Högskolan i Skövde

Abstract

Sepsis is a serious clinical condition that is characterized by a systemic inflammatory response syndrome resulting from a known or suspected infection. The major clinical symptoms involve an abnormal WBC count, elevated body temperature, respiration and pulse rate. Reported cases with high mortality rate range between 13 - 20 million. In order to treat Sepsis, the detection of bacteria in blood culture is extremely crucial. Treating patients with broad spectrum antibiotics is usually related to adverse effects, drug resistance, increased mortality, and high cost. In the past decades, researches had detected that E. coli and S. aureus are the major role players that cause sepsis. These microbes are molecularly tested by methods like MALDI TOF, FISH and Microarrays.  

In this analysis, DNA fluorescence in situ hybridization (FISH) assessment for the identification of S. aureus, one of the most frequent blood pathogens, was optimized in the labs of Högskolan i Skövde. As a result, the growth of S. aureus was observed very carefully, optimizing the FISH procedure for gram positive bacteria was done and the sensitivity, stability and specificity of the DNA probe were examined under variant conditions like the continuous decrease in the bacteria cells number and utilizing a mixture of different types of bacteria cells. 

Barley loose smut caused by the fungal pathogen Ustilago nuda is a global concern with detrimental effects on barley production. Early detection of this infection is vital for effective disease supervision. However, current seed health testing protocols suffer from limitations in terms of time and efficiency. The present research work aimed to produce a method using qPCR for simultaneous screening of barley and U. nuda. A set of primers, 1F and 1R, was employed for the detection of rRNA operon internal transcribed spacer 1 sequence from U. nuda and a part of the COX1 gene, present in barley seeds, was selected as an internal control for comparison with U. nuda. A specific 79 bp target amplicon from a part of the COX1 gene was successfully amplified using COX1 F and COX1 R primers, and cloned into a vector for standard curve generation. However, attempts to replicate the previously published qPCR method by bachelor researcher for U. nuda internal transcribed spacer 1 sequence detection using 1F and 1R primers were unsuccessful. Several efforts were made to reproduce the results, but amplification was not observed. Further optimization, including literature review, primers and probe optimization is required to improve this method. The successful amplification of a part of the COX1 gene in both normal and infected samples underscore its potential as a reliable internal control. However, further research is necessary to refine the detection of U. nuda. This study underscores the need for continuous advancements in disease screening methodologies to meet global market demands.

Co-occurrence of abiotic stresses, especially drought and salinity, is a natural phenomenon in field conditions and is worse for crop production than any single stress. Nowadays, rigorous methods of meta-analysis and systems biology have made it possible to perform cross-study comparisons of single stress experiments, which can uncover main overlapping mechanisms underlying tolerance to combined stress. In this study, a meta-analysis of RNA-Seq data was conducted to obtain the overlapping gene network of drought and salinity stresses in barley (Hordeum vulgare L.), which identified Rubisco activase A (RcaA) as a hub gene in the dual-stress response. Thereafter, a greenhouse experiment was carried out using two barley genotypes with different abiotic stress tolerance and evaluated several physiochemical properties as well as the expression profile and protein activity of RcaA. Finally, machine learning analysis was applied to uncover relationships among combined stress tolerance and evaluated properties. We identified 441 genes which were differentially expressed under both drought and salinity stress. Results revealed that the photosynthesis pathway and, in particular, the RcaA gene are major components of the dual-stress responsive transcriptome. Comparative physiochemical and molecular evaluations further confirmed that enhanced photosynthesis capability, mainly through regulation of RcaA expression and activity as well as accumulation of proline content, have a significant association with combined drought and salinity stress tolerance in barley. Overall, our results clarify the importance of RcaA in combined stress tolerance and may provide new insights for future investigations. 

The freshwater mussel Anodonta anatina is a common mollusc found in rivers and lakes in Europe. Due to its lifestyle as an immobile substrate filter feeder, it is only exposed to pollutions which passes by it, thus the mussels are viable to be used as a bioindicator for the analysis of local aquatic health. With the recent decades of advancements in nucleotide sequencing platforms, transcriptomic analysis could be utilized to make an assessment of the quality of water, as the transcriptome of organisms respond to the presence of toxins and changes in the environment. The aim of this project was to develop a method to isolate and sequence the transcriptome of the hepatopancreas of the freshwater mussel A. anatina with an Oxford MinION nanopore sequencer. The method used two samples collected from a previous study where one mussel, named “unexposed sample 05H” for this thesis, was used as a control while the second sample, named “exposed sample 08H”, was exposed to 100 μg Cu2+/L. The method processed 80 mg of hepatopancreatic tissue and measured a 99.7 % drop in RNA during the five different protocols utilized. The method produced viable data, and the web based bioinformatic program NanoPipe managed to identify homologous sequences to the genome of the Mediterranean mussel Mytilus galloprovincialis. Due to a low number of samples, no comparative study of coppers effect on the transcriptome could be made.

A combination of site-directed mutagenesis and NMR chemical shift perturbation analysis of backbone and side-chain protons has been used to characterize the transient complex of the photosynthetic redox proteins plastocyanin and cytochrome f. To elucidate the importance of charged residues on complex formation, the complex of cytochrome f and E43Q/D44N or E59K/E60Q spinach plastocyanin double mutants was studied by full analysis of the (1)H chemical shifts by use of two-dimensional homonuclear NMR spectra. Both mutants show a significant overall decrease in chemical shift perturbations compared with wild-type plastocyanin, in agreement with a large decrease in binding affinity. Qualitatively, the E43Q/D44N mutant showed a similar interaction surface as wild-type plastocyanin. The interaction surface in the E59K/E60Q mutant was distinctly different from wild type. It is concluded that all four charged residues contribute to the affinity and that residues E59 and E60 have an additional role in fine tuning the orientation of the proteins in the complex.

Many regions of the world are facing the problem with arsenic toxicity. Arsenic contamination has become a considerable threat to the environment triggering various big health issues for every life in that contaminated environment. Lysinibacillus sphaericus (B1-CDA) is an arsenic tolerant strain of bacteria that has been reported and characterized before by the researchers of the University of Skövde, Sweden. The bacteria were found to contain many arsenic responsive genes such as arsB, arsC, and arsR which are responsible for arsenic tolerance in the bacterium. The main focus of the current study was to characterize one of the arsB genes (gene-4251) of Lysinibacillus sphaericus B1-CDA by in silico and in vitro analyses in order to determine the molecular function of this gene. The in silico studies conducted by using the Iterative Threading Assembly and Refinement (I-TASSER) server predicted the tertiary structure of the ArsB protein and suggested that this protein is an intrinsic component of the membrane which primarily helps in the binding of metal ions and liberation of metabolic energy. To validate this predictive results, several in vitro experiments were performed. For complementation studies, the arsB gene was cloned from L. sphaericus B1-CDA and transferred to an arsB knock-out mutant of Escherichia coli JW3469-1. Both, the transgenic and mutant strains were grown under the arsenic stress of 50 mM for 96 hrs followed by measuring their growth and arsenic tolerance after every 24 hrs. Statistical analysis confirmed that there was a significant difference in growth between the transgenic and the mutant E. coli strains. The ICP-MS (Inductive Coupled Plasma-Mass Spectroscopy) analysis revealed that after 24 hrs of culture, the arsenic content in the cell-free broth of transgenic strain was reduced from 50 mM to 9.10 mM (81.8%), whereas the reduction in arsenic content by the mutant strain was from 50 mM to 9.80 mM (80.2%). These results suggest that the arsB gene is partly involved in the accumulation of arsenic inside the cells and this feature could be used for a large scale removal of arsenic from the contaminated environment.

