Family of cytochrome c7-type proteins from Geobacter sulfurreducens: structure of one cytochrome c7 at 1.45 A resolution

The structure of a cytochrome c(7) (PpcA) from Geobacter sulfurreducens was determined by X-ray diffraction at 1.45 A resolution; the R factor is 18.2%. The protein contains a three-heme core that is surrounded by 71 amino acid residues. An unusual feature of this cytochrome is that it has 17 lysine residues, but only nine hydrophobic residues that are larger than alanine. The details of the structure are described and compared with those of cytochrome c(7) from Desulfuromonas acetoxidans and with cytochromes c(3). The two cytochrome c(7) molecules have sequences that are 46% identical, but the arrangements of the hemes in the two structures differ; the rms deviation of all alpha-carbons is 2.5 A. These cytochromes can reduce various metal ions. The reduction site of the chromate ion in D. acetoxidans is occupied by a sulfate ion in the crystal structure of PpcA. We identified four additional homologues of cytochrome c(7) in the G. sulfurreducens genome and three polymers of c(7)-type domains. Of the polymers, two have four repeats and one has nine repeats. On the basis of sequence alignments, one of the hemes in each of the cytochrome c(7)-type domains does not have the bis-histidine coordination. The packing of the molecules in the crystal structure of PpcA suggests that the polymers have an elongated conformation and might form a "nanowire".     

Glenohumeral elevation-dependent influence of anterior glenohumeral capsular lesions on passive axial humeral rotation

The purpose of this study was to assess the effect of standardized anterior glenohumeral capsular lesions on axial humeral rotation in a full arc of glenohumeral elevation. Using a testing apparatus, the range of internal and external humeral rotation was assessed in an arc of glenohumeral elevation in the scapular plane with steps of 15 degrees in six isolated shoulder joint specimens. Cutting of the glenohumeral joint capsule 1 cm laterally from, and parallel to the glenoid rim was performed in seven steps of 1 cm till the anterior capsule was cut. Capsular lesions were made in three ways: from inferior, from superior and from the middle of the capsule. Anterior capsular lesions resulted in significant increase of external humeral rotation. This occurred particularly at 15-60 degrees glenohumeral elevation. Lesions of the inferior part of the capsule mainly increased external rotation at 30-60 degrees glenohumeral elevation, lesions of the superior part mainly in lower elevation angles and lesions of the middle more gradually in the range till 60 degrees of glenohumeral elevation. Cutting of the anterior glenohumeral capsule barely increased passive axial humeral rotation at elevation angles over 60 degrees. Above 60 degrees glenohumeral elevation, tightening of the inferior posterior glenohumeral joint capsule prevented both internal and, increasingly, external humeral rotation. From these observations it is concluded that increased external rotation correlates with progressive anterior capsular lesions, mainly below 60 degrees glenohumeral elevation. To assess anterior glenohumeral capsular lesions in patients, axial humeral rotation tests should probably not exceed 60 degrees glenohumeral elevation, i.e. 90 degrees thoracohumeral elevation.     

Analysing the effect of candidate genes on complex traits: an application in multiple sclerosis

The conventional approach of candidate gene studies in complex diseases is to look at the effect of one gene at a time. However, as the outcome of chronic diseases is influenced by a large number of alleles, simultaneous analysis is needed. We demonstrate the application of multivariate regression and cluster analysis to a multiple sclerosis (MS) dataset with genotypes for 489 patients at 11 candidate genes selected on their involvement in the immune response. Using multivariate regression, we observed that different sets of genes were associated with different disease characteristics that reflect different aspects of disease. Out of 15 polymorphisms, we identified one that contributed to the severity of disease. In addition, the set of 15 polymorphisms was predictive for yearly increase in lesion volume as seen on T1-weighted MRI (p=0.044). From this set, no individual polymorphisms could be identified after adjustment for multiple hypotheses testing. By means of a cluster analysis, we aimed to identify subgroups of patients with different pathogenic subtypes of MS on the basis of their genetic profile. We constructed genetic profiles from the genotypes at the 11 candidate genes. The approach proved to be feasible. We observed three clusters in the sample of patients. In this study, we observed no significant differences in the usual clinical and MRI outcome measures between the different clusters. However, a number of consistent trends indicated that this clustering might be related to the course of disease. With a larger number of genes regulating the course of disease, we may be able to identify clinically relevant clusters. The analyses are easily implemented and will be applicable to candidate gene studies of complex traits in general.     

