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This study aimed to determine the compatibility of pre-fermented sugar beet pulp to support the growth of Pleurotus ostreatus mycelium in submerged fermentation. The goal was to create a meat alternative based on mycelial-fermented pulp. It was further explored whether pre-fermentation with lactic acid bacteria (LAB) on the pulp increased meat-like properties, such as aroma, springiness, and hardness, in the final product. Three strains were selected from a high throughput screening of 105 plant-derived LAB based on their acidification and metabolite production in the pulp. Two homofermentative strains (Lactococcus lactis) and one heterofermentative strain (Levilactobacillus brevis) were selected based on their low ethanol production, high lactic acid production, and overall acidification of the pulp. Mycelium of P. ostreatus was grown in submerged fermentations on the pre-fermented pulp, and the biomass was removed by centrifugation. The fungal strain consumed all available sugars and acids and released arabinose to the media. Volatiles were detected using GC-MS, and a large increase in concentrations of hexanal, 1-octen-3-ol, and 2-octenal was measured. Concentration of 1-octen-3-ol was lower in the pre-fermented samples vs. the non-pre-fermented. LC-MS amino acid analysis showed the presence of all essential amino acids on day 0 and 7 of fermentation. The highest concentration of amino acids was for glutamic acid/glutamine and aspartic acid/asparagine. A decrease in all amino acids after 7 days of fungal fermentation was measured for all fermentations. The decrease was more significant for pre-fermented samples. This was also confirmed through a total protein determination, except for samples pre-fermented with Lactococcus lactis strain NFICC142 which increased in total protein content after fungal fermentation. The protein digestibility increased after fungal fermentation, and the highest increase was seen for non-pre-fermented samples. The springiness of the fermented product indicated similarities to meat alternatives, while the hardness was much lower than other meat alternatives. The results indicate that dried sugar beet pulp can be used for submerged cultivation of P. ostreatus, but that pre-fermentation does not improve the physical or nutritional properties of the end product significantly, except for an increased protein content for NFICC142 pre-fermented media. This is the first known attempt to use LAB and P. ostreatus in mixed fermentation to produce fungal mycelium, as well as the first attempt at using SBP in a liquid fermentation for mycelial production of P. ostreatus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The production of plant-based dairy alternatives has been majorly focused on the improvement of sensorial, technological and nutritional properties, to be able to mimic and replace milk-based fermented products. The presence of off-flavours and antinutrients, the lack of production of dairy-like flavours or the metabolic inaccessibility of plant proteins are some of the challenges to overcome to generate plant-based dairy alternatives. However, in the present study, it is demonstrated how the synergistic effect of two LAB strains, when cocultured, can simultaneously solve those challenges when fermenting in four different plant-based raw materials: soy, pea, oat, and potato drinks (SPOP). The fermentation was performed through the mono- and co-culture of the two LAB strains isolated from Apis mellifera (honeybee): Leuconostoc pseudomesenteroides NFICC 2004 and Lactococcus lactis NFICC 2005. Firstly, the coculture of both strains demonstrated to increase the acidification rate of the four plant matrices. Moreover, L. pseudomesenteroides (LP) demonstrated to in situ produce high concentrations of mannitol when fructose was present as C-source. Furthermore, L. pseudomesenteroides, which encoded for PII-proteinase, demonstrated to break down SPOP proteins, releasing free amino acids that were used by L.lactis (LL) for growth and metabolism. Lastly, the analysis of their co-metabolic volatile performance showed the principal ability of removal of the main off-flavours found in SPOP, such as hexanal, 1-octen-3-ol, 2-pentylfuran, pentanal, octanal, heptanal, and nonanal, mainly led by L. pseudomesenteroides, as well as the production of dairy-like flavours, such as diacetyl and 3-methyl-1-butanol, triggered by L. lactis metabolism. Overall, these findings endorsed the use of honeybee isolated strains as starter cultures, demonstrated the potential of coupling genotypes and phenotypes of multiple strains to improve the organoleptic properties suggesting a potential of combining plant-based matrices for the generation of future high-quality plant-based dairy alternatives., Competing Interests: Declaration of competing interest There are not any kind of conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Brewers' spent grain (BSG) is a major side-stream from the beer industry, with an annual estimated production of 39 million tons worldwide. Due to its high nutritional value, high abundance and low price, it has been proposed as an ingredient in human food. Here we investigated the ability of different lactic acid bacteria to produce the flavor molecule acetoin in liquid BSG extract, in order to broaden the possibilities of utilization of BSG in human food. All the investigated lactic acid bacteria (LAB) covering the Leuconostoc, Lactobacillus and Lactoccocus species were able to convert the fermentable sugars in liquid BSG into acetoin. Production levels varied significantly between the different LAB species, with Leuconostoc pseudomesenteroides species reaching the highest titers of acetoin with only acetate as the main byproduct, while also being the fastest consumer of the fermentable sugars present in liquid BSG. Surprisingly, the currently best investigated LAB for acetoin production, L. lactis, was unable to consume the maltose fraction of liquid BSG and was therefore deemed unfit for full conversion of the sugars in BSG into acetoin. The production of acetoin in Leu. pseudomesenteroides was pH dependent as previously observed in other LAB, and the conversion of BSG into acetoin was scalable from shake flasks to 1 L bioreactors. While all investigated LAB species produced acetoin under aerobic conditions, Leu. pseudomesenteroides was found to be an efficient and scalable organism for bioconversion of liquid BSG into a safe acetoin rich food additive., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Anders Peter Watjen reports financial support was provided by European Institute of Innovation & Technology Climate Knowledge and Innovation Community UK & Ireland., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The popularity of quinoa seeds has increased in the last decade due to their high nutritional value and natural gluten-free composition. Consumption of new proteins may pose a risk of introducing new allergies. In the present study the immunogenicity and sensitising capacity of quinoa proteins were assessed in a dose-response experiment in Brown Norway rats in comparison to proteins from spinach and peanut. Cross-reactivity between quinoa proteins and known allergens was evaluated by in silico analyses followed by analyses with 11 selected protein extracts and their anti-sera by means of ELISAs and immunoblotting. Further, an in vitro simulated gastro-duodenal digestion was performed. Quinoa proteins were found to have an inherent medium to high immunogenicity and sensitising capacity, being able to induce specific IgG1 and IgE levels higher than spinach but lower than peanut and elicit reactions of clinical relevance similar to peanut. Quinoa proteins were generally shown to resist digestion and retain capacity to bind quinoa-specific antibodies. Quinoa proteins were shown to be cross-reactive with peanut and tree nut allergens as high sequence homology and antibody cross-binding were demonstrated. Present study suggests that quinoa pose a medium to high level of allergenicity that should be further investigated in human studies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: NPHK and SG are employees at ALK. KLB has ongoing collaboration with ALK. Authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We have mapped a locus on chromosome 7p22.3-7p15.3 spanning a 22.4 Mb region for ulcerative colitis (UC) by whole genome linkage analyses of a large Danish family. The family represent three generations with UC segregating as an autosomal dominant trait with variable expressivity. The whole-genome scan resulted in a logarithm of odds score (LOD score) of Z = 3.31, and a whole genome sequencing (WGS) of two affected excluded disease-causing mutations in the protein coding genes. Two rare heterozygote variants, rs182281985:G>A and rs541426369:G>A, both with low allele frequencies (MAF A:0.0001, gnomAD ver3.1.2), were found in clusters of ChiP-seq transcription factors binding sites close to the AHR (aryl hydrocarbon receptor) gene and the UC associated SNP rs1077773:G>A. Testing the two SNPs in a promoter reporter assay for regulatory activity revealed that rs182281985:G>A influenced the AHR promoter. These results suggest a regulatory region that include rs182281985:G>A close to the UC GWAS SNP rs1077773:G>A and further demonstrate evidence that the AHR gene on the 7p-tel region is a candidate susceptible gene for UC., (© 2023. The Author(s), under exclusive licence to European Society of Human Genetics.)

