Cerebrospinal Fluid Protein Markers Indicate Neuro-Damage in SARS-CoV-2-Infected Nonhuman Primates.

Neurologic manifestations are among the most frequently reported complications of COVID-19. However, given the paucity of tissue samples and the highly infectious nature of the etiologic agent of COVID-19, we have limited information to understand the neuropathogenesis of COVID-19. Therefore, to better understand the impact of COVID-19 on the brain, we used mass-spectrometry-based proteomics with a data-independent acquisition mode to investigate cerebrospinal fluid (CSF) proteins collected from two different nonhuman primates, Rhesus Macaque and African Green Monkeys, for the neurologic effects of the infection. These monkeys exhibited minimal to mild pulmonary pathology but moderate to severe central nervous system (CNS) pathology. Our results indicated that CSF proteome changes after infection resolution corresponded with bronchial virus abundance during early infection and revealed substantial differences between the infected nonhuman primates and their age-matched uninfected controls, suggesting these differences could reflect altered secretion of CNS factors in response to SARS-CoV-2-induced neuropathology. We also observed the infected animals exhibited highly scattered data distributions compared to their corresponding controls indicating the heterogeneity of the CSF proteome change and the host response to the viral infection. Dysregulated CSF proteins were preferentially enriched in functional pathways associated with progressive neurodegenerative disorders, hemostasis, and innate immune responses that could influence neuroinflammatory responses following COVID-19. Mapping these dysregulated proteins to the Human Brain Protein Atlas found that they tended to be enriched in brain regions that exhibit more frequent injury following COVID-19. It, therefore, appears reasonable to speculate that such CSF protein changes could serve as signatures for neurologic injury, identify important regulatory pathways in this process, and potentially reveal therapeutic targets to prevent or attenuate the development of neurologic injuries following COVID-19.

Serum Cell-Free DNA-based Detection of Mycobacterium avium Complex Infection.

Rationale: Mycobacterium avium complex (MAC) is the most common cause of nontuberculous mycobacterial (NTM) pulmonary disease (PD), which exhibits increasing global incidence. Current microbiologic methods routinely used in clinical practice lack sensitivity and have long latencies, leading to delays in diagnosis and treatment initiation and evaluation. A clustered regularly interspaced short palindromic repeats (CRISPR)-based assay that measures MAC cell-free DNA (cfDNA) concentrations in serum could provide a rapid means to detect MAC infection and monitor response to antimicrobial treatment. Objectives: To develop and optimize a CRISPR MAC assay for MAC infection detection and to evaluate its diagnostic and prognostic performance in two MAC disease cohorts. Methods: MAC cfDNA serum concentrations were measured in individuals with diagnoses of MAC disease or who had bronchiectasis or chronic obstructive pulmonary disease diagnoses without histories of NTM PD or NTM-positive sputum cultures. Diagnostic performance was analyzed using pretreatment serum from two cohorts. Serum MAC cfDNA changes during MAC PD treatment were evaluated in a subset of patients with MAC PD who received macrolide-based multidrug regimens. Measurements and Main Results: The CRISPR MAC assay detected MAC cfDNA in MAC PD with 97.6% (91.6-99.7%) sensitivity and 97.6% (91.5-99.7%) specificity overall. Serum MAC cfDNA concentrations markedly decreased after MAC-directed treatment initiation in patients with MAC PD who demonstrated MAC culture conversion. Conclusions: This study provides preliminary evidence for the utility of a serum-based CRISPR MAC assay to rapidly detect MAC infection and monitor the response to treatment.

Monocrystalline Labeling Enables Stable Plasmonic Enhancement for Isolation-Free Extracellular Vesicle Analysis.

