Engineering of insulin receptor isoform-selective insulin analogues

Background

The insulin receptor (IR) exists in two isoforms, A and B, and the isoform expression pattern is tissue-specific. The C-terminus of the insulin B chain is important for receptor binding and has been shown to contact the IR just adjacent to the region where the A and B isoforms differ. The aim of this study was to investigate the importance of the C-terminus of the B chain in IR isoform binding in order to explore the possibility of engineering tissue-specific/liver-specific insulin analogues.

Methodology/Principal Findings

Insulin analogue libraries were constructed by total amino acid scanning mutagenesis. The relative binding affinities for the A and B isoform of the IR were determined by competition assays using scintillation proximity assay technology. Structural information was obtained by X-ray crystallography. Introduction of B25A or B25N mutations resulted in analogues with a 2-fold preference for the B compared to the A isoform, whereas the opposite was observed with a B25Y substitution. An acidic amino acid residue at position B27 caused an additional 2-fold selective increase in affinity for the receptor B isoform for analogues bearing a B25N mutation. Furthermore, the combination of B25H with either B27D or B27E also resulted in B isoform-preferential analogues (2-fold preference) even though the corresponding single mutation analogues displayed no differences in relative isoform binding affinity.

Conclusions/Significance

We have discovered a new class of IR isoform-selective insulin analogues with 2–4-fold differences in relative binding affinities for either the A or the B isoform of the IR compared to human insulin. Our results demonstrate that a mutation at position B25 alone or in combination with a mutation at position B27 in the insulin molecule confers IR isoform selectivity. Isoform-preferential analogues may provide new opportunities for developing insulin analogues with improved clinical benefits.     

Conflicting Notions on Violence and PTSD in the Military: Institutional and Personal Narratives of Combat-Related Illness

Research indicates that soldiers struggling with PTSD under-utilize mental health care. Quantitative studies of barriers to care point to the importance of soldiers’ beliefs about mental health and mental health interventions in their care-seeking behavior, yet these studies still struggle to understand the particular beliefs involved and the ways they impact care-seeking behavior. This preliminary study makes a start in examining these questions through qualitative literature analysis. It maps out dominant messages surrounding PTSD in military mental health interventions, and explores how they can both shape and conflict with soldiers’ personal notions. It does so by analyzing these messages and notions as institutional and personal (illness) narratives. Institutional military PTSD-narratives, which draw on mainstream scientific and clinical models, appear to communicate contradictory notions on the meanings of violence and its psychological consequences, often without acknowledging these contradictions. As such, these narratives seem to shape struggles of soldiers, both within themselves and with the military institution. The identified conflicts indicate, contrary to the individualizing and decontextualizing focus of dominant PTSD-understandings, that soldiers’ struggles also have social and moral dimensions. This has important implications for both research into PTSD-interventions and understandings of PTSD as such.     

Conjugative transfer facilitates stable maintenance of IncP-1 plasmid pKJK5 in Escherichia coli cells colonizing the gastrointestinal tract of the germfree rat

Quantitative determination of IncP-1 plasmid loss from Escherichia coli cells colonizing the gastrointestinal tracts of germfree rats was achieved by flow cytometry. Results show that the plasmid's ability to conjugate counteracts plasmid loss and is thus an important mechanism for the stable maintenance of IncP-1 plasmids within the gastrointestinal environment.     

Recombinant expression and characterization of a reducing-end xylose-releasing exo-oligoxylanase from Bifidobacterium adolescentis

The family 8 glycoside hydrolase (RexA) from Bifidobacterium adolescentis was expressed in Escherichia coli. The recombinant enzyme was characterized as a reducing-end xylose-releasing exo-oligoxylanase. Apart from giving insights into this new class of enzymes, knowledge of the RexA enzyme helps to postulate a mechanism for the B. adolescentis breakdown of prebiotic xylooligosaccharides.     

Targeted molecular engineering of a family 11 endoxylanase to decrease its sensitivity towards Triticum aestivum endoxylanase inhibitor types

The Bacillus subtilis endoxylanase XynA (BSXY) is frequently used to improve the functionality of arabinoxylan-containing material in cereal based industries. The presence of endogenous Triticum aestivum xylanase inhibitors (TAXI-I and TAXI-II) in wheat is a real concern as they have a direct negative impact on the efficiency of this enzyme. Here, we used the recently determined structure of the complex between TAXI-I and an endoxylanase of Aspergillus niger to develop inhibitor-insensitive BSXY variants by site-directed mutagenesis of strategically chosen amino acids. We either induced steric hindrance to reject the inhibitors or interrupted key interactions with the inhibitors in the endoxylanase substrate-binding groove. The first strategy was successfully applied to position G12 where G12W combined inhibition insensitivity with unharmed catalytic performance. Variants from the second strategy showed altered inhibitor sensitivities concomitant with changes in enzyme activities and allowed to gain insight in the binding-mode of both TAXI-I and TAXI-II with BSXY.     

