Combined lifestyle factors and the risk of LADA and type 2 diabetes - Results from a Swedish population-based case-control study

Aims: We investigated the risk of latent autoimmune diabetes in adults (LADA) and type 2 diabetes in relation to a healthy lifestyle, the proportion of patients attributable to an unhealthy lifestyle, and the influence of family history of diabetes (FHD) and genetic susceptibility. Methods: The population-based study included incident LADA (n = 571), type 2 diabetes (n = 1962), and matched controls (n = 2217). A healthy lifestyle was defined by BMI < 25 kg/m2, moderate-to-high physical activity, a healthy diet, no smoking, and moderate alcohol consumption. We estimated odds ratios (OR) with 95% confidence intervals (CIs) adjusted for age, sex, education, and FHD. Results: Compared to a poor/moderate lifestyle, a healthy lifestyle was associated with a reduced risk of LADA (OR 0.51, CI 0.34-0.77) and type 2 diabetes (OR 0.09, CI 0.05-0.15). A healthy lifestyle conferred a reduced risk irrespective of FHD and high-risk HLA genotypes. Having a BMI < 25 kg/m2 conferred the largest risk reduction for both LADA (OR 0.54, CI 0.43-0.66) and type 2 diabetes (OR 0.12, CI 0.10-0.15) out of the individual items. Conclusion: People with a healthy lifestyle, especially a healthy body weight, have a reduced risk of LADA including those with genetic susceptibility to diabetes. (C) 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Peer reviewe

Consumption of red meat, genetic susceptibility, and risk of LADA and type 2 diabetes

Purpose Red meat consumption is positively associated with type 1 (T1D) and type 2 (T2D) diabetes. We investigated if red meat consumption increases the risk of latent autoimmune diabetes in adults (LADA) and T2D, and potential interaction with family history of diabetes (FHD), HLA and TCF7L2 genotypes. Methods Analyses were based on Swedish case-control data comprising incident cases of LADA (n = 465) and T2D (n = 1528) with matched, population-based controls (n = 1789; n = 1553 in genetic analyses). Multivariable-adjusted ORs in relation to self-reported processed and unprocessed red meat intake were estimated by conditional logistic regression models. Attributable proportion (AP) due to interaction was used to assess departure from additivity of effects. Results Consumption of processed red meat was associated with increased risk of LADA (per one servings/day OR 1.27, 95% CI 1.07-1.52), whereas no association was observed for unprocessed red meat. For T2D, there was no association with red meat intake once BMI was taken into account. The combination of high (> 0.3 servings/day vs. less) processed red meat intake and high-risk HLA-DQB1 and -DRB1 genotypes yielded OR 8.05 (95% CI 4.86-13.34) for LADA, with indications of significant interaction (AP 0.53, 95% CI 0.32-0.73). Results were similar for the combination of FHD-T1D and processed red meat. No interaction between processed red meat intake and FHD-T2D or risk variants of TCF7L2 was seen in relation to LADA or T2D. Conclusion Consumption of processed but not unprocessed red meat may increase the risk of LADA, especially in individuals with FHD-T1D or high-risk HLA genotypes.Peer reviewe

Interaction Between Overweight and Genotypes of HLA, TCF7L2, and FTO in Relation to the Risk of Latent Autoimmune Diabetes in Adults and Type 2 Diabetes

Objective: We investigated potential interactions between body mass index (BMI) and genotypes of human leukocyte antigen (HLA), TCF7L2-rs7903146, and FTO-rs9939609 in relation to the risk of latent autoimmune diabetes in adults (LADA) and type 2 diabetes. Methods: We pooled data from two population-based studies: (i) a Swedish study with incident cases of LADA [positive for glutamic acid decarboxylase autoantibodies (GADA); n = 394) and type 2 diabetes (negative for GADA; n = 1290) and matched controls without diabetes (n = 2656) and (ii) a prospective Norwegian study that included incident cases of LADA (n = 131) and type 2 diabetes (n = 1901) and 886,120 person-years of follow-up. Analyses were adjusted for age, sex, physical activity, and smoking. Interaction between overweight (BMI >= 25 kg/m(2)) and HLA/TCF7L2/FTO high-risk genotypes was assessed by attributable proportion due to interaction (AP). Results: The combination of overweight and high-risk genotypes of HLA, TCF7L2, and FTO was associated with pooled relative risk (RRpooled) of 7.59 (95% CI, 5.27 to 10.93), 2.65 (95% CI, 1.97 to 3.56), and 2.21 (95% CI, 1.60 to 3.07), respectively, for LADA, compared with normal-weight individuals with low/intermediate genetic risk. There was a significant interaction between overweight and HLA (AP, 0.29; 95% CI, 0.10 to 0.47), TCF7L2 (AP, 0.31; 95% CI, 0.09 to 0.52), and FTO (AP, 0.38; 95% CI, 0.15 to 0.61). The highest risk of LADA was seen in overweight individuals homozygous for the DR4 genotype [RR, 26.76 (95% CI, 15.42 to 46.43); AP, 0.58 (95% CI, 0.32 to 0.83) (Swedish data)]. Overweight and TCF7L2 also significantly interacted in relation to type 2 diabetes (AP, 0.26; 95% CI, 0.19 to 0.33), but no interaction was observed with high-risk genotypes of HLA or FTO. Conclusions: Overweight interacts with HLA high-risk genotypes but also with genes associated with type 2 diabetes in the promotion of LADA.Peer reviewe

