A Microphysiological Device to Model the Choriodecidual Interface Immune Status during Pregnancy.

During human pregnancy the chorion (fetal) lines decidua (maternal) creating the feto-maternal interface. Despite their proximity, resident decidual immune cells remain quiescent during gestation and do not invade the chorion. Infection and infiltration of activated immune cells toward the chorion are often associated with preterm birth. However, the mechanisms that maintain choriodecidual immune homeostasis or compromise immune barrier functions remain unclear. To understand these processes, a two-chamber microphysiological system (MPS) was created to model the human choriodecidual immune interface under normal and infectious conditions in vitro. This MPS has outer (fetal chorion trophoblast cells) and inner chambers (maternal decidual + CD45+ cells [70:30 ratio]) connected by microchannels. Decidual cells were treated with LPS to mimic maternal infection, followed by immunostaining for HLA-DR and HLA-G, immune panel screening by imaging cytometry by time of flight, and immune regulatory factors IL-8 and IL-10, soluble HLA-G, and progesterone (ELISA). LPS induced a proinflammatory phenotype in the decidua characterized by a decrease in HLA-DR and an increase in IL-8 compared with controls. LPS treatment increased the influx of immune cells into the chorion, indicative of chorionitis. Cytometry by time of flight characterized immune cells in both chambers as active NK cells and neutrophils, with a decrease in the abundance of nonproinflammatory cytokine-producing NK cells and T cells. Conversely, chorion cells increased progesterone and soluble HLA-G production while maintaining HLA-G expression. These results highlight the utility of MPS to model choriodecidual immune cell infiltration and determine the complex maternal-fetal crosstalk to regulate immune balance during infection.

Phenotype-Based Threat Assessment.

Bacterial pathogen identification, which is critical for human health, has historically relied on culturing organisms from clinical specimens. More recently, the application of machine learning (ML) to whole-genome sequences (WGSs) has facilitated pathogen identification. However, relying solely on genetic information to identify emerging or new pathogens is fundamentally constrained, especially if novel virulence factors exist. In addition, even WGSs with ML pipelines are unable to discern phenotypes associated with cryptic genetic loci linked to virulence. Here, we set out to determine if ML using phenotypic hallmarks of pathogenesis could assess potential pathogenic threat without using any sequence-based analysis. This approach successfully classified potential pathogenetic threat associated with previously machine-observed and unobserved bacteria with 99% and 85% accuracy, respectively. This work establishes a phenotype-based pipeline for potential pathogenic threat assessment, which we term PathEngine, and offers strategies for the identification of bacterial pathogens.

High-Efficiency Interdigitated Electrode-Based Droplet Merger for Enabling Error-Free Droplet Microfluidic Systems.

Merging two droplets into a droplet to add and mix two contents is one of the common droplet microfluidic functions with droplet generation and sorting, performing broad ranges of biological and chemical assays in droplets. However, traditional droplet-merging techniques often encounter unsynchronized droplets, causing overmerging or mis-merging, and unwanted merging outside of the desired zone. This is more severe when the incoming droplets to be merged are polydisperse in their sizes, often observed in assays that require long-term incubation, elevated-temperature, and/or multiple droplet processing steps. Here, we developed an interdigitated electrode (IDE)-based droplet merger consisting of a droplet autosynchronizing channel and a merging channel. The autosynchronizing channel provides >95% merging efficiency even when 20% polydispersity in the droplet size exists. The highly localized and enhanced dielectrophoretic force generated by the IDEs on the channel bottom allows droplet merging at an extremely low voltage (4.5 V) and only locally at the IDE region. A systematic evaluation of how various design and operation parameters of the IDE merger, such as IDE finger dimensions, dielectric coating layer thickness, droplet size, and droplet flow speed impact the performance was conducted. The optimized device showed consistent performance even when operating for up to 100 h consecutively at high throughput (100 droplets/s). The presented technology has been integrated into a droplet microfluidics workflow to test the lytic activities of bacteriophage on bacterial host cells with 100% merging efficiency. We expect this function to be integrated into droplet microfluidic systems performing broad ranges of high-throughput chemical and biological assays.

Development of oxidative stress-associated disease models using feto-maternal interface organ-on-a-chip.

