Design, fabrication, and characterization of kinetic-inductive force sensors for scanning probe applications.

We describe a transducer for low-temperature atomic force microscopy based on electromechanical coupling due to a strain-dependent kinetic inductance of a superconducting nanowire. The force sensor is a bending triangular plate (cantilever) whose deflection is measured via a shift in the resonant frequency of a high-Q superconducting microwave resonator at 4.5 GHz. We present design simulations including mechanical finite-element modeling of surface strain and electromagnetic simulations of meandering nanowires with large kinetic inductance. We discuss a lumped-element model of the force sensor and describe the role of an additional shunt inductance for tuning the coupling to the transmission line used to measure the microwave resonance. A detailed description of our fabrication is presented, including information about the process parameters used for each layer. We also discuss the fabrication of sharp tips on the cantilever using focused electron beam-induced deposition of platinum. Finally, we present measurements that characterize the spread of mechanical resonant frequency, the temperature dependence of the microwave resonance, and the sensor's operation as an electromechanical transducer of force.

Selection and validation of reference genes for quantifying gene expression in the Gill's mealybug.

The Gill's mealybug, Ferrisia gilli Gullan, (Hemiptera: Pseudococcidae) has emerged as a major pest of pistachio in California. Because F. gilli is only relatively recently described, there are no validated reference genes to normalize the expression data from real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in this species. We selected and validated 8 commonly used reference genes (RPS8, TBP, UBQE2, RPL7, RPL5, RPL40, RPLP1, and HEL) for expression stability in F. gilli. These genes were evaluated in 5 different geographical populations of F. gilli collected from organic and conventionally grown pistachio orchards. Candidate reference genes were also evaluated in F. gilli fed with 4 plant hosts: pistachio, almond, grapes, and lima beans. The stability of candidate genes was analyzed using 4 software algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. Three genes RPS8, RPL40, and RPL7 encoding for ribosomal proteins were identified as the most stable across the treatments and thus were recommended for normalizing the qRT-PCR data. These findings will support resistance monitoring, molecular toxicology, and functional genomics research in F. gilli.

Multipartite Entanglement in a Microwave Frequency Comb.

Significant progress has been made with multipartite entanglement of discrete qubits, but continuous variable systems may provide a more scalable path toward entanglement of large ensembles. We demonstrate multipartite entanglement in a microwave frequency comb generated by a Josephson parametric amplifier subject to a bichromatic pump. We find 64 correlated modes in the transmission line using a multifrequency digital signal processing platform. Full inseparability is verified in a subset of seven modes. Our method can be expanded to generate even more entangled modes in the near future.

Baseline Susceptibility and Evidence of Resistance to Acetamiprid in Gill's Mealybug, Ferrisia gilli Gullan (Hemiptera: Pseudococcidae).

Gill's mealybug, Ferrisia gilli (Gullan) (Hemiptera: Pseudococcidae), is a major pest of pistachio in California. Insecticide treatment is the primary control method and acetamiprid is widely used to control this pest. However, there have been numerous reports of control failures for F. gilli after field applications of recommended insecticides in recent years. The purpose of this study was to develop a method for routine monitoring of F. gilli susceptibility and quantify current levels of F. gilli susceptibility to acetamiprid. A leaf-dip bioassay method using lima bean leaves was established and baseline susceptibility responses of 5 field populations were determined. Lethal concentrations to kill 50% of population (LC50) for second instar nymphs at 48 h ranged from 0.367 to 2.398 µg(AI)ml-1 of acetamiprid. Similarly, lethal concentrations to kill 90% of population (LC90) for second instar nymphs at 48 h ranged from 2.887 to 10.752 µg(AI)ml-1 of acetamiprid. The F. gilli population collected from Hanford area showed up to 6.5-fold significantly decreased mortality to acetamiprid compared to other populations. The resistance identified in this study, although relatively low, indicates that there has been repeated pressure to select for acetamiprid resistance and resistance levels can further magnify if effective management steps are not taken. The baseline susceptibility established in this study can be used to investigate potential cause of recent acetamiprid failures against F. gilli. In the long-term, results of this study will support the development of resistance management strategies by monitoring shifts in the susceptibility of F. gilli populations.

