A novel acetabular alignment guide for THR using selective anatomic landmarks on the pelvis

This paper presents a novel approach for acetabular alignment during the implant of a prosthetic hip joint in a natural pelvis. The alignment instrument uses selective anatomic bony landmarks on the pelvis, which are accessible in surgery, to guide the placement of the acetabular component in the appropriate orientation. A closed form solution, involving both a forward and reverse analysis, is presented to relate the parameters of the device with the abduction and anteversion angles. Using mathematical models, this device should allow the surgeon to place the acetabular component with an orientation between 10.9 degrees and 19.1 degrees anteversion and 35.7 degrees and 44.3 degrees abduction with 95% confidence in a male/left specimen for the commonly accepted target of 15 degrees anteversion and 40 degrees abduction. This device is currently being used successfully by one of the authors in THR surgery.     

Two Routes of Metabolic Cross-Feeding between Bifidobacterium adolescentis and Butyrate-Producing Anaerobes from the Human Gut

Dietary carbohydrates have the potential to influence diverse functional groups of bacteria within the human large intestine. Of 12 Bifidobacterium strains of human gut origin from seven species tested, four grew in pure culture on starch and nine on fructo-oligosaccharides. The potential for metabolic cross-feeding between Bifidobacterium adolescentis and lactate-utilizing, butyrate-producing Firmicute bacteria related to Eubacterium hallii and Anaerostipes caccae was investigated in vitro. E. hallii L2-7 and A. caccae L1-92 failed to grow on starch in pure culture, but in coculture with B. adolescentis L2-32 butyrate was formed, indicating cross-feeding of metabolites to the lactate utilizers. Studies with [¹³C]lactate confirmed carbon flow from lactate, via acetyl coenzyme A, to butyrate both in pure cultures of E. hallii and in cocultures with B. adolescentis. Similar results were obtained in cocultures involving B. adolescentis DSM 20083 with fructo-oligosaccharides as the substrate. Butyrate formation was also stimulated, however, in cocultures of B. adolescentis L2-32 grown on starch or fructo-oligosaccharides with Roseburia sp. strain A2-183, which produces butyrate but does not utilize lactate. This is probably a consequence of the release by B. adolescentis of oligosaccharides that are available to Roseburia sp. strain A2-183. We conclude that two distinct mechanisms of metabolic cross-feeding between B. adolescentis and butyrate-forming bacteria may operate in gut ecosystems, one due to consumption of fermentation end products (lactate and acetate) and the other due to cross-feeding of partial breakdown products from complex substrates.     

Animal models of ischemic stroke: balancing experimental aims and animal care

Animal models of ischemic stroke are examples of an induced model that can present challenges from the perspectives of protocol review and animal management. The review presented here will include a brief summary of the current state of knowledge about clinical stroke; a general synopsis of important unanswered research questions that justify use of animal stroke models; an overview of various animal models of ischemic stroke, including strengths and limitations; and a discussion of animal care issues relative to ischemic stroke models. Good communication and interactive education among primary investigators, laboratory animal veterinarians and caretakers, and institutional animal care and use committee members are critical in achieving a balance between research objectives and animal care issues when using animal stroke models.     

Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction.

Rheumatoid arthritis is a chronic inflammatory joint disease, leading to cartilage and bone destruction. In this study, we investigated the effects of local IL-4 application, introduced by a recombinant human type 5 adenovirus vector, in the knee joint of mice with collagen-induced arthritis. One intraarticular injection with an IL-4-expressing virus caused overexpression of IL-4 in the mouse knee joint. Enhanced onset and aggravation of the synovial inflammation were found in the IL-4 group. However, despite ongoing inflammation, histologic analysis showed impressive prevention of chondrocyte death and cartilage erosion. In line with this, chondrocyte proteoglycan synthesis was enhanced in the articular cartilage. This was quantified with ex vivo 35S-sulfate incorporation in patellar cartilage and confirmed by autoradiography on whole knee joint sections. Reduction of cartilage erosion was further substantiated by lack of expression of the stromelysin-dependent cartilage proteoglycan breakdown neoepitope VDIPEN in the Ad5E1 mIL-4-treated knee joint. Reduced metalloproteinase activity was also supported by markedly diminished mRNA expression of stromelysin-3 in the synovial tissue. Histologic analysis revealed marked reduction of polymorphonuclear cells in the synovial joint space in the IL-4-treated joints. This was confirmed by immunolocalization studies on knee joint sections using NIMP-R14 staining and diminished mRNA expression of macrophage-inflammatory protein-2 in the synovium tissue. mRNA levels of TNF-alpha and IL-1beta were suppressed as well, and IL-1beta and nitric oxide production by arthritic synovial tissue were strongly reduced. Our data show an impressive cartilage-protective effect of local IL-4 and underline the feasibility of local gene therapy with this cytokine in arthritis.     

