Remote blood sampling offers multiple advantages over traditional clinic-based blood sampling studies, including greater patient inclusion, more frequent sampling, and broader geographical reach. Combining remote blood sampling with transcriptomic analysis opens potential in translational applications for capturing acute and dynamic immune responses to various exposures. In this study, we establish the feasibility of homeRNA, a capillary blood collection and RNAlater-based stabilization kit, for use in downstream total RNA-sequencing applications via capturing a lipopolysaccharide (LPS)-induced inflammatory response. We also compared the baseline gene expression profiles and induced inflammatory response following LPS stimulation between homeRNA-stabilized samples and venous blood stabilized with RNAlater or PAXgene. We found that homeRNA was successfully able to capture an inflammatory response to LPS, specifically targeting various cytokines (e.g., IL6, IL12B, IL1B), chemokines (e.g., CCL3, CXCL10, CCL4), and other transcriptional factors in the toll-like receptor pathway, the primary pathway activated during LPS stimulation. Importantly, we also found that homeRNA captured a LPS-induced inflammatory response comparable to that of venous blood samples stabilized with either RNAlater or PAXgene. Overall, this work demonstrates that the homeRNA platform is compatible with downstream total RNA-sequencing analysis and can capture transcriptomic immune responses to a known stimulus which are analogous to results in traditional stabilized venous blood samples.

Blood biomarkers are central to monitoring disease progression and evaluating treatment responses, yet traditional venipuncture captures a single physiological snapshot in time and becomes burdensome with repeated sampling. Remote blood self-sampling offers a path toward longitudinal, decentralized monitoring, but maintaining protein integrity from draw to analysis remains a critical challenge. Here, we optimized pre-analytical blood collection and stabilization parameters to maintain protein levels at the time of collection for use with remote sampling technology. First, we optimized blood collection time with Tasso remote self-sampling devices to minimize interference from clotting, finding that a 2.5 min collection time best reduces clot formation while collecting enough blood. Next, we found that Protein Plus, a commercial protein stabilizer, limited hemolysis (a metric for stabilizer efficacy) in venous blood for up to 5 days at 25°C-35°C and for 1 day at 40°C. In addition, we optimized the stabilizer volume and acceptable blood volume range for self-sampling as the stabilizer efficacy is impacted by the stabilizer to blood ratio and collection volume can vary with remote self-sampling devices. Finally, we incubated stabilized blood samples collected via Tasso device at 25°C-35°C for 72 h, mimicking a 2-day shipping period. Using a panel of 21 inflammatory proteins, we found that Protein Plus limited intracellular protein release for various proteins (e.g., VEGF-A, CCL11, and IL-8), inhibited protein degradation for CCL2, and enabled minimal hemolysis. These results support Protein Plus as a viable stabilization strategy for remote blood collection technology targeting longitudinal inflammatory protein monitoring.

Stereolithography (SLA) 3D printing has become increasingly popular for fabricating microfluidic devices, with applications including hydrogel patterning and tissue modeling. In open-channel systems with surface tension-driven flow, 3D-printer-induced discrepancies in channel surface texture can significantly impact fluid flow and device performance. While previous work has focused on comparing different 3D printing methods for microchannel fabrication, the effect of device orientation during SLA printing on microchannel morphology and capillary-driven flow has not been systematically evaluated. Furthermore, there is minimal research elucidating the influence of channel surface texture on the flow of biologically relevant hydrogel precursors commonly used in organ-on-a-chip applications. Herein, we investigated the impact of print orientation on channel morphology, fluid wetting behavior, and fluid flow by comparing laser SLA-based parts where the length of the channel was tilted at 0°, 15°, 45°, or 90° during printing. We demonstrated that channel floor surface texture is greatly affected by print orientation: the highest axial surface roughness was measured in 15° printed channels, and the highest axial surface tortuosity-which describes the real length along the surface-was measured in 45° printed channels. Print angles of 15° and 45° also resulted in asymmetric roughness of the channel floor, which caused asymmetric wetting of glycerol solution. Surface tension-driven flow of glycerol solution, agarose precursor solution, and collagen precursor solution was affected by print orientation, in which the 45° printed flow devices had slowest flow for all test fluids. Root mean square roughness was not a reliable predictor of slower flow; instead, surface tortuosity should be considered. Potential alternatives to better theoretically model how print angle-induced surface texture affects open-channel flow are discussed as well. These findings provide a framework of fabrication considerations for laser SLA printing of open microchannels that can also be applied to other layer-by-layer, vat photopolymerization-based 3D printing technologies.