Measurement and control of a superconducting quantum processor with a fully integrated radio-frequency system on a chip.

We describe a digital microwave platform called Presto, designed for measurement and control of multiple quantum bits (qubits) and based on the third-generation radio-frequency system on a chip. Presto uses direct digital synthesis to create signals up to 9 GHz on 16 synchronous output ports, while synchronously analyzing responses on 16 input ports. Presto has 16 DC-bias outputs, four inputs and four outputs for digital triggers or markers, and two continuous-wave outputs for synthesizing frequencies up to 15 GHz. Scaling to a large number of qubits is enabled through deterministic synchronization of multiple Presto units. A Python application programming interface configures a firmware for synthesis and analysis of pulses, coordinated by an event sequencer. The analysis integrates template matching (matched filtering) and low-latency (184-254 ns) feedback to enable a wide range of multi-qubit experiments. We demonstrate Presto's capabilities with experiments on a sample consisting of two superconducting qubits connected via a flux-tunable coupler. We show single-shot readout and active reset of a single qubit; randomized benchmarking of single-qubit gates showing 99.972% fidelity, limited by the coherence time of the qubit; and calibration of a two-qubit iSWAP gate.

CD44 receptor targeted nanoparticles augment immunity against tuberculosis in mice.

We describe a role of CD44-mediated signaling during host-defense against tuberculosis (TB) using a mouse model of TB and studies in M. tuberculosis (Mtb) infected human macrophage (MФ). Liposomes targeting CD44 using thioaptamers (CD44TA-LIP) were designed and tested as new vaccines to boost host immunity in TB. CD44TA-LIP enhanced killing of Mtb in human MФ, which correlated with an increased production of pro-inflammatory cytokines IL-1ß, TNF-α and IL-12. CD44TA-LIP activated MФ showed an enhanced MHC-II dependent antigen presentation to CD4 T-cells. Inhibition of cellular proliferation and cytoskeleton rearrangement pathways downstream of CD44 signaling abrogated CD44TA-LIP-induced antimicrobial effects. Blockade of inflammatory pathways also reduced antigen presentation by MФ and activation of CD4 T cells. Mtb infected MФ treated with CD44TA-LIP exhibited increased nitric oxide and HßD2 defensin peptide production. Among Mtb infected mice with increased lung and spleen loads of organisms, intranasal administration of CD44TA-LIP led to a ten-fold reduction of colony forming units of Mtb and elevated IFN-γ + CD4, effector, central and resident memory T cells. Biodistribution studies demonstrated that CD44TA-LIP preferentially accumulated in the lungs and were associated with CD11b + cells. CD44TA-LIP treated mice showed no weight loss or increased liver LDH levels. This study highlights the importance of CD44-mediated signaling in host-defense during TB and the therapeutic potential of CD44TA-LIP.

Single-cell analysis of human glioma and immune cells identifies S100A4 as an immunotherapy target.

A major rate-limiting step in developing more effective immunotherapies for GBM is our inadequate understanding of the cellular complexity and the molecular heterogeneity of immune infiltrates in gliomas. Here, we report an integrated analysis of 201,986 human glioma, immune, and other stromal cells at the single cell level. In doing so, we discover extensive spatial and molecular heterogeneity in immune infiltrates. We identify molecular signatures for nine distinct myeloid cell subtypes, of which five are independent prognostic indicators of glioma patient survival. Furthermore, we identify S100A4 as a regulator of immune suppressive T and myeloid cells in GBM and demonstrate that deleting S100a4 in non-cancer cells is sufficient to reprogram the immune landscape and significantly improve survival. This study provides insights into spatial, molecular, and functional heterogeneity of glioma and glioma-associated immune cells and demonstrates the utility of this dataset for discovering therapeutic targets for this poorly immunogenic cancer.

Pheromone Deployment Strategies for Mating Disruption of a Vineyard Mealybug.

