The value of animal models in predicting genetic susceptibility to complex diseases such as rheumatoid arthritis

For a long time, genetic studies of complex diseases were most successfully conducted in animal models. However, the field of genetics is now rapidly evolving, and human genetics has also started to produce strong candidate genes for complex diseases. This raises the question of how to continue gene-finding attempts in animals and how to use animal models to enhance our understanding of gene function. In this review we summarize the uses and advantages of animal studies in identification of disease susceptibility genes, focusing on rheumatoid arthritis. We are convinced that animal genetics will remain a valuable tool for the identification and investigation of pathways that lead to disease, well into the future.     

PHOTOSYSTEM II PROTEIN33, a Protein Conserved in the Plastid Lineage,Is Associated with the Chloroplast Thylakoid Membrane and Provides Stability to Photosystem II Supercomplexes in Arabidopsis

Photosystem II (PSII) is a multiprotein complex that catalyzes the light-driven water-splitting reactions of oxygenic photosynthesis. Light absorption by PSII leads to the production of excited states and reactive oxygen species that can cause damage to this complex. Here, we describe Arabidopsis (Arabidopsis thaliana) At1g71500, which encodes a previously uncharacterized protein that is a PSII auxiliary core protein and hence is named PHOTOSYSTEM II PROTEIN33 (PSB33). We present evidence that PSB33 functions in the maintenance of PSII-light-harvesting complex II (LHCII) supercomplex organization. PSB33 encodes a protein with a chloroplast transit peptide and one transmembrane segment. In silico analysis of PSB33 revealed a light-harvesting complex-binding motif within the transmembrane segment and a large surface-exposed head domain. Biochemical analysis of PSII complexes further indicates that PSB33 is an integral membrane protein located in the vicinity of LHCII and the PSII CP43 reaction center protein. Phenotypic characterization of mutants lacking PSB33 revealed reduced amounts of PSII-LHCII supercomplexes, very low state transition, and a lower capacity for nonphotochemical quenching, leading to increased photosensitivity in the mutant plants under light stress. Taken together, these results suggest a role for PSB33 in regulating and optimizing photosynthesis in response to changing light levels.PSII is a multiprotein complex in plants with 31 identified polypeptides (Wegener et al., 2011; Pagliano et al., 2013). It is associated with an extrinsic trimeric light-harvesting complex (LHC), forming the PSII-LHCII supercomplex. The PSII complex performs a remarkable biochemical reaction, the oxidation of water using light energy from the sun, which profoundly contributes to the overall biomass accumulation in the biosphere (Barber et al., 2004). Consequently, the stability and functional integrity of the PSII-LHCII supercomplex is crucially important for photosynthetic function. The energy of a photon, either absorbed directly by PSII or indirectly via energy transfer from adjacent antenna chlorophyll (Chl) molecules, excites the PSII reaction center P680. The excited state, P680*, can transfer an electron to pheophytin, producing the most powerful oxidant known in biology, P680⁺, which can remove electrons from water. Excessive input of excitation energy into PSII saturates the electron transfer system and causes either acceptor or donor site limitation in the complex. This results in increased production of reactive oxygen species (ROS): singlet oxygen at the PSII donor side and superoxide at the acceptor side (Munné-Bosch et al., 2013). Several protective mechanisms have been documented that decrease the production of singlet oxygen at the PSII donor side in photosynthetic eukaryotes. Notably, reducing energy transfer from LHC to PSII via nonphotochemical quenching (NPQ) is a key avoidance mechanism (Ruban and Murchie, 2012).Despite years of intensive study of PSII structure and function, new proteins that are associated with the PSII complex continue to be discovered, including an increasing number involved in the stability and organization of PSII-LHCII supercomplexes (García-Cerdán et al., 2011; Lu et al., 2011a; Wegener et al., 2011). Two complementary approaches (Merchant et al., 2007; Lu et al., 2008, 2011b; Ajjawi et al., 2010) that utilize phylogenomics (GreenCut) and large-scale phenotypic mutant screening (Chloroplast 2010 Project; http://www.plastid.msu.edu/) were employed by our groups to discover novel plant proteins with roles in photosynthesis. GreenCut identifies proteins found only in photosynthetic organisms, and it is likely that many of them are involved in biochemical processes associated with the structure, assembly, or function of the photosynthetic apparatus and the chloroplast that houses it (Merchant et al., 2007; Karpowicz et al., 2011). The Chloroplast 2010 Project was a large-scale reverse-genetic mutant screen in which thousands of homozygous Arabidopsis (Arabidopsis thaliana) transfer DNA (T-DNA) insertion lines were analyzed for defects in the rise and decay kinetics of Chl fluorescence (Lu et al., 2008, 2011a, 2011b; Ajjawi et al., 2010).The GreenCut and Chloroplast 2010 approaches both identified the Arabidopsis At1g71500 locus as encoding a protein of unknown function with potential relevance to photosynthesis. In this work, we demonstrate that plant lines carrying three independent mutations at this locus display severe light-induced photoinhibition due to a less stable supramolecular organization of PSII. Biochemical analyses revealed that this protein is associated with PSII complexes, and since the last described PSII protein was called PHOTOSYSTEM II PROTEIN32 (PSB32), we named the gene PSB33. The nuclear genome-encoded PSB33 is predicted to have a chloroplast transit peptide and a transmembrane domain. The biochemical analyses presented below indicate that PSB33 is required for the proper interaction and stability of PSII-LHCII supercomplexes and, in turn, in regulating photosynthesis in response to fluctuating light levels.     

