Role for Caspase-8 in the Release of IL-1ß and Active Caspase-1 from Viable Human Monocytes during Toxoplasma gondii Infection.

Monocytes are actively recruited to sites of infection and produce the potent proinflammatory cytokine IL-1ß. We previously showed that IL-1ß release during Toxoplasma gondii infection of primary human monocytes requires the NLRP3 inflammasome and caspase-1 but is independent of gasdermin D and pyroptosis. To investigate mechanisms of IL-1ß release, we generated caspase-1, -4, -5, or -8 knockout (KO) THP-1 monocytic cells. Genetic ablation of caspase-1 or -8, but not caspase-4 or -5, decreased IL-1ß release during T. gondii infection without affecting cell death. In contrast, TNF-α and IL-6 secretion were unperturbed in caspase-8 KO cells during T. gondii infection. Dual pharmacological inhibition of caspase-8 and RIPK1 in primary monocytes also decreased IL-1ß release without affecting cell viability or parasite infection. Caspase-8 was also required for the release of active caspase-1 from T. gondii-infected cells and for IL-1ß release during infection with the related apicomplexan parasite Neospora caninum. Surprisingly, caspase-8 deficiency did not impair synthesis or cleavage of pro-IL-1ß, but resulted in the retention of mature IL-1ß within cells. Generation of gasdermin E KO and ATG7 KO THP-1 cells revealed that the release of IL-1ß was not dependent on gasdermin E or ATG7. Collectively, our data indicate that during T. gondii Infection of human monocytes, caspase-8 functions in a novel gasdermin-independent mechanism controlling IL-1ß release from viable cells. This study expands on the molecular pathways that promote IL-1ß in human immune cells and provides evidence of a role for caspase-8 in the mechanism of IL-1ß release during infection.

Efficacy of antiviral therapy and host-virus interactions visualised using serial liver sampling with fine-needle aspirates.

Background & Aims: Novel therapies for chronic hepatitis B (CHB), such as RNA interference, target all viral RNAs for degradation, whereas nucleoside analogues are thought to block reverse transcription with minimal impact on viral transcripts. However, limitations in technology and sampling frequency have been obstacles to measuring actual changes in HBV transcription in the liver of patients starting therapy. Methods: We used elective liver sampling with fine-needle aspirates (FNAs) to investigate the impact of treatment on viral replication in patients with CHB. Liver FNAs were collected from patients with CHB at baseline and 12 and 24 weeks after starting tenofovir alafenamide treatment. Liver FNAs were subjected to single-cell RNA sequencing and analysed using the Viral-Track method. Results: HBV was the only viral genome detected and was enriched within hepatocytes. The 5' sequencing technology identified protein-specific HBV transcripts and showed that tenofovir alafenamide therapy specifically reduced pre-genomic RNA transcripts with little impact on HBsAg or HBx transcripts. Infected hepatocytes displayed unique gene signatures associated with an immunological response to viral infection. Conclusions: Longitudinal liver sampling, combined with single-cell RNA sequencing, captured the dynamic impact of antiviral therapy on the replication status of HBV and revealed host-pathogen interactions at the transcriptional level in infected hepatocytes. This sequencing-based approach is applicable to early-stage clinical studies, enabling mechanistic studies of immunopathology and the effect of novel therapeutic interventions. Impact and Implications: Infection-dependent transcriptional changes and the impact of antiviral therapy on viral replication can be measured in longitudinal human liver biopsies using single-cell RNA sequencing data.

Longitudinal liver sampling in patients with chronic hepatitis B starting antiviral therapy reveals hepatotoxic CD8+ T cells.