The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity.

PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC' sheet, which is flexible and completely lacks a C '' strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC' sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1.ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The 3-4-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the 3-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T cells with antigen-presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1 and reveal that potent inhibitory signaling can be initiated by weakly interacting receptors.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) constitute an appealing tool for drug discovery, disease modeling, and cardiotoxicity screening. However, their physiological immaturity, resembling CMs in the late fetal stage, limits their utility. Herein, we have developed a novel, scalable cell culture medium designed to enhance the maturation of hPSC-CMs. This medium facilitates a metabolic shift towards fatty acid utilization and augments mitochondrial function by targeting Acetyl-CoA carboxylase 2 (ACC2) with a specific small molecule inhibitor. Our findings demonstrate that this maturation protocol significantly advances the metabolic, structural, molecular and functional maturity of hPSC-CMs at various stages of differentiation. Furthermore, it enables the creation of cardiac microtissues with superior structural integrity and contractile properties. Notably, hPSC-CMs cultured in this optimized maturation medium display increased accuracy in modeling a hypertrophic cardiac phenotype following acute endothelin-1 induction and show a strong correlation between in vitro and in vivo target engagement in drug screening efforts. This approach holds promise for improving the utility and translatability of hPSC-CMs in cardiac disease modeling and drug discovery. 

Myosin is vital for body movement and heart contractility. Mutations in MYH7, encoding slow/ß-cardiac myosin heavy chain, are an important cause of hypertrophic and dilated cardiomyopathy, as well as skeletal muscle disease. A dominant missense mutation (R1845W) in MYH7 has been reported in several unrelated cases with myosin storage myopathy. We have developed a Drosophila model for a myosin storage myopathy in order to investigate the dose-dependent mechanisms underlying the pathological roles of R1845W mutation. This study shows that higher expression level of the mutated allele is concomitant with severe impairment of muscle function and progressively disrupted muscle morphology. The impaired muscle morphology associated with the mutant allele was supressed by expression of Abba/Thin, an E3 ubiquitin ligase.This Drosophila model recapitulates pathological features seen in myopathy patients with the R1845W mutation and severe ultrastructural abnormalities including extensive loss of thick filaments with selective A-band loss and preservation of I-band and Z-disks were observed in indirect flight muscles of flies with exclusive expression of mutant myosin. Further, the impaired muscle morphology associated with the mutant allele was supressed by expression of Abba/Thin, an E3 ubiquitin ligase. These findings suggest that modification of ubiquitin proteasome system may be beneficial in myosin storage myopathy by reducing the impact of MYH7 mutation in patients.

Breast cancer is the most common cancer in female population worldwide. In addition to mutations, the breast tumor microenvironment especially the tumor cell - stroma interactions through extracellular matrix components and multiple growth factors have been shown to promote tumor progression. Among those, increases in both TGF-β (transforming growth factor beta) activities and periostin expression were associated with tumor cell survival, proliferation and metastasis. TGF-β role in breast cancer progression including its ability to promote periostin expression has been extensively studied. In contrast, the role of periostin in cancer progression remains to be fully understood. Thus, the present study aimed to determine whether TGF-β and periostin have effect on each other’s expressions in breast tumor and stroma cells using in vitro cell models. Through Western blot analyses and ELISAs, the periostin and TGF-β expressions of both stroma and tumor cells were analyzed following TGF-β and periostin treatments, respectively. The results indicate that TGF-β treatments led to significant increase in periostin expression in fibroblasts (p<0.05). In addition, periostin was differentially expressed by human breast cancer cells following TGF-β1 treatment. The TGF-β activities involved activation of pSMAD2 in both L929 fibroblasts and MCF10A mammary cells. Taken together, all experimental data indicate that within the breast tumor TGF-β and periostin likely participate in a regulation loop. Whether this putative regulation loop is critical to metastasis remains to be determined. Should periostin play a critical role in breast cancer progression, it could become a specific target in the preventive and/or therapeutic development of breast cancer patients.

This study characterizes different MTP1 (metal tolerance protein)/ZAT (zinc transporter) homologs involved in zinc (Zn) homeostasis in plants. BLAST analysis of AtMTP1 protein against 15 plant species showed 21 MTP1 homologs. These MTP1 protein homologs generally contain ~400 residues, six transmembrane helices and cation transmembrane transporter activity (GO:0008324), which can be utilized in predicting Zn uptake and tolerance mechanisms. These MTP1 genes having 1 exon are located on chromosomes 2, 7, and 14. Motifs contain conserved sequences of 41–50 residues belonging to the cation efflux family, which may help target binding sites and transcription factors. Further, AtMTP1 shows close similarities with Glycine max and Medicago trunculata. Interactome analysis suggests the association of AtMTP1 with cadmium/zinc-transporting ATPase and ZIP metal ion transporter. The AtMTP1 network is predominantly connected to cadmium/zinc-transporting ATPase (heavy metal ATPase 2, HMA2; heavy metal ATPase 3, HMA3; heavy metal ATPase 4, HMA4), cation efflux protein (MTP11), and metal tolerance protein C3 (AT4G58060). The Genevestigator predicts the high expression potential of AtMTP1 in the apical root during senescence, seedling, and bolting stages in an association with 11 co-expressed genes, mainly linked to estradiol toxicity and heat stress. Besides, AtMTP1 protein homologs possess conserved N-glyco motifs and physicochemical properties. The similarity and interactions of AtMTP1 gene with other genes suggest that Zn homeostasis in plants is associated with the regulation of different genes. These findings may advance our understanding to further develop plants capable of maintaining Zn homeostasis under adverse conditions. © 2021 Elsevier Inc.

As life expectancy increases globally, so does the prevalence of age-related diseases, some of which are more difficult to adapt to and accommodate for than others. In particular, neurodegenerative disorders are among those for which adaptations are more complex, often requiring long-term care. Alzheimer’s disease is a neurodegenerative disorder linked with atrophy of certain cognition related brain regions, causing severe memory, and cognitive function loss. A major hypothesis behind Alzheimer’s disease, upon which most pharmaceutical therapies are based, proposes its cause as the degeneration of cholinergic neurons. Nerve growth factor is a biomolecule found to stimulate the generation, protection, and regeneration of cholinergic neurons. Synthesis of nerve growth factor has been found to be promoted by hericenones and erinacines, bioactive compounds originally extracted from the mycelium of the Lion’s Mane Mushroom (Hericium erinaceus); however, direct supplementation with H. erinaceus has also yielded positive results. In animal models H. erinaceus has enhanced Nerve growth factor levels, increased neuronal survival, promoted hippocampal neurogenesis, decreased amyloid plaque build-up, and improved behavioural outcomes. Human trials showed improvements in cognitive function scores, short-term memory, and visual contrast sensitivity. Phytotherapeutic remedies such as these have long been used across a multitude of cultures, however, now with quantitative scientific evidence supporting their benefits, their implementation into clinical therapies is being explored. Though there is still room for further research, H. erinaceus shows a promising future as a potential pharmaceutical therapy for Alzheimer’s disease and other cognitive impairments.