The Proton Trap Technology—Toward High Potential Quinone‐Based Organic Energy Storage

An organic cathode material based on a copolymer of poly(3,4‐ethylenedioxythiophene) containing pyridine and hydroquinone functionalities is described as a proton trap technology. Utilizing the quinone to hydroquinone redox conversion, this technology leads to electrode materials compatible with lithium and sodium cycling chemistries. These materials have high inherent potentials that in combination with lithium give a reversible output voltage of above 3.5 V (vs Li^0/+) without relying on lithiation of the material, something that is not showed for quinones previously. Key to success stems from coupling an intrapolymeric proton transfer, realized by an incorporated pyridine proton donor/acceptor functionality, with the hydroquinone redox reactions. Trapping of protons in the cathode material effectively decouples the quinone redox chemistry from the cycling chemistry of the anode, which makes the material insensitive to the nature of the electrolyte cation and hence compatible with several anode materials. Furthermore, the conducting polymer backbone allows assembly without any additives for electronic conductivity. The concept is demonstrated by electrochemical characterization in several electrolytes and finally by employing the proton trap material as the cathode in lithium and sodium batteries. These findings represent a new concept for enabling high potential organic materials for the next generation of energy storage systems.     

Hydrogenases and Hydrogen Metabolism of Cyanobacteria

Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect—the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included.     

Reduced Expression of the Polymeric Immunoglobulin Receptor in Pancreatic and Periampullary Adenocarcinoma Signifies Tumour Progression and Poor Prognosis

The polymeric immunoglobulin receptor (pIgR) is a key component of the mucosal immune system that mediates epithelial transcytosis of immunoglobulins. High pIgR expression has been reported to correlate with a less aggressive tumour phenotype and an improved prognosis in several human cancer types. Here, we examined the expression and prognostic significance of pIgR in pancreatic and periampullary adenocarcinoma. The study cohort encompasses a consecutive series of 175 patients surgically treated with pancreaticoduodenectomy for pancreatic and periampullary adenocarcinoma in Malmö and Lund University Hospitals, Sweden, between 2001–2011. Tissue microarrays were constructed from primary tumours (n = 175) and paired lymph node metastases (n = 105). A multiplied score was calculated from the fraction and intensity of pIgR staining. Classification and regression tree analysis was used to select the prognostic cut-off. Unadjusted and adjusted hazard ratios (HR) for death and recurrence within 5 years were calculated. pIgR expression could be evaluated in 172/175 (98.3%) primary tumours and in 96/105 (91.4%) lymph node metastases. pIgR expression was significantly down-regulated in lymph node metastases as compared with primary tumours (p = 0.018). Low pIgR expression was significantly associated with poor differentiation grade (p<0.001), perineural growth (p = 0.027), lymphatic invasion (p = 0.016), vascular invasion (p = 0.033) and infiltration of the peripancreatic fat (p = 0.039). In the entire cohort, low pIgR expression was significantly associated with an impaired 5-year survival (HR = 2.99, 95% confidence interval (CI) 1.71–5.25) and early recurrence (HR = 2.89, 95% CI 1.67–4.98). This association remained significant for survival after adjustment for conventional clinicopathological factors, tumour origin and adjuvant treatment (HR = 1.98, 95% CI 1.10–3.57). These results demonstrate, for the first time, that high tumour-specific pIgR expression signifies a more favourable tumour phenotype and that low expression independently predicts a shorter survival in patients with pancreatic and periampullary cancer. The mechanistic basis for the putative tumour suppressing properties of pIgR in these cancers merits further study.     

A rapid gas chromatography-mass spectrometry method for quantification of alkylresorcinols in human plasma

Alkylresorcinols (ARs) are phenolic lipids that among foods are found almost exclusively in whole grain and bran products based on wheat and rye. They have been suggested to be used as selective biomarkers of whole grain wheat and rye intake and, thus, may serve as an alternative/complement to commonly used dietary assessment methods in epidemiological studies. For such investigations where analysis of large numbers of valuable samples is required, rapid, sensitive, and repeatable methods are essential. In this article, we describe a rapid and sensitive gas chromatography-mass spectrometry (GC-MS) method for quantification of AR homologues C17:0, C19:0, C21:0, C23:0, and C25:0 in human plasma. The method uses Oasis MAX solid phase extraction cartridges for sample cleanup. A plasma sample of 0.2 ml could be used without preincubation with water. Samples in the range of 7 to 8750 nmol total AR/L were successfully analyzed with the method described, and the average total AR recovery within the reported range was 92 ± 12%. The within- and between-day precision values of total AR concentration in a quality control sample, determined as the coefficients of variation, were on average 7 and 10%, respectively. Approximately 30 to 50 samples could be analyzed within 1 day. The improved GC-MS method presented can be used for rapid analysis of ARs in relatively small sample volumes.     