Unidirectional Na(+) and Ca (2+) fluxes in two euryhaline teleost fishes, Fundulus heteroclitus and Oncorhynchus mykiss, acutely submitted to a progressive salinity increase

Na⁺ and Ca²⁺ regulation were compared in two euryhaline species, killifish (normally estuarine-resident) and rainbow trout (normally freshwater-resident) during an incremental salinity increase. Whole-body unidirectional fluxes of Na⁺ and Ca²⁺, whole body Na⁺ and Ca²⁺, and plasma concentrations (trout only), were measured over 1-h periods throughout a total 6-h protocol of increasing salinity meant to simulate a natural tidal flow. Killifish exhibited significant increases in both Na⁺ influx and efflux rates, with efflux slightly lagging behind efflux up to 60% SW, but net Na⁺ balance was restored by the time killifish reached 100% SW. Whole body Na⁺ did not change, in agreement with the capacity of this species to tolerate daily salinity fluctuations in its natural habitat. In contrast, rainbow trout experienced a dramatic increase in Na⁺ influx (50-fold relative to FW values), but not Na⁺ efflux between 40 and 60% SW, resulting in a large net loading of Na⁺ at higher salinities (60–100% SW), and increases in plasma Na⁺ and whole body Na⁺ at 100% SW. Killifish were in negative Ca²⁺ balance at all salinities, whereas trout were in positive Ca²⁺ balance throughout. Ca²⁺ influx rate increased two- to threefold in killifish at 80 and 100% SW, but there were no concomitant changes in Ca²⁺ efflux. Ca²⁺ flux rates were affected to a larger degree in trout, with twofold increases in Ca²⁺ influx at 40% SW and sevenfold increases at 100% SW. Again, there was no change in Ca²⁺ efflux with salinity, so plasma Ca²⁺ concentration increased in 100% SW. As the killifish is regularly submitted to increased salinity in its natural environment, it is able to rapidly activate changes in unidirectional fluxes in order to ensure ionic homeostasis, in contrast to the trout.     

Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression

The majority of people infected with hepatitis C virus (HCV) fail to generate or maintain a T-cell response effective for viral clearance. Evidence from murine chronic viral infections shows that expression of the coinhibitory molecule PD-1 predicts CD8+ antiviral T-cell exhaustion and may contribute to inadequate pathogen control. To investigate whether human CD8+ T cells express PD-1 and demonstrate a dysfunctional phenotype during chronic HCV infection, peripheral and intrahepatic HCV-specific CD8+ T cells were examined. We found that in chronic HCV infection, peripheral HCV-specific T cells express high levels of PD-1 and that blockade of the PD-1/PD-L1 interaction led to an enhanced proliferative capacity. Importantly, intrahepatic HCV-specific T cells, in contrast to those in the periphery, express not only high levels of PD-1 but also decreased interleukin-7 receptor alpha (CD127), an exhausted phenotype that was HCV antigen specific and compartmentalized to the liver, the site of viral replication.     

Synaptic differentiation is defective in mice lacking acetylcholine receptor beta-subunit tyrosine phosphorylation

Agrin activates MuSK, a receptor tyrosine kinase expressed in skeletal muscle, leading to tyrosine phosphorylation of the acetylcholine receptor (AChR) beta-subunit and clustering of AChRs. The importance of AChR beta-subunit tyrosine phosphorylation in clustering AChRs and regulating synaptic differentiation is poorly understood. We generated mice with targeted mutations in the three intracellular tyrosines of the AChR beta-subunit (AChR-beta(3F/3F)). Mice lacking AChR beta-subunit tyrosine phosphorylation thrive postnatally and have no overt behavioral defects, indicating that AChR beta-subunit tyrosine phosphorylation is not essential for the formation of neuromuscular synapses. Nonetheless, the size of synapses and the density of synaptic AChRs are reduced in AChR- beta(3F/3F) mutant mice. Moreover, synapses are structurally simplified and the organization of postjunctional folds is aberrant in mice lacking tyrosine phosphorylation of the AChR beta-subunit. Furthermore, mutant AChRs cluster poorly in response to agrin and are readily extracted from the cell surface of cultured myotubes by non-ionic detergent. These data indicate that tyrosine phosphorylation of the AChR beta-subunit has an important role in organizing AChRs and regulating synaptic differentiation.     

