinea pig serum (Biojet Service) as a source of complement and incubated at 37°C for 1 h with shaking. LB broth was then added and the mixture incubated for 3 h at 37°C without shaking before the mixture was plated out on blood agar plates. Single colonies were checked with serum agglutination and selected Inaba strains were sequenced with the primers wbeT 1 and wbeT 2. The strain MS1489 had a mutated wbeT gene and stably expressed the Inaba phenotype as tested by agglutination.A modified method of Bhaskaran V. cholerae bacteria. The challenge experiments were performed, in principle as described previously 6 colony–forming units, cfu) of LB cultured early log phase V. cholerae O1 bacteria pre–incubated for 30 minutes with pooled heat–inactivated immune serum from orally vaccinated or as control unvaccinated CD1 mice diluted 1∶30 in PBS. Each serum pool was tested for protective activity against both a V. cholerae O1 El Tor Ogawa strain (X25049) and its isogenic Inaba strain (MS1489). Inoculated infants were kept separate from dams at 26°C and carefully monitored for 48 hours, after which surviving animals were euthanized. Animals that during the course of the experiments showed clinical signs of server disease i.e. change of skin coloration from pink to blue or grey, labored breathing and persistent recumbency, were defined as moribund and immediately euthanized; but statistically defined as dead one hour later. The proportions of remaining live to total challenged mice  at different time points were calculated, and Kaplan–Meier type survival curves were plotted and compared using the log–rank tests; P–values were adjusted for multiple comparisons using the Bonferroni correction method Pregnant female Swiss outbred CD1 mice were purchased from Charles River Laboratories (Sweden). Three to four days after birth infant mice weighing between 2.3 and 2.7 grams were separated from their mothers, randomly grouped in 9–10 mice per group and placed at 26°C for 6 hours before oral inoculation with virulent All animals were housed under specific–pathogen–free conditions and all treatments and procedures performed in accordance with the Swedish Animal Welfare Act (1988∶534) and the Animal Welfare Ordinance (1988∶539). Approval for the study was given by the Ethical Committee for Laboratory Animals in Gothenburg, Sweden . Humane endpoints were used in all animal experiments to minimize animal suffering.Adult mice were monitored daily for the first 3 days after immunization and then at least 3 days a week during the course of each experiment. The mice were humanely sacrificed if they had more than 10% weight loss, signs of apathy, or loss of fur. The adult animals were anesthetized with isofluoran and sacrificed by cutting the pulmonary aorta. Adult mice were immunized via the non–invasive oral route or by an injection into the abdominal cavity. The adult mice were kept in covered cages with 2–10 mice in each cage with free access at all times to water, food and light. The infant mouse survival experiment had an endpoint at 48 hours post–infection; before that infected moribund animals could be identified by clinical signs of severe disease, i.e. labored breathing, persistent recumbency and change of skin coloration from pink to blue or grey and these mice were euthanized to avoid prolonged suffering. The infant mice were sacrificed by rapid decapitation using a sharp scalpel. Blood was immediately drained by placing the carcass on filter paper. Infected infant mice were monitored at least every sixth hour from 0–18 hours post–infection and at least every second hour between 18 and 48 hours post–infection. In addition to two specially trained staff from our laboratory an experienced animal caretaker was involved in the monitoring of the infant mice. The infant mice were infected via the non–invasive oral route using a non–cutting, round–tip stainless steel feeding needle. In agreement with our animal ethics permit for infant mice (227/12) no chemical agents were administered during the course of the experiments. To the best of our knowledge no publications exist that describes the use of any analgesics and anaesthetics during the course of the challenge experiments.


We investigated whether beliefs about addiction being a ‘disease’ or ‘brain disease’, and holding certain beliefs about addiction aetiology, are associated with public views about addicted persons and support for different types of treatment, coerced treatment and punishment for addiction.Data were collected as part of the 2012 Queensland Social Survey, a computer assisted telephone interview of 1263 residents of Queensland, Australia. Participants were presented with scenarios of two addicted males, one who was addicted to heroin and the other addicted to alcohol. Participants were then asked a series of questions for both characters.2 = 273.90, p < 0.001). Being 35 years of age or older , heroin: OR = 0.49 (0.28-0.85)) and having 15 or more years of education , heroin: 0.55 (0.40-0.75)) predicted less support for coerced treatment. 31.7% of respondents agreed heroin use should be punished by imprisonment and being 35 years of age or older (OR = 0.51 (0.33-0.80)) predicted lack of support. The sample agreed that an alcohol or heroin dependent person would suffer career damage , marriage breakdown  and get in trouble with the law . Respondents expressed more comfort with encountering alcohol rather than heroin addicted persons in the workplace or at a dinner party. Beliefs that addiction was a ‘brain disease’ or a ‘disease’ did not predict any of these attitudes. Beliefs about addiction aetiology were inconsistent predictors of outcomes measured.There was widespread support for all treatment modalities . There was less support for coerced treatment for alcohol than heroin addiction (alcohol: 41%, heroin: 71%, χAge and educational attainment were the most consistent predictors of stigmatising beliefs and beliefs about coercion and punishment. Beliefs that addiction is a ‘disease’ or a ‘brain disease’ were not associated with an overall reduction in beliefs about stigma, coercion or punishment. Beliefs in different causes of addiction were not consistent predictors of beliefs about stigma, coercion or punishment. Neuroscience research on the effects of chronic addictive drug use on brain function has been used to argue that addiction is a chronic relapsing ‘brain disease’ ,2. In paThe implications of this model for how addicted individuals are viewed and treated are hotly contested. Advocates argue that acceptance of the brain disease view will reduce the stigmatisation that many people with an addiction experience by showing that an inability to refrain from using drugs arises not from personal qualities such as moral ‘weakness’ but as a result of drugs ‘hijacking’ the brain’s reward system . The hopCritics of the brain disease model of addiction argue that it will fail to deliver these benefits or perhaps produce more negative consequences for addicted persons. Some argue that placing responsibility for addiction on the individual’s ‘brain’ neglects the role of the social environment – ranging from family to workplace, to broader structural and cultural dynamics – as a contributor to addiction. They argue that the brain disease model individualises responsibility and may increase funding for health programs that focus on ‘medical’ aspects of addiction at the expense of social ones . Some deincrease stigmatisation  and calculating the mean score  . PositivFollowing exploratory data analysis, the demographic variables included as predictors of attitudes in the logistic regressions we conducted were: gender ; age ; and education . The binary variable ‘familiarity’ was based on answers to questions about personal experience with addiction. Those who had or knew someone who had had a problem with alcohol/heroin use were classed as having familiarity with addiction . 