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For in vivo evaluation in sheep, the adherence module was incorporated into a modified IVR design consisting of a standard silicone pod-IVR [18, 19] containing no drug pods with the module inserted into an additional hollow tubular compartment added to span the center of the ring. The central compartment configuration, rather than embedding the electronics module in the outer silicone ring, was used for the initial sheep study to allow facile access to the circuit components during testing. Devices were programmed, powered, and placed in Sleep mode, and then inserted into the central compartment and sealed with silicone adhesive.
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study
| 99.94 |
The adherence-monitoring IVR was evaluated in a Rambouillet X Columbia sheep at the Colorado State University Preclinical Surgical Research Laboratory (Fort Collins, CO, USA). A schedule of ring insertion and removal was is shown in Table 1. The IVR was immersed in warm water bath (50°C) to initiate data logging, and remained in the warm bath until insertion in the sheep vaginal cavity. The IN/OUT status of the IVR was measured and stored in non-volatile memory on a 1.00 min interval, using a reference temperature of 34.4°C. The insertion/removal pattern was followed for 29 hours, whereupon the ring was removed and logging stopped by cutting through the ring to sever the battery connection to the PCB. The insertion and removal periods were based on the workflow in the animal facility during the two days of the study, and were designed to provide both short and long insertion and removal periods with minimal disturbance to the animal. The stored IN/OUT data stream was downloaded from the device to a PC using the Microchip PICkit3 programmer USB interface, and the data in hexadecimal format was converted to a series of binary (0 or 1) values indicating IVR OUT or IN.
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study
| 100.0 |
The sheep study was performed according to NIH guidelines at Colorado State University, a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Protocols were approved by Institutional Animal Care and Use Committees at Colorado State University (Fort Collins, CO, USA; Animal Welfare Assurance Number: A3572-01) under approval #12-3704A. Implantation of IVRs was performed under minimal physical restraint without causing undue stress or pain to the sheep. Sheep were flipped, hooded and hobbled, and placed in lateral recumbency. The perineal region was prepped with multiple scrubs of povidine iodine alternated with isopropyl alcohol. The device was inserted into the cranial vagina using a sterile gloved finger lubricated with medical-grade lubricant gel. A non-absorbable nylon suture tied to the IVR was allowed to extend outside of the vulva to indicate that the ring had not been expelled, and to aid in removal of the ring during removal/reinsertion procedures. Sheep were minimally restrained while the IVR was removed from the vagina using a suture traction. Devices were re-inserted using similar methods. The study was non-terminal.
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study
| 99.94 |
The PIC12F683 microcontroller (Microchip Technology, Inc., Chandler, AZ, USA) was chosen as the basis of the adherence-monitoring IVR design. The PIC12F683 is a miniature 8-pin, Flash-based, 8-bit CMOS microcontroller, with a size of 4 mm × 4 mm × 0.9 mm in the DFN surface-mount package. The PIC12F683 has numerous features making it a good choice for this application, including: low-power sleep and operating modes to preserve battery life (50 nA standby current; 11 μA operating current at 32 kHz), analog comparator with on-chip programmable voltage reference, external oscillator functionality for precision long-term timing stability, six I/O pins individually programmable for sensor input or LED indicator output, and 256 byte non-volatile EEPROM memory capable of storing 2048 1-bit IN/OUT readings. The adherence monitor consists of the following functional modules (Fig 2A):
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other
| 99.9 |
(a) Diagram of adherence IVR electronics module showing major functional components (boxes) and dataflow (arrows). (b) Plot showing relationships between reference temperature, comparator reference voltage (Vref), and the 4-bit Vref generator parameter. Each point represents a reference temperature that is set in software by the generator 4-bit value (0–15). Here a generator value of 11 is set to select a reference temperature of 34°C. Dotted lines indicate Vref values selected for IN/OUT determination and “Logging On” “Logging Off” functionality in the in vitro and in vivo testing of adherence IVR devices.
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other
| 99.9 |
Battery: The device can operate with a voltage input range of 2.0 to 5.5 V. Two silver oxide coin cell batteries (SR416) provided a nominal 3.1 V input (Vdd) voltage. For debugging and testing, the electronics module was powered by a variable DC power supply, and power for PIC12 microcontroller programming and data retrieval is provided through the programming/data interface connection.
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other
| 99.94 |
Programming/Data Interface. A 5-pin interface is used for microcontroller programming and data transfer from the IVR to a PC for retrieval of logged status values. For the prototype device described here, all programming and data transfer operations utilized the Microchip PICkit 3 programming interface and the Microchip MPLabX Integrated Development Environment (IDE).
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other
| 99.94 |
External crystal clock. A precision quartz crystal tuning fork provides a stable clock to drive all device timing. The crystal’s 32.768 kHz oscillations are counted into the PIC12 16-bit incrementing counter so that one timer rollover occurs every 2 s. This rollover initiates a wake from the microcontroller sleep state (wake every 2 sec), and all device timing (e.g. measurement interval) is a multiple of 16-bit timer cycles. The 200 ppm accuracy of the quartz crystal leads to timing accuracy of 6 ms h-1 (4.3 s per 30 days).
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other
| 99.9 |
LED output. The general purpose I/O pin (GP2) is connected to an LED, allowing visual indication of device status, temperature measurement, and EEPROM writing. LED output is configured and enabled in software and allows for LED indicators in debugging mode, and the ability to turn off LED functionality programmatically to decrease device power consumption during long-term use.
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other
| 99.94 |
Temperature sensor. A 10 kΩ precision thermistor temperature sensor in a resistive divider circuit provides a voltage input (Vth) to the PIC12 comparator (GP1, CIN-) that is proportional to device temperature. The magnitude of Vth is a fraction of the battery voltage Vdd and varies linearly with thermistor temperature. The comparator compares this voltage to an internally generated reference voltage (Vref). Comparator output is high (1) when Vth > Vref and low (0) when Vth < Vref. The value of Vref is set programmatically with a 4-bit parameter, allowing a range of reference temperatures to be used for implementing various device functions such as starting or stopping data logging to internal memory.
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other
| 99.9 |
The device’s embedded software was written in C using the Microchip XC8 compiler in the MPLAB X Integrated Development Environment (IDE). A code flow chart is shown in Fig 3. The compiled program is downloaded to the device using the PICkit 3 programmer. The software implements three main functions: measuring and saving to memory the 1-bit value representing device IN or OUT, enabling data logging, and disabling data logging. All are based on comparison of Vth to a Vref generated in the microcontroller. Following completion of the initialization routine, the device enters a low-power sleep mode in a continuous loop waiting for 16-bit timer rollover. At each rollover (65538 counts at 32.768 kHz = 2 s), the device wakes and evaluates three options: (1) run the log comparator output (IN or OUT status) subroutine (green in Fig 3), (2) run the logging on/off subroutine (blue in Fig 3), or (3) return to sleep. The time interval for checking logging on/off status and logging comparator output is set in the program code. For prototype evaluation, the logging on/off subroutine was run every 2 rollovers (4 sec), and the data logging interval was varied from 10 s (5 wake cycles) to 5 min (150 wake cycles) depending on the test conditions.
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other
| 99.7 |
Initially, a status bit indicating logging disabled prevents the execution of the logging (green) subroutine, and at wake the device queries if it is time to enter the logging on/off (blue) subroutine. If yes, the device measures Vth and compares it to the “Logging On” Vref value. The relationship between Vref and temperature is shown in Fig 2B. If Vth > Vref for a set number of wake cycles (typically 20 sec), data logging is enabled prior to returning to the sleep state. This has the effect of allowing data logging to be switched on by heating the device above the temperature corresponding to the “Logging On” Vref value (typically 50°C). Once logging is enabled, subsequent wake cycles will run the green data logging subroutine at the appropriate sampling interval set in the program. When logging is enabled, each blue subroutine measures Vth, compares it to the “Logging Off” Vref (typically 5°C), and turns off logging if Vth < Vref. In typical real-world applications, the “Logging Off” functionality is not used and data logging is stopped by removing power (cutting the ring and battery connection).
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other
| 99.8 |
When logging is enabled and the appropriate number of wake cycles have been completed to reach the set logging interval, the data logging (green) subroutine is entered. The program sets appropriate Vref for determining IN/OUT status (typically ~35°C) at one comparator input and reads Vth at the other. The output is “1” of Vth > Vref (IN) and “0” if Vth < Vref (OUT). This 1-bit value is stored in the current data byte. When 8 IN/OUT bits are accumulated, the data byte is written to non-volatile EEPROM memory. The built-in PIC12F683 EEPROM size of 256 bytes allows storage of 2048 IN/OUT readings. Additionally, the LED may be flashed to indicate a measurement or other device status. The current implementation uses a short LED flash to indicate power on, a 3 sec LED on to indicate logging started or stopped, and a short flash to indicate a logged data value. More complex LED output enabled for debugging has included a double flash for “IN” measurement and single flash for “OUT” measurement with three flashes indicating a successful data write to EEPROM. The LED functionality may be enabled or disabled programmatically in software. During long-term testing and in vivo evaluation, the LED functionality is minimized to preserve battery life.
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other
| 99.9 |
The in vitro performance of an adherence-monitoring IVR undergoing multiple temperature cycles between 25°C and 40°C to simulate repeated insertion and removal over 100 h (4.2 days) is shown in Fig 4. The device accurately monitors the IVR status (temperature relative to 34.4°C) over 33 simulated IN/OUT cycles and 1192 measurements made on a 5 min interval. This is the equivalent of one IVR removal and replacement every 1.5 days for 50 days using a 1 h measurement cycle. The measurement frequency (1s –days) and reference temperatures (0–45° C) are set in software and cover a broad range of values appropriate for laboratory evaluation and long-term clinical use. The temporal accuracy of the device is shown in the expansion plot (Fig 4), where the simulated IVR replacements (temperature ~ 40°C) at approximately 6, 48, and 96 hours are captured at the same time by each of the three devices under test.
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study
| 100.0 |
Response of adherence IVR over 100 h using a simulated insertion/removal cycle of 2.5 h in and 0.5 h out (lower plot) using a programmed temperature test apparatus. Data represent the IN/OUT status (0 or 1) logged at a 5 min interval. The upper plot shows expansion of data at 6, 48, and, 96 h for three adherence IVRs measuring simultaneously. Vertical dotted lines indicate transition to “IN” temperature for each cycle shown. The temporal alignment of “IN” measurements does not vary between the three devices over 96 h of measurement. The time required for the device to respond to a simulated insertion or removal event was determined using the temperature-time data shown in Fig 5. The device was exposed to three repeated step changes in temperature between 28°C and 37°C, and response was calculated as the time required for the device to measure the range of 10% to 90% of the total 9°C temperature change (28.9°C and 36.1°C). For the step from 28°C to 37°C (simulated insertion), the 10%-90% time was 1.84 ± 0.006 min. For the step from 37°C to 28°C (simulated removal), the 90% to 10% time was 1.96 ± 0.096 min.
