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1. Rapid detection of pathogenic E. coli based on CRISPR Cas system

   https://doi.org/10.3389/fmicb.2024.1423478  

   Rathore, Pallavi; Basnet, Ashesh; Kilonzo-Nthenge, Agnes; Dumenyo, Korsi; Yadegari, Zeinab; Taheri, Ali (June 2024, Frontiers in Microbiology)

   Access to safe and nutritious food is critical for maintaining life and supporting good health. Eating food that is contaminated with pathogens leads to serious diseases ranging from diarrhea to cancer. Many foodborne infections can cause long-term impairment or even death. Hence, early detection of foodborne pathogens such as pathogenicEscherichia colistrains is essential for public safety. Conventional methods for detecting these bacteria are based on culturing on selective media and following standard biochemical identification. Despite their accuracy, these methods are time-consuming. PCR-based detection of pathogens relies on sophisticated equipment and specialized technicians which are difficult to find in areas with limited resources. Whereas CRISPR technology is more specific and sensitive for identifying pathogenic bacteria because it employs programmable CRISPR-Cas systems that target particular DNA sequences, minimizing non-specific binding and cross-reactivity. In this project, a robust detection method based on CRISPR-Cas12a sensing was developed, which is rapid, sensitive and specific for detection of pathogenicE. coliisolates that were collected from the fecal samples from adult goats from 17 farms in Tennessee. Detection reaction contained amplified PCR products for the pathogenic regions, reporter probe, Cas12a enzyme, and crRNA specific to three pathogenic genes—stx1, stx2, and hlyA. The CRISPR reaction with the pathogenic bacteria emitted fluorescence when excited under UV light. To evaluate the detection sensitivity and specificity of this assay, its results were compared with PCR based detection assay. Both methods resulted in similar results for the same samples. This technique is very precise, highly sensitive, quick, cost effective, and easy to use, and can easily overcome the limitations of the present detection methods. This project can result in a versatile detection method that is easily adaptable for rapid response in the detection and surveillance of diseases that pose large-scale biosecurity threats to human health, and plant and animal production. 

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2. Associations between Antiretrovirals and Cognitive Function in Women with HIV

   https://doi.org/10.1007/s11481-020-09910-1  

   Rubin, Leah H.; Li, Yuliang; Fitzgerald, Kathryn C.; Dastgheyb, Raha; Spence, Amanda B.; Maki, Pauline M.; Sharma, Anjali; Gustafson, Deborah R.; Milam, Joel; Weber, Kathleen M.; et al (January 2020, Journal of Neuroimmune Pharmacology)

   Cognitive complications persist in antiretroviral therapy(ART)-treated people with HIV. However, the pattern and severity of domain- specific cognitive performance is variable and may be exacerbated by ART-mediated neurotoxicity. 929 women with HIV(WWH) from the Women’s Interagency HIV Study who were classified into subgroups based on sociodemographic and longitudinal behavioral and clinical data using semi-parametric latent class trajectory modelling. Five subgroups were comprised of: 1) well-controlled HIV with vascular comorbidities(n = 116); 2) profound HIV legacy effects(CD4 nadir <250 cells/μL; n = 275); 3) primarily <45 year olds with hepatitis C(n = 165); 4) primarily 35–55 year olds(n = 244), and 5) poorly-controlled HIV/substance use(n = 129). Within each subgroup, we fitted a constrained continuation ratio model via penalized maximum likelihood to examine adjusted associations between recent ART agents and cognition. Most drugs were not associated with cognition. However, among the few drugs, non- nucleoside reverse transcriptase inhibitor (NNRTIs) and protease inhibitors(PIs) were most commonly associated with cognition, followed by nucleoside reverse transcriptase inhibitors(NRTIs) and integrase inhibitors(IIs). Directionality of ART-cognition associa- tions varied by subgroup. Better psychomotor speed and fluency were associated with ART for women with well-controlled HIV with vascular comorbidities. This pattern contrasts women with profound HIV legacy effects for whom poorer executive function and fluency were associated with ART. Motor function was associated with ART for younger WWH and primarily 35–55 year olds. Memory was associated with ART only for women with poorly-controlled HIV/substance abuse. Findings demonstrate interindividual variability in ART-cognition associations among WWH and highlight the importance of considering sociodemographic, clinical, and behavioral factors as an underlying contributors to cognition. 

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3. Limits to detecting epistasis in the fitness landscape of HIV

   https://doi.org/10.1371/journal.pone.0262314  

   Biswas, Avik; Haldane, Allan; Levy, Ronald M. (January 2022, PLOS ONE)

   Gallicchio, Emilio (Ed.)