62% of respondents had themselves experienced or had someone close to them experience problematic alcohol use. 19% of respondents reported having familiarity with problematic heroin use. In both cases, it was mostly someone close to the participant rather than the participant who had experienced problematic substance use .‘Biological’: indicating participants’ agreement that addiction is caused by i) chemistry in the brain; or is ii) genetic/inherited;‘Socio-environmental’: including i) the way he was raised; or ii) stress;‘Personal qualities’: including i) psychological problems; ii) addictive personality; or iii) ‘bad character’.As described previously , we creaResponses to the four and five-point scales for agreement, likelihood and helpfulness were re-coded into binary variables  as follows: disagree/strongly disagree = negative = 0, agree/strongly agree = affirmative = 1; unlikely/very unlikely = negative = 0, very likely/quite likely = affirmative = 1; not helpful = negative = 0, very helpful/somewhat helpful = affirmative = 1.Don’t know/unsure responses to these questions  were discarded from analysis. Overall high rates of agreement relating to treatment made logistic regression analysis infeasible for these questions. The two questions from the AMIQ pertaining to ‘comfort’ were combined, by adding the dichotomized variables together, to create a comfort scale .There was strong support for all treatment modalities for both the heroin and alcohol addicted characters Table . Partici2 = 273.90, p < 0.001). Beliefs about whether addiction was a disease or a brain disease were not associated with support for coerced treatment (Table There was significantly greater support for forcing Peter into treatment for his heroin addiction (71%) than for forcing John into treatment for his alcohol addiction 41%)  to agree that John should be forced into treatment. In the case of heroin addiction, those 35 years of age and older were 0.49 times as likely as those aged 18–34  to agree that Peter should be forced into treatment. Having 15 or more years of education was associated with lower support for coerced treatment in the case of both alcohol  and heroin addiction  compared to those with 1–12 years of education.People who had experienced their own, or another’s alcohol addiction were 0.72 times less likely than those with no familiarity with alcohol addiction to agree that John should be coerced into treatment . No association was evident between familiarity with heroin addiction and views on coerced treatment. Females were 0.73 times less likely than males to agree that Peter should be forced into treatment for his heroin addiction (OR = 0.54-0.99). We observed no association between gender and beliefs about coerced alcohol addiction treatment.Approximately one-third (31.7%) of the sample agreed that Peter should go to prison for his heroin addiction. Table t-test, p < 0.001).Table Low variation in ratings on the AMIQ items mean that it was not possible to assess associations between demographic factors and stigmatising beliefs about addiction. Answers to questions about the extent to which people felt comfortable interacting with addicted persons in the workplace or at a dinner party showed more variation than answers to questions about the possible negative consequences of addiction. We therefore modelled these separately Table .Table 7Persons with more familiarity with alcohol addiction were more comfortable with an alcohol addicted person . The same was the case for people who agreed that alcohol addiction had biological causes . People who were neutral or did not know if alcohol addiction had social-environmental causes were more likely to be comfortable than those who disagreed . People who agreed that alcohol addiction was caused by personal qualities were less likely to feel comfortable with an alcohol addicted person than those who disagreed .Different factors predicted the level of comfort people felt with a heroin addicted person. Females were less likely to report feeling comfortable with a heroin addicted person  and those with 15 or more years of education were marginally more likely to feel comfortable with someone who had a heroin addiction . People who were neutral or did not know whether heroin addiction had social-environmental causes were more likely to feel comfortable with a heroin addicted person than those who disagreed . Those who agreed that heroin addiction had social-environmental causes were also more likely to feel comfortable with a heroin addicted person than those who disagreed .Our findings show that public acceptance of the brain disease model of addiction did not reliably predict, either positively or negatively, the attitudinal outcomes we measured. This corroborates our previous qualitative research, which suggested that neuroscientific understandings of addiction were added to, and incorporated into, older ideas about addictions, making it unlikely that new ideas about addiction will produce dramatic shifts in the public’s beliefs about addiction and its treatment ,16. It aAll forms of treatment except medication were endorsed on average by more than 90% of participants. Medication was supported by more than 80% of participants. Participants were highly supportive of all treatment options for both alcohol and heroin addiction, suggesting that the public favours a ‘try anything and everything’ approach.Predictions that understanding addiction as a brain disease would be associated with greater or lesser support for the use of coerced medical treatment of addicted individuals were not supported by our findings ,6. InsteSupport for the belief that addiction is a brain disease did not predict agreement with imprisoning someone addicted to heroin. Support for the belief that addiction is a disease was on the borderline of significance in predicting beliefs about imprisonment for heroin addiction. Support for addiction as a disease or a brain disease was not associated with attitudes towards coerced addiction treatment. Age and education level were more consistent predictors of support for coerced treatment across alcohol and heroin, and age also predicted agreement with imprisonment. This suggests that, overall, life experience (i.e. being 35 years of age or older) and one’s broader knowledge shaped less punitive attitudes towards addicted persons. Women were less likely than men to support coerced treatment of heroin addiction but not alcohol addiction. There was no gender difference in beliefs about imprisonment for heroin use.Methodological differences between this and previous studies of stigma in addiction limit the ability to make exact quantitative comparisons. However, our pattern of results were consistent with previous studies using the AMIQ , where tFamiliarity with alcohol addiction was associated with increased comfort with someone addicted to alcohol. This pattern did not hold for heroin. This fits with mixed evidence shown in the literature: familiarity can, but does not necessarily, increase comfort levels ,27. BeinHaving 15 of more years of education was barely significant in predicting comfort with someone addicted to heroin. Educational attainment is not a factor that has received much attention in the literature to date. The lack of a clear pattern in the roles of familiarity, gender and