The volume of point of care (POC) testing continues to grow steadily due to the increased availability of easy-to-use devices, thus making it possible to deliver less costly care closer to the patient site in a shorter time relative to the central laboratory services. A novel class of molecules called microRNAs have recently gained attention in healthcare management for their potential as biomarkers for human diseases. The increasing interest of miRNAs in clinical practice has led to an unmet need for assays that can rapidly and accurately measure miRNAs at the POC. However, the most widely used methods for analyzing miRNAs, including Northern blot-based platforms, in situ hybridization, reverse transcription qPCR, microarray, and next-generation sequencing, are still far from being used as ideal POC diagnostic tools, due to considerable time, expertize required for sample preparation, and in terms of miniaturizations making them suitable platforms for centralized labs. In this review, we highlight various existing and upcoming technologies for miRNA amplification and detection with a particular emphasis on the POC testing industries. The review summarizes different miRNA targets and signals amplification-based assays, from conventional methods to alternative technologies, such as isothermal amplification, paper-based, oligonucleotide-templated reaction, nanobead-based, electrochemical signaling-based, and microfluidic chip-based strategies. Based on critical analysis of these technologies, the possibilities and feasibilities for further development of POC testing for miRNA diagnostics are addressed and discussed.

Low-density arrays for quantitative real-time PCR (qPCR) are increasingly being used as an experimental technique for miRNA expression profiling. As with gene expression profiling using microarrays, data from such experiments needs effective analysis methods to produce reliable and high-quality results. In the pre-processing of the data, one crucial analysis step is normalization, which aims to reduce measurement errors and technical variability among arrays that might have arisen during the execution of the experiments. However, there are currently a number of different approaches to choose among and an unsuitable applied method may induce misleading effects, which could affect the subsequent analysis steps and thereby any conclusions drawn from the results. The choice of normalization method is hence an important issue to consider. In this study we present the comparison of a number of data-driven normalization methods for TaqMan low-density arrays for qPCR and different descriptive statistical techniques that can facilitate the choice of normalization method. The performance of the normalization methods was assessed and compared against each other as well as against standard normalization using endogenous controls. The results clearly show that the data-driven methods reduce variation and represent robust alternatives to using endogenous controls.

Nuclear receptors (NRs) are ligand-activated transcription factors that regulate gene expression when bound to specific DNA sequences. Crosstalk between steroid NR systems has been studied for understanding the development of hormone-driven cancers but not to an extent at a genetic level. This study aimed to investigate crosstalk between steroid NRs in conserved intron and exon sequences, with a focus on steroid NRs involved in prostate cancer etiology. For this purpose, we evaluated conserved intron and exon sequences among all 49 members of the NR Superfamily (NRS) and their relevance as regulatory sequences and NR-binding sequences. Sequence conservation was found to be higher in the first intron (35%), when compared with downstream introns. Seventy-nine percent of the conserved regions in the NRS contained putative transcription factor binding sites (TFBS) and a large fraction of these sequences contained splicing sites (SS). Analysis of transcription factors binding to putative intronic and exonic TFBS revealed that 5 and 16%, respectively, were NRs. The present study suggests crosstalk between steroid NRs, e.g., vitamin D, estrogen, progesterone, and retinoic acid endocrine systems, through cis-regulatory elements in conserved sequences of introns and exons. This investigation gives evidence for crosstalk between steroid hormones and contributes to novel targets for steroid NR regulation. 

The Toll/interleukin 1 receptor (TIR) domain is a region found in the cytoplasmic tails of members of the Toll-like receptor/interleukin-1 receptor superfamily. The domain is essential for signaling and is also found in the adaptor proteins Mal (MyD88 adaptor-like) and MyD88, which function to couple activation of the receptor to downstream signaling components. Experimental structures of two Toll/interleukin 1 receptor domains reveal a alpha-beta-fold similar to that of the bacterial chemotaxis protein CheY, and other evidence suggests that the adaptors can make heterotypic interactions with both the receptors and themselves. Here we show that the purified TIR domains of Mal and MyD88 can form stable heterodimers and also that Mal homodimers and oligomers are dissociated in the presence of ATP. To identify structural features that may contribute to the formation of signaling complexes, we produced models of the TIR domains from human Toll-like receptor 4 (TLR4), Mal, and MyD88. We found that although the overall fold is conserved the electrostatic surface potentials are quite distinct. Docking studies of the models suggest that Mal and MyD88 bind to different regions in TLRs 2 and 4, a finding consistent with a cooperative role of the two adaptors in signaling. Mal and MyD88 are predicted to interact at a third non-overlapping site, suggesting that the receptor and adaptors may form heterotetrameric complexes. The theoretical model of the interactions is supported by experimental data from glutathione S-transferase pull-downs and co-immunoprecipitations. Neither theoretical nor experimental data suggest a direct role for the conserved proline in the BB-loop in the association of TLR4, Mal, and MyD88. Finally we show a sequence relationship between the Drosophila protein Tube and Mal that may indicate a functional equivalence of these two adaptors in the Drosophila and vertebrate Toll pathways.

Background: Hydroxylated biphenyls are currently recognized as secondary pollutants that are hazardous to animals and humans. Bacterial degradation is the most effective method for the degradation of hydroxylated biphenyls. Several strains capable of degrading polychlorinated biphenyls have been described, which also degrade hydroxylated biphenyls.

Objectives: 1) To study the biodegradative properties of the Rhodococcus opacus strain KT112-7 towards mono-hydroxylated biphenyls. 2) To analyze the genome of the Rhodococcus opacus strain KT112-7. 3) To identify the genetic basis for the unique biodegradative potential of the Rhodococcus opacus strain KT112-7.

Methods: A genome analysis of the strain KT112-7 was conducted based on whole-genome sequencing using various programs and databases (Velvet, CONTIGuator, RAST, KEGG) for annotation and identification of protein-coding sequences. The strain KT112-7 was cultivated in a K1 mineral medium supplemented with mono-hydroxy biphenyls or mono-hydroxybenzoic acids as the carbon source. For the growth test mono-hydroxybiphenyls or mono-hydroxybenzoic acids were dosed at concentrations of 0.5 g/L and 1.0 g/L correspondently, and the bacterial growth was monitored by the optical density. For the biodegradative activity test, mono-hydroxybiphenyls were dosed at a concentration of 0.1 g/L in vials, inoculated with late exponential phase bacteria previously acclimated on biphenyl. Compound analysis was performed using GC-MS, HPLC, and spectrophotometry.

Results: It was found that the genome of strain KT112-7 consists of a chromosome and 2 plasmids. Biphenyl degradation genes (bph genes) were identified on plasmid PRHWK1 and the chromosome, as well as hydroxybenzoic acid degradation genes on the chromosome. The strain KT112-7 was shown to degrade mono-hydroxylated biphenyls to basal metabolic compounds of the cell, with the highest destructive activity observed towards 3- and 4-hydroxylated biphenyls (98%).

Conclusion: The Rhodococcus opacus strain KT112-7 is characterized by genetic systems that contribute to its high biodegradative potential towards mono-hydroxylated biphenyls and their metabolites. Thus, the strain KT112-7 is promising for use in hydroxybiphenyl degradation technologies.

