Quantitation of class IA PI3Ks in mice reveals p110-free-p85s and isoform-selective subunit associations and recruitment to receptors.

Class IA PI3Ks have many roles in health and disease. The rules that govern intersubunit and receptor associations, however, remain unclear. We engineered mouse lines in which individual endogenous class IA PI3K subunits were C-terminally tagged with 17aa that could be biotinylated in vivo. Using these tools we quantified PI3K subunits in streptavidin or PDGFR pull-downs and cell lysates. This revealed that p85α and ß bound equivalently to p110α or p110ß but p85α bound preferentially to p110δ. p85s were found in molar-excess over p110s in a number of contexts including MEFs (p85ß, 20%) and liver (p85α, 30%). In serum-starved MEFs, p110-free-p85s were preferentially, compared with heterodimeric p85s, bound to PDGFRs, consistent with in vitro assays that demonstrated they bound PDGFR-based tyrosine-phosphorylated peptides with higher affinity and co-operativity; suggesting they may act to tune a PI3K activation threshold. p110α-heterodimers were recruited 5-6× more efficiently than p110ß-heterodimers to activated PDGFRs in MEFs or to PDGFR-based tyrosine-phosphorylated peptides in MEF-lysates. This suggests that PI3Kα has a higher affinity for relevant tyrosine-phosphorylated motifs than PI3Kß. Nevertheless, PI3Kß contributes substantially to acute PDGF-stimulation of PIP3 and PKB in MEFs because it is synergistically, and possibly sequentially, activated by receptor-recruitment and small GTPases (Rac/CDC42) via its RBD, whereas parallel activation of PI3Kα is independent of its RBD. These results begin to provide molecular clarity to the rules of engagement between class IA PI3K subunits in vivo and past work describing "excess p85," p85α as a tumor suppressor, and differential receptor activation of PI3Kα and PI3Kß.

Proposed Scheme to Generate Bright Entangled Photon Pairs by Application of a Quadrupole Field to a Single Quantum Dot.

Entangled photon sources are crucial for quantum optics, quantum sensing, and quantum communication. Semiconductor quantum dots generate on-demand entangled photon pairs via the biexciton-exciton cascade. However, the pair of photons are emitted isotropically in all directions, thus limiting the collection efficiency to a fraction of a percent. Moreover, strain and structural asymmetry in quantum dots lift the degeneracy of the intermediate exciton states in the cascade, thus degrading the measured entanglement fidelity. Here, we propose an approach for generating a pair of entangled photons from a semiconductor quantum dot by application of a quadrupole electrostatic potential. We show that the quadrupole electric field corrects for the spatial asymmetry of the excitonic wave function for any quantum dot dipole orientation and fully erases the fine-structure splitting without compromising the spatial overlap between electrons and holes. Our approach is compatible with nanophotonic structures such as microcavities and nanowires, thus paving the way towards a deterministic source of entangled photons with high fidelity and collection efficiency.

The innominate tubercle of the femur: Application to anterior surgical approaches to the hip.

There has recently been an increase in popularity of the direct anterior approach (DAA) hip arthroplasty, due to the muscle sparing nature of its interneural intervals, with the innominate tubercle being used as a lateral reference point for the femoral neck osteotomy. However, there is very little information in the literature on this rather enigmatic structure, with no evidence as to whether it is a consistent and reliable marker, or if it varies significantly in the population. In this study, data were gathered from 79 pairs of adult, post-medieval skeletal femora to investigate the effects of sex, age, femoral side, femoral length, femoral neck length, and femoral neck-shaft angle on the width, length, and height of the innominate tubercle. The sex, age, and date of death of the individuals had been recorded. Statistical analysis included canonical correlation and multivariate multiple regression. We found that there was no statistical significance or correlation between the width, length, or height of the tubercle with respect to any of the variables investigated. These results suggest that the innominate tubercle does not differ markedly between individuals in the Caucasian population, and, is therefore, a reliable landmark for femoral neck osteotomy during DAA hip arthroplasty. We present what we believe to be a definitive survey of the variability of the innominate tubercle in a Caucasian population. Clin. Anat. 30:578-584, 2017. © 2017 Wiley Periodicals, Inc.

StayFuse for proximal interphalangeal joint fusion.