The mealybug, Planococcus ficus (Signoret), is a primary vineyard pest in California and other grape-growing regions throughout the World. Mating disruption programs are commercially available to manage Pl. ficus, but widespread adoption has been limited, in part, by high costs compared with insecticide programs. To improve mating disruption economic effectiveness, different deployment technologies (passive, aerosol, and microencapsulated formulations) were individually examined. Adult male Pl. ficus captures in pheromone traps and categorical ratings of vine infestation or crop damage suggest that all deployment strategies lowered mealybug densities or damage. Using passive dispensers, deployment rates of 310 and 465 per ha lowered Pl. ficus crop damage similar to 615 per ha, a rate commonly used in commercial operations; reduced rates would lower product and deployment costs. Meso dispensers, containing more a.i., deployed at 35 per ha did not have a treatment impact, but a microencapsulated formulation and aerosol canisters lowered male flight captures and/or crop damage. Male mealybug flight activity was greatest from 0500-1100 hr, which coincided with temperatures >16° and <32°C. These restricted times and temperatures suggest programable dispensers might allow pheromone deployment to coincide only with flight patterns. A large field trial using passive dispensers found greater treatment separation after 3 yr of pheromone deployment. Discrepancies in results among vineyards may be related to Pl. ficus density, but combined results from all trials suggest that different deployment technologies can be used to impact Pl. ficus densities and damage, even at reduced rates, especially with continued use over multiple seasons.

Treatment Thresholds for Tetranychus pacificus (Acari: Tetranychidae) in California Almonds Based on Monitoring for the Natural Enemy Scolothrips sexmaculatus (Thysanoptera: Thripidae).

Optimization of integrated pest management for Pacific spider mite, Tetranychus pacificus McGregor, in California almonds requires that miticide applications be delayed as long as possible to promote biological control, while still occurring before exponential mite increases cause defoliation. We used data from twelve untreated almond orchards between 2006 and 2019 to determine that decisions to treat T. pacificus should be made on the first monitoring date with 1.4 mites per leaf (38.1% of leaves infested) to ensure that miticide applications are made prior to reaching the treatment threshold of 5.4 per leaf an average of 4.7 d later. Modifications to this threshold based on biological control were made using capture rates of sixspotted thrips, Scolothrips sexmaculatus Pergande, on yellow adhesive traps from 14 almond orchards between 2016 and 2019. Data showed that T. pacificus density remained unchanged when there were 0.42 S. sexmaculatus per trap per week for every one T. pacificus per leaf. For orchards where T. pacificus density has exceeded action and treatment thresholds, we developed a less accurate, but more practical, model that does not require counting mites on leaves. On average, T. pacificus density remained unchanged for seven and 14 d if there were an average of five and three S. sexmaculatus per trap per week, respectively. Growers who adopt the action and treatment thresholds presented herein have the potential to avoid unnecessary miticide use, maximize reliance on biological control, and promote the long-term sustainability of California's almond industry.

Field Biology of Scolothrips sexmaculatus (Thysanoptera: Thripidae) as a Predator of Tetranychus pacificus (Acari: Tetranychidae) in California Almonds.

Biological control of tetranychid spider mites in California almonds has predominantly been associated with predatory mites in the family Phytoseiidae. However, changes in production practices, including shifts to more sustainable chemical control programs, have resulted in a trophic shift leading to the predominance of six-spotted thrips, Scolothrips sexmaculatus Pergande, as the primary mite predator. Over a 3-yr period, we studied S. sexmaculatus biology and relationship to Tetranychus pacificus McGregor in almond orchards in the southern San Joaquin Valley of California. Scolothrips sexmaculatus had two periods of activity in response to increases in T. pacificus density, one in the spring and one in the summer. Sex ratios were consistently skewed to more than 90% females captured on yellow sticky traps, with females producing eggs from May to October. Female thrips were larger than males, and both sexes were larger from June to September when temperatures were warmer and food was more abundant. In response to spider mite outbreaks, S. sexmaculatus increased from approximately 2/trap/wk to an average of 1,272/trap/wk over a 5-wk period, representing a population doubling time of 4.0 d. Over this time, we described trends in T. pacificus and S. sexmaculatus density that are consistent with population regulation through predator-prey relationships by a specialist predator.