Leuconostoc spp. is often regarded as the flavor producer, responsible for the production of acetoin and diacetyl in dairy cheese. In this study, we investigate seven plant-derived Leuconostoc strains, covering four species, in their potential as a lyophilized starter culture for flavor production in fermented soy-based cheese alternatives. We show that the process of lyophilization of Leuconostoc can be feasible using a soy-based lyoprotectant, with survivability up to 63% during long term storage. Furthermore, the storage in this media improves the subsequent growth in a soy-based substrate in a strain specific manner. The utilization of individual raffinose family oligosaccharides was strain dependent, with Leuconostoc pseudomesenteroides NFICC99 being the best consumer. Furthermore, we show that all investigated strains were able to produce a range of volatile flavor compounds found in dairy cheese products, as well as remove certain dairy off-flavors from the soy-based substrate like hexanal and 2-pentylfuran. Also here, NFICC99 was strain producing most cheese-related volatile flavor compounds, followed by Leuconostoc mesenteroides NFICC319. These findings provide initial insights into the development of Leuconostoc as a potential starter culture for plant-based dairy alternatives, as well as a promising approach for generation of stable, lyophilized cultures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lactic acid bacteria (LAB) have an extracellular proteolytic system that includes a multi-domain, cell envelope protease (CEP) with a subtilisin homologous protease domain. These CEPs have different proteolytic activities despite having similar protein sequences. Structural characterization has previously been limited to CEP homologs of dairy- and human-derived LAB strains, excluding CEPs of plant-derived LAB strains. CEP structures are a challenge to determine experimentally due to their large size and attachment to the cell envelope. This study aims to clarify the prevalence and structural diversity of CEPs by using the structure prediction software AlphaFold 2. Domain boundaries are clarified based on a comparative analysis of 21 three-dimensional structures, revealing novel domain architectures of CEP homologs that are not necessarily restricted to specific LAB species or ecological niches. The C-terminal flanking region of the protease domain is divided into fibronectin type-III-like domains with various structural traits. The analysis also emphasizes the existence of two distinct domains for cell envelope attachment that are preceded by an intrinsically disordered cell wall spanning domain. The domain variants and their combinations provide CEPs with different stability, proteolytic activity, and potentially adhesive properties, making CEPs targets for steering proteolytic activity with relevance for both food development and human health.

Allergic diseases are broadly classified as IgE-mediated type-I hypersensitivity immune reactions due to exposure to typically harmless substances known as allergens. These allergenic substances activate antigen presenting cells, which further triggers T-helper 2 cells immune response and class switch B-cells for synthesis of allergen-specific IgE, followed by classical activation of inflammatory mast cells and eosinophils, which releases preformed mediators involved in the cascade of allergic symptoms. However, the role of Mesenchymal stem cells (MSCs) in tissue repair ability and immunomodulation, makes them as an appropriate tool for treatment of various allergic diseases. Several clinical and preclinical studies show that MSCs could be a promising alternative therapy to allergic diseases. Further, short chain fatty acids, produced from gut microbes by breaking down complex fibre-rich foods, acts through G-coupled receptor mediated activation of MSCs, and their role as key players involved in amelioration of allergic inflammation needs further investigation. Therefore, there is a need for understating the role of SCFAs on the activation of MSCs, which might shed light on the development of new therapeutic regime in allergy treatment. In summary, this review focuses on the underlying of therapeutic role of MSCs in different allergic diseases and the prospects of SCFA and MSC therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Fermentation of plant-based milk alternatives (PBMAs), including nut-based products, has the potential to generate new foods with improved sensorial properties. In this study, we screened 593 lactic acid bacteria (LAB) isolates from herbs, fruits and vegetables for their ability to acidify an almond-based milk alternative. The majority of the strongest acidifying plant-based isolates were identified as Lactococcus lactis, which were found to lower the pH of almond milk faster than dairy yoghurt cultures. Whole genome sequencing (WGS) of 18 plant-based Lc. lactis isolates revealed the presence of sucrose utilisation genes (sacR, sacA, sacB and sacK) in the strongly acidifying strains (n = 17), which were absent in one non-acidifying strain. To confirm the importance of Lc. lactis sucrose metabolism in efficient acidification of nut-based milk alternatives, we obtained spontaneous mutants defective in sucrose utilisation and confirmed their mutations by WGS. One mutant containing a sucrose-6-phosphate hydrolase gene (sacA) frameshift mutation was unable to efficiently acidify almond, cashew and macadamia nut milk alternatives. Plant-based Lc. lactis isolates were heterogeneous in their possession of the nisin gene operon near the sucrose gene cluster. The results of this work show that sucrose-utilising plant-based Lc. lactis have potential as starter cultures for nut-based milk alternatives., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Ulcerative colitis (UC) is a disorder with unknown etiology, and animal models play an essential role in studying its molecular pathophysiology. Here, we aim to identify common conserved pathological UC-related gene expression signatures between humans and mice that can be used as treatment targets and/or biomarker candidates. Methods: To identify differentially regulated protein-coding genes and non-coding RNAs, we sequenced total RNA from the colon and blood of the most widely used dextran sodium sulfate Ulcerative colitis mouse. By combining this with public human Ulcerative colitis data, we investigated conserved gene expression signatures and pathways/biological processes through which these genes may contribute to disease development/progression. Results: Cross-species integration of human and mouse Ulcerative colitis data resulted in the identification of 1442 genes that were significantly differentially regulated in the same direction in the colon and 157 in blood. Of these, 51 genes showed consistent differential regulation in the colon and blood. Less known genes with importance in disease pathogenesis, including SPI1, FPR2, TYROBP, CKAP4, MCEMP1, ADGRG3, SLC11A1, and SELPLG, were identified through network centrality ranking and validated in independent human and mouse cohorts. Conclusion: The identified Ulcerative colitis conserved transcriptional signatures aid in the disease phenotyping and future treatment decisions, drug discovery, and clinical trial design., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yarani, Palasca, Doncheva, Anthon, Pilecki, Svane, Mirza, Litman, Holmskov, Bang-Berthelsen, Vilien, Jensen, Gorodkin and Pociot.)