A study of DNA interactive proteins and their origin from thermophilic Siphoviridae phages

This thesis covers the results of studies on the amino acid sequence, three-dimensional structure, and activities of two types of DNA-interactive proteins: Holliday Junction resolving enzyme (represented by Hjc_15-6 isolated from phage Tth15-6) and DNA Polymerase I (represented by PolI_G20c, isolated from phage G20c), including studies on their homologous counterparts. As these proteins originate from two different, but related phages that infect Thermus thermophilus, the studies also include the evolutionary relationship between these and other related phages at genomic level. The novel phage Tth15-6 was isolated, its genome sequenced, and from the sequence data, a large number of genes were selected. The selected genes were cloned and expressed in Escherichia coli and the resulting production levels of soluble target protein were analysed. The DNA polymerase encoded in phage Tth15-6, PolI_15-6, was considered interesting, due to its relatively low sequence conservation compared to previously studied candidates from the same family of polymerases. It was, however, not possible to produce the protein in its soluble form in the selected E. coli production host. Therefore, a gene encoding a homologous protein, PolI_G20c, was identified, cloned and expressed, that resulted in soluble production of the target protein. After purification, PolI_G20c was crystallised, but its structure could not be solved by molecular replacement, using available structures in the Protein Data Bank, PDB. However, a fragment of a homologous protein, ExnV1, was also produced and crystallised. In this case, it was possible to solve the three-dimensional structure, which was subsequently used as a template, allowing the determination of the three-dimensional structure of PolI_G20c. The gene encoding the Holliday junction resolving enzyme, Hjc_15-6, was also cloned, and the enzyme was produced, purified and crystallised. In this case, production of a selenium derivatized variant was successful. Crystallisation of both variants, allowed determination of the three-dimensional structure. Studies were also made on how Hjc_15-6 interacts with branched DNA structures and single peptides in conjunction with polymerases and polymerase reactions. Analysis of the genome sequence of phage Tth15-6, showed that the identified open reading frames in many cases lacked homologues of known function. This sequence divergence makes it difficult to find related candidates of known function using traditional bioinformatics. However, the organisation of genes in the whole genome may be a valuable tool for this purpose. The evolutionary relationship between phage Tth15-6, phage G20c, and other related phages, were also reflected in the deduced amino acid sequences of the enzymes Hjc_15-6 and PolI_G20c. A three-part sequence signature motif of Hjc_15-6 differed from corresponding motif in earlier reported Hj resolving enzymes. It was thus proposed that this novel sequence signature is common among Hj resolving enzymes originating from the related phages. Furthermore, Hjc_15-6 is the first Hj resolving enzyme originating from a phage that could be classified as an archaeal type Hj resolving enzyme, regarding both its amino acid sequence, and its structure. This is the first time an archaeal type Hj resolving enzyme originating from a phage is reported. The structure of PolI_G20c and ExnV1 revealed a new structural motif that has not previously been reported among type 1 polymerases. However, based on the amino acid sequence analysis, this motif may also occur in enzymes from related phages. Finally, the studies show that Hjc_15-6 has unusual features not reported among Hj resolving enzymes elsewhere. Therefore, Hjc_15-6 was studied further, both regarding its capability to cleave DNA oligomers, and its function in conjunction with polymerases and polymerase reactions. It was revealed that Hjc_15-6 is capable of cleaving branched products from isothermal polymerase reactions based on strand replacement. It was also shown that it was possible to induce a polymerase reaction with neither DNA template nor primers present, when Hjc_15-6 was present. Moreover, Hjc_15-6 may form large networks of DNA, something that was studied in real-time under an electron microscope. Hence, Hjc_15-6 may be a valuable tool for DNA origami with a wide variety of applications. This thesis contains four research papers along with introducing chapters for the reader's orientation including brief presentations of the content in the Papers, followed by a chapter of Concluding remarks where the most important findings in the articles are presented, followed by a chapter of Future prospects, Acknowledgements and References