Management of Navel Orangeworm (Lepidoptera: Pyralidae) Using Four Commercial Mating Disruption Systems in California Almonds.

The navel orangeworm, Amyelois transitella (Walker), is the most significant pest of California almonds. Direct feeding on the kernel by the larvae causes reductions in salable crop, crop quality, and exportability. Pheromone mating disruption (MD) targeting navel orangeworm is a relatively new technique with the potential to improve management. In 2017, we used replicated ~16-ha plots to compare the efficacy of four commercial MD systems (CheckMate, Cidetrak, Isomate, and Semios) for their relative impacts on the number of navel orangeworm in monitoring traps and crop quality. From 2017 to 2018, we conducted nine direct comparison studies in 16 to 40 ha almond orchards to compare conventional pest management programs to programs incorporating pheromone MD systems. Across all studies, MD reduced male moth captures in pheromone traps by >94%. In the efficacy study, use of mating disruption led to 35% and 53% reductions in kernel damage in Nonpareil and pollinizer cultivars, respectively, and an average increase in crop value of $370 ha-1. In the direct comparison, kernel damage to Nonpareil and pollinizer cultivars was reduced by 65% and 78%, respectively, resulting in an average increase in crop value of $357 ha-1. Economic analyses showed that increases in crop returns exceeded the costs of implementing MD systems with the break-even point ranging from 0.86 to 1.06% of kernel damage. These results suggest that adding MD to an existing navel orangeworm management program is a cost-effective way to reduce damage while promoting sustainable pest management practices.

Acly promotes metabolic reprogramming and induction of IRF4 during early CD8+ T cell activation.

CD8+ T cells, a fundamental part of the adaptive immune system, employ cytotoxic responses important for targeting pathogenic bacteria, viruses, and tumor cells. During early activation, CD8+ T cells undergo many changes in metabolism and gene expression. The bridge between epigenetic and metabolic influences on gene expression and cell fate has yet to be fully understood. Here, we investigated the importance of ATP citrate lyase (Acly), an enzyme involved in both metabolism and histone acetylation, for early stages of CD8+ T cell activation. We performed polyclonal activation of murine CD8+ T cells in vitro in the presence or absence of the Acly inhibitor BMS303141. We found that inhibiting Acly during early activation results in decreased expression of early activation markers. Consistent with impaired early activation, we found that inhibition also resulted in increased uptake of fatty acids and decreased glucose uptake without changing mitochondrial ATP levels. On an epigenetic and transcriptional level, early stage Acly inhibition specifically downregulated promoter histone H3 acetylation (H3ac) and expression of the key transcription factor IRF4; however, global levels of H3ac remained similar. Most importantly, the study was able to highlight the importance of Acly in early stages of CD8+ T cell activation and histone regulation.

Evaluation of Sticky Traps for Monitoring Scolothrips sexmaculatus (Thysanoptera: Thripidae) and Stethorus punctum (Coleoptera: Coccinellidae) as Predators of Spider Mites in California Almonds.

Changes in almond production practices have caused sixspotted thrips, Scolothrips sexmaculatus Pergande, and the coccinellid beetle, Stethorus punctum LeConte, to replace phytoseiid mites as the dominant predators of spider mites in California almonds. We conducted a series of field studies to evaluate nine commercially available adhesive traps for S. sexmaculatus and S. punctum and found that the yellow strip trap was the most effective, least expensive, and easy to use. At peak population levels, individual cards routinely caught >1,000 S. sexmaculatus and >100 S. punctum trap-1 week-1. We documented that larger traps collected more S. sexmaculatus, and more S. sexmaculatus per square area, suggesting that the trap surface was attractive. We determined the number of traps needed to have 50, 70, and 90% confidence that the averages of trap captures were within 10, 30, and 50% of the population mean. Two yellow strip traps per orchard provided 90% confidence that trap captures were within 50% of the population mean if there were an average of 16.0 S. sexmaculatus trap-1 week-1 or 7.9 S. punctum trap-1 week-1. Populations required to attain the same levels of confidence using four traps per orchard were 3.9 S. sexmaculatus and 2.9 S. punctum trap-1 week-1. We conclude that use of the yellow strip trap to monitor for natural enemies, in combination with leaf samples for spider mites, has the potential to improve integrated pest management programs for spider mites, and assist future research to understand the biology and phenology of both predator species.