Accumulation of activated immune cells results in nonspecific hepatocyte killing in chronic hepatitis B (CHB), leading to fibrosis and cirrhosis. This study aims to understand the underlying mechanisms in humans and to define whether these are driven by widespread activation or a subpopulation of immune cells. We enrolled CHB patients with active liver damage to receive antiviral therapy and performed longitudinal liver sampling using fine-needle aspiration to investigate mechanisms of CHB pathogenesis in the human liver. Single-cell sequencing of total liver cells revealed a distinct liver-resident, polyclonal CD8+ T cell population that was enriched at baseline and displayed a highly activated immune signature during liver damage. Cytokine combinations, identified by in silico prediction of ligand-receptor interaction, induced the activated phenotype in healthy liver CD8+ T cells, resulting in nonspecific Fas ligand-mediated killing of target cells. These results define a CD8+ T cell population in the human liver that can drive pathogenesis and a key pathway involved in their function in CHB patients.

Robotic automation of droplet microfluidics.

Droplet microfluidics enables powerful analytic capabilities but often requires workflows involving macro- and microfluidic processing steps that are cumbersome to perform manually. Here, we demonstrate the automation of droplet microfluidics with commercial fluid-handling robotics. The workflows incorporate common microfluidic devices including droplet generators, mergers, and sorters and utilize the robot's native capabilities for thermal control, incubation, and plate scanning. The ability to automate microfluidic devices using commercial fluid handling will speed up the integration of these methods into biological workflows.

Single-Cell Protein Profiling by Microdroplet Barcoding and Next-Generation Sequencing.

DNA barcoding of individual cells combined with next-generation sequencing enables high-throughput parallel analysis of biomolecules at the single-cell level. Encoding protein identity with DNA barcoding of specific antibody binders achieves sequencing-based protein quantitation by converting protein signals into DNA signals. Here, we describe how to prepare DNA-barcoded antibodies and connect protein identities to cellular identities using droplet microfluidics. This approach allows for multiplex single-cell protein analysis compatible with single-cell transcriptomic and mutational profiling methods.

US Tobacco 21 is Paving the Way for a Tobacco Endgame.

The battle against tobacco usage is being fought on all fronts. On December 19, 2019, a measure to raise the minimum age to buy tobacco products to 21 from 18 was passed by the United States Congress and signed by President Donald Trump. This instated banning the sale of all tobacco products and electronic cigarettes to anyone in the US under the age of 21. This follows the raising of the age to buy tobacco in California to 21 in 2016. According to the California Tobacco Control Program: in 2016, roughly 10% of high-school students were smoking cigarettes, but by 2018, only 2%. The percentage of retailers selling tobacco to underaged youth dropped dramatically. These data show that the CA Tobacco 21 law was effective in decreasing the obtainability and usage of tobacco by youth. We expect that US Tobacco 21 will be similarly effective in reducing tobacco use by youth leading to less tobacco addiction in the US.

Unhealthy Lifestyle and Gut Dysbiosis: A Better Understanding of the Effects of Poor Diet and Nicotine on the Intestinal Microbiome.

The study of the intestinal or gut microbiome is a newer field that is rapidly gaining attention. Bidirectional communication between gut microbes and the host can impact numerous biological systems regulating immunity and metabolism to either promote or negatively impact the host's health. Habitual routines, dietary choices, socioeconomic status, education, host genetics, medical care and environmental factors can all contribute to the composition of an individual's microbiome. A key environmental factor that may cause negative outcomes is the consumption of nicotine products. The effects of nicotine on the host can be exacerbated by poor dietary choices and together can impact the composition of the gut microbiota to promote the development of metabolic disease including non-alcoholic fatty liver disease. This review explores the contribution of nicotine, poor dietary choices and other unhealthy lifestyle factors to gut dysbiosis.

Discovery of Stable and Selective Antibody Mimetics from Combinatorial Libraries of Polyvalent, Loop-Functionalized Peptoid Nanosheets.

