#5 - Identifying cellular mechano-biological responses to PEG-based hydrogels

Cells sense and respond to mechanical stimuli from their external environment through a process called mechanotransduction. Focal adhesions are integrin-containing, multiprotein structures through which mechanical force is transmitted between the extracellular matrix and the interacting cell. Cells convert the transmitted force into biological responses including migration, proliferation and differentiation. The Garcia lab has previously engineered an integrin-specific hydrogel system resulting in changes in mesenchymal stem cell (MSC) gene expression, secretome, and ultimately regenerative capacity in a murine bone repair model. However, the mechano-biological mechanism driving this cell response to varying hydrogel biophysical and biochemical properties has yet to be studied. Here we have developed relationships between various hydrogel properties and cellular responses (cell adhesion, YAP localization, cell area, and cell shape). We engineered PEG-based hydrogel systems with two different polymerization chemistries, malemide and norbornene, to explore the effect of hydrogel chemistry on MSC cell adhesion and spreading. Using rheology, we demonstrated that hydrogel mechanical properties can be tuned by altering the weight percent of PEG macromer, while adhesion ligand type and density had no effect on hydrogel mechanical properties. PEG-4MAL gels were used for the remaining studies because that chemistry resulted in improved spreading and cell adhesion over norbornene hydrogels. By varying the density of RGD, the adhesive ligand for αvβ3 integrin, on the hydrogels, we showed that higher RGD densities resulted in greater YAP nuclear localization. We sought to understand the mechano-biological signaling pathway involved in YAP nuclear localization by inhibiting ROCK and FAK, proteins critical in mechanosensing via focal adhesion complexes. The inhibition of ROCK, and FAK decreased cell spread area, increased cell circularity and decreased YAP nuclear localization. Taken together this data demonstrates that external signals from PEG-based hydrogels as well as the intracellular signaling cascades involving ROCK and FAK can modulate YAP mechanosensing in MSCs

Exercise-induced mechanical hypoalgesia in musculotendinous tissues of the lateral elbow

The aim of this study was to investigate mechanical sensitivity responses at the lateral elbow to repeated weekly bouts of low load exercise in healthy subjects. Thirteen young men (n = 6) and women participated in 4 weeks of exercise. Arms were randomly allocated to an eccentric-only exercise protocol (ECC: 5 sets of 20 contractions) or to a concentric–eccentric protocol (CON-ECC: 5 sets of 10 eccentric/10 concentric contractions) performed at 30% maximal wrist extension force. Arms were exercised consecutively within each supervised weekly session. Quantitative measures of pressure pain threshold (PPT) recorded at three sites and maximal force for grip and wrist extension were assessed at baseline, and immediately pre/post exercise at each session. Muscle endurance during 100 maximal grip contractions force was assessed at baseline and one week following the final exercise session. Results showed that regardless of protocol, repeated low load exercise resulted in a time-dependent increase in PPT at all sites post exercise Weeks 3 and 4 and persisting at follow up Week 5 (P \u3c 0.02). No significant difference between protocols was evident for any measure. Muscle force and endurance were not significantly augmented compared with baseline. In conclusion mechanical hypoalgesia is induced by repeated low load exercises regardless of exercise mode, and this may prove beneficial if replicated clinically

Investigations into Asymmetric Post-Metallocene Group 4 Complexes for the Synthesis of Highly Regioirregular Polypropylene

A series of asymmetric post-metallocene group 4 complexes based on a modular anilide(pyridine)phenoxide framework have been synthesized and tested for propylene polymerization activity. These complexes, upon activation with methylaluminoxane (MAO), produce highly regioirregular and stereoirregular polypropylene with moderate to good activities. Surprisingly, modification of the anilide R-group substituent from 1-phenethyl to benzyl or adamantyl did not significantly change the polymer microstructure as determined by ^(13)C NMR spectroscopy. Although polymer molecular weights and polydispersities vary with propylene pressure, temperature, and activator, regio- and stereoirregularity were also found to be relatively insensitive to these variables. When the polymerization is conducted at 70 °C under dihydrogen, partial decomposition to a highly active catalyst that produces an isotactic microstructure occurs; the undecomposed catalyst continues to produce highly regioirregular and stereoirregular polypropylene under these conditions