Oxidative stress (OS) and inflammation arising from cellular derangements at the fetal membrane-decidual interface (feto-maternal interface [FMi]) is a major antecedent to preterm birth (PTB). However, it is impractical to study OS-associated FMi disease state during human pregnancy, and thus it is difficult to develop strategies to reduce the incidences of PTB. A microfluidic organ-on-chip model (FMi-OOC) that mimics the in vivo structure and functions of FMi in vitro was developed to address this challenge. The FMi-OOC contained fetal (amnion epithelial, mesenchymal, and chorion) and maternal (decidua) cells cultured in four compartments interconnected by arrays of microchannels to allow independent but interconnected co-cultivation. Using this model, we tested the effects of OS and inflammation on both fetal (fetal → maternal) and maternal (maternal → fetal) sides of the FMi and determined their differential impact on PTB-associated pathways. OS was induced using cigarette smoke extract (CSE) exposure. The impacts of OS were assessed by measuring cell viability, disruption of immune homeostasis, epithelial-to-mesenchymal transition (EMT), development of senescence, and inflammation. CSE propagated (LC/MS-MS analysis for nicotine) over a 72-hour period from the maternal to fetal side, or vice versa. However, they caused two distinct pathological effects on the maternal and fetal cells. Specifically, fetal OS induced cellular pathologies and inflammation, whereas maternal OS caused immune intolerance. The pronounced impact produced by the fetus supports the hypothesis that fetal inflammatory response is a mechanistic trigger for parturition. The FMi disease-associated changes identified in the FMi-OOC suggest the unique capability of this in vitro model in testing in utero conditions.

Uniform sized cancer spheroids production using hydrogel-based droplet microfluidics: a review.

Three-dimensional (3D) cell culture models have been extensively utilized in various mechanistic studies as well as for drug development studies as superior in vitro platforms than conventional two-dimensional (2D) cell culture models. This is especially the case in cancer biology, where 3D cancer models, such as spheroids or organoids, have been utilized extensively to understand the mechanisms of cancer development. Recently, many sophisticated 3D models such as organ-on-a-chip models are emerging as advanced in vitro models that can more accurately mimic the in vivo tissue functions. Despite such advancements, spheroids are still considered as a powerful 3D cancer model due to the relatively simple structure and compatibility with existing laboratory instruments, and also can provide orders of magnitude higher throughput than complex in vitro models, an extremely important aspects for drug development. However, creating well-defined spheroids remain challenging, both in terms of throughputs in generation as well as reproducibility in size and shape that can make it challenging for drug testing applications. In the past decades, droplet microfluidics utilizing hydrogels have been highlighted due to their potentials. Importantly, core-shell structured gel droplets can avoid spheroid-to-spheroid adhesion that can cause large variations in assays while also enabling long-term cultivation of spheroids with higher uniformity by protecting the core organoid area from external environment while the outer porous gel layer still allows nutrient exchange. Hence, core-shell gel droplet-based spheroid formation can improve the predictivity and reproducibility of drug screening assays. This review paper will focus on droplet microfluidics-based technologies for cancer spheroid production using various gel materials and structures. In addition, we will discuss emerging technologies that have the potential to advance the production of spheroids, prospects of such technologies, and remaining challenges.

A dynamic flow fetal membrane organ-on-a-chip system for modeling the effects of amniotic fluid motion.

Fetal membrane (amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic fluid subtly rocks back and forth, and thus, the innermost amnion epithelial cells are continuously exposed to low levels of shear stress from fluid undulation. Here, we tested the impact of fluid motion on amnion epithelial cells (AECs) as a bearer of force impact and their potential vulnerability to cytopathologic changes that can destabilize fetal membrane functions. A previously developed amnion membrane (AM) organ-on-chip (OOC) was utilized but with dynamic flow to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 h to mimic fluid motion. A static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control representing pathophysiological changes. The impacts of fluidic motion were evaluated by measuring cell viability, cellular transition, and inflammation. Additionally, scanning electron microscopy (SEM) imaging was performed to observe microvilli formation. The results show that regardless of the applied flow rate, AECs and AMCs maintained their viability, morphology, innate meta-state, and low production of pro-inflammatory cytokines. E-cadherin expression and microvilli formation in the AECs were upregulated in a flow rate-dependent fashion; however, this did not impact cellular morphology or cellular transition or inflammation. OS treatment induced a mesenchymal morphology, significantly higher vimentin to cytokeratin 18 (CK-18) ratio, and pro-inflammatory cytokine production in AECs, whereas AMCs did not respond in any significant manner. Fluid motion and shear stress, if any, did not impact AEC cell function and did not cause inflammation. Thus, when using an amnion membrane OOC model, the inclusion of a dynamic flow environment is not necessary to mimic in utero physiologic cellular conditions of an amnion membrane.