Shifting human subjects research from research sites to participants' homes removes barriers to participation. Previously, we developed homeRNA, a kit for stabilization of RNA in self-collected blood using a custom-engineered tube containing RNA stabilizer fluid. The stabilized RNA is extracted and used for downstream gene expression analysis. Here, we introduce homeRNAmax, which improves our original design by interfacing with a commercially available blood collection tube (BD Microtainer), allowing homeRNAmax to be used with any blood collection method that uses this tube and doubling the possible sample volume that can be collected and stabilized compared to the original homeRNA. Through a pilot study (n = 19 participants), we show that homeRNAmax (with the Tasso+ blood collection device) produces RNA samples of sufficient quality (mean RIN = 7.8) and yield (mean yield = 1.93 μg) for downstream analysis and can reach participants across the United States, who generally (n = 17/19) found the homeRNAmax kit easy to use. We also present RNA integrity data from an ongoing longitudinal clinical study using homeRNAmax in rheumatology (mean RIN = 7.1). A key aspect of the homeRNA and homeRNAmax platforms is a fluidic feature that prevents the RNA stabilizer from spilling, for which we developed a theoretical model. In brief, fluid in the tube is suspended due to a balance of pressures; an increase in air volume within the tube reduces the air pressure above the fluid, creating a small vacuum, and preventing fluid leakage. Overall, we show that homeRNAmax is a user-friendly, effective tool for remote blood RNA stabilization.

Importance:Remote sampling technologies are invaluable for protecting both participants and researchers when studying highly infectious diseases. When leveraged for longitudinal studies, remote sampling with transcriptomic readouts is a powerful tool for studying the host immune response. Additionally, remote study flexibility circumvents common barriers to research participation including length of commute, transportation, and scheduling, thereby expanding access to clinical research. Objective:In this work, we investigate the effectiveness of a remote study model for reaching women from underrepresented, underserved, and underreported (U3) populations. We sought to recruit individuals who qualify as underrepresented in clinical research, who are located in rural areas, or who come from disadvantaged backgrounds per the NIH definition. Design:In this longitudinal study, U3 women positive for COVID-19 were enrolled and followed over the course of 6 months. In the first month of the infection, participants (n = 40) self-collected a set of 5 nasal swabs, 5 homeRNA-stabilized blood samples, and 2 additional unstabilized blood samples at first and last sampling. Sampling time points were spaced 5 days apart, so that the total of the 5 time points was completed within 25 days. homeRNA is a platform for remote self-collection of blood samples with subsequent RNA stabilization. A subset of participants likely to develop post-acute sequelae of COVID-19 (PASC) and their age-matched controls were selected to self-collect an additional set of 5 nasal swabs and 5 homeRNA-stabilized blood samples during month 3 of study participation, with the same sampling frequency. All participants were resurveyed at months 4, 5, and 6 about their symptoms. Participants also completed surveys at each sampling and a more comprehensive survey about study experience after each set of 5 time points. Setting:This was a fully remote study with all sampling supplies and instructions shipped to the participants. Participants self-collected blood and nasal swabs at home and shipped these back to our lab for further processing. Surveys were administered electronically using REDCap. Participants:For this study, we enrolled women who were 18 or older, met the NIH criteria for U3, and who had tested positive for SARS-CoV-2 within a week of enrollment. Further, we excluded protected populations including individuals who were pregnant and/or incarcerated. Of the 334 individuals who completed the screening process, 65 were invited into the study based on the eligibility criteria and balancing age, race/ethnicity, and state of residence to closely correspond to the demographics of the United States. Of the 65 invited individuals, 40 were fully enrolled in the study and 39 completed all study components. Main Outcomes and Measures:Prior to the study, we proposed that the increased flexibility of a remote study design would allow for participation of populations underrepresented in clinical research. The primary measurements planned for this study consisted of usability data and general experience in a longitudinal study. These data were collected by self report using electronically administered surveys. The Consolidated Framework for Implementation Research (CFIR), a well-established implementation science framework, was used to guide the development of questions about usability and study experience. Results:40 women were recruited from 19 states, with diverse racial backgrounds (62% White, 15% Black or African American, 10% Asian, 5% American Indian or Alaska Native, 5% Other, 3% More than one race), a mostly even age distribution (26% ages 20 - 29, 15% ages 30 - 39, 31% ages 40 - 49, 28% ages 50+), and most of whom (80%) are categorized as having a disadvantaged background per the NIH. Survey responses show high satisfaction with the study, where all participants who completed the study (100%, n = 39/39) indicating that they would be willing to participate in a similar study again, with most (n = 32/39) indicating a willingness to participate for up to 4 years with around 15 samples collected per year. We note that 4 years was the longest time period that participants were able to select in their surveys, suggesting that participants may be willing to participate for even longer periods. Most (>90%) either agreed or strongly agreed that all components of the kit were easy to use. Conclusions and Relevance:The high retention (98%, n = 39/40) and satisfaction of participants in this study indicates the utility of a remote study design for longitudinal research. We also find that study topic, flexibility of study, and positive interactions with the study team are important factors for participant recruitment and retention. This work suggests that the increased flexibility of a fully remote design enables engagement of individuals who may otherwise be excluded from clinical research.

