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Abstract Current antibody-based immunotherapy depends on tumor antigen shedding for proper T cell priming. Here we select a novel human CD40 agonistic drug candidate and generate a bispecific antibody, herein named BiA9*2_HF, that allows for rapid antibody-peptide conjugate formation. The format is designed to facilitate peptide antigen delivery to CD40 expressing cells combined with simultaneous CD40 agonistic activity. In vivo, the selected bispecific antibody BiA9*2_HF loaded with peptide cargos induces improved antigen-specific proliferation of CD8+ (10-15 fold) and CD4+ T cells (2-7 fold) over control in draining lymph nodes. In both virus-induced and neoantigen-based mouse tumor models, BiA9*2_HF demonstrates therapeutic efficacy and elevated safety profile, with complete tumor clearance, as well as measured abscopal impact on tumor growth. The BiA9*2_HF drug candidate can thus be utilized to tailor immunotherapeutics for cancer patients.

Abstract Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.

Abstract Background Genomic rearrangements in cancer cells can create fusion genes that encode chimeric proteins or alter the expression of coding and non-coding RNAs. In some cancer types, fusions involving specific kinases are used as targets for therapy. Fusion genes can be detected by whole genome sequencing (WGS) and targeted fusion panels, but RNA sequencing (RNA-Seq) has the advantageous capability of broadly detecting expressed fusion transcripts. Results We developed a pipeline for validation of fusion transcripts identified in RNA-Seq data using matched WGS data from The Cancer Genome Atlas (TCGA) and applied it to 910 tumors from 11 different cancer types. This resulted in 4237 validated gene fusions, 3049 of them with at least one identified genomic breakpoint. Utilizing validated fusions as true positive events, we trained a machine learning classifier to predict true and false positive fusion transcripts from RNA-Seq data. The final precision and recall metrics of the classifier were 0.74 and 0.71, respectively, in an independent dataset of 249 breast tumors. Application of this classifier to all samples with RNA-Seq data from these cancer types vastly extended the number of likely true positive fusion transcripts and identified many potentially targetable kinase fusions. Further analysis of the validated gene fusions suggested that many are created by intrachromosomal amplification events with microhomology-mediated non-homologous end-joining. Conclusions A classifier trained on validated fusion events increased the accuracy of fusion transcript identification in samples without WGS data. This allowed the analysis to be extended to all samples with RNA-Seq data, facilitating studies of tumor biology and increasing the number of detected kinase fusions. Machine learning could thus be used in identification of clinically relevant fusion events for targeted therapy. The large dataset of validated gene fusions generated here presents a useful resource for development and evaluation of fusion transcript detection algorithms.

Abstract We report the application of ancestral sequence reconstruction on coronavirus spike protein, resulting in stable and highly soluble ancestral scaffold antigens (AnSAs). The AnSAs interact with plasma of patients recovered from COVID-19 but do not bind to the human angiotensin-converting enzyme 2 (ACE2) receptor. Cryo-EM analysis of the AnSAs yield high resolution structures (2.6–2.8 Å) indicating a closed pre-fusion conformation in which all three receptor-binding domains (RBDs) are facing downwards. The structures reveal an intricate hydrogen-bonding network mediated by well-resolved loops, both within and across monomers, tethering the N-terminal domain and RBD together. We show that AnSA-5 can induce and boost a broad-spectrum immune response against the wild-type RBD as well as circulating variants of concern in an immune organoid model derived from tonsils. Finally, we highlight how AnSAs are potent scaffolds by replacing the ancestral RBD with the wild-type sequence, which restores ACE2 binding and increases the interaction with convalescent plasma.

