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.     

CRISPR interference of nucleotide biosynthesis improves production of a single-domain antibody in Escherichia coli

Growth decoupling can be used to optimize the production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference to increase the production of a single-domain antibody (sdAb) by inhibiting growth during production. First, we screened 21 sgRNA targets in the purine and pyrimidine biosynthesis pathways and found that the repression of 11 pathway genes led to the increased green fluorescent protein production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve the production of two versions of an expression-optimized sdAb. Proteomics analysis of the sdAb-producing pyrF, pyrG, and cmk growth decoupling strains showed significantly decreased RpoS levels and an increase of ribosome-associated proteins, indicating that the growth decoupling strains do not enter stationary phase and maintain their capacity for protein synthesis upon growth inhibition. Finally, sdAb production was scaled up to shake-flask fermentation where the product yield was improved 2.6-fold compared to the control strain with no sgRNA target sequence. An sdAb content of 14.6% was reached in the best-performing pyrG growth decoupling strain.     

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.     

Cell cycle regulators cyclin D1 and CDK4/6 have estrogen receptor-dependent divergent functions in breast cancer migration and stem cell-like activity

Cyclin D1 and its binding partners CDK4/6 are essential regulators of cell cycle progression and are implicated in cancer progression. Our aim was to investigate a potential regulatory role of these proteins in other essential tumor biological characteristics. Using a panel of breast cancer cell lines and primary human breast cancer samples, we have demonstrated the importance of these cell cycle regulators in both migration and stem-like cell activity. siRNA was used to target cyclin D1 and CDK4/6 expression, having opposing effects on both migration and stem-like cell activity dependent upon estrogen receptor (ER) expression. Inhibition of cyclin D1 or CDK4/6 increases or decreases migration and stem-like cell activity in ER−ve (ER-negative) and ER+ve (ER-positive) breast cancer, respectively. Furthermore, overexpressed cyclin D1 caused decreased migration and stem-like cell activity in ER−ve cells while increasing activity in ER+ve breast cancer cells. Treatment of breast cancer cells with inhibitors of cyclin D1 and CDK4/6 (Flavopiridol/PD0332991), currently in clinical trials, mimicked the effects observed with siRNA treatment. Re-expression of ER in two ER−ve cell lines was sufficient to overcome the effects of either siRNA or clinical inhibitors of cyclin D1 and CDK4/6.   In conclusion, cyclin D1 and CDK4/6 have alternate roles in regulation of migration and stem-like cell activity. Furthermore, these effects are highly dependent upon expression of ER. The significance of these results adds to our general understanding of cancer biology but, most importantly, could be used diagnostically to predict treatment response to cell cycle inhibition in breast cancer.     

THE EVOLUTION OF LOCOMOTOR RHYTHMICITY IN TETRAPODS

Differences in rhythmicity (relative variance in cycle period) among mammal, fish, and lizard feeding systems have been hypothesized to be associated with differences in their sensorimotor control systems. We tested this hypothesis by examining whether the locomotion of tachymetabolic tetrapods (birds and mammals) is more rhythmic than that of bradymetabolic tetrapods (lizards, alligators, turtles, salamanders). Species averages of intraindividual coefficients of variation in cycle period were compared while controlling for gait and substrate. Variance in locomotor cycle periods is significantly lower in tachymetabolic than in bradymetabolic animals for datasets that include treadmill locomotion, non‐treadmill locomotion, or both. When phylogenetic relationships are taken into account the pooled analyses remain significant, whereas the non‐treadmill and the treadmill analyses become nonsignificant. The co‐occurrence of relatively high rhythmicity in both feeding and locomotor systems of tachymetabolic tetrapods suggests that the anatomical substrate of rhythmicity is in the motor control system, not in the musculoskeletal components.     

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.     

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.