BACKGROUND: Proximal interphalangeal (PIP) joint fusion is a commonly performed procedure for lesser-toe deformities. There are various techniques described to accomplish it. We report the results of PIP joint fusion carried out with an intramedullary fusion device in 150 consecutive toes. The aim of our study was to evaluate the outcomes of PIP joint fusion with this technique. METHOD: A total of 150 toes in 140 consecutive patients who underwent PIP joint fusions of the lesser toes with a StayFuse implant were included in our study. The mean age of the patients was 69.5 years, and the mean follow-up was 18 months. Clinical, radiologic, and subjective evaluations as well as preoperative and postoperative American Orthopaedic Foot and Ankle Society (AOFAS) scores were carried out. RESULTS: Of the PIP joints, 95.3% were clinically asymptomatic, but the radiologic fusion was 73%. The mean preoperative AOFAS score improved from 22.9 to 81.6 at follow-up. There were implant-related complications in 8 toes. Ninety-five percent of the patients were satisfied with the procedure, and 3.3% of the patients needed revision surgery. CONCLUSION: This technique maintained PIP joint alignment and provided rotational and angular stability with high patient satisfaction and low complication and reoperation rates. We conclude that this is a reproducible technique and an alternative for PIP joint fusions. LEVEL OF EVIDENCE: Level IV, retrospective case series.

Enhanced photonic crystal cavity-waveguide coupling using local slow-light engineering.

This Letter introduces an enhanced cavity-waveguide coupling architecture based upon slow-light engineering in a two-port photonic crystal system. After analyzing the system transmittance using coupled-mode theory, the system is probed experimentally and shown to have increased transmittance due to the enhanced cavity-waveguide coupling. Such a coupling architecture may facilitate next-generation planar lightwave circuitry such as onchip quantum information processing or high precision light-matter sensing applications.

Filamentation and focusing of electron beams due to interactions with plasma waves.

Results of numerical modeling of the interaction of an electron beam propagating across relativistic plasma waves indicate that electron beam filamentation and focusing may occur under certain conditions. The model is based on solving the relativistic equation of motion in three dimensions for the individual electrons in a tenuous Gaussian beam, as they pass through a relativistic plasma wave. Several electron beam and plasma wave parameters were varied, and the results are summarized. One of the results is that the spacing of the electron beam filaments correlates with the wavelength of the plasma wave. The electron beam filaments appear as vertical slabs after the beam exits the plasma waves. The electron beam also compresses to a focus after it exits the plasma, and the focal distance depends on several parameters including the electron beam energy and phase velocity of the relativistic plasma wave. It is suggested that these focusing and filamentation phenomena may be the basis for diagnostics schemes for laser plasma interactions. The parameters used in the model were electron beam energies in the 5-50 keV range and plasma wave properties typical for the beat-wave produced by CO2 lasers, which correspond to the facilities available in our laboratory. The limitations of these results to lower energy density beam and plasma regimes and to higher energy density regimes will be discussed.

Selective epitaxy of semiconductor nanopyramids for nanophotonics.

We present a detailed study of the parameters which affect the geometrical perfection of nanopyramids used for the site-selected nucleation of quantum dots. Through an understanding of crystal facet formation, we demonstrate that undesirable high index planes can be suppressed using carefully optimized lithography together with properly orientated source fluxes in the growth reactor. High quality InP nanopyramids are reported with individual InAs/InP quantum dots positioned with high precision. This represents an important milestone for the fabrication of complex quantum dot based nanophotonic devices.

Friction transfer of polytetrafluoroethylene (PTFE) to produce nanoscale features and influence cellular response in vitro.

A large number of cell types are known to respond to chemical and topographical patterning of substrates. Friction transfer of polytetrafluoroethylene (PTFE) onto substrates has been shown to produce continuous, straight, parallel nanofibres. Ammonia plasma treatment can be used to defluorinate the PTFE, decreasing the dynamic contact angle. Fibroblast and epithelial cells were elongated and oriented with their long axis parallel to the fibres, both individually and in clusters. The fibres restricted cell migration. Cell alignment was slightly reduced on the plasma-treated fibres. These results indicated that although surface topography can affect cellular response, surface chemistry also mediates the extent of this response.