Abstract Background Oestrogen receptor alpha (ER) is involved in cell growth and proliferation and functions as a transcription factor, a transcriptional coregulator, and in cytoplasmic signalling. It affects, for example, bone, endometrium, ovaries and mammary epithelium. It is a key biomarker in clinical management of breast cancer, where it is used as a prognostic and treatment‐predictive factor, and a therapeutical target. Several ER isoforms have been described, but transcript annotation in public databases is incomplete and inconsistent, and functional differences are not well understood. Methods We have analysed short‐ and long‐read RNA sequencing data from breast tumours, breast cancer cell lines, and normal tissues to create a comprehensive annotation of ER transcripts and combined it with experimental studies of full‐length protein and six alternative isoforms. Results The isoforms have varying transcription factor activity, subcellular localisation, and response to the ER‐targeting drugs tamoxifen and fulvestrant. Antibodies differ in ability to detect alternative isoforms, which raises concerns for the interpretation of ER‐status in routine pathology. Conclusions Future work should investigate the effects of alternative isoforms on patient survival and therapy response. An accurate annotation of ER isoforms will aid in interpretation of clinical data and inform functional studies to improve our understanding of the ER in health and disease.

Abstract Background In cancer, genomic rearrangements can create fusion genes that either combine protein-coding sequences from two different partner genes or place one gene under the control of the promoter of another gene. These fusion genes can act as oncogenic drivers in tumor development and several fusions involving kinases have been successfully exploited as drug targets. Expressed fusions can be identified in RNA sequencing (RNA-Seq) data, but fusion prediction software often has a high fraction of false positive fusion transcript predictions. This is problematic for both research and clinical applications. Results We describe a method for validation of fusion transcripts detected by RNA-Seq in matched whole-genome sequencing (WGS) data. Our pipeline uses discordant read pairs to identify supported fusion events and analyzes soft-clipped read alignments to determine genomic breakpoints. We have tested it on matched RNA-Seq and WGS data for both tumors and cancer cell lines and show that it can be used to validate both new predicted gene fusions and experimentally validated fusion events. It was considerably faster and more sensitive than using BreakDancer and Manta, software that is instead designed to detect many different types of structural variants on a genome-wide scale. Conclusions We have developed a fast and very sensitive pipeline for validation of gene fusions detected by RNA-Seq in matched WGS data. It can be used to identify high-quality gene fusions for further bioinformatic and experimental studies, including validation of genomic breakpoints and studies of the mechanisms that generate fusions. In a clinical setting, it could help find expressed gene fusions for personalized therapy.

Abstract Chimeric antigen receptor (CAR)-T cell therapy is rapidly advancing as cancer treatment, however, designing an optimal CAR remains challenging. A single-chain variable fragment (scFv) is generally used as CAR targeting moiety, wherein the complementarity-determining regions (CDRs) define its specificity. We report here that the CDR loops can cause CAR clustering, leading to antigen-independent tonic signalling and subsequent CAR-T cell dysfunction. We show via CARs incorporating scFvs with identical framework and varying CDR sequences that CARs may cluster on the T cell surface, which leads to antigen-independent CAR-T cell activation, characterized by increased cell size and interferon (IFN)-γ secretion. This results in CAR-T cell exhaustion, activation-induced cell death and reduced responsiveness to target-antigen-expressing tumour cells. CDR mutagenesis confirms that the CAR-clustering is mediated by CDR-loops. In summary, antigen-independent tonic signalling can be induced by CDR-mediated CAR clustering, which could not be predicted from the scFv sequences, but could be tested for by evaluating the activity of unstimulated CAR-T cells.

