Biology subjects

Chang, M. M.

Publications and source records attributed to Chang, M. M..

11 recordsLinked to original sources

bioRxiv2025

An Optimized Product-Enhanced Reverse Transcriptase Assay for Sensitive and Quantitative Detection of HIV Viral Load and Phenotypic Drug Resistance

The World Health Organization (WHO) recommends Human Immunodeficiency Virus (HIV) drug resistance testing (DRT), but current tests are too complex and expensive for routine use. Genotypic DRT is challenging to interpret because of the growing list of mutations responsible for HIV drug resistance. Although phenotypic DRT is simpler to interpret, it requires slow and labor-intensive viral culture. Phenotypic tests that measure the activity of isolated HIV enzymes are faster and less labor-intensive, but none have yet met the 2023 WHO Target Product Profile (TPP) for HIV DRT. Here we present an optimized Product-Enhanced Reverse Transcriptase (PERT) assay for sensitive and quantitative detection of HIV viral load and drug resistance. PERT combines complementary DNA (cDNA) synthesis by HIV-Reverse Transcriptase (HIV-RT) with cDNA amplification and detection by quantitative PCR (qPCR). We established sensitive detection down to 10 HIV-RT molecules in a 25 {micro}L sample corresponding to a viral load of [~]10 HIV RNA copies/mL. We demonstrated the assays feasibility for phenotypic DRT using lamivudine-5-triphosphate (3TC-TP)--to which the M184V mutation confers high-level resistance--and quantified 3TC-TP inhibition using the difference in cDNA produced between drug and no-drug conditions. We surpassed WHO minimal analytical sensitivity requirements (20% low-abundance variant detection) by differentiating 10% M184V HIV-RT fractions in heterogenous mixtures (1,000 total HIV-RT, viral load [~]2,500 copies RNA/mL). Finally, we showed that PERT could detect resistance to existing and emerging RT inhibitors including tenofovir-diphosphate (TFV-DP), doravirine (DOR), and islatravir (ISL)-TP. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/691922v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@afec9borg.highwire.dtl.DTLVardef@1d6c6eeorg.highwire.dtl.DTLVardef@19e4082org.highwire.dtl.DTLVardef@9d49e6_HPS_FORMAT_FIGEXP M_FIG C_FIG The Product-Enhanced Reverse Transcriptase (PERT) assay measures HIV-RT activity by its ability to synthesize complementary DNA, providing a phenotypic measurement of HIV viral load (by quantity of HIV-RT enzyme) and susceptibility to RT inhibitors (by level of inhibition of HIV-RT activity). IMPORTANCEAlthough antiretroviral therapy can effectively treat and prevent HIV infection, treatment efficacy and global control of the HIV epidemic are threatened by rising rates of HIV drug resistance. Inexpensive and decentralized HIV DRT could facilitate surveillance efforts to understand the prevalence of HIV drug resistance in local and global contexts. This need is especially timely and pressing considering anticipated increased rates of HIV acquisition and drug resistance due to the reductions in global HIV services driven by recent funding cuts. Our optimized PERT assay for simultaneous viral load and phenotypic drug resistance quantification is fast ([~]2 hours), sensitive, and accurate. We can also leverage existing RT-qPCR instruments used for viral load measurement to significantly improve access to HIV drug resistance monitoring and tailored regimen selection.

bioengineering↗

bioRxiv2025

REverse-transcriptase ACTivity with CRISPR (REACTR) Assay for Ganciclovir Triphosphate Monitoring