educational level in predicting comfort suggest that more research needs to be done in order to understand these factors and their implications.extent to which addiction is considered voluntary, by comparison to mental illnesses like schizophrenia. They advocate for a more detailed understanding of the factors underpinning ‘social’ versus ‘biological’ causal beliefs [Our findings have implications for the communication of information about addiction. The variability in our findings across the drugs alcohol and heroin raises questions about the benefits of formulating messages about the nature of addiction in general. Our findings suggest that we may need to reduce negative views of heroin users and the challenge the public’s tendency to believe that coerced treatment of this group of drug users is justifiable and/or effective . Our fin beliefs . It coul beliefs . Examini beliefs .per se. Three of the questions are quite similar to the symptoms of substance use disorders specified in the DSM-5 [Methodological differences limit our ability to directly compare our findings on stigma with the previous literature . Our quehe DSM-5 . Thus, rThe primary purpose of this study was to provide a snapshot of the Australian public’s views on the treatment and stigma of addiction, and to do so in a way that yielded results of policy relevance. Latent Class Analysis could be used to achieve a more nuanced understanding of, and address theoretical questions about, the relationships between causal beliefs and stigma ,34. In pThe overall high level of agreement among respondents in the use of all treatment modalities restricted our ability to undertake statistical analyses to compare beliefs about addiction aetiology and beliefs about treatment. Additionally, we did not ask questions about the public’s belief in the effectiveness of treatments. Further research exploring the public’s beliefs about treatment efficacy is needed.As we have reported elsewhere, the low response rate and bias in the sample towards older and more educated individuals limits the generalisability of our findings . A low rOur study does not support the more extreme predictions made about either the benefits or the harms of public acceptance of ‘brain disease’ explanations of addiction. The findings suggest a need to communicate messages that destigmatise drug dependent persons, particularly those addicted to heroin. On our data, communication of information about biological causes and understandings of heroin addiction may not yield the large benefits predicted by proponents of this view, although our data suggest a possible benefit from communicating messages about the biology of alcohol addiction.


Recent decades have witnessed a burgeoning interest in improving health and health systems in low and middle–income countries (LMIC). With the increase in program funding came parallel increases in the number of university programs in the US and Europe offering concentrations or degrees in a global health field –6. The cHow satisfactory is the numerical balance between job aspirants and job openings? Are the trend lines of aspirants and openings similar or divergent? Are we at risk of having too many job seekers or too few?How good is the qualitative match between employer needs and training program outputs? Which competencies are in short supply –12?What are the contributions, and liabilities, of short–term trainee and volunteer participation in the workforce? How do their contributions fit into the larger picture of the global health workforce ,10–12?Answering these questions will take substantial effort through carefully structured investigations to provide reliable answers. In this article, we present a limited pilot study through a targeted web–based job posting review that does not attempt to answer all these questions but sheds some light on the current landscape of employment opportunities in program management, clinical, and public health–related aspects of global health in international settings.The investigative team consisted of five physicians, one with a doctorate in psychology, and another with a doctoral degree in Public Health. The team convened in March 2013. Review of online job postings occurred between November 2013 and January 2014. Websites with employment opportunities in global health were identified using the Google search engine. The terms, “Global Health Work,” “Global Health Jobs,” “Global Health Job Opportunities,” “Global Health Workforce,” “Global Health Hiring” were searched in August 2013.These searches returned a large number of results with potential sources of job information. From this sizeable response, for feasibility and efficiency’s sake, an initial cohort of 14 websites were selected, limited to English language websites primarily affiliated with organizations in North America, and (if the site permitted access) to a regularly available and rotating list of job postings. A similar consideration in prioritizing this initial pilot list was the Google “PageRank” of each site. Page Rank is an objective measure of a citation’s importance that corresponds with users subjective idea of importance . Over thThe investigative team developed a standardized selection and coding tool using a shared online document matrix. The tool allowed for easy categorization of a number of factors related to the job in question. 26 websites in total were selected for inclusion. Each investigator was assigned one high traffic website with frequent job postings and another with lower traffic and fewer postings. The six investigators then each reviewed a subset of the websites during two 6–week sampling periods. Each investigator retrieved a minimum of 10 job postings during each sampling period. The results were then tabulated and underwent basic statistical analysis.In this limited, but wide–reaching review of online job postings that included 178 employment opportunities from 26 websites, key findings included:67% (119/178) of the positions were in non–governmental organizations (NGOs) in both developed countries and LMICs. When combined with multinational organizations such as the World Health Organization (WHO) and the World Bank, the two employer types accounted for 89% (158/178) of the total (14% (25/178) of the positions involved clinical disciplines primarily medicine. (50% (89/178) of job posts included the request for applicants to have the kind of knowledge and skills normally acquired in schools of public health offering courses relevant to global health.(51% (91/178) of the listed opportunities required at least a Master’s degree level of qualification or doctoral degree  (84% (149/178) of the positions were program–related. Program–related jobs included planning, program direction, finance, management and other supportive functions (not depicted but subcategorized in The majority of program–related jobs were identified to be at the senior program management and direction level (58% (87/149)  studies to gain a better understanding of the content and the characteristics of global health (and related) training programs; (2) studies to understand the match between employer needs and applicant qualifications; (3) analyses of the likely trends and stability of the global health job market; (4) surveys of the Global South host countries to determine if training among visitors from the Global North adequately meets their needs; (5) analyses of the intersections between domestic and international employment opportunities, training and career paths. We welcome exchanging views with others interested in learning more about the global health workforce.