Plant-based food products are generating a growing interest as part of the ongoing transition to a primarily plant-based diet, which makes demands to the quality, functionality, and health properties of plant proteins. Microbes used for traditional food fermentations such as lactic acid bacteria (LAB) and fungi (yeasts and molds) carry out enzymatic changes on their protein substrates by which technological and sensorial characteristics can be improved. The literature on extracellular proteases targeting plant proteins, on the other hand, is scattered with only a narrow representation of plants even for traditionally plant-based products. Therefore, this review aims to explore the current state of knowledge regarding the application potential of microbial extracellular proteases targeting plant proteins, with a focus on traditional applied food microbes. Plant proteins are targeted by proteolytic microbes of both animal and plant origins, and their proteases show a wide range of activities. Extracellular microbial proteases can hydrolyze specific protein-based allergens and even reduce the toxicity of plant proteins. Additionally, microbial assisted proteolysis can improve plant protein digestibility by increasing availability of peptides and amino acids. This catabolic process will change the organoleptic characteristics of fermented plant proteins, and the release of bioactive peptides can provide additional functionalities to the plant matrix. The proteolytic activity is determined by the microbial strain, and it can be quite substrate selective, which is why proteases may be overlooked by the prevalent use of casein as substrate in proteolytic screenings. The synergetic effects of LAB and fungal species consortia can facilitate and steer plant protein hydrolysis by which co-fermentation may increase or change the properties of plant protein hydrolysates. Microbes do not necessarily require extracellular proteases because endogenous proteases in a plant-matrix may meet the microbial amino acid requirements. However, extracellular proteases have the potential to provide central properties to diverse food-matrixes by which the full proteolytic potential of food microbes needs to be explored in order to facilitate the development of high-quality plant-based food products., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Accelerated evolution of any portion of the genome is of significant interest, potentially signaling positive selection of phenotypic traits and adaptation. Accelerated evolution remains understudied for structured RNAs, despite the fact that an RNA's structure is often key to its function. RNA structures are typically characterized by compensatory (structure-preserving) basepair changes that are unexpected given the underlying sequence variation, i.e., they have evolved through negative selection on structure. We address the question of how fast the primary sequence of an RNA can change through evolution while conserving its structure. Specifically, we consider predicted and known structures in vertebrate genomes. After careful control of false discovery rates, we obtain 13 de novo structures (and three known Rfam structures) that we predict to have rapidly evolving sequences-defined as structures where the primary sequences of human and mouse have diverged at least twice as fast (1.5 times for Rfam) as nearby neutrally evolving sequences. Two of the three known structures function in translation inhibition related to infection and immune response. We conclude that rapid sequence divergence does not preclude RNA structure conservation in vertebrates, although these events are relatively rare., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Plant-based foods with desirable texture and nutritional value have attracted considerable interest from consumers. In order to meet the growing demand for more sustainable and health-focused products, new sources for plant-based products are needed. In this study, we aimed to develop an innovative plant-based dessert based on the underutilized crop chufa tubers ( Cyperus esculentus) . The chufa extract was fermented with plant-adapted lactic acid bacteria and formulated with the purpose of imitating the Danish summer dessert "cold butter-milk soup". The effect of various bacterial fermentations and formulations on steady and oscillatory rheology, stability, dry matter, pH, and sugar profile of the product were studied and compared to a commercial cold buttermilk soup sample. A strain of Leuconostoc mesenteroides was found to create the most similar taste to a commercial sample. By adding lemon juice, sucrose, xanthan gum, and vanilla to the fermented chufa drink, the drink was found to mimic the pH, texture, acid profile, and stability of a commercial dairy-based sample, while containing a lower concentration of carbohydrates.

Nourishment of the growing human population requires new and alternative food sources, preferable produced without occupying new land areas. Cultivation of seaweed presents an opportunity, however, a major obstacle is sustainable preservation. Fermentation has been used for centuries to preserve vegetables, e.g., to produce kimchi based on cabbage. This study investigated changes in the microbiota, characteristics (pH, organic acids and water soluble carbohydrates) and food safety of raw shredded Alaria esculenta and Saccharina latissima during fermentation by the natural microbiota with or without addition of a Lactiplantibacillus plantarum starter culture. The Lb. plantarum fermented products retained a high Shannon diversity index, indicating a partially unsuccessful fermentation. Lb. plantarum performed better in A. esculenta causing pH to drop to below 4.6, a critical limit for control of growth of Clostridium botulinum, within 2 days compared to 7 days for S. latissima. Natural fermentation by the endogenous microbiota resulted in unsafe products with high final pH values (4.8-5.2), presence of unwanted organic acids, such as butyric acid, and in the case of A. esculenta sustenance of inoculated Listeria monocytogenes. Fermentation of A. esculenta and S. latissima by Lb. plantarum is a promising preservation method. However, future work is needed to optimise the process, by investigation of the use of different starter cultures, seaweed pre-treatments (blanching, freezing, etc.) and adjuvants (i.e., addition of sugars, minerals and similar) to promote growth of the starter culture and ensure the fermented products are safe to eat., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Lactococcus lactis has great potential for high-yield production of mannitol, which has not yet been fully realized. In this study, we characterize how the mannitol genes in L. lactis are organized and regulated and use this information to establish efficient mannitol production. Although the organization of the mannitol genes in L. lactis was similar to that in other Gram-positive bacteria, mtlF and mtlD , encoding the enzyme IIA component (EIIA mtl ) of the mannitol phosphotransferase system (PTS) and the mannitol-1-phosphate dehydrogenase, respectively, were separated by a transcriptional terminator, and the mannitol genes were found to be organized in two transcriptional units: an operon comprising mtlA , encoding the enzyme IIBC component (EIIBC mtl ) of the mannitol PTS, mtlR , encoding a transcriptional activator, and mtlF , as well as a separately expressed mtlD gene. The promoters driving expression of the two transcriptional units were somewhat similar, and both contained predicted catabolite responsive element ( cre ) genes. The presence of carbon catabolite repression was demonstrated and was shown to be relieved in stationary-phase cells. The transcriptional activator MtlR ( mtlR ), in some Gram-positive bacteria, is repressed by phosphorylation by EIIA mtl , and when we knocked out mtlF , we indeed observed enhanced expression from the two promoters, which indicated that this mechanism was in place. Finally, by overexpressing the mtlD gene and using stationary-phase cells as biocatalysts, we attained 10.1 g/liter mannitol with a 55% yield, which, to the best of our knowledge, is the highest titer ever reported for L. lactis. Summing up, the results of our study should be useful for improving the mannitol-producing capacity of this important industrial organism. IMPORTANCE Lactococcus lactis is the most studied species of the lactic acid bacteria, and it is widely used in various food fermentations. To date, there have been several attempts to persuade L. lactis to produce mannitol, a sugar alcohol with important therapeutic and food applications. Until now, to achieve mannitol production in L. lactis with significant titer and yield, it has been necessary to introduce and express foreign genes, which precludes the use of such strains in foods, due to their recombinant status. In this study, we systematically characterize how the mannitol genes in L. lactis are regulated and demonstrate how this impacts mannitol production capability. We harnessed this information and managed to establish efficient mannitol production without introducing foreign genes.

With emerging interests in heterologous production of proteins such as antibodies, growth factors, nanobodies, high-quality protein food ingredients, etc. the demand for efficient production hosts increases. Corynebacterium glutamicum is an attractive industrial host with great secretion capacity to produce therapeutics. It lacks extracellular protease and endotoxin activities and easily achieves high cell density. Therefore, this study focuses on improving protein production and secretion in C. glutamicum with the use of droplet-based microfluidic (DBM) high throughput screening. A library of C. glutamicum secreting β-glucosidase was generated using chemical mutagenesis coupled with DBM screening of 200,000 mutants in just 20 min. Among 100 recovered mutants, 16 mutants exhibited enhanced enzyme secretion capacity, 13 of which had unique mutation profiles. Whole-genome analysis showed that approximately 50-150 SNVs had occurred on the chromosome per mutant. Functional enrichment analysis of genes with non-synonymous mutations showed overrepresentation of genes involved in protein synthesis and secretion relevant biological processes, such as DNA and ribosome RNA synthesis, protein secretion and energy turnover. Two mutants JCMT1 and JCMT8 exhibited the highest secretion with a six and a fivefold increase in the β-glucosidase activity in the supernatant, respectively, relative to the reference strain JC0190. After plasmid curing, a new plasmid with the gene encoding α-amylase was cloned into these two mutants. The new strains SB024 and SB025 also exhibited a five and a sixfold increase in α-amylase activity in the supernatant, respectively, relative to the reference strain SB023. The results demonstrate how DBM screening can serve as a powerful development tool to improve cell factories for the production and secretion of heterologous proteins., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Balasubramanian, Chen, Wigneswaran, Bang-Berthelsen and Jensen.)