Photosynthesis is a process in which photons from sunlight excite chlorophylls in the thylakoid membranes of plants, algae and cyanobacteria. The photo-oxidised reaction centre chlorophyll P700 is re-reduced by an electron transferred from the soluble, blue copper protein plastocyanin. The oxidised plastocyanin dissociates and binds to the cytochrome b6f complex, where it accepts an electron and a new redox cycle can begin. Plastocyanin has three regions of importance for the interaction with its redox partners, a hydrophobic patch and two acidic (negatively charged) patches. Electrostatic interactions between opposite charges are important for the association and the specificity and stability of the formed complexes.

In this work the interactions with photosystem 1 and cytochrome c have been studied using mutants of plastocyanin. The mutations introduced in the small acidic patch and position 88 of plastocyanin had small effects on the binding to photosystem 1 as compared to the weak binding reported for mutants in the large acidic patch. The affinity was increased by the Glu60Gln, Glu60Lys and Asp61Lys mutations and a more efficient electron transfer was observed for the Gln88Lys mutation. The association between Pc mutated in the small acidic patch and cytochrome c was weakened and the rearrangement hindered by lysines in positions 59 and 60.

The development of an efficient expression system for spinach plastocyanin in the bacterium Escherichia coli made it possible to produce sufficient amounts of isotopically labelled plastocyanin for NMR experiments. This technique was used for solving the structure of the complex between plastocyanin and cytochrome f. The hydrophobic patch on plastocyanin binds to an area close to the heme on cytochrome f. Electrostatic interactions between opposite charges on the two proteins are also important. The short distance from the heme to the copper ligand His87 suggests an electron transfer from the heme via Tyr1 or Phe4 on cytochrome f.

The involvement of specific amino-acid residues in copper binding or folding of plastocyanin has also been examined by site-directed mutagenesis. The copper-binding histidines have been replaced by other amino acids, but no blue protein could be produced. The stability of the different redox forms of copper plastocyanin as well as the zinc protein has also been determined by guanidinium-induced unfolding.

Cytochrome f and plastocyanin are redox partners in the photosynthetic electron-transfer chain. Electron transfer from cytochrome f to plastocyanin occurs in a specific short-lived complex. To obtain detailed information about the binding interface in this transient complex, the effects of binding on the backbone and side-chain protons of plastocyanin have been analyzed by mapping NMR chemical-shift changes. Cytochrome f was added to plastocyanin up to 0.3 M equiv, and the plastocyanin proton chemical shifts were measured. Out of approximately 500 proton resonances, 86% could be observed with this method. Nineteen percent demonstrate significant chemical-shift changes and these protons are located in the hydrophobic patch (including the copper ligands) and the acidic patches of plastocyanin, demonstrating that both areas are part of the interface in the complex. This is consistent with the recently determined structure of the complex [Ubbink, M., Ejdebäck, M., Karlsson, B. G., and Bendall, D. S. (1998) Structure 6, 323-335]. The largest chemical-shift changes are found around His87 in the hydrophobic patch, which indicates tight contacts and possibly water exclusion from this part of the protein interface. These results support the idea that electron transfer occurs via His87 to the copper in plastocyanin and suggest that the hydrophobic patch determines the specificity of the binding. The chemical-shift changes in the acidic patches are significant but small, suggesting that the acidic groups are involved in electrostatic interactions but remain solvent exposed. The existence of small differences between the present data and those used for the structure may imply that the redox state of the metals in both proteins slightly affects the structure of the complex. The chemical-shift mapping is performed on unlabeled proteins, making it an efficient way to analyze effects of mutations on the structure of the complex.

Spinach plastocyanin has been expressed in Escherichia coli and exported to the periplasmic space. The effects of codon usage, expression system, growth length, and temperature on expression levels in LB medium were investigated. A stretch of codons, rare in E. coli, was identified and replaced with highly expressed codons, increasing the yield by at least 20%. Plastocyanin was more efficiently expressed under the T7 promoter than under the lac promoter. Maximum yields were obtained at 37 degrees C when growing the cells for 16 h after induction. The optimized expression system produced 38 mg holoprotein per liter culture. In this system it was also possible to express plastocyanin in minimal medium, at a yield of 10 mg per liter. N-terminal sequencing and mass spectrometry showed that plastocyanin was correctly processed. The expressed plastocyanin was purified to homogeneity, as shown by an A278/A597 ratio of 1.0, and together with amino acid analysis and the determination of oxidized and total copper contents, both the absorption coefficients for epsilon 278 and for epsilon 597 were determined to be 4700 M-1 cm-1.

Aquatic organisms are constantly at risk of being exposed to potentially harmful chemical compounds of natural or anthropogenic origin. Biological life can for instance respond to chemical stressors by changes in gene expression, and thus, certain gene transcripts can potentially function as biomarkers, i.e. early warnings, of toxicity and chemical stress. A major challenge for biomarker application is the extrapolation of transcriptional data to potential effects at the organism level or above. Importantly, successful biomarker use also requires basal understanding of how to distinguish actual responses from background noise. The aim of this thesis is, based on response magnitude and variation, to evaluate the biomarker potential in a set of putative transcriptional biomarkers of general toxicity and chemical stress.

Specifically, I addressed a selection of six transcripts involved in cytoprotection and oxidative stress: catalase (cat), glutathione-S-transferase (gst), heat shock proteins 70 and 90 (hsp70, hsp90), metallothionein (mt) and superoxide dismutase (sod). Moreover, I used metal exposures to serve as a proxy for general chemical stress, and due to their ecological relevance and nature as sedentary filter-feeders, I used bivalves as study organisms.

In a series of experiments, I tested transcriptional responses in the freshwater duck mussel, Anodonta anatina, exposed to copper or an industrial wastewater effluent, to address response robustness and sensitivity, and potential controlled (e.g. exposure concentration) and random (e.g. gravidness) sources of variation. In addition, I performed a systematic review and meta-analysis on transcriptional responses in metal exposed bivalves to (1) evaluate what responses to expect from arbitrary metal exposures, (2) assess the influence from metal concentration (expressed as toxic unit), exposure time and analyzed tissue, and (3) address potential impacts from publication bias in the scientific literature.

Response magnitudes were generally small in relationship to the observed variation, both for A. anatina and bivalves in general. The expected response to an arbitrary metal exposure would generally be close to zero, based on both experimental observations and on the estimated impact from publication bias. Although many of the transcripts demonstrated concentration-response relationships, large background noise might in practice obscure the small responses even at relatively high exposures. As demonstrated in A. anatina under copper exposure, this can be the case already for single species under high resolution exposures to single pollutants. As demonstrated by the meta-regression, this problem can only be expected to increase further upon extrapolation between different species and exposure scenarios, due to increasing heterogeneity and random variation. Similar patterns can also be expected for time-dependent response variation, although the meta-regression revealed a general trend of slightly increasing response magnitude with increasing exposure times.

In A. anatina, gravidness was identified as a source of random variability that can potentially affect the baseline of most assessed biomarkers, particularly when quantified in gills. Response magnitudes and variability in this species were generally similar for selected transcripts as for two biochemical biomarkers included for comparison (AChE, GST), suggesting that the transcripts might not capture early warnings more efficiently than other molecular endpoints that are more toxicologically relevant. Overall, high concentrations and long exposure durations presumably increase the likelihood of a detectable transcriptional response, but not to an extent that justifies universal application as biomarkers of general toxicity and chemical stress. Consequently, without a strictly defined and validated application, this approach on its own appears unlikely to be successful for future environmental risk assessment and monitoring. Ultimately, efficient use of transcriptional biomarkers might require additional implementation of complementary approaches offered by current molecular techniques.