Gender mainstreaming - from policy to management : An analysis on Swedish reform cooperation in Bosnia and Herzegovina regarding issues of gender equality linked to air pollution, within the framework of gender mainstreaming and Sweden’s Feminist Foreign Policy

The Swedish Environmental Protection Agency (SEPA) recognized a need for external help to implement a gender perspective in their project on improving air quality in Bosnia and Herzegovina (BiH) extending from 2018-2022, which this thesis is a contribution to. To collect empirical material for the analysis, a cluster interview was conducted on site in Sarajevo with female representatives from ten CSOs working with gender equality nationally, in addition to a household survey targeted at six different cities in BiH through social media with the help of SEPA’s partnering ad agency network. The empirical findings of this study hence consist of local representations of problems regarding gender equality and its links to environmental issues, which were subsequently used to put the goals and problem representations present in the Swedish Foreign Service action plan for feminist foreign policy 2019-2022 and the Strategy for Sweden’s reform cooperation in the Western Balkans and Turkey 2021-2027 in context. This was specifically done with the help of Carol Bacchi’s policy analysis approach “What’s the Problem (Represented to be). Throughout the analysis, it became evident that there was a gap between the discourse of gender equality problems on site in BiH versus how gender equality problems were represented in the analysed Swedish policies steering the project. It also became evident that there was a gap between the horizontal goals of gender equality in these policies, and the resources available at SEPA for the actual implementation.  The conclusion of the analysis implicates the occurrence of shortcomings in Swedish reform cooperation when mainstreaming gender into environmental policies, and that this could potentially lead to efforts reproducing and entrenching gendered and socio-economic inequity.  By exemplifying the relevance of implementing a context-based gender perspective in this specific case, this thesis is hoped to encourage improving the prerequisites for mainstreaming gender into all international reform cooperation projects and applying the Swedish Feminist Foreign Policy accurately.

Gender mainstreaming - from policy to management : An analysis on Swedish reform cooperation in Bosnia and Herzegovina regarding issues of gender equality linked to air pollution, within the framework of gender mainstreaming and Sweden’s Feminist Foreign Policy

The Swedish Environmental Protection Agency (SEPA) recognized a need for external help to implement a gender perspective in their project on improving air quality in Bosnia and Herzegovina (BiH) extending from 2018-2022, which this thesis is a contribution to. To collect empirical material for the analysis, a cluster interview was conducted on site in Sarajevo with female representatives from ten CSOs working with gender equality nationally, in addition to a household survey targeted at six different cities in BiH through social media with the help of SEPA’s partnering ad agency network. The empirical findings of this study hence consist of local representations of problems regarding gender equality and its links to environmental issues, which were subsequently used to put the goals and problem representations present in the Swedish Foreign Service action plan for feminist foreign policy 2019-2022 and the Strategy for Sweden’s reform cooperation in the Western Balkans and Turkey 2021-2027 in context. This was specifically done with the help of Carol Bacchi’s policy analysis approach “What’s the Problem (Represented to be). Throughout the analysis, it became evident that there was a gap between the discourse of gender equality problems on site in BiH versus how gender equality problems were represented in the analysed Swedish policies steering the project. It also became evident that there was a gap between the horizontal goals of gender equality in these policies, and the resources available at SEPA for the actual implementation.  The conclusion of the analysis implicates the occurrence of shortcomings in Swedish reform cooperation when mainstreaming gender into environmental policies, and that this could potentially lead to efforts reproducing and entrenching gendered and socio-economic inequity.  By exemplifying the relevance of implementing a context-based gender perspective in this specific case, this thesis is hoped to encourage improving the prerequisites for mainstreaming gender into all international reform cooperation projects and applying the Swedish Feminist Foreign Policy accurately.