Three-dimensional (3D) cell culture can leverage the precise arrangement of materials, known as patterning, to generate physiologically relevant tissue-like structures. Hydrogels are widely used in 3D cell culture due to their ability to mimic the properties of biological extracellular matrix networks. In this tutorial review, we discuss the use of microfluidic systems to control fluid movement and placement within fabricated microchannels to pattern hydrogel precursors in 3D through the use of capillary flow. Such systems offer unique advantages in their ability to create complex biomimetic structures, organs-on-a-chip, and microphysiological systems with high spatial resolution and relatively small volumes of hydrogel material. We first discuss the fundamental principles behind capillary pinning and aspiration-mediated patterning. We then review literature describing the development and applications of three different types of microfluidic systems - closed, semi-open, and open - and describe how different patterning techniques are applied to each system. We also discuss modular microfluidic systems, in which multiple classes of microfluidic systems are combined together for complex and biomimetic modeling of biological systems. In each section, we provide synthesis and critical analysis of established and novel techniques to draw connections across diverse papers in literature. Finally, we offer our perspectives on the advantages of microfluidic systems for hydrogel patterning and the future of the field. Taken together, microfluidic flow-based patterning is an exciting tool for microphysiological systems and other 3D cell culture models that are poised to transform our understanding of basic biological mechanisms and provide new opportunities for studying diverse phenomena in physiologically relevant tissue models.

Extracellular vesicles (EVs) are promising biomarkers for disease detection using a 'liquid biopsy' approach, in which they are enriched and analyzed directly from biofluids. However, implementing EV biomarker technologies in the clinic remains limited by the need for practical and patient-centric biofluid collection methods that are compatible with downstream EV processing and analysis. While saliva offers a non-invasive source of EVs, its complexity and heterogeneity-cells, debris, and other non-EV proteins-can present hurdles when using traditional analytical platforms. Here, we present the CandyCollect, a lollipop-inspired sampling device with open microfluidic channels, as a patient-friendly approach for rapid salivary EV capture. CandyCollect simplifies sample preparation by effectively pre-concentrating EVs in oxygen-plasma treated open microfluidic channels. In this proof-of-principle study, we show that following a 3-5 minute-oral sampling period, EVs collected by the CandyCollect can be released with high purity within minutes and subsequently quantified and analyzed for cargo content. We observed consistent EV capture across repeated collections within individuals and expected variability across healthy participants. Additionally, single and pooled collections of EVs from a healthy participant resulted in a concordant protein profile. Overall, the CandyCollect is a new platform for rapid, non-invasive salivary EV collection and analysis for clinical diagnostics.