Allergen-specific immunotherapy (IT) is emerging as a viable avenue for the treatment of food allergies. Clinical trials currently investigate raw or slightly processed foods as therapeutic agents, as trials using food-grade agents can be performed without the strict regulations to which conventional drugs are subjected. However, this limits the ability of standardization and may affect clinical trial outcomes and reproducibility. Herein, we provide an overview of methods used in the production of immunotherapeutic agents for the treatment of food allergies, including processed foods, allergen extracts, recombinant allergens, and synthetic peptides, as well as the physical and chemical processes for the reduction of protein allergenicity. Commercial interests currently favor producing standardized drug-grade allergen extracts for therapeutic use, and clinical trials are ongoing. In the near future, recombinant production could replace purification strategies since it allows the manufacturing of pure, native allergens or sequence-modified allergens with reduced allergenicity. A recurring issue within this field is the inadequate reporting of production procedures, quality control, product physicochemical characteristics, allergenicity, and immunological properties. This information is of vital importance in assessing therapeutic standardization and clinical safety profile, which are central parameters for the development of future therapeutic agents.

Mannitol can be obtained as a by-product of certain heterolactic lactic acid bacteria, when grown on substrates containing fructose. Lactococcus lactis , a homolactic lactic acid bacterium, normally does not form mannitol but can be persuaded into doing so by expressing certain foreign enzyme activities. In this study, we find that L. lactis has an inherent capacity to form mannitol from glucose. By adaptively evolving L. lactis or derivatives blocked in NAD + regenerating pathways, we manage to accelerate growth on mannitol. When cells of the adapted strains are resuspended in buffer containing glucose, 4-58% of the glucose metabolized is converted into mannitol, in contrast to nonadapted strains. The highest conversion was obtained for a strain lacking all major NAD + regenerating pathways. Mannitol had an inhibitory effect on the conversion, which we speculated was due to the mannitol uptake system. After its inactivation, 60% of the glucose was converted into mannitol by cells suspended in glucose buffer. Using a two-stage setup, where biomass first was accumulated by aerated culturing, followed by a nonaerated phase (static conditions), it was possible to obtain 6.1 g/L mannitol, where 60% of the glucose had been converted into mannitol, which is the highest yield reported for L. lactis .

Lactococcus lactis is globally used in food fermentation. Genomics is useful to investigate speciation and differential occurrence of (un)desired gene functions, often related to mobile DNA. This study investigates L. lactis for putative chromosomal mobile genetic elements through comparative genomics, and analyses how they contribute to chromosomal variation at strain level. Our work identified 95 loci that may range over 10% of the chromosome size when including prophages, and the loci display a marked differential occurrence in the analysed strains. Analysis of differential transcriptomics data revealed how mobile genetic elements may impact the host physiology in response to conditional changes. This insight in the genetic variation of mobile genetic elements in L. lactis holds potential to further identify important functions related to food and biotechnology applications within this important species., (© FEMS 2019.)

Purpose: To identify the mutation for Volkmann cataract ( CTRCT8 ) at 1p36.33., Methods: The genes in the candidate region 1p36.33 were Sanger and parallel deep sequenced, and informative single nucleotide polymorphisms (SNPs) were identified for linkage analysis. Expression analysis with reverse transcription polymerase chain reaction (RT-PCR) of the candidate gene was performed using RNA from different human tissues. Quantitative transcription polymerase chain reaction (qRT-PCR) analysis of the GNB1 gene was performed in affected and healthy individuals. Bioinformatic analysis of the linkage regions including the candidate gene was performed., Results: Linkage analysis of the 1p36.33 CCV locus applying new marker systems obtained with Sanger and deep sequencing reduced the candidate locus from 2.1 Mb to 0.389 Mb flanked by the markers STS-22AC and rs549772338 and resulted in an logarithm of the odds (LOD) score of Z = 21.67. The identified mutation, rs763295804, affects the donor splice site in the long non-coding RNA gene RP1-140A9.1 (ENSG00000231050). The gene including splice-site junctions is conserved in primates but not in other mammalian genomes, and two alternative transcripts were shown with RT-PCR. One of these transcripts represented a lens cell-specific transcript. Meta-analysis of the Cross-Linking-Immuno-Precipitation sequencing (CLIP-Seq) data suggested the RNA binding protein (RBP) eIF4AIII is an active counterpart for RP1-140A9.1 , and several miRNA and transcription factors binding sites were predicted in the proximity of the mutation. ENCODE DNase I hypersensitivity and histone methylation and acetylation data suggest the genomic region may have regulatory functions., Conclusions: The mutation in RP1-140A9.1 suggests the long non-coding RNA as the candidate cataract gene associated with the autosomal dominant inherited congenital cataract from CCV. The mutation has the potential to destroy exon/intron splicing of both transcripts of RP1-140A9.1 . Sanger and massive deep resequencing of the linkage region failed to identify alternative candidates suggesting the mutation in RP1-140A9.1 is causative for the CCV phenotype.

Accurate prediction of regional lymph node metastases (LNM) in endoscopically resected pT1 colorectal cancer (CRC) is crucial in treatment stratification for subsequent radical surgery. Several miRNAs have been linked to CRC invasion and metastasis, including the oncogenic miR-17/92 cluster, and expression levels might have predictive value in the risk assessment of early metastatic progression in CRC. We performed global miRNA microarray using tissue samples from the invasive front of pT1 CRC and investigated associations of the miR-17/92 cluster and presence of LNM. In total, 56 matched pT1 CRCs were thoroughly clinicopathologically characterized, and miRNA microarrays were performed on invasive front tissue samples. Global miRNA intensities were screened using paired t-tests between pT1pN+ and pT1pN0. Associations between miR-17/92 and histopathological features were analyzed using general linear models and tumor cell adjusted expression intensities. miR-17-3p and miR-92a were significantly higher expressed in the invasive front of tumors with LNM compared to those without, corresponding to 1.53-fold higher expression of miR-17-3p (95%CI: 1.04-2.24, P = .030) and 1.28-fold higher expression of miR-92a (95%CI: 1.01-1.68, P = .042). An inverse association between miR-19a and presence of high-grade tumor budding was observed (1.55-fold, 95%CI: 1.13-2.12, P = .008). We provide evidence for associations between early regional LNM and high expression levels of the miR-17/92 cluster members: miR-17-3p and miR-92a, in the invasive front of CRC. Our results support a role for the miR-17/92 cluster in early metastatic progression of CRC and calls for further investigation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization, and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for conserved RNA structures (CRSs), leveraging structure-based, rather than sequence-based, alignments. After careful correction for sequence identity and GC content, we predict ∼516,000 human genomic regions containing CRSs. We find that a substantial fraction of human-mouse CRS regions (1) colocalize consistently with binding sites of the same RNA binding proteins (RBPs) or (2) are transcribed in corresponding tissues. Additionally, a CaptureSeq experiment revealed expression of many of our CRS regions in human fetal brain, including 662 novel ones. For selected human and mouse candidate pairs, qRT-PCR and in vitro RNA structure probing supported both shared expression and shared structure despite low abundance and low sequence identity. About 30,000 CRS regions are located near coding or long noncoding RNA genes or within enhancers. Structured (CRS overlapping) enhancer RNAs and extended 3' ends have significantly increased expression levels over their nonstructured counterparts. Our findings of transcribed uncharacterized regulatory regions that contain CRSs support their RNA-mediated functionality., (© 2017 Seemann et al.; Published by Cold Spring Harbor Laboratory Press.)