Modeling ascending Ureaplasma parvum infection through the female reproductive tract using vagina-cervix-decidua-organ-on-a-chip and feto-maternal interface-organ-on-a-chip.

Genital mycoplasmas can break the cervical barrier and cause intraamniotic infection and preterm birth. This study developed a six-chamber vagina-cervix-decidua-organ-on-a-chip (VCD-OOC) that recapitulates the female reproductive tract during pregnancy with culture chambers populated by vaginal epithelial cells, cervical epithelial and stromal cells, and decidual cells. Cells cultured in VCD-OOC were characterized by morphology and immunostaining for cell-specific markers. We transferred the media from the decidual cell chamber of the VCD-OOC to decidual cell chamber in feto-maternal interface organ-on-a-chip (FMi-OOC), which contains the fetal membrane layers. An ascending Ureaplasma parvum infection was created in VCD-OOC. U. parvum was monitored for 48 h post-infection with their cytotoxicity (LDH assay) and inflammatory effects (multiplex cytokine assay) in the cells tested. An ascending U. parvum infection model of PTB was developed using CD-1 mice. The cell morphology and expression of cell-specific markers in the VCD-OOC mimicked those seen in lower genital tract tissues. U. parvum reached the cervical epithelial cells and decidua within 48 h and did not cause cell death in VCD-OOC or FMi-OOC cells. U. parvum infection promoted minimal inflammation, while the combination of U. parvum and LPS promoted massive inflammation in the VCD-OOC and FMi-OOC cells. In the animal model, U. parvum vaginal inoculation of low-dose U. parvum did not result in PTB, and even a high dose had only some effects on PTB (20%). However, intra-amniotic injection of U. parvum resulted in 67% PTB. We report the colonization of U. parvum in various cell types; however, inconsistent, and low-grade inflammation across multiple cell types suggests poor immunogenicity induced by U. parvum.

High-aspect-ratio three-dimensional polymer and metallic microstructure microfabrication using two-photon polymerization.

Creating micrometer-resolution high-aspect-ratio three-dimensional (3D) structures remain very challenging despite significant microfabrication methods developed for microelectromechanical systems (MEMS). This is especially the case when such structures are desired to be metallic to support electronic applications. Here, we present a microfabrication process that combines two-photon-polymerization (2PP) printing to create a polymeric high-aspect-ratio three-dimensional structure and electroless metal plating that selectively electroplates only the polymeric structure to create high-aspect-ratio 3D metallic structures having micrometer-resolution. To enable this, the effect of various 2PP processing parameters on SU-8 photoresist microstructures were first systematically studied. These parameters include laser power, slicing/hatching distances, and pre-/post-baking temperature. This optimization resulted in a maximum aspect ratio (height to width) of ~ 12. Following this polymeric structure printing, electroless plating using Tollens' Reagent were utilized to selectively coat silver particles only on the polymeric structure, but not on the silicon substrate. The final 3D metallic structures were evaluated in terms of their resistivity, reproducibly showing resistivity of ~ 10-6 [Ω·m]. The developed 3D metallic structure microfabrication process can be further integrated with conventional 2D lithography to achieve even more complex structures. The developed method overcomes the limitations of current MEMS fabrication processes, allowing a variety of previously impossible metallic microstructures to be created.

Polyethylene Degradation by a Rhodococcous Strain Isolated from Naturally Weathered Plastic Waste Enrichment.