Control of fluids is a hallmark of microfluidic systems and fundamental for the successful application of microfluidic devices. Trigger valves use geometric features to autonomously control the release of fluids in microfluidic devices. Our previous work has adapted geometries used in closed trigger valve systems to enable use in open systems, allowing for open microfluidic devices with up to three trigger valves. Here, we focus on the parallel co-flows produced by sequential release of trigger valves and present a model that predicts their layer widths as a function of the geometric characteristics of the different side channels of each trigger valve. We show layered co-flows with widths as low as 50 microns. Additionally, we expand the use of trigger valves in open microfluidic devices by incorporating 1) varied step heights, 2) devices with up to seven trigger valves, and 3) use of varied fluids and plastics. To validate the implementation and use of these trigger valves in open systems, we have developed a theoretical framework to compare predicted outcomes (i.e., fluid travel distance, velocity, and layering width) with our experimental values. This theoretical work offers applications in various fields, including hydrogel patterning for 3D cell culture, organ-on-a-chip models, at-home sample preparation, and autonomous microfluidic systems for biosensing.

Free-standing tissue structures tethered between pillars are powerful mechanobiology tools for studying cell contraction. To model interfaces ubiquitous in natural tissues and upgrade existing single-region suspended constructs, we developed Suspended Tissue Open Microfluidic Patterning (STOMP), a method to create multi-regional suspended tissues. STOMP uses open microfluidics and capillary pinning to pattern subregions within free-standing tissues, facilitating the study of complex tissue interfaces, such as diseased-healthy boundaries (e.g., fibrotic-healthy) and tissue-type interfaces (e.g., bone-ligament). We observed altered contractile dynamics in fibrotic-healthy engineered heart tissues compared to single-region tissues and differing contractility in bone-ligament enthesis constructs compared to single-tissue periodontal ligament models. STOMP is a versatile platform - surface tension-driven patterning removes material requirements common with other patterning methods (e.g., shear-thinning, photopolymerizable) allowing tissue generation in multiple geometries with native extracellular matrices and advanced four-dimensional (4D) materials. STOMP combines the contractile functionality of suspended tissues with precise patterning, enabling dynamic and spatially controlled studies.

We created CandyCollect, a lollipop-inspired, child-friendly saliva sampling device that provides enhanced comfort compared to common throat and mouth swabs. The device has an open microchannel and a functionalized surface that effectively captures and stores the saliva and pathogens. We recently demonstrated the ability to combine the CandyCollect with rapid antigen detection tests (RADTs) for fast and accessible detection of group A Streptococcus (GAS). However, the current procedure necessitates manual steps to elute and transfer captured antigens from the lollipop to RADTs, which relies on the user to follow complex instructions, increasing the likelihood of error. In this work, we developed the CandyCollect open-to-closed (O2C) microfluidic system to automate antigen elution and streamline detection with RADTs. The O2C system securely seals the open microchannels of the CandyCollect device to create a continuous closed channel for seamless delivery of elution reagents to release antigens at the press of a button for subsequent lateral flow detection. Our system leverages and combines the unique benefits of both open channel and closed channel microfluidics into one platform, further enhancing the potential of both methods. We performed experiments with GAS bacteria spiked into saliva and compared the manual and O2C elution methods. Both the manual and O2C methods showed clear test lines on the RADT strips at clinically relevant GAS concentrations (between 5 × 105 and 109 CFU mL-1). The O2C provided comparable results to the manual procedure in a more convenient form factor. The O2C platform has the potential to enable user-friendly screening for respiratory pathogens by minimally trained users in decentralized settings.