Sensitive detection of extracellular vesicles (EVs) as emerging biomarkers has shown great promises for disease diagnosis. Plasmonic metal nanostructures conjugated with molecules that bind specific biomarker targets are widely used for EVs sensing but involve tradeoffs between particle-size-dependent signal intensity and conjugation efficiency. One solution to this problem would be to induce nucleation on nanoparticles that have successfully bound a target biomarker to permit in situ nanoparticle growth for signal amplification, but approaches that are evaluated to date require harsh conditions or lack nucleation specificity, prohibiting their effective use with most biological specimens. This study describes a one-step in situ strategy to induce monocrystalline copper shell growth on gold nanorod probes without decreasing signal by disrupting probe-target interactions or lipid bilayer integrity to enable EV biomarker detections. This approach increases the detected nanoparticle signal about two orders of magnitude after a 10 min copper nanoshell growth reaction. This has significant implications for improved disease detection, as indicated by the ability of a novel immunoassay using this approach to detect low abundance EVs carrying a pathogen-derived biomarker, after their direct capture from serum, to facilitate the diagnosis of tuberculosis cases in a diagnostically challenging pediatric cohort.

Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants.

Six ebolavirus species are reported to date, including human pathogens Bundibugyo virus (BDBV), Ebola virus (EBOV), Sudan virus (SUDV), and Taï Forest virus (TAFV); non-human pathogen Reston virus (RESTV); and the plausible Bombali virus (BOMV). Since there are differences in the disease severity caused by different species, species identification and viral burden quantification are critical for treating infected patients timely and effectively. Here we developed an immunoprecipitation-coupled mass spectrometry (IP-MS) assay for VP40 antigen detection and quantification. We carefully selected two regions of VP40, designated as peptide 8 and peptide12 from the protein sequence that showed minor variations among Ebolavirus species through MS analysis of tryptic peptides and antigenicity prediction based on available bioinformatic tools, and generated high-quality capture antibodies pan-specific for these variant peptides. We applied this assay to human plasma spiked with recombinant VP40 protein from EBOV, SUDV, and BDBV and virus-like particles (VLP), as well as EBOV infected NHP plasma. Sequence substitutions between EBOV and SUDV, the two species with highest lethality, produced affinity variations of 2.6-fold for p8 and 19-fold for p12. The proposed IP-MS assay differentiates four of the six known EBV species in one assay, through a combination of p8 and p12 data. The IP-MS assay limit of detection (LOD) using multiple reaction monitoring (MRM) as signal readout was determined to be 28 ng/mL and 7 ng/mL for EBOV and SUDV respectively, equivalent to ~1.625-6.5×105 Geq/mL, and comparable to the LOD of lateral flow immunoassays currently used for Ebola surveillance. The two peptides of the IP-MS assay were also identified by their tandem MS spectra using a miniature MALDI-TOF MS instrument, greatly increasing the feasibility of high specificity assay in a decentralized laboratory.

Sensitive Blood-Based Detection of HIV-1 and Mycobacterium tuberculosis Peptides for Disease Diagnosis by Immuno-Affinity Liquid Chromatography-Tandem Mass Spectrometry: A Method Development and Proof-of-Concept Study.

BACKGROUND: Novel approaches that allow early diagnosis and treatment monitoring of both human immunodeficiency virus-1 (HIV-1) and tuberculosis disease (TB) are essential to improve patient outcomes. METHODS: We developed and validated an immuno-affinity liquid chromatography-tandem mass spectrometry (ILM) assay that simultaneously quantifies single peptides derived from HIV-1 p24 and Mycobacterium tuberculosis (Mtb) 10-kDa culture filtrate protein (CFP10) in trypsin-digested serum derived from cryopreserved serum archives of cohorts of adults and children with/without HIV and TB. RESULTS: ILM p24 and CFP10 results demonstrated good intra-laboratory precision and accuracy, with recovery values of 96.7% to 104.6% and 88.2% to 111.0%, total within-laboratory precision (CV) values of 5.68% to 13.25% and 10.36% to 14.92%, and good linearity (r2 > 0.99) from 1.0 to 256.0 pmol/L and 0.016 to 16.000 pmol/L, respectively. In cohorts of adults (n = 34) and children (n = 17) with HIV and/or TB, ILM detected p24 and CFP10 demonstrated 85.7% to 88.9% and 88.9% to 100.0% diagnostic sensitivity for HIV-1 and TB, with 100% specificity for both, and detected HIV-1 infection earlier than 3 commercial p24 antigen/antibody immunoassays. Finally, p24 and CFP10 values measured in longitudinal serum samples from children with HIV-1 and TB distinguished individuals who responded to TB treatment from those who failed to respond or were untreated, and who developed TB immune reconstitution inflammatory syndrome. CONCLUSIONS: Simultaneous ILM evaluation of p24 and CFP10 results may allow for early TB and HIV detection and provide valuable information on treatment response to facilitate integration of TB and HIV diagnosis and management.