miRNAs are small 22-nucleotide RNAs that can post-transcriptionally regulate gene expression. It has been proposed that dietary plant miRNAs can enter the human bloodstream and regulate host transcripts; however, these findings have been widely disputed. We here conduct the first comprehensive meta-study in the field, surveying the presence and abundances of cross-species miRNAs (xenomiRs) in 824 sequencing data sets from various human tissues and body fluids. We find that xenomiRs are commonly present in tissues (17%) and body fluids (69%); however, the abundances are low, comprising 0.001% of host human miRNA counts. Further, we do not detect a significant enrichment of xenomiRs in sequencing data originating from tissues and body fluids that are exposed to dietary intake (such as liver). Likewise, there is no significant depletion of xenomiRs in tissues and body fluids that are relatively separated from the main bloodstream (such as brain and cerebro-spinal fluids). Interestingly, the majority (81%) of body fluid xenomiRs stem from rodents, which are a rare human dietary contribution but common laboratory animals. Body fluid samples from the same studies tend to group together when clustered by xenomiR compositions, suggesting technical batch effects. Last, we performed carefully designed and controlled animal feeding studies, in which we detected no transfer of plant miRNAs into rat blood, or bovine milk sequences into piglet blood. In summary, our comprehensive computational and experimental results indicate that xenomiRs originate from technical artifacts rather than dietary intake., (© 2017 Kang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Background: Beneficial roles for glucagon-like peptide 1 (GLP-1)/GLP-1R signaling have recently been described in diseases, where low-grade inflammation is a common phenomenon. We investigated the effects of GLP-1 in Brunner's glands and duodenum with abundant expression of GLP-1 receptors, as well as GLP-1 effect on colonic inflammation., Methods: RNA from Brunner's glands of GLP-1R knockout and wild-type mice were subjected to full transcriptome profiling. Array results were validated by quantitative reverse transcription polymerase chain reaction in wild-type mice and compared with samples from inflammatory bowel disease (IBD) patients and controls. In addition, we performed a detailed investigation of the effects of exogenous liraglutide dosing in a T-cell driven adoptive transfer (AdTr) colitis mouse model., Results: Analyses of the Brunner's gland transcriptomes of GLP-1R knockout and wild-type mice identified 722 differentially expressed genes. Upregulated transcripts after GLP-1 dosing included IL-33, chemokine ligand 20 (CCL20), and mucin 5b. Biopsies from IBD patients and controls, as well as data from the AdTr model, showed deregulated expression of GLP-1R, CCL20, and IL-33 in colon. Circulating levels of GLP-1 were found to be increased in mice with colitis. Finally, the colonic cytokine levels and disease scores of the AdTr model indicated reduced levels of colonic inflammation in liraglutide-dosed animals., Conclusions: We demonstrate that IL-33, GLP-1R, and CCL20 are deregulated in human IBD, and that prophylactic treatment with 0.6 mg/kg liraglutide improves disease in AdTr colitis. In addition, GLP-1 receptor agonists upregulate IL-33, mucin 5b, and CCL20 in murine Brunner's glands. Taken together, our data indicate that GLP-1 receptor agonists affect gut homeostasis in both proximal and distal parts of the gut.

As microRNAs (miRs) are gaining increasing attention as key regulators of cellular processes, expressional quantification is widely applied. However, in the processing of relatively quantified data, the importance of testing the stability of several reference mRNAs and/or miRs and choosing among these for normalization is often overlooked, potentially leading to biased results. Here, we have optimized the purification of miR-enriched total RNA from pancreatic insulin-producing INS-1 cells. Additionally, we optimized and analyzed miR expression by a qPCR-based microarray and by specific qPCR and tested the stability of candidate reference mRNAs and miRs. Hence, this study gives a widely applicable example on how to easily and systematically test and decide how to normalize miR quantification. We suggest that caution in the interpretation of miR quantification studies that do not comprise stability analysis should be exerted.

In the last decade, there has been an explosion in both the number of and knowledge about miRNAs associated with both type 1 and type 2 diabetes. Even though we are presently in the initial stages of understanding how this novel class of posttranscriptional regulators are involved in diabetes, recent studies have demonstrated that miRNAs are important regulators of the islet transcriptome, controlling apoptosis, differentiation and proliferation, as well as regulating unique islet and beta-cell functions and pathways such as insulin expression, processing and secretion. Furthermore, a large number of miRNAs have been linked to diabetogenic processes induced by elevated levels of glucose, free fatty acids and inflammatory cytokines. Thus, miRNAs are novel therapeutic targets with the potential of protecting the beta-cell, and there is proof of principle that miRNA antagonists, so-called antagomirs, are effective in vivo for other disorders. miRNAs are exported out of cells in exosomes, raising the intriguing possibility of cell-to-cell communication between distant tissues via miRNAs and that miRNAs can be used as biomarkers of beta-cell function, mass and survival. The purpose of this review is to provide a status on how miRNAs control beta-cell function and viability in health and disease., (Copyright © 2015 John Wiley & Sons, Ltd.)

Background: microRNAs (miRNAs) are important regulators of translation and have been implicated in the pathogenesis of a number of cardiovascular diseases, including stroke, and suggested as possible prognostic biomarkers. Our aim was to identify miRNAs that are differentially regulated in cerebral arteries after subarachnoid hemorrhage (SAH), using a rat injection model of SAH and a qPCR-based screen of 728 rat miRNAs. Additionally, serum was analyzed for a possible spill-over to the circulation of regulated miRNAs from the vessel walls., Results: We identified 482 different miRNAs expressed in cerebral arteries post-SAH. Two miRNAs, miR-30a and miR-143, were significantly upregulated in cerebral arteries after SAH when compared to sham-operated animals. However, none of these exhibited significantly altered serum levels after SAH versus post-sham surgery. The most robust upregulation was seen for miR-143, which has several predicted targets and is a strong regulator of vascular morphology. We hypothesize that miR-30a and miR-143 may play a role in the vascular wall changes seen after SAH., Conclusions: We report that miR-30a and miR-143 in the cerebral arteries show significant changes over time after SAH, but do not differ from sham-operated rats at 24 h post-SAH. Although this finding suggests interesting novel possible mechanisms involved in post-SAH cerebrovascular changes, the lack of regulation of these miRNAs in serum excludes their use as blood-borne biomarkers for cerebrovascular changes following SAH.