Sperm cryopreservation is important for many individuals across the globe. Recent studies show that vitrification is a valuable approach for maintaining sperm quality after freeze-thawing processes and requires sub-microliter to microliter volumes. A major challenge for the adoption of vitrification in fertility laboratories is the ability to pipet small volumes of sample. Here, we present an open droplet generator that leverages open-channel microfluidics to passively generate sub-microliter to microliter volumes of purified human sperm samples and preserves sperm kinematics. We conclude that our platform is compatible with human sperm, an important foundation for future implementation of vitrification in fertility laboratories.

The CandyCollect is a lollipop-inspired open-fluidic oral sampling device designed to provide a comfortable user sampling experience. We demonstrate that the CandyCollect device can be coupled to a rapid antigen detection test (RADT) kit designed for Group A Streptococcus (GAS). Through in vitro experiments with pooled saliva spiked with Streptococcus pyogenes, we tested various reagents and elution volumes to optimize the RADT readout from CandyCollect device samples. The resulting optimized protocol uses the kit-provided reagents and lateral flow assay (LFA) while replacing the kit's pharyngeal swab with the CandyCollect device, reducing the elution solution volume, and substituting the tube used for elution to accommodate the CandyCollect device. Positive test results were detected by eye with bacterial concentrations as low as the manufacturer's "minimal detection limit" of 1.5 × 105 CFU/mL. LFA strips were also scanned and semiquantified with image analysis software to determine the signal-to-baseline ratio (SBR) and categorize positive test results without human bias. We tested our optimized protocol for integrating CandyCollect and RADT using CandyCollect clinical samples from pediatric patients (n = 6) who were previously diagnosed with GAS pharyngitis via pharyngeal swabs tested with RADT as part of their clinical care. The LFA results of these CandyCollect devices and interspersed negative controls were determined by independent observers with positive results obtained in four of the six participants on at least one LFA replicate. Taken together, our results show that CandyCollect devices from children with GAS pharyngitis can be tested by using LFA rapid tests.

Introduction Effective community-based disease management is essential for public health. In low- and middle-income countries, sustainable strategies for timely diagnosis and treatment are a research priority. This study aims to assess the feasibility of a non-invasive saliva self-sampling method, paired with digitally linked molecular point-of-care diagnostics, for detecting respiratory infections among paediatric patients in the Tshwane District, South Africa.Methods and analysis A field study will be conducted at Steve Biko Academic Hospital to compare saliva collection using the CandyCollect lollipop device and standard mouth swabs. The spiral groove of the lollipop device captures pathogens, which are stored in DNA/RNA preservation media and later analysed using quantitative PCR and commercially available rapid antigen tests. The multiplex respiratory pathogen panel, based on TaqMan real-time PCR technology, targets key paediatric pathogens including Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, respiratory syncytial virus (RSV) and influenza A/B. Nucleic acids will be extracted using standard viral extraction kits and analysed following manufacturer protocols. Internal controls will be included in each qPCR run, and samples with CT values below defined thresholds will be considered positive. Rapid antigen tests will detect common pathogens such as influenza A/B, RSV and SARS-CoV-2 for comparative analysis. User experience and acceptability will be assessed via child-friendly and caregiver surveys following sample collection. The study will be implemented in two phases: diagnostic performance evaluation and user feedback assessment. The protocol is aligned with the Standard Protocol Items: Recommendations for Interventional Trials 2013 checklist.Ethics and dissemination Ethical approval has been granted by the University of Pretoria (509/2023) and the Gauteng Department of Health (GP_202406_032). The study is registered in the Pan African Clinical Trial Registry (PACTR202411743094783). Findings will be disseminated through peer-reviewed journals, conferences and stakeholder briefings. The study complies with South Africa’s Protection of Personal Information Act. Data collection is scheduled from November 2024 to February 2025, with project completion expected within 1 year.Trial registration number Pan African Clinical Trial Registry (PACTR202411743094783).

