Mechanistic basis of branch-site selection in filamentous bacteria

Many filamentous organisms, such as fungi, grow by tip-extension and by forming new branches behind the tips. A similar growth mode occurs in filamentous bacteria, including the genus Streptomyces, although here our mechanistic understanding has been very limited. The Streptomyces protein DivIVA is a critical determinant of hyphal growth and localizes in foci at hyphal tips and sites of future branch development. However, how such foci form was previously unknown. Here, we show experimentally that DivIVA focus-formation involves a novel mechanism in which new DivIVA foci break off from existing tip-foci, bypassing the need for initial nucleation or de novo branch-site selection. We develop a mathematical model for DivIVA-dependent growth and branching, involving DivIVA focus-formation by tip-focus splitting, focus growth, and the initiation of new branches at a critical focus size. We quantitatively fit our model to the experimentally-measured tip-to-branch and branch-to-branch length distributions. The model predicts a particular bimodal tip-to-branch distribution results from tip-focus splitting, a prediction we confirm experimentally. Our work provides mechanistic understanding of a novel mode of hyphal growth regulation that may be widely employed.     

The diagnostic challenge of progressive pseudorheumatoid dysplasia (PPRD): a review of clinical features, radiographic features, and WISP3 mutations in 63 affected individuals

Progressive pseudorheumatoid dysplasia (PPRD) is a genetic, non-inflammatory arthropathy caused by recessive loss of function mutations in WISP3 (Wnt1-inducible signaling pathway protein 3; MIM 603400), encoding for a signaling protein. The disease is clinically silent at birth and in infancy. It manifests between the age of 3 and 6 years with joint pain and progressive joint stiffness. Affected children are referred to pediatric rheumatologists and orthopedic surgeons; however, signs of inflammation are absent and anti-inflammatory treatment is of little help. Bony enlargement at the interphalangeal joints progresses leading to camptodactyly. Spine involvement develops in late childhood and adolescence leading to short trunk with thoracolumbar kyphosis. Adult height is usually below the 3rd percentile. Radiographic signs are relatively mild. Platyspondyly develops in late childhood and can be the first clue to the diagnosis. Enlargement of the phalangeal metaphyses develops subtly and is usually recognizable by 10 years. The femoral heads are large and the acetabulum forms a distinct "lip" overriding the femoral head. There is a progressive narrowing of all articular spaces as articular cartilage is lost. Medical management of PPRD remains symptomatic and relies on pain medication. Hip joint replacement surgery in early adulthood is effective in reducing pain and maintaining mobility and can be recommended. Subsequent knee joint replacement is a further option. Mutation analysis of WISP3 allowed the confirmation of the diagnosis in 63 out of 64 typical cases in our series. Intronic mutations in WISP3 leading to splicing aberrations can be detected only in cDNA from fibroblasts and therefore a skin biopsy is indicated when genomic analysis fails to reveal mutations in individuals with otherwise typical signs and symptoms. In spite of the first symptoms appearing in early childhood, the diagnosis of PPRD is most often made only in the second decade and affected children often receive unnecessary anti-inflammatory and immunosuppressive treatments. Increasing awareness of PPRD appears to be essential to allow for a timely diagnosis.     

Optimized protocol for the isolation of spleen-resident murine neutrophils

Neutrophils are an important cellular component of the innate immune system that provides immediate protection to the host from infection. Neutrophil infiltration into inflamed peripheral tissues during infection is beneficial for immunity through phagocytosis of microbes, the release of antimicrobial factors, and secretion of proinflammatory cytokines. Recent reports further suggest that spleen-infiltrating neutrophils play a role in the adaptive immune response by providing survival signals to B cells. However, neutrophils may have detrimental effects on immunity in inflammatory diseases where their recruitment to lymphoid tissues and activation occur abnormally. To determine the contribution of neutrophils that reside in secondary lymphoid tissues to adaptive immunity, direct evaluation of the functional properties of tissue-resident neutrophils is required. We have developed a modified magnetic bead isolation approach for purifying neutrophils from inflamed spleens of autoimmune-prone mice by negative selection. Using this approach, we yielded neutrophils with greater than 90% purity without compromising cell viability. Equally important, the isolation procedure had little effect on the activation of neutrophils and did not impair phagocytic function. Thus, isolation of spleen-resident neutrophils by this optimized approach could be useful for interrogating the functional role of murine neutrophils in normal and abnormal immune responses. ? 2012 International Society for Advancement of Cytometry.     

Two decades of biomonitoring polar bear health in Greenland: a review

Background

Different animal models are used as fracture models in orthopaedic research prior to implant use in humans, although biomechanical forces can differ to a great extend between species due to variable anatomic conditions, particularly with regard to the gait. The rabbit is an often used fracture model, but biomechanical data are very rare. The objective of the present study was to measure axial forces, bending moments, and bending axis directly in the rabbit tibia in vivo. The following hypothesis was tested: Axial forces and bending moments in the mid-diaphysis of rabbit tibia differ from other experimental animals or indirectly calculated data.