Abstract Background To address the reactivity and affinity against histidyl-transfer RNA synthetase (HisRS) autoantigen of anti-Jo1 autoantibodies from serum and bronchoalveolar lavage fluid (BALF) in patients with idiopathic inflammatory myopathies/anti-synthetase syndrome (IIM/ASSD). To investigate the associations between the reactivity profile and clinical data over time. Methods Samples and clinical data were obtained from (i) 25 anti-Jo1+ patients (19 sera with 16 longitudinal samples and 6 BALF/matching sera at diagnosis), (ii) 29 anti-Jo1− patients (25 sera and 4 BALF/matching sera at diagnosis), and (iii) 27 age/gender-matched healthy controls (24 sera and 3 BALF/matching sera). Reactivity towards HisRS full-length (HisRS-FL), three HisRS domains (WHEP, antigen binding domain (ABD), and catalytic domain (CD)), and the HisRS splice variant (SV) was tested. Anti-Jo1 IgG reactivity was evaluated by ELISA and western blot using IgG purified from serum by affinity chromatography. In paired serum-BALF, anti-Jo1 IgG and IgA reactivity was analyzed by ELISA. Autoantibody affinity was measured by surface plasmon resonance using IgG purified from sera. Correlations between autoantibody reactivity and clinical data were evaluated at diagnosis and longitudinally. Results Anti-Jo1 IgG from serum and BALF bound HisRS-FL, WHEP, and SV with high reactivity at the time of diagnosis and recognized both conformation-dependent and conformation-independent HisRS epitopes. Anti-HisRS-FL IgG displayed high affinity early in the disease. At the time of IIM/ASSD diagnosis, the highest autoantibody levels against HisRS-FL were found in patients ever developing interstitial lung disease (ILD) and arthritis, but with less skin involvement. Moreover, the reactivity of anti-WHEP IgG in BALF correlated with poor pulmonary function. Levels of autoantibodies against HisRS-FL, HisRS domains, and HisRS splice variant generally decreased over time. With some exceptions, longitudinal anti-HisRS-FL antibody levels changed in line with ILD activity. Conclusion High levels and high-affinity anti-Jo1 autoantibodies towards HisRS-FL were found early in disease in sera and BALF. In combination with the correlation of anti-HisRS-FL antibody levels with ILD and ILD activity in longitudinal samples as well as of anti-WHEP IgG in BALF with poor pulmonary function, this supports the previously raised hypothesis that the lung might have a role in the immune reaction in anti-Jo1-positive patients.

Abstract Background Heterobeltiosis is the phenomenon when the hybrid’s performance is superior to its best performing parent. Banana (Musa spp. AAA) breeding is a tedious, time-consuming process, taking up to two decades to develop a consumer acceptable hybrid. Exploiting heterobeltiosis in banana breeding will help to select breeding material with high complementarity, thus increasing banana breeding efficiency. The aim of this study was therefore to determine and document the level of heterobeltiosis of bunch weight and plant stature in the East African highland bananas, in order to identify potential parents that can be used to produce offspring with desired bunch weight and stature after a few crosses. Results This research found significant progressive heterobeltiosis in cross-bred ‘Matooke’ (highland cooking) banana hybrids, also known as NARITAs, when grown together across years with their parents and grandparents in Uganda. Most (all except 4) NARITAs exhibited positive heterobeltiosis for bunch weight, whereas slightly more than half of them had negative heterobeltiosis for stature. The secondary triploid NARITA 17 had the highest heterobeltiosis for bunch weight: 249% versus its ‘Matooke’ grandparent and 136% against its primary tetraploid parent. Broad sense heritability (across three cropping cycles) for yield potential and bunch weight were high (0.84 and 0.76 respectively), while that of plant stature was very low (0.0035). There was a positive significant correlation (P

Abstract Background Avocado is an important cash crop in Tanzania, however its genetic diversity is not thoroughly investigated. This study was undertaken to explore the genetic diversity of avocado in the southern highlands using microsatellite markers. A total of 226 local avocado trees originating from seeds were sampled in eight districts of the Mbeya, Njombe and Songwe regions. Each district was considered as a population. The diversity at 10 microsatellite loci was investigated. Results A total of 167 alleles were detected across the 10 loci with an average of 16.7 ± 1.3 alleles per locus. The average expected and observed heterozygosity were 0.84 ± 0.02 and 0.65 ± 0.04, respectively. All but two loci showed a significant deviation from the Hardy-Weinberg principle. Analysis of molecular variance showed that about 6% of the variation was partitioned among the eight geographic populations. Population FST pairwise comparisons revealed lack of genetic differentiation for the seven of 28 population pairs tested. The principal components analysis (PCA) and hierarchical cluster analysis showed a mixing of avocado trees from different districts. The model-based STRUCTURE subdivided the trees samples into four major genetic clusters. Conclusion High diversity detected in the analysed avocado germplasm implies that this germplasm is a potentially valuable source of variable alleles that might be harnessed for genetic improvement of this crop in Tanzania. The mixing of avocado trees from different districts observed in the PCA and dendrogram points to strong gene flow among the avocado populations, which led to population admixture revealed in the STRUCTURE analysis. However, there is still significant differentiation among the tree populations from different districts that can be utilized in the avocado breeding program.

