An Investigation of Micro and Nanomanufactured Polymer Substrates to Direct Stem Cell Response for Biomedical Applications

The rapidly advancing field of micro and nano-manufacturing is continuously offering novel advantages to existing technologies. Micro-injection molding provides a unique opportunity to create substrates capable of controlling the mechanical environment in stem cell culture in a high throughput industrially relevant manner. The modification of such polymer surfaces to match the target surface stiffness of relatively more compliant biological tissues necessitates the movement towards higher aspect ratio smaller dimension features. The requirements provide a significant manufacturing challenge which is approaching a solution. The development of high aspect ratio large feature density polymer microarrays requires the synergistic optimization of design, material, mold tooling, and processing. A conventional mold base with steel inserts and controllable resistance heating was assembled to incorporate interchangeable inserts with microfeatured silicon inlays. Ultraviolet (UV) lithography with dry etching was used to impart microfeatures into silicon wafers with a variety of different geometries containing aspect ratios ranging from 0.92 to 6. Multiple polymer resins, including polystyrene (HIPS, PS), low density polyethylene (LDPE), cyclic olefin copolymer (COC), and thermoplastic polyurethane (TPU), were used to test replication and cellular response to materials with different bulk stiffness and topography-modified surface stiffness. The maximum achieved microfeature aspect ratio was 9.3 (high impact polystyrene), owed to tensile stretching during part ejection. For non-stretched substrates, the maximum molded aspect ratio was 4.5 (LDPE) and highest replication quotient (RQ = feature height / tooling feature depth) was 0.97 (COC). The maximum aspect ratio molded with consistent features across the entire surface was 2.1 (TPU).Parameters shown to enhance replication were mold temperature (Tmold = Tg was a critical replication transition point), injection velocity at higher mold temperatures, holding time, holding pressure, and nozzle temperature. The importance of certain parameters was material dependent, but mold temperature consistently had a relatively large impact.A concern that was addressed for a high density array of microfeatures was the consistency of replication, which is vital for the intended application and seldom address in published literature. Increased consistency was attained through strategic placement of temperature control, modification of the main cavity design, and optimized silicon tooling with reduced microcavity nanoroughness.Silicon tooling was fabricated with the initial objective being to achieve high aspect ratio negative features. However, with the realization of molding and demolding limitations, the tooling microfeature profiles were altered to include a taper and reduction of sidewall scalloping. Sophisticated methods of dry etching were used, in which a novel etching technique known as passivation compensation, was utilized to manufacture microchannels containing low levels of roughness, a well-controlled tapered profile, and the prospect of high aspect ratios. With the new tooling, topography consistency was dramatically enhanced for both COC and TPU, with Taguchi orthogonal array optimization leading to RQs of 0.82 (aspect ratio = 2) and 0.85 (aspect ratio = 2.1), respectively.Water contact angle (WCA) measurements for both COC and TPU generally increased with an increase in surface roughness (dictated by microfeature dimensions), reaching WCA measurements of 139.8o and 141.1o, respectively. WCA hysteresis appeared to increase with roughness up to a critical value for COC while continuing to increase for TPU with a transition, which is thought to be the result of material properties. Moreover, hydrophobic surfaces containing high levels of hysteresis were attributed to the petal effect associated with hierarchical surface structures. Hydrophobicity has been shown to be related to biological cell behavior, and thus is an effective characterization technique to measure interfacial properties.Simulation of the injection molding process using conventional methods was used to describe general conditions present at microchannel inlets. The sprue gate and an increase in plate thickness gave the microfeatured region additional time to fill microfeatures prior to generation of a frozen layer. The delayed solidification is attributed to the low thermal conductivity associated with the polymer melt.A cell sensing model was developed based on the mechanical interaction between cell and substrate. The model provides a useful design map by which nanofeatured polymer geometry and material choice can be made to achieve a particular apparent surface stiffness. Bending mechanics were simulated for a few specific examples, providing an indication of the limitations associated with using higher aspect ratio nanostructures. A bending example was applied to a manufactured tapered pillar to note the stiffness reduction achieved through use of the substrates molded during the current study. Cell culture studies showed that the presence of topography had a dramatic effect on cellular morphology and on stress fiber thickness, causing an increase in thickness compared to flat controls. The cytoskeletal re-arrangements occurring may be indicative of a differentiation event, and future results will indicate whether that is the case. Unconventional morphology was observed in the presence of low aspect ratio COC microtopography, ranging from alignment with the micropattern to a circular conformation where adhesion is taking place exclusively in the middle of the cell. Micromolding of tensile bars was conducted to better understand the processing effects on mechanical and thermal properties of microscale molded components. Such results could provide useful general trends for the consideration of mechanical properties of molded microfeatures being exposed to cellular mechanical traction forces, especially considering the extreme processing conditions necessary to fill increasingly small and high aspect ratio features.Results revealed that the mechanical properties of COC is largely unaffected by a wide range processing conditions, but is reduced to approximately 41% of the value obtained from traditional tensile test results. TPU showed a dramatic dependence on molding properties, with higher injection velocities and lower mold temperatures resulting in reduced elastic modulus. Simulation was used to further elucidate the cause for varying properties. Microscale elastic modulus average values approximately 31% higher compared to traditional tensile test results. Trends in thermal properties were not apparent, and were difficult to detect from relatively weak melting peaks. The use of two different polymer lots elicited drastically different results, prompting the further investigation of the differences. Crystallinity, viscosity, and chemical bond structure was found to be very different from one lot to the other.The successful fabrication of uniform tapered microfeatures with middle range aspect ratios were manufactured, and the robust mold design and the tooling fabrication method provides a blueprint for achieving higher aspect ratios with a significant level of fidelity in the future. The enhanced macro and microscale mold design, combined with a deeper understanding of processing induced mechanical thermal microscale properties, can be used to tailor the substrate bio-interface properties to the desired mechanical structure for controllable hMSC behavior