Polyethylene (PE) is the most widely produced synthetic polymer and the most abundant plastic waste worldwide due to its recalcitrance to biodegradation and low recycle rate. Microbial degradation of PE has been reported, but the underlying mechanisms are poorly understood. Here, we isolated a Rhodococcus strain A34 from 609 day enriched cultures derived from naturally weathered plastic waste and identified the potential key PE degradation enzymes. After 30 days incubation with A34, 1% weight loss was achieved. Decreased PE molecular weight, appearance of C-O and C═O on PE, palmitic acid in the culture supernatant, and pits on the PE surface were observed. Proteomics analysis identified multiple key PE oxidation and depolymerization enzymes including one multicopper oxidase, one lipase, six esterase, and a few lipid transporters. Network analysis of proteomics data demonstrated the close relationships between PE degradation and metabolisms of phenylacetate, amino acids, secondary metabolites, and tricarboxylic acid cycles. The metabolic roadmap generated here provides critical insights for optimization of plastic degradation condition and assembly of artificial microbial communities for efficient plastic degradation.

Body mass index and self-rated oral health in Korean adults in 2017.

OBJECTIVE: To investigate the association between obesity and self-rated oral health (SROH). This study examined the cross-sectional associations between body mass index (BMI) and SROH in Korean adults. MATERIALS AND METHODS: This study used data from 217 304 adults (100 110 men and 117 194 women aged > 19 years) from the 2017 Korean Community Health Survey. Participants were categorised into six ordinal groups based on BMI: underweight (<18.5 kg/m2 ), normal weight (18.5-22.9 kg/m2 ), overweight (23.0-24.9 kg/m2 ), obese-I (25.0-27.4 kg/m2 ), obese-II (27.5-29.9 kg/m2 ) or obese-III (≥30.0 kg/m2 ). SROH was assessed using responses to the question, "How do you rate your oral health, including your teeth and gums?" rated on a 5-point scale. SROH was categorised as "good" (reported as "fair," "good" or "very good") or "poor" or "very poor." Age- and sex-stratified associations between BMI categories and poor SROH were assessed using ordinal logistic regression analysis with sampling weights. RESULTS: The age-adjusted odds ratio (OR) for poor SROH according to BMI levels was lowest in the overweight group in both men and women. In men, the OR for poor SROH was 2.03 (99% confidence interval [CI], 1.72-2.39) in the underweight group, 1.17 (99% CI, 1.17-1.25) in the normal group, 1.05 (99% CI, 0.98-1.13) in the obese-I group, 1.08 (99% CI, 0.98-1.18) in the obese-II group and 1.36 (99% CI, 1.20-1.55) in the obese-III group. In women, the OR was 1.18 (99% CI, 1.07-1.31) in the underweight group, 1.01 (99% CI, 0.95-1.07) in the normal group, 1.07(99% CI, 0.99-1.16) in the obese-I group, 1.16 (99% CI, 1.04-1.30) in the obese-II group and 1.39 (99% CI, 1.20-1.62) in the obese-III group. From the restricted cubic spline models in both sexes, BMI showed a J-shaped association with poor and very poor SROH in men and women. In a stratified analysis by age group and sex, men and older women in the underweight group had poorer SROH than those in overweight group. CONCLUSION: In a nationally representative sample of Korean adults, there was a J-shaped association between BMI and poor SROH, with the highest risk in the underweight group amongst men and in the obese-III group amongst women. Furthermore, in men and women over 65 years of age, underweight and obesity were associated with poorer SROH.

Microfluidic Dielectrophoretic Method Enables On-Demand Spatial Arrangement of Bacteria-Encapsulated Agarose Gel Microparticles.

Microbial interactions within a natural or engineered consortium of microbes play an important role in the functions of the consortium. Better understanding these interactions is also important for engineering microbial consortia for specific applications. As such, tools that can enable investigating microbial interactions are highly valuable. One aspect of microbial interactions that impacts community formation is how the spatial organization of individual microbes impacts interactions leading to community formation. Here, we report the development of a tool that can manipulate the spatial organization of microorganisms to investigate the role of these interactions in community formation. Our developed microfluidic platform utilizes dielectrophoretic (DEP) force to perform on-demand spatial arrangement of microorganism-encapsulated agarose gel microparticles. To demonstrate this concept, three gel microparticle manipulators composed of three independently controllable DEP electrodes were utilized for the on-demand spatial arrangement of a specific combination of microparticles, each containing Escherichia coli cells expressing red fluorescence protein, green fluorescent protein, or blank content. The spatially arranged microparticles suspended in carrier oil were first trapped in a downstream particle trapping structure to form a defined microparticle array, followed by the application of an electric field to disrupt the carrier oil barrier so that all gel microparticles were within the same aqueous solution while the individual gel microparticles remain intact, thereby maintaining their spatial arrangements. We demonstrated that this method can be utilized to generate various arrays with differing number of "spacer microparticles", which were blank microparticles, between the two different E. coli-containing microparticles, enabling precise control over spatial distances between the two different cell populations. This method paves the way for more easily investigating bacterial interactions, especially those that depend on their spatial arrangement such as where cell-cell communication plays a major role.

