Men who inject opioids exhibit altered tRNA-Gly-GCC isoforms in semen.

In addition to their role in protein translation, tRNAs can be cleaved into shorter, biologically active fragments called tRNA fragments (tRFs). Specific tRFs from spermatocytes can propagate metabolic disorders in second generations of mice. Thus, tRFs in germline cells are a mechanism of epigenetic inheritance. It has also been shown that stress and toxins can cause alterations in tRF patterns. We were therefore interested in whether injecting illicit drugs, a major stressor, impacts tRFs in germline cells. We sequenced RNA from spermatocytes and from semen-derived exosomes from people who inject illicit drugs (PWID) and from non-drug using controls, both groups of unknown fertility status. All PWID injected opioids daily, but most also used other illicit drugs. The tRF cleavage products from Gly-GCC tRNA were markedly different between spermatocytes from PWID compared to controls. Over 90% of reads in controls mapped to shorter Gly-GCC tRFs, while in PWID only 45% did. In contrast, only 4.1% of reads in controls mapped to a longer tRFs versus 45.6% in PWID. The long/short tRF ratio was significantly higher in PWID than controls (0.23 versus 0.16, P = 0.0128). We also report differential expression of a group of small nucleolar RNAs (snoRNAs) in semen-derived exosomes, including, among others, ACA14a, U19, and U3-3. Thus, PWID exhibited an altered cleavage pattern of tRNA-Gly-GCC in spermatocytes and an altered cargo of snoRNAs in semen-derived exosomes. Participants were not exclusively using opioids and were not matched with controls in terms of diet, chronic disease, or other stressors, so our finding are not conclusively linked to opioid use. However, all individuals in the PWID group did inject heroin daily. Our study indicates a potential for opioid injection and/or its associated multi-drug use habits and lifestyle changes to influence epigenetic inheritance.

Rapid single-molecule digital detection of protein biomarkers for continuous monitoring of systemic immune disorders.

Digital protein assays have great potential to advance immunodiagnostics because of their single-molecule sensitivity, high precision, and robust measurements. However, translating digital protein assays to acute clinical care has been challenging because it requires deployment of these assays with a rapid turnaround. Herein, we present a technology platform for ultrafast digital protein biomarker detection by using single-molecule counting of immune-complex formation events at an early, pre-equilibrium state. This method, which we term "pre-equilibrium digital enzyme-linked immunosorbent assay" (PEdELISA), can quantify a multiplexed panel of protein biomarkers in 10 µL of serum within an unprecedented assay incubation time of 15 to 300 seconds over a 104 dynamic range. PEdELISA allowed us to perform rapid monitoring of protein biomarkers in patients manifesting post-chimeric antigen receptor T-cell therapy cytokine release syndrome, with ∼30-minute sample-to-answer time and a sub-picograms per mL limit of detection. The rapid, sensitive, and low-input volume biomarker quantification enabled by PEdELISA is broadly applicable to timely monitoring of acute disease, potentially enabling more personalized treatment.

Direct kinetic fingerprinting and digital counting of single protein molecules.

The sensitive and accurate quantification of protein biomarkers plays important roles in clinical diagnostics and biomedical research. Sandwich ELISA and its variants accomplish the capture and detection of a target protein via two antibodies that tightly bind at least two distinct epitopes of the same antigen and have been the gold standard for sensitive protein quantitation for decades. However, existing antibody-based assays cannot distinguish between signal arising from specific binding to the protein of interest and nonspecific binding to assay surfaces or matrix components, resulting in significant background signal even in the absence of the analyte. As a result, they generally do not achieve single-molecule sensitivity, and they require two high-affinity antibodies as well as stringent washing to maximize sensitivity and reproducibility. Here, we show that surface capture with a high-affinity antibody combined with kinetic fingerprinting using a dynamically binding, low-affinity fluorescent antibody fragment differentiates between specific and nonspecific binding at the single-molecule level, permitting the direct, digital counting of single protein molecules with femtomolar-to-attomolar limits of detection (LODs). We apply this approach to four exemplary antigens spiked into serum, demonstrating LODs 55- to 383-fold lower than commercially available ELISA. As a real-world application, we establish that endogenous interleukin-6 (IL-6) can be quantified in 2-µL serum samples from chimeric antigen receptor T cell (CAR-T cell) therapy patients without washing away excess serum or detection probes, as is required in ELISA-based approaches. This kinetic fingerprinting thus exhibits great potential for the ultrasensitive, rapid, and streamlined detection of many clinically relevant proteins.