A Plasma Membrane Localization Signal in the HIV-1 Envelope Cytoplasmic Domain Prevents Localization at Sites of Vesicular Stomatitis Virus Budding and Incorporation into VSV Virions

AbstractPrevious studies showed that the HIV-1 envelope (Env) protein was not incorporated into vesicular stomatitis virus (VSV) virions unless its cytoplasmic tail was replaced with that of the VSV glycoprotein (G). To determine whether the G tail provided a positive incorporation signal for Env, or if sequences in the Env tail prevented incorporation, we generated mutants of Env with its 150-amino-acid tail shortened to 29, 10, or 3 amino acids (Envtr mutants). Cells infected with VSV recombinants expressing these proteins or an Env-G tail hybrid showed similar amounts of Env protein at the surface. The Env-G tail hybrid or the Envtr3 mutant were incorporated at the highest levels into budding VSV virions. In contrast, the Envtr29 or Envtr10 mutants were incorporated poorly. These results defined a signal preventing incorporation within the 10 membrane-proximal amino acids of the Env tail. Confocal microscopy revealed that this signal functioned by causing localization of human immunodeficiency virus type 1 Env to plasma membrane domains distinct from the VSV budding sites, where VSV proteins were concentrated

Detection and characterization of 0.5–8 MeV neutrons near Mercury: Evidence for a solar origin

Data from the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) Neutron Spectrometer (NS) have been used to identify energetic neutrons (0.5–8 MeV energy) associated with solar events that occurred on 4 June 2011. Multiple lines of evidence, including measurements from the NS and the MESSENGER Gamma-Ray Spectrometer, indicate that the detected neutrons have a solar origin. This evidence includes a lack of time-coincident, energetic (>45 MeV) charged particles that could otherwise create local neutrons from nearby spacecraft material and a lack of proton-induced gamma rays that should be seen if energetic protons were present. NS data cannot rule out the presence of lower-energy ions (<30 MeV) that can produce local neutrons. However, the ion spectral shape required to produce the measured neutron count rate locally is softer than any known ion spectral shape. The neutron energy spectrum shows a relative enhancement in the energy range 0.8–3 MeV compared with cosmic-ray-generated neutrons from the spacecraft or Mercury. The spectral shape of the measured neutron fluence spectrum is consistent with a previously modeled fluence spectrum of neutrons that originate at the Sun and are propagated through the MESSENGER spacecraft to the NS. These measurements provide strong evidence for a solar origin of the detected neutrons and suggest that a large number of low-energy threshold ion evaporation reactions were taking place on the Sun during the neutron event

Long-term correction of ornithine transcarbamylase deficiency by WPRE-mediated overexpression using a helper-dependent adenovirus

The urea cycle disorders (UCDs) are important models for developing gene replacement therapy for liver diseases. Long-term correction of the most common UCD, ornithine transcarbamylase (OTC) deficiency, has yet to be achieved in clinical or preclinical settings. The single human clinical trial using early-generation adenovirus (Ad) failed to show any biochemical correction. In adult OTC-deficient mice, an E1/E2-deleted Ad vector expressing the mouse OTC gene, but not the human, was only transiently therapeutic. By using post-transcriptional overexpression in the context of the less immunogenic helper-dependent adenoviral vector, we achieved metabolic correction of adult OTC-deficient mice for \u3e6 months. Demonstrating this result were normalized orotic aciduria, normal hepatic enzyme activity, and elevated OTC RNA and protein levels in the absence of chronic hepatotoxicity. Overexpressing the human protein may have overcome two potential mechanisms accounting for poor cross-species complementation: a kinetic block at the level of mitochondrial import or a dominant negative effect by the mutant polypeptide. These data represent an important approach for treating human inborn errors of hepatocyte metabolism like the UCDs that require high-level transduction and gene expression for clinical correction