Methods

A minifixateur system with 4 force sensors was developed and attached to rabbit tibia (n = 4), which were subsequently ostectomised. Axial forces, bending moments and bending angles were calculated telemetrically during weight bearing in motion between 6 and 42 days post operation.

Results

Highest single values were 201% body weight [% bw] for axial forces and 409% bw cm for bending moments. Whereas there was a continous decrease in axial forces over time after day 10 (P = 0.03 on day 15), a decrease in bending moments was inconsistent (P = 0.03 on day 27). High values for bending moments were frequently, but not consistently, associated with high values for axial forces.

Conclusion

Axial forces in rabbit tibia exceeded axial forces in sheep, and differed from indirectly calculated data. The rabbit is an appropriate fracture model because axial loads and bending moments in rabbit tibia were more closely to human conditions than in sheep tibia as an animal model.     

Genotyping success of historical Eurasian lynx (Lynx lynx L.) samples

Historical samples, like tanned hides and trophy skulls, can be extremely important for genetic studies of endangered or elusive species. Selection of a sampling protocol that is likely to provide sufficient amount and quality of DNA with a minimum damage to the original specimen is often critical for a success of the study. We investigated microsatellite genotyping success of DNA isolated from three different types of Eurasian lynx historical samples. We analysed a total of 20 microsatellite loci in 106 historical samples from the endangered Dinaric lynx population, established from re-introduction of three pairs of lynx in 1973 from Slovakian Carpathians. Of the three tested sample types, turbinal bone and septum from the nasal cavity of the trophy skulls had the lowest percentage of samples successfully genotyped for all 20 microsatellite loci. Footpad samples, collected using a cork drill, exhibited better results in polymerase chain reaction amplification and genotyping than samples of footpad epidermis cut with a scalpel. We report simple and efficient sampling protocols, which could be widely applied for future studies utilizing historical samples.     

DYNAMIC TRANSMISSION,HOST QUALITY,AND POPULATION STRUCTURE IN A MULTIHOST PARASITE OF BUMBLEBEES

The evolutionary ecology of multihost parasites is predicted to depend upon patterns of host quality and the dynamics of transmission networks. Depending upon the differences in host quality and transmission asymmetries, as well as the balance between intra‐ and interspecific transmission, the evolution of specialist or generalist strategies is predicted. Using a trypanosome parasite of bumblebees, we ask how host quality and transmission networks relate to parasite population structure across host species, and thus the potential for the evolution of specialist strains adapted to different host species. Host species differed in quality, with parasite growth varying across host species. Highly asymmetric transmission networks, together with differences in host quality, likely explain local population structure of the parasite across host species. However, parasite population structure across years was highly dynamic, with parasite populations varying significantly from one year to the next within individual species at a given site. This suggests that, while host quality and transmission may provide the opportunity for short‐term host specialization by the parasite, repeated bottlenecking of the parasite, in combination with its own reproductive biology, overrides these smaller scale effects, resulting in the evolution of a generalist parasite.     

Development of polymorphic microsatellite markers for the dung fly (Sepsis cynipsea)

The polyandrous fly Sepsis cynipsea has been used extensively in studies of sexual selection and local adaptation. We isolated and characterized 11 novel microsatellite markers for S. cynipsea from a genomic library and screened 32 flies for polymorphism. All microsatellite markers show high allelic diversity with an average of 9.64 alleles per locus. Two microsatellites were found likely to be X-linked. These novel markers will significantly advance studies of sexual selection and evolutionary genetics of S. cynipsea and related species, especially given the low numbers of markers currently available in this family.     

A Proteomic View of an Important Human Pathogen – Towards the Quantification of the Entire Staphylococcus aureus Proteome

The genome sequence is the “blue-print of life,” but proteomics provides the link to the actual physiology of living cells. Because of their low complexity bacteria are excellent model systems to identify the entire protein assembly of a living organism. Here we show that the majority of proteins expressed in growing and non-growing cells of the human pathogen Staphylococcus aureus can be identified and even quantified by a metabolic labeling proteomic approach. S. aureus has been selected as model for this proteomic study, because it poses a major risk to our health care system by combining high pathogenicity with an increasing frequency of multiple antibiotic resistance, thus requiring the development of new anti-staphylococcal therapy strategies. Since such strategies will likely have to target extracellular and surface-exposed virulence factors as well as staphylococcal survival and adaptation capabilities, we decided to combine four subproteomic fractions: cytosolic proteins, membrane-bound proteins, cell surface-associated and extracellular proteins, to comprehensively cover the entire proteome of S. aureus. This quantitative proteomics approach integrating data ranging from gene expression to subcellular localization in growing and non-growing cells is a proof of principle for whole-cell physiological proteomics that can now be extended to address physiological questions in infection-relevant settings. Importantly, with more than 1700 identified proteins (and 1450 quantified proteins) corresponding to a coverage of about three-quarters of the expressed proteins, our model study represents the most comprehensive quantification of a bacterial proteome reported to date. It thus paves the way towards a new level in understanding of cell physiology and pathophysiology of S. aureus and related pathogenic bacteria, opening new avenues for infection-related research on this crucial pathogen.