We tested concentration-dependence of selected gene transcripts (cat, gst, hsp70, hsp90, mt and sod) for evaluation as biomarkers of chemical stress. Contrary to the common approach of factorial designs and few exposure concentrations, we used regression across a high-resolution concentration series. Specifically, freshwater mussels (Anodonta anatina) were acutely (96 h) exposed to Cu (13 nominal concentrations, measuring 0.13–1 600 µg/L), and transcripts were measured by RT-qPCR. In digestive glands, cat, hsp90 and mt decreased with water Cu (p < 0.05), but response magnitudes saturated at < 2-fold decreases. In gills, gst, hsp70, hsp90 and mt increased with water Cu (p < 0.05). While hsp70, hsp90 and mt exceeded 2-fold increases within the exposure range, high Cu concentrations were required (38–160 µg/L). Although gill responses were generally more robust compared to digestive glands, overall small response magnitudes and moderate sensitivity may set limit for potential application as general biomarkers of chemical stress.

Through a systematic review and a series of meta-analyses, we evaluated the general responsiveness of putative transcriptional biomarkers of general toxicity and chemical stress. We targeted metal exposures performed on bivalves under controlled laboratory conditions, and selected six transcripts associated with general toxicity for evaluation: catalase (cat), glutathione-S-transferase (gst), heat shock proteins 70 and 90 (hsp70, hsp90), metallothionein (mt) and superoxide dismutase (sod). Transcriptional responses (n = 396) were extracted from published scientific articles (k = 22) and converted to log response ratios (lnRRs). By estimating toxic units (TUs), we normalized different metal exposures to a common scale, as a proxy of concentration. Using Bayesian hierarchical random effect models, we then tested the effects of metal exposure on lnRR, both for metal exposure in general and in meta-regressions using TU and exposure time as independent variables. Corresponding analyses were also repeated with transcript and tissue as additional moderators. Observed patterns were similar for general as for transcript- and tissue-specific responses. The expected overall response to arbitrary metal exposure was a lnRR of 0.50, corresponding to a 65 % increase relative a non-exposed control. However, when accounting for publication bias, the estimated ‘true’ response showed no such effect. Furthermore, expected response magnitude increased slightly with exposure time, but there was little support for general monotonic concentration-dependence with regards to TU. Altogether, this work reveals potential limitations that need consideration prior to applying the selected transcripts as biomarkers in environmental risk assessment. This article is protected by copyright. All rights reserved. Environ Toxicol Chem 2022;00:0–0. 

Correspondence between evolution and development has been discussed for more than two centuries. Recent work reveals that phylogeny-ontogeny correlations are indeed present in developmental transcriptomes of eukaryotic clades with complex multicellularity. Nevertheless, it has been largely ignored that the pervasive presence of phylogeny-ontogeny correlations is a hallmark of development in eukaryotes. This perspective opens a possibility to look for similar parallelisms in biological settings where developmental logic and multicellular complexity are more obscure. For instance, it has been increasingly recognized that multicellular behavior underlies biofilm formation in bacteria. However, it remains unclear whether bacterial biofilm growth shares some basic principles with development in complex eukaryotes. Here we show that the ontogeny of growing Bacillus subtilis biofilms recapitulates phylogeny at the expression level. Using time-resolved transcriptome and proteome profiles, we found that biofilm ontogeny correlates with the evolutionary measures, in a way that evolutionary younger and more diverged genes were increasingly expressed toward later timepoints of biofilm growth. Molecular and morphological signatures also revealed that biofilm growth is highly regulated and organized into discrete ontogenetic stages, analogous to those of eukaryotic embryos. Together, this suggests that biofilm formation in Bacillus is a bona fide developmental process comparable to organismal development in animals, plants, and fungi. Given that most cells on Earth reside in the form of biofilms and that biofilms represent the oldest known fossils, we anticipate that the widely adopted vision of the first life as a single-cell and free-living organism needs rethinking.

Plant hormones are responsible for plant growth and adaptation to the environment. Among them the most important are cytokinins. Plants undergo gene silencing processes called homology-dependent gene silencing processes. In Arabidopsis there are two homology-dependent gene silencing genes that were chosen for further study, namely AtHOG1 and AtHOG2. Transgenic plants were generated previously with ten different constructs containing AtHOG1 or AtHOG2 genes and were used in this study. Some of the constructs had GFP attached so that the protein expressed could be visualised in a confocal microscope. Transgenic plants generated were T1 and T2 generations. Their DNA was extracted from leaves. By means of PCR transgenic plants were identified. There were 147 samples. Among them there were 39 positiveswith BAR primers and 32 positives with construct specific primers. The localisation of the HOG2 protein was observed in a confocal microscope. Seeds used were T3 generation and were obtained from the lab. HOG2 protein was found to be localised in cell membrane, root tip and chloroplasts.

1α,25-dihydroxyvitamin D3 (1,25D3 ) is a highly potential anti-cancerous agent for prevention and treatment of prostate cancer, the most commonly diagnosed cancer type of males in western countries. A recent study by our laboratory, demonstrates that LNCaP cancer cells treated with 1,25D3, evoked dose-dependent activation of the JNK/SAPK MAPK signaling pathway within 10 minutes after hormone treatment, indicative of membrane-initiated steroid signaling (MISS) by 1,25D3. This confirms previous reports on intestinal-, chondrocyte- and osteoblast cells, where 1,25D3 operates through pharmacologically distinct nuclear-initiated mechanisms (NISS) and plasma membrane-initiated mechanisms. NISS is mediated via the vitamin D receptor (nVDR) and MISS is mediated through 1,25D3-MARRS (PDIA3, 1,25D3-membraneassociated rapid response steroid binding protein) or nVDR. The aims of the present study were to investigate the mechanisms of MISS evoked effects on alkaline phosphatase (ALP) and activation of the JNK/SAPK by 1,25D3, and the involvement of PDIA3 in 1,25D3 initiated activation of the JNK/SAPK signaling pathway. Furthermore, 1,25D3-treated LNCaP cells were transfected with siRNA against PDIA3 and phosphorylated JNK/SAPK was estimated by western analysis. Western analysis and ALP-assays demonstrated rapid activation of both JNK/SAPK as well as ALP. Silencing of PDIA3 did not affect 1,25D3 mediated activation of JNK/SAPK, suggesting that PDIA3 is not involved in the 1,25D3-initiated activation of the JNK/SAPK signaling pathway.

The human type I interferon (IFN) genes are clustered on human chromosome 9p21 and the mouse genes are located in the region of conserved synteny on mouse chromosome 4. We have identified two novel mouse Ifna genes (Ifna12, Ifna13) and Ifnl2 (IFN-like 2, a homologue of Limitin/IFN-like 1). Another type I IFN gene was designated Ifne1. Mouse Ifne1 was expressed in ovaries and uterus but not in tissues of hematopoietic origin. IFN-epsilon1 has general structural characteristics of a type I IFN. These studies represent the first detailed annotation of the mouse type I IFN locus, and the products of these novel genes may have important functions in reproduction and host defense.