The ability of antibodies to bind a wide variety of analytes with high specificity and high affinity makes them ideal candidates for therapeutic and diagnostic applications. However, the poor stability and high production cost of antibodies have prompted exploration of a variety of synthetic materials capable of specific molecular recognition. Unfortunately, it remains a fundamental challenge to create a chemically diverse population of protein-like, folded synthetic nanostructures with defined molecular conformations in water. Here we report the synthesis and screening of combinatorial libraries of sequence-defined peptoid polymers engineered to fold into ordered, supramolecular nanosheets displaying a high spatial density of diverse, conformationally constrained peptoid loops on their surface. These polyvalent, loop-functionalized nanosheets were screened using a homogeneous Förster resonance energy transfer (FRET) assay for binding to a variety of protein targets. Peptoid sequences were identified that bound to the heptameric protein, anthrax protective antigen, with high avidity and selectivity. These nanosheets were shown to be resistant to proteolytic degradation, and the binding was shown to be dependent on the loop display density. This work demonstrates that key aspects of antibody structure and function-the creation of multivalent, combinatorial chemical diversity within a well-defined folded structure-can be realized with completely synthetic materials. This approach enables the rapid discovery of biomimetic affinity reagents that combine the durability of synthetic materials with the specificity of biomolecular materials.

Uniform, Large-Area, Highly Ordered Peptoid Monolayer and Bilayer Films for Sensing Applications.

The production of atomically defined, uniform, large-area 2D materials remains as a challenge in materials chemistry. Many methods to produce 2D nanomaterials suffer from limited lateral film dimensions, lack of film uniformity, or limited chemical diversity. These issues have hindered the application of these materials to sensing applications, which require large-area uniform films to achieve reliable and consistent signals. Furthermore, the development of a 2D material system that is biocompatible and readily chemically tunable has been a fundamental challenge. Here, we report a simple, robust method for the production of large-area, uniform, and highly tunable monolayer and bilayer films, from sequence-defined peptoid polymers, and their application as highly selective molecular recognition elements in sensor production. Monolayers and bilayer films were produced on the centimeter scale using Langmuir-Blodgett methods and exhibited a high degree of uniformity and ordering as evidenced by atomic force microscopy, electron diffraction, and grazing incidence X-ray scattering. We further demonstrated the utility of these films in sensing applications by employing the biolayer interferometry technique to detect the specific binding of the pathogen derived proteins, shiga toxin and anthrax protective antigen, to peptoid-coated sensors.

Role of KASH domain lengths in the regulation of LINC complexes.

The linker of the nucleoskeleton and cytoskeleton (LINC) complex is formed by the conserved interactions between Sad-1 and UNC-84 (SUN) and Klarsicht, ANC-1, SYNE homology (KASH) domain proteins, providing a physical coupling between the nucleoskeleton and cytoskeleton that mediates the transfer of physical forces across the nuclear envelope. The LINC complex can perform distinct cellular functions by pairing various KASH domain proteins with the same SUN domain protein. For example, in Caenorhabditis elegans, SUN protein UNC-84 binds to two KASH proteins UNC-83 and ANC-1 to mediate nuclear migration and anchorage, respectively. In addition to distinct cytoplasmic domains, the luminal KASH domain also varies among KASH domain proteins of distinct functions. In this study, we combined in vivo C. elegans genetics and in silico molecular dynamics simulations to understand the relation between the length and amino acid composition of the luminal KASH domain, and the function of the SUN-KASH complex. We show that longer KASH domains can withstand and transfer higher forces and interact with the membrane through a conserved membrane proximal EEDY domain that is unique to longer KASH domains. In agreement with our models, our in vivo results show that swapping the KASH domains of ANC-1 and UNC-83, or shortening the KASH domain of ANC-1, both result in a nuclear anchorage defect in C. elegans.

Particle-Templated Emulsification for Microfluidics-Free Digital Biology.

The compartmentalization of reactions in monodispersed droplets is valuable for applications across biology. However, the requirement of microfluidics to partition the sample into monodispersed droplets is a significant barrier that impedes implementation. Here, we introduce particle-templated emulsification, a method to encapsulate samples in monodispersed emulsions without microfluidics. By vortexing a mixture of hydrogel particles and sample solution, we encapsulate the sample in monodispersed emulsions that are useful for most droplet applications. We illustrate the method with ddPCR and single cell culture. The ability to encapsulate samples in monodispersed droplets without microfluidics should facilitate the implementation of compartmentalized reactions in biology.