Abstract Background During breast cancer progression, the epithelial to mesenchymal transition has been associated with metastasis and endocrine therapy resistance; however, the underlying mechanisms remain elusive. To gain insight into this process, we studied the transition undergone by MCF7-derived cells, which is driven by the constitutive nuclear expression of a MKL1 variant devoid of the actin-binding domain (MKL1 ΔN200). We characterized the adaptive changes that occur during the MKL1-induced cellular model and focused on regulation of translation machinery and metabolic adaptation. Methods We performed a genome-wide analysis at the transcriptional and translational level using ribosome profiling complemented with RNA-Seq and analyzed the expression of components of the translation machinery and enzymes involved in energy metabolism. NGS data were correlated with metabolomic measurements and quantification of specific mRNAs extracted from polysomes and western blots. Results Our results reveal the expression profiles of a luminal to basal-like state in accordance with an epithelial to mesenchymal transition. During the transition, the synthesis of ribosomal proteins and that of many translational factors was upregulated. This overexpression of the translational machinery appears to be regulated at the translational level. Our results indicate an increase of ribosome biogenesis and translation activity. We detected an extensive metabolic rewiring occurring in an already “Warburg-like” context, in which enzyme isoform switches and metabolic shunts indicate a crucial role of HIF-1α along with other master regulatory factors. Furthermore, we detected a decrease in the expression of enzymes involved in ribonucleotide synthesis from the pentose phosphate pathway. During this transition, cells increase in size, downregulate genes associated with proliferation, and strongly upregulate expression of cytoskeletal and extracellular matrix genes. Conclusions Our study reveals multiple regulatory events associated with metabolic and translational machinery adaptation during an epithelial mesenchymal-like transition process. During this major cellular transition, cells achieve a new homeostatic state ensuring their survival. This work shows that ribosome profiling complemented with RNA-Seq is a powerful approach to unveil in-depth global adaptive cellular responses and the interconnection among regulatory circuits, which will be helpful for identification of new therapeutic targets.

Abstract Background Accurate classification of breast cancer using gene expression profiles has contributed to a better understanding of the biological mechanisms behind the disease and has paved the way for better prognostication and treatment prediction. Results We found that miRNA profiles largely recapitulate intrinsic subtypes. In the case of HER2-enriched tumors a small set of miRNAs including the HER2-encoded mir-4728 identifies the group with very high specificity. We also identified differential expression of the miR-99a/let-7c/miR-125b miRNA cluster as a marker for separation of the Luminal A and B subtypes. High expression of this miRNA cluster is linked to better overall survival among patients with Luminal A tumors. Correlation between the miRNA cluster and their precursor LINC00478 is highly significant suggesting that its expression could help improve the accuracy of present day’s signatures. Conclusions We show here that miRNA expression can be translated into mRNA profiles and that the inclusion of miRNA information facilitates the molecular diagnosis of specific subtypes, in particular the clinically relevant sub-classification of luminal tumors.

Fusion gene research traditionally focuses on fusions that result in hybrid proteins or promoter switching events. Here, the authors demonstrate enrichment of fusions in miRNA host genes in breast cancer, highlighting that disparate fusions could have convergent impact on miRNA.

MicroRNAs (miRNAs) are ∼22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples. Here, we develop a scalable protocol for the rapid and economical preparation of a large number of small RNA sequencing libraries using dual indexing for multiplexing. Combined with the use of off-the-shelf reagents, more samples can be sequenced simultaneously on large-scale sequencing platforms at a considerably lower cost per sample. Sample preparation is simplified by pooling libraries prior to gel purification, which allows for the selection of a narrow size range while minimizing sample variation. A comparison with publicly available data from benchmarking of miRNA analysis platforms showed that this method captures absolute and differential expression as effectively as commercially available alternatives.

Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks specific diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reflect differences in underlying pathogenesis. Our objective was to obtain protein profiles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratification. Plasma samples from a cross-sectional study of well-characterized SLE patients (n = 379) and matched population controls (n = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann–Whitney U-test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. We identified Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest p-values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (padjusted = 3 × 10−9, 3 × 10−6, and 5 × 10−6 respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). IRF5 was up-regulated while SPDEF was found to be down-regulated in SLE patients. Unsupervised clustering of all investigated proteins identified three molecular subgroups among SLE patients, characterized by (1) high levels of rheumatoid factor-IgM, (2) low IRF5, and (3) high IRF5. IRF5 expressing microparticles were analyzed by flow cytometry in a subset of patients to confirm the presence of IRF5 in plasma and detection of extracellular IRF5 was further confirmed by immunoprecipitation-mass spectrometry (IP-MS). Interestingly IRF5, a known genetic risk factor for SLE, was detected extracellularly and suggested by unsupervised clustering analysis to differentiate between SLE subgroups. Our results imply a set of circulating molecules as markers of possible pathogenic importance in SLE. We believe that these findings could be of relevance for understanding the pathogenesis and diversity of SLE, as well as for selection of patients in clinical trials.

East African highland bananas (EAHB) were regarded as sterile. Their screening for female fertility with “Calcutta 4” as male parent revealed that 37 EAHB were fertile. This was the foundation for the establishment of the EAHB crossbreeding programs by the International Institute of Tropical Agriculture (IITA) and the National Agricultural Research Organization (NARO) in Uganda in the mid-1990s. The aim of this study was to assess the progress and efficiency of the EAHB breeding program at IITA, Sendusu in Uganda. Data on pollinations, seeds generated and germinated, plus hybrids selected between 1995 and 2015 were analyzed. Pollination success and seed germination percentages for different cross combinations were calculated. The month of pollination did not result in significantly different (P = 0.501) pollination success. Musa acuminata subsp. malaccensis accession 250 had the highest pollination success (66.8%), followed by the cultivar “Rose” (66.6%) among the diploid males. Twenty-five EAHB out of 41 studied for female fertility produced up to 305 seeds per pollinated bunch, and were therefore deemed fertile. The percentage of seed germination varied among crosses: 26% for 2x × 4x, 23% for 2x × 2x, 11% for 3x × 2x, and 7% for 4x × 2x. Twenty-seven NARITA hybrids (mostly secondary triploids ensuing from the 4x × 2x) were selected for further evaluation in the East African region. One so far –“NARITA 7”– was officially released to farmers in Uganda. Although pollination of EAHB can be conducted throughout the year, the seed set and germination is low. Thus, further research on pollination conditions and optimization of embryo culture protocols should be done to boost seed set and embryo germination, respectively. More research in floral biology and seed germination as well as other breeding strategies are required to increase the efficiency of the EAHB breeding program.

Pancreatic ductal adenocarcinoma (PDAC) is a disease where detection preceding clinical symptoms significantly increases the life expectancy of patients. In this study, a recombinant antibody microarray platform was used to analyze 213 Chinese plasma samples from PDAC patients and normal control (NC) individuals. The cohort was stratified according to disease stage, i.e. resectable disease (stage I/II), locally advanced (stage III) and metastatic disease (stage IV). Support vector machine analysis showed that all PDAC stages could be discriminated from controls and that the accuracy increased with disease progression, from stage I to IV. Patients with stage I/II PDAC could be discriminated from NC with high accuracy based on a plasma protein signature, indicating a possibility for early diagnosis and increased detection rate of surgically resectable tumors.

Heterosis, or hybrid vigour, is the superiority of the hybrid for a certain trait over the mean of its two parents. Heterobeltiosis is a form of heterosis where the hybrid is superior to its best parent. Banana breeding is a tedious, time-consuming process, taking up to two decades to develop a hybrid. Understanding heterosis in banana breeding will contribute to selecting right breeding materials for further crossing, thus increasing banana breeding efficiency. Here we document heterobeltiosis by using the recently bred NARITA ‘Matooke’ hybrids and their ancestors. NARITA hybrids, their parents (4x and 2x), grandparents (3x and 2x), and local 3x ‘Matooke’ cultivar checks were planted in a rectangular lattice design with two replications. Yield and other agronomic data were collected at flowering and harvest. The NARITAs were compared with their 3x ‘Matooke’ grandmothers. Heterobeltiosis on bunch weight was calculated with the data of 3 cycles. All the NARITAs showed heterobeltiosis for bunch weight. NARITA 17 had the highest grandparent heterobeltiosis (ca. 250%). Genetic gains due to crossbreeding were determined for fruit yield considering three generations: matooke cultigen (C0), primary tetraploid hybrids (C1) and secondary tetraploid hybrids (C2). The average genetic gain (from C0 to C2) rates for bunch weight (kg) and yield potential (t ha−1 year−1) were 1.4% and 1.3% per year, respectively.