Remote research studies are an invaluable tool for reaching populations with limited access to large medical centers or universities. To expand the remote study toolkit, we previously developed homeRNA, which allows for at-home self-collection and stabilization of blood and demonstrated the feasibility of using homeRNA in high temperature climates. Here, we expand upon this work through a systematic study exploring the effects of high temperature on RNA integrity (represented as RNA Integrity Number, RIN) through in-lab and field experiments. Compared to the frozen controls (overall mean RIN of 8.2, n = 8), samples kept at 37 °C for 2, 4, and 8 days had mean RINs of 7.6, 5.9, and 5.2 (n = 3), respectively, indicating that typical shipping conditions (∼2 days) yield samples suitable for downstream RNA sequencing. Shorter time intervals (6 h) resulted in minimal RNA degradation (median RIN of 6.4, n = 3) even at higher temperatures (50 °C) compared to the frozen control (mean RIN of 7.8, n = 3). Additionally, we shipped homeRNA-stabilized blood from a single donor to 14 states and back during the summer with continuous temperature probes (7.1 median RIN, n = 42). Samples from all locations were analyzed with 3' mRNA-seq to assess differences in gene counts, with the data suggesting that there was no preferential degradation of transcripts as a result of different shipping times, temperatures, and regions. Overall, our data support that homeRNA can be used in elevated temperature conditions, enabling decentralized sample collection for telemedicine, global health, and clinical research.

Shifting human subjects research from research sites to participants' homes removes barriers to participation, including transportation and scheduling difficulties. Previously, we developed homeRNA, a kit for immediate stabilization of RNA in self-collected blood using a custom-engineered tube containing RNA stabilizer fluid. The stabilized RNA is extracted and used for downstream gene expression analysis. Here, we introduce homeRNAmax, which improves our original design by interfacing with a commercially available blood collection tube (BD Microtainer), allowing homeRNAmax to be used with any blood collection method that uses this tube and doubling the possible sample volume that can be collected and stabilized compared to the original homeRNA. Through a pilot study (n=19 participants), we show that homeRNAmax (with the Tasso+ blood collection device) produces RNA samples of sufficient quality (mean RIN=7.8) and yield (mean yield=1.93 μg) for downstream analysis and can reach participants across the United States, who generally (n=17/19) found the homeRNAmax kit easy to use. A key aspect of the homeRNA and homeRNAmax platforms is a fluidic feature that prevents the RNA stabilizer from spilling, however our previous work had not yet fully characterized the mechanism of this feature. Here, we developed a theoretical model of the spill-resistant feature. In brief, fluid in the tube is suspended due to a balance of pressures; an increase in air volume within the tube reduces the air pressure above the fluid, creating a small vacuum, and preventing fluid leakage. Overall, we show that homeRNAmax is a user-friendly, effective tool for remote blood RNA stabilization.

The Lucas-Washburn-Rideal law is commonly applied to describe capillary flow dynamics in closed or open channels, microporous media, such as paper pads and fiber threads or even granulous soil. It assumes a viscous flow regime where capillary forces are counteracted by friction with the solid structure, a valid assumption given the small flow velocities and device dimensions. However, scenarios exist outside the viscous regime, where inertial effects become significant, meaning capillary and friction forces do not fully balance. One well-documented case is the transient inertial regime at the onset of capillary motion. With the advancement of capillary devices, other configurations also raise the possibility of inertia influencing flow behavior. This study introduces a criterion to identify inertial contributions in capillary-driven flows in spatially varying geometries within open or closed channels and demonstrates how the Bosanquet equation can account for inertial effects in rectangular open-channel configurations.

