Rapid isolation of viable circulating tumor cells from patient blood samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of >50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.     

Subcutaneous bioavailability of therapeutic antibodies as a function of FcRn binding affinity in mice

The neonatal Fc receptor (FcRn) plays an important and well-known role in immunoglobulin G (IgG) catabolism; however, its role in the disposition of IgG after subcutaneous (SC) administration, including bioavailability, is relatively unknown. To examine the potential effect of FcRn on IgG SC bioavailability, we engineered three anti-amyloid β monoclonal antibody (mAb) reverse chimeric mouse IgG2a (mIgG2a) Fc variants (I253A.H435A, N434H and N434Y) with different binding affinities to mouse FcRn (mFcRn) and compared their SC bioavailability to that of the wild-type (WT) mAb in mice. Our results indicated that the SC bioavailability of mIgG2a was affected by mFcRn-binding affinity. Variant I253A.H435A, which did not bind to mFcRn at either pH 6.0 or pH 7.4, had the lowest bioavailability (41.8%). Variant N434Y, which had the greatest increase in binding affinity at both pH 6.0 and pH 7.4, had comparable bioavailability to the WT antibody (86.1% vs. 76.3%), whereas Variant N434H, which had modestly increased binding affinity at pH 6.0 to mFcRn and affinity comparable to the WT antibody at pH 7.4, had the highest bioavailability (94.7%). A semi-mechanism-based pharmacokinetic model, which described well the observed data with the WT antibody and variant I253A.H435A, is consistent with the hypothesis that the decreased bioavailability of variant I253A.H435A was due to loss of the FcRn-mediated protection from catabolism at the absorption site. Together, these data demonstrate that FcRn plays an important role in SC bioavailability of therapeutic IgG antibodies.Key words: monoclonal antibody, FcRn, binding affinity, subcutaneous bioavailability, semi-mechanism-based pharmacokinetic model     

Crucial role for membrane fluidity in proliferation of primitive cells

The cell wall is a defining structural feature of the bacterial subkingdom. However, most bacteria are capable of mutating into a cell-wall-deficient "L-form" state, requiring remarkable physiological and structural adaptations. L-forms proliferate by an unusual membrane deformation and scission process that is independent of the conserved and normally essential FtsZ based division machinery, and which may provide a model for the replication of primitive cells. Candidate gene screening revealed no requirement for the cytoskeletal systems that might actively drive membrane deformation or scission. Instead, we uncovered a crucial role for branched-chain fatty acid (BCFA) synthesis. BCFA-deficient mutants grow and undergo pulsating shape changes, but membrane scission fails, abolishing the separation of progeny cells. The failure in scission is associated with a reduction in membrane fluidity. The results identify a step in L-form proliferation and demonstrate that purely biophysical processes may have been sufficient for proliferation of primitive cells.     

R-Spondin potentiates Wnt/β-catenin signaling through orphan receptors LGR4 and LGR5

The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/β-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced β-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced β-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated β-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.     

Coordinating environmental genomics and geochemistry reveals metabolic transitions in a hot spring ecosystem

We have constructed a conceptual model of biogeochemical cycles and metabolic and microbial community shifts within a hot spring ecosystem via coordinated analysis of the "Bison Pool" (BP) Environmental Genome and a complementary contextual geochemical dataset of ~75 geochemical parameters. 2,321 16S rRNA clones and 470 megabases of environmental sequence data were produced from biofilms at five sites along the outflow of BP, an alkaline hot spring in Sentinel Meadow (Lower Geyser Basin) of Yellowstone National Park. This channel acts as a >22 m gradient of decreasing temperature, increasing dissolved oxygen, and changing availability of biologically important chemical species, such as those containing nitrogen and sulfur. Microbial life at BP transitions from a 92 °C chemotrophic streamer biofilm community in the BP source pool to a 56 °C phototrophic mat community. We improved automated annotation of the BP environmental genomes using BLAST-based Markov clustering. We have also assigned environmental genome sequences to individual microbial community members by complementing traditional homology-based assignment with nucleotide word-usage algorithms, allowing more than 70% of all reads to be assigned to source organisms. This assignment yields high genome coverage in dominant community members, facilitating reconstruction of nearly complete metabolic profiles and in-depth analysis of the relation between geochemical and metabolic changes along the outflow. We show that changes in environmental conditions and energy availability are associated with dramatic shifts in microbial communities and metabolic function. We have also identified an organism constituting a novel phylum in a metabolic "transition" community, located physically between the chemotroph- and phototroph-dominated sites. The complementary analysis of biogeochemical and environmental genomic data from BP has allowed us to build ecosystem-based conceptual models for this hot spring, reconstructing whole metabolic networks in order to illuminate community roles in shaping and responding to geochemical variability.     