Generation and characterization of human Fetal membrane and Decidual cell lines for reproductive biology experiments†.

Human fetal membrane and maternal decidua parietalis form one of the major feto-maternal interfaces during pregnancy. Studies on this feto-maternal interface is limited as several investigators have limited access to the placenta, and experience difficulties to isolate and maintain primary cells. Many cell lines that are currently available do not have the characteristics or properties of their primary cells of origin. Therefore, we created, characterized the immortalized cells from primary isolates from fetal membrane-derived amnion epithelial cells, amnion and chorion mesenchymal cells, chorion trophoblast cells and maternal decidua parietalis cells. Primary cells were isolated from a healthy full-term, not in labor placenta. Primary cells were immortalized using either a HPV16E6E7 retroviral or a SV40T lentiviral system. The immortalized cells were characterized for the morphology, cell type-specific markers, and cell signalling pathway activation. Genomic stability of these cells was tested using RNA seq, karyotyping, and short tandem repeats DNA analysis. Immortalized cells show their characteristic morphology, and express respective epithelial, mesenchymal and decidual markers similar to that of primary cells. Gene expression of immortalized and primary cells were highly correlated (R = 0.798 to R = 0.974). Short tandem repeats DNA analysis showed in the late passage number (>P30) of cell lines matched 84-100% to the early passage number (

Stress signaler p38 mitogen-activated kinase activation: a cause for concern?

Oxidative stress (OS) induced activation of p38 mitogen-activated kinase (MAPK) and cell fate from p38 signaling was tested using the human fetal membrane's amnion epithelial cells (AEC). We created p38 KO AEC using the CRISPR/Cas9 approach and tested cell fate in response to OS on an AEC-free fetal membrane extracellular matrix (ECM). Screening using image CyTOF indicated OS causing epithelial-mesenchymal transition (EMT). Further testing revealed p38 deficiency prevented AEC senescence, EMT, cell migration, and inflammation. To functionally validate in vitro findings, fetal membrane-specific conditional KO (cKO) mice were developed by injecting Cre-recombinase encoded exosomes intra-amniotically into p38αloxP/loxP mice. Amnion membranes from p38 cKO mice had reduced senescence, EMT, and increased anti-inflammatory IL-10 compared with WT animals. Our study suggested that overwhelming activation of p38 in response to OS inducing risk exposures can have an adverse impact on cells, cause cell invasion, inflammation, and ECM degradation detrimental to tissue homeostasis.

Organ-on-chip of the cervical epithelial layer: A platform to study normal and pathological cellular remodeling of the cervix.

Damage to the cervical epithelial layer due to infection and inflammation is associated with preterm birth. However, the individual and/or collective roles of cervical epithelial layers in maintaining cervical integrity remain unclear during infection/inflammation. To determine the intercellular interactions, we developed an organ-on-chip of the cervical epithelial layer (CE-OOC) composed of two co-culture chambers connected by microchannels, recapitulating the ectocervical and endocervical epithelial layers. Further, we tested the interactions between cells from each distinct region and their contributions in maintaining cervical integrity in response to LPS and TNFα stimulations. The co-culture of ectocervical and endocervical cells facilitated cellular migration of both epithelial cells inside the microchannels. Compared to untreated controls, both LPS and TNFα increased apoptosis, necrosis, and senescence as well as increased pro-inflammatory cytokine productions by cervical epithelial cells. In summary, the CE-OOC established an in vitro model that can recapitulate the ectocervical and the endocervical epithelial regions of the cervix. The established CE-OOC may become a powerful tool in obstetrics and gynecology research such as in studying cervical remodeling during pregnancy and parturition and the dynamics of cervical epithelial cells in benign and malignant pathology in the cervix.