Evaluation of a serum-based antigen test for tuberculosis in HIV-exposed infants: a diagnostic accuracy study.

BACKGROUND: Non-sputum methods are urgently needed to improve tuberculosis diagnosis and treatment monitoring in children. This study evaluated the ability of a serum assay quantifying a species-specific peptide of the Mycobacterium tuberculosis CFP-10 virulence factor via nanotechnology and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to diagnose tuberculosis in HIV-infected and HIV-uninfected infants. METHODS: Serum CFP-10 peptide signal was blinded evaluated in cryopreserved sera of 519 BCG-immunized, HIV-exposed infants (284 HIV-infected, 235 HIV-uninfected) from a multi-center randomized placebo-controlled isoniazid prophylaxis trial conducted in southern Africa between 2004 and 2008, who were followed up to 192 weeks for Mtb infection and TB. Children were classified as confirmed, unconfirmed, or unlikely tuberculosis cases using 2015 NIH diagnostic criteria for pediatric TB. RESULTS: In HIV-infected infants, CFP-10 signal had 100% sensitivity for confirmed TB (5/5, 95% CI, 47.8-100) and 83.7% sensitivity for unconfirmed TB (36/43, 95% CI 69.3-93.2), with 93.1% specificity (203/218, 95% CI 88.9-96.1). In HIV-uninfected infants, CFP-10 signal detected the single confirmed TB case and 75.0% of unconfirmed TB cases (15/20; 95% CI 50.9-91.3), with 96.2% specificity (177/184, 95% CI, 92.3-98.5). Serum CFP-10 achieved 77% diagnostic sensitivity for confirmed and unconfirmed TB (13/17, 95% CI, 50-93%) at ≤ 24 weeks pre-diagnosis, and both CFP-10-positivity and concentration declined following anti-TB therapy initiation. CONCLUSIONS: Serum CFP-10 signal exhibited high diagnostic sensitivity and specificity for tuberculosis in HIV-infected and HIV-uninfected infants and potential utility for early TB detection and monitoring of anti-TB treatment responses.

Serum-Based Diagnosis of Pediatric Tuberculosis by Assay of Mycobacterium tuberculosis Factors: a Retrospective Cohort Study.

Diagnosis of pediatric tuberculosis (TB) is often complicated by its nonspecific symptoms, paucibacillary nature, and the need for invasive specimen collection techniques. However, a recently reported assay that detects Mycobacterium tuberculosis virulence factors in serum can diagnose various TB manifestations, including paucibacillary TB cases, in adults with good sensitivity and specificity. The current study examined the ability of this M. tuberculosis biomarker assay to diagnose pediatric TB using archived cryopreserved serum samples drawn from children ≤18 years of age who were screened for suspected TB as part of a prospective population-based active surveillance study. In this analysis, any detectable level of either of the M. tuberculosis virulence factors CFP-10 and ESAT-6 was considered direct evidence of TB. Serum samples from 105 children evaluated for TB (55 TB cases and 50 close contacts without TB) were analyzed. The results of this analysis yielded sensitivity of 85.5% (95% confidence interval [CI], 73.3 to 93.5). Similar diagnostic sensitivities were observed for culture-positive (87.5%; 95% CI, 67.6 to 97.3) and culture-negative (83.9%; 95% CI, 66.3 to 94.5) TB cases and for culture negative pulmonary (77.8%; 95% CI, 40.0 to 97.2) and extrapulmonary (86.4%; 95% CI, 65.1 to 97.1) TB cases. These results suggest that serum biomarker analysis holds significant promise for rapid and sensitive diagnosis of pediatric TB cases, including extrapulmonary or paucibacillary TB cases. The ability to use frozen samples for this analysis should also permit assays to be performed at central sites, without a requirement for strict timelines for sample analysis.

