Recombinant human proteoglycan 4 lowers inflammation and atherosclerosis susceptibility in female low-density lipoprotein receptor knockout mice.

Recombinant human proteoglycan 4 (rhPRG4) is a macromolecular mucin-like glycoprotein that is classically studied as a lubricant within eyes and joints. Given that endogenously produced PRG4 is present within atherosclerotic lesions and genetic PRG4 deficiency increases atherosclerosis susceptibility in mice, in the current study we investigated the anti-atherogenic potential of chronic rhPRG4 treatment. Female low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet for 6 weeks and injected three times per week intraperitoneally with 0.5 mg rhPRG4 or PBS as control. Treatment with rhPRG4 was associated with a small decrease in plasma-free cholesterol levels, without a change in cholesteryl ester levels. A marked increase in the number of peritoneal foam cells was detected in response to the peritoneal rhPRG4 administration, which could be attributed to elevated peritoneal leukocyte MSR1 expression levels. However, rhPRG4-treated mice exhibited significantly smaller aortic root lesions of 278 ± 21 × 103 µm2 compared with 339 ± 15 × 103 µm2 in the aortic root of control mice. The overall decreased atherosclerosis susceptibility coincided with a shift in the monocyte and macrophage polarization states towards the patrolling and anti-inflammatory M2-like phenotypes, respectively. Furthermore, rhPRG4 treatment significantly reduced macrophage gene expression levels as well as plasma protein levels of the pro-inflammatory/pro-atherogenic cytokine TNF-alpha. In conclusion, we have shown that peritoneal administration and subsequent systemic exposure to rhPRG4 beneficially impacts the inflammatory state and reduces atherosclerosis susceptibility in mice. Our findings highlight that PRG4 is not only a lubricant but also acts as an anti-inflammatory agent. KEY POINTS: Endogenously produced proteoglycan 4 is found in atherosclerotic lesions and its genetic deficiency in mice is associated with enhanced atherosclerosis susceptibility. In this study we investigated the anti-atherogenic potential of chronic treatment with recombinant human PRG4 in hypercholesterolaemic female low-density lipoprotein receptor knockout mice. We show that recombinant human PRG4 stimulates macrophage foam cell formation, but also dampens the pro-inflammatory state of monocyte/macrophages, eventually leading to a significant reduction in plasma TNF-alpha levels and a lowered atherosclerosis susceptibility. Our findings highlight that peritoneal recombinant human PRG4 treatment can execute effects both locally and systemically and suggest that it will be of interest to study whether rhPRG4 treatment is also able to inhibit the progression and/or induce regression of previously established atherosclerotic lesions.

SARS-CoV-2 recombinase polymerase amplification assay with lateral flow readout and duplexed full process internal control.

Nucleic acid amplification tests for the detection of SARS-CoV-2 have been an important testing mechanism for the COVID-19 pandemic. While these traditional nucleic acid diagnostic methods are highly sensitive and selective, they are not suited to home or clinic-based uses. Comparatively, rapid antigen tests are cost-effective and user friendly but lack in sensitivity and specificity. Here we report on the development of a one-pot, duplexed reverse transcriptase recombinase polymerase amplification SARS-CoV-2 assay with MS2 bacteriophage as a full process control. Detection is carried out with either real-time fluorescence or lateral flow readout with an analytical sensitivity of 50 copies per reaction. Unlike previously published assays, the RNA-based MS2 bacteriophage control reports on successful operation of lysis, reverse transcription, and amplification. This SARS-CoV-2 assay features highly sensitive detection, visual readout through an LFA strip, results in less than 25 minutes, minimal instrumentation, and a useful process internal control to rule out false negative test results.

Impact of Clinician Subjectivity on the Assessment of Dry Eye Disease Prevalence in a UK Public Health Care Patient Population.