Geranyl diphosphate synthase is required for biosynthesis of gibberellins

Geranyl diphosphate synthase (GPS) is generally considered to be responsible for the biosynthesis of monoterpene precursors only. However, reduction of LeGPS expression in tomato (Lycopersicon esculentum) by virus-induced gene silencing resulted in severely dwarfed plants. Further analysis of these dwarfed plants revealed a decreased gibberellin content, whereas carotenoid and chlorophyll levels were unaltered. Accordingly, the phenotype could be rescued by application of gibberellic acid. The dwarfed phenotype was also obtained in Arabidopsis thaliana plants transformed with RNAi constructs of AtGPS. These results link geranyl diphosphate (GPP) to the gibberellin biosynthesis pathway. They also demand a re-evaluation of the role of GPS in precursor synthesis for other di-, tri-, tetra- and/or polyterpenes and their derivatives.     

Regulatory impact on insect biotechnology and pest management

The application of insect biotechnology is promising for the development of environmentally compatible pest management solutions. As we have refined and enhanced genetic engineering techniques in several insect species that cause significant economic loss and public health injury, it has become clear that insect biotechnology will move forward as one of the key tools of pest management in agriculture and in the human environment. Well characterized genetic elements can be manipulated toward specific aims and maintain a viable insect, albeit one with diminished capacity to exchange genetic material, vector a virus or bacterium, or complete its life cycle. Despite this degree of knowledge and precision, there remain unanswered questions regarding environmental fate, release and public acceptance of this technology. The uncertainty surrounding any novel technology inevitably increases the level of regulatory scrutiny associated with its use. Although the term “insect biotechnology” has many connotations, it certainly includes the genetic modification of symbiotic or commensally associated microbes as a means of delivering a trait (e.g. a toxin) to manage plant and human diseases and insect pests. The distinction between this paratransgenic approach and direct genetic modification of insect pests is an important one biologically as well as from a regulatory standpoint. The regulatory framework for microbial applications to agriculture is in many instances in place; however, we must strive to forge the development of guidelines and regulations that will foster deployment of insect biotechnologies.     

Social Contact Networks and Disease Eradicability under Voluntary Vaccination

Certain theories suggest that it should be difficult or impossible to eradicate a vaccine-preventable disease under voluntary vaccination: Herd immunity implies that the individual incentive to vaccinate disappears at high coverage levels. Historically, there have been examples of declining coverage for vaccines, such as MMR vaccine and whole-cell pertussis vaccine, that are consistent with this theory. On the other hand, smallpox was globally eradicated by 1980 despite voluntary vaccination policies in many jurisdictions. Previous modeling studies of the interplay between disease dynamics and individual vaccinating behavior have assumed that infection is transmitted in a homogeneously mixing population. By comparison, here we simulate transmission of a vaccine-preventable SEIR infection through a random, static contact network. Individuals choose whether to vaccinate based on infection risks from neighbors, and based on vaccine risks. When neighborhood size is small, rational vaccinating behavior results in rapid containment of the infection through voluntary ring vaccination. As neighborhood size increases (while the average force of infection is held constant), a threshold is reached beyond which the infection can break through partially vaccinated rings, percolating through the whole population and resulting in considerable epidemic final sizes and a large number vaccinated. The former outcome represents convergence between individually and socially optimal outcomes, whereas the latter represents their divergence, as observed in most models of individual vaccinating behavior that assume homogeneous mixing. Similar effects are observed in an extended model using smallpox-specific natural history and transmissibility assumptions. This work illustrates the significant qualitative differences between behavior–infection dynamics in discrete contact-structured populations versus continuous unstructured populations. This work also shows how disease eradicability in populations where voluntary vaccination is the primary control mechanism may depend partly on whether the disease is transmissible only to a few close social contacts or to a larger subset of the population.