Measurement of dielectric properties of cells at single-cell resolution using electrorotation.

Dielectric properties of a cell are biophysical properties of high interest for various applications. However, measuring these properties accurately is not easy, which can be exemplified by the large variations in reported dielectric properties of the same cell types. This paper presents a method for measuring the dielectric properties of cells at high frequency, especially lipid-producing microalgae, at single-cell resolution, by integrating an electrorotation-based dielectric property measurement method with a negative dielectrophoretic (nDEP) force-based single-cell trapping method into a single device. In this method, a four-electrode nDEP structure was used to trap a single cell in an elevated position in the center of another four-electrode structure that can apply electrorotational force. By measuring the speed of cell rotation under different applied electrorotation frequencies and fitting the results into a theoretical core-shell cell model, the dielectric properties of cells, including membrane capacitance and cytoplasm conductivity, could be obtained. This system was applied to measure the dielectric properties of lipid-accumulating microalga Chlamydomonas reinhardtii strain Sta6 by applying an electrorotation signal of up to 100 MHz. By utilizing a broad frequency range and expanding the measurement spectra to a high frequency region, increased accuracy in fitting the dielectric parameters to a theoretical model was possible, especially the cytoplasm conductivity. The developed method can be used in various applications, such as screening microalgae based on their lipid production capabilities, separating cells of different dielectric properties, identifying different cell types, as well as conducting basic biophysical analyses of cellular properties.

Calreticulin is a Critical Cell Survival Factor in Malignant Neoplasms.

Calreticulin (CRT) is a high-capacity Ca2+ protein whose expression is up-regulated during cellular transformation and is associated with disease progression in multiple types of malignancies. At the same time, CRT has been characterized as an important stress-response protein capable of inducing immunogenic cell death (ICD) when translocated to the cell surface. It remains unclear why CRT expression is preserved by malignant cells during the course of transformation despite its immunogenic properties. In this study, we identify a novel, critical function of CRT as a cell survival factor in multiple types of human solid-tissue malignancies. CRT knockdown activates p53, which mediates cell-death response independent of executioner caspase activity and accompanied full-length poly ADP ribose polymerase (PARP) cleavage. Mechanistically, we show that down-regulation of CRT results in mitochondrial Ca2+ overload and induction of mitochondria permeability transition pore (mPTP)-dependent cell death, which can be significantly rescued by the mPTP inhibitor, Cyclosporin A (CsA). The clinical importance of CRT expression was revealed in the analysis of the large cohort of cancer patients (N = 2,058) to demonstrate that high levels of CRT inversely correlates with patient survival. Our study identifies intracellular CRT as an important therapeutic target for tumors whose survival relies on its expression.

Cell Washing and Solution Exchange in Droplet Microfluidic Systems.

Water-in-oil emulsion droplet microfluidic systems have been extensively developed, and currently, almost all cell handling steps can be conducted in this format. An exception is the cell washing and solution exchange step, which is commonly utilized in many conventional cell assays. This paper presents an in-droplet cell washing and solution exchange technology that utilizes dielectrophoretic (DEP) force to move all cells to one side of a droplet, followed by asymmetrical splitting of the droplet to obtain a small daughter droplet that contains all or most of the cells, and then finally merges this cell-concentrated droplet with a new droplet that contains the desired solution. These sequential droplet manipulation steps were integrated into a single platform, where up to 88% of the original solution in the droplet could be exchanged with the new solution while keeping cell loss to less than 5%. Two application examples were demonstrated using the developed technology. In the first example, green microalga Chlamydomonas reinhardtii cells were manipulated using negative DEP force to exchange the regular culture medium with a nitrogen-limited medium to induce lipid production. In the second example, Salmonella enterica cells were manipulated using positive DEP force to replace fluorescent dye that models fluorescent cell stains that contribute to high background noise in fluorescence-based droplet content detection with fresh buffer solution, significantly improving the droplet content detection sensitivity. Since the cell washing step is one of the most frequently utilized steps in many cell biology assays, we expect that the developed technology can significantly broaden the type of assay that can be conducted in droplet microfluidic format.

Sub-second heat inactivation of coronavirus using a betacoronavirus model.