The genetics of rheumatoid arthritis and the need for animal models to find and understand the underlying genes

The causes of rheumatoid arthritis (RA) are largely unknown. However, RA is most probably a multifactorial disease with contributions from genetic and environmental factors. Searches for genes that influence RA have been conducted in both human and experimental model materials. Both types of study have confirmed the polygenic inheritance of the disease. It has become clear that the features of RA complicate the human genetic studies. Animal models are therefore valuable tools for identifying genes and determining their pathogenic role in the disease. This is probably the fastest route towards unravelling the pathogenesisis of RA and developing new therapies.     

Studies of cytochrome c oxidase-driven H(+)-coupled phosphate transport catalyzed by the Saccharomyces cerevisiae Pho84 permease in coreconstituted vesicles

The proton-coupled Pho84 phosphate permease of Saccharomyces cerevisiae, overexpressed as a histidine-tagged chimera in Escherichia coli, was detergent-solubilized, purified, and reconstituted into proteoliposomes. Proteoliposomes containing the Pho84 protein were fused with proteoliposomes containing purified cytochrome c oxidase from beef heart mitochondria. Both components of the coreconstituted system were functionally incorporated in tightly sealed membrane vesicles in which the cytochrome c oxidase-generated electrochemical proton gradient could drive phosphate transport via the proton-coupled Pho84 permease. The metal dependency of transport indicates that a metal-phosphate complex is the translocated substrate.     

Reidentification of the female sex pheromone of the Indian meal moth, Plodia interpunctella: evidence for a four-component pheromone blend

Pheromone gland extracts from calling female Plodia interpunctella contained at least seven compounds that consistently elicited electroantennographic responses from male antennae upon gas chromatographic analysis. Three of these compounds were found to be the previously identified gland constituents, i.e., (Z,E)-9,12-tetradecadienyl acetate (Z9,E12-14:OAc), (Z,E)-9,12-tetradecadienal (Z9,E12-14:Ald) and (Z,E)-9,12-tetradecadienol (Z9,E12-14:OH). A fourth EAD-active compound was identified as (Z)-9-tetradecenyl acetate (Z9-14:OAc). The homologue (Z)-11-hexadecenyl acetate (Z11-16:OAc) was also identified in the extracts, but showed no EAD activity. The identity of all five compounds was confirmed by comparison of GC retention times and mass spectra with those of synthetic standards. In flight tunnel tests there were no significant differences in response of male P. interpunctella to the bait containing all four EAD-active compounds and the responses to female gland extacts. A behavioural assay of different two-compound blends in the flight tunnel showed that only addition of the corresponding aldehyde to the major pheromone component Z9,E12-14:OAc raised the male response. A subtractive assay, however, revealed that the exclusion of any of the compounds from the complete four-compound blend reduced its activity significantly. We thus conclude that the female-produced sex pheromone of P. interpunctella consists of at least four components, i.e., Z9,E12-14:OAc, Z9,E12-14:Ald, Z9,E12-14:OH and Z9-14:OAc.In a field trapping test performed in a storage facility, the four-component blend attracted significantly more males of P. interpunctella than traps baited with Z9,E12-14:OAc alone. In contrast, the highest number of Ephestia kuehniella males was found in the traps baited with this major component, suggesting that the secondary pheromone components contribute to the species specificity of the blend.     

RAF2 is a RuBisCO assembly factor in Arabidopsis thaliana

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the reaction between gaseous carbon dioxide (CO₂) and ribulose‐1,5‐bisphosphate. Although it is one of the most studied enzymes, the assembly mechanisms of the large hexadecameric RuBisCO is still emerging. In bacteria and in the C4 plant Zea mays, a protein with distant homology to p terin‐4α‐c arbinolamine d ehydratase (PCD) has recently been shown to be involved in RuBisCO assembly. However, studies of the homologous PCD‐like protein (RAF2, RuBisCO assembly factor 2) in the C3 plant Arabidopsis thaliana (A. thaliana) have so far focused on its role in hormone and stress signaling. We investigated whether A. thalianaRAF2 is also involved in RuBisCO assembly. We localized RAF2 to the soluble chloroplast stroma and demonstrated that raf2 A. thaliana mutant plants display a severe pale green phenotype with reduced levels of stromal RuBisCO. We concluded that the RAF2 protein is probably involved in RuBisCO assembly in the C3 plant A. thaliana.