Datura quids at Pinwheel Cave, California, provide unambiguous confirmation of the ingestion of hallucinogens at a rock art site.

While debates have raged over the relationship between trance and rock art, unambiguous evidence of the consumption of hallucinogens has not been reported from any rock art site in the world. A painting possibly representing the flowers of Datura on the ceiling of a Californian rock art site called Pinwheel Cave was discovered alongside fibrous quids in the same ceiling. Even though Native Californians are historically documented to have used Datura to enter trance states, little evidence exists to associate it with rock art. A multianalytical approach to the rock art, the quids, and the archaeological context of this site was undertaken. Liquid chromatography-mass spectrometry (LC-MS) results found hallucinogenic alkaloids scopolamine and atropine in the quids, while scanning electron microscope analysis confirms most to be Datura wrightii Three-dimensional (3D) analyses of the quids indicate the quids were likely masticated and thus consumed in the cave under the paintings. Archaeological evidence and chronological dating shows the site was well utilized as a temporary residence for a range of activities from Late Prehistory through Colonial Periods. This indicates that Datura was ingested in the cave and that the rock painting represents the plant itself, serving to codify communal rituals involving this powerful entheogen. These results confirm the use of hallucinogens at a rock art site while calling into question previous assumptions concerning trance and rock art imagery.

Xylella fastidiosa and Glassy-Winged Sharpshooter Population Dynamics in the Southern San Joaquin Valley of California.

Xylella fastidiosa is a vector-transmitted bacterial plant pathogen that affects a wide array of perennial crops, including grapevines (Pierce's disease). In the southern San Joaquin Valley of California, epidemics of Pierce's disease of grapevine were associated with the glassy-winged sharpshooter, Homalodisca vitripennis. During the growing season, rates of X. fastidiosa spread in vineyards are affected by changes in pathogen distribution within chronically infected grapevines and by vector population dynamics. Grapevines chronically infected with X. fastidiosa rarely tested positive for the pathogen prior to July, suggesting vector acquisition of X. fastidiosa from grapevines increases as the season progresses. This hypothesis was supported by an increase in number of X. fastidiosa-positive glassy-winged sharpshooters collected from vineyards during July through September. Analysis of insecticide records indicated that vineyards in the study area were typically treated with a systemic neonicotinoid in spring of each year. As a result, abundance of glassy-winged sharpshooters was typically low in late spring and early summer, with abundance of glassy-winged sharpshooter adults increasing in late June and early July of each year. Collectively, the results suggest that late summer is a crucial time for X. fastidiosa secondary spread in vineyards in the southern San Joaquin Valley, because glassy-winged sharpshooter abundance, number of glassy-winged sharpshooters testing positive for X. fastidiosa, and grapevines with detectable pathogen populations were all greatest during this period.

Rigor and Reproducibility of Cytometry Practices for Immuno-Oncology: A multifaceted challenge.