Sensory Computing and Object Processing Entity: Assistive Robotics for Healthcare

Team SCOPE has created an assistive robot for healthcare delivery. The robot is mobile, responds to spoken commands, and possesses Artificial Intelligence (AI). It extracts meanings about the patient’s health from conversations and visual interactions. It summarizes these observations into reports that could be merged with the patient’s Electronic Health Records (EHRs). This process aids healthcare professionals in delivering better care by augmenting attendance, increasing accuracy of patient information collection, aiding in diagnosis, streamlining data collection, and automating the process of ingesting and incorporating this information into EHR systems. SCOPE’s solution uses cloud-based AI services along with local processing. Using VEX Robotics parts and an Arduino microcontroller, SCOPE created a mobile platform for the robot. The robotic platform implements basic motions and obstacle avoidance. These separate systems are integrated using a Java master program, Node-Red, and IBM Watson cloud services. The resulting AI can be expanded for different applications within healthcare delivery

Trametinib versus standard of care in patients with recurrent low-grade serous ovarian cancer (GOG 281/LOGS): a randomised, open-label phase 2/3 trial

BACKGROUND: Low-grade serous carcinoma of the ovary or peritoneum is characterised by MAPK pathway aberrations and its reduced sensitivity to chemotherapy relative to high-grade serous carcinoma. We compared the MEK inhibitor trametinib to physician's choice standard of care in patients with recurrent low-grade serous carcinoma. METHODS: This international, randomised, open-label, multicentre, phase 2/3 trial was done at 84 hospitals in the USA and UK. Eligible patients were aged 18 years or older with recurrent low-grade serous carcinoma and measurable disease, as defined by Response Evaluation Criteria In Solid Tumors version 1.1, had received at least one platinum-based regimen, but not all five standard-of-care drugs, and had received an unlimited number of previous regimens. Patients with serous borderline tumours or tumours containing low-grade serous and high-grade serous carcinoma were excluded. Eligible patients were randomly assigned (1:1) to receive either oral trametinib 2 mg once daily (trametinib group) or one of five standard-of-care treatment options (standard-of-care group): intravenous paclitaxel 80 mg/m(2) by body surface area on days 1, 8, and 15 of every 28-day cycle; intravenous pegylated liposomal doxorubicin 40–50 mg/m(2) by body surface area once every 4 weeks; intravenous topotecan 4 mg/m(2) by body surface area on days 1, 8, and 15 of every 28-day cycle; oral letrozole 2·5 mg once daily; or oral tamoxifen 20 mg twice daily. Randomisation was stratified by geographical region (USA or UK), number of previous regimens (1, 2, or ≥3), performance status (0 or 1), and planned standard-of-care regimen. The primary endpoint was investigator-assessed progression-free survival while receiving randomised therapy, as assessed by imaging at baseline, once every 8 weeks for 15 months, and then once every 3 months thereafter, in the intention-to-treat population. Safety was assessed in patients who received at least one dose of study therapy. This trial is registered with ClinicalTrials.gov, NCT02101788, and is active but not recruiting. FINDINGS: Between Feb 27, 2014, and April 10, 2018, 260 patients were enrolled and randomly assigned to the trametinib group (n=130) or the standard-of-care group (n=130). At the primary analysis, there were 217 progression-free survival events (101 [78%] in the trametinib group and 116 [89%] in the standard-of-care group). Median progression-free survival in the trametinib group was 13·0 months (95% CI 9·9–15·0) compared with 7·2 months (5·6–9·9) in the standard-of-care group (hazard ratio 0·48 [95% CI 0·36–0·64]; p<0·0001). The most frequent grade 3 or 4 adverse events in the trametinib group were skin rash (17 [13%] of 128), anaemia (16 [13%]), hypertension (15 [12%]), diarrhoea (13 [10%]), nausea (12 [9%]), and fatigue (ten [8%]). The most frequent grade 3 or 4 adverse events in the standard-of-care group were abdominal pain (22 [17%]), nausea (14 [11%]), anaemia (12 [10%]), and vomiting (ten [8%]). There were no treatment-related deaths. INTERPRETATION: Trametinib represents a new standard-of-care option for patients with recurrent low-grade serous carcinoma. FUNDING: NRG Oncology, Cancer Research UK, Target Ovarian Cancer, and Novartis