Monitoring the production of inclusion bodies during fermentation and enzyme-linked immunosorbent assay analysis of intact inclusion bodies using cryogel minicolumn plates

A novel minicolumn chromatgraphic method to monitor the production of inclusion bodies during fermentation and anenzyme-linked immunosorbent assay (ELISA) system allowing direct analysis of the particles with surface-displayed antigens are described. A 33-kDa protein containing 306 amino acids with three sulfur bridges produced its inclusion bodies wits labeled with polyclonal antibodies against 15 amino acid (anti-A15) and 17 amino acid (anti-B17) residues at the N- and C-terminal ends of the protein, respectively. Labeled particles were bound to macroporous Monolithic protein A-cryogel adsorbents inserted into the open-ended wells of a 96-well plate (referred to as protein A-cryogel minicolumn plate). The concept behind this application is that the binding degree of inclusion bodies from lysed fermentation broth to the cryogel minicolumns increases with an increase in their concentration during fermentation. The technique allowed LIS to monitor the increase in the production levels of the inclusion bodies as the fermentation process progressed. The system also has a built-in quality parameter to ensure that the target protein has been fully expressed. Alternatively, inclusion bodies immobilized on phenyl-cryogel minicolumn plate were used in indirect ELISA based on anti-A15 and anti-B17 antibodies against terminal amino acid residues displayed oil the surface of inclusion bodies. Drainage-protected properties of the cryogel minicolumns allow performance of successive reactions with tested immunoglobulin G (IgG) samples and enzyme-conjugated secondary I-G and of enzymatic reaction within the adsorbent. (c) 2006 Elsevier Inc. All rights reserved

Antioxidant Nutrients and Risk of Latent Autoimmune Diabetes in Adults and Type 2 Diabetes : A Swedish Case-Control Study and Mendelian Randomization Analysis

Antioxidant vitamins C and E are inversely associated with type 1 diabetes (T1D). We investigated if antioxidants are also associated with latent autoimmune diabetes in adults (LADA), with low (LADAlow) and high (LADAhigh) autoantibody levels, type 2 diabetes (T2D), and estimates of beta cell function (HOMA-B) and insulin resistance (HOMA-IR). We used Swedish case-control data with incident cases of LADA (n = 584) and T2D (n = 1989) and matched population-based controls (n = 2276). Odds ratios (OR) and 95% confidence intervals (CI) were calculated per one standard deviation higher beta-carotene, vitamin C, vitamin E, selenium, and zinc intakes. Two-sample Mendelian randomization (MR) analyses assessed causality between genetically predicted circulating antioxidants and LADA, T1D, and T2D, using summary statistics from genome-wide association studies. Among the antioxidants, vitamins C and E were inversely associated with LADAhigh (OR 0.84, CI 0.73, 0.98 and OR 0.80, CI 0.69, 0.94 respectively), but not with LADAlow or T2D. Vitamin E was also associated with higher HOMA-B and lower HOMA-IR. MR analyses estimated an OR of 0.50 (CI 0.20, 1.25) for the effect of vitamin E on T1D, but did not support causal relationships between antioxidants and either LADA or T2D. In conclusion, vitamin E may have a protective effect on autoimmune diabetes, possibly through preserved beta cell function and less insulin resistance

Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies

A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B.. 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices

Antioxidant Nutrients and Risk of Latent Autoimmune Diabetes in Adults and Type 2 Diabetes : A Swedish Case-Control Study and Mendelian Randomization Analysis

Antioxidant vitamins C and E are inversely associated with type 1 diabetes (T1D). We investigated if antioxidants are also associated with latent autoimmune diabetes in adults (LADA), with low (LADA(low)) and high (LADA(high)) autoantibody levels, type 2 diabetes (T2D), and estimates of beta cell function (HOMA-B) and insulin resistance (HOMA-IR). We used Swedish case-control data with incident cases of LADA (n = 584) and T2D (n = 1989) and matched population-based controls (n = 2276). Odds ratios (OR) and 95% confidence intervals (CI) were calculated per one standard deviation higher beta-carotene, vitamin C, vitamin E, selenium, and zinc intakes. Two-sample Mendelian randomization (MR) analyses assessed causality between genetically predicted circulating antioxidants and LADA, T1D, and T2D, using summary statistics from genome-wide association studies. Among the antioxidants, vitamins C and E were inversely associated with LADA(high) (OR 0.84, CI 0.73, 0.98 and OR 0.80, CI 0.69, 0.94 respectively), but not with LADA(low) or T2D. Vitamin E was also associated with higher HOMA-B and lower HOMA-IR. MR analyses estimated an OR of 0.50 (CI 0.20, 1.25) for the effect of vitamin E on T1D, but did not support causal relationships between antioxidants and either LADA or T2D. In conclusion, vitamin E may have a protective effect on autoimmune diabetes, possibly through preserved beta cell function and less insulin resistance.Peer reviewe