World Health Organization Global Estimates and Regional Comparisons of the Burden of Foodborne Disease in 2010

Illness and death from diseases caused by contaminated food are a constant threat to public health and a significant impediment to socio-economic development worldwide. To measure the global and regional burden of foodborne disease (FBD), the World Health Organization (WHO) established the Foodborne Disease Burden Epidemiology Reference Group (FERG), which here reports their first estimates of the incidence, mortality, and disease burden due to 31 foodborne hazards. We find that the global burden of FBD is comparable to those of the major infectious diseases, HIV/AIDS, malaria and tuberculosis. The most frequent causes of foodborne illness were diarrheal disease agents, particularly norovirus and Campylobacter spp. Diarrheal disease agents, especially non-typhoidal Salmonella enterica, were also responsible for the majority of deaths due to FBD. Other major causes of FBD deaths were Salmonella Typhi, Taenia solium and hepatitis A virus. The global burden of FBD caused by the 31 hazards in 2010 was 33 million Disability Adjusted Life Years (DALYs); children under five years old bore 40% of this burden. The 14 subregions, defined on the basis of child and adult mortality, had considerably different burdens of FBD, with the greatest falling on the subregions in Africa, followed by the subregions in South-East Asia and the Eastern Mediterranean D subregion. Some hazards, such as non-typhoidal S. enterica, were important causes of FBD in all regions of the world, whereas others, such as certain parasitic helminths, were highly localised. Thus, the burden of FBD is borne particularly by children under five years old–although they represent only 9% of the global population–and people living in low-income regions of the world. These estimates are conservative, i.e., underestimates rather than overestimates; further studies are needed to address the data gaps and limitations of the study. Nevertheless, all stakeholders can contribute to improvements in food safety throughout the food chain by incorporating these estimates into policy development at national and international levels.

Summary Points

• Thirty-one foodborne hazards caused 600 (95% uncertainty interval [UI] 420–960) million foodborne illnesses and 420,000 (95% UI 310,000–600,000) deaths in 2010.
• The global burden of FBD caused by the 31 hazards studied was 33 (95% UI 25–46) million DALYs in 2010.
• The most frequent causes of foodborne illness were diarrheal disease agents; particularly norovirus and Campylobacter spp.
• Foodborne diarrheal disease agents, particularly non-typhoidal Salmonella enterica, caused 230,000 (95% UI 160,000–320,000) deaths
• Other major causes of FBD deaths were Salmonella Typhi, Taenia solium, hepatitis A virus and aflatoxin.
• 40% of the FBD burden was among children under 5 years old.
• The African (AFR), South-East Asian (SEAR) and Eastern Mediterranean (EMR) D subregions had the highest FBD burden.
• Diarrheal disease agents were the leading cause of FBD burden in most subregions, and non-typhoidal Salmonella enterica caused an important burden in all subregions, particularly in the subregions in Africa.
• Other main causes of diarrheal FBD burden were enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli and Vibrio cholerae in low-income subregions, and Campylobacter spp. in high-income subregions.
• The burden of aflatoxin was high in the AFR D, Western Pacific (WPR) B and SEAR B subregions, whereas dioxins caused the highest burden in SEAR D, EMR D and European (EUR) A and C subregions.
• In the South-East Asian subregions, there was a considerable burden of Salmonella Typhi; the burden of Opisthorchis spp. was concentrated in the SEAR B region, where the seafoodborne trematodes Paragonimus spp. and Clonorchis sinensis were also important.
• In Central and South American (AMR B and AMR D) subregions, T. solium and Toxoplasma gondii contributed significantly to the FBD burden.
• These estimates should inform policy development at national and international levels to improve food safety throughout the food chain.

     

Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis

Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six different primer pairs targeting 16S rRNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal samples that had been frozen compared to identical samples that had not. This effect was further supported by qPCR analysis of bacterial groups within these two phyla. The results demonstrate that storage conditions of fecal samples may adversely affect the determined Firmicutes to Bacteroidetes ratio, which is a frequently used biomarker in gut microbiology.     