In addition to glucose which most commonly supplies carbon units for DNL, fructose is also a profoundly lipogenic substrate that can drive DNL, important when considering the increasing use of fructose in corn syrup as a sweetener. In the context of disease, DNL is thought to contribute to the pathogenesis of non‐alcoholic fatty liver disease, a common condition often associated with the metabolic syndrome and consequent insulin resistance. Whether DNL plays a significant role in the pathogenesis of insulin resistance is yet to be fully elucidated, but it may be that the prevalent products of this synthetic process induce some aspect of hepatic insulin resistance.Hepatic  I.de novo lipogenesis (DNL) is a fundamental biosynthetic pathway within the liver, contributing to the lipids that are stored and secreted by hepatocytes  (Donnelly II.DNL is the synthesis of fatty acid (FA) chains from acetyl‐CoA subunits produced during glycolysis  (Bianchi 161) at the β‐ketoacyl synthase (KS) active site  active site  active site, and further reduction by NADPH‐dependent enoyl‐reductase (EnR)  Wakil, . This gevia activity of the thioesterase (TE) domain of FAS  synthesis FAs are incorporated through the initial acylation, by acyl‐CoA, of glycerol‐3‐phosphate, generating lysophosphatidic acid (LPA). This step is catalysed by glycerol‐phosphate acyl transferase (GPAT)  are complexes containing both lipids and proteins, and serve as the export vehicle for lipids from hepatocytes, hence representing an important fate of lipids produced during hepatic DNL. They are composed of an external monolayer of mostly phospholipids (PLs) and unesterified cholesterol encasing a neutral lipid core (mainly TGs)  membrane   and carbohydrate response element binding protein (ChREBP). These two pathways are activated by increased insulin signalling and increased glucose concentrations, respectively; both induced by feeding via an incompletely elucidated pathway. It results in proteolytic release of the active form of SREBP1c from the Golgi membrane, where the membrane‐bound immature form resides, allowing it to translocate to the nucleus, promoting transcription of lipogenic genes. This proteolytic activation of SREBP1c occurs as a result of its interactions with two membrane proteins of the ER, SREBP cleavage‐activated protein (SCAP) and insulin‐induced gene (INSIG) /protein kinase B (PKB) pathway, one resulting in the phosphorylation of the nascent SREBP1c itself, the other in the activation of the liver X receptor (LXR), predominantly the LXRα isoform in liver  (Chawla (2)via the insulin‐independent glucose transporter family protein, GLUT2  or adenosine monophosphate‐activated kinase (AMPK) phosphorylation sites, which leads to dissociation from 14‐3‐3 protein in the cytosol, translocation to the nucleus and activation of genes containing the carbohydrate response element (ChoRE) , using glucose metabolites as substrates, both increase ChREBP activity et al., et al., As discussed above ACC catalyses the synthesis of malonyl‐CoA in DNL, providing the monomer from which FAs are synthesised. It thus provides an efficient control point to limit rates of DNL. ACC is present in two major isoforms in mammals: ACC1 and ACC2 , lower in Caucasians (33%) and lower still in African Americans (24%) (Browning VI.i) hypertriglyceridaemia; (ii) low serum high‐density lipoprotein (HDL)–cholesterol concentrations; (iii) hypertension; (iv) elevated fasting glycaemia; and (v) enlarged waist circumference  , suggesting an independent role of NAFLD in the development of T2DM beyond initial insulin resistance /PI3K/PKB/FoxO1 to suppress gluconeogenesis is dysfunctional, but signalling via SREBP1c is maintained. Both ob/ob and lipodystrophic mice develop severe insulin resistance due to impaired insulin signalling failing to suppress gluconeogenesis and hepatic steatosis  mice develop severe insulin resistance, but lack the hepatic steatosis, suggesting divergent signalling pathways in the stimulation of lipogenesis and inhibition of gluconeogenesis  that translocates fatty acyl derivatives into the mitochondrial matrix for β‐oxidation (Topping & Mayes, et al., et al., et al., Under this paradigm the majority of fructose generates triose phosphates which undergo gluconeogenesis to be stored as glycogen (Tappy & Lê, X.et al., et al., et al., et al., The gut microbiome has come under much scrutiny in recent years due to potential interplay between bacterial overgrowth, intestinal wall permeability and the development of NAFLD (Abu‐Shanab & Quigley, et al., et al., et al., In relation to the gut mircobiome, fructose‐feeding‐induced NAFLD in mice can be ameliorated by TLR4‐inactivating mutations (Spruss et al., et al., It should be noted that not all gut microbiota promote fatty liver disease; some strains act as ‘probiotics’ reducing the deleterious effects of other gut microflora (Eslamparast XI.The role of DNL within NAFLD may prove a fruitful area of research. It appears to be a component of the pathological basis of the hugely prevalent NAFLD, which may be an important and under acknowledged risk factor for T2DM.Future studies should attempt to clarify whether endogenous metabolites or lipids constitute simple and effective biomarkers of this process.It may be that DNL provides a suitable pharmacological target for early intervention to prevent the onset of T2DM. This would prove hugely beneficial in the developed world where the burdens of over‐nutrition are underpinning unprecedented increases in obesity, prediabetes and overt T2DM.It is clear that the pathological basis of T2DM and the metabolic syndrome are not solely based on increased rates of hepatic lipogenesis, but that DNL is a contributor to the overall paradigm of these metabolic diseases.Investigators should not consider the role of DNL in isolation, as an exclusive cause of insulin resistance, NAFLD and T2DM, but as a part of the total pathogenic disturbance that can lead to a failure of insulin secretion and signalling.


