Recombinant plasmids with genes for the biosynthesis of alkaline phosphatase of Escherichia coli

Summary Restriction maps of several recombinant plasmids representing a section of the E. coli K12 chromosome 35,000 bp in size with the genes phoA, proC and phoB were prepared. The orientation of phoA and the exact position of its N-terminal end on this map were determined by identifying a subfragment which carried the phoA promoter and by determining the nucleotide sequence of a 160 bp portion of this subfragment comprising the codons for the N-terminal end of pre-alkaline phosphatase. From this DNA sequence the leader sequence of alkaline phosphatase which consists of 21 amino acids was derived.     

Periosteum stimulates subchondral bone densification in autologous chondrocyte transplantation in a sheep model

In this sheep study, we have tested the hypothesis that an osteogenic response is triggered in the subchondral bone by periosteum implanted in full thickness cartilage defects and can be prevented by replacing the periosteum by a cell-free collagen type I/III membrane. Two 7-mm diameter osteochondral defects were made in the trochlea groove and in the medial femoral condyle of one of the knees in each of 15 adult sheep. The animals were divided into three groups (n=5): a control group with untreated cartilage defects, a group treated with autologous chondrocyte transplantation (ACT) and periosteum, and a group treated with ACT in combination with a collagen I/III membrane cover. Histological examination was performed 1 year later. The optical density of the subchondral bone in the histological sections was measured with digital imaging software. There was a dramatic, statistically significant (P<0.0001; power=1) increase in bone density of 45%–70% under defects that were treated with the periosteal cover, compared with the collagen membrane and control groups, which displayed the same bone density. There was no difference in the cartilaginous reparative tissue in the defects in the three groups. Periosteum thus stimulates the remodelling process in subchondral bone. Stiffening of the subchondral bone can lead to degeneration of the overlying reparative cartilaginous tissue because of an increase in the mechanical stress in the tissue. These findings warrant evaluation of subchondral bone changes in patients treated by ACT and the correlation of these changes with clinical outcome.     

C4 Photosynthesis Promoted Species Diversification during the Miocene Grassland Expansion

Identifying how organismal attributes and environmental change affect lineage diversification is essential to our understanding of biodiversity. With the largest phylogeny yet compiled for grasses, we present an example of a key physiological innovation that promoted high diversification rates. C₄ photosynthesis, a complex suite of traits that improves photosynthetic efficiency under conditions of drought, high temperatures, and low atmospheric CO₂, has evolved repeatedly in one lineage of grasses and was consistently associated with elevated diversification rates. In most cases there was a significant lag time between the origin of the pathway and subsequent radiations, suggesting that the ‘C₄ effect’ is complex and derives from the interplay of the C₄ syndrome with other factors. We also identified comparable radiations occurring during the same time period in C₃ Pooid grasses, a diverse, cold-adapted grassland lineage that has never evolved C₄ photosynthesis. The mid to late Miocene was an especially important period of both C₃ and C₄ grass diversification, coincident with the global development of extensive, open biomes in both warm and cool climates. As is likely true for most “key innovations”, the C₄ effect is context dependent and only relevant within a particular organismal background and when particular ecological opportunities became available.     

Evaluation of Argos Telemetry Accuracy in the High-Arctic and Implications for the Estimation of Home-Range Size

Animal tracking through Argos satellite telemetry has enormous potential to test hypotheses in animal behavior, evolutionary ecology, or conservation biology. Yet the applicability of this technique cannot be fully assessed because no clear picture exists as to the conditions influencing the accuracy of Argos locations. Latitude, type of environment, and transmitter movement are among the main candidate factors affecting accuracy. A posteriori data filtering can remove “bad” locations, but again testing is still needed to refine filters. First, we evaluate experimentally the accuracy of Argos locations in a polar terrestrial environment (Nunavut, Canada), with both static and mobile transmitters transported by humans and coupled to GPS transmitters. We report static errors among the lowest published. However, the 68^th error percentiles of mobile transmitters were 1.7 to 3.8 times greater than those of static transmitters. Second, we test how different filtering methods influence the quality of Argos location datasets. Accuracy of location datasets was best improved when filtering in locations of the best classes (LC3 and 2), while the Douglas Argos filter and a homemade speed filter yielded similar performance while retaining more locations. All filters effectively reduced the 68^th error percentiles. Finally, we assess how location error impacted, at six spatial scales, two common estimators of home-range size (a proxy of animal space use behavior synthetizing movements), the minimum convex polygon and the fixed kernel estimator. Location error led to a sometimes dramatic overestimation of home-range size, especially at very local scales. We conclude that Argos telemetry is appropriate to study medium-size terrestrial animals in polar environments, but recommend that location errors are always measured and evaluated against research hypotheses, and that data are always filtered before analysis. How movement speed of transmitters affects location error needs additional research.     