Morphology,Molecules, and Monogenean Parasites: An Example of an Integrative Approach to Cichlid Biodiversity

The unparalleled biodiversity of Lake Tanganyika (Africa) has fascinated biologists for over a century; its unique cichlid communities are a preferred model for evolutionary research. Although species delineation is, in most cases, relatively straightforward, higher-order classifications were shown not to agree with monophyletic groups. Here, traditional morphological methods meet their limitations. A typical example are the tropheine cichlids currently belonging to Simochromis and Pseudosimochromis. The affiliations of these widespread and abundant cichlids are poorly understood. Molecular work suggested that genus and species boundaries should be revised. Moreover, previous morphological results indicated that intraspecific variation should be considered to delineate species in Lake Tanganyika cichlids. We review the genera Simochromis and Pseudosimochromis using an integrative approach. Besides a morphometric study and a barcoding approach, monogenean Cichlidogyrus (Platyhelminthes: Ancyrocephalidae) gill parasites, often highly species-specific, are used as complementary markers. Six new species are described. Cichlidogyrus raeymaekersi sp. nov., C. muterezii sp. nov. and C. banyankimbonai sp. nov. infect S. diagramma. Cichlidogyrus georgesmertensi sp. nov. was found on S. babaulti and S. pleurospilus, C. franswittei sp. nov. on both S. marginatus and P. curvifrons and C. frankwillemsi sp. nov. only on P. curvifrons. As relatedness between Cichlidogyrus species usually reflects relatedness between hosts, we considered Simochromis monotypic because the three Cichlidogyrus species found on S. diagramma belonged to a different morphotype than those found on the other Simochromis. The transfer of S. babaulti, S. marginatus, S. pleurospilus and S. margaretae to Pseudosimochromis was justified by the similarity of their Cichlidogyrus fauna and the intermediate morphology of S. margaretae. Finally parasite data also supported the synonymy between S. pleurospilus and S. babaulti, a species that contains a large amount of geographical morphological variation.     

Problematic barcoding in flatworms: A case-study on monogeneans and rhabdocoels (Platyhelminthes)

Some taxonomic groups are less amenable to mitochondrial DNA barcoding than others. Due to the paucity of molecular information of understudied groups and the huge molecular diversity within flatworms, primer design has been hampered. Indeed, all attempts to develop universal flatworm-specific COI markers have failed so far. We demonstrate how high molecular variability and contamination problems limit the possibilities for barcoding using standard COI-based protocols in flatworms. As a consequence, molecular identification methods often rely on other widely applicable markers. In the case of Monogenea, a very diverse group of platyhelminth parasites, and Rhabdocoela, representing one-fourth of all free-living flatworm taxa, this has led to a relatively high availability of nuclear ITS and 18S/28S rDNA sequences on GenBank. In a comparison of the effectiveness in species assignment we conclude that mitochondrial and nuclear ribosomal markers perform equally well. In case intraspecific information is needed, rDNA sequences can guide the selection of the appropriate (i.e. taxon-specific) COI primers if available.     

Plasmid transfer from Pseudomonas putida to the indigenous bacteria on alfalfa sprouts: characterization,direct quantification,and in situ location of transconjugant cells

The transfer of the plasmids pJKJ5 and TOL (pWWO) from Pseudomonas putida to the indigenous bacterial community on alfalfa sprouts was studied. Tagging with fluorescent protein markers allowed direct quantification of the introduced donor bacteria and of indigenous bacteria that had received the plasmids. The sprouts were observed for 9 days; during this time alfalfa seeds, inoculated with donor bacteria, developed to edible and subsequently decaying sprouts. The first transconjugants were detected on day 6 after donor inoculation and occurred at frequencies of 3.4 x 10(-4) and 2.0 x 10(-6) transconjugant cells per donor cell for pKJK5::gfp and TOL::gfp, respectively. Confocal laser scanning microscopy revealed that the sprouts were heavily colonized with donors and that most transconjugants were located around the hypocotyl and root areas. Randomly selected members of the indigenous bacterial community from both inoculated and uninoculated sprouts, as well as a representative part of the community that had received the plasmids, were characterized by polymorphisms of PCR-amplified ribosomal DNA (rDNA) spacer regions between the 16S and 23S genes, followed by partial 16S rDNA sequencing. This showed that the initially dominating genera Erwinia and Paenibacillus were gradually replaced by Pseudomonas on the fully developed sprouts. Transconjugants carrying either of the investigated plasmids mainly belonged to the genera Pseudomonas and ERWINIA: The numbers of transconjugant cells did not reach detectable levels until 6 days after the onset of germination, at which point these species constituted the majority of the indigenous bacteria. In conclusion, the alfalfa sprouts provided an environment that allowed noteworthy frequencies of plasmid transfer from P. putida in the absence of selective pressure that could favor the presence of the investigated plasmids.