MicroRNAs (miRNAs) are involved in the pathogenesis of many human diseases, including some neurological disorders. Recently, we have reported dysregulated miRNAs in plasma from patients with central hypersomnias including type 1 and type 2 narcolepsy, and idiopathic hypersomnia. This study addressed whether miRNA levels are altered in the cerebrospinal fluid (CSF) of patients with central hypersomnias. We conducted high-throughput analyses of miRNAs in CSF from patients using quantitative real-time polymerase chain reaction panels. We identified 13, 9, and 11 miRNAs with a more than two-fold change in concentration in CSF from patients with type 1 and type 2 narcolepsy and idiopathic hypersomnia, respectively, compared with matched healthy controls. Most miRNAs differed in more than one of the sleep disorders. However, all miRNAs were detected at low levels in CSF and varied between individuals. None of them showed significant differences in concentrations between groups after correcting for multiple testing, and none could be validated in an independent cohort. Nevertheless, approximately 60% of the most abundant miRNAs in the profile reported here have previously been identified in the CSF of healthy individuals, showing consistency with previous miRNA profiles found in CSF. In conclusion, we were not able to demonstrate distinct levels or patterns of miRNAs in CSF from central hypersomnia patients., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Study Objectives: MicroRNAs (miRNAs) have been implicated in the pathogenesis of human diseases including neurological disorders. The aim is to address the involvement of miRNAs in the pathophysiology of central hypersomnias including autoimmune narcolepsy with cataplexy and hypocretin deficiency (type 1 narcolepsy), narcolepsy without cataplexy (type 2 narcolepsy), and idiopathic hypersomnia., Design: We conducted high-throughput analysis of miRNA in plasma from three groups of patients-with type 1 narcolepsy, type 2 narcolepsy, and idiopathic hypersomnia, respectively-in comparison with healthy controls using quantitative real-time polymerase chain reaction (qPCR) panels., Setting: University hospital based sleep clinic and research laboratories., Patients: Twelve patients with type 1 narcolepsy, 12 patients with type 2 narcolepsy, 12 patients with idiopathic hypersomnia, and 12 healthy controls., Measurements and Results: By analyzing miRNA in plasma with qPCR we identified 50, 24, and 6 miRNAs that were different in patients with type 1 narcolepsy, type 2 narcolepsy, and idiopathic hypersomnia, respectively, compared with healthy controls. Twenty miRNA candidates who fulfilled the criteria of at least two-fold difference and p-value < 0.05 were selected to validate the miRNA changes in an independent cohort of patients. Four miRNAs differed significantly between type 1 narcolepsy patients and healthy controls. Levels of miR-30c, let-7f, and miR-26a were higher, whereas the level of miR-130a was lower in type 1 narcolepsy than healthy controls. The miRNA differences were not specific for type 1 narcolepsy, since the levels of the four miRNAs were also altered in patients with type 2 narcolepsy and idiopathic hypersomnia compared with healthy controls., Conclusion: The levels of four miRNAs differed in plasma from patients with type 1 narcolepsy, type 2 narcolepsy and idiopathic hypersomnia suggesting that alterations of miRNAs may be involved in the pathophysiology of central hypersomnias., (© 2014 Associated Professional Sleep Societies, LLC.)

Over 40 susceptibility loci have been identified for type 1 diabetes (T1D). Little is known about how these variants modify disease risk and progression. Here, we combined in vitro and in vivo experiments with clinical studies to determine how genetic variation of the candidate gene cathepsin H (CTSH) affects disease mechanisms and progression in T1D. The T allele of rs3825932 was associated with lower CTSH expression in human lymphoblastoid cell lines and pancreatic tissue. Proinflammatory cytokines decreased the expression of CTSH in human islets and primary rat β-cells, and overexpression of CTSH protected insulin-secreting cells against cytokine-induced apoptosis. Mechanistic studies indicated that CTSH exerts its antiapoptotic effects through decreased JNK and p38 signaling and reduced expression of the proapoptotic factors Bim, DP5, and c-Myc. CTSH overexpression also up-regulated Ins2 expression and increased insulin secretion. Additionally, islets from Ctsh(-/-) mice contained less insulin than islets from WT mice. Importantly, the TT genotype was associated with higher daily insulin dose and faster disease progression in newly diagnosed T1D patients, indicating agreement between the experimental and clinical data. In line with these observations, healthy human subjects carrying the T allele have lower β-cell function, which was evaluated by glucose tolerance testing. The data provide strong evidence that CTSH is an important regulator of β-cell function during progression of T1D and reinforce the concept that candidate genes for T1D may affect disease progression by modulating survival and function of pancreatic β-cells, the target cells of the autoimmune assault.

The Cd247 gene encodes for a transmembrane protein important for the expression and assembly of TCR/CD3 complex on the surface of T lymphocytes. Down-regulation of CD247 has functional consequences in systemic autoimmunity and has been shown to be associated with Type 1 Diabetes in NOD mouse. In this study, we have utilized the wealth of high-throughput sequencing data produced during the Encyclopedia of DNA Elements (ENCODE) project to identify spatially conserved regulatory elements within the Cd247 gene from human and mouse. We show the presence of two transcription factor binding sites, supported by histone marks and ChIP-seq data, that specifically have features of an enhancer and a promoter, respectively. We also identified a putative long non-coding RNA from the characteristically long first intron of the Cd247 gene. The long non-coding RNA annotation is supported by manual annotations from the GENCODE project in human and our expression quantification analysis performed in NOD and B6 mice using qRT-PCR. Furthermore, 17 of the 23 SNPs already known to be implicated with T1D were observed within the long non-coding RNA region in mouse. The spatially conserved regulatory elements identified in this study have the potential to enrich our understanding of the role of Cd247 gene in autoimmune diabetes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Type 1 Diabetes (T1D) is an autoimmune disease where local release of cytokines such as IL-1β and IFN-γ contributes to β-cell apoptosis. To identify relevant genes regulating this process we performed a meta-analysis of 8 datasets of β-cell gene expression after exposure to IL-1β and IFN-γ. Two of these datasets are novel and contain time-series expressions in human islet cells and rat INS-1E cells. Genes were ranked according to their differential expression within and after 24 h from exposure, and characterized by function and prior knowledge in the literature. A regulatory network was then inferred from the human time expression datasets, using a time-series extension of a network inference method. The two most differentially expressed genes previously unknown in T1D literature (RIPK2 and ELF3) were found to modulate cytokine-induced apoptosis. The inferred regulatory network is thus supported by the experimental validation, providing a proof-of-concept for the proposed statistical inference approach., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background and Aim. Hepatitis B e antigen positive (HBeAg-positive) children are at high risk of severe complications such as hepatocellular carcinoma and cirrhosis. Liver damage is caused by the host immune response to infected hepatocytes, and we hypothesise that specific microRNAs play a role in this complex interaction between virus and host. The study aimed to identify microRNAs with aberrant plasma expressions in HBeAg-positive children and with liver-specific target genes. Methods. By revisiting our previous screen of microRNA plasma levels in HBeAg-positive and HBeAg-negative children with chronic hepatitis B (CHB) and in healthy controls, candidate microRNAs with aberrant plasma expressions in HBeAg-positive children were identified. MicroRNAs targeting liver-specific genes were selected based on bioinformatics analysis and validated by qRT-PCR using plasma samples from 34 HBeAg-positive, 26 HBeAg-negative, and 60 healthy control children. Results. Thirteen microRNAs showed aberrant plasma expressions in HBeAg-positive children and targeted liver-specific genes. In particular, three microRNAs were upregulated and one was downregulated in HBeAg-positive children compared to HBeAg-negative and healthy control children, which showed equal levels. Conclusion. The identified microRNAs might impact the progression of CHB in children. Functional studies are warranted, however, to elucidate the microRNAs' role in the immunopathogenesis of childhood CHB.