Oestrous synchronization, ovarian superovulation and intraspecific transfers from a closed breeding colony of inbred SLA miniature pigs.

The inbred SLA miniature pig is a unique animal model developed for organ transplantation studies and pre-clinical experimental purposes. Reported oestrous synchronization and superovulation treatments were examined in two SLA haplotypes (AA and DD) to allow collection of embryos for both practical embryo transfer and experimental technologies from a closed breeding colony. Pre-puberal miniature pigs were poor responders to oestrous synchronization treatments, while post-puberal sows were equivalent to commercial sows. Following superovulation, the ovulation number (corpora .hemorrhagica) was higher (p < 0.05) in the cycling sows when compared with non-cycling sows. Ovulations were equivalent to commercial pre-puberal gilts and non-cycling sows (p > 0.05). No difference in ovulation number between haplotypes was observed, which differs from the previous report (DD>AA). Collection of zygotes for pronuclear injection was the highest in the non-cycling post-puberal miniature pig group (p < 0.05), although significantly lower when compared with the commercial pig treatment groups (p < 0.05). The incidence of cystic endometrial hyperplasia in our colony was equivalent to rates observed in commercial pigs. Pronuclear visualization following centrifugation was the highest in the non-cycling miniature sow group and approximates to about 25% of ovulations and about half the rate observed in the commercial pigs (50%). Miniature pig embryos transferred between SLA haplotypes and transfer of DD embryos to commercial pigs resulted in live births at a higher efficiency than previously reported. This study demonstrates the feasibility of undertaking assisted reproductive technologies in a closed breeding colony of inbred SLA miniature pigs without compromise to the breeding programmes.

CRISPR-Cas9-mediated labelling of the C-terminus of human laminin ß1 leads to secretion inhibition.

OBJECTIVES: The laminins (LM) are a family of basement membranes glycoproteins with essential roles in supporting epithelia, endothelia, nerves and muscle adhesion, and in regulating a range of processes including cell migration, stem cell maintenance and differentiation. However, surprisingly little is known about the mechanisms of turnover and remodelling of LM networks due to lack of appropriate tools to study these processes at the necessary resolution. Recently, the nematode C. elegans ortholog of human the LMß1 chain was labelled at the C-terminus with the photoconvertible fluorophore Dendra2. Here we used genome editing to establish a similar system in a mammalian cell line as proof of concept for future mammalian models. RESULTS: CRISPR-Cas9 was used to introduce the Dendra2 sequence at the C-terminus of LMß1 in the human lung adenocarcinoma cell line A549. Despite expression of the tagged protein within cells, no detectable LMß1-Dendra2 protein was deposited to the extracellular matrices or conditioned media of edited cells. Moreover, the edited cells displayed reduced proliferation rates. Together, these data suggest that, in humans, addition of C-terminal Dendra2 tag to LMß1 inhibits LM secretion, and is not a viable approach for use in animal models.

Mefloquine (WR 142,490) in the treatment of human malaria.

Mefloquine hydrochloride, a new 4-quinolinemethanol, was administered as a single oral dose to 47 volunteers infected with malaria. Treatment resulted in rapid clearence of fever and parasitemia. No recrudescence of parasites was observed after treatment of chloroquine-sensitive infections of Plasmodium falciparum. More significantly, in nonimmune persons with chloroquine-resistant infections, 1 gram of mefloquine cured 10 of 12 patients and 1.5 grams cured all 8 patients who received this dose of the drug. The marked activity of a single dose of mefloquine against chloroquine-resistant strains of Plasmodium falciparum suggests that this agent may be more useful than currently available drugs are for the treatment of drug-resistant malaria.

A gellan-based fluid gel carrier to enhance topical spray delivery.