Heat treatment denatures viral proteins that comprise the virion, making the virus incapable of infecting a host. Coronavirus (CoV) virions contain single-stranded RNA genomes with a lipid envelope and four proteins, three of which are associated with the lipid envelope and thus are thought to be easily denatured by heat or surfactant-type chemicals. Prior studies have shown that a temperature as low as 75°C with a treatment duration of 15 min can effectively inactivate CoV. The degree of CoV heat inactivation greatly depends on the length of heat treatment time and the temperature applied. With the goal of finding whether sub-second heat exposure of CoV can sufficiently inactivate CoV, we designed and developed a simple fluidic system that can measure sub-second heat inactivation of CoV. The system is composed of a stainless-steel capillary immersed in a temperature-controlled oil bath followed by an ice bath, through which virus solution can flow at various speeds. Flowing virus solution at different speeds, along with temperature control and monitoring system, allows the virus to be exposed to the desired temperature and treatment durations with high accuracy. Using mouse hepatitis virus, a betacoronavirus, as a model CoV system, we identified that 71.8°C for 0.51 s exposure is sufficient to obtain >5 Log10 reduction in viral titer (starting titer: 5 × 107 PFU/ml), and that when exposed to 83.4°C for 1.03 s, the virus was completely inactivated (>6 Log10 reduction).

Physical distancing messages targeting youth on the social media accounts of Canadian public health entities and the use of behavioral change techniques.

INTRODUCTION: Physical distancing (PD) is an important public health strategy to reduce the transmission of COVID-19 and has been promoted by public health authorities through social media. Although youth have a tendency to engage in high-risk behaviors that could facilitate COVID-19 transmission, there is limited research on the characteristics of PD messaging targeting this population on social media platforms with which youth frequently engage. This study examined social media posts created by Canadian public health entities (PHEs) with PD messaging aimed at youth and young adults aged 16-29 years and reported behavioral change techniques (BCTs) used in these posts. METHODS: A content analysis of all social media posts of Canadian PHEs from Facebook, Twitter, Instagram and YouTube were conducted from April 1st to May 31st, 2020. Posts were classified as either implicitly or explicitly targeting youth and young adults. BCTs in social media posts were identified and classified based on Behavior Change Technique Taxonomy version 1 (BCTTv1). Frequency counts and proportions were used to describe the data. RESULTS: In total, 319 youth-targeted PD posts were identified. Over 43% of the posts originated from Ontario Regional public health units, and 36.4 and 32.6% of them were extracted from Twitter and Facebook, respectively. Only 5.3% of the total posts explicitly targeted youth. Explicit posts were most frequent from federal PHEs and posted on YouTube. Implicit posts elicited more interactions than explicit posts regardless of jurisdiction level or social media format. Three-quarters of the posts contained at least one BCT, with a greater portion of BCTs found within implicit posts (75%) than explicit posts (52.9%). The most common BCTs from explicit posts were instructions on how to perform a behavior (25.0%) and restructuring the social environment (18.8%). CONCLUSIONS: There is a need for more PD messaging that explicitly targets youth. BCTs should be used when designing posts to deliver public health messages and social media platforms should be selected depending on the target population.

A Gel-Based Separation-Free Point-of-Care Device for Whole Blood Glucose Detection.

This article introduces a gel-based separation-free point-of-care (POC) device for whole blood glucose colorimetric detection. Enzymes and a chromogenic substrate needed for colorimetric detection of glucose were entrapped in a photopolymerized poly(ethylene) glycol diacrylate (PEG-DA) hydrogel that was cast-molded into a circular shape. Our method enables colorimetric detection without the need for preseparation of blood plasma as the nanometer-scale three-dimensional porous structure of the hydrogel allows the diffusion of small analytes such as glucose while blocking the much larger blood cells. Our method requires less enzymatic concentration and, hence, offers a cost-saving benefit. In addition, PEG-DA also acts as an enzyme stabilizer, and the shelf-life testing result shows that enzyme activity can be maintained in PEG-DA over a long period of time. The concept of this simple, cost-effective method was demonstrated by the colorimetric detection of blood glucose directly from human whole bloodthout any sample preparation steps. The results were compared with those of a spectrophotometry method and showed relative error ranging from 5 to 19%, and less than 9% when compared with a commercial glucose meter. The presented method has the potential to be broadly utilized for other whole blood biomolecule analyses in POC testing applications.