A molecular model for LINC complex regulation: activation of SUN2 for KASH binding.

Linkers of the nucleoskeleton and cytoskeleton are key molecular complexes that span the nuclear envelope (NE) and provide a direct linkage between the nucleoskeleton and cytoskeleton. Two major components of these complexes are members of the SUN and KASH protein families that interact in the perinuclear space to allow the transmission of mechanochemical signals across the NE. Structural details of the mammalian SUN domain protein SUN2 have established that SUN2 must form a trimer to bind to KASH, and that this trimerization is mediated through two predicted coiled-coil regions of the protein, CC1 and CC2, which precede the SUN domain. Recent crystallographic data suggest that CC2-SUN formed an unexpected autoinhibited monomer unable to bind to KASH. These structural insights raise the question of how full-length SUN2 transitions from a monomer to a trimer inside the NE. In this study we used a computational approach to model a fragment of SUN2 containing CC1, CC2, and the SUN domain. We observed the dynamics of these modeled structures using ∼1 µs molecular dynamics simulations and showed that the interplay between CC1 and CC2 may be sufficient for the release of CC2-SUN2 from its autoinhibited state. Additionally, using our models and gel filtration analysis, we show the involvement of an E452 residue on CC1 in the monomer--trimer transition of SUN2. Intriguingly, mutations in this residue have been seen in muscular dystrophy-associated SUN2 variants. Finally, we propose a Ca2+-dependent monomer-trimer transition of SUN2.

Single-Cell RT-PCR in Microfluidic Droplets with Integrated Chemical Lysis.

Droplet microfluidics can identify and sort cells using digital reverse transcription polymerase chain reaction (RT-PCR) signals from individual cells. However, current methods require multiple microfabricated devices for enzymatic cell lysis and PCR reagent addition, making the process complex and prone to failure. Here, we describe a new approach that integrates all components into a single device. The method enables controlled exposure of isolated single cells to a high pH buffer, which lyses cells and inactivates reaction inhibitors but can be instantly neutralized with RT-PCR buffer. Using our chemical lysis approach, we distinguish individual cells' gene expression with data quality equivalent to more complex two-step workflows. Our system accepts cells and produces droplets ready for amplification, making single-cell droplet RT-PCR faster and more reliable.

Bulk double emulsification for flow cytometric analysis of microfluidic droplets.

Droplet microfluidics is valuable for applications in chemistry and biology, but generates massive numbers of droplets that must be analyzed and sorted. Here, we describe a simple approach to bulk double emulsify microfluidic emulsions for analysis and sorting with commercial flow cytometers. We illustrate the method by using it to identify droplets based on nucleic acid content. Though simple, our method provides a general approach for analyzing and sorting microfluidic droplets without custom microfluidic double emulsifiers or sorters.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding.

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Measurement of copy number variation in single cancer cells using rapid-emulsification digital droplet MDA.

Uniform amplification of low input DNA is important for applications across biology, including single-cell genomics, forensic science, and microbial and viral sequencing. However, the requisite biochemical amplification methods are prone to bias, skewing sequence proportions and obscuring signals relating to copy number. Digital droplet multiple displacement amplification enables uniform amplification, but requires expert knowledge of microfluidics to generate monodisperse emulsions. In addition, existing microfluidic methods are tedious and labor intensive for preparing many samples. Here, we introduce rapid emulsification multiple displacement amplification, a method to generate monodisperse droplets with a hand-held syringe and hierarchical droplet splitter. While conventional microfluidic devices require >10 minutes to emulsify a sample, our system takes tens of seconds and yields data of equivalent quality. We demonstrate the approach by using it to accurately measure copy number variation in single cancer cells.