Stellar Collisions and Ultracompact X-ray Binary Formation

(abridged) We report the results of SPH calculations of parabolic collisions between a subgiant or slightly evolved red-giant star and a neutron star (NS). Such collisions are likely to form ultracompact X-ray binaries (UCXBs) observed today in old globular clusters. In particular, we compute collisions of a 1.4 Msun NS with realistically modelled parent stars of initial masses 0.8 and 0.9 Msun, each at three different evolutionary stages (corresponding to three different radii R). The distance of closest approach for the initial orbit varies from 0.04 R (nearly head-on) to 1.3 R (grazing). These collisions lead to the formation of a tight binary, composed of the NS and the subgiant or red-giant core, embedded in an extremely diffuse common envelope (CE) typically of mass ~0.1 to 0.3 Msun. Our calculations follow the binary for many hundreds of orbits, ensuring that the orbital parameters we determine at the end of the calculations are close to final. Some of the fluid initially in the envelope of the (sub)giant, from 0.003 to 0.023 Msun in the cases we considered, is left bound to the NS. The eccentricities of the resulting binaries range from about 0.2 for our most grazing collision to about 0.9 for the nearly head-on cases. In almost all the cases we consider, gravitational radiation alone will cause sufficiently fast orbital decay to form a UCXB within a Hubble time, and often on a much shorter timescale. Our hydrodynamics code implements the recent SPH equations of motion derived with a variational approach by Springel & Hernquist and by Monaghan. Numerical noise is reduced by enforcing an analytic constraint equation that relates the smoothing lengths and densities of SPH particles. We present tests of these new methods to help demonstrate their improved accuracy.Comment: 41 pages, 17 figures, accepted by Ap

Cis-Acting Regulation of Brain-Specific ANK3 Gene Expression by a Genetic Variant Associated with Bipolar Disorder

Several genome-wide association studies (GWAS) for bipolar disorder (BD) have found a strong association of the Ankyrin3 (ANK3) gene. This association spans numerous linked single nucleotide polymorphisms (SNPs) in a ~250 kb genomic region overlapping ANK3. The associated region encompasses predicted regulatory elements as well as two of six validated alternative first exons, which encode distinct protein domains at the N-terminus of the protein also known as ankyrin-G (AnkG). Using RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RLM-RACE) to identify novel transcripts in conjunction with a highly sensitive, exon-specific multiplexed mRNA expression assay, we detected differential regulation of distinct ANK3 transcription start sites (TSSs) and coupling of specific 5’ ends with 3’ mRNA splicing events in post-mortem human brain and human stem cell-derived neural progenitors and neurons. Furthermore, allelic variation at the BD–associated SNP rs1938526 correlated with a significant difference in cerebellar expression of a brain-specific ANK3 transcript. These findings suggest a brain-specific cis-regulatory transcriptional effect of ANK3 may be relevant to BD pathophysiology

Current management of treatment-induced bone loss in women with breast cancer treated in the United Kingdom

New therapeutic options in breast cancer have improved survival but consequently increase the relevance of late complications. Ovarian suppression/ablation and aromatase inhibitors (AI) in the adjuvant setting have improved outcome, but have clinically important adverse effects on bone health. However, investigation and management of cancer treatment-induced bone loss (CTIBL) is poorly defined with no national guidance. In 2004, a questionnaire was sent to over 500 breast surgeons and oncologists who treat breast cancer within the United Kingdom. The questionnaire evaluated access to bone densitometry and specialist expertise as well as attitudes to investigation of CTIBL and anticipated changes in the use of AI for postmenopausal early breast cancer. A total of 354 completed questionnaires were received, 47 from clinicians not currently treating breast cancer. Of the 307 evaluable questionnaires, 164 (53%) were from breast surgeons, 112 (36%) from clinical oncologists and 31 (10%) from medical oncologists. Although most respondents recognised that CTIBL was the responsibility of the treating breast team, investigations for CTIBL are limited even though most had adequate access to bone densitometry; 98 (32%) had not requested a DXA scan in the last 6 months and 224 (73%) had requested fewer than five scans. In all, 235 (76%) were not routinely investigating patients on AI for bone loss. A total of 277 (90%) felt that their practice would benefit from national guidelines to manage these patients, and the majority (59%) had little or no confidence in interpreting DXA results and advising on treatment. This questionnaire has highlighted clear deficiencies in management of CTIBL in early breast cancer. The development of national guidelines for the management of these patients and educational initiatives for breast teams are urgently required