Targeted next generation sequencing identifies clinically actionable mutations in patients with melanoma

Somatic sequencing of cancers has produced new insight into tumorigenesis, tumor heterogeneity, and disease progression, but the vast majority of genetic events identified are of indeterminate clinical significance. Here, we describe a NextGen sequencing approach to fully analyzing 248 genes, including all those of known clinical significance in melanoma. This strategy features solution capture of DNA followed by multiplexed, high‐throughput sequencing and was evaluated in 31 melanoma cell lines and 18 tumor tissues from patients with metastatic melanoma. Mutations in melanoma cell lines correlated with their sensitivity to corresponding small molecule inhibitors, confirming, for example, lapatinib sensitivity in ERBB4 mutant lines and identifying a novel activating mutation of BRAF. The latter event would not have been identified by clinical sequencing and was associated with responsiveness to a BRAF kinase inhibitor. This approach identified focal copy number changes of PTEN not found by standard methods, such as comparative genomic hybridization (CGH). Actionable mutations were found in 89% of the tumor tissues analyzed, 56% of which would not be identified by standard‐of‐care approaches. This work shows that targeted sequencing is an attractive approach for clinical use in melanoma.     

Human Cytomegalovirus Vaccine Based on the Envelope gH/gL Pentamer Complex

Human Cytomegalovirus (HCMV) utilizes two different pathways for host cell entry. HCMV entry into fibroblasts requires glycoproteins gB and gH/gL, whereas HCMV entry into epithelial and endothelial cells (EC) requires an additional complex composed of gH, gL, UL128, UL130, and UL131A, referred to as the gH/gL-pentamer complex (gH/gL-PC). While there are no established correlates of protection against HCMV, antibodies are thought to be important in controlling infection. Neutralizing antibodies (NAb) that prevent gH/gL-PC mediated entry into EC are candidates to be assessed for in vivo protective function. However, these potent NAb are predominantly directed against conformational epitopes derived from the assembled gH/gL-PC. To address these concerns, we constructed Modified Vaccinia Ankara (MVA) viruses co-expressing all five gH/gL-PC subunits (MVA-gH/gL-PC), subsets of gH/gL-PC subunits (gH/gL or UL128/UL130/UL131A), or the gB subunit from HCMV strain TB40/E. We provide evidence for cell surface expression and assembly of complexes expressing full-length gH or gB, or their secretion when the corresponding transmembrane domains are deleted. Mice or rhesus macaques (RM) were vaccinated three times with MVA recombinants and serum NAb titers that prevented 50% infection of human EC or fibroblasts by HCMV TB40/E were determined. NAb responses induced by MVA-gH/gL-PC blocked HCMV infection of EC with potencies that were two orders of magnitude greater than those induced by MVA expressing gH/gL, UL128-UL131A, or gB. In addition, MVA-gH/gL-PC induced NAb responses that were durable and efficacious to prevent HCMV infection of Hofbauer macrophages, a fetal-derived cell localized within the placenta. NAb were also detectable in saliva of vaccinated RM and reached serum peak levels comparable to NAb titers found in HCMV hyperimmune globulins. This vaccine based on a translational poxvirus platform co-delivers all five HCMV gH/gL-PC subunits to achieve robust humoral responses that neutralize HCMV infection of EC, placental macrophages and fibroblasts, properties of potential value in a prophylactic vaccine.