Purpose: To understand the impact of subjectivity on diagnosis rates of dry eye disease (DED) in an unbiased population. Patients and Methods: A multicenter study enrolled 818 subjects with complete report forms (465 females, 67.1 ± 16.7 years, 353 males, 65.0 ± 15.9 years). Subjects were evaluated for staining, TBUT, tear osmolarity, meibomian gland disease, and OSDI. Results: Physicians diagnosed 48.7% of subjects as having DED, ranging from 42.9% to 62.3% between sites. Positivity rates for staining (≥ grade 1) ranged from 41.3% to 84.1% (mean = 0.8 ± 0.9 grade), TBUT (<10s) ranged from 39.1% to 61.6% (mean = 10.4 ± 6.6 seconds), osmolarity (>308 mOsm/L) ranged from 63.7% to 72.4% (mean = 319.7 ± 20.8), MGD grading ranged from 28.9% to 51.3% (mean = 0.5 ± 0.7), and symptoms measured by OSDI ranged from 57.6% to 71.0% (mean = 23.5 ± 20.5) between sites. Tear osmolarity was the most consistent between sites (max/min positivity = 114%), followed by OSDI (123%), TBUT (158%), MGD (178%), and staining (204%). DED markers were uncorrelated (average r2 = 0.05 ± 0.07). A substantial number of subjects (N = 110) exhibited positive symptoms (OSDI = 32.4 ± 15.7) and hyperosmolarity (338.1 ± 20.1 mOsm/L) but no other obvious signs of DED (MGD grade = 0.2 ± 0.4, TBUT = 13.5 ± 7.0 seconds, staining grade = 0.4 ± 0.5). Conclusion: Subjective signs of DED varied considerably, whereas objective measurements of OSDI and osmolarity were the most consistent between sites. A large proportion of subjects exhibited high symptoms and hyperosmolarity but no other obvious signs of dry eye disease, most of whom were undiagnosed by clinical assessment without access to the osmolarity measurement.

A structural and functional comparison between two recombinant human lubricin proteins: Recombinant human proteoglycan-4 (rhPRG4) vs ECF843.

Proteoglycan 4 (PRG4, lubricin) is a mucin-like glycoprotein present on the ocular surface that has both boundary lubricating and anti-inflammatory properties. Full-length recombinant human PRG4 (rhPRG4) has been shown to be clinically effective in improving signs and symptoms of dry eye disease (DED). In vitro, rhPRG4 has been shown to reduce inflammation-induced cytokine production and NFκB activity in corneal epithelial cells, as well as to bind to and inhibit MMP-9 activity. A different form of recombinant human lubricin (ECF843), produced from the same cell line as rhPRG4 but manufactured using a different process, was recently assessed in a DED clinical trial. However, ECF843 did not significantly improve signs or symptoms of DED compared to vehicle. Initial published characterization of ECF843 showed it had a smaller hydrodynamic diameter and was less negatively charged than native PRG4. Further examination of the structural and functional properties of ECF843 and rhPRG4 could contribute to the understanding of what led to their disparate clinical efficacy. Therefore, the objective of this study was to characterize and compare rhPRG4 and ECF843 in vitro, both biophysically and functionally. Hydrodynamic diameter and charge were measured by dynamic light scattering (DLS) and zeta potential, respectively. Size and molecular weight was determined for individual species by size exclusion chromatography (SEC) with in-line DLS and multi-angle light scattering (MALS). Bond structure was measured by Raman spectroscopy, and sedimentation properties were measured by analytical ultracentrifugation (AUC). Functionally, MMP-9 inhibition was measured using a commercial MMP-9 activity kit, coefficient of friction was measured using an established boundary lubrication test at a latex-glass interface, and collagen 1-binding ability was measured by quart crystal microbalance with dissipation (QCMD). Additionally, the ability of rhPRG4 and ECF843 to inhibit urate acid crystal formation and cell adhesion was assessed. ECF843 had a significantly smaller hydrodynamic diameter and was less negatively charged than rhPRG4, as assessed by DLS and zeta potential. Size was further explored with SEC-DLS-MALS, which indicated that while rhPRG4 had 3 main peaks, corresponding to monomer, dimer, and multimer as expected, ECF843 had 2 peaks that were similar in size and molecular weight compared to rhPRG4's monomer peak and a third peak that was significantly smaller in both size and molar mass than the corresponding peak of rhPRG4. Raman spectroscopy demonstrated that ECF843 had significantly more disulfide bonds, which are functionally determinant structures, relative to the carbon-carbon backbone compared to rhPRG4, and AUC indicated that ECF843 was more compact than rhPRG4. Functionally, ECF843 was significantly less effective at inhibiting MMP-9 activity and functioning as a boundary lubricant compared to rhPRG4, as well as being slower to bind to collagen 1. Additionally, ECF843 was significantly less effective at inhibiting urate acid crystal formation and at preventing cell adhesion. Collectively, these data demonstrate ECF843 and rhPRG4 are significantly different in both structure and function. Given that a protein's structure sets the foundation for its interactions with other molecules and tissues in vivo, which ultimately determine its function, these differences most likely contributed to the disparate DED clinical trial results.