Pain outcomes in patients with bone metastases from advanced cancer: assessment and management with bone-targeting agents

Bone metastases in advanced cancer frequently cause painful complications that impair patient physical activity and negatively affect quality of life. Pain is often underreported and poorly managed in these patients. The most commonly used pain assessment instruments are visual analogue scales, a single-item measure, and the Brief Pain Inventory Questionnaire-Short Form. The World Health Organization analgesic ladder and the Analgesic Quantification Algorithm are used to evaluate analgesic use. Bone-targeting agents, such as denosumab or bisphosphonates, prevent skeletal complications (i.e., radiation to bone, pathologic fractures, surgery to bone, and spinal cord compression) and can also improve pain outcomes in patients with metastatic bone disease. We have reviewed pain outcomes and analgesic use and reported pain data from an integrated analysis of randomized controlled studies of denosumab versus the bisphosphonate zoledronic acid (ZA) in patients with bone metastases from advanced solid tumors. Intravenous bisphosphonates improved pain outcomes in patients with bone metastases from solid tumors. Compared with ZA, denosumab further prevented pain worsening and delayed the need for treatment with strong opioids. In patients with no or mild pain at baseline, denosumab reduced the risk of increasing pain severity and delayed pain worsening along with the time to increased pain interference compared with ZA, suggesting that use of denosumab (with appropriate calcium and vitamin D supplementation) before patients develop bone pain may improve outcomes. These data also support the use of validated pain assessments to optimize treatment and reduce the burden of pain associated with metastatic bone disease

Assessment of Barotrauma Resulting from Rapid Decompression of Depth Acclimated Juvenile Chinook Salmon Bearing Radio Telemetry Transmitters

A multifactor study was conducted by Battelle for the US Army Corps of Engineers to assess the significance of the presence of a radio telemetry transmitter on the effects of rapid decompression from simulated hydro turbine passage on depth acclimated juvenile run-of-the-river Chinook salmon. Study factors were: (1) juvenile chinook salmon age;, subyearling or yearling, (2) radio transmitter present or absent, (3) three transmitter implantation factors: gastric, surgical, and no transmitter, and (4) four acclimation depth factors: 1, 10, 20, and 40 foot submergence equivalent absolute pressure, for a total of 48 unique treatments. Exposed fish were examined for changes in behavior, presence or absence of barotrauma injuries, and immediate or delayed mortality. Logistic models were used to test hypotheses that addressed study objectives. The presence of a radio transmitter was found to significantly increase the risk of barotrauma injury and mortality at exposure to rapid decompression. Gastric implantation was found to present a higher risk than surgical implantation. Fish were exposed within 48 hours of transmitter implantation so surgical incisions were not completely healed. The difference in results obtained for gastric and surgical implantation methods may be the result of study design and the results may have been different if tested fish had completely healed surgical wounds. However, the test did simulate the typical surgical-release time frame for in-river telemetry studies of fish survival so the results are probably representative for fish passing through a turbine shortly following release into the river. The finding of a significant difference in response to rapid decompression between fish bearing radio transmitters and those not implies a bias may exist in estimates of turbine passage survival obtained using radio telemetry. However, the rapid decompression (simulated turbine passage) conditions used for the study represented near worst case exposure for fish passing through turbines. At this time, insufficient data exist about the distribution of river-run fish entering turbines, and particularly, the distribution of fish passing through turbine runners, to extrapolate study findings to the population of fish passing through FCRPS turbines. This study is the first study examining rapid decompression study to include acclimation depth as an experimental factor for physostomous fish. We found that fish acclimated to deeper depth were significantly more vulnerable to barotrauma injury and death. Insufficient information about the distribution of fish entering turbines and their depth acclimation currently exists to extrapolate these findings to the population of fish passing through turbines. However, the risk of barotrauma for turbine-passed fish could be particularly high for subyearling Chinook salmon that migrate downstream at deeper depths late in the early summer portion of the outmigration. Barotrauma injuries led to immediate mortality delayed mortality and potential mortality due to increased susceptibility to predation resulting from loss of equilibrium or swim bladder rupture