Background and Aim: Children with chronic hepatitis B (CHB) are at high risk of progressive liver disease. It is suggested that a newly-identified panel of 16 microRNAs is important in the pathogenesis of CHB in children. Subviral hepatitis B surface antigen (HBsAg) particles are produced in large excess over infectious virions. Interestingly, circulating HBsAg particles have been shown to carry microRNAs. A thorough characterisation of the identified microRNAs and HBsAg over time in plasma from children with CHB may provide useful information about the natural course of childhood CHB., Patients and Methods: A cohort of 42 children with CHB was followed over time. Three to five blood samples were obtained from each child at minimum intervals of half a year; in total 180 blood samples. Plasma levels of the 16 microRNAs previously identified were analysed by quantitative real-time polymerase-chain-reaction. Plasma HBsAg was quantified using ARCHITECT® HBsAg assay., Results: The presence of 14/16 plasma microRNAs in children with CHB was confirmed. All 14 microRNAs were significantly differentially expressed in different immunological phases of the disease. MicroRNA plasma levels were highest in immune-tolerant children, lower in immune-active children, and reached the lowest values in immune-inactive children, p<0.001. Plasma levels of four microRNAs decreased significantly over time in immune-tolerant and immune-active children whereas the microRNA plasma levels were stable in immune-inactive children, p<0.004. HBsAg quantity was positively correlated with plasma levels of 11/14 microRNAs, p<0.004., Conclusion: This is the first study to characterise plasma microRNAs and HBsAg over time in children with CHB. Our data suggest that plasma levels of selected microRNAs and HBsAg are inversely correlated with immunological control of CHB in children. Further studies are, however, needed to advance the understanding of microRNAs and HBsAg in the pathogenesis of CHB in children.

Type 1 diabetes is considered an autoimmune disease characterised by specific T cell-mediated destruction of the insulin-producing beta cells. Yet, except for insulin, no beta cell-specific antigens have been discovered. This may imply that the autoantigens in type 1 diabetes exist in modified forms capable of specifically triggering beta cell destruction. In other immune-mediated diseases, autoantigens targeted by the immune system have undergone post-translational modification (PTM), thereby creating tissue-specific neo-epitopes. In a similar manner, PTM of beta cell proteins might create beta cell-specific neo-epitopes. We suggest that the current paradigm of type 1 diabetes as a classical autoimmune disease should be reconsidered since the immune response may not be directed against native beta cell proteins. A modified model for the pathogenetic events taking place in islets leading to the T cell attack against beta cells is presented. In this model, PTM plays a prominent role in triggering beta cell destruction. We discuss literature of relevance and perform genetic and human islet gene expression analyses. Both direct and circumstantial support for the involvement of PTM in type 1 diabetes exists in the published literature. Furthermore, we report that cytokines change the expression levels of several genes encoding proteins involved in PTM processes in human islets, and that there are type 1 diabetes-associated polymorphisms in a number of these. In conclusion, data from the literature and presented experimental data support the notion that PTM of beta cell proteins may be involved in triggering beta cell destruction in type 1 diabetes. If the beta cell antigens recognised by the immune system foremost come from modified proteins rather than native ones, the concept of type 1 diabetes as a classical autoimmune disease is open for debate.

Background and Aim: Children chronically infected with hepatitis B virus (HBV) are at high risk of progressive liver disease. However, no treatment is available that is consistently effective in curing chronic hepatitis B (CHB) in children. Improved understanding of the natural course of disease is warranted. Identification of specific microRNA (miRNA) profiles in children chronically infected with HBV may provide insight into the pathogenesis of CHB and lead to advances in the management of children with CHB., Patients and Methods: MiRNA PCR panels were employed to screen plasma levels of 739 miRNAs in pooled samples from HBeAg positive, HBeAg negative, and healthy children. The three groups' plasma miRNA profiles were compared, and aberrantly expressed miRNAs were identified. The identified miRNAs were then validated. Individual RT-qPCRs were performed on plasma from 34 HBeAg positive, 26 HBeAg negative, and 60 healthy children., Results: A panel of 16 plasma miRNAs were identified as aberrantly expressed in HBeAg positive and HBeAg negative children (p<0.001). Levels of all of the miRNAs were upregulated in HBeAg positive children compared with in HBeAg negative children. A positive correlation was furthermore found between plasma levels of the identified miRNAs and HBV DNA (p<0.001)., Conclusion: We are the first to investigate the plasma miRNA profile of children chronically infected with HBV. Our data indicates the existence of a relationship between abundance of circulating miRNAs and immunological stages in the natural course of disease. Certain miRNAs may contribute to the establishment and maintenance of CHB in children. Further studies are warranted to advance understanding of miRNAs in the pathogenesis of CHB, hopefully leading to the identification of future therapeutic targets.

Genome-wide association studies (GWAS) have heralded a new era in susceptibility locus discovery in complex diseases. For type 1 diabetes, >40 susceptibility loci have been discovered. However, GWAS do not inevitably lead to identification of the gene or genes in a given locus associated with disease, and they do not typically inform the broader context in which the disease genes operate. Here, we integrated type 1 diabetes GWAS data with protein-protein interactions to construct biological networks of relevance for disease. A total of 17 networks were identified. To prioritize and substantiate these networks, we performed expressional profiling in human pancreatic islets exposed to proinflammatory cytokines. Three networks were significantly enriched for cytokine-regulated genes and, thus, likely to play an important role for type 1 diabetes in pancreatic islets. Eight of the regulated genes (CD83, IFNGR1, IL17RD, TRAF3IP2, IL27RA, PLCG2, MYO1B, and CXCR7) in these networks also harbored single nucleotide polymorphisms nominally associated with type 1 diabetes. Finally, the expression and cytokine regulation of these new candidate genes were confirmed in insulin-secreting INS-1 β-cells. Our results provide novel insight to the mechanisms behind type 1 diabetes pathogenesis and, thus, may provide the basis for the design of novel treatment strategies.

This study aims to identify key miRNAs in circulation, which predict ongoing beta-cell destruction and regeneration in children with newly diagnosed Type 1 Diabetes (T1D). We compared expression level of sera miRNAs from new onset T1D children and age-matched healthy controls and related the miRNAs expression levels to beta-cell function and glycaemic control. Global miRNA sequencing analyses were performed on sera pools from two T1D cohorts (n = 275 and 129, resp.) and one control group (n = 151). We identified twelve upregulated human miRNAs in T1D patients (miR-152, miR-30a-5p, miR-181a, miR-24, miR-148a, miR-210, miR-27a, miR-29a, miR-26a, miR-27b, miR-25, miR-200a); several of these miRNAs were linked to apoptosis and beta-cell networks. Furthermore, we identified miR-25 as negatively associated with residual beta-cell function (est.: -0.12, P = 0.0037), and positively associated with glycaemic control (HbA1c) (est.: 0.11, P = 0.0035) 3 months after onset [corrected]. In conclusion this study demonstrates that miR-25 might be a "tissue-specific" miRNA for glycaemic control 3 months after diagnosis in new onset T1D children and therefore supports the role of circulating miRNAs as predictive biomarkers for tissue physiopathology and potential intervention targets.