State the Purpose:Obtaining high-quality samples to diagnose streptococcal pharyngitis in pediatric patients is challenging due to discomfort associated with traditional pharyngeal swabs. This may cause reluctance to go to the clinic, inaccurate diagnosis, or inappropriate treatment for children with sore throats. Here, we determined the efficacy of CandyCollect, a lollipop-inspired open-microfluidic pathogen collection device, to capture Group A Streptococcus (GAS) and compare user preference for CandyCollect, conventional pharyngeal swabs, or mouth swabs in children with pharyngitis and their caregivers. Results:All child participants (30/30) were positive for GAS by qPCR on both the mouth swab and CandyCollect. Caregivers ranked CandyCollect as a good sampling method overall (27/30), and all caregivers (30/30) would recommend CandyCollect for children 5 years and older. Twenty-three of 30 children "really like" the taste and 24/30 would prefer to use CandyCollect if a future test were needed. All caregivers (30/30) and most children (28/30) would be willing to use CandyCollect at home. Conclusion:All participants tested positive for GAS on all three collection methods (pharyngeal swab, mouth swab, and CandyCollect). While both caregivers and children like CandyCollect, some caregivers would prefer a shorter collection time. Future work includes additional studies with larger cohorts presenting with pharyngitis of unknown etiology and shortening collection time while maintaining the attractive form of the device.

Malassezia yeasts are commensal microorganisms found in human and animal skin. Species of Malassezia have been connected to skin and opportunistic infections, where certain microenvironmental conditions are required in the host for the pathogenic processes to occur. We present the analysis of the volatile space of Malassezia pachydermatis grown at three pH values (5.7, 9.7, and 12.4) by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS). Since changes in pH also affect the growth media and the volatile organic compounds (VOCs) produced by it, media blanks at the three pHs were analyzed, with 5 replicates of each of the 6 samples. Following data collection, GC×GC-TOFMS chromatograms were analyzed by Fisher ratio software that found 566 analytes, out of which 288 were tentatively identified with a mass spectrum match value (MV) ≥ 800 based upon a NIST library search. A signal pattern for each of the 566 analytes was obtained by averaging the replicates, and two metrics (R and RSD) were calculated for each signal pattern. The R metric was defined to focus upon the differences between analyte signals of media blanks and M. pachydermatis by taking away the influence of pH changes, while the RSD metric was defined to evaluate only the influence of pH. Based on the R metric magnitude, the analytes were split into 3 categories: media analytes consumed by M. pachydermatis, analytes at similar concentration at a given pH in the media and M. pachydermatis, and analytes produced in M. pachydermatis only. Many of the M. pachydermatis produced analytes were already shown to be produced by other yeast species and shown to have biological significance when the pH is varied. Further, there is evidence of some bioconversions between the consumed analytes discovered versus the analytes produced. We also verified our classification results using a support vector machine (SVM) model, where cross-validation provided a very promising outcome with true positive rate (TPR) and true negative rate (TNR) both being over 0.95 and the error being below 0.03 (or 3%).

Transcriptomic responses to wildfire smoke are difficult to study given the unpredictability of wildfires and the challenges of collecting blood during active disasters. To overcome these challenges, we developed a flexible study design leveraging homeRNA, our at-home blood collection and RNA stabilization kit. Between June 2021 and April 2022, 58 participants across 10 U.S. states collected 635 blood samples before, during, and after wildfire events. This responsive approach captured three exposure groups: high exposure in Okanogan County, Washington, medium exposure from transported smoke, and low exposure. During the 10-month study, 93% of participants (n=54/58) returned at least 6 samples. In a preliminary exploratory analysis, we analyzed 770 genes with a Nanostring panel from nine participants (6 high, 3 low-medium exposure) using the BloodGen3 framework. In the high exposure participants, we observed trends toward overexpression of inflammation (inflammation aggregates A33 and A35, and modules M13.1 and M13.12), with concurrent underexpression of adaptive immune responses (lymphocytic aggregates A1 and A6, B cell module M13.18, T cell modules M16.24 and M15.38). This study establishes that homeRNA enables flexible, responsive sampling during disasters, overcoming traditional logistical barriers to capture time-sensitive biological data across dispersed populations.