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study
| 100.0 |
(a) Temperature measured by the thermistor as a function of time during three cycles of alternating between two controlled temperature chambers maintained at 28°C and 37°C, respectively. (b) Zoomed data for first cycle showing individual temperature readings collected at 10 s intervals. The lower and upper gray dotted lines indicate the values at 10% and 90%, respectively, of the full-scale measured temperature change.
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study
| 97.94 |
A single device was evaluated for 29 h over a series of three insertion/removal events in the vaginal cavity of a sheep. Fig 6 shows the IVR response to the sequence of events. The timing of the events is clearly captured by the device as shown by the alignment of the recorded data with the symbols indicating each insertion or removal.
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study
| 99.94 |
Device response as a function of time for a series of insertion and removal periods in a sheep vaginal model. The black trace is the logged data (1 min logging interval) retrieved from the device at the study conclusion. The actual time for removal and insertion events is indicated by the symbols above the black trace. The gray trace shows the temperature as a function of time for the three ring dispositions: water bath (50°C) sheep vaginal temperature measured at the start of the study (39°C), and ambient laboratory temperature (25°C).
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study
| 100.0 |
The discordant results observed among HIV PrEP clinical trials clearly indicate the importance of adherence in determining successful prevention outcomes . Sustained, topical delivery of ARV agents and microbicides using IVRs has been suggested for improving adherence; however, clinical data to support this assertion are limited [20, 21]. Real-time assessment of adherence during IVR clinical trials could allow quantitative measurement of the relationship between IVR use and HIV prevention efficacy. Until recently, no methods for real-time monitoring of IVR adherence had been reported. Boyd, et al. demonstrated proof-of-principle for using IVR temperature as a surrogate measure of adherence . They employed a commercially available temperature logging device inserted in the vaginal cavity to continuously monitor device temperature, and compared it to intravaginal (body) temperature to determine periods of device insertion and removal. These devices store over 5000 temperature measurements for later retrieval and have a measurement accuracy of 0.2°C. The data collected during a 7 day macaque study using these devices embedded in segments of silicone tubing showed that device temperature can accurately track insertion and removal patterns even in the presence of normal vaginal temperature fluctuations. The $340 cost per device, however, limits the ability to incorporate these devices into IVRs for widespread use, particularly in resource-limited settings. Additionally, these are specialized devices from a single vendor, with a risk of limited availability due to market or other circumstances.
|
review
| 99.7 |
The device described here offers a low-cost alternative for real-time temperature-based IVR adherence monitoring. The design is based on the widely used 8-bit Microchip PIC12 microcontroller platform. The circuit uses 8 discrete surface-mount components on a single PCB, with a total cost for components, PCB fabrication, and assembly of US$6 in quantities of 5000, including batteries. This is the cost for the device only and does not include IVR manufacture. A number of characteristics and advantages of the PIC12F683 microcontroller are described above. Use of the PIC12 internal comparator and non-volatile memory enabled the IVRs to use only 8 circuit components and achieve the smallest possible PCB size. The PIC12 data memory is limited to 256 bytes and required the use of a novel data reduction scheme to achieve storage of 2048 IVR status values for retrieval following IVR use. Our approach was to compare a measured voltage proportional to IVR temperature to a reference temperature to determine IVR insertion or removal status, but to store the data in bytes containing eight sequential 1-bit representations of OUT (0) or IN (1) instead of actual temperature values. Data files are retrieved from the device in hexadecimal format, and direct conversion to binary values results in a sequence of 0 and 1 values indicating IVR OUT or IN, respectively, at each predetermined logging interval. An important aspect of the device design is the ability to programmatically set the reference temperature through a simple software setting. For the studies described here, a reference temperature of 34.4°C was used. This temperature was selected based on the temporal variation in macaque vaginal temperature described by Boyd et al. and the normal sheep body temperature measured in this study and by others, with a margin for error to prevent false readings of IVR removal. The reference temperature will need to be optimized in clinical trial to accommodate differences between human and sheep basal temperatures.
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study
| 100.0 |
A second device functionality that enables the simple, compact circuit design is the use of temperature readings to control device functions. As shown in the program flow diagram (Fig 3), data logging is started from the initial device sleep state when the measured thermistor voltage is above a reference value set programmatically. A reference corresponding to 50°C was used to initiate logging. Logging may be started by simply placing the device in a cup of hot water for approximately 1 minute. Water is used here as a convenience, and any method of raising the device temperature to 50°C or more would work equally well. Similarly, logging is stopped when the thermistor voltage is below a programmatically set “stop” threshold, easily achieved by exposing the device to a low temperature such as a cup of ice water or a refrigerator. A simpler method of stopping logging that may be more useful in a trial setting is to simply cut the IVR elastomer to sever the battery wires and remove device power. The stored data is retained in non-volatile memory in the unpowered device and may be retrieved through the program/data interface as described above.
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other
| 99.9 |
A limitation of using temperature as a surrogate measure of adherence is that in some climates, temperatures may exceed the reference temperature, and even body temperature at times. Local temperatures at clinical sites used in the ASPIRE and Ring Study clinical trials of the dapivirine IVR are typically not high enough to be above the 34.4°C reference temperature used in these adherence IVR studies. Average high temperatures during the summer months approach this limit in some areas of South Africa and Zimbabwe, and high temperatures for individual summer days may exceed the reference or body temperatures. Issues associated with high local temperatures can be mitigated to allow retrieval of usable adherence data even in cases where ambient temperatures exceed the reference temperature. An IVR that is removed and stored at ambient temperature will record diurnal temperature variations if the daily ambient high temperature exceeds the reference temperature. It would be rare for the low temperature, even on the hottest days, to be above a 34–35°C reference temperature. This diurnal pattern will be distinct, and can be correlated with historical temperature records or temperatures measured at clinical sites as part of a trial protocol.
|
study
| 99.56 |
The adherence-monitoring IVR design is a modification of the established pod-IVR platform that was developed specifically for controlled vaginal delivery of drug combinations. The pod-IVR has been applied to delivery of small molecule ARV drugs for HIV [18, 19, 23–25] and HSV [18, 26] prevention as well as to vaginal delivery of monoclonal antibodies and bacterial probiotics . The pod-IVR consists of a silicone elastomer scaffold containing individual embedded drug pods that act as independent delivery devices. The adherence-monitoring module may be incorporated in the same manner as the drug pods, and drug release from the device will be unaffected. The dimensions of the prototype adherence module described here was chosen to be small enough to fit within a pod-IVR containing an expanded section as shown in Fig 1A, but large enough to allow manual soldering rework and other modifications during device development to be easily carried out. The electronics layout has subsequently been further miniaturized to a circuit board size of 14 mm × 5.1 mm that will fit within the dimensions of a standard pod-IVR (8 mm cross-sectional diameter, 56 mm outer ring diameter). This is also the size of the dapivirine IVR that has recently completed Phase 3 clinical trial . Future efforts will focus on further miniaturization of the electronics module and the incorporation of adherence-monitoring functionality into pod-IVRs delivering ARV combinations.
|
study
| 99.94 |
The in vitro and in vivo studies reported here demonstrate feasibility of the adherence monitoring approach and device; however, there are a number of development and regulatory hurdles associated with successful incorporation of the monitoring device in a combination product delivering one or more antiretroviral agents for HIV prophylaxis. The next steps in device development will focus on miniaturization of the electronics module and long term (≥28 days) in vitro and in vivo studies. The current devices are capable of this longer monitoring time. With a memory capacity of 2048 values, logging with a 30 min data interval allows continuous adherence monitoring for more than 42 days, and a 1 h interval increases this time to 85 days.
|
study
| 99.94 |
The regulatory pathway for a combination product consisting of an IVR delivering one or more antiretroviral agents modified with the adherence module is more complex than that of a drug or device alone. For incorporation of the adherence monitor into an IVR for HIV prevention, there are two primary regulatory concerns: (1) safety of the adherence module itself, and (2) demonstrating that addition of the adherence module does not negatively affect the safety, stability, or dreg release characteristics of the IVR. Because the primary mode of action (the mode expected to have the greatest overall therapeutic effect) of the combination device is antiviral drug delivery, the FDA Center for Drug Evaluation and Research (CDER) has regulatory jurisdiction, and clinical evaluation will take place under an Investigator New Drug (IND) application using a series of clinical trials (Phase 1, Phase 2, Phase 3).The adherence module is being developed primarily, but not exclusively, for incorporation into the pod-IVR platform that is currently in early clinical evaluation [26, 29]. Initial clinical evaluation of an adherence IVR to demonstrate feasibility would be conducted on the device without drug product under an Investigational Device Exemption (IDE) if the device is determined to be Significant Risk (SR). A SR designation for the adherence monitoring IVR is likely because it is an implantable device maintaining mucosal contact for more than 28 days. For initial clinical studies lasting fewer than 28 days, however, it is likely that the device would be designated Non-significant Risk (NSR), and evaluation could proceed without an IDE and only approval by the clinical investigator’s Institutional Review Board (IRB). This initial evaluation without drug product would provide a foundation of safety data to use in preparing the combination product IND as well as serve as Phase 1 clinical evaluation of an IVR that could be used in placebo groups in future clinical trials.
|
review
| 98.4 |
Adherence monitoring in HIV prevention trials has traditionally been limited to participant self-report or tracking of study materials (pill count or return of used gel applicators), approaches that have led to inaccurate assessment of true product use [8–10]. The ASPIRE dapivirine IVR trial incorporated an objective measure of adherence to IVR use in a large scale study in sub-Saharan Africa [30, 31]. Returned IVRs were tested for residual dapivirine levels to determine the amount of drug released. The distribution of residual dapivirine values was bimodal with one peak at 24–25 mg residual DPV indicating IVRs were unused, and a second peak at 20–21 mg indicating IVRs were used for 28 days. Using a length-adjusted analysis of the residual DPV data, seroconverting subjects were binned into four adherence groups based on the clinic visit with the lowest adherence for the three visits prior to HIV detection. A strong correlation of protection with adherence group was observed (% risk reduction, incidence per 100 person-years): no use (11%, 4.9), bottom third (29%, 3.1), middle third (58%, 1.9), top third (92%, 0.4). The placebo group exhibited an incidence of 4.7 per 100 person-years. Plasma levels of dapivirine was also measured quarterly as an additional measure of IVR use; however, insertion of the ring for 8 hours or more before a clinic visit resulted in identical plasma levels to 28 days of continuous use. Although the residual dapivirine levels only provided a snapshot of total IVR use over one month and no temporal use data, the ASPIRE data indicate that even imperfect and incomplete objective adherence data can lend valuable insight into patterns of adherence and the relationship between adherence and product efficacy in a clinical setting. The IVR use pattern obtained from the adherence module developed here would allow a much more detailed analysis than was conducted in ASPIRE, including specific time periods of IVR use, total drug dose during use period, self-report of IVR use and sexual activity, and HIV infection status to extract the key pharmacokinetic-pharmacodynamic relationships that result in HIV prevention efficacy.
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study
| 99.94 |
Incorporation into a clinical trial can follow several approaches. (1) For a Phase III (efficacy) trial, all active and placebo IVRs can incorporate adherence devices. This will allow determination of adherence and investigation of product efficacy in both perfect adherence and non-adherence scenarios. Additionally, both placebo and active arms of trials can be monitored for adherence to determine patterns of adherence in different groups. Because residual dapivirine in returned IVRs and plasma dapivirine levels were surrogate measures of adherence in ASPIRE, no method for determining IVR use in placebo groups was available. Significant differences in adherence between active and placebo groups may indicate inadequate study blinding, poor randomization of study groups, or other trial design issues. (2) Adherence devices may be incorporated into a subset of Phase 3 study IVRs, both active and placebo. This will allow sub-group adherence data to be extrapolated to the larger study population in studies where it may be logistically difficult to include adherence monitoring for all study participants. Data from temperature-based adherence monitoring can also be combined with plasma drug levels and residual drug in used IVRs to provide temporal IVR use data to increase accuracy of adherence assessment. (3) Devices can be incorporated into IVRs in Phase 1 and Phase 2 trials as a “perfect” measure of adherence that can be used to evaluate non-objective adherence measures and to develop additional adherence endpoints, such as compounds adsorbed into the IVR from vaginal fluids, drug levels in blood or other relevant compartments, and residual drug in used IVRs.
|
study
| 98.75 |
A miniature, low-cost IVR device using readily available electronic components, can provide accurate and quantitative determination of adherence to IVR-based HIV prevention methods. Adherence has been shown to be a primary factor in PrEP and microbicide clinical trial failures, and adherence monitoring may allow more accurate evaluation of product efficacy, identify factors influencing product use, and enable early interventions to ensure successful preventative therapy. Based on these initial in vitro and in vivo results, this adherence-monitoring IVR merits further development and evaluation in preclinical and clinical studies.
|
study
| 87.94 |
Stimuli-responsive polymers play an important role in many applications, such as diagnostics, smart optical systems, and tissue engineering, as well as in micro-electromechanical systems, biosensors, textiles, and coatings . Shape memory polymers (SMPs) are one type of important stimulus-responsive polymer that can adopt temporary shapes and recover their original shapes upon exposure to external stimuli . Although the basic shape memory behaviours of SMPs are focused on elongation, bending, and swelling, through proper material structure and stimuli design, complicated shape memory behaviours such as walking, rotation, and vibration can also be achieved. The general mechanism of the shape memory effect (SME) is attributed to the reversible switching structure of SMPs. This reversible switching structure may be responsive to stimuli such as heat, electricity, magnetic field, light, and Ph . Thus, SMPs have received the increased attention of researchers for many potential applications.
|
review
| 99.7 |
To date, numerous SMPs based on various structures have been developed, including polyurethane, polystyrene copolymer, and epoxy-based polymers. Among them, the thermal-induced shape memory polyurethane (SMPU) has been mostly used in practice because of its competitive mechanical and shape memory properties, e.g., high specific strength, high parameters of shape fixity and shape recovery, and a transition temperature that can be approximately tuned to room and body temperature . A variety of SMPUs have been reported with a wide range of activation temperatures and applications, such as medical devices, heat shrinkable packages, mechanical actuators, sensors, and self-deployable structures. However, common SMPUs cannot satisfy the growing trend of new multi-functional materials. Recently, the important development of shape memory polymer composites (SMPCs) with different fillers has significantly broadened the varieties and functionalities of SMPs, which significantly expand their applications . For example, spatially controlled electro-active shape recovery is achieved in CNT-filled SMPCs . Selective light heating is reported in gold nanoparticle-filled SMPCs . Multiple-SMEs have also been developed in the norbornyl-polyhedral oligomeric silsesquioxanes (POSS) copolymer . These studies indicate that the investigation of SMPCs is one of the most important research directions in the field of SMPs and polymer composites. It provides a new way to prepare polymer composites with diversified shape memory properties and more functionality .
|
review
| 99.9 |
As important functional fillers, liquid crystals (LC) are widely used to fabricate various liquid-crystalline polymer composites because they combine the basic properties of conventional polymers with those of LC . For example, Finkelmann et al. have prepared an LC shape memory elastomer, which can be stimulated with photo-illumination by incorporating azo groups into the LC . Additionally, Samit V. Ahir et al. have synthesized a new tri-block copolymer with a large central block composed of main-chain nematic units to retain the director alignment, and the resulting LC copolymers show a significant two-way SME . These previous studies have primarily focused on liquid crystalline elastomers or well-defined LC copolymers. Recently, we have developed a facile and versatile method for the preparation of LC-SMPCs. Several series of LC-SMPU composites with both LC properties and shape memory properties were successfully prepared by mixing various SMPU matrixes with 4-n-hexadecyloxybenzoic acid (HOBA) . Our recent investigations also demonstrated that supramolecular LC-SMPCs based on 4-octyldecyloxybenzoic acid (OOBA) and the supramolecular SMPUs exhibit triple-SMEs and quadruple-SMEs . It was thus proposed that the LC-SMPCs might be good candidates for many potential applications in the field of artificial intelligence. In order to further understand the LC-SMPCs completely and promote their future applications, the significant effects of HOBA and OOBA mesogens on the glass transition and crystal-melting transition of the soft segments have been reported in the previous HOBA-SMPU and supramolecular OOBA-SMPU systems. However, the effect of LC mesogens on the hard segment of SMPU remains unclear. We would like to present a series of investigations about the LC-SMPCs with different LC mesogens in this study.
|
study
| 100.0 |
Therefore, a new kind of LC-SMPCs, termed SMPU-OOBAm, were prepared with PEG-based SMPU containing about 40 wt % hard segment as the polymer matrix, and OOBA mesogen as the LC fillers. This study investigates the effect of OOBA on the structure, morphology, and properties of the SMPU-OOBAm. Notably different from previous systems, the present LC-SMPCs show a successful combination of the liquid crystalline properties, triple-shape memory effects, and self-healing properties. In particular, the self-healing mechanism may be notably different from the common self-healing materials. Thus, this study may be useful in order to fundamentally examine the multi-functional properties, multi-responsiveness, and efficient actuating method of SMPs and polymer composites.
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study
| 100.0 |
Extra pure grade polyethylene glycol (Mn = 6000, PEG6000), was dried before usage at 80 °C under 0.1–0.2 MPa for 6 h. 4,4-Diphenylmethane diisocyanate (MDI, analytical grade), 1,4-butanediol (BDO, analytical grade), 4-octocyloxy benzoic acid (OOBA, analytical grade) and dimethylformamide (DMF, high performance liquid chromatography grade, solvent) were purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China) and were used without further purification.
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other
| 99.7 |
Based on the compositions shown in Table 1, a certain quantity of OOBA was added to the SMPU/DMF solution containing approximately 3.0 g of PEG-based SMPU resin. Under strong mechanical stirring at 80 °C for 2 h, SMPU and OOBA were sufficiently mixed to obtain a homogenous solution-phase mixture. Then, the SMPU-OOBAm composites for the following tests were obtained by casting the mixture onto a Teflon pan, which was placed at 80 °C for 24 h and further dried at 80 °C for 24 h. The samples were coded as P1–P5 (SMPU-OOBAm, m = 0.2, 0.4, 0.6, 0.8, and 1.0, which is the molar ratio of OOBA/MDI).
|
study
| 100.0 |
Fourier transformation infrared (FT-IR) spectra were scanned from smooth polymer films with a thickness of 0.2 mm using a Nicolet 6700 FT-IR spectrometer (Nicolet, Madison, WI, USA) by the FT-IR attenuated total reflection (ATR) method. Ten scans at 4 cm−1 resolution were signal averaged and stored as data files for further analysis.
|
study
| 99.94 |
The surface morphology of the samples was examined using scanning electron microscopy (SEM, SU-70, Hitachi, Tokyo, Japan), 20 kV, equipped with an energy dispersive X-ray detector for elemental composition. Prior to scanning, the samples were coated with a thin layer of gold. Fraction morphology was scanned after the samples were fractured with liquid nitrogen.
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study
| 99.94 |
X-ray diffraction (XRD) experiments were performed on a BRUKER AXS D8 Advance diffractometer (BRUKER-AXS, Karlsruhe, Germany) with a 40 kV FL tube as the X-ray source (Cu Kα) and a Lynxeye-Xe detector. The applied voltage is 40 kV. Film samples were used in this experiment.
|
study
| 99.75 |
Thermo Gravimetric Analyzer (TGA) spectra were recorded on a computer-controlled TG Q50 system (TA, New Castle, PA, USA) after drying at 100 °C, under the following operational conditions: heating rate 10 °C/min, temperature range 100–600 °C, sample weight about 5.0 mg, using the film sample in platinum crucibles, 60 mL/min N2 flow.
|
study
| 99.9 |
Differential scanning calorimetry (DSC) measurements were carried out with a TA Q200 (TA, New Castle, PA, USA) instrument having nitrogen as the purged gas. Indium and zinc standards were used for calibration. Both heating scan rate and cooling. The scan rate is 10 °C/min. The second heating curves were used for analysis.
|
study
| 99.7 |
Dynamic mechanical analysis (DMA) curves of the samples were determined using a Perkin-Elmer DMA (TA, New Castle, PA, USA) at 1 Hz and at a heating rate of 2 °C/min. The change in modulus and the recovery stress were measured with a DMS module and TMA module, respectively.
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study
| 99.94 |
Mesophases were identified, and the phase transition temperatures were determined using a Zeiss-Axioscope polarised optical microscope (POM) (Mshot, Guangzhou, China) equipped with a Linkam-THMS-600 variable-temperature stage at a scan rate of 2 °C/min. The samples were heated from 25 to 160 °C and cooled from 160 to 25 °C.
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study
| 99.94 |
Thermally induced triple-shape memory behaviours were examined via thermo-mechanical analysis using a TA Instruments DMA Q800 system (TA, New Castle, PA, USA) with tension clamps in controlled-force mode. Samples were dried at 100 °C for 24 h and cut into rectangular pieces of 10 mm × 2.0 mm × 0.5 mm.
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study
| 99.94 |
Isothermal crystallization experiments were performed with a TA-Q200 DSC instrument (TA, New Castle, PA, USA) using nitrogen as the purge gas, according to the procedure described in the literature. The sample (4–6 mg) was initially heated to 200 °C at a rate of 10 °C/min and held for 5 min to remove the thermal history of the crystallizable phase. Subsequently, the sample was rapidly cooled (−60 °C/min) to a designated crystallization temperature (Tc) and held at this temperature until the end of the exothermic crystallization. The heat flow during the isothermal crystallization process was recorded as a function of time. Tc was chosen as 137 °C in this experiment. The amounts of heat generated during the development of the crystal phase were recorded and analysed according to the typical equation used for evaluating the degree of relative crystallinity (Xt): (1)Xt=∫t0t(dHdt)dt∫t0t=∝(dHdt)dt where t0 and t = ∞ are the times at which the sample reached isothermal conditions (as indicated by a flat baseline after an initial spike in the thermal curve) and the time at which the dominant sharp exothermic peak ends, respectively. H is the enthalpy of crystallization at time t. After isothermal crystallization, the sample was heated to 200 °C, and the crystal melting temperature (Tm), indicated by the maximum of the endothermic peak, was recorded.
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study
| 100.0 |
Figure 1 shows the FT-IR spectra of SMPU, OOBA, and SMPU-OOBAm composites (e.g., P1, P2, P3, P4, P5). In the spectrum of OOBA (Figure 1a), the C=O stretching vibration was detected at approximately 1683 cm−1, which suggest the formation of hydrogen bond associations. Thus, OOBA molecules may form dimers and create a long lath-like structure with a three-ring core, which is the basic structure of mesogen, to form the LC phase. In the spectrum of SMPU, the N–H stretching vibration is detected at approximately 3323 cm−1 and 3286 cm−1, whereas the C–N stretching vibration occurs at 1533 cm−1 and 1531 cm−1. In the SMPU-OOBAm composites (e.g., P4), the N–H stretching vibration shifts to a low wavenumber, which implies the formation of a hydrogen bond between the OOBA and the hard segment of SMPU. Additionally, P4 shows a new frequency at approximately 1685 cm−1, which can be ascribed to the C=O stretching vibration of OOBA. The frequency at approximately 1166 cm−1 also likely results from the C–O stretching vibration of OOBA. These results suggest that OOBA has successfully incorporated into SMPU and has formed an SMPU-OOBAm composite. Figure 1b also shows that all SMPU-OOBAm composites have similar FT-IR results to P4. The peak density at 1685 cm−1 obviously increases from P1 to P5, which confirms the increased OOBA content. In the previous literature, the LC phase was lost when LC mesogens containing pyridine moieties were attached to the SMPU containing carboxyl groups through hydrogen bonding between pyridine and COOH . However, in this work, the SMPU-OOBAm composites tend to keep the intrinsic LC properties of OOBA because of their dimer structure. The first advantage of the SMPU-OOBAm composites is that the liquid crystalline properties of OOBA can be used in the form of polymeric materials for many applications, being more conducive to the applications of liquid crystal materials.
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study
| 100.0 |
The surface morphology of the SMPU-OOBAm composites was investigated systematically via SEM and XRD. As shown in Figure 2, the SEM images of the pure SMPU exhibit a smooth surface with no hole, but the surface becomes rough after OOBA is incorporated into SMPU (Figure 2b–d). When the OOBA-content increases, more cracks are observed. These broken surfaces demonstrate that the SMPU-OOBAm forms a two-phase separated structure (Figure 2c,d): the OOBA phase and the SMPU matrix. For example, for P5, some self-assembled crystals are observed, which the free OOBA molecules form, but the other OOBA molecules could not be precisely estimated because they are wrapped into a notably small size by the SMPU matrix. It has been confirmed that the incorporated OOBA enters the polyurethane matrix and self-assembles into the free OOBA phase in the composites. Unlike the previous SMPU-HOBA systems, OOBA does not form individual lamellar crystals in the SMPU-OOBAm composites, and the OOBA molecules have better dispersion in the SMPU matrix. This might affect the structure, morphology, and properties of the SMPU greatly.
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study
| 100.0 |
Wide-angle X-ray diffraction (WAXD) experiments were used to further study the morphology of the SMPU-OOBAm composites. As shown in Figure 3a, only two weak, broad peaks are observed in the pure SMPU at 19.5° and 23.8°, assigned to the (120) and (132) crystal faces, represents the crystal of polyethylene glycol, which indicates that the pure SMPU contains only a semi-crystalline soft phase and an amorphous hard phase. In addition, the pure OOBA shows many sharp peaks in the range of 10°~35°, which indicates a complete crystalline structure of OOBA . When the OOBA is incorporated into the SMPU, nearly all crystalline peaks of OOBA remain detected, e.g., P2 and P5 (see Figure 3a), and the intensity of these crystalline peaks increases when the OOBA content increases (see Figure 3b). The crystalline peaks of the PEG soft phase are also intensified with the increase in OOBA content. This result indicates that OOBA promotes the crystallinity of the soft phase of SMPU. However, the intensity of the crystalline peaks in SMPU-OOBAm is not as strong as that of pure OOBA, which implies a reduced crystallinity of OOBA. Moreover, a new peak at 16.2° is observed; which also intensifies with an increase of OOBA-content. According to the literature , we know that pure OOBA can form two different crystals (α and β crystalline phases) at low temperature. Therefore, this new peak may be ascribed to the second crystal type (β phase) of OOBA. Thus, the crystallizability of the first crystal type (α phase) of OOBA is restrained, and OOBA subsequently tends to form a second β phase when the OOBA content increases. This phenomenon can also be reflected in the thermal properties and phase transitions.
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study
| 100.0 |
The thermal properties of the SMPU-OOBAm composites were investigated by TGA and DSC. The TG curves show that all samples appear to decompose in two stages, which corresponds to the decomposition of the soft (at 200–300 °C) and hard segments (at 350–450 °C) of the SMPU matrix (see Figure 4a). The TG results also show that the decomposition temperatures of the SMPU-OOBAm composites increase when the OOBA-content increases, which implies that the decomposition of SMPU-OOBAm is restrained when OOBA is incorporated. However, when the OOBA-content increases, the decomposition quantity in the first stage increases because of the increased decomposition of OOBA. Additionally, a broad weak peak is observed at about 210 °C on the DTG curves. This result demonstrates that the thermal stability of the composites at the first stage improves (see Figure 4b). The improved thermal stability make the composites good polymeric materials.
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study
| 100.0 |
The second DSC heating curves demonstrate that the pure SMPU shows an exothermic peak at −20 °C and an obvious endothermic peak at 50 °C (see Figure 5a), which correspond to the cold crystallization and crystal-melting temperature of the soft phase, respectively. Pure OOBA shows four obvious endothermic peaks (Figure 5a). Among them, the first two peaks at 78 °C and 101 °C can be attributed to the crystal-melting transitions of the α and β crystalline phases, respectively. The β crystal-melting transition also indicates that OOBA enters the smectic phase. The third peak at 107 °C indicates a smectic-nematic phase transition, and the fourth peak at 148 °C suggests a phase transition from the nematic phase to the isotropic phase. When OOBA is incorporated into the SMPU matrix, the two crystal-melting transitions in the composites (e.g., P3) at 49 °C and 74 °C can be attributed to the crystal-melting of the soft segment of SMPU and the α phase of OOBA, respectively. Meanwhile, a new weak exothermic peak which can be attributed to the β crystal-melting transition appears at 89 °C. When the molar ratio of OOBA/MDI is lower than 0.4 (m ≤ 0.4), the β crystal-melting transitions cannot be detected on the second DSC heating curve (see Figure 5b), because the SMPU polymer chain affects the order structure of OOBA. In addition, another weak exothermic peak is detected at 183 °C and is attributed to the crystal melting of the hard segment of SMPU. This result indicates that the crystallizability of the hard segment of SMPU is also enhanced, because the OOBA crystals provide the nuclear seeds of crystallization for the hard phase. However, the cooling curves demonstrate that pure SMPU shows two weak peaks at 105 °C and −14 °C (see Figure 5c), which are the crystallization temperatures of the hard and soft segments of SMPU, respectively; whereas SMPU-OOBAm shows a series of endothermic peaks upon cooling. For P5, the lowest and highest endothermic peaks at 15 °C and 133.6 °C are attributed to the crystallization temperatures of the soft phase and hard phase of SMPU, and these crystallization temperatures are also higher than those of pure SMPU. When the OOBA-content increases, the crystallization temperature of the soft phase of SMPU shifts to a higher temperature range. It is confirmed again that the soft phase of composites with higher OOBA-content has higher crystallinity. In addition, the endothermic peaks at 37 °C should be attributed to the crystallization temperature of the OOBA phase, whereas the endothermic peak at 80 °C shows the phase transition from the isotropic phase to the LC phase. These phase transitions are also verified by the following POM investigations.
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study
| 100.0 |
The influence of heat treatment was also investigated in this study. In Figure 5d, the effect of OOBA in the composites on the thermal properties during the heat treatment was investigated with DSC by heating sample P3 to 100, 120, 140, 160, and 180 °C. When P3 was thermally treated at 120 °C and 140 °C, the cold crystallization peak of the soft phase of SMPU was detected at −11 °C. However, the peak disappears after the thermal treatment at 100 °C, 160 °C, and 180 °C. A possible reason is that the movement of the polymer chains is frozen at low temperature (<100 °C), and the composites cannot form cold crystallization; the higher treatment temperature (≥160 °C) can promote the movement of the polymer chains and cause a complete crystallization of the soft segments when they cool to a low temperature. Thus, there is no cold crystallization in the second heating process. In addition, Figure 5d demonstrates that the crystallization temperatures of both the soft and hard phases of SMPU shift to higher temperatures when the thermal treatment temperature increases. This result also demonstrates that the heat treatment can promote the crystallizability of the soft and hard segments of SMPU. However, the heat treatment results show that the crystal melting transition of OOBA is maintained at 74 °C in all composites. Thus, OOBA can aggregate into an isolated phase and retain its intrinsic LC properties in the SMPU-OOBAm composites.
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study
| 100.0 |
For understanding the crystallization behaviors of the hard phase in the SMPU-OOBAm composites, the isothermal crystallization kinetics were investigated using DSC analysis. Detailed calculations are provided in Section 2.5. The crystallization temperature was fixed at 137 °C in this experiment. Figure 6a shows that the exothermic peak of SMPU-OOBAm widens when the OOBA-content increases during the isothermal crystallization process, which suggests the increased crystallization time. According to Equation (1), the relationships between relative crystallinity (Xt) and time are shown in Figure 6b. The curves exhibit an S pattern. The crystallization rate of the hard phase is slow during the initial stage and during the later period of crystallization. Moreover, the slope of the curves decreases when the OOBA-content increases. Thus, the crystallization rate of the hard phase decreases when the OOBA-content increases. The Avrami equation (Equation (2)) was used to denote the complete crystallization behaviours of the SMPU-OOBAm composites. Generally, and for convenient analysis, the Avrami equation is converted to Equation (3) via linearization. Figure 6c presents the dependence of log{−ln(1 − X(t))} on log(t). It was found that log{−ln(1 − X(t))} is linearly related to log(t), but the curve slightly deviates during the later period of crystallization. This deviation implies the existence of a secondary crystallization, which should be attributed to the phase transformation from sphero-crystal to lamella-crystal. Finally, based on the original data and fitting a straight line, the kinetic parameters n, K, and t1/2 of the isothermal crystallization were easily calculated. The results are summarized in Table 2. The n value of all samples is ~1.3, which further indicates that the crystallization mechanism of the hard segment of SMPU in all composites is the nucleated mechanism. The crystallization rate decreases because the K value decreases when the OOBA-content increases. One possible reason for this is that the increased OOBA content might affect the aggregation of the hard segment of SMPU, though they provide more crystal seeds.
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study
| 100.0 |
Dynamical mechanical properties can also be used to investigate phase transitions by characterizing the changes of the storage modulus. Figure 7 shows the DMA curves of SMPU-OOBAm composites. All of the samples have a large glassy modulus (E’g) below −30 °C, whereas the storage modulus gradually decreases because of the glass transition of the soft segment of SMPU. The first significant storage modulus occurs when the temperature increases to 46 °C, because of the crystal-melting of the soft segment of SMPU. Thereafter, SMPU enters the rubber state. The DMA curves demonstrate that the rubber modulus at 46~60 °C in the SMPU-OOBAm composites tends to be less than those of SMPU. This result again confirms that the OOBA crystals reinforce the crystallinity of the soft segment of SMPU, and act as crystal fillers in the rubber state. The second storage modulus change occurs above 77 °C, particularly in samples with higher OOBA-content. This modulus change should be ascribed to the crystal melting transition of OOBA because it exactly corresponds to the Tm of OOBA. This observation is also notably consistent with the DSC and POM results. Thus, OOBA is another isolated phase that controls the storage modulus. The OOBA isolated phase provides a second reversible phase to the shape fixation and shape recovery. Therefore, SMPU-OOBAm composites may have a triple-shape memory effect.
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study
| 100.0 |
The liquid-crystalline properties and the phase transition behaviors of SMPU-OOBAm composites were investigated by POM. As we known, OOBA exhibits two crystalline forms, followed by smectic and nematic LC phases upon heating. Typical POM textures of OOBA upon heating are shown in Figure 8. At low temperatures, OOBA forms a stable crystalline phase (see Figure 8a). When the temperature increases to 112 °C, the colourful crystalline texture turns into a smectic LC phase (see Figure 8b). When the temperature continuously increases, the smectic LC texture converts to the nematic LC phase at 135 °C (see Figure 8c). As is known, an amorphous substance has no birefringence, and the field of vision is dark. The OOBA has good liquidity in the nematic phase. Thus, it is hard to get a whole continuous nematic texture at 135 °C. Therefore, the dark areas seen in Figure 8c resulted from either the isotropic phase of part of the OOBA or the amorphous substrate. When the temperature reaches 139 °C, the LC texture begins to disappear and the field of vision becomes dark (Figure 8d). This is the isotropic temperature of OOBA, which exactly corresponds to the exothermic peak on the DSC curve (see Figure 5a).
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study
| 100.0 |
For the SMPU-OOBAm composites, sample P5 has a bright abundant crystalline texture at 28 °C (Figure 9a) because the crystals of the soft segment of SMPU and OOBA mixed together. When the temperature increases to over 50 °C, part of the bright crystalline texture disappears (see Figure 9b), and the field of vision becomes darker, which suggests the crystal melting of the soft segment of SMPU. When the temperature reaches 76 °C, the composites enter the smectic phase (see Figure 9c). This result is consistent with the DSC curve (see Figure 5b). At 95 °C, the POM image shows the nematic phase, which suggests the phase transition from smectic phase to nematic phase because of the OOBA. When the temperature reaches 106 °C (see Figure 9e), the nematic phase gradually disappears. Compared with pure OOBA, the LC phase transition temperatures shift to lower temperatures in all composites. These results confirm that the aggregation of OOBA mesogen was restrained by the SMPU matrix, which significantly affected the LC phase transitions. In addition, the POM images also demonstrate that some weak crystalline texture is maintained above 180 °C (see Figure 9f), which implies the crystal formation of the hard segment of SMPU. These phenomena are also identified on the DSC curves.
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study
| 100.0 |
Additionally, a comparison study of the POM textures of the composites is shown in Figure 10. The results demonstrate that all composites showed similar LC phase transitions with relatively lower temperatures than pure OOBA. In the composites with lower OOBA contents (e.g., P2 and P4), the OOBA phase tended to disperse into the isolated OOBA phase in an SMPU matrix. As shown in Figure 9, the black phase is the SMPU phase, and the bright phase is the OOBA LC phase. When the OOBA-content was higher, the OOBA phase could also form a continuous OOBA phase in the composites. This result confirms that the crystalline behaviours and LC properties of OOBA are maintained in all SMPU-OOBAm composites. It is thus confirmed again that SMPU-OOBAm composites can make the OOBA mesogens be used in many polymeric materials applications, including flexible displays, electronic papers, etc.
|
study
| 100.0 |
Triple-shape memory behaviours of SMPU-OOBAm composites were examined in a thermo-mechanical analysis using a TA Instruments DMA800 with tension clamps in controlled-force mode. In this experiment, the rectangular samples were first elongated at 100 °C, and then fixed at 80 °C, while the second elongation and fixation were carried out at 80 °C and 0 °C, respectively. Thereafter, the two step shape recovery is tested each for 40 min at 80 °C and 100 °C, respectively. The strain deformation, strain fixation, and strain recovery process were recorded for analysis. Figure 11 presents the strain-time-temperature curves of P1, P2, P3, and P4, respectively. The PEG-based SMPU was reported to show only dual-shape memory effects in previous studies. However, Figure 11 demonstrates that all composites show a two-step shape recovery with typical triple-shape memory effects. For example, in sample P1, after 62% strain is deformed at 100 °C, only 38% strain is fixed at 80 °C because of the higher fixation temperature, which is ascribed to the crystallization of the OOBA crystals. However, more than 98% strain fixation is obtained when the temperature decreases to 0 °C because of the further crystallization of the soft phase of the SMPU matrix. During the strain recovery process, P1 recovers 62.6% strain at 80 °C, and the remaining 13.8% strain is quickly recovered when the temperature increases to 100 °C. Finally, more than 90% of the total strain is recovered after the temperature is maintained at 100 °C for approximately 20 min. The strain recovery temperature in the second step corresponds exactly to the crystal-melting temperature of OOBA (76 °C) as mentioned in the DSC and POM results. However, when the OOBA-content increases, the strain recovery ratio in the second step tends to reduce from 90% (P1), 79% (P2), and 78% (P3) to 77% (P4). One possible reason for this is that OOBA has lubricated the SMPU polymer chains as a plasticizer. Although the crystallizability of the hard segment of SMPU is enhanced, the OOBA molecules in the composites may destroy the entire network structure of SMPU, which results in permanent deformation. The shape recovery thus decreases as the OOBA-content increases. However, all composites show good triple-SME. Both the strain fixation and strain recovery are programmable. Therefore, the SMPU-OOBAm can potentially be used in smart optical devices, electronic devices, and sensors.
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study
| 100.0 |
The SMPU-OOBAm composites were also found to show self-healing properties in this work. As shown in Figure 12, a specimen with a H pattern was prepared using sample P2. First, the sample was cut into two parts before testing (Figure 12), which were subsequently placed together. When the temperature was increased to 80 °C for 10 min, the sample became soft because of the crystal-melting of the soft segment of SMPU. When heated to 100 °C, the gap between the two cut parts gradually decreased. When the temperature reached 130 °C and the samples had been treated for 10 min, the two cut parts joined together completely (see Figure 12c). After cooling to room temperature, the healed sample was obtained (see Figure 12d). Thus, SMPU-OOBAm exhibits thermal-induced self-healing properties. According to the described structure and morphological analysis, the self-healing process is a thermal-induced self-healing mechanism, which can be explained as follows: the OOBA forms an isolated phase, and the SMPU matrix provides the backbone. When the temperature increases to 100 °C, the crystalline OOBA enters the LC state, and a good fluidity is obtained in OOBA molecules at 130 °C. Thus, the OOBA molecules bleed from the SMPU matrix and spontaneously enter the gap. After cooling to room temperature, the OOBA molecules in the gap form crystals via the crystallization process. Therefore, the gap between two cut parts can be sealed. Therefore, the SMPU-OOBAm composites might also have many promise self-healing applications in the fields of flexible printed circuit, self-protecting textiles, and self-healing concrete.
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study
| 100.0 |
This paper describes a new type of LC-SMPU composites, which were prepared with PEG-based SMPU and OOBA mesogens. The effect of OOBA on the structure, morphology, and properties of the material was carefully investigated. The results demonstrate that the LC-SMPU composites have liquid crystalline properties, triple-shape memory properties, and self-healing properties. The incorporated OOBA promotes the crystallizability of both the soft and hard segments of SMPU. However, the isothermal crystallization kinetics revealed that the crystallization rate of the hard segment decreases when the OOBA-content increases. Additionally, the SEM confirmed that the SMPU-OOBAm composites form a two-phase separated structure, which was comprised of an SMPU phase and an OOBA phase. DMA further reveals that the composites show two-step modulus changes upon heating. Therefore, the SMPU-OOBAm composites exhibit triple-shape memory behaviour, but the shape recovery ratio decreases with the increase in OOBA-content. Because of the crystalline behaviours and LC properties of OOBA that are maintained in all SMPU-OOBAm composites, the SMPU-OOBAm composites have good self-healing properties, due to the heating-induced “bleeding” of OOBA in the liquid crystalline state and its subsequent crystallization upon cooling. Therefore, the SMPU-OOBAm composites have promising applications in smart optical devices, smart electronic devices, and smart sensors.
|
study
| 100.0 |
Circulating tumor DNA (ctDNA) is cell-free DNA (cfDNA) that is released from dying/dead cancer cells into the blood stream. It is a biomarker of cancer and is expected to have wide applications, such as the early detection of cancer and monitoring of drug resistance . However, some characteristics of ctDNA make it poorly suited for use in diagnostic applications. One milliliter of blood contains cfDNA from one to several thousand genomes as fragments with an average size of 170 base pairs. The rare mutations from cancer cells must be detected among the vast amount of DNA from normal cells and quantitated.
|
review
| 94.94 |
Digital PCR and next-generation sequencing (NGS) are becoming the technologies of choice for detecting cancer variants. However, the high sequencing error rate of current NGS platforms is a major problem. In addition, sequencing multiple sites or genomic regions dramatically increases the number of false positives. The introduction of molecular barcodes [3–7] is likely to solve this problem. So called “barcoding” involves the labeling of DNA fragments with unique oligonucleotides, typically 10–15 bases long, which enables reads from the same DNA fragment to be grouped together. Constructing a consensus read from the grouped reads eliminates sequencing errors as well as errors introduced during PCR. However, molecular barcodes cannot detect base substitutions in genomic DNA introduced by DNA damage, and somatic mutations that preexist at a low frequency in the cells of healthy individuals also make it difficult to discriminate ctDNA from cfDNA originating from normal cells. Thus, to use NGS for diagnostic purposes, such variants must be removed.
|
review
| 99.2 |
We previously developed a high-fidelity sequencing method that utilizes molecular barcodes called the non-overlapping integrated read sequencing system (NOIR-SS) . NOIR-SS is distinct from other methods that utilize molecular barcodes owing to its ability for absolute quantitation of cancer mutations. Since a substantial fraction (or majority) of barcode tags include PCR/sequencing errors, removal of erroneous barcode tags is crucial for absolute quantitation of cancer mutations. NOIR-SS removes erroneous barcode tags during data analysis.
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study
| 99.94 |
Since surgical resection of localized tumors improves overall survival, early detection of pancreatic cancer would have substantial health benefits . For early detection, high specificity is especially important. In this study, we devised a NOIR-SS-based assay for a panel of genes related to pancreatic cancer. In the first step, variants, including those present in healthy individuals, are identified. Therefore, we developed a bioinformatic filter to remove variants that are unlikely to be somatic mutations in cancer tissues. The results showed that the filter completely removed such inappropriate variants. The performance of the assay and filter system was validated using an independent cohort of patients with intraductal papillary mucinous neoplasm (IPMN) and pancreatic cancer. Sequencing based on molecular barcodes along with the filter we developed is likely to eliminate most variants not specific to cancer cells, while maintaining sensitivity comparable to that of conventional digital PCR/deep sequencing.
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study
| 100.0 |
Blood samples were obtained from patients with pancreatic cancer or IPMN between January 2012 and February 2016 at the Osaka Medical Center for Cancer and Cardiovascular Diseases. Plasma preparation and DNA extraction were performed as described previously . Tissue samples were obtained using endoscopic ultrasound-guided fine-needle aspiration. Written informed consent was obtained from all the patients. This study was approved by the ethics committee of the O Osaka Medical Center for Cancer and Cardiovascular Diseases.
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study
| 99.94 |
The targeted regions of genes related to pancreatic cancer are shown in S1 Table. The sequences of the adapters and primers are shown in S2 Table. A 30-base-long adapter sequence, including the primer sequence for Ion Torrent sequencing, was joined to a 5-base sequence for indexing individuals, a 12-base sequence for indexing molecules, and a 20-base spacer at the 3ʹ end.
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study
| 100.0 |
Library construction for NOIR-SS was performed according to a modification of a previously described procedure . For each plasma sample, we prepared two separate reaction mixtures with two gene-specific primers (shown with a “1” or “2” suffix in S2 Table) since our PCR system did not allow the use of primer pairs. Cell-free DNA obtained from approximately 1 ml of whole blood (average 4.5 ng, median 3.1 ng).was end-repaired in a 15 μL reaction containing 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP, 0.4 mM dNTPs, 2.4 units of T4 DNA polymerase (Takara Bio, Kusatu, Japan), 7.5 units of T4 polynucleotide kinase (NEB, Ipswich, MA, USA), and 0.5 units of KOD DNA polymerase (Toyobo, Osaka, Japan), which was incubated for 30 minutes at 25°C and then for 20 minutes at 75°C. Adapters tagged with the 12-nucleotide barcode sequence were ligated in a 20 μL end-repair reaction containing 0.5 μL of 10× T4 DNA ligase buffer (NEB), 40 pmol of adapter, and 2000 units of T4 DNA ligase (NEB), which was incubated at 25°C for 15 minutes. The ligation products were purified twice with 1.2× volumes of AMPure XP beads (Beckman Coulter, Brea, CA, USA). The purification beads were then mixed with 20 μL of the linear amplification reaction mix (1× Q5 Reaction Buffer [NEB], 0.2 mM dNTPs, 6 μM gene-specific primer mix [suffix “1” or “2” in S2 Table], and 0.4 units of Q5 Hot Start High-Fidelity DNA Polymerase [NEB]). After the AMPure XP beads were removed, the amplification was performed in the reaction: denaturation at 98°C for 30 seconds, and then 15 cycles of 10 seconds at 98°C and 2 minutes at 65°C. Then, 1.2 μL of 100 μM T_PCR_A was added to the reaction, and the mixture was incubated as follows: 15 cycles of 10 seconds at 98°C, 30 seconds at 65°C, and 30 seconds at 72°C. The amplification products were purified once with 1.2× volumes of AMPure XP, and recovered in 20 μL of 0.1× TE. Three microliters of the purified products were added to two tubes containing PCR amplification solution (20 μL each): 1× High Fidelity PCR Buffer (Thermo Fisher Scientific, Waltham, MA, USA), 0.2 mM dNTPs, 2 mM MgSO4, 0.5 μM T_PCR_A, 0.5 μM nested-primer-mix (suffix “L” or “H” in S2 Table), and 0.4 units of Platinum Taq DNA Polymerase, High Fidelity (Thermo Fisher Scientific). The thermal cycling program was as follows: 2 minutes of denaturation at 95°C followed by 25 (nested-primer-mix with suffix “H”) or 30 (nested-primer-mix with suffix “L”) cycles of 15 seconds at 95°C and 1 minute at 63°C. The amplification products were purified with 1.2× volumes of AMPure XP beads. The product concentration was determined using the Qubit dsDNA HS Assay Kit or Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific).
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study
| 100.0 |
Massively parallel sequencing was performed using an Ion Torrent Proton sequencer (Thermo Fisher Scientific) according to the manufacturer’s protocol. Torrent Suite (Thermo Fisher Scientific) was used to convert the raw data into base calls and to extract the FASTQ files of the sequence reads.
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other
| 99.9 |
Reads in FASTQ format were sorted using the 5-base indices for the assignment of individuals. The sequences between the 5-base indices and spacer sequences were obtained and used as molecular barcode tags. Reads with a total length, including the spacer and the sequence following it, >50 bases were aligned to target regions using BWA-MEM , whereas reads with short mapped ends (<40 bases) were discarded. Reads with the same barcode sequences were grouped together; erroneous barcode tags were detected and removed as described previously . Consensus sequences for reads with the same barcode were created using VarScan as described previously . When more than 85% of reads had the same base at a position, this base was selected as the consensus. For variant detection, we applied a Poisson distribution model to calculate the sequencing error as previously described . We evaluated each target region for the presence of variant(s), setting P = 10−4 as the threshold for detection. We evaluated each base position in codons 12 and 13 of the KRAS gene at the specified threshold value . Common SNP sites and error-prone sites were not considered in our analysis. We used Genome Reference Consortium human genome build 37 (GRCh37/hg19) as the reference genome.
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study
| 100.0 |
We used NOIR-SS to target regions of eight pancreatic cancer-related genes: KRAS, TP53, SMAD4, CTNNB1, CDKN2A, GNAS, HRAS, and NRAS. The total size of the target regions was 2.8 kb. In the original version of NOIR-SS , barcode-tagged adapters were attached to the template DNA molecules after digestion with restriction enzymes. Since this procedure limited the choice of the target regions, here we attached the barcode-tagged adapters directly to the undigested ends of the cfDNA. As end positions of cfDNAs were variable, target sizes ranged from 110 to 260 bp, including adapter sequences. For library construction, we amplified target regions with approximately 4.5 ng of cell-free DNA using the adapter and a mixture of gene-specific primers after a linear amplification step with only the gene-specific primers. The reaction scheme is presented in Fig 1. The library was subjected to NGS on an Ion Torrent sequencer. Sequence reads were grouped using molecular barcodes. After removal of erroneous sequences, high quality sequence data were used to build a consensus sequence for each read group. The sequencing depth was at least 100 per molecule. The average number of sequenced molecules was 900 per target region.
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study
| 100.0 |
Cell-free DNA is end-repaired, phosphorylated and ligated to adapters including barcode sequences. DNA fragments with the adapters are subjected to linear amplification using multiple gene-specific primers. To construct sequencing libraries, the products are subjected to PCR using primers including sequences indispensable for the Ion Torrent sequencer.
|
other
| 99.9 |
The first data set was obtained from a cohort of 12 healthy individuals and 57 patients with pancreatic cancer. The variant detection results are summarized in the top-half of Table 1. In the samples from healthy individuals, 12 variants were detected (S3 Table). Although variants present in less than 1% of the reads do not affect the analysis in conventional NGS applications, such as whole genome or exome sequencing, they are a serious problem in the detection of ctDNA. In our case, 5 out of 12 healthy individuals were variant-positive (Table 1, top right). Therefore, direct use of variants identified by NOIR-SS is not appropriate for the diagnosis of pancreatic cancer. Therefore, we developed a filter to remove variants present in healthy individuals. To develop our variant filter, we used the first set of 12 healthy individuals and 57 patients with pancreatic cancer as “training data,” as is the norm when solving classification problems in the field of statistics.
|
study
| 100.0 |
All known cancer-specific mutations are stored in a public database called the Catalogue of Somatic Mutations in Cancer (COSMIC) . Recent large-scale efforts to characterize cancer genomes, such as the international cancer genome consortium and the cancer genome atlas , are assumed to have identified most mutations originating in primary tumors. However, 10 of the 12 variants identified in the healthy individuals were not cataloged in COSMIC. Tumor-specific mutations have biological activities, and their base positions should be restricted to hot spots. On the contrary, non-functional variants are likely to occur randomly. To handle this, we made the following two assumptions: 1) COSMIC includes all somatic mutations present in cancer tissues; 2) low-frequency entries in COSMIC may arise from artifacts such as DNA damage or PCR/sequencing errors. A recent report supports the appropriateness of the second assumption . Accordingly, we removed variants not cataloged in COSMIC (version 78) and single-entry variants, except variants in TP53. Since a larger number of somatic mutations have been cataloged for the coding region of TP53, a more stringent criterion was appropriate. Accordingly, we removed TP53 variants with less than 10 entries. Thus, of the 16 TP53 variants identified in patients with pancreatic cancer in the first data set, we removed five variants with no entry, one with a single entry, and three with 2–9 entries in COSMIC. This bioinformatic process was designated as the CV78 filter. The CV78 filter removed all variants present in the healthy individuals (Table 1, top). The sensitivity of the assay to detect pancreatic cancer patients, namely the fraction of variant-positive cancer patients, was decreased from 54.4% to 33.3% with the CV78 filter. Since insertion/deletion errors were uncommon in our results (one insertion and one deletion), we excluded them from the analysis.
|
study
| 100.0 |
For 10 patients with pancreatic cancer, both plasma and tumor samples were available. Their variants identified with NOIR-SS are shown in Table 2. Among the 35 variants identified, six were only detected in plasma samples, and all six of these variants were removed by the CV78 filter. To examine whether these variants were derived from leucocytes, we sequenced the corresponding target regions of leucocyte DNA. As shown in S4 Table, no variants were found in leucocytes. As variants in cfDNA are derived from dead/dying cells, it may be difficult to deduce those sequences from living cells. The CV78 filter also discriminated variants present in normal cells from those present in tumor cells. However, it should be noted that six variants present in the tumor samples were also removed by the CV78 filter, indicating the possible loss of true mutations. Except for variants identified in the sample from patient P188, the frequencies of all variant alleles were low. This indicates that these variants do not represent genetic abnormalities present in the majority of the cell population.
|
study
| 100.0 |
The entire process, i.e., the identification of variants with NOIR-SS and subsequent filtering of variants for cancer-specificity, was validated on an independent sample set. We designated it as the second sample set, and it included plasma samples from 20 patients with IPMN and 86 patients with pancreatic cancer. Plasma samples from patients with pancreatic cancer included in the second set were obtained later than those included in the first data set, except for the plasma samples with paired tissue samples. All the samples in the second set were assayed and analyzed only after development of the CV78 filter.
|
study
| 100.0 |
IPMN is a benign neoplasm that grows within the pancreatic duct, and therefore is unlikely to release cfDNA into the blood stream. KRAS mutations are rarely detected in the plasma of patients with benign neoplasms . Since a significant proportion of IPMN cases progress to pancreatic cancer , differentiation of IPMN from pancreatic cancer may have substantial clinical benefits.
|
other
| 98.75 |
Initially, 10 out of 20 patients with IPMN were variant-positive; however, after CV78 filtering, only one patient was variant-positive (the bottom section of Table 1). In contrast, 32 out of 86 patients with pancreatic cancer were variant-positive after CV78 filtering. The CV78 filter decreased the assay sensitivity from 72.1% to 37.2%; the relative reduction in assay sensitivity was similar to that of the first data set. Use of the filter increased the identification of variants present in metastatic cancer, which would have otherwise remained obscure. The major characteristics of the first sample set were reproduced by the second sample set.
|
study
| 100.0 |
Variants identified by NOIR-SS were classified according to their presence within specific genes (Table 3). Filtered variants were defined as those selected by the CV78 filter. A characteristic common to the first and the second sample sets was the high recovery rate of KRAS mutations with the CV78 filter. This is due to mutational hotspots present within codons 12 and 13. There was no significant difference in the recovery rates (i.e., the percentage of selected variants) between the first and the second sample sets.
|
study
| 100.0 |
Since there was no significant difference between the results from the first and second sample sets, subsequent analyses were performed by combining data from both sample sets. To examine the contribution of individual genes (after application of the CV78 filter), we plotted the cumulative number of variant-positive patients identified for each gene (Fig 2). In Fig 2A, the genes are arranged along the x-axis in descending order of the number of variant-positive patients identified. Conversely, in Fig 2B, the genes are arranged along the x-axis in descending order of the number of variant-positive patients identified per gene. Fig 2A shows that the highest number of variant-positive patients had variants in KRAS (explained below). In addition, only a small number of variant-positive patients was removed after applying the CV78 filter. Fig 2B shows that the numbers of selected and removed variant-positive patients increased as the number of genes increased. Using variants in all genes except KRAS identified the same number of filtered variant-positive patients as variants in KRAS only, and the numbers of variant-positive patients after filtering and their proportions among the total number of patients (143) were as follows: all genes, 51 (35.7%); KRAS, 37 (25.9%); other genes excluding KRAS, 37 (25.9%).
|
study
| 100.0 |
A, Genes are in descending order according to the number of variants. B, Genes are in ascending order according to the number of variants. The y-axis shows the number of patients, and the x-axis shows genes in order of the number of variant-positive patients identified. Solid line: patients identified by filtered variants; broken line: patients removed by CV78 filtering.
|
study
| 99.5 |
A statistical analysis of the base substitution patterns is shown in S5 Table. Among the filtered variants, 23.3% were G>T/C>A transversions and 62.8% were C>T/G>A transitions. Among the variants removed by the CV78 filter, 32.2% were G>T/C>A transversions and 40.6% were C>T/G>A transitions. These transversions and transitions were the major mutations detected in both data sets. Their dominance among the filtered variants suggests that tumor-specific mutations likely arise via oxidation/deamination and are repaired by cell proliferation.
|
study
| 100.0 |
All samples were displayed on a scatter plot, with the number of sequenced molecules on the x-axis and number of variant molecules on the y-axis (Fig 3). Variant allele frequencies are indicated by broken diagonal lines. The variant allele frequency of filtered and removed variants ranged from 0.25–76.1% and from 0.35–45.9%, respectively. However, the distribution patterns between the two types of variants were different. The variant allele frequency of filtered variants varied widely in their respective proportions, from more than 10% to less than 1% of sequenced molecules. In contrast, the removed variants rarely made up more than 10% of all sequenced molecules, and most were approximately 1%. More than two-thirds of variants isolated from tissue samples showed high variant allele frequency.
|
study
| 100.0 |
The x-axis shows the number of sequenced molecules, and the y-axis shows the number of molecules in which a variant was detected. The diagonal broken lines indicate variant allele frequencies. Closed circles: filtered variants; Cross marks: variants removed by CV78 filtering; Diamonds: variants in tumor tissue.
|
other
| 92.56 |
Finally, we compared the performance of the method developed in this study to that of conventional deep sequencing. Since the presence of KRAS mutations is used as the standard for identifying ctDNA in patients with pancreatic cancer, we compared the ability of our method to detect KRAS mutations to that of conventional deep sequencing. In the first analysis, we analyzed our sequence data without using the molecular barcodes, thus regarding them as conventional deep sequencing data. In the second analysis, we utilized the data from a large-scale case-control study of a population of European descent by Le Calvez-Kelm et al. conducted using deep sequencing. Sensitivity and specificity were respectively defined as the proportion of KRAS mutation-positive samples among samples from patients of pancreatic cancer and the proportion of KRAS mutation-negative samples among samples from the control population. The control population in the first analysis included healthy individuals as well as IPMN patients. The respective calculated sensitivity and specificity were as follows: NOIR-SS/CV78 (our data), 25.9% and 100%; deep sequencing (our data), 30.8% and 96.9%; and deep sequencing (data from Le Calvez-Kelm et al.), 20.9% and 96.3%. Two-by-two tables and representative parameters are shown in S6 Table. In comparison, the sensitivity of two previous studies that used droplet digital PCR [20, 21] was 32.0%. These two studies did not report the specificity of their method using control populations.
|
study
| 100.0 |
Molecular barcode technology eliminates PCR/sequencing errors and generally achieves an error rate of approximately 10−5, regardless of platform and assay conditions [3–5]. Thus, this technology is likely to be indispensable for NGS of cfDNA. However, it is not effective for removing artifacts introduced before PCR, such as those introduced by damage to genomic DNA during or before sample preparation. In addition, preexisting somatic mutations in normal cells make it difficult to discriminate ctDNA from cfDNA originating from normal cells. Current knowledge regarding the prevalence of mutations in nuclear DNA is based on living cells, and comparatively little is known about mutations in DNA entering the circulation from dead/dying cells. Somatic mutations in normal cells might become more frequent just before death, especially under stress conditions, such as cancer.
|
study
| 99.94 |
We identified a number of variants in healthy individuals and patients with pancreatic cancer using NOIR-SS. Then, we applied a bioinformatic filter, called the CV78 filter, to select ctDNA variants based on the COSMIC database. Variants with numbers of entries exceeding a preset threshold were selected and regarded as somatic mutations specific to the tumor tissue; the other variants were discarded. We achieved near-complete elimination of variants present in control samples, and complete elimination of variants not present in tumor samples. The combined application of NOIR-SS and the CV78 filter is likely to successfully identify somatic mutations specific to ctDNA. Based on the evidence presented here, we support conducting a larger study for confirmation.
|
study
| 100.0 |
One of the benefits of using ctDNA as a biomarker is that it allows tracking of new mutations potentially involved in disease progression and/or drug resistance in metastatic lesions . Such mutations may not be in the COSMIC database because of the difficulty in sampling metastatic lesions. However, it is important to note that our approach is diagnostic, and therefore is not appropriate for the research described in this paragraph.
|
review
| 93.0 |
The CV78 filter was calibrated to remove all non-specific variants. It removed all low-frequency variants, irrespective of whether they were a genuine mutation or an error. As a result, a few of the somatic mutations detected in tumor tissues were also removed by the CV78 filter. Thus, the current filter thresholds might be too stringent, and there is some room for improvement. A different approach for evaluating whether a variant is a mutation or an error would involve referencing normal DNA sequences deposited in in-house databases [5, 23]. The integrated digital error suppression (iDES) technique includes such a bioinformatic step . Since iDES is not based on previous knowledge of tumor-specific mutations, it is suitable for exploratory studies. Although the iDES identifies errors due to DNA damage to some extent, it by itself, cannot remove mutations present in normal cells.
|
study
| 99.94 |
Most techniques, including NOIR-SS, cannot discriminate true mutations from variants introduced by DNA damage. In contrast, duplex sequencing can discriminate variants present in both strands (mutations) of the DNA and those present in only a single strand (DNA damage). Therefore, comprehensive analysis of cfDNA from patients with cancer using duplex sequencing would be highly beneficial for understanding the origin of variants. However, duplex sequencing is not suitable for diagnostic use, because it requires a vast amount of DNA . Generally, it is more important to discriminate mutations occurring in the tumor tissue from mutations occurring in normal tissue, than it is to discriminate between genuine mutations and DNA damage.
|
review
| 99.2 |
This study demonstrated the benefit of using NOIR-SS and the CV78 filter for the detection of somatic mutations specific to ctDNA. However, the sensitivity of the assay does not suffice for early detection. We extracted plasma DNA from 1 mL whole blood samples for each sequencing reaction; therefore, the sample volume could be increased. Increasing the yield of plasma DNA may be especially beneficial for NOIR-SS, as the sensitivity of detection increases as the number of sequenced molecules increases. As pointed out previously , the low levels of ctDNA prevent early diagnosis of cancer. We should continue to improve our assay technique to overcome this limitation and to detect all molecules present in plasma.
|
study
| 100.0 |
The NISTmAb Reference Material (RM) 8671 is intended as a product-agnostic control to support biopharmaceutical innovation, and was therefore developed under a quality system of similar rigor to marketed biotechnology products. Quality of biotechnology products is defined as the suitability of the drug substance or drug product for its intended use . This term includes product quality attributes such as quantity, identity, biological activity, and purity. Appropriate specification setting to ensure quality for biotechnology products must be underpinned by rigorous characterization of physicochemical, biophysical, and immunochemical properties . The most immediate utility of the NISTmAb is for physicochemical/biophysical analysis technology development, method control, and harmonization of best practices. An expansive and continuously growing historical characterization dataset has been compiled as a foundation for these intended uses [3–8], a subset of which has been refined and qualified for quality monitoring [9–12], and used herein for quality attribute value assignment as part of the NISTmAb lifecycle management plan.
|
review
| 73.06 |
An RM must be established to be fit for its intended use in measurement or in examination of nominal properties, sufficiently homogeneous and stable with respect to specified properties, and available long-term with consistent product quality attributes to serve as a sustainable industry standard. Property values may be assigned to a given RM through a variety of measurement modes, the results of which influence the value designation (certified, reference, or informational as described in) [10, 13]. A series of method-specific protocols, each of which has been recognized by the community as common measurement methods for mAbs, were qualified to be suitable for NISTmAb measurement in the preceding papers of this series using the in-house NISTmAb primary sample (PS 8670) [9–12]. The physicochemical property values were then assigned to each individual lot of RM 8671 using the qualified methods and a stratified sampling and analysis plan, thereby allowing simultaneous evaluation of intra-lot homogeneity. The following discussion also shows inter-lot homogeneity with respect to each property value through analysis of three lots of RM 8671. The consistent performance of each lot provides confidence that the designated process and lifecycle management plan will produce consistent product quality attributes for current and future lots of RM 8671.
|
study
| 99.94 |
The NISTmAb RM 8671 is intended to be a widely available metric used to evaluate current capabilities and compare emerging technologies to define analytical and biophysical capabilities. Each individual lot of RM 8671, shown herein to demonstrate minimal variation in product quality attributes, is accompanied by a lot-specific Report of Investigation that describes the properties of the RM and its intended use. The current publication series is a thorough expansion on the RM 8671 Report of Investigation, and affords an opportunity to report more detailed procedures and results obtained from the value assignment exercise. This comprehensive report is a necessity to demonstrate RM quality, fully describe current state-of-the-art measurement capabilities, and serves as a means to continually evaluate best practices, promote innovative approaches, and inform regulatory paradigms as technology advances.
|
other
| 99.75 |
Vialing of NISTmAb primary sample (PS) 8670 and RM 8671 (lots 14HB-D-001, 14HB-D-002, and 14HB-D-003) are described in the first publication of this series . Samples from each RM 8671 lot for homogeneity and value assignment, thaw/freeze (T/F) stability, and accelerated stability analysis were reserved as indicated in the Electronic Supplementary Material (ESM).
|
study
| 53.2 |
To establish homogeneity and assign reference values, ten vials were analyzed from each of lot 14HB-D-001, 14HB-D-002, and 14HB-D-003 for a total of thirty vials (no intra-vial replicates). Thaw/freeze stability with respect to concentration was assessed using vials from lot 14HB-D-001 that had undergone 1 or 5 thaw/freeze cycles at either −80 °C or −20 °C, for a total of four (4) vials. The zero time point and three vials from the 1, 7, and 28 day time points were evaluated from each accelerated stability temperature (4 °C, room temperature, and 40 °C).
|
study
| 100.0 |
Reference value assignment was performed using a nominal 0.5 mm path length cuvette from Starna Cells, Inc. (Atascadero, CA) and constructed from Spectrosil fused silica. Primary decadic attenuance measurements, which is analogous to absorbance except without light scatter correction, of the mAb were measured using the NIST Material Measurement Laboratory (MML) Transfer Spectrophotometer (Cary 6000i spectrometer (Agilent)). Traceability is to the decadic logarithm of the derived International System of Units (SI unit) of regular spectral transmittance (expressed as decadic attenuance) via the national reference instrument for optical absorbance (HAS-2) [14, 15]. Traceability is achieved using transfer standards SRMs 2034 Holmium Oxide Solution Wavelength Standard (240 nm to 650 nm); 930, 1930 and 2930 Glass Filters for Spectrophotometry; and 2031 Metal-on Fused--Silica Neutral Density Filters (250 nm to 635 nm). Calibration of the wavelength scale was done using SRM 2034. The Cary instrument was allowed to warm up for 30 min before measurements were taken to stabilize the UV lamp. The instrument was operated in double beam mode with a decreased slit height, temperature of 22 °C, and a spectral bandwidth of 0.8 nm, which are the typical operation conditions used to match the HAS II reference spectrophotometer. The decadic attenuance spectra were measured from 240 nm to 340 nm, at a scan rate of 20 nm/min and wavelength interval of 0.5 nm.
|
study
| 100.0 |
Decadic attenuance measurements at 280 nm (D280) of the samples were made using the same cuvette as the blank (formulation buffer prepared as indicated in ESM). Note that the NISTmAb sample was measured directly without further dilution or other preparation steps. The Blank D280 was subtracted from each RM sample D280 to provide a Corrected D280 (Dcorr). Concentration of the NISTmAb was determined utilizing a theoretical extinction coefficient (ε) of 1.42 (mL mg−1 cm−1) . The equation used for calculation of the concentration (C) was1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ C=\frac{D_{corr}}{\varepsilon \bullet b} $$\end{document}C=Dcorrε∙bwith a path length (b) of 0.05092 cm.
|
study
| 100.0 |
Capillary zone electrophoresis (CZE), size exclusion chromatography (SEC), reducing sodium dodecyl sulfate capillary electrophoresis (rCE-SDS), and non-reducing sodium dodecyl sulfate capillary electrophoresis (nrCE-SDS) were performed according to the qualified methods as described in publications 2 and 3 of this series [11, 12]. To establish homogeneity and assign reference values, one 150 μL vial fraction (prepared as described in the ESM) was analyzed from three racks (racks 1, 50, 90) of each lot of RM 8671 (n = 3 vials per lot). The standard protocol for each lot was to perform triplicate repeatability on one vial and individual measurements on two additional vials for physicochemical value assignment. In some cases, deviations from this sampling sequence were required to allow analysis of all three lots within 24 h as described in ESM. Thaw/Freeze stability samples were analyzed from lot 14HB-D-001 vials that had undergone 1 or 5 thaw/freeze cycles at either −80 °C or −20 °C, for a total of 4 samples. Accelerated stability samples were analyzed from lot 14HB-D-001 vials that had undergone incubation at 4 °C, room temperature, or 40 °C for zero, 7, or 28 days for a total of 7 samples; an additional time point of 1 day at each temperature was analyzed by SEC. Method performance was evaluated on the day of analysis by injections of the method-specific instrument quality control standard (IQ) and PS 8670 as the system suitability standard that bracketed injections of RM 8671. The generic injection sequence was: Blank - IQ - PS 8670 - test samples (no more than 10) - IQ - PS 8670 – Blank, and was repeated as necessary to analyze all samples. Instrument qualification and system suitability controls were required to pass method performance criteria as outlined during method qualification [11, 12]. Data analysis for each method was performed as described in the requisite qualification paper [9, 11, 12]. Combined standard uncertainty (uc) for RM 8671 samples were calculated as described in ESM. The combined standard uncertainty is intended to represent, at the level of one standard deviation, the effect of the combined components of uncertainty including Type A measurement uncertainty and Type B components related to intermediate precision as observed during method qualification.
|
study
| 100.0 |
Flow imaging (FI) and dynamic light scattering (DLS) were performed according to the optimized methods described in publication 3 of this series . To establish homogeneity and assign informational values, six vials (two obtained from each rack numbered 1, 50, and 90) from each of lot 14HB-D-001, 14HB-D-002 and 14HB-D-003 were analyzed by FI. Three vials from each lot 14HB-D-001, 14HB-D-002 and 14HB-D-003 were used for homogeneity assessment by DLS. A total of twelve vials were evaluated from the 14HB-D-001 lot that had undergone 1 or 5 thaw/freeze cycles at either −80 °C or at −20 °C. The same number of vials containing PS 8670, stressed in an identical fashion, was also used for comparison. A total of 22 vials from the 14HB-D-001 lot were used for a stability study. This sample set contained the following: four zero time point vials, six vials stored at 4 °C for 7 days or 28 days, six vials stored at room temperature for 7 days or 28 days, and six vials stored at 40 °C for 7 days or 28 days. The formulation buffer (described in ESM) alone was also stressed under the same conditions and assessed for subvisible particle concentration. Method performance was evaluated on the day of analysis by injection of the method-specific instrument quality control standard (IQ) and PS 8670 as the system suitability standard. Data collection and analysis for each method was performed as described previously .
|
study
| 100.0 |
Peptide mapping using reversed phase ultrahigh performance liquid chromatography coupled to UV-Visible and tandem mass spectrometry detection (LC-UV-MS/MS) was used to confirm the primary amino acid sequence. LC-UV-MS/MS was performed according to the method as described in publication 4 of this series . To confirm identity, one 150 μL vial fraction (prepared as described in ESM) was analyzed from rack 1 of each lot of RM 8671 (14HB-D-001, 14HB-D-002, 14HB-D-003). Method performance was evaluated on the day of analysis by parallel sample preparation and analysis of PS 8670. Data collection and analysis for each method was performed as described previously .
|
study
| 100.0 |
Concentration of protein material is often determined using UV-Visible spectrophotometry wherein the measured absorbance is assumed to be equivalent to the decadic attenuance. The decadic attenuance, D, is computed as the negative logarithm (base 10) of the transmittance, and is analogous to absorbance except for the inclusion of scattering and luminescence effects upon the radiant power exiting the sample . Concentrations reported herein are based on decadic attenuance at 280 nm (D280) with no scatter or luminescence correction. Although the use of decadic attenuance results in an “apparent concentration,” it is most reflective of the experiments commonly performed by the end user (e.g. no scatter or luminescence correction). It should be noted, however, that the D320 has been reported as a means to correct for scattering at 280 nm . Proteins absorb little to no light at 320 nm, and therefore the D320 represents scattered rather than absorbed light. Correction at 280 nm is performed via the Rayleigh scattering equation (intensity of scattered light is proportional to λ−4). This correction becomes increasingly important under formulation conditions inducing colloidal instability. In the current study, D320 was determined to be 0.00222, 0.00287, and 0.00184 (14HB-D-001, 14HB-D-002, 14HB-D-003, respectively) absorbance units which represents a small contribution and is in agreement with the low level of particulates observed using flow imaging as discussed below.
|
study
| 100.0 |
Representative spectra for the Formulation Buffer (∙∙∙∙∙∙) and NISTmAb RM 8671 (∙ − ∙ − ∙ − ∙) can be seen in Fig. 1. Figure 1 also depicts a representative blank-subtracted (corrected) NISTmAb spectrum (───). This spectrum displays decadic attenuance characteristic of a protein, with an apex of absorption at ≈280 nm.Fig. 1Representative spectra obtained for the Formulation Buffer blank (∙∙∙∙∙∙), NISTmAb RM 8671 14HB-D-002 (∙ − ∙ − ∙ − ∙), and the blank-subtracted (corrected) NISTmAb spectrum (───)
|
study
| 100.0 |
The corrected decadic attenuance at 280 nm (Dcorr) for each sample was calculated by subtracting the mean D280 blank measurement from each sample D280 measurement. Intra-vial measurement repeatability was evaluated by performing triplicate analyses from the same vial of lot 14HB-D-001 and resulted in a Coefficient of Variation (CV) of 0.065% (based on the standard deviation of the measured decadic attenuance, SDD). The inter-vial homogeneity (n = 10 vials per lot) resulted in a CV of 0.090%, 0.176%, and 0.137% (based on SDD for lots D-001, D-002, and D-003 respectively); only slightly larger than the intra-vial precision. There was no apparent trend in the data with respect to vial/rack position within a lot or when plotted against the sequence in which the samples were prepared. Similar results were found for each of the three lots, indicating the vial filling process was homogeneous across all racks from an individual lot.
|
study
| 100.0 |
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