High-frequency temperature monitoring at home using a wearable device: A case series of early fever detection and antibiotic administration for febrile neutropenia with bacteremia.

We present a case series of three febrile episodes in neutropenic pediatric cancer patients who wore a Food and Drug Administration approved high-frequency temperature monitoring (HFTM) wearable device (WD) at home. The WD detected fever events when temperature monitoring by thermometer did not detect fever or was not feasible to perform. Two of the episodes were associated with bloodstream infections and the WD detected fevers 5 and 12 h prior to fevers detected by thermometer, triggering earlier medical evaluation and more prompt administration of antibiotics. These observations provide a basis for future investigation of home-based HFTM to improve infection-related outcomes in pediatric oncology.

BaiHui: cross-species brain-specific network built with hundreds of hand-curated datasets.

MOTIVATION: Functional gene networks, representing how likely two genes work in the same biological process, are important models for studying gene interactions in complex tissues. However, a limitation of the current network-building scheme is the lack of leveraging evidence from multiple model organisms as well as the lack of expert curation and quality control of the input genomic data. RESULTS: Here, we present BaiHui, a brain-specific functional gene network built by probabilistically integrating expertly-hand-curated (by reading original publications) heterogeneous and multi-species genomic data in human, mouse and rat brains. To facilitate the use of this network, we deployed a web server through which users can query their genes of interest, visualize the network, gain functional insight from enrichment analysis and download network data. We also illustrated how this network could be used to generate testable hypotheses on disease gene prioritization of brain disorders. AVAILABILITY AND IMPLEMENTATION: BaiHui is freely available at: http://guanlab.ccmb.med.umich.edu/BaiHui/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Targeted exome analysis identifies the genetic basis of disease in over 50% of patients with a wide range of ataxia-related phenotypes.

PURPOSE: To examine the impact of a targeted exome approach for the molecular diagnosis of patients nationwide with a wide range of ataxia-related phenotypes. METHODS: One hundred and seventy patients with ataxia of unknown etiology referred from clinics throughout the United States and Canada were studied using a targeted exome approach. Patients ranged in age from 2 to 88 years. Analysis was focused on 441 curated genes associated with ataxia and ataxia-like conditions. RESULTS: Pathogenic and suspected diagnostic variants were identified in 88 of the 170 patients, providing a positive molecular diagnostic rate of 52%. Forty-six different genes were implicated, with the six most commonly mutated genes being SPG7, SYNE1, ADCK3, CACNA1A, ATP1A3, and SPTBN2, which accounted for >40% of the positive cases. In many cases a diagnosis was provided for conditions that were not suspected and resulted in the broadening of the clinical spectrum of several conditions. CONCLUSION: Exome sequencing with targeted analysis provides a high-yield approach for the genetic diagnosis of ataxia-related conditions. This is the largest targeted exome study performed to date in patients with ataxia and ataxia-like conditions and represents patients with a wide range of ataxia phenotypes typically encountered in neurology and genetics clinics.

Mutations in VPS13D lead to a new recessive ataxia with spasticity and mitochondrial defects.