SBD) is common in dioecious taxa, and understanding variation in the direction and magnitude of SBD across taxa has been a persistent challenge. We took a comparative, laboratory‐based approach using 16 groups (species or strains) of bean beetles  to test two predictions that emerge from dominant hypotheses for the evolution of SBD: (1) groups that suffer greater costs of inbreeding should exhibit greater SBD in favor of either sex (inbreeding avoidance hypothesis) and (2) groups with stronger local mate competition should exhibit greater male bias in dispersal (kin competition avoidance hypothesis). We used laboratory experiments to quantify SBD in crawling dispersal, the fitness effects of inbreeding, and the degree of polygyny , a proxy for local mate competition. While we found that both polygyny and male‐biased dispersal were common across bean beetle groups, consistent with the kin competition avoidance hypothesis, quantitative relationships between trait values did not support the predictions. Across groups, there was no significant association between SBD and effects of inbreeding nor SBD and degree of polygyny, using either raw values or phylogenetically independent contrasts. We discuss possible limitations of our experimental approach for detecting the predicted relationships, as well as reasons why single‐factor hypotheses may be too simplistic to explain the evolution of SBD.Understanding the selective forces that shape dispersal strategies is a fundamental goal of evolutionary ecology and is increasingly important in changing, human‐altered environments. Sex‐biased dispersal ( The study of dispersal is important for many reasons. Among them are the predictions of expansion by invasive species , dispersal is commonly sex‐biased  that emerge from theory. First, we predicted that taxa that suffer greater costs of inbreeding should exhibit greater sex bias in dispersal. Because inbreeding alone does not select for a direction of dispersal bias in the absence of sex‐specific costs . We interpret the degree of polygyny  as a proxy for local mate competition, because competition between related males for mating opportunities should increase with potential for single males to fertilize multiple females. We then used Bayesian statistical methods and phylogenetically independent contrasts (PICs) to test the strength of the predicted trait relationships.Acanthoscelides obtectus, oviposits near but not on beans). Larvae develop inside the bean, and adults emerge, mate and die without requiring any additional food or water. The complete life cycle takes ca. 32 days under our laboratory conditions (27.5°C and a 16:8 h photoperiod). Adults are sexually dimorphic and distinguishable by shape, size, and coloration. All beetles used in our experiments were raised on black‐eyed peas (Vigna unguiculata).Bean beetles complete their life cycle entirely on dry beans. For most species, eggs are deposited on the surface of beans . The C. maculatus populations were collected from localities distributed worldwide (this species is a cosmopolitan pest of legumes) and are known from previous work to exhibit significant genetic and phenotypic differentiation  and nuclear (28S rRNA) markers under a general time reversible model of evolution with gamma‐distributed rate variation (GTR+G model) in a Bayesian framework , each of which contained 5 g of black‐eyed peas. For each dispersal trial, ten newly emerged (<48 h old) beetles  were released into the center patch of the array and allowed to disperse in either direction. Trials ran for 24 h, after which the patch locations of females and males were recorded . Each group had a minimum of 6 trials, for a minimum of 30 observed dispersal distances for each sex . We distributed the total number of dispersing beetles across multiple trials to minimize potential for local density to influence dispersal distance.(logeμFμM). This quantity takes positive values for female bias, negative values for male bias, and a value of zero for identical dispersal between the sexes. We fit dispersal kernels to data using Bayesian statistical software  to sex‐specific dispersal data . The Poisson is an appropriate kernel for discrete landscapes as it relies on a single parameter for the mean and variance of dispersal distance, providing a convenient metric to compare female and male kernels. We characterize the direction and degree of sex bias using the log ratio of mean female dispersal distance to mean male dispersal distance C. phaseoli, which was not included in the inbreeding experiment (it was unavailable at the time of the experiment). The offspring of these crosses were counted and weighed. We focus here on offspring number as the response variable, which assumes that any inbreeding depression is manifest in survival from the egg stage through adult eclosion and emergence. Results were qualitatively identical when we instead analyzed offspring mass.We created experimental crosses within and between maternal families to quantify the effects of inbreeding across groups. We employed a block design following Fox et al. , where eFor each block, we estimated inbreeding depression as offspring production in the two inbred crosses minus offspring production in the two outbred crosses and divided the difference by the outbred value  across groups. We quantified mating system indirectly by examining how experimental variation in sex ratio affected one generation of population growth. The population growth of more polygynous groups should be less affected by female‐biased sex ratios, because fewer males would be needed to fertilize all females. We used a response surface experimental design (Miller and Inouye Nt+1) was proportional to the harmonic mean of female (Ft) and male (Mt) densities in the starting generation: We fit to these data a two‐sex demographic model in which recruitment in the next generation , per capita population growth is maximized at a 1:1 sex ratio, where every male has a single female mate. Under this condition, competition among males for access to females, and hence local mate competition, should be weak or absent as long as the birth sex ratio is also 1:1, as it is in bean beetles  corresponding to the joint posterior probability distribution of the trait values. We tested the statistical significance of the correlations by asking whether