Nanotip Ambient Ionization Mass Spectrometry.

A method called nanotip ambient ionization mass spectrometry (NAIMS) is described, which applies high voltage between a tungsten nanotip and a metal plate to generate a plasma in which ionized analytes on the surface of the metal plate are directed to the inlet and analyzed by a mass spectrometer. The dependence of signal intensity is investigated as a function of the tip-to-plate distance, the tip size, the voltage applied at the tip, and the current. These parameters are separately optimized to achieve sensitivity or high spatial resolution. A partially observable Markov decision process is used to achieve a stabilized plasma as well as high ionization efficiency. As a proof of concept, the NAIMS technique has been applied to phenanthrene and caffeine samples for which the limits of detection were determined to be 0.14 fmol for phenanthrene and 4 amol for caffeine and to a printed caffeine pattern for which a spatial resolution of 8 ± 2 µm, and the best resolution of 5 µm, was demonstrated. The limitations of NAIMS are also discussed.

Performance of chemically modified plastic blood collection tubes.

OBJECTIVE: The objective of this study was to compare newly-modified and aged chemoPET tubes, which contain no problematic surfactants, with commercially available serum blood collection tubes (BCTs) for use in analysis of cortisol, total triiodothyronine (TT3), total thyroxine (TT4), and routine clinical chemistry analytes in serum from apparently healthy volunteers and pooled quality control (QC) specimens. MATERIALS AND METHODS: Blood specimens collected from 60 apparently healthy volunteers (18 males, 42 females) and pooled QC specimens poured into seven different BCTs were analyzed by a trained phlebotomist. Cortisol, TT3, and TT4 levels were measured on an Immulite 1000 instrument and routine chemistry tests were analyzed on a Siemens RxL instrument. The significance of differences between chemoPET and other BCT types compared to glass tubes were assessed by Student's paired t-test or repeated measures ANOVA or their non-parametric equivalents. The BCT-related biases (deviation from glass tubes) in analyte concentrations were compared with the current desirable allowable bias, derived from biological variation. Serum analyte concentrations in the different BCTs that exceeded their respective significant change limits were considered clinically significant. RESULTS: No statistically and/or clinically significant differences were noted in the analyte concentrations from serum specimens and pooled QC material when our newly modified and aged chemoPET tubes were compared to glass and other BCTs. CONCLUSIONS: The chemoPET tubes described here may be a suitable alternative to serum BCTs that contain problematic surfactants known to interfere with some clinical assays on the Immulite 1000 and RxL instruments.

Patterning microfluidic device wettability with spatially-controlled plasma oxidation.

Microfluidic devices can form double emulsions with uniform properties, but require cumbersome fabrication steps to pattern their wettability. We demonstrate spatially-controlled plasma oxidation to create wettability patterns for forming double emulsions. Our method performs comparably to chemical techniques but is simpler, more reliable, and scalable to patterning large arrays of drop makers.

Miniaturized Antimicrobial Susceptibility Test by Combining Concentration Gradient Generation and Rapid Cell Culturing.

Effective treatment of bacterial infection relies on timely diagnosis and proper prescription of antibiotic drugs. The antimicrobial susceptibility test (AST) is one of the most crucial experimental procedures, providing the baseline information for choosing effective antibiotic agents and their dosages. Conventional methods, however, require long incubation times or significant instrumentation costs to obtain test results. We propose a lab-on-a-chip approach to perform AST in a simple, economic, and rapid manner. Our assay platform miniaturizes the standard broth microdilution method on a microfluidic device (20 × 20 mm) that generates an antibiotic concentration gradient and delivers antibiotic-containing culture media to eight 30-nL chambers for cell culture. When tested with 20 µL samples of a model bacterial strain (E. coli ATCC 25922) treated with ampicillin or streptomycin, our method allows for the determination of minimum inhibitory concentrations consistent with the microdilution test in three hours, which is almost a factor of ten more rapid than the standard method.