OBJECTIVE: To identify novel causes of recessive ataxias, including spinocerebellar ataxia with saccadic intrusions, spastic ataxias, and spastic paraplegia. METHODS: In an international collaboration, we independently performed exome sequencing in 7 families with recessive ataxia and/or spastic paraplegia. To evaluate the role of VPS13D mutations, we evaluated a Drosophila knockout model and investigated mitochondrial function in patient-derived fibroblast cultures. RESULTS: Exome sequencing identified compound heterozygous mutations in VPS13D on chromosome 1p36 in all 7 families. This included a large family with 5 affected siblings with spinocerebellar ataxia with saccadic intrusions (SCASI), or spinocerebellar ataxia, recessive, type 4 (SCAR4). Linkage to chromosome 1p36 was found in this family with a logarithm of odds score of 3.1. The phenotypic spectrum in our 12 patients was broad. Although most presented with ataxia, additional or predominant spasticity was present in 5 patients. Disease onset ranged from infancy to 39 years, and symptoms were slowly progressive and included loss of independent ambulation in 5. All but 2 patients carried a loss-of-function (nonsense or splice site) mutation on one and a missense mutation on the other allele. Knockdown or removal of Vps13D in Drosophila neurons led to changes in mitochondrial morphology and impairment in mitochondrial distribution along axons. Patient fibroblasts showed altered morphology and functionality including reduced energy production. INTERPRETATION: Our study demonstrates that compound heterozygous mutations in VPS13D cause movement disorders along the ataxia-spasticity spectrum, making VPS13D the fourth VPS13 paralog involved in neurological disorders. Ann Neurol 2018.

ctDNA transiting into urine is ultrashort and facilitates noninvasive liquid biopsy of HPV+ oropharyngeal cancer.

BACKGROUNDTransrenal cell-free tumor DNA (TR-ctDNA), which transits from the bloodstream into urine, has the potential to enable noninvasive cancer detection for a wide variety of nonurologic cancer types.MethodsUsing whole-genome sequencing, we discovered that urine TR-ctDNA fragments across multiple cancer types are predominantly ultrashort (<50 bp) and, therefore, likely to be missed by conventional ctDNA assays. We developed an ultrashort droplet digital PCR assay to detect TR-ctDNA originating from HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) and confirmed that assaying ultrashort DNA is critical for sensitive cancer detection from urine samples.ResultsTR-ctDNA was concordant with plasma ctDNA for cancer detection in patients with HPV+ OPSCC. As proof of concept for using urine TR-ctDNA for posttreatment surveillance, in a small longitudinal case series, TR-ctDNA showed promise for noninvasive detection of recurrence of HPV+ OPSCC.ConclusionOur data indicate that focusing on ultrashort fragments of TR-ctDNA will be important for realizing the full potential of urine-based cancer diagnostics. This has implications for urine-based detection of a wide variety of cancer types and for facilitating access to care through at-home specimen collections.FundingNIH grants R33 CA229023, R21 CA225493; NIH/National Cancer Institute grants U01 CA183848, R01 CA184153, and P30CA046592; American Cancer Society RSG-18-062-01-TBG; American Cancer Society Mission Boost grant MBGI-22-056-01-MBG; and the A. Alfred Taubman Medical Research Institute.

Impacts of Salmonella enteritidis infection on liver transcriptome in broilers.

Salmonella enterica serovar enteritidis is an enteric bacterium that can contaminate chicken eggs and meat, resulting in production losses and consumer illness. To provide insight into the systemic metabolic effects of S. enteritidis infection, liver samples were harvested 10-days postinfection from broiler hens. Hepatic global gene expression levels were assessed using a chicken 44K Agilent microarray. Forty-four genes were differentially expressed at a significance level of q value < 0.05. One hundred eighty-three genes were differentially expressed at a suggestive significance level of q value < 0.1. A predominance of downregulation existed among significantly differentially expressed genes. Cell cycle and metabolism networks were created from the differentially expressed genes. Mitochondria-mediated apoptosis, electron transport, peptidase activity, vein constriction, cell differentiation, IL-2 signaling, Jak-Stat signaling, B-cell receptor signaling, GDP/GTP exchange, and protein recycling were among the functions of the differentially expressed genes that were down-regulated in response to S. enteritidis. The effects of S. enteritidis infection on the liver transcriptome profiles of broilers reflect a predominance of downregulation of genes with cell cycle and metabolic functions. The most pronounced response was the downregulation of genes that function in metabolic pathways, inflammation, and mitochondria-mediated apoptosis. These results provide insight into important systemic metabolic mechanisms that are active in the chicken liver in response to S. enteritidis infection at 10-days postinfection.

Development of a high-performance multi-probe droplet digital PCR assay for high-sensitivity detection of human papillomavirus circulating tumor DNA from plasma.

OBJECTIVES: To develop a high-performance droplet digital PCR (ddPCR) assay capable of enhancing the detection of human papillomavirus (HPV) circulating tumor DNA (ctDNA) in plasma from patients with HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC). MATERIALS AND METHODS: Plasma samples from subjects with HPV+ OPSCC were collected. We developed a high-performance ddPCR assay designed to simultaneously target nine regions of the HPV16 genome. RESULTS: The new assay termed 'ctDNA HPV16 Assessment using Multiple Probes' (CHAMP- 16) yielded significantly higher HPV16 counts compared to our previously validated 'Single-Probe' (SP) assay and a commercially available NavDx® assay. Analytical validation demonstrated that the CHAMP-16 assay had a limit of detection (LoD) of 4.1 copies per reaction, corresponding to < 1 genome equivalent (GE) of HPV16. When tested on plasma ctDNA from 21 patients with early-stage HPV+ OPSCC and known HPV16 ctDNA using the SP assay, all patients were positive for HPV16 ctDNA in both assays and the CHAMP-16 assay displayed 6.6-fold higher HPV16 signal on average. Finally, in a longitudinal analysis of samples from a patient with recurrent disease, the CHAMP-16 assay detected HPV16 ctDNA signal âˆ¼ 20 months prior to the conventional SP assay. CONCLUSION: Increased HPV16 signal detection using the CHAMP-16 assay suggests the potential for detection of recurrences significantly earlier than with conventional ddPCR assays in patients with HPV16+ OPSCC. Critically, this multi-probe approach maintains the cost-benefit advantage of ddPCR over next generation sequencing (NGS) approaches, supporting the cost-effectiveness of this assay for both large population screening and routine post-treatment surveillance.

Strong concordance between transcriptomic patterns of spleen and peripheral blood leukocytes in response to avian pathogenic Escherichia coli infection.

Avian pathogenic Escherichia coli (APEC) causes morbidity in chickens and exhibits zoonotic potential. Understanding host transcriptional responses to infection aids the understanding of protective mechanisms and serves to inform future colibacillosis control strategies. Transcriptomes of spleen and peripheral blood leukocytes (PBLs) of the same individual birds in response to APEC infection were compared to identify common response patterns and connecting pathways. More than 100 genes in three contrasts examining pathology and infection status were significantly differentially expressed in both tissues and similarly regulated. Tissue-specific differences in catalytic activity, however, appear between birds with mild and severe pathology responses. Early expression differences, between birds with severe pathology and uninfected controls, in the mitogen-activated protein kinase pathway in PBLs precede spleen responses in the p53 and cytokine-cytokine receptor pathways. Tissue bianalysis is useful in identifying genes and pathways important to the response to APEC, whose role might otherwise be underestimated in importance.

Attomolar Sensitivity in Single Biomarker Counting upon Aqueous Two-Phase Surface Enrichment.

From longstanding techniques like enzyme-linked immunosorbent assay (ELISA) to modern next-generation sequencing, many of the most sensitive and specific biomarker detection assays require capture of the analyte at a surface. While surface-based assays provide advantages, including the ability to reduce background by washing away excess reagents and/or increase specificity through analyte-specific capture probes, the limited efficiency of capture from dilute solution often restricts assay sensitivity to the femtomolar-to-nanomolar range. Although assays for many nucleic acid analytes can decrease limits of detection (LODs) to the subfemtomolar range using polymerase chain reaction, such amplification may introduce biases, errors, and an increased risk of sample cross-contamination. Furthermore, many analytes cannot be amplified easily, including short nucleic acid fragments, epigenetic modifications, and proteins. To address the challenge of achieving subfemtomolar LODs in surface-based assays without amplification, we exploit an aqueous two-phase system (ATPS) to concentrate target molecules in a smaller-volume phase near the assay surface, thus increasing capture efficiency compared to passive diffusion from the original solution. We demonstrate the utility of ATPS-enhanced capture via single molecule recognition through equilibrium Poisson sampling (SiMREPS), a microscopy technique previously shown to possess >99.9999% detection specificity for DNA mutations but an LOD of only ∼1-5 fM. By combining ATPS-enhanced capture with a Förster resonance energy transfer (FRET)-based probe design for rapid data acquisition over many fields of view, we improve the LOD ∼ 300-fold to <10 aM for an EGFR exon 19 deletion mutation. We further validate this ATPS-assisted FRET-SiMREPS assay by detecting endogenous exon 19 deletion molecules in cancer patient blood plasma.

Improved prediction of radiation pneumonitis by combining biological and radiobiological parameters using a data-driven Bayesian network analysis.

Grade 2 and higher radiation pneumonitis (RP2) is a potentially fatal toxicity that limits efficacy of radiation therapy (RT). We wished to identify a combined biomarker signature of circulating miRNAs and cytokines which, along with radiobiological and clinical parameters, may better predict a targetable RP2 pathway. In a prospective clinical trial of response-adapted RT for patients (n = 39) with locally advanced non-small cell lung cancer, we analyzed patients' plasma, collected pre- and during RT, for microRNAs (miRNAs) and cytokines using array and multiplex enzyme linked immunosorbent assay (ELISA), respectively. Interactions between candidate biomarkers, radiobiological, and clinical parameters were analyzed using data-driven Bayesian network (DD-BN) analysis. We identified alterations in specific miRNAs (miR-532, -99b and -495, let-7c, -451 and -139-3p) correlating with lung toxicity. High levels of soluble tumor necrosis factor alpha receptor 1 (sTNFR1) were detected in a majority of lung cancer patients. However, among RP patients, within 2 weeks of RT initiation, we noted a trend of temporary decline in sTNFR1 (a physiological scavenger of TNFα) and ADAM17 (a shedding protease that cleaves both membrane-bound TNFα and TNFR1) levels. Cytokine signature identified activation of inflammatory pathway. Using DD-BN we combined miRNA and cytokine data along with generalized equivalent uniform dose (gEUD) to identify pathways with better accuracy of predicting RP2 as compared to either miRNA or cytokines alone. This signature suggests that activation of the TNFα-NFκB inflammatory pathway plays a key role in RP which could be specifically ameliorated by etanercept rather than current therapy of non-specific leukotoxic corticosteroids.

Spleen transcriptome response to infection with avian pathogenic Escherichia coli in broiler chickens.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) is detrimental to poultry health and its zoonotic potential is a food safety concern. Regulation of antimicrobials in food-production animals has put greater focus on enhancing host resistance to bacterial infections through genetics. To better define effective mechanism of host resistance, global gene expression in the spleen of chickens, harvested at two times post-infection (PI) with APEC, was measured using microarray technology, in a design that will enable investigation of effects of vaccination, challenge, and pathology level. RESULTS: There were 1,101 genes significantly differentially expressed between severely infected and non-infected groups on day 1 PI and 1,723 on day 5 PI. Very little difference was seen between mildly infected and non-infected groups on either time point. Between birds exhibiting mild and severe pathology, there were 2 significantly differentially expressed genes on day 1 PI and 799 on day 5 PI. Groups with greater pathology had more genes with increased expression than decreased expression levels. Several predominate immune pathways, Toll-like receptor, Jak-STAT, and cytokine signaling, were represented between challenged and non-challenged groups. Vaccination had, surprisingly, no detectible effect on gene expression, although it significantly protected the birds from observable gross lesions. Functional characterization of significantly expressed genes revealed unique gene ontology classifications during each time point, with many unique to a particular treatment or class contrast. CONCLUSIONS: More severe pathology caused by APEC infection was associated with a high level of gene expression differences and increase in gene expression levels. Many of the significantly differentially expressed genes were unique to a particular treatment, pathology level or time point. The present study not only investigates the transcriptomic regulations of APEC infection, but also the degree of pathology associated with that infection. This study will allow for greater discovery into host mechanisms for disease resistance, providing targets for marker assisted selection and advanced drug development.

Machine learning-based cytokine microarray digital immunoassay analysis.

Serial measurement of a large panel of protein biomarkers near the bedside could provide a promising pathway to transform the critical care of acutely ill patients. However, attaining the combination of high sensitivity and multiplexity with a short assay turnaround poses a formidable technological challenge. Here, the authors develop a rapid, accurate, and highly multiplexed microfluidic digital immunoassay by incorporating machine learning-based autonomous image analysis. The assay has achieved 12-plexed biomarker detection in sample volume <15 µL at concentrations < 5 pg/mL while only requiring a 5-min assay incubation, allowing for all processes from sampling to result to be completed within 40 min. The assay procedure applies both a spatial-spectral microfluidic encoding scheme and an image data analysis algorithm based on machine learning with a convolutional neural network (CNN) for pre-equilibrated single-molecule protein digital counting. This unique approach remarkably reduces errors facing the high-capacity multiplexing of digital immunoassay at low protein concentrations. Longitudinal data obtained for a panel of 12 serum cytokines in human patients receiving chimeric antigen receptor-T (CAR-T) cell therapy reveals the powerful biomarker profiling capability. The assay could also be deployed for near-real-time immune status monitoring of critically ill COVID-19 patients developing cytokine storm syndrome.

Human papilloma virus circulating tumor DNA assay predicts treatment response in recurrent/metastatic head and neck squamous cell carcinoma.

Despite the rising incidence of human papillomavirus related (HPV+) oropharyngeal squamous cell carcinoma (OPSCC), treatment of metastatic disease remains palliative. Even with new treatments such as immunotherapy, response rates are low and can be delayed, while even mild tumor progression in the face of an ineffective therapy can lead to rapid death. Real-time biomarkers of response to therapy could improve outcomes by guiding early change of therapy in the metastatic setting. Herein, we developed and analytically validated a new droplet digital PCR (ddPCR)-based assay for HPV16 circulating tumor DNA (ctDNA) and evaluated plasma HPV16 ctDNA for predicting treatment response in metastatic HPV+ OPSCC. We found that longitudinal changes HPV16 ctDNA correlate with treatment response and that ctDNA responses are observed earlier than conventional imaging (average 70 days, range: 35-166). With additional validation in multi-site studies, this assay may enable early identification of treatment failure, allowing patients to be directed promptly toward clinical trials or alternative therapies.

Monitoring Health Care Workers at Risk for COVID-19 Using Wearable Sensors and Smartphone Technology: Protocol for an Observational mHealth Study.

BACKGROUND: Health care workers (HCWs) have been working on the front lines of the COVID-19 pandemic with high risks of viral exposure, infection, and transmission. Standard COVID-19 testing is insufficient to protect HCWs from these risks and prevent the spread of disease. Continuous monitoring of physiological data with wearable sensors, self-monitoring of symptoms, and asymptomatic COVID-19 testing may aid in the early detection of COVID-19 in HCWs and may help reduce further transmission among HCWs, patients, and families. OBJECTIVE: By using wearable sensors, smartphone-based symptom logging, and biospecimens, this project aims to assist HCWs in self-monitoring COVID-19. METHODS: We conducted a prospective, longitudinal study of HCWs at a single institution. The study duration was 1 year, wherein participants were instructed on the continuous use of two wearable sensors (Fitbit Charge 3 smartwatch and TempTraq temperature patches) for up to 30 days. Participants consented to provide biospecimens (ie, nasal swabs, saliva swabs, and blood) for up to 1 year from study entry. Using a smartphone app called Roadmap 2.0, participants entered a daily mood score, submitted daily COVID-19 symptoms, and completed demographic and health-related quality of life surveys at study entry and 30 days later. Semistructured qualitative interviews were also conducted at the end of the 30-day period, following completion of daily mood and symptoms reporting as well as continuous wearable sensor use. RESULTS: A total of 226 HCWs were enrolled between April 28 and December 7, 2020. The last participant completed the 30-day study procedures on January 16, 2021. Data collection will continue through January 2023, and data analyses are ongoing. CONCLUSIONS: Using wearable sensors, smartphone-based symptom logging and survey completion, and biospecimen collections, this study will potentially provide data on the prevalence of COVID-19 infection among HCWs at a single institution. The study will also assess the feasibility of leveraging wearable sensors and self-monitoring of symptoms in an HCW population. TRIAL REGISTRATION: ClinicalTrials.gov NCT04756869; https://clinicaltrials.gov/ct2/show/NCT04756869. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/29562.