BackgroundCytomegalovirus (CMV) can cause severe disease and death in infants and immunocompromised people. Although effective at treating CMV, the first-line drug - ganciclovir (GCV) - has high rates of pharmacokinetic variability which leads to significant rates of underexposure or toxicity. Intracellular concentrations of ganciclovir triphosphate (GCV-TP) - GCVs active anabolite - are associated with dose-dependent neutropenia and could enable dose individualization to improve treatment efficacy and reduce adverse effects. However, GCV-TP is currently measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) which is impractical for routine use, especially in resource-limited settings, because of its high cost, labor-intensiveness, and need for specialized equipment. To address this gap, we adapted the REverse transcriptase ACTivity with CRISPR (REACTR) assay to measure GCV-TP. MethodsWe leveraged our earlier work using REACTR to measure reverse transcriptase (RT) inhibitors used to treat and prevent human immunodeficiency virus (HIV) because GCV-TP serendipitously also inhibits HIV RT. We designed custom DNA templates, primers, and CRISPR complexes to accurately measure GCV-TP spiked into buffer and blood. We evaluated the assays analytical performance with 40 dried blood spots from infants with congenital CMV. ResultsREACTR reproducibly measured clinically relevant GCV-TP concentrations using a simple workflow and equipment that are readily available in many clinical laboratories. REACTR measurements of clinical samples correlated with LC-MS/MS GCV-TP measurements (r = -0.7891; p<0.0001). ConclusionsThis study highlights the potential of REACTR as a rapid and accessible alternative to LC-MS/MS for therapeutic drug monitoring of GCV.

bioengineering↗

bioRxiv2025

Optimizing Bispecific Antibody Expression via Multi-Omics Analysis and Vector Redesign

Bispecific antibodies are a growing class of therapeutics that simultaneously engage two targets. However, their complex molecular structures pose challenges for production in Chinese hamster ovary cells, the current industry standard for biologics manufacturing. Here we present a case study of three IgG-scFv format bsAbs expressed in CHO cells, in which one candidate exhibited markedly lower titers despite high sequence homology to the other two. Using multi-omics analysis (RNA sequencing, splicing prediction, codon optimization assessment, and motif screening) to investigate potential causes, we identified several likely mechanisms for poor expression, including aberrant splicing motifs, ribosome pausing sites, and suboptimal codon usage. Through targeted protein and DNA sequence engineering, we generated a revised variant with an 11-fold increase in stable expression titers. This work demonstrates that integrating sequence-level bioinformatic and synthetic biology diagnostics can directly improve manufacturability, providing a generalizable framework for resolving hidden expression liabilities in complex biologics.

bioengineering↗

bioRxiv2025

CandyCollect Open to Closed (O2C) Microfluidic System for Rapid and User-Centric Detection of Group A Streptococcus

We created CandyCollect, a lollipop-inspired, child-friendly saliva sampling device that provides enhanced comfort compared to common throat and mouth swabs. The device has an open microchannel and a functionalized surface that effectively captures and stores the saliva and pathogens. We recently demonstrated the ability to combine the CandyCollect with rapid antigen detection tests (RADTs) for fast and accessible detection of group A streptococcus (GAS). However, the current procedure necessitates manual steps to elute and transfer captured antigens from the lollipop to RADTs, which relies on the user to follow complex instructions, increasing the likelihood of error. In this work, we developed the CandyCollect Open to Closed (O2C) microfluidic system to automate antigen elution and streamline detection with RADTs. The O2C system securely seals the open microchannels of the CandyCollect device to create a continuous closed channel for seamless delivery of elution reagents to release antigens at the press of a button for subsequent lateral flow detection. Our system leverages and combines the unique benefits of both open channel and closed channel microfluidics into one platform, further enhancing the potential of both methods. We performed experiments with GAS bacteria spiked into saliva and compared the manual and O2C elution methods. Both the manual and O2C methods showed clear test lines on the RADT strips at clinically relevant GAS concentrations (between 5 x105 and 109 CFU/mL). The O2C provided comparable or better results than the manual procedure in a more convenient form factor. The O2C platform has the potential to enable user-friendly screening for respiratory pathogens by minimally trained users in decentralized settings.

bioengineering↗

bioRxiv2024

Rapid enzymatic assay for antiretroviral drug monitoring using CRISPR-Cas12a enabled readout