Two-hundred and twenty-six old avocado trees (Persea americana Mill) derived from seeds were selected from eight districts of the Mbeya, Njombe and Songwe regions in Tanzania. The tree, leaf, fruit and seed characteristics were studied using the descriptors for avocado (Persea spp.) from the International Plant Genetic Resources Institute. Cross tabulation and Chi-square tests were conducted in order to assess the distribution of traits between districts and altitude ranges. Principle coordinate analysis (PCoA) and hierarchical cluster analysis (HCA) were used to assess variation of traits within and among districts. Various morphological features were observed among the samples which point to the existence of the Mexican, Guatemalan and West Indian avocado races in Tanzania. The biplot from PCoA revealed extensive variation between the sampled trees at the district level but no clear groupings of the samples based on geographic location. Likewise, dendrograms ensuing from the neighbor−joining and Wards methods displayed that the avocado samples from the same district and even region differed considerably. This morphological trait variation suggests high diversity that may help in planning germplasm management and conservation, as well as breeding strategies in the future.

In vitro generation of antibodies often requires variable domain sequence evolution to adapt the protein in terms of affinity, specificity, or developability. Such antibodies, including those that are of interest for clinical development, may have their origins in a diversity of immunoglobulin germline genes. Others and we have previously shown that antibodies of different origins tend to evolve along different, preferred trajectories. Apart from substitutions within the complementary determining regions, evolution may also, in a germline gene-origin-defined manner, be focused to residues in the framework regions, and even to residues within the protein core, in many instances at a substantial distance from the antibody’s antigen-binding site. Examples of such germline origin-defined patterns of evolution are described. We propose that germline gene-preferred substitution patterns offer attractive alternatives that should be considered in efforts to evolve antibodies intended for therapeutic use with respect to appropriate affinity, specificity, and product developability. We also hypothesize that such germline gene-origin-defined in vitro evolution hold potential to result in products with limited immunogenicity, as similarly evolved antibodies will be parts of conventional, in vivo-generated antibody responses and thus are likely to have been seen by the immune system in the past.

High-yielding rice cultivars with good processing quality and rich in nutrition suitable to a changing climate are of particular importance for future rice-based food production. Here, seven Rwandan rice cultivars were grown in a climate chamber of the biotron facility at the Swedish University of Agricultural Sciences, to be evaluated for their grain yield, nutritional composition, and dough mixing properties. Two different levels of inorganic fertilizer were applied weekly from the seedling stage until flowering. Significant differences for grain yield and quality attributes were found between cultivars. Jyambere showed significantly the highest yield while Ingwizabukungu, Nemeyubutaka, and Jyambere were high in mineral elements content. Ndamirabahinzi and Mpembuke had the highest levels of TPC and TAC. Generally, the lower fertilizer dose resulted in a better performance of the cultivars for both yield and quality attributes. Significantly higher content of Fe, Ca, and Ba was found in grains from the moderate fertilizer dose, whereas K, Na, P, S, Zn, Cd, and Pb increased in grains from the higher fertilizer dose. The cultivar Ndamirabahinzi showed less variability of evaluated characters across fertilizer doses. The results from this study may be used for rice breeding of cultivars with high yield and good grain quality.