The rapid advancement of immunotherapy strategies has created a need for technologies that can reliably and reproducibly identify rare populations, detect subtle changes in modulatory signals, and assess antigenic expression patterns that are time-sensitive. Accomplishing these tasks requires careful planning and the employment of tools that provide greater sensitivity and specificity without demanding extensive time. Flow Cytometry has earned its place as a preferred analysis platform. This technology offers a flexible path to the interrogation of protein expression patterns and detection of functional properties in cell populations of interest. Mass Cytometry is a newcomer technology that has generated significant interest in the field. By incorporating mass spectrometry analysis to the traditional principles of flow cytometry, this innovative tool promises to significantly expand the ability to detect multiple proteins on a single cell. The use of these technologies in a manner that is consistent and reproducible through multiple sample sets demands careful attention to experiment design, reagent selection, and instrumentation. Whether applying flow or mass cytometry, reaching successful, reliable results involves many factors. Sample preparation, antibody titrations, and appropriate controls are major biological considerations that impact cytometric analysis. Additionally, instrument voltages, lasers, and run quality assessments are essential for ensuring comparability and reproducibility between analyses. In this article, we aim to discuss the critical aspects that impact flow cytometry, and will touch on important considerations for mass cytometry as well. Focusing on their relevance to immunotherapy studies, we will address the importance of appropriate sample processing and will discuss how selection of suitable panels, controls, and antibodies must follow a carefully designed plan. We will also comment on how educated use of instrumentation plays a significant role in the reliability and reproducibility of results.Through this work, we hope to contribute to the effort toward establishing higher standards for rigor and reproducibility of cytometry practices by researchers, operators, and general cytometry users employing cytometry-based assays in their work. © 2019 International Society for Advancement of Cytometry.

Intermodulation spectroscopy as an alternative to pump-probe for the measurement of fast dynamics at the nanometer scale.

We present an alternative approach to pump-probe spectroscopy for measuring fast charge dynamics with an atomic force microscope (AFM). Our approach is based on coherent multifrequency lock-in measurement of the intermodulation between a mechanical drive and an optical or electrical excitation. In response to the excitation, the charge dynamics of the sample is reconstructed by fitting a theoretical model to the measured frequency spectrum of the electrostatic force near resonance of the AFM cantilever. We discuss the time resolution, which in theory is limited only by the measurement time, but in practice is of order 1 ns for standard cantilevers and imaging speeds. We verify the method with simulations and demonstrate it with a control experiment, achieving a time resolution of 30 ns in ambient conditions, limited by thermal noise.

Calibrating torsional eigenmodes of micro-cantilevers for dynamic measurement of frictional forces.

Non-invasive thermal noise calibration of both torsional and flexural eigenmodes is performed on numerous cantilevers of 10 different types. We show that for all tipless and short-tipped cantilevers, the ratio of torsional to flexural mode stiffness is given by the ratio of their resonant frequency times a constant, unique to that cantilever type. By determining this constant, we enable a calibration of the torsional eigenmode, starting from a calibration of the flexural eigenmode. Our results are well motivated from beam theory, and we verify them with finite element simulation.

Reduced Susceptibility of Homalodisca vitripennis (Hemiptera: Cicadellidae) to Commonly Applied Insecticides.

Pest management for the glassy-winged sharpshooter, Homalodisca vitripennis Germar (Hemiptera: Cicadellidae), in Kern County, California relies on the application of insecticides. These treatments have contributed to low H. vitripennis field counts since applications were initiated in 2001. However, densities have been high in recent years despite continued management, prompting efforts to evaluate the susceptibility of current populations to insecticides. H. vitripennis adults were subjected to bioassays with five commonly applied insecticides, and the results were compared to baseline toxicities determined in 2002. Two neonicotinoids, imidacloprid and thiamethoxam, were evaluated using systemic uptake bioassays. Contact toxicities of the neonicotinoid acetamiprid and pyrethroids bifenthrin and fenpropathrin were estimated using leaf dip bioassays. Dose-mortality responses were analyzed by probit analysis. For each compound, there was no significant difference in annual LC50 values determined over 2 yr. Compared to baseline toxicities, acetamiprid and bifenthrin were found to be significantly less toxic to H. vitripennis. The LC50 values of these two compounds increased sevenfold and 152-fold, respectively. Tests with the neonicotinoids revealed a trend of decreasing susceptibility levels within each season followed by reversion back to early season LC50 estimates in the following year. In addition, data showed seasonal and site variation in susceptibility to imidacloprid, possibly due to differential applications in nearby fields.