Tumor-derived exosomes (TDEs): How to avoid the sting in the tail.

Exosomes are abundantly secreted extracellular vesicles that accumulate in the circulation and are of great interest for disease diagnosis and evaluation since their contents reflects the phenotype of their cell of origin. Tumor-derived exosomes (TDEs) are of particular interest for cancer diagnosis and therapy, since most tumor demonstrate highly elevated exosome secretion rates and provide specific information about the genotype of a tumor and its response to treatment. TDEs also contain regulatory factors that can alter the phenotypes of local and distant tissue sites and alter immune cell functions to promote tumor progression. The abundance, information content, regulatory potential, in vivo half-life, and physical durability of exosomes suggest that TDEs may represent a superior source of diagnostic biomarkers and treatment targets than other materials currently under investigation. This review will summarize current information on mechanisms that may differentially regulate TDE biogenesis, TDE effects on the immune system that promote tumor survival, growth, and metastasis, and new approaches understudy to counteract or utilize TDE properties in cancer therapies.

Quantification of circulating Mycobacterium tuberculosis antigen peptides allows rapid diagnosis of active disease and treatment monitoring.

Tuberculosis (TB) is a major global health threat, resulting in an urgent unmet need for a rapid, non-sputum-based quantitative test to detect active Mycobacterium tuberculosis (Mtb) infections in clinically diverse populations and quickly assess Mtb treatment responses for emerging drug-resistant strains. We have identified Mtb-specific peptide fragments and developed a method to rapidly quantify their serum concentrations, using antibody-labeled and energy-focusing porous discoidal silicon nanoparticles (nanodisks) and high-throughput mass spectrometry (MS) to enhance sensitivity and specificity. NanoDisk-MS diagnosed active Mtb cases with high sensitivity and specificity in a case-control study with cohorts reflecting the complexity of clinical practice. Similar robust sensitivities were obtained for cases of culture-positive pulmonary TB (PTB; 91.3%) and extrapulmonary TB (EPTB; 92.3%), and the sensitivities obtained for culture-negative PTB (82.4%) and EPTB (75.0%) in HIV-positive patients significantly outperformed those reported for other available assays. NanoDisk-MS also exhibited high specificity (87.1-100%) in both healthy and high-risk groups. Absolute quantification of serum Mtb antigen concentration was informative in assessing responses to antimycobacterial treatment. Thus, a NanoDisk-MS assay approach could significantly improve the diagnosis and management of active TB cases, and perhaps other infectious diseases as well.

Rapid Lipid-Based Approach for Normalization of Quantum-Dot-Detected Biomarker Expression on Extracellular Vesicles in Complex Biological Samples.

Extracellular vesicles (EVs) are of considerable interest as tumor biomarkers because tumor-derived EVs contain a broad array of information about tumor pathophysiology. However, current EV assays cannot distinguish between EV biomarker differences resulting from altered abundance of a target EV population with stable biomarker expression, altered biomarker expression in a stable target EV population, or effects arising from changes in both parameters. We now describe a rapid nanoparticle- and dye-based fluorescent immunoassay that can distinguish among these possibilities by normalizing EV biomarker levels to EV abundance. In this approach, EVs are captured from complex samples (e.g., serum), stained with a lipophilic dye, and hybridized with antibody-conjugated quantum dot probes for specific EV surface biomarkers. EV dye signal is used to quantify EV abundance and normalize EV surface biomarker expression levels. EVs from malignant and nonmalignant pancreatic cell lines exhibited similar staining, and probe-to-dye ratios did not change with EV abundance, allowing direct analysis of normalized EV biomarker expression without a separate EV quantification step. This EV biomarker normalization approach markedly improved the ability of serum levels of two pancreatic cancer biomarkers, EV EpCAM and EV EphA2, to discriminate pancreatic cancer patients from nonmalignant control subjects. The streamlined workflow and robust results of this assay are suitable for rapid translation to clinical applications and its modular design permits it to be rapidly adapted to quantitate other EV biomarkers by the simple expedient of swapping the antibody-conjugated quantum dot probes for those that recognize a different disease-specific EV biomarker.