Arsenic (As) is a phytotoxic element causing health hazards. This work investigates whether and how silicon (Si) alleviates As toxicity in wheat. The addition of Si under As-stress significantly improved morphophysiological characteristics, total protein, and membrane stability compared to As-stressed plants, suggesting that Si does have critical roles in As detoxification in wheat. Analysis of arsenate reductase activity and phytosiderophore (PS) release reveals their no involvement in the Si-mediated alleviation of As in wheat. Furthermore, Si supplementation in As-stressed plants showed a significant increase of As in roots but not in shoots compared with the plants grown under As stress. Further, gene expression analysis of two chelating molecules, TaPCS1 (phytochelatin synthase) and TaMT1 (metallothionein synthase) showed significant induction due to Si application under As stress compared with As-stressed plants. It is consistent with the physiological observations and suggests that alleviation of As toxicity in rice might be associated with As sequestration in roots leading to reduced As translocation in shoots. Furthermore, increased catalase, peroxidase, and glutathione reductase activities in roots imply the active involvement of reactive oxygen species scavenging for protecting wheat plants from As-induced oxidative injury. The study provides mechanistic evidence on the beneficial effect of Si on As toxicity in wheat plants.

Background: Cardiovascular diseases (CVDs) and cancers are the major causes of chronic arsenic exposure-related morbidity and mortality. Matrix metalloproteinase-2 (MMP-2) and −9 (MMP-9) are deeply involved in the pathogenesis of CVDs and cancers. This study has been designed to evaluate the interactions of arsenic exposure with serum MMP-2 and MMP-9 concentrations especially in relation to the circulating biomarkers of CVDs.

Methods: A total of 373 human subjects, 265 from arsenic-endemic and 108 from non-endemic areas in Bangladesh were recruited for this study. Arsenic concentrations in the specimens were measured by inductively coupled plasma mass spectroscopy (ICP-MS) and serum MMPs were quantified by immunoassay kits.

Results: Serum MMP-2 and MMP-9 concentrations in arsenic-endemic population were significantly (p < 0.001) higher than those in non-endemic population. Both MMPs showed significant positive interactions with drinking water (rs = 0.208, p < 0.001 for MMP-2; rs = 0.163, p <0.01 for MMP-9), hair (rs= 0.163, p < 0.01 for MMP-2; rs = 0.173, p < 0.01 for MMP-9) and nail (rs= 0.160, p < 0.01 for MMP-2; rs = 0.182, p < 0.001 for MMP-9) arsenic of the study subjects. MMP-2 concentrations were 1.02, 1.03 and 1.05 times, and MMP-9 concentrations were 1.03, 1.06 and 1.07 times greater for 1 unit increase in log-transformed water, hair and nail arsenic concentrations, respectively, after adjusting for covariates (age, sex, BMI, smoking habit and hypertension). Furthermore, both MMPs were increased dose-dependently when the study subjects were split into three (≤10, 10.1-50 and > 50 μg/L) groups based on the regulatory upper limit of water arsenic concentration set by WHO and Bangladesh Government. MMPs were also found to be significantly (p < 0.05) associated with each other. Finally, the concentrations of both MMPs were correlated with several circulating markers related to CVDs.

Conclusions: This study showed the significant positive associations and dose–response relationships of arsenic exposure with serum MMP-2 and MMP-9 concentrations. This study also showed the interactions of MMP-2 and MMP-9 concentrations with the circulating markers of CVDs suggesting the MMP-2 and MMP-9 -mediated mechanism of arsenic-induced CVDs.

This is a comparative study of the photoinduced (so-called forward) electron-transfer reaction 3Zncyt/pc(II) --> Zncyt+/pc(I), between the triplet state of zinc cytochrome c (3Zncyt) and cupriplastocyanin [pc(II)], and the thermal (so-called back) electron-transfer reaction Zncyt+/pc(I) --> Zncyt/pc(II), between the cation (radical) of zinc cytochrome c (Zncyt+) and cuproplastocyanin [pc(I)], which follows it. Both reactions occur between associated (docked) reactants, and the respective unimolecular rate constants are kF and kB. Our previous studies showed that the forward reaction is gated by a rearrangement of the diprotein complex. Now we examine the back reaction and complare the two. We study the effects of temperature (in the range 273.3-302.9 K) and viscosity (in the range 1.00-17.4 cP) on the rate constants and determine enthalpies (DeltaH), entropies (DeltaS), and free energies (DeltaG) of activation. We compare wild-type spinach plastocyanin, the single mutants Tyr83Leu and Glu59Lys, and the double mutant Glu59Lys/Glu60Gln. The rate constant kB for wild-type spinach plastocyanin and its mutants markedly depends on viscosity, an indication that the back reaction is also gated. The activation parameters DeltaH and DeltaS show that the forward and back reactions have similar mechanisms, involving a rearrangement of the diprotein complex from the initial binding configuration to the reactive configuration. The rearrangements of the complexes 3Zncyt/pc(II) and Zncyt+/pc(I) that gate their respective reactions are similar but not identical. Since the back reaction of all plastocyanin variants is faster than the forward reaction, the difference in free energy between the docking and the reactive configuration is smaller for the back reaction than for the forward reaction. This difference is explained by the change in the electrostatic potential on the plastocyanin surface as Cu(II) is reduced to Cu(I). It is the smaller DeltaH that makes DeltaG smaller for the back reaction than for the forward reaction.

The unimolecular rate constant for the photoinduced electron-transfer reaction 3Zncyt/pc(II) --> Zncyt+/pc(I) within the electrostatic complex of zinc cytochrome c and spinach cupriplastocyanin is kF. We report the effects on kF of the following factors, all at pH 7.0: 12 single mutations on the plastocyanin surface (Leu12Asn, Leu12Glu, Leu12Lys, Asp42Asn, Asp42Lys, Glu43Asn, Glu59Gln, Glu59Lys, Glu60Gln, Glu60Lys, Gln88Glu, and Gln88Lys), the double mutation Glu59Lys/Glu60Gln, temperature (in the range 273.3-302.9 K), and solution viscosity (in the range 1. 00-116.0 cP) at 283.2 and 293.2 K. We also report the effects of the plastocyanin mutations on the association constant (Ka) and the corresponding free energy of association (DeltaGa) with zinc cytochrome c at 298.2 K. Dependence of kF on temperature yielded the activation parameters DeltaH, DeltaS, and DeltaG. Dependence of kF on solution viscosity yielded the protein friction and confirmed the DeltaG values determined from the temperature dependence. The aforementioned intracomplex reaction is not a simple electron-transfer reaction because donor-acceptor electronic coupling (HAB) and reorganizational energy (lambda), obtained by fitting of the temperature dependence of kF to the Marcus equation, deviate from the expectations based on precedents and because kF greatly depends on viscosity. This last dependence and the fact that certain mutations affect Ka but not kF are two lines of evidence against the mechanism in which the electron-transfer step is coupled with the faster, but thermodynamically unfavorable, rearrangement step. The electron-transfer reaction is gated by the slower, and thus rate determining, structural rearrangement of the diprotein complex; the rate constant kF corresponds to this rearrangement. Isokinetic correlation of DeltaH and DeltaS parameters and Coulombic energies of the various configurations of the Zncyt/pc(II) complex consistently show that the rearrangement is a facile configurational fluctuation of the associated proteins, qualitatively the same process regardless of the mutations in plastocyanin. Correlation of kF with the orientation of the cupriplastocyanin dipole moment indicates that the reactive configuration of the diprotein complex involves the area near the residue 59, between the upper acidic cluster and the hydrophobic patch. Kinetic effects and noneffects of plastocyanin mutations show that the rearrangement from the initial (docking) configuration, which involves both acidic clusters, to the reactive configuration does not involve the lower acidic cluster and the hydrophobic patch but involves the upper acidic cluster and the area near the residue 88.