Ecosystem carbon and nitrogen gains following 27 years of grazing management in a semiarid alluvial valley.

Soils in semiarid riparian ecosystems have large carbon (C) stocks that promote water and nutrient availability for productive plant communities consumed by grazing animals. Changes to riparian hydrologic conditions caused by channel incision result in different edaphic conditions and a greater abundance of upland plant species that may be associated with lower soil C stocks. Using riparian meadows alongside Maggie Creek in central Nevada, we show that 27 years of modified grazing practices can repair ecosystem processes and increase the C stocks. We compared C and nitrogen (N) stocks (of soils and plant biomass) on floodplains, terraces, and uplands of reaches where grazing was either modified or excluded to reaches where no changes to grazing practices were made. Grazing management allowed beaver to establish, improving hydrology and lengthening the growing season. These changes allowed C and N to accumulate on geomorphic surfaces that extended from the stream channel to the surrounding hillslopes. A stoichiometric relationship between C and N shows carbon sequestration can reduce nutrient runoff to nearby waterways and may depend on nitrogen availability. Gains in ecosystem carbon ranged from 93 to 452 g C m-2 y-1 and were dominated by increases in soil C. Gains in soil C occurred across the full depth range measured (0-45 cm) and were comparable to those found in restored wetlands and meadows located in more humid ecosystems. Carbon gains exhibited substantial variability caused by microtopography and plant community composition. While grazing exclusion resulted in the largest gains in ecosystem C, managed grazing that limited consumption of riparian plants increased ecosystem C relative to reaches where management wasn't changed. We demonstrate that managed grazing that maintains ecosystem process is compatible with projects aimed at increasing soil carbon in semiarid riparian rangelands.

REverSe TRanscrIptase chain termination (RESTRICT) for selective measurement of nucleotide analogs used in HIV care and prevention.

Sufficient drug concentrations are required for efficacy of antiretroviral drugs used in HIV care and prevention. Measurement of nucleotide analogs, included in most HIV medication regimens, enables monitoring of short- and long-term adherence and the risk of treatment failure. The REverSe TRanscrIptase Chain Termination (RESTRICT) assay rapidly infers the concentration of intracellular nucleotide analogs based on the inhibition of DNA synthesis by HIV reverse transcriptase enzyme. Here, we introduce a probabilistic model for RESTRICT and demonstrate selective measurement of multiple nucleotide analogs using DNA templates designed according to the chemical structure of each drug. We measure clinically relevant concentrations of tenofovir diphosphate, emtricitabine triphosphate, lamivudine triphosphate, and azidothymidine triphosphate with agreement between experiment and theory. RESTRICT represents a new class of activity-based assays for therapeutic drug monitoring in HIV care and could be extended to other diseases treated with nucleotide analogs.