Clinical Evaluation of a Blood Assay to Diagnose Paucibacillary Tuberculosis via Bacterial Antigens.

BACKGROUND: The diagnosis of active tuberculosis (TB) cases primarily relies on methods that detect Mycobacterium tuberculosis (Mtb) bacilli or their DNA in patient samples (e.g., mycobacterial culture and Xpert MTB/RIF assays), but these tests have low clinical sensitivity for patients with paucibacillary TB disease. Our goal was to evaluate the clinical performance of a newly developed assay that can rapidly diagnose active TB cases by direct detection of Mtb-derived antigens in patients' blood samples. METHODS: Nanoparticle (NanoDisk)-enriched peptides derived from the Mtb virulence factors CFP-10 (10-kDa culture factor protein) and ESAT-6 (6-kDa early secretory antigenic target) were analyzed by high-throughput mass spectrometry (MS). Serum from 294 prospectively enrolled Chinese adults were analyzed with this NanoDisk-MS method to evaluate the performance of direct serum Mtb antigen measurement as a means for rapid diagnosis of active TB cases. RESULTS: NanoDisk-MS diagnosed 174 (88.3%) of the study's TB cases, with 95.8% clinical specificity, and with 91.6% and 85.3% clinical sensitivity for culture-positive and culture-negative TB cases, respectively. NanoDisk-MS also exhibited 88% clinical sensitivity for pulmonary and 90% for extrapulmonary TB, exceeding the diagnostic performance of mycobacterial culture for these cases. CONCLUSIONS: Direct detection and quantification of serum Mtb antigens by NanoDisk-MS can rapidly and accurately diagnose active TB in adults, independent of disease site or culture status, and outperform Mycobacterium-based TB diagnostics.

Antigen 85B peptidomic analysis allows species-specific mycobacterial identification.

BACKGROUND: Nontuberculous mycobacteria (NTM)-mediated infections are a growing cause of worldwide morbidity, but lack of rapid diagnostics for specific NTM species can delay the initiation of appropriate treatment regimens. We thus examined whether mass spectrometry analysis of an abundantly secreted mycobacterial antigen could identify specific NTM species. METHODS: We analyzed predicted tryptic peptides of the major mycobacterial antigen Ag85B for their capacity to distinguish Mycobacterium tuberculosis and three NTM species responsible for the majority of pulmonary infections caused by slow-growing mycobacterial species. Next, we analyzed trypsin-digested culture supernatants of these four mycobacterial species by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect candidate species-specific Ag85B peptides, the identity of which were validated by LC-MS/MS performed in parallel reaction monitoring mode. RESULTS: Theoretical tryptic digests of the Ag85B proteins of four common mycobacterial species produced peptides with distinct sequences, including two peptides that could each identify the species origin of each Ag85B protein. LC-MS/MS analysis of trypsinized culture supernatants of these four species detected one of these species-specific signature peptides in each sample. Subsequent LC-MS/MS analyses confirmed these results by targeting these species-specific Ag85B peptides. CONCLUSIONS: LC-MS/MS analysis of Ag85B peptides from trypsin-digested mycobacterial culture supernatants can rapidly detect and identify common mycobacteria responsible for most pulmonary infections caused by slow-growing mycobacteria, and has the potential to rapidly diagnose pulmonary infections caused by these mycobacteria through direct analysis of clinical specimens.

Rapid diagnosis of new and relapse tuberculosis by quantification of a circulating antigen in HIV-infected adults in the Greater Houston metropolitan area.

BACKGROUND: HIV-associated immune defects inhibit tuberculosis (TB) diagnosis, promote development of extrapulmonary TB and paucibacillary pulmonary TB cases with atypical radiographic features, and increase TB relapse rates. We therefore assessed the diagnostic performance of a novel assay that directly quantitates serum levels of the Mycobacterium tuberculosis (Mtb) virulence factor 10-kDa culture filtrate protein (CFP-10) to overcome limitations associated with detecting Mtb bacilli in sputum or tissue biopsies. METHODS: This study analyzed HIV-positive adults enrolled in a large, population-based TB screening and surveillance project, the Houston Tuberculosis Initiative, between October 1995 and September 2004, and assigned case designations using standardized criteria. Serum samples were trypsin-digested and immunoprecipitated for an Mtb-specific peptide of CFP-10 that was quantified by liquid chromatography-mass spectrometry for rapid and sensitive TB diagnosis. RESULTS: Among the 1053 enrolled patients, 110 met all inclusion criteria; they included 60 tuberculosis cases (12 culture-negative TB), including 9 relapse TB cases, and 50 non-TB controls, including 15 cases with history of TB. Serum CFP-10 levels diagnosed 89.6% (77.3-96.5) and 66.7% (34.9-90.1) of culture-positive and culture-negative TB cases, respectively, and exhibited 88% (75.7-95.5) diagnostic specificity in all non-TB controls. Serum antigen detection and culture, respectively, identified 85% (73.4-92.9) and 80.0% (67.3-88.8) of all 60 TB cases. CONCLUSIONS: Quantitation of the Mtb virulence factor CFP-10 in serum samples of HIV-infected subjects diagnosed active TB cases with high sensitivity and specificity and detected cases missed by the gold standard of Mtb culture. These results suggest that serum CFP-10 quantitation holds great promise for the rapid diagnosis of suspected TB cases in patients who are HIV-infected.

Noise Reduction Method for Quantifying Nanoparticle Light Scattering in Low Magnification Dark-Field Microscope Far-Field Images.

Nanoparticles have become a powerful tool for cell imaging and biomolecule, cell and protein interaction studies, but are difficult to rapidly and accurately measure in most assays. Dark-field microscope (DFM) image analysis approaches used to quantify nanoparticles require high-magnification near-field (HN) images that are labor intensive due to a requirement for manual image selection and focal adjustments needed when identifying and capturing new regions of interest. Low-magnification far-field (LF) DFM imagery is technically simpler to perform but cannot be used as an alternate to HN-DFM quantification, since it is highly sensitive to surface artifacts and debris that can easily mask nanoparticle signal. We now describe a new noise reduction approach that markedly reduces LF-DFM image artifacts to allow sensitive and accurate nanoparticle signal quantification from LF-DFM images. We have used this approach to develop a "Dark Scatter Master" (DSM) algorithm for the popular NIH image analysis program ImageJ, which can be readily adapted for use with automated high-throughput assay analyses. This method demonstrated robust performance quantifying nanoparticles in different assay formats, including a novel method that quantified extracellular vesicles in patient blood sample to detect pancreatic cancer cases. Based on these results, we believe our LF-DFM quantification method can markedly decrease the analysis time of most nanoparticle-based assays to impact both basic research and clinical analyses.

A Panorama of Extracellular Vesicle Applications: From Biomarker Detection to Therapeutics.

Extracellular vesicles (EVs) secreted by all cell types are involved in the cell-to-cell transfer of regulatory factors that influence cell and tissue phenotypes in normal and diseased tissues. EVs are thus a rich source of biomarker targets for assays that analyze blood and urinary EVs for disease diagnosis. Sensitive biomarker detection in EVs derived from specific cell populations is a key major hurdle when analyzing complex biological samples, but innovative approaches surveyed in this Perspective can streamline EV isolation and enhance the sensitivity of EV detection procedures required for clinical application of EV-based diagnostics and therapeutics, including nanotechnology and microfluidics, to achieve EV characterizations. Finally, this Perspective also outlines opportunities and challenges remaining for clinical translation of EV-based assays.

Climate change, its impact on emerging infectious diseases and new technologies to combat the challenge.

ABSTRACTImproved sanitation, increased access to health care, and advances in preventive and clinical medicine have reduced the mortality and morbidity rates of several infectious diseases. However, recent outbreaks of several emerging infectious diseases (EIDs) have caused substantial mortality and morbidity, and the frequency of these outbreaks is likely to increase due to pathogen, environmental, and population effects driven by climate change. Extreme or persistent changes in temperature, precipitation, humidity, and air pollution associated with climate change can, for example, expand the size of EID reservoirs, increase host-pathogen and cross-species host contacts to promote transmission or spillover events, and degrade the overall health of susceptible host populations leading to new EID outbreaks. It is therefore vital to establish global strategies to track and model potential responses of candidate EIDs to project their future behaviour and guide research efforts on early detection and diagnosis technologies and vaccine development efforts for these targets. Multi-disciplinary collaborations are demanding to develop effective inter-continental surveillance and modelling platforms that employ artificial intelligence to mitigate climate change effects on EID outbreaks. In this review, we discuss how climate change has increased the risk of EIDs and describe novel approaches to improve surveillance of emerging pathogens that pose the risk for EID outbreaks, new and existing measures that could be used to contain or reduce the risk of future EID outbreaks, and new methods to improve EID tracking during further outbreaks to limit disease transmission.

Nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2), a key regulator of the antioxidant response to protect against atherosclerosis and nonalcoholic steatohepatitis.

Tissue oxidative stress is a common hallmark of atherosclerosis and non-alcoholic steatohepatitis (NASH), 2 conditions linked epidemiologically and pathophysiologically. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is the master regulator of inducible antioxidant responses, that can attenuate cellular injury from oxidative stress induced by obesity and other redox insults. Nrf2 expression and activation is reduced in mouse and human vessels that harbor accelerated atherosclerosis and in livers with histologic criteria of NASH. Systemic antioxidants have thus been attractive therapeutic targets, but clinical trials have been largely unsuccessful in improving cardiovascular health. Macrophage-selective Nrf2 activation may, however, provide an approach to reduce vascular and hepatocyte injury without the complications of systemic antioxidants, since macrophages play key roles in the development and progression of both atherosclerosis and NASH. In this article, we review the common mechanisms of oxidative stress and inflammation in atherosclerosis and NASH, and discuss the role of Nrf2 in vascular and hepatocyte protection.

Myeloid deletion of nuclear factor erythroid 2-related factor 2 increases atherosclerosis and liver injury.

OBJECTIVE: To determine the impact of hematopoietic deletion of nuclear factor- (erythroid-derived 2) like 2 factor (Nrf2) on the development of atherosclerosis and liver injury in an obese, hypercholesterolemic mouse model. METHODS AND RESULTS: Two-month-old male low-density lipoprotein receptor-deficient mice were lethally irradiated and transplanted with either wild type or Nrf2-deficient (Nrf2(-/-)) bone marrow cells. At 3 months of age, mice were placed on an obesogenic high-fat diet (HFD), high-cholesterol diet for 7 months. Despite no differences in body weight, body fat percentage, liver fat, plasma glucose, lipids, or insulin, the HFD-fed Nrf2(-/-) bone marrow recipients had increased proinflammatory vascular gene expression, a significant increase in atherosclerosis area (18% versus 28%; P=0.018) and lesion complexity, and a marked increase in liver fibrosis. The acceleration of vascular and liver injury may arise from enhanced macrophage migration, inflammation, and oxidative stress resulting from myeloid Nrf2 deficiency. CONCLUSIONS: Myeloid-derived Nrf2 activity attenuates atherosclerosis development and liver inflammation and fibrosis associated with obesity. Prevention of oxidative stress in macrophage and other myeloid lineage cells may be an important therapeutic target to reduce inflammation-driven complications of obesity.

CRISPR-based assays for low-resource settings.

CRISPR-based assays can be adopted as ultrasensitive molecular diagnostics in resource-limited settings, but point-of-care applications must address additional requirements. Here, we discuss the major obstacles for developing these assays and offer insights into how to surmount them.

Outlook for CRISPR-based tuberculosis assays now in their infancy.

Tuberculosis (TB) remains a major underdiagnosed public health threat worldwide, being responsible for more than 10 million cases and one million deaths annually. TB diagnosis has become more rapid with the development and adoption of molecular tests, but remains challenging with traditional TB diagnosis, but there has not been a critical review of this area. Here, we systematically review these approaches to assess their diagnostic potential and issues with the development and clinical evaluation of proposed CRISPR-based TB assays. Based on these observations, we propose constructive suggestions to improve sample pretreatment, method development, clinical validation, and accessibility of these assays to streamline future assay development and validation studies.