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photo-bleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.

Modulating the activities of costimulatory molecules controlling immune responses holds considerable promise for immunotherapy. CTLA4Ig (abatacept), a soluble version of the T cell-expressed membrane receptor CTLA-4, is approved for the treatment of rheumatoid arthritis. Like natural CTLA-4 molecules, CTLA4Ig ligates B7-1 and B7-2 on antigen presenting cells, preventing CD28-mediated costimulation of T cells. However. CTLA4Ig can also prevent ligation of CTLA-4, potentially blocking vital inhibitory signals, thereby augmenting immunity. There have been no quantitative analyses of the likely effects of CTLA4Ig on costimulatory interactions at the immunological synapse. We present a mathematical model, based on rigorous biophysical and expression data, for simulating the effects of abatacept and a mutated derivative, LEA29Y, on the synaptic interactions of CD28 and CTLA-4. The simulations reveal an unexpectedly large window within which CD28, but not CTLA-4, ligation is blocked by CTLA4Ig, perhaps explaining the efficacy of abatacept at the recommended therapeutic dose (10 mg/kg) and its relative safety. However, the simulations suggest that the present dosing regimen is close to the maximum theoretically safe dose. The simulations also show that, within the therapeutic window, LEA29Y enhances the interaction of CTLA-4 with the more potent of its two native ligands, B7-1. They also suggest that CTLA-4 ligation by B7-1 could, in principle, be enhanced by further decreasing the off-rate of CTLA4Ig for binding to B7-2. Our findings therefore offer molecular explanations for why LEA29Y might prove to be more effective than abatacept in a clinical setting, and suggest ways in which its therapeutic efficacy could be further optimised. (C) 2011 Elsevier Ltd. All rights reserved.

Lactic acid bacteria isolated from unconventional sources are often attractive targets in the quest for obtaining better probiotics. In the present study, 16 members of the genus Lactobacillus, isolated from 3 different sources in western India, viz., plants, fermented foods and beverages, and human feces, were evaluated for their probiotic and bioactive properties. The isolates were closely related to Lactobacillus fermentum, Lactobacillus pentosus, and mainly Lactobacillus plantarum. The isolates were tolerant to bile salt, acidic pH and pancreatin, although pancreatin tolerance was generally low. Cellular extracts of several isolates displayed antioxidant activity, while cell-free supernatants displayed antibacterial activity against human pathogens. Antioxidant activity of Lactobacilli of human origin was higher than those from vegetables or fermented foods and beverages. L. plantarum AG40V prevented spoilage of fresh-cut fruits, vegetables and sprouted mung-beans. Lactobacilli from all sources displayed equal probiotic potential and those of human origin displayed superior antioxidant activity over others.

Cadmium (Cd) is one of the most phytotoxic elements causing an agricultural problem and human health hazards. This work investigates whether and how silicon (Si) ameliorates Cd toxicity in Alfalfa. The addition of Si in Cd-stressed plants caused significant improvement in morpho-physiological features as well as total protein and membrane stability, indicating that Si does have critical roles in Cd detoxification in Alfalfa. Furthermore, Si supplementation in Cd stressed plants showed a significant decrease in Cd and Fe concentrations in both roots and shoots compared with Cd-stressed plants, revealing that Si-mediated tolerance to Cd stress is associated with Cd inhibition in Alfalfa. Results also showed no significant changes in the  expression of two metal chelators [MsPCS1 (phytochelatin synthase) and MsMT2  (metallothionein)] and PC (phytochelatin) accumulation, indicating that there may be no metal sequestration or change in metal sequestration following Si application under Cd stress in  Alfalfa. We further performed a targeted study on the effect of Si on Fe uptake mechanisms. We observed the consistent reduction in Fe reductase activity, expression of Fe-related genes [MsIRT1 (Fe transporter), MsNramp1 (metal transporter) and OsFRO1 (ferric chelate reductase] and Fe chelators (citrate and malate) by Si application to Cd stress in roots of Alfalfa. These results support that limiting Fe uptake through the down-regulation of Fe acquisition mechanisms confers Si-mediated alleviation of Cd toxicity in Alfalfa. Finally, an increase of catalase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD) activities along with elevated methionine and proline subjected to Si application might play roles, at least in part, to reduce H2O2 and to provide antioxidant defense against Cd stress in Alfalfa. The study shows evidence of the effect of Si on alleviating Cd toxicity in Alfalfa and can be further extended for phytoremediation of Cd toxicity in plants.

Approximately 30 million years ago, ancestors of fungus-growing termites started an obligate mutualistic relationship with a Basidiomycete fungus Termitomyces. The success of this obligate relation is the division of labour and reliance on termite caste gut microbial symbionts. Termites workers maintain Termitomyces fungal garden with their workforce and dual gut passage while the soldier caste protects the colony from predators. The fungal garden concurrently provides enough food for the colony members. Royal pair (a king and a queen) is the centralised caste in the colony, and they control the colony population by their massive reproduction, but their gut community composition remains unexplored. This project aimed to characterise the gut microbes associated with royal pairs of a fungus‐growing termite species Macrotermes natalensis. Four colonies were explored using high throughput sequencing of 16S rRNA gene amplicon dataset. The high-throughput sequence result showed that royal gut microbiotas were comprised of a lower number of bacterial taxa than sterile caste (workers and soldiers). This less number of bacterial taxa suggested that the royal pair gut was completely decoupled from the sterile castes gut, which indicates that the royal pair were possibly provided with a unique diet. The study also showed diversity in bacterial genus-level OTUs of royal pairs in all four colonies which indicated that there is a diet variation between the king and queen. The media predicting strategy could facilitate future cultivation efforts for targeted royal pair gut bacterial strains.

Most organisms naturally accumulating trehalose upon stress produce the sugar in a two-step process by the action of the enzymes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). Transgenic plants overexpressing TPS have shown enhanced drought tolerance in spite of minute accumulation of trehalose, amounts believed to be too small to provide a protective function. However, overproduction of TPS in plants has also been found combined with pleiotropic growth aberrations. This paper describes three successful strategies to circumvent such growth defects without loosing the improved stress tolerance. First, we introduced into tobacco a double construct carrying the genes TPS1 and TPS2 (encoding TPP) from Saccharomyces cerevisiae. Both genes are regulated by an Arabidopsis RuBisCO promoter from gene AtRbcS1A giving constitutive production of both enzymes. The second strategy involved stress-induced expression by fusing the coding region of ScTPS1 downstream of the drought-inducible Arabidopsis AtRAB18 promoter. In transgenic tobacco plants harbouring genetic constructs with either ScTPS1 alone, or with ScTPS1 and ScTPS2 combined, trehalose biosynthesis was turned on only when the plants experienced stress. The third strategy involved the use of AtRbcS]A promoter together with a transit peptide in front of the coding sequence of ScTPS1, which directed the enzyme to the chloroplasts. This paper confirms that the enhanced drought tolerance depends on unknown ameliorated water retention as the initial water status is the same in control and transgenic plants and demonstrates the influence of expression of heterologous trehalose biosynthesis genes on Arabidopsis root development.