Rapid detection of hepatitis C virus using recombinase polymerase amplification.

Over 71 million people are infected with hepatitis C virus (HCV) worldwide, and approximately 400,000 global deaths result from complications of untreated chronic HCV. Pan-genomic direct-acting antivirals (DAAs) have recently become widely available and feature high cure rates in less than 12 weeks of treatment. The rollout of DAAs is reliant on diagnostic tests for HCV RNA to identify eligible patients with viremic HCV infections. Current PCR-based HCV RNA assays are restricted to well-resourced central laboratories, and there remains a prevailing clinical need for expanded access to decentralized HCV RNA testing to provide rapid chronic HCV diagnosis and linkage to DAAs in outpatient clinics. This paper reports a rapid, highly accurate, and minimally instrumented assay for HCV RNA detection using reverse transcription recombinase polymerase amplification (RT-RPA). The assay detects all HCV genotypes with a limit of detection of 25 copies per reaction for genotype 1, the most prevalent in the United States and worldwide. The clinical sensitivity and specificity of the RT-RPA assay were both 100% when evaluated using 78 diverse clinical serum specimens. The accuracy, short runtime, and low heating demands of RT-RPA may enable implementation in a point-of-care HCV test to expand global access to effective treatment via rapid chronic HCV diagnosis.

Quantitative isothermal amplification on paper membranes using amplification nucleation site analysis.

Quantitative nucleic acid amplification tests (qNAATs) are critical in treating infectious diseases, such as in HIV viral load monitoring or SARS-CoV-2 testing, in which viral load indicates viral suppression or infectivity. Quantitative PCR is the gold standard tool for qNAATs; however, there is a need to develop point-of-care (POC) qNAATs to manage infectious diseases in outpatient clinics, low- and middle-income countries, and the home. Isothermal amplification methods are an emerging tool for POC NAATs as an alternative to traditional PCR-based workflows. Previous works have focused on relating isothermal amplification bulk fluorescence signals to input copies of target nucleic acids for sample quantification with limited success. In this work, we show that recombinase polymerase amplification (RPA) reactions on paper membranes exhibit discrete fluorescent amplification nucleation sites. We demonstrate that the number of nucleation sites can be used to quantify HIV-1 DNA and viral RNA in less than 20 minutes. An image-analysis algorithm quantifies nucleation sites and determines the input nucleic acid copies in the range of 67-3000 copies per reaction. We demonstrate a mobile phone-based system for image capture and onboard processing, illustrating that this method may be used at the point-of-care for qNAATs with minimal instrumentation.

Restoring function: Positive responses of carbon and nitrogen to 20 years of hydrologic restoration in montane meadows.

Montane meadows are highly productive ecosystems that contain high densities of soil carbon (C) and nitrogen (N). However, anthropogenic disturbances that have led to channel incision and disconnected floodplain hydrology have altered the C balance of many meadows, converting them from net C sinks to net sources of C to the atmosphere. Restoration efforts designed to reconnect floodplain hydrology may slow rates of soil C loss from degraded meadows and restore the conditions for C sequestration and N immobilization, yet questions remain about the long-term impact of such efforts. Here, we used a 22-year meadow restoration chronosequence to measure the decadal impact of hydrologic restoration on aboveground and belowground C and N stocks and concentrations. Increases in herbaceous vegetation biomass preceded changes in soil C stocks, with the largest gains occurring belowground. Root biomass (0-15 cm) increased at a rate of 270.3 g m-2 year-1 and soil C stocks (0-15 cm) increased by 232.9 g C m-2 year-1 across the chronosequence. Increases in soil C concentration (2.99 g C kg-1 year-1 ) were tightly coupled with increases in soil N concentration (0.21 g N kg-1 year-1 ) and soil C:N did not vary with time since restoration. Fourier transform infrared spectroscopy results showed that the fraction of labile aliphatic C-H and carboxylate C-O (COO) compounds in the soil increased with the age of restoration and were positively correlated with soil C and N concentrations. Our results demonstrate that restoration of floodplain hydrology in montane meadows has significant impacts on belowground C and N stocks, soil C and N concentration, and soil C chemistry within the first two decades following restoration.