Autologous cell transplantation was introduced to clinical practice nearly four decades ago to enhance burn wound re-epithelialisation. Autologous cultured or uncultured cells are often delivered to the surface in saline-like suspensions. This delivery method is limited because droplets of the sprayed suspension form upon deposition and run across the wound bed, leading to uneven coverage and cell loss. One way to circumvent this problem would be to use a gel-based material to enhance surface retention. Fibrin systems have been explored as co-delivery system with keratinocytes or as adjunct to 'seal' the cells following spray delivery, but the high costs and need for autologous blood has impeded its widespread use. Aside from fibrin gel, which can exhibit variable properties, it has not been possible to develop a gel-based carrier that solidifies on the skin surface. This is because it is challenging to develop a material that is sprayable but gels on contact with the skin surface. The manuscript reports the use of an engineered carrier device to deliver cells via spraying, to enhance retention upon a wound. The device involves shear-structuring of a gelling biopolymer, gellan, during the gelation process; forming a yield-stress fluid with shear-sensitive behaviours, known as a fluid gel. In this study, a formulation of gellan gum fluid gels are reported, formed with from 0.75 or 0.9% (w/v) polymer and varying the salt concentrations. The rheological properties and the propensity of the material to wet a surface were determined for polymer modified and non-polymer modified cell suspensions. The gellan fluid gels had a significantly higher viscosity and contact angle when compared to the non-polymer carrier. Viability of cells was not impeded by encapsulation in the gellan fluid gel or spraying. The shear thinning property of the material enabled it to be applied using an airbrush and spray angle, distance and air pressure were optimised for coverage and viability. STATEMENT OF SIGNIFICANCE: Spray delivery of skin cells has successfully translated to clinical practice. However, it has not yet been widely accepted due to limited retention and disputable cell viability in the wound. Here, we report a method for delivering cells onto wound surfaces using a gellan-based shear-thinning gel system. The viscoelastic properties allow the material to liquefy upon spraying and restructure rapidly on the surface. Our results demonstrate reduced run-off from the surface compared to currently used low-viscosity cell carriers. Moreover, encapsulated cells remain viable throughout the process. Although this paper studies the encapsulation of one cell type, a similar approach could potentially be adopted for other cell types. Our data supports further studies to confirm these results in in vivo models.

Rheometry and associated techniques for blood coagulation studies.

This review considers various rheometrical approaches that have been adopted to study blood coagulation, with special reference to the rheological assessment of clotting time and studies of the evolution of viscoelasticity during the course of fibrin polymerization and cross-linking. The significance of the Gel Point in blood coagulation studies is discussed as a common feature of many of these studies in that they attempt to detect a liquid-to-solid transition during coagulation. Coagulation studies based on various forms of complex shear modulus measurements are considered, the latter being based principally on controlled stress and controlled strain rheometers. Also considered are the long established technique of thromboelastography and several emerging techniques such as wave propagation measurements, free oscillation rheometry, quartz crystal microbalance measurements and surface plasmon resonance.

Studies of whole blood coagulation by oscillatory shear, thromboelastography and free oscillation rheometry.

We report studies of the coagulation of samples of whole human blood by oscillatory shear techniques, including Fourier Transform Mechanical Spectroscopy (FTMS). These techniques are used herein to identify the Gel Point of coagulating blood in terms of the Chambon-Winter Gel Point criterion which provides a rheometrical basis for detecting the establishment of an incipient clot. A comparison of the results of FTMS with those obtained from measurements involving a Thromboelastograph (TEG) and a Free Oscillation Rheometer (FOR) indicate that the latter techniques are not capable of detecting the incipient clot, whose establishment occurs several minutes prior to TEG or FOR-based assessments of clot formation time. The results of the present study suggest that FTMS is a useful tool in blood clotting research, being capable of providing a global coagulation profile in addition to detecting the instant of incipient clot formation.

Neurosarcoidosis presenting as acute infarction on diffusion-weighted MR imaging: summary of radiologic findings.

A 36-year-old woman presented with acute-onset right lower extremity paresthesias, dysarthria, right facial droop, and right hemiparesis. CT and MR imaging of the brain revealed extensive white matter disease and left basal ganglia infarction with dural and leptomeningeal enhancement. Differential considerations included vasculitis, granulomatous disease, and neoplasm. Chest, abdomen, and pelvis CTs were normal. Right temporal lobe biopsy revealed noncaseating granulomatous inflammation consistent with neurosarcoidosis.

Annexin-enriched osteoblast-derived vesicles act as an extracellular site of mineral nucleation within developing stem cell cultures.