Background: Several approaches have been developed for miRNA target prediction, including methods that incorporate expression profiling. However the methods are still in need of improvements due to a high false discovery rate. So far, none of the methods have used independent component analysis (ICA). Here, we developed a novel target prediction method based on ICA that incorporates both seed matching and expression profiling of miRNA and mRNA expressions. The method was applied on a cellular model of type 1 diabetes., Results: Microarray profiling identified eight miRNAs (miR-124/128/192/194/204/375/672/708) with differential expression. Applying ICA on the mRNA profiling data revealed five significant independent components (ICs) correlating to the experimental conditions. The five ICs also captured the miRNA expressions by explaining > 97% of their variance. By using ICA, seven of the eight miRNAs showed significant enrichment of sequence predicted targets, compared to only four miRNAs when using simple negative correlation. The ICs were enriched for miRNA targets that function in diabetes-relevant pathways e.g. type 1 and type 2 diabetes and maturity onset diabetes of the young (MODY)., Conclusions: In this study, ICA was applied as an attempt to separate the various factors that influence the mRNA expression in order to identify miRNA targets. The results suggest that ICA is better at identifying miRNA targets than negative correlation. Additionally, combining ICA and pathway analysis constitutes a means for prioritizing between the predicted miRNA targets. Applying the method on a model of type 1 diabetes resulted in identification of eight miRNAs that appear to affect pathways of relevance to disease mechanisms in diabetes.

Background: Prolonged periods of high glucose exposure results in human islet dysfunction in vitro. The underlying mechanisms behind this effect of high glucose are, however, unknown. The polypyrimidine tract binding protein (PTB) is required for stabilization of insulin mRNA and the PTB mRNA 3'-UTR contains binding sites for the microRNA molecules miR-133a, miR-124a and miR-146. The aim of this study was therefore to investigate whether high glucose increased the levels of these three miRNAs in association with lower PTB levels and lower insulin biosynthesis rates., Methodology/principal Findings: Human islets were cultured for 24 hours in the presence of low (5.6 mM) or high glucose (20 mM). Islets were also exposed to sodium palmitate or the proinflammatory cytokines IL-1beta and IFN-gamma, since saturated free fatty acids and cytokines also cause islet dysfunction. RNA was then isolated for real-time RT-PCR analysis of miR-133a, miR-124a, miR-146, insulin mRNA and PTB mRNA contents. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. Synthetic miR-133a precursor and inhibitor were delivered to dispersed islet cells by lipofection, and PTB was analyzed by immunoblotting following culture at low or high glucose. Culture in high glucose resulted in increased islet contents of miR-133a and reduced contents of miR-146. Cytokines increased the contents of miR-146. The insulin and PTB mRNA contents were unaffected by high glucose. However, both PTB protein levels and insulin biosynthesis rates were decreased in response to high glucose. The miR-133a inhibitor prevented the high glucose-induced decrease in PTB and insulin biosynthesis, and the miR-133a precursor decreased PTB levels and insulin biosynthesis similarly to high glucose., Conclusion: Prolonged high-glucose exposure down-regulates PTB levels and insulin biosynthesis rates in human islets by increasing miR-133a levels. We propose that this mechanism contributes to hyperglycemia-induced beta-cell dysfunction.

The aim of this study was to characterize two monoclonal antibodies (F6A11 and F109-D12) generated against Pdx1 (pancreatic and duodenal homeobox-1), a homeodomain transcription factor, which is critical for pancreas formation as well as for normal pancreatic beta cell function. For production of monoclonal antibodies, we immunized Robertsonian POSF (RBF)mice with a GST-Pdx1 fusion protein containing a 68-amino acid C-terminal fragment of rat Pdx1. These monoclonal antibodies detect Pdx1 by western blotting and allow immunohistochemical detection of Pdx1 in both mouse and rat tissue. F6A11 and F109-D12 produce IHC staining patterns indistinguishable from that obtained with highly specific polyclonal Pdx1 antisera raised in rabbits and goats, when applied to embryonic or adult mouse pancreatic tissue. In contrast to previously generated polyclonal anti-Pdx1 antisera, we also demonstrate that F6A11 works for intracellular fluorescence activated cell sorting (FACS) staining of Pdx1. By using F6A11, we characterize the induction of Pdx1 in the Doxycycline (DOX) inducible insulinoma cell line INSralphabeta-Pdx1 and follow the reduction of Pdx1 after removing Dox. Finally, we show that induction of exogenous Pdx1 leads to a reduction in endogenous Pdx1 levels, which suggests that a negative feedback loop is involved in maintaining correct levels of Pdx1 in the cell.

Aims/hypothesis: The suppressor of cytokine signalling 1 (SOCS1) is a natural inhibitor of cytokine and insulin signalling pathways and may also play a role in obesity. In addition, SOCS1 is considered a candidate gene in the pathogenesis of both type 1 diabetes (T1D) and type 2 diabetes (T2D). The objective was to perform mutation analysis of SOCS1 and to test the identified variations for association to T2D-related quantitative traits, T2D or T1D., Methods: Mutation scanning was performed by direct sequencing in 27 white Danish subjects. Genotyping was carried out by TaqMan allelic discrimination. A total of more than 8100 individuals were genotyped., Results: Eight variations were identified in the 5' untranslated region (UTR) region. Two of these had allele frequencies below 1% and were not further examined. The six other variants were analysed in groups of T1D families (n = 1461 subjects) and T2D patients (n = 1430), glucose tolerant first-degree relatives of T2D patients (n = 212) and normal glucose tolerant (NGT) subjects. The rs33977706 polymorphism (-820G > T) was associated with a lower body mass index (BMI) (p = 0.004). In a second study (n = 4625 NGT subjects), significant associations of both the rs33977706 and the rs243330 (-1656G > A) variants to obesity were found (p = 0.047 and p = 0.015) respectively. The rs33977706 affected both binding of a nuclear protein to and the transcriptional activity of the SOCS1 promoter, indicating a relationship between this polymorphism and gene regulation., Conclusions/interpretation: This study demonstrates that functional variations in the SOCS1 promoter may associate with alterations in BMI in the general white population.

Background: Beta-cells are extremely rich in zinc and zinc homeostasis is regulated by zinc transporter proteins. beta-cells are sensitive to cytokines, interleukin-1beta (IL-1beta) has been associated with beta-cell dysfunction and -death in both type 1 and type 2 diabetes. This study explores the regulation of zinc transporters following cytokine exposure., Methods: The effects of cytokines IL-1beta, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) on zinc transporter gene expression were measured in INS-1-cells and rat pancreatic islets. Being the more sensitive transporter, we further explored ZnT8 (Slc30A8): the effect of ZnT8 over expression on cytokine induced apoptosis was investigated as well as expression of the insulin gene and two apoptosis associated genes, BAX and BCL2., Results: Our results showed a dynamic response of genes responsible for beta-cell zinc homeostasis to cytokines: IL-1beta down regulated a number of zinc-transporters, most strikingly ZnT8 in both islets and INS-1 cells. The effect was even more pronounced when mixing the cytokines. TNF-alpha had little effect on zinc transporter expression. IFN-gamma down regulated a number of zinc transporters. Insulin expression was down regulated by all cytokines. ZnT8 over expressing cells were more sensitive to IL-1beta induced apoptosis whereas no differences were observed with IFN-gamma, TNF-alpha, or a mixture of cytokines., Conclusion: The zinc transporting system in beta-cells is influenced by the exposure to cytokines. Particularly ZnT8, which has been associated with the development of diabetes, seems to be cytokine sensitive.