HIV pre-exposure prophylaxis adherence test using reverse transcription isothermal amplification inhibition assay.

Current HIV antiretroviral therapy (ART) or pre-exposure prophylaxis (PrEP) therapy adherence monitoring relies on either patient self-reported adherence or monitored drug dispensing, which are not reliable. We report a proof-of-concept adherence monitoring assay which directly measures nucleotide reverse transcriptase inhibitor (NRTI) concentration using a reverse transcription isothermal amplification inhibition assay. We measure the concentration of Tenofovir diphosphate (TFV-DP) - an NRTI that functions as a deoxyadenosine triphosphate (dATP) analog and long-term adherence marker for PrEP - by measuring the inhibition of the reverse transcription of an RNA template. The completion or inhibition of reverse transcription is evaluated by recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification assay commonly used for point-of-care diagnostics. We present and validate a model that predicts the amplification probability as a function of dATP and TFV-DP concentrations, nucleotide insertion sites on the RNA template, and RNA template concentration. The model can be used to rationally design and optimize the assay to operate at clinically relevant TFV-DP concentrations. We provide statistical analysis that demonstrates how the assay may be used as a qualitative or semi-quantitative tool for measuring adherence to NRTI drugs and used to support patient compliance. Due to its simple instrumentation and short runtime (<1 hour), this assay has the potential for implementation in low-complexity laboratories or point-of-care settings, which may improve access to ART and PrEP adherence monitoring.

Patient-level costing analysis of paediatric short bowel syndrome care in a specialist tertiary centre.

PURPOSE: To undertake a pilot study estimating patient-level costs of care for paediatric short bowel syndrome (SBS) from the healthcare provider perspective. METHODS: A pilot group of patients with anatomical SBS was selected at a single specialist tertiary centre in the United Kingdom. The Patient Level Information and Costing System (PLICS) was used to extract costing data for all hospital-based activities related to SBS, from the implementation of PLICS in 2016 to April 2021. Patient-specific and pooled data were reported descriptively in per patient-year terms. RESULTS: Five patients had full PLICS data available for the 5-year study period and 2 patients had 4 years of data. The median cost for hospital care of SBS was £52,834 per patient-year (range £1804-£331,489). The key cost drivers were inpatient beds, pharmacy, and staffing costs, which made up > 60% of annual costs. In the first 3 years following index admission (n = 2), there was a steady decline in the annual cost of care to a level comparable with patients with established SBS. CONCLUSION: Patient-level cost of care analysis for SBS is feasible using PLICS. Hospital-related costs vary widely between and within individual patients over time. Key drivers of cost are related to complications of SBS.

Quantitative Isothermal Amplification on Paper Membranes using Amplification Nucleation Site Analysis.

Quantitative nucleic acid amplification tests (qNAATs) are critical in treating infectious diseases, such as in HIV viral load monitoring or SARS-CoV-2 testing, in which viral load indicates viral suppression or infectivity. Quantitative PCR is the gold standard tool for qNAATs; however, there is a need to develop point-of-care (POC) qNAATs to manage infectious diseases in outpatient clinics, low- and middle-income countries, and the home. Isothermal amplification methods are an emerging tool for POC NAATs as an alternative to traditional PCR-based workflows. Previous works have focused on relating isothermal amplification bulk fluorescence signals to input copies of target nucleic acids for sample quantification with limited success. In this work, we show that recombinase polymerase amplification (RPA) reactions on paper membranes exhibit discrete fluorescent amplification nucleation sites. We demonstrate that the number of nucleation sites can be used to quantify HIV-1 DNA and RNA in less than 20 minutes. An image-analysis algorithm quantifies nucleation sites and determines the input nucleic acid copies in the range of 67-3,000 copies per reaction. We demonstrate a mobile phone-based system for image capture and onboard processing, illustrating that this method may be used at the point-of-care for qNAATs with minimal instrumentation.