Associations of insulin and insulin-like growth factors with physical performance in old age in the Boyd Orr and Caerphilly studies

Objective

Insulin and the insulin-like growth factor (IGF) system regulate growth and are involved in determining muscle mass, strength and body composition. We hypothesised that IGF-I and IGF-II are associated with improved, and insulin with worse, physical performance in old age.

Methods

Physical performance was measured using the get-up and go timed walk and flamingo balance test at 63–86 years. We examined prospective associations of insulin, IGF-I, IGF-II and IGFBP-3 with physical performance in the UK-based Caerphilly Prospective Study (CaPS; n = 739 men); and cross-sectional insulin, IGF-I, IGF-II, IGFBP-2 and IGFBP-3 in the Boyd Orr cohort (n = 182 men, 223 women).

Results

In confounder-adjusted models, there was some evidence in CaPS that a standard deviation (SD) increase in IGF-I was associated with 1.5% faster get-up and go test times (95% CI: −0.2%, 3.2%; p = 0.08), but little association with poor balance, 19 years later. Coefficients in Boyd Orr were in the same direction as CaPS, but consistent with chance. Higher levels of insulin were weakly associated with worse physical performance (CaPS and Boyd Orr combined: get-up and go time = 1.3% slower per SD log-transformed insulin; 95% CI: 0.0%, 2.7%; p = 0.07; OR poor balance 1.13; 95% CI; 0.98, 1.29; p = 0.08), although associations were attenuated after controlling for body mass index (BMI) and co-morbidities. In Boyd Orr, a one SD increase in IGFBP-2 was associated with 2.6% slower get-up and go times (95% CI: 0.4%, 4.8% slower; p = 0.02), but this was only seen when controlling for BMI and co-morbidities. There was no consistent evidence of associations of IGF-II, or IGFBP-3 with physical performance.

Conclusions

There was some evidence that high IGF-I and low insulin levels in middle-age were associated with improved physical performance in old age, but estimates were imprecise. Larger cohorts are required to confirm or refute the findings.     

Dominance of native grasses leads to community convergence in wetland restoration

Wetland restoration is a pressing conservation priority, but there are few replicated field studies that provide a scientific foundation for these activities. We conducted a 3-year, replicated field experiment to examine the effectiveness of initial site preparation techniques (combinations of solarization, herbicide, tilling, and thermal weed control) in restoring native plant biodiversity to an agricultural field in a former wetland prairie in Oregon, USA. Post-treatment, plots were sown with a typical restoration mix of native graminoids and forbs. Treatments were compared to three high-quality managed reference wetlands and the adjacent agricultural field. Site preparation treatments varied in their effectiveness in suppressing extant vegetation and eliminating the residual seed bank. After 1 year, the solarization and fall herbicide application treatments were the most effective at reducing exotic cover. However, after 3 years, plant community composition converged in all treatments due to a loss of annual species and increasing dominance of native perennial bunchgrasses. Plant community composition became more similar to the reference wetlands each year, but diversity and richness diverged, apparently due to a trade-off between the cover of the dominant native bunchgrasses and diversity. Successional theory offers insights into how priority effects and competitive inhibition may influence community trajectories, and offers a useful model for restoring plant communities with high native diversity and dominance. Finding ways to mitigate the tradeoff between native plant cover and diversity by actively managing successional trajectories is an important challenge in wetland restoration that deserves further investigation.