   The rapid evolution of HIV is constrained by interactions between mutations which affect viral fitness. In this work, we explore the role of epistasis in determining the mutational fitness landscape of HIV for multiple drug target proteins, including Protease, Reverse Transcriptase, and Integrase. Epistatic interactions between residues modulate the mutation patterns involved in drug resistance, with unambiguous signatures of epistasis best seen in the comparison of the Potts model predicted and experimental HIV sequence “prevalences” expressed as higher-order marginals (beyond triplets) of the sequence probability distribution. In contrast, experimental measures of fitness such as viral replicative capacities generally probe fitness effects of point mutations in a single background, providing weak evidence for epistasis in viral systems. The detectable effects of epistasis are obscured by higher evolutionary conservation at sites. While double mutant cycles in principle, provide one of the best ways to probe epistatic interactions experimentally without reference to a particular background, we show that the analysis is complicated by the small dynamic range of measurements. Overall, we show that global pairwise interaction Potts models are necessary for predicting the mutational landscape of viral proteins. 

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4. A click chemistry amplified nanopore assay for ultrasensitive quantification of HIV-1 p24 antigen in clinical samples

   https://doi.org/10.1038/s41467-022-34273-x  

   Wei, Xiaojun; Wang, Xiaoqin; Zhang, Zehui; Luo, Yuanyuan; Wang, Zixin; Xiong, Wen; Jain, Piyush K.; Monnier, John R.; Wang, Hui; Hu, Tony Y.; et al (December 2022, Nature Communications)

   Abstract Despite major advances in HIV testing, ultrasensitive detection of early infection remains challenging, especially for the viral capsid protein p24, which is an early virological biomarker of HIV-1 infection. Here, To improve p24 detection in patients missed by immunological tests that dominate the diagnostics market, we show a click chemistry amplified nanopore (CAN) assay for ultrasensitive quantitative detection. This strategy achieves a 20.8 fM (0.5 pg/ml) limit of detection for HIV-1 p24 antigen in human serum, demonstrating 20~100-fold higher analytical sensitivity than nanocluster-based immunoassays and clinically used enzyme-linked immunosorbent assay, respectively. Clinical validation of the CAN assay in a pilot cohort shows p24 quantification at ultra-low concentration range and correlation with CD4 count and viral load. We believe that this strategy can improve the utility of p24 antigen in detecting early infection and monitoring HIV progression and treatment efficacy, and also can be readily modified to detect other infectious diseases. 

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5. An Optimized Probe‐Based qPCR Assay for the Detection and Monitoring of the Invasive Lionfish ( Pterois volitans ) in the Atlantic

   https://doi.org/10.1002/edn3.70078  

   Viehl, Katherine; Khalid, Zain; Greiner‐Ferris, Kathryn; Taub, Eli; Amirthalingam, Pavithiran; Kumar, Girish; Marciante, Victoria; Gaither, Michelle R (March 2025, Environmental DNA)

   ABSTRACT The Indo‐Pacific lionfish,Pterois volitans,is an invasive species in the western Atlantic. Since its introduction to Florida in the early 1980s, populations have surged with lionfish now found from North Carolina to Venezuela. As their range expands, these generalist predators threaten native fauna, and while they are primarily a marine species, their tolerance for low salinity conditions may allow them to expand into sensitive estuarine habitats undetected. Traditional approaches for tracking invasive species such as direct observation or trapping are impractical over large spatial scales, making environmental DNA (eDNA) an attractive alternative. Molecular assays, such as those employing quantitative polymerase chain reaction (qPCR), amplify low copy number DNA fragments in environmental samples and are increasingly employed as a complement to traditional methods for the detection of invasive species. Currently, there is one published PCR assay for the detection of lionfish eDNA. However, the specificity of this assay is unverified, and the critical performance parameters limit of detection (LOD) and limit of quantification (LOQ) have not been established. Here we evaluate the efficacy of this assay and show that it is likely to result in false negatives in the western Atlantic. As an alternative, we developed a new TaqMan probe‐based qPCR assay that is species‐specific forP. volitansand highly sensitive with a LOD of 12 copies per reaction and a LOQ of 598 copies per reaction. While our assay does not amplify the closely relatedP. miles, which was also introduced in the western Atlantic, the low prevalence of this species in the invasive population means our assay is effective for most monitoring purposes. We conclude that our assay is a robust method for the detection of lionfish and can be employed in any habitat, offering new opportunities for controlling the spread of invasive lionfish. 

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