the 95% credible intervals included zero. For the inbreeding/dispersal correlation, we used the absolute value of the log ratio of mean female‐to‐mean male dispersal distances (|logeμFμM|), thus capturing bias in either direction. Each draw from the joint posterior, representing one MCMC iteration, was associated with one pair of correlation coefficients (for the two trait pairs). By collecting all draws from the joint posterior, we are better able to capture uncertainty in the trait correlations. The alternative approach – calculating a single correlation coefficient for one set of trait values (means) – would assume that traits are invariant across individuals and measured without error. Thus, the Bayesian approach allowed us to propagate uncertainty in the underlying trait values to uncertainty in the correlation coefficient.We predicted positive correlations across groups between sex‐biased dispersal and the negative effects of inbreeding , and between sex‐biased dispersal and the degree of polygyny . To test these correlations, we estimated all traits within a single Bayesian framework, then estimated the posterior probability distribution of Pearson correlation coefficients (http://ape-package.ird.fr/) was used to calculate independent contrasts for the variables. To confirm correct standardization of contrasts (evolution consistent with a Brownian motion model), we used diagnostic scripts included in the R package picante (http://ib.berkeley.edu/courses/ib200b/scripts/ diagnostics_v3.R) which suggested an untransformed tree was appropriate. For the inbreeding analysis, we excluded C. phaseoli.In addition to the raw trait correlations, we also used phylogenetically independent contrasts (PICs) to control for any biases in the correlation results due to shared evolutionary history . The resulting distribution of correlation coefficients therefore reflects uncertainty in the strength of the relationship given uncertainty in trait estimation and controlling for phylogeny (but not accounting for uncertainty in the phylogeny itself).logeμFμM. Note that because this analysis used Bayesian posterior trait means, there was no replication at the level of beetle groups . Therefore, we could not include random effects associated with phylogenetic relationships, because these effects are indistinguishable from residual variance. The analysis therefore does not account for phylogeny and should be interpreted with appropriate caution. We used likelihood ratio tests to determine whether the two explanatory traits, alone or in combination, provided a better fit than the null model.Finally, to test whether combinations of polygyny and inbreeding depression could better explain observed sex‐biased dispersal than either factor alone, we fit linear models to group mean trait values. We fit candidate models that included inbreeding depression  and degree of polygyny  as predictor variables . The response variable was the log ratio of female‐to‐male dispersal A. obtectus) to significantly male‐biased in ten other groups, including most populations of C. maculatus and the species C. phaseoli. There was no significant dispersal bias  in five groups  to a maximum of 125 (in C. maculatus (Benin)), with a mean of 55. However, only three of the groups we tested (C. maculatus (IITA), C. rhodesianus and Z. subfasciatus) exhibited significant inbreeding depression . One group (C. maculatus (UVBF)) showed a positive response to inbreeding .We also found variation across groups in the effects of inbreeding on offspring recruitment Fig. B. OffsprZ. subfasciatus) was a strong outlier, with an estimated harem size of 125 females and a very wide credible interval  based on raw  trait values Fig. A and B. Likewise, we found no evidence for an association between polygyny and male‐biased dispersal based on raw trait values Fig. C. The meP > 0.25).Finally, consistent with the pairwise, phylogenetically controlled correlation results, we found no support for linear models that included combined effects of inbreeding and polygyny on sex‐biased dispersal. Among five candidate models that included effects of inbreeding and degree of polygyny as predictor variables, alone or in combination, the null model (no influence of either trait) received the most AIC support (AIC weight = 0.46) and no other model provided a significant improvement based on likelihood ratio tests . Better understanding of the natural history of this species could help determine whether there is reason to expect local resource competition as an important evolutionary force. In general, the selective environments of bean beetles' recent evolutionary histories (infestation of stored grains or field crops) and their status as pests suggest that resources have not been strongly limiting. For this reason, we continue to expect greater potential for kin competition between males (for mates) than females (for oviposition resources), although mate competition is clearly not a sufficient explanation for the patterns of variation that we observed.One limitation of our approach is that we focus on local mate competition between related males as the sole source of kin competition. Resource competition between related females is an unmeasured factor that would act in the opposite direction, favoring female‐biased dispersal. Indeed, we found evidence for female‐biased dispersal in one species (Recent theory identifies additional demographic factors, not necessarily tied to inbreeding or kin competition, that can contribute to dispersal differences between the sexes. For animals dispersing to locate mates, costs of dispersal would favor only one sex dispersing, such that the other avoids unnecessary dispersal costs (Meier et al. Our laboratory‐based approach with “captive” populations of bean beetles provided several advantages regarding taxonomic breadth of the study and power to estimate the values of traits that are very difficult to rigorously quantify in natural populations. However, this approach also involved drawbacks that may have clouded our ability to detect the hypothesized relationships. Our study organisms have been maintained in a laboratory environment for many generations. The selective regime of the laboratory may have altered dispersal behavior relative to wild beetles. For example, the costs and benefits of dispersal behavior