Alcohol biosensing by polyamidoamine (PAMAM)/cysteamine/alcohol oxidase‐modified gold electrode

A highly stable and sensitive amperometric alcohol biosensor was developed by immobilizing alcohol oxidase (AOX) through Polyamidoamine (PAMAM) dendrimers on a cysteamine‐modified gold electrode surface. Ethanol determination is based on the consumption of dissolved oxygen content due to the enzymatic reaction. The decrease in oxygen level was monitored at ?0.7 V vs. Ag/AgCl and correlated with ethanol concentration. Optimization of variables affecting the system was performed. The optimized ethanol biosensor showed a wide linearity from 0.025 to 1.0 mM with 100 s response time and detection limit of (LOD) 0.016 mM. In the characterization studies, besides linearity some parameters such as operational and storage stability, reproducibility, repeatability, and substrate specificity were studied in detail. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 67% of its initial sensitivity was kept after 1 month storage at 4°C. The analytical characteristics of the system were also evaluated for alcohol determination in flow injection analysis (FIA) mode. Finally, proposed biosensor was applied for ethanol analysis in various alcoholic beverage as well as offline monitoring of alcohol production through the yeast cultivation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Links between plant species’ spatial and temporal responses to a warming climate

To generate realistic projections of species’ responses to climate change, we need to understand the factors that limit their ability to respond. Although climatic niche conservatism, the maintenance of a species’s climatic niche over time, is a critical assumption in niche-based species distribution models, little is known about how universal it is and how it operates. In particular, few studies have tested the role of climatic niche conservatism via phenological changes in explaining the reported wide variance in the extent of range shifts among species. Using historical records of the phenology and spatial distribution of British plants under a warming climate, we revealed that: (i) perennial species, as well as those with weaker or lagged phenological responses to temperature, experienced a greater increase in temperature during flowering (i.e. failed to maintain climatic niche via phenological changes); (ii) species that failed to maintain climatic niche via phenological changes showed greater northward range shifts; and (iii) there was a complementary relationship between the levels of climatic niche conservatism via phenological changes and range shifts. These results indicate that even species with high climatic niche conservatism might not show range shifts as instead they track warming temperatures during flowering by advancing their phenology.     

Speciation and Introgression between Mimulus nasutus and Mimulus guttatus

Mimulus guttatus and M. nasutus are an evolutionary and ecological model sister species pair differentiated by ecology, mating system, and partial reproductive isolation. Despite extensive research on this system, the history of divergence and differentiation in this sister pair is unclear. We present and analyze a population genomic data set which shows that M. nasutus budded from a central Californian M. guttatus population within the last 200 to 500 thousand years. In this time, the M. nasutus genome has accrued genomic signatures of the transition to predominant selfing, including an elevated proportion of nonsynonymous variants, an accumulation of premature stop codons, and extended levels of linkage disequilibrium. Despite clear biological differentiation, we document genomic signatures of ongoing, bidirectional introgression. We observe a negative relationship between the recombination rate and divergence between M. nasutus and sympatric M. guttatus samples, suggesting that selection acts against M. nasutus ancestry in M. guttatus.     

A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing

Background

CRISPR-Cas9 is a revolutionary genome editing technique that allows for efficient and directed alterations of the eukaryotic genome. This relatively new technology has already been used in a large number of ‘loss of function’ experiments in cultured cells. Despite its simplicity and efficiency, screening for mutated clones remains time-consuming, laborious and/or expensive.

Results

Here we report a high-throughput screening strategy that allows parallel screening of up to 96 clones, using next-generation sequencing. As a proof of principle, we used CRISPR-Cas9 to disrupt the coding sequence of the homeobox gene, Evx1 in mouse embryonic stem cells. We screened 67 CRISPR-Cas9 transfected clones simultaneously by next-generation sequencing on the Ion Torrent PGM. We were able to identify both homozygous and heterozygous Evx1 mutants, as well as mixed clones, which must be identified to maintain the integrity of subsequent experiments.

Conclusions

Our CRISPR-Cas9 screening strategy could be widely applied to screen for CRISPR-Cas9 mutants in a variety of contexts including the generation of mutant cell lines for in vitro research, the generation of transgenic organisms and for assessing the veracity of CRISPR-Cas9 homology directed repair. This technique is cost and time-effective, provides information on clonal heterogeneity and is adaptable for use on various sequencing platforms.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1002) contains supplementary material, which is available to authorized users.     

Sexuality and Genetic Identity in the Agaricus Section Arvenses

Twelve wild collections and one commercial strain were used to characterize breeding systems and to develop molecular identities in the Arvenses section of the genus Agaricus, which includes the “horse mushroom” A. arvensis. Two morphotypes were identified based on macro- and micromorphological features. However, not all collections could be delimited by conventional taxonomic characters. Sequencing of the small subunit intergenic spacer (ITS) region (368 to 370 bp) of the rRNA genes clearly resolved the 13 collections into two clusters consistent with the identified morphotypes. Single-spore progenies and mating type testers were established and used to test intra- and interstock compatibility. The two compatibility groups identified were consistent with ITS clusters. Compatibility group I stocks readily interbred within the constraints of a unifactorial heterothallic system with a multiallelic mating type factor. Compatibility group II had a more restricted breeding pattern, and interactions were difficult to predict on the basis of mating type. Morphological data, ITS sequences, and the ability to interbreed suggest that these collections are part of a complex of interrelated species. Single-spore, homokaryotic isolates from both compatibility groups were able to fruit in compost culture, and two of the collections may represent natural homokaryotic fruiting. We conclude that species from the section Arvenses have versatile unifactorial heterothallic life cycles that permit both interbreeding and homokaryotic fruiting.