There is a great need for targeted protein assays with the capacity of sensitive measurements in complex samples such as plasma or serum, not the least for clinical purposes. Proteomics keeps generating hundreds of biomarker candidates that need to be transferred towards true clinical application through targeted verification studies and towards clinically applicable analysis formats. The immunoaffinity assay AFFIRM (AFFInity sRM) combines the sensitivity of recombinant single chain antibodies (scFv) for targeted protein enrichment with a specific mass spectrometry readout through selected reaction monitoring (SRM) in an automated workflow. Here we demonstrate a 100 times improved detection capacity of the assay down to pg/ml range through the use of oriented antibody immobilization to magnetic beads. This was achieved using biotin-tagged scFv coupled to streptavidin coated magnetic beads, or utilizing the FLAG tag for coupling to anti-FLAG antibody coated magnetic beads. An improved multiplexing capacity with an 11-plex setup was also demonstrated compared to a previous 3-plex setup, which is of great importance for the analysis of panels of biomarker targets.

B cells produce antibodies, key effector molecules in health and disease. They mature their properties, including their affinity for antigen, through hypermutation events; processes that involve, e.g., base substitution, codon insertion and deletion, often in association with an isotype switch. Investigations of antibody evolution define modes whereby particular antibody responses are able to form, and such studies provide insight important for instance for development of efficient vaccines. Antibody evolution is also used in vitro for the design of antibodies with improved properties. To better understand the basic concepts of antibody evolution, we analyzed the mutational paths, both in terms of amino acid substitution and insertions and deletions, taken by antibodies of the IgG isotype. The analysis focused on the evolution of the heavy chain variable domain of sets of antibodies, each with an origin in 1 of 11 different germline genes representing six human heavy chain germline gene subgroups. Investigated genes were isolated from cells of human bone marrow, a major site of antibody production, and characterized by next-generation sequencing and an in-house bioinformatics pipeline. Apart from substitutions within the complementarity determining regions, multiple framework residues including those in protein cores were targets of extensive diversification. Diversity, both in terms of substitutions, and insertions and deletions, in antibodies is focused to different positions in the sequence in a germline gene-unique manner. Altogether, our findings create a framework for understanding patterns of evolution of antibodies from defined germline genes.

Since the early 1980s remarkable progress has been made in understanding the role of the HER2 locus in carcinogenesis, but many details of its regulatory network are still elusive. We recently reported the finding of 367 new human microRNA (miRNA) genes of which one, mir-4728, is encoded in an intron of the HER2 gene. Here, we confirm that the HER2 oncogene is a bi-functional locus encoding the membrane receptor and a functional miRNA gene. We further show that miR-4728-3p has alternative functionalities depending on the region used for interaction with its target; the canonical seed between nucleotides 2-8 or a novel, more internal seed shifted to nucleotides 6-12. Analysis of public data shows that this internal seed region, although rare compared to the far more abundant canonical 2-8 seed interaction, can also direct targeted down-regulation by other miRNAs. Through the internal seed, miR-4728-3p regulates expression of estrogen receptor alpha, an interaction that would have remained undetected if classic rules for miRNA-target interaction had been applied. In summary, we present here an alternative mode of miRNA regulation and demonstrate this dual function of the HER2 locus, linking the two major biomarkers in breast cancer.

Biology, chemistry and physics might be taught as separate subjects, but sometimes they are taught as combined with one another or with other subjects. Nationally and internationally the concept integrated science teaching lacks a uniform definition. To find out more about how practicing teachers define and perform integrated teaching, interviews were undertaken with five teachers, teaching science in school year 7 to 9 (age 13-16) in the Swedish compulsory school. Two ways of integrating science teaching were found; one combining the science subjects and one combining the science subjects with other school subjects. Results showed differences in the ways of teaching integrated science, in the interpretation of what the teachers meant by science integrating teaching and which obstacles and possibilities they saw. Visions about working with integrated science in the future and how these visions correspond to teacher’s reality is also discussed.

In this paper we report a study about two teachers’ intentions with integrated science in lower secondary school and how these intentions turn out in their classrooms. The study is based on intention alanalysis of interviews and classrooms observations. The teachers’ intentions are a result of internal and external factors. Internal factors for organizing teaching as integrated science were to let the students get a holistic understanding of the science content, to be able to apply knowledge and to see the relevance in their daily lives. External factors were school organization and syllabuses. In practice the teachers clearly connected to the students’ daily life while the holistic approach did not seem to be as important. In classroom reality the external factors became important and made the teachers change their actions.