in a laboratory context are both likely to be minimal, in which case selection on dispersal would be very weak and dispersal traits may change simply due to drift. Such an influence of the recent selective environment may override any historical contributions of inbreeding depression or kin competition to dispersal evolution. Previous studies have used some of the same beetle populations to test the adaptive significance of trait variation across groups (Rankin and Arnqvist C. maculatus increased with environmental stress and that, across diverse groups, inbreeding depression may be difficult to detect in benign environments. Thus, a more challenging plant host or abiotic context may yield different quantitative estimates for the effects of inbreeding in these groups.It is also likely that maintenance in laboratory culture has altered the consequences of inbreeding depression relative to conditions in natural populations. The genetic bottleneck associated with establishment of the laboratory stocks, followed by many generations of inbreeding, likely purged much of the genetic load in our focal populations. Indeed, we found very little evidence for negative effects of inbreeding Fig. B, consisIn summary, our work provides a thorough and phylogenetically explicit investigation of sex‐biased dispersal across related species and populations. The results add to our growing understanding of variability in the direction and magnitude of sex‐biased dispersal. Yet, the evolutionary forces that may explain this variability across bean beetles remain elusive and merit continuing study.None declared.


Hum Brain Mapp 36:633–642, 2015. © 2014 The Authors. Human Brain Mapping Published by Wiley Periodicals, Inc.Recent reports of multivariate machine learning (ML) techniques have highlighted their potential use to detect prognostic and diagnostic markers of pain. However, applications to date have focussed on acute experimental nociceptive stimuli rather than clinically relevant pain states. These reports have coincided with others describing the application of arterial spin labeling (ASL) to detect changes in regional cerebral blood flow (rCBF) in patients with on‐going clinical pain. We combined these acquisition and analysis methodologies in a well‐characterized postsurgical pain model. The principal aims were (1) to assess the classification accuracy of rCBF indices acquired prior to and following surgical intervention and (2) to optimise the amount of data required to maintain accurate classification. Twenty male volunteers, requiring bilateral, lower jaw third molar extraction (TME), underwent ASL examination prior to and following individual left and right TME, representing presurgical and postsurgical states, respectively. Six ASL time points were acquired at each exam. Each ASL image was preceded by visual analogue scale assessments of alertness and subjective pain experiences. Using all data from all sessions, an independent Gaussian Process binary classifier successfully discriminated postsurgical from presurgical states with 94.73% accuracy; over 80% accuracy could be achieved using half of the data . This work demonstrates the concept and feasibility of time‐efficient, probabilistic prediction of clinically relevant pain at the individual level. We discuss the potential of ML techniques to impact on the search for novel approaches to diagnosis, management, and treatment to complement conventional patient self‐reporting.  ASLArterial Spin LabelingfMRIFunctional Magnetic Resonance ImagingGPGaussian ProcessGPCGaussian Process ClassificationLOOCVLeave‐one‐out Cross ValidationMLMachine LearningMPCMultivariate Pattern ClassificationPETPositron Emission TomographyrCBFRegional Cerebral Blood FlowTMEThird Molar ExtractionDespite decades of effort and investment, effective pain management remains an unmet need worldwide . The T2 weighted image was skull stripped using a brain extraction tool [FSL‐BET] and the resulting brain‐only image was coregistered with the average ASL image and used as a mask to exclude extra‐cerebral signal [SPM‐CO‐REGISTER]. A nonlinear transformation was calculated between the mean ASL image and a custom ASL template in the standardized, stereotaxic co‐ordinates of the Montreal Neurological Institute (MNI) [SPM‐NORMALISE]. The raw images were then transformed to MNI space in one interpolation step. The resulting images were smoothed with an 8 mm full width at half maximum isotropic Gaussian kernel. To account for the intersubject variability of global blood perfusion values, all normalized, smoothed images were scaled to have a median value of 1,000. This scaling was performed to ensure that global differences in CBF values did not confound later classification.Image analysis was carried out using a combination of tools from FSL v4.1.6  and Microsoft Excel for Macintosh 2011 (http://www.microsoft.com/mac).VAS scores of perceived pain and alertness were averaged for each subject and session. Between‐session differences in pain and alertness were investigated using one‐way repeated measures ANOVA, with session  used as the within‐subjects factor. Post‐hoc 1One subject was excluded from this analysis due to the presence of an image artifact in ASL data acquired during S4.discrimination of the postsurgical from presurgical state. To maximise sensitivity in this analysis, we used all presurgical  and postsurgical ASL data from both third molar extractions (TME);classification between left, compared to right‐sided postsurgical states;discrimination of the postsurgical from follow‐up pain‐free scans acquired 2 weeks following the second TME. We also examined the effects of using data from multiple ASL scans. On each side we then repeated the GPC analysis five times, progressively reducing the number of ASL scans available to train the classifier from six scans per session to one, performed in reverse acquisition order. As the purpose of this analysis was to determine the effect of the number of scans acquired on classification accuracy, the average of the remaining ASL images were used in both the training and testing phases.Independent binary GPCs [Rasmussen et al., http://www.brainmap.co.uk), which relies on the GPML toolbox for GPC inference (http://www.gaussianprocess.org/gpml).A detailed description of the GPC approach used in this work has been provided elsewhere [Marquand et al., All classifiers were embedded within a leave‐one‐out cross‐validation (LOOCV) framework to estimate the generalisation performance of each classifier for novel data points. This was achieved by repeatedly repartitioning the data into a test set  and a training set  such that each subject was excluded once. Predictions derived from the test data were then used to compute the sensitivity and specificity of the classifier. These were defined respectively as the proportion of postsurgical or presurgical scans correctly classified across all LOOCV cycles. The sensitivity and specificity were then averaged to derive a balanced accuracy measure quantifying the overall performance of each classifier.P‐value for each classifier was derived by computing the proportion of random permutations achieving higher balanced accuracy than the nonpermuted classifier. To account for multiple comparisons in analysis (iii), the P‐values derived from the permutation test were corrected using Holm's Step‐Down procedure [Holm, A permutation test was used to assess whether each classifier exceeded the accuracy that would be predicted by chance (50%). To achieve this, the entire LOOCV procedure was repeated 1,000 times after randomly permuting the class labels in a manner accommodating the repeated‐measures experimental design . The balanced accuracy was computed for each permutation and a re Holm, .Z or T‐statistical maps, it is important to reiterate that they should be interpreted as a pattern, rather than clusters of individual brain regions. As whole‐brain gray matter contributed to covariance estimates used by the classifier, g‐maps were not thresholded.For this application, it is desirable to know how blood flow distribution differs between experimental classes, so a mapping approach that enables direct visualization of the relative class distribution was used. Under this approach, the coefficient scores at each voxel represent the relative difference between experimental classes in the context of the entire pattern. They are constructed by projecting the training data onto the vector that defines the direction of maximal difference between experimental classes. In other words, this vector describes the mean of the posterior distribution in the input  space. Further details can be found in [Marquand et al., P < 0.0001) , postsurgical left and right sessions did not differ from one another . There were no effects of session on VAS indices of alertness  .In the first analysis, we combined all ASL datasets from presurgical and postsurgical scanning sessions on left and right teeth to classify postsurgical from presurgical states. GPC correctly discriminated between states with 94.73% accuracy (classifier significance = P > 0.439).Analysis (ii) assessed the performance of the GP classifier at separating left and right postsurgical images from each other. Classifier accuracy was 52.5%, just above chance and not significantly predictive (P < 0.001); and for the right side classification accuracy was 93% (P < 0.001). Figure In analysis (iii), we assessed classification accuracy between ASL images acquired during the pain‐free follow‐up session (S6) and postsurgical states for left and right sides separately, to mimic the constraints of a cross‐over trial design. When TME was performed on the left side, postsurgical ASL data could be discriminated from the pain‐free follow‐up session Fig. a with 85P < 0.01) discrimination of the pain‐free state at follow‐up from postsurgery states on both left and right sides with an accuracy of 80% or greater. Using only the first ASL image acquired did not result in significantly accurate classification of either left or right‐sided postsurgical state. We visualized the effects of systematic data reduction on classification accuracy as a simple line segment plot  was performed on rCBF endpoints derived from ASL‐MRI. The technique is rapid, efficient, and ethical, minimising the amount of time patients suffering pain must endure the MRI experience. 80% classification accuracy or greater could be derived from as few as two ASL scans, equivalent to a total scanning session of approximately 15 min. Classification performance was similar for both left and right‐sided postsurgical pain. Although the multivariate pattern of brain regions underlying classification was physiologically plausible, compared with previous MRI investigations [Wasan et al., It is clear that there is no difficulty in discriminating the presurgical from postsurgical states, either using neuroimaging or conventional self‐reported endpoints. That said, the TME model provides an ideal test‐bed for the development of ASL as a robust, ethical means of eliciting on‐going pain secondary to tissue trauma [Howard et al., Reports describing conventional mass‐univariate approaches to studying pain using ASL, provide encouraging reading for future pain and therapeutic research [Rosa and Seymour, Potentially these clinical investigations may also be used in concert with MPC techniques to provide diagnostic/prognostic predictions but to the best of our knowledge these studies have yet to be performed. Reports of MPC analyses in the field of pain research have largely focussed on conventional “evoked‐response” BOLD fMRI, reporting discrimination between experimentally induced painful and nonpainful stimulation using laser, electric, and thermal stimulations  have been utilized, largely in the absence of an alternative mechanism for measuring pain [Mogil and Crager, In summary, we have demonstrated how state of the art MPC methods can be applied to ASL data to provide single‐subject predictions regarding the probability of an individual experiencing moderate to severe postoperative on‐going pain. The acquisition phase relies only on the consent of the individual, is rapid and requires no other experimental intervention. Importantly, the output of MPC can be generalized to new subjects. These findings represent tentative, but vital, steps toward the goals of personalized medicine in health care and evaluation of novel therapeutics. These methods must now be applied in two areas, examining individuals with persistent pain, and predicting prognostic outcomes. We contend that neuroimaging may, finally, be taking steps toward providing a complementary measure to self‐report for pain measurement.