HIV detection from human serum with paper-based isotachophoretic RNA extraction and reverse transcription recombinase polymerase amplification.

The number of people living with HIV continues to increase with the current total near 38 million, of which about 26 million are receiving antiretroviral therapy (ART). These treatment regimens are highly effective when properly managed, requiring routine viral load monitoring to assess successful viral suppression. Efforts to expand access by decentralizing HIV nucleic acid testing in low- and middle-income countries (LMICs) has been hampered by the cost and complexity of current tests. Sample preparation of blood samples has traditionally relied on cumbersome RNA extraction methods, and it continues to be a key bottleneck for developing low-cost POC nucleic acid tests. We present a microfluidic paper-based analytical device (µPAD) for extracting RNA and detecting HIV in serum, leveraging low-cost materials, simple buffers, and an electric field. We detect HIV virions and MS2 bacteriophage internal control in human serum using a novel lysis and RNase inactivation method, paper-based isotachophoresis (ITP) for RNA extraction, and duplexed reverse transcription recombinase polymerase amplification (RT-RPA) for nucleic acid amplification. We design a specialized ITP system to extract and concentrate RNA, while excluding harsh reagents used for lysis and RNase inactivation. We found the ITP µPAD can extract and purify 5000 HIV RNA copies per mL of serum. We then demonstrate detection of HIV virions and MS2 bacteriophage in human serum within 45-minutes.

Pilot evaluation of an enzymatic assay for rapid measurement of antiretroviral drug concentrations.

OBJECTIVE: Maintaining adequate drug adherence is crucial to ensure the HIV prevention benefits of pre-exposure prophylaxis (PrEP). We developed an enzymatic assay for rapidly measuring tenofovir-diphosphate (TFV-DP) concentrations-a metabolite that indicates long-term PrEP adherence. SETTING: The study was conducted at the Madison HIV Clinic at Harborview Medical Center in Seattle. METHODS: We enrolled adults receiving standard oral PrEP, and individuals not receiving any antiretrovirals. We measured TFV-DP concentrations in diluted whole blood using our novel REverSe TRanscrIptase Chain Termination (RESTRICT) assay, based on inhibition of HIV reverse transcriptase (RT) enzyme. Blood samples were diluted in water, DNA templates, nucleotides, RT, and intercalating dye added, and results measured with a fluorescence reader-stronger fluorescence indicated higher RT activity. We compared RESTRICT assay results to TFV-DP concentrations from matched dried blood spot samples measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) using ≥ 700 fmol/punch TFV-DP as a threshold for adequate adherence (≥ 4 doses/week). RESULTS: Among 18 adults enrolled, 4 of 7 participants receiving PrEP had TFV-DP levels ≥ 700 fmol/punch by LC-MS/MS. RESTRICT fluorescence correlated with LC-MS/MS measurements (r = - 0.845, p < 0.0001). Median fluorescence was 93.3 (95% confidence interval [CI] 90.9 to 114) for samples < 700 fmol/punch and 54.4 (CI 38.0 to 72.0) for samples ≥ 700 fmol/punch. When calibrated to an a priori defined threshold of 82.7, RESTRICT distinguished both groups with 100% sensitivity and 92.9% specificity. CONCLUSIONS: This novel enzymatic assay for measuring HIV reverse transcriptase activity may be suitable for distinguishing TFV-DP concentrations in blood that correspond to protective PrEP adherence.

Elimination of Oxygen Interference in the Electrochemical Detection of Monochloramine, Using In Situ pH Control at Interdigitated Electrodes.