Maintaining efficacy of human immunodeficiency virus (HIV) medications is challenging among children because of dosing difficulties, the limited number of approved drugs, and low rates of medication adherence. Drug level feedback (DLF) can support dose optimization and timely interventions to prevent treatment failure, but current tests are heavily instrumented and centralized. We developed the REverse-transcriptase ACTivity-crispR (REACTR) assay for rapid measurement of HIV drugs based on the extent of DNA synthesis by HIV reverse transcriptase. CRISPR-Cas enzymes bind to synthesized DNA, triggering collateral cleavage of quenched reporters and generating fluorescence. We measured azidothymidine triphosphate (AZT-TP), a key drug in pediatric HIV treatment, and investigated the impact of assay time and DNA template length on REACTRs sensitivity. REACTR selectively measured clinically relevant AZT-TP concentrations in the presence of genomic DNA and peripheral blood mononuclear cell lysate. REACTR has the potential to enable rapid point-of-care HIV DLF to improve pediatric HIV care.

bioengineering↗

bioRxiv2024

REverse transcriptase ACTivity (REACT) assay for point-of-care measurement of established and emerging antiretrovirals for HIV treatment and prevention

Maintaining adequate levels of antiretroviral (ARV) medications is crucial for the efficacy of HIV treatment and prevention regimens. Monitoring ARV levels can predict or prevent adverse health outcomes like treatment failure or drug resistance. However, conventional ARV measurement using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is slow, expensive, and centralized delaying clinical and behavioral interventions. We previously developed a rapid enzymatic assay for measuring nucleotide reverse transcriptase inhibitors (NRTIs) - the backbone of HIV treatment and prevention regimens - based on the drugs termination of DNA synthesis by HIV reverse transcriptase (RT) enzyme. Here we expand our work to include non-nucleoside reverse transcriptase inhibitors (NNRTIs) - an ARV class used in established and emerging HIV treatment and prevention regimens. We demonstrate that the REverse Transcriptase ACTivity (REACT) assay can detect NNRTIs including medications used in oral and long-acting/extended-release HIV treatment and prevention. We demonstrate that REACT can measure NNRTIs spiked in either buffer or diluted plasma, and that fluorescence can be measured on both a traditional plate reader and an inexpensive portable reader that can be deployed in point-of-care (POC) settings. REACT measured clinically relevant concentrations of five NNRTI spiked in aqueous buffer. REACT measurements showed excellent agreement between the plate reader and the portable reader, with a high correlation in both aqueous buffer (Pearsons r=0.9807, P < 0.0001) and diluted plasma (Pearsons r = 0.9681, P < 0.0001). REACT has the potential to provide rapid measurement of NNRTIs in POC settings and may help to improve HIV treatment and prevention outcomes.

bioengineering↗

bioRxiv2023

A transcriptome-wide meta-analysis reveals lack of cancer-cell intrinsic determinants of response to immune checkpoint blockade

Immune-checkpoint therapy (ICB) has conferred significant and durable clinical benefit to some cancer patients. However, most patients do not respond to ICB, and reliable biomarkers of ICB response are needed to improve patient stratification. Here, we performed a transcriptome-wide meta-analysis across 1,486 tumors from ICB-treated patients and tumors with expected ICB outcomes based on microsatellite status. Using a robust transcriptome deconvolution approach, we inferred cancer and stroma-specific gene expression differences and identified cell-type specific features of ICB response across cancer types. Consistent with current knowledge, stromal expression of CXCL9, CXCL13, and IFNG were the top determinants of favorable ICB response. In addition, we identified a group of potential immune-suppressive genes, including FCER1A, associated with poor response to ICB. Strikingly, PD-L1 expression in stromal cells, but not cancer cells, is correlated with ICB response across cancer types. Furthermore, the unbiased transcriptome-wide analysis failed to identify cancer-cell intrinsic features of ICB response conserved across tumor types. Overall, our results challenge the prevailing dogma that cancer cells present tissue-agnostic molecular markers that modulate immune activity and ICB treatment response. These results have implications for the development of improved ICB treatments and diagnostics.

cancer biology↗

bioRxiv2021

Integrative Epigenomic and High-Throughput Functional Enhancer Profiling Reveals Determinants of Enhancer Heterogeneity in Gastric Cancer

BackgroