Allogeneic chimeric antigen receptor-T cells with CRISPR-disrupted programmed death-1 checkpoint exhibit enhanced functional fitness.

BACKGROUND AIMS: Therapeutic disruption of immune checkpoints has significantly advanced the armamentarium of approaches for treating cancer. The prominent role of the programmed death-1 (PD-1)/programmed death ligand-1 axis for downregulating T cell function offers a tractable strategy for enhancing the disease-modifying impact of CAR-T cell therapy. METHODS: To address checkpoint interference, primary human T cells were genome edited with a next-generation CRISPR-based platform (Cas9 chRDNA) by knockout of the PDCD1 gene encoding the PD-1 receptor. Site-specific insertion of a chimeric antigen receptor specific for CD19 into the T cell receptor alpha constant locus was implemented to drive cytotoxic activity. RESULTS: These allogeneic CAR-T cells (CB-010) promoted longer survival of mice in a well-established orthotopic tumor xenograft model of a B cell malignancy compared with identically engineered CAR-T cells without a PDCD1 knockout. The persistence kinetics of CB-010 cells in hematologic tissues versus CAR-T cells without PDCD1 disruption were similar, suggesting the robust initial debulking of established tumor xenografts was due to enhanced functional fitness. By single-cell RNA-Seq analyses, CB-010 cells, when compared with identically engineered CAR-T cells without a PDCD1 knockout, exhibited fewer Treg cells, lower exhaustion phenotypes and reduced dysfunction signatures and had higher activation, glycolytic and oxidative phosphorylation signatures. Further, an enhancement of mitochondrial metabolic fitness was observed, including increased respiratory capacity, a hallmark of less differentiated T cells. CONCLUSIONS: Genomic PD-1 checkpoint disruption in the context of allogeneic CAR-T cell therapy may provide a compelling option for treating B lymphoid malignancies.

Label-free DNA quantification using isothermal amplification on an exposed core optical fiber microfluidic platform.

Isothermal amplification technology has triggered a surge in research due to its compatibility with small and portable equipment, simplicity, and high efficiency, especially in light of the COVID-19 pandemic where reliable widescale testing is critical to outbreak management. In this paper, a label-free isothermal deoxyribonucleic acid (DNA) amplification method based on refractive index (RI) quantification is proposed and demonstrated for the first time by combining optical fiber sensing, microfluidics, and isothermal amplification. A highly RI-sensitive Mach-Zehnder (MZ) interference is formed by splicing a short length of an exposed-core fiber between two lengths of a single-mode fiber while the microfluidic liquid channel on the exposed side of the ECF is filled with target DNA and the amplification solution. Real-time quantitative measurement of the target DNA is then realized by monitoring the change in RI of the solution during the isothermal DNA amplification process. The experimental results show that the platform successfully realizes real-time label-free monitoring of isothermal amplification of 0.16 aM DNA samples. This method is a breakthrough for applications in the fields of DNA detection and quantification where simple operation, rapid detection, portability, small size, high selectivity, and high sensitivity are required.

Prevalence and clinical associations of mitral and aortic regurgitation in patients with aortic stenosis.

BACKGROUND: Most guidelines directing clinicians to manage valve disease are directed at single valve lesions. Limited data exists to direct our understanding of how concomitant valve disease impacts the left ventricle (LV). METHODS: We identified 2817 patients with aortic stenosis (AS) from the echocardiography laboratory database between September 2012 and June 2018 who had a LV ejection fraction (EF) ≥50%. LV mass, LV mass index, LV systolic pressure (systolic blood pressure + peak aortic gradient). Covariates were collected from the electronic medical record. Multi-variate analysis of covariance was used to generate adjusted comparisons. RESULTS: Our population was 66% female, 17% African-American with a mean age of 65 years. Of note, 7.3% were noted to have significant (moderate/severe) aortic regurgitation (AR), and 11% had significant (moderate/severe) mitral regurgitation (MR). Adjusting for covariates at different levels, significant MR had a much stronger association with heart failure compared to those with significant AR (p < .001 vs. p = .313, respectively) at all levels of adjustment. Both significant mitral and AR exhibited an association with increasing left ventricular mass, even with adjustment for baseline demographics and clinical features (p < .001 vs. p = .007, respectively). CONCLUSION: In patients with AS, 16% also experience at least moderate MR or AR. Further, significant MR has a stronger association with heart failure than significant AR, even though both increase left ventricular mass. Those with moderate AS and significant MR or AR experience similar or higher levels of heart failure compared to severe AS without regurgitation. Mixed valve disease merits further studies to direct longitudinal management.

Machine learning for sensing with a multimode exposed core fiber specklegram sensor.

Fiber specklegram sensors (FSSs) traditionally use statistical methods to analyze specklegrams obtained from fibers for sensing purposes, but can suffer from limitations such as vulnerability to noise and lack of dynamic range. In this paper we demonstrate that deep learning improves the analysis of specklegrams for sensing, which we show here for both air temperature and water immersion length measurements. Two deep neural networks (DNNs); a convolutional neural network and a multi-layer perceptron network, are used and compared to a traditional correlation technique on data obtained from a multimode fiber exposed-core fiber. The ability for the DNNs to be trained against a random noise source such as specklegram translations is also demonstrated.

Temperature compensated fiber optic magnetic sensor based on the combination interference principle.

In this paper, a highly sensitive temperature compensated fiber optic magnetic field sensor by Sagnac and Mach-Zehnder combination interference (SMZI) is proposed and verified. The sensing structure relies on microstructured exposed core fiber (ECF) filled with ethanol and magnetic fluid (MF). The refractive index of MF and ethanol is affected by the magnetic field and temperature (MFT). SMZI is based on the multimode and birefringence characteristics of ECF. The measurement principle is that the spectra of Sagnac interference and Mach-Zehnder interference have respective sensitivities to the MFT. The magnetic sensitivity can reach 1.17 nm/mT, and the temperature sensitivity is up to -1.93 nm/°C. At the same time, the sensor has good repeatability and low detection limits of 0.41 mT and 0.25°C, respectively. It not only solves the cross-influence of temperature but also makes the spectral analysis more intuitive. The sensor has a broad development prospect in the application of MFT detection.

In Situ Temperature-Compensated DNA Hybridization Detection Using a Dual-Channel Optical Fiber Sensor.

A multifunction, high-sensitivity, and temperature-compensated optical fiber DNA hybridization sensor combining surface plasmon resonance (SPR) and Mach-Zehnder interference (MZI) has been designed and implemented. We demonstrate, for the first time to our knowledge, the dual-parameter measurement of temperature and refractive index (RI) by simultaneously using SPR and MZI in a simple single-mode fiber (SMF)-no-core fiber (NCF)-SMF structure. The experimental results show RI sensitivities of 930 and 1899 nm/RIU and temperature sensitivities of 0.4 and -1.4 nm/°C for the MZI and SPR, respectively. We demonstrate a sensitivity matrix used to simultaneously detect both parameters, solving the problem of temperature interference of RI variation-based biosensors. In addition, the sensor can also distinguish biological binding events by detecting the localized RI changes at the fiber's surface. We realize label-free sensing of DNA hybridization detection by immobilizing probe DNA (pDNA) onto the fiber as the probe to capture complementary DNA (cDNA). The experimental results show that the sensor can qualitatively detect cDNA after temperature compensation, and the limit of detection (LOD) of the sensor reaches 80 nM. The proposed sensor has advantages of high sensitivity, real time, low cost, temperature compensation, and low detection limit and is suitable for in situ monitoring, high-precision sensing of DNA molecules, and other related fields, such as gene diagnosis, kinship judgment, environmental monitoring, and so on.

Whispering gallery mode excitation using exposed-core fiber.

Whispering gallery modes (WGMs) in micro-resonators are of interest due to their high Q-factors. Ultra-thin fiber tapers are widely deployed to couple light into micro-resonators but achieving stable and practical coupling for out-of-lab use remains challenging. Here, a new WGM coupling scheme using an exposed-core silica fiber (ECF) is proposed, which overcomes the challenge of using fragile fiber tapers. Microspheres are deposited onto the exposed channel for excitation via the evanescent field of the fiber's guided modes. The outer jacket of the ECF partially encapsulates the microspheres, protecting them from external physical disturbance. By varying the mode launching conditions in this few-mode ECF, in combination with a Fano resonance effect, we demonstrate a high degree of tunability in the reflection spectrum. Furthermore, we show multi-particle WGM excitation, which could be controlled to occur either simultaneously or separately through controlling the ECF mode launching conditions. This work can bring value towards applications such as optical switches and modulators, multiplexed/distributed biosensing, and multi-point lasing, integrated in a single optical fiber device that avoids fiber post-processing.

Evaluation of the durability of long-lasting insecticidal nets in Guatemala.

BACKGROUND: Insecticide-treated bed nets (ITNs) are widely used for the prevention and control of malaria. In Guatemala, since 2006, ITNs have been distributed free of charge in the highest risk malaria-endemic areas and constitute one of the primary vector control measures in the country. Despite relying on ITNs for almost 15 years, there is a lack of data to inform the timely replacement of ITNs whose effectiveness becomes diminished by routine use. METHODS: The survivorship, physical integrity, insecticide content and bio-efficacy of ITNs were assessed through cross-sectional surveys conducted at 18, 24 and 32 months after a 2012 distribution of PermaNet® 2.0 in a malaria focus in Guatemala. A working definition of 'LLIN providing adequate protection' was developed based on the combination of the previous parameters and usage of the net. A total of 988 ITNs were analysed (290 at 18 months, 349 at 24 months and 349 at 32 months). RESULTS: The functional survivorship of bed nets decreased over time, from 92% at 18 months, to 81% at 24 months and 69% at 32 months. Independent of the time of the survey, less than 80% of the bed nets that were still present in the household were reported to have been used the night before. The proportion of bed nets categorized as "in good condition" per World Health Organization (WHO) guidelines of the total hole surface area, diminished from 77% to 18 months to 58% at 32 months. The portion of ITNs with deltamethrin concentration less than 10 mg/m2 increased over time. Among the bed nets for which bioassays were conducted, the percentage that met WHO criteria for efficacy dropped from 90% to 18 months to 52% at 32 months. The proportion of long-lasting insecticidal nets (LLINs) providing adequate protection was 38% at 24 months and 21% at 32 months. CONCLUSIONS: At 32 months, only one in five of the LLINs distributed in the campaign provided adequate protection in terms of survivorship, physical integrity, bio-efficacy and usage. Efforts to encourage the community to retain, use, and properly care for the LLINs may improve their impact. Durability assessments should be included in future campaigns.

Evaluation of the durability and use of long-lasting insecticidal nets in Nicaragua.

BACKGROUND: Vector control for malaria prevention relies most often on the use of insecticide-treated bed net (ITNs) and indoor residual spraying. Little is known about the longevity of long-lasting insecticidal nets (LLINs) in the Americas. The physical integrity and insecticide retention of LLINs over time were monitored after a bed net distribution campaign to assess community practices around LLIN care and use in Waspam, northeastern Nicaragua. METHODS: At least 30 nets were collected at 6, 12, 24, and 36 months post distribution. Physical integrity was measured by counting holes and classifying nets into categories (good, damaged, and too torn) depending on a proportionate hole index (pHI). Insecticide bioefficacy was assessed using cone bioassays, and insecticide content measured using a cyanopyrethroid field test (CFT). RESULTS: At 6 months, 87.3 % of LLINs were in good physical condition, while by 36 months this decreased to 20.6 %, with 38.2 % considered 'too torn.' The median pHI increased from 7 at the 6-month time point to 480.5 by 36 months. After 36 months of use, median mortality in cone bioassays was 2 % (range: 0-6 %) compared to 16 % (range: 2-70 %) at 6 months. There was a decrease in the level of deltamethrin detected on the surface of the LLINs with 100 % of tested LLINs tested at 12 months and 24 months crossing the threshold for being considered a failed net by CFT. CONCLUSIONS: This first comprehensive analysis of LLIN durability in Central America revealed rapid loss of chemical bioefficacy and progressive physical damage over a 36-month period. Use of these findings to guide future LLIN interventions in malaria elimination settings in Nicaragua, and potentially elsewhere in the Americas, could help optimize the successful implementation of vector control strategies.

Single-peak fiber Bragg gratings in suspended-core optical fibers.

Femtosecond laser inscribed fiber Bragg gratings in pure-silica suspended-core optical fibers have previously been demonstrated as a promising platform for high temperature sensing. However, the density of gratings that could be written on a single fiber was limited by undesired reflections associated with higher order modes in these high numerical aperture fibers. This resulted in a complex, broadband reflection spectrum with limited multiplexing capability. In this work we utilize modifications to the fine structure of the suspended core optical fibers to fine tune the relative confinement loss of the optical fiber modes, thus reducing the contribution from such higher order modes. The effects of these changes on mode propagation are modeled, giving a range of fibers with different confinement loss properties which can be tailored to the specific length scale of a desired application. We achieve single-peak reflections from individual fiber Bragg gratings, significantly improving performance for multipoint sensing and demonstrate this technique by writing 20 gratings onto a single fiber.

Multimode exposed core fiber specklegram sensor.

The inception of photonic crystal fibers (PCFs) allowed for unprecedented tailoring of waveguide properties for specialty sensing probes. Exposed core microstructured fibers (ECFs) represent a natural evolution of the PCF design for practical liquid and gas sensing. Until now, to the best of our knowledge, only single-mode or few-modes ECFs have been explored. In this Letter, we demonstrate a highly multimode ECF with a lateral access that extends throughout the whole length of the fiber. The ECF is operated as a fiber specklegram sensor for assessing properties of fluids and interrogated using a simple and low-cost setup. The probe exhibits a refractive index resolution and sensitivity of at least 4.6×10-4 refractive index units (RIUs) and -10.97RIU-1, respectively. A maximum temperature resolution up to 0.017°C with a -0.20∘C-1 temperature sensitivity over the 23°C-28°C range and a liquid level sensing resolution up to 0.12 mm with -0.015mm-1 sensitivity over the 0.0-50.0 mm bathed the length range in water.

Attrition, physical integrity and insecticidal activity of long-lasting insecticidal nets in sub-Saharan Africa and modelling of their impact on vectorial capacity.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the primary malaria prevention and control intervention in many parts of sub-Saharan Africa. While LLINs are expected to last at least 3 years under normal use conditions, they can lose effectiveness because they fall out of use, are discarded, repurposed, physically damaged, or lose insecticidal activity. The contributions of these different interrelated factors to durability of nets and their protection against malaria have been unclear. METHODS: Starting in 2009, LLIN durability studies were conducted in seven countries in Africa over 5 years. WHO-recommended measures of attrition, LLIN use, insecticidal activity, and physical integrity were recorded for eight different net brands. These data were combined with analyses of experimental hut data on feeding inhibition and killing effects of LLINs on both susceptible and pyrethroid resistant malaria vectors to estimate the protection against malaria transmission-in terms of vectorial capacity (VC)-provided by each net cohort over time. Impact on VC was then compared in hypothetical scenarios where one durability outcome measure was set at the best possible level while keeping the others at the observed levels. RESULTS: There was more variability in decay of protection over time by country than by net brand for three measures of durability (ratios of variance components 4.6, 4.4, and 1.8 times for LLIN survival, use, and integrity, respectively). In some countries, LLIN attrition was slow, but use declined rapidly. Non-use of LLINs generally had more effect on LLIN impact on VC than did attrition, hole formation, or insecticide loss. CONCLUSIONS: There is much more variation in LLIN durability among countries than among net brands. Low levels of use may have a larger impact on effectiveness than does variation in attrition or LLIN degradation. The estimated entomological effects of chemical decay are relatively small, with physical decay probably more important as a driver of attrition and non-use than as a direct cause of loss of effect. Efforts to maximize LLIN impact in operational settings should focus on increasing LLIN usage, including through improvements in LLIN physical integrity. Further research is needed to understand household decisions related to LLIN use, including the influence of net durability and the presence of other nets in the household.

All-fiber all-optical quantitative polymerase chain reaction (qPCR).

Quantitative polymerase chain reaction (qPCR), the real-time amplification and measurement of a targeted DNA molecule, has revolutionized the biological sciences and is routinely applied in areas such as medical diagnostics, forensics, and agriculture. Despite widescale use of qPCR technology in the lab, the availability of low-cost and high-speed portable systems remains one of the barriers to routine in-field implementation. Here we propose and demonstrate a potential solution using a photonics-based qPCR system. By using an all-optical approach, we achieve ultra-fast temperature response with real-time temperature feedback using nanoliter scale reaction volumes. The system uses a microcavity to act as a nanoliter scale reaction vessel with a laser-driven and optically monitored temperature cycling system for ultrafast thermal cycling and incorporates an all-fiber fluorescence excitation/detection system to achieve real-time, high sensitivity fluorescence monitoring of the qPCR process. Further, we demonstrate the potential of the system to operate as a label-free qPCR system through direct optical measurement of the sample refractive index. Due to advantages in portability and fabrication simplicity, we anticipate that this platform technology will offer a new strategy for fundamental techniques in biochemistry applications, such as point-of-care and remote diagnostics.

Quantum noise limited nanoparticle detection with exposed-core fiber.

Label-free biosensors are important tools for clinical diagnostics and for studying biology at the single molecule level. The development of optical label-free sensors has allowed extreme sensitivity but can expose the biological sample to photodamage. Moreover, the fragility and complexity of these sensors can be prohibitive to applications. To overcome these problems, we develop a quantum noise limited exposed-core fiber sensor providing robust platform for label-free biosensing with a natural path toward microfluidic integration. We demonstrate the detection of single nanoparticles down to 25 nm in radius with optical intensities beneath known biophysical damage thresholds.

Tunable multi-wavelength third-harmonic generation using exposed-core microstructured optical fiber.

We demonstrate that exposed-core microstructured optical fibers offer multiple degrees of freedom for tailoring third-harmonic generation through the core diameter, input polarization, and nanofilm deposition. Varying these parameters allows control of the phase-matching position between an infrared pump wavelength and the generated visible wavelengths. In this Letter, we show how increasing the core diameter over previous experiments (2.57 µm compared to 1.85 µm) allows the generation of multiple wavelengths, which can be further controlled by rotating the input pump polarization and the deposition of dielectric nanofilms. This can lead to highly tailorable light sources for applications such as spectroscopy or nonlinear microscopy.

Soft-glass imaging microstructured optical fibers.

We demonstrate the fabrication of multi-core (imaging) microstructured optical fiber via soft-glass preform extrusion through a 3D printed titanium die. The combination of extrusion through 3D printed dies and structured element (capillary) stacking allows for unprecedented control of the optical fiber geometry. We have exploited this to demonstrate a 100 pixel rectangular array imaging microstructured fiber. Due to the high refractive index of the glass used (n = 1.62), such a fiber can theoretically have a pixel pitch as small as 1.8 µm. This opens opportunities for ultra-small, high-resolution imaging fibers fabricated from diverse glass types.

Nanofilm-induced spectral tuning of third harmonic generation.

Intermodal third-harmonic generation using waveguides is an effective frequency conversion process due to the combination of long interaction lengths and strong modal confinement. Here we introduce the concept of tuning the third harmonic phase-matching condition via the use of dielectric nanofilms located on an open waveguide core. We experimentally demonstrate that tantalum oxide nanofilms coated onto the core of an exposed core fiber allow tuning the third harmonic wavelength over 30 nm, as confirmed by qualitative simulations. Due to its generic character, the presented tuning scheme can be applied to any form of exposed core waveguide and will find applications in fields including microscopy, biosensing, and quantum optics.

Cell cycle-dependent adaptor complex for ClpXP-mediated proteolysis directly integrates phosphorylation and second messenger signals.

The cell-division cycle of Caulobacter crescentus depends on periodic activation and deactivation of the essential response regulator CtrA. Although CtrA is critical for transcription during some parts of the cell cycle, its activity must be eliminated before chromosome replication because CtrA also blocks the initiation of DNA replication. CtrA activity is down-regulated both by dephosphorylation and by proteolysis, mediated by the ubiquitous ATP-dependent protease ClpXP. Here we demonstrate that proteins needed for rapid CtrA proteolysis in vivo form a phosphorylation-dependent and cyclic diguanylate (cdG)-dependent adaptor complex that accelerates CtrA degradation in vitro by ClpXP. The adaptor complex includes CpdR, a single-domain response regulator; PopA, a cdG-binding protein; and RcdA, a protein whose activity cannot be predicted. When CpdR is unphosphorylated and when PopA is bound to cdG, they work together with RcdA in an all-or-none manner to reduce the Km of CtrA proteolysis 10-fold. We further identified a set of amino acids in the receiver domain of CtrA that modulate its adaptor-mediated degradation in vitro and in vivo. Complex formation between PopA and CtrA depends on these amino acids, which reside on alpha-helix 1 of the CtrA receiver domain, and on cdG binding by PopA. These results reveal that each accessory factor plays an essential biochemical role in the regulated proteolysis of CtrA and demonstrate, to our knowledge, the first example of a multiprotein, cdG-dependent proteolytic adaptor.

Direct core structuring of microstructured optical fibers using focused ion beam milling.

We demonstrate the use of focused ion beam milling to machine optical structures directly into the core of microstructured optical fibers. The particular fiber used was exposed-core microstructured optical fiber, which allowed direct access to the optically guiding core. Two different designs of Fabry-Perot cavity were fabricated and optically characterized. The first cavity was formed by completely removing a section of the fiber core, while the second cavity consisted of a shallow slot milled into the core, leaving the majority of the core intact. This work highlights the possibility of machining complex optical devices directly onto the core of microstructured optical fibers using focused ion beam milling for applications including environmental, chemical, and biological sensing.

Interferometric high temperature sensor using suspended-core optical fibers.

We propose and experimentally demonstrate, for the first time to our knowledge, high temperature fiber sensing using the multimode interference effect within a suspended-core microstructured optical fiber (SCF). Interference fringes were found to red-shift as the temperature increased and vice versa. Temperature sensing up to 1100°C was performed by measuring the wavelength shifts of the fringes after fast Fourier transform (FFT) filtering of the spectra. In addition, phase monitoring at the dominant spatial frequency in the Fourier spectrum was used as an interrogation method to monitor various temperature-change scenarios over a period of 80 hours. Our proposed high temperature fiber sensor is simple, cost-effective, and can operate at temperatures beyond 1000°C.