Disinfection of water systems by chloramination is a method frequently used in North America as an alternative to chlorination. In such a case, monochloramine is used as the primary chlorine source for disinfection. Regular monitoring of the residual concentrations of this species is essential to ensure adequate disinfection. An amperometric sensor for monochloramine would provide fast, reagent-free analysis; however, the presence of dissolved oxygen in water complicates sensor development. In this work, we used in-situ pH control as a method to eliminate oxygen interference by conversion of monochloramine to dichloramine. Unlike monochloramine, the electrochemical reduction of dichloramine occurs outside the oxygen reduction potential window and is therefore not affected by the oxygen concentration. Potential sweep methods were used to investigate the conversion of monochloramine to dichloramine at pH 3. The pH control method was used to calibrate monochloramine concentrations between 1 and 10 ppm, with a detection limit of 0.03 ppm. Tests were carried out in high alkalinity samples, wherein it was found that the sensitivity of this method effectively remained unchanged. Monochloramine was also quantified in the presence of common interferents (copper, phosphate, and iron) which also had no significant impact on the analysis.

Ambient Discrimination, Victimization, and Suicidality in a Non-Probability U.S. Sample of LGBTQ Adults.

Individuals who identify as lesbian, gay, bisexual, transgender, or queer (LGBTQ) are at a higher risk for suicidality compared to the general population. A growing body of research has investigated this risk, particularly with attention to systemic factors such as discrimination and harassment. Unfortunately, research has only examined the impact of direct discrimination on suicidality and has neglected to examine how ambient discrimination (i.e., witnessing or being made aware of discriminatory behaviors directed at someone other than yourself in your group) relates to suicidality. Additionally, although some links exist between discrimination and suicidality, the mechanisms by which these are related are understudied. This study aimed to address these gaps by exploring the effect of ambient discrimination on suicidal ideation and examining psychological pain as a mediator in this relationship. Data were collected from a sample of 200 LGBTQ-identified individuals (M age = 35 years; 53.5% female; 86% White). Results of independent t tests and a one-way multivariate ANOVA revealed greater vulnerability for ambient/direct discrimination and psychache among individuals identifying as transgender, queer, and other. Regression and mediation analyses revealed that while both ambient and direct discrimination predicted suicidal ideation, only direct discrimination accounted for unique variance in the outcome; however, both ambient and direct discrimination contributed unique variance to psychological pain, which fully mediated their relationships to suicidal ideation. Results of this study may begin to provide insight into the pathways of risk and points of intervention for suicidality in the LGBTQ community.

Nucleic acid sample preparation from whole blood in a paper microfluidic device using isotachophoresis.

Nucleic acid amplification tests (NAATs) are a crucial diagnostic and monitoring tool for infectious diseases. A key procedural step for NAATs is sample preparation: separating and purifying target nucleic acids from crude biological samples prior to nucleic acid amplification and detection. Traditionally, sample preparation has been performed with liquid- or solid-phase extraction, both of which require multiple trained user steps and significant laboratory equipment. The challenges associated with sample preparation have limited the dissemination of NAAT point-of-care diagnostics in low resource environments, including low- and middle-income countries. We report on a paper-based device for purification of nucleic acids from whole blood using isotachophoresis (ITP) for point-of-care NAATs. We show successful extraction and purification of target nucleic acids from large volumes (33 µL) of whole human blood samples with no moving parts and few user steps. Our device utilizes paper-based buffer reservoirs to fully contain the liquid ITP buffers and does not require complex filling procedures, instead relying on the natural wicking of integrated paper membranes. We perform on-device blood fractionation via filtration to remove leukocytes and erythrocytes from our sample, followed by integrated on-paper proteolytic digestion of endogenous plasma proteins to allow for successful isotachophoretic extraction. Paper-based isotachophoresis purifies and concentrates target nucleic acids that are added directly to recombinase polymerase amplification (RPA) reactions. We show consistent amplification of input copy concentrations of as low as 3 × 103 copies nucleic acid per mL input blood with extraction and purification taking only 30 min. By employing a paper architecture, we are able to incorporate these processes in a single, robust, low-cost design, enabling the direct processing of large volumes of blood, with the only intermediate user steps being the removal and addition of tape. Our device represents a step towards a simple, fully integrated sample preparation system for nucleic acid amplification tests at the point-of-care.

The influence of soil age on ecosystem structure and function across biomes.

The importance of soil age as an ecosystem driver across biomes remains largely unresolved. By combining a cross-biome global field survey, including data for 32 soil, plant, and microbial properties in 16 soil chronosequences, with a global meta-analysis, we show that soil age is a significant ecosystem driver, but only accounts for a relatively small proportion of the cross-biome variation in multiple ecosystem properties. Parent material, climate, vegetation and topography predict, collectively, 24 times more variation in ecosystem properties than soil age alone. Soil age is an important local-scale ecosystem driver; however, environmental context, rather than soil age, determines the rates and trajectories of ecosystem development in structure and function across biomes. Our work provides insights into the natural history of terrestrial ecosystems. We propose that, regardless of soil age, changes in the environmental context, such as those associated with global climatic and land-use changes, will have important long-term impacts on the structure and function of terrestrial ecosystems across biomes.

Enzymatic and Chemical-Based Methods to Inactivate Endogenous Blood Ribonucleases for Nucleic Acid Diagnostics.

There are ongoing research efforts into simple and low-cost point-of-care nucleic acid amplification tests (NATs) addressing widespread diagnostic needs in resource-limited clinical settings. Nucleic acid testing for RNA targets in blood specimens typically requires sample preparation that inactivates robust blood ribonucleases (RNases) that can rapidly degrade exogenous RNA. Most NATs rely on decades-old methods that lyse pathogens and inactivate RNases with high concentrations of guanidinium salts. Herein, we investigate alternatives to standard guanidinium-based methods for RNase inactivation using an activity assay with an RNA substrate that fluoresces when cleaved. The effects of proteinase K, nonionic surfactants, SDS, dithiothreitol, and other additives on RNase activity in human serum are reported. Although proteinase K has been widely used in protocols for nuclease inactivation, it was found that high concentrations of proteinase K are unable to eliminate RNase activity in serum, unless used in concert with denaturing concentrations of SDS. It was observed that SDS must be combined with proteinase K, dithiothreitol, or both for irreversible and complete RNase inactivation in serum. This work provides an alternative chemistry for inactivating endogenous RNases for use in simple, low-cost point-of-care NATs for blood-borne pathogens.

Enzymatic Assay for Rapid Measurement of Antiretroviral Drug Levels.

Poor adherence to pre-exposure prophylaxis (PrEP) and antiretroviral therapy (ART) can lead to human immunodeficiency virus (HIV) acquisition and emergence of drug-resistant infections, respectively. Measurement of antiviral drug levels provides objective adherence information that may help prevent adverse health outcomes. Gold-standard drug-level measurement by liquid chromatography/mass spectrometry is centralized, heavily instrumented, and expensive and is thus unsuitable and unavailable for routine use in clinical settings. We developed the REverSe TRanscrIptase Chain Termination (RESTRICT) assay as a rapid and accessible measurement of drug levels indicative of long-term adherence to PrEP and ART. The assay uses designer single-stranded DNA templates and intercalating fluorescent dyes to measure complementary DNA (cDNA) formation by reverse transcriptase in the presence of nucleotide reverse transcriptase inhibitor drugs. We optimized the RESTRICT assay using aqueous solutions of tenofovir diphosphate (TFV-DP), a metabolite that indicates long-term adherence to ART and PrEP, at concentrations over 2 orders of magnitude above and below the clinically relevant range. We used dilution in water as a simple sample preparation strategy to detect TFV-DP spiked into whole blood and accurately distinguished TFV-DP drug levels corresponding to low and high PrEP adherences. The RESTRICT assay is a fast and accessible test that could be useful for patients and clinicians to measure and improve ART and PrEP adherence.