Endometrial cancer (EC) is the most abundant female gynaecologic malignancy, ranking fourth in incidence among invasive tumors in women. Hormone-related (estrogen-dependent) EC is the prevalent subtype and accounts for approximately 75% of these cancers. Females of the BDII inbred rat strain are extremely prone to endometrial adenocarcinoma, (EAC) and approximately 90% of virgin females spontaneously develop EAC during their life span. Thus, these rats serve as a useful model for the genetic analysis of this malignancy. In the present work, gene expression profiling, by means of cDNA microarrays, was performed on cDNA from endometrial tumor cell lines and from cell lines derived from nonmalignant lesions/normal tissues of the endometrium without specific findings (WSF). We identified numerous genes differentially expressed between endometrial cell lines and WSFs employing clustering analysis and statistical inference analysis. Many of the genes identified are located within or close to the chromosomal regions earlier identified to be associated with EAC susceptibility and development. Several of the genes identified are involved in pathways commonly altered in carcinogenesis, such as the TGF-pathway.

The NF-kappa B transcription factor protein family has diverse cellular and biological functions, and posttranslational modification is important to regulate these functions. An important site of phosphorylation of NF-kappa B p65 subunit is at serine-536 (phospho-Ser536-p65), and this phosphorylation is involved in regulation of transcriptional activity, nuclear localization, and protein stability. In this study, we investigated expression of phospho-Ser536-p65 in colorectal cancers and its relationships with clinicopathological factors. The expression of phospho-Ser536-p65 was examined by immunohistochemistry in 203 primary colorectal cancers, 156 normal mucosa specimens, and 18 metastases in the lymph nodes. The expression of phospho-Ser536-p65 increased from normal mucosa to primary tumor (p < 0.0001). Further, the increased expression of phospho-Ser536-p65 in the cytoplasm of the primary tumors correlated with worse survival of the patients independently of gender, age, tumor location, stage, and differentiation (p = 0.04; hazard ratio, 1.89; 95% CI 1.03-3.47). The NF-kappa B p65 subunit phosphorylated at serine-536 is an independent prognostic factor in colorectal cancer patients.

The innate immune system are a cooperation of many components – receptors being one of them. Both membrane-bound and cytosolic receptors play a large role in the defence system against pathogens and danger. NLRP3 is a receptor which assembles a protein complex called inflammasome in response to cytosolic stress and is responsible for many autoimmune diseases if it malfunctions. The activation of the NLRP3 inflammasome leads to secretion of inflammatory cytokines and in many cases to programmed cell death. The structure, function and activation of the NLRP3 inflammasome is still not fully understood and the urge to understand the mechanisms behind are important for future medical improvements. The aim was to anchor the NLRP3 inflammasome by the cell membrane - By Overlap PCR, the NLRP3 cDNA was fused extracellular and trans-membrane parts of the TLR4 cDNA to anchor the NLRP3 to the membrane and in turn analyse the inflammasome with LPI™ technology. Multiple primers and a TLR4 nucleotide were designed and the NLRP3 was amplified with specific overhangs by PCR. The fusion protein was successfully linked together by Overlap PCR but not confirmed by sequencing. The gene fusion demands high quality primers for amplification and further evaluation must be made to the details of the laboratory. To anchor the protein complex to the cell membrane, continue to be of full importance and can be an asset in many structural studies and biopharmaceuticals trials.

Objective. The aim of this study was to identify changes in inflammatory molecules in the blood (plasma) of patients with chronic prostatitis/chronic pelvic syndrome (CP/CPPS) compared with controls. Altered levels indicate a systemic component by possible involvement of the prostate and/or the inner pelvic floor musculature. Material and methods. In 32 patients with CP/CPPS and 37 controls, blood plasma levels of testosterone, macrophage migration inhibitory factor (MIF), tumour necrosis factor-alpha (TNF-alpha), TNF-beta, interleukin-2 (IL-2) and IL-1 beta were measured by enzyme-linked immunosorbent assay. Cortisol in saliva samples was measured in the morning and late evening. All participants answered a questionnaire regarding their health profile. Results. Significantly higher levels of MIF (p = 0.012) were detected in patients. The testosterone level was, contrary to other studies, little lower in patients (p = 0.014; age adjusted). When controls with health issues and patients with a parallel disease were excluded, the MIF and TNF-alpha levels were higher in the patients (p = 0.007, p = 0.016, respectively) than in controls, and the testosterone was slightly lower in patients (p = 0.047). Conclusions. The findings show an immune response extending to the circulatory system, in which MIF makes a significant contribution to CP/CPPS. This study also indicates TNF-alpha as a circulatory component when excluding subjects with concomitant diseases. Both MIF and TNF-alpha have previously been highlighted for other diseases related to chronic pain and here also for CP/CPPS. These results provide further insights into the immunological basis of CP/CPPS.

Background: Transcription factors (TFs) are the upstream regulators that orchestrate gene expression, and therefore a centrepiece in bioinformatics studies. While a core strategy to understand the biological context of genes and proteins includes annotation enrichment analysis, such as Gene Ontology term enrichment, these methods are not well suited for analysing groups of TFs. This is particularly true since such methods do not aim to include downstream processes, and given a set of TFs, the expected top ontologies would revolve around transcription processes.

Results: We present the TFTenricher, a Python toolbox that focuses specifically at identifying gene ontology terms, cellular pathways, and diseases that are over-represented among genes downstream of user-defined sets of human TFs. We evaluated the inference of downstream gene targets with respect to false positive annotations, and found an inference based on co-expression to best predict downstream processes. Based on these downstream genes, the TFTenricher uses some of the most common databases for gene functionalities, including GO, KEGG and Reactome, to calculate functional enrichments. By applying the TFTenricher to differential expression of TFs in 21 diseases, we found significant terms associated with disease mechanism, while the gene set enrichment analysis on the same dataset predominantly identified processes related to transcription.

Conclusions and availability: The TFTenricher package enables users to search for biological context in any set of TFs and their downstream genes. The TFTenricher is available as a Python 3 toolbox at https://github.com/rasma774/Tftenricher, under a GNU GPL license and with minimal dependencies.

Prediction algorithms for protein or gene structures, including transcription factor binding from sequence information, have been transformative in understanding gene regulation. Here we ask whether human transcriptomic profiles can be predicted solely from the expression of transcription factors (TFs). We find that the expression of 1600 TFs can explain >95% of the variance in 25,000 genes. Using the light-up technique to inspect the trained NN, we find an over-representation of known TF-gene regulations. Furthermore, the learned prediction network has a hierarchical organization. A smaller set of around 125 core TFs could explain close to 80% of the variance. Interestingly, reducing the number of TFs below 500 induces a rapid decline in prediction performance. Next, we evaluated the prediction model using transcriptional data from 22 human diseases. The TFs were sufficient to predict the dysregulation of the target genes (rho = 0.61, P < 10−216). By inspecting the model, key causative TFs could be extracted for subsequent validation using disease-associated genetic variants. We demonstrate a methodology for constructing an interpretable neural network predictor, where analyses of the predictors identified key TFs that were inducing transcriptional changes during disease.