The application of extracellular vesicles (EVs) as natural delivery vehicles capable of enhancing tissue regeneration could represent an exciting new phase in medicine. We sought to define the capacity of EVs derived from mineralising osteoblasts (MO-EVs) to induce mineralisation in mesenchymal stem cell (MSC) cultures and delineate the underlying biochemical mechanisms involved. Strikingly, we show that the addition of MO-EVs to MSC cultures significantly (P < 0.05) enhanced the expression of alkaline phosphatase, as well as the rate and volume of mineralisation beyond the current gold-standard, BMP-2. Intriguingly, these effects were only observed in the presence of an exogenous phosphate source. EVs derived from non-mineralising osteoblasts (NMO-EVs) were not found to enhance mineralisation beyond the control. Comparative label-free LC-MS/MS profiling of EVs indicated that enhanced mineralisation could be attributed to the delivery of bridging collagens, primarily associated with osteoblast communication, and other non-collagenous proteins to the developing extracellular matrix. In particular, EV-associated annexin calcium channelling proteins, which form a nucleational core with the phospholipid-rich membrane and support the formation of a pre-apatitic mineral phase, which was identified using infrared spectroscopy. These findings support the role of EVs as early sites of mineral nucleation and demonstrate their value for promoting hard tissue regeneration.

Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer.

Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localize to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequisite for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knock-in of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, cyclin-dependent kinase (CDK)7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knock-in mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine-resistant breast cancer mediated by ER mutations.

The effect of silica nanoparticulate coatings on serum protein adsorption and cellular response.

Serum protein adsorption on colloidal silica surfaces was investigated using a quartz crystal microbalance with dissipation (QCM-D) monitoring. The amount of serum proteins adsorbed on colloidal silica-coated surfaces was not significantly different from the control silica surfaces, with the exception of 21nm colloidal silica which experienced significantly less (P<0.05) fibrinogen adsorption compared with control silica. The adhesion and proliferation of human endothelial cells (C11STH) on nano-scale colloidal silica surfaces were significantly reduced compared with control silica surfaces, suggesting that the conformation of adsorbed proteins on the colloidal silica surfaces plays a role in modulating the amount of cell binding. Fibronectin is one of the main extracellular matrix proteins involved in endothelial cell attachment to biomaterial surfaces. There was reduced binding of a monoclonal anti-fibronectin antibody, that reacted specifically with the cell-binding fragment, to fibronectin-coated colloidal silica surfaces compared with control silica surfaces. This suggests that the fibronectin adsorbed on the colloidal silica-coated surfaces was conformationally changed compared with control silica reducing the availability of the cell-binding domain of fibronectin.

Structural views of phosphoinositide-specific phospholipase C: signalling the way ahead.

Recent structural studies of mammalian phosphoinositide-specific phospholipase C (PI-PLC) have begun to shed light on the mechanism whereby this family of effector enzymes is able to hydrolyze phospholipid substrates to yield second messengers. PI-PLC isozymes employ a variety of modules (PH domain, EF-hand domain, SH2 domain, SH3 domain and C2 domain) that are common in proteins involved in signal transduction to reversibly interact with membranes and protein components of the signalling pathways.

Crystal structure of a diabody, a bivalent antibody fragment.

BACKGROUND: Diabodies are dimeric antibody fragments. In each polypeptide, a heavy-chain variable domain (VH) is linked to a light-chain variable domain (VL) but unlike single-chain Fv fragments, each antigen-binding site is formed by pairing of one VH and one VL domain from the two different polypeptides. Diabodies thus have two antigen-binding sites, and can be bispecific. Direct structural evidence is lacking for the connections and dimeric interactions between the two polypeptides of the diabody. RESULTS: The 2.6 A resolution structure has been determined for a bivalent diabody with a flexible five-residue polypeptide linker between the (amino-terminal) VH and (carboxy-terminal) VL domains. The asymmetric unit of the crystal consists of four polypeptides comprising two diabodies; for one of these polypeptides the linker can be traced between the VH and VL domains. Within each diabody the two associated VH and VL domains make back-to-back interactions through the VH domains, and there is an extensive VL-VL interface between the two diabodies in the asymmetric unit. CONCLUSIONS: The structure of the diabody is very similar to that which had been predicted by molecular modelling. Diabodies directed against cell-surface antigens should be capable of bringing together two cells, such as in cell-targeted therapy, because the two antigen-binding sites of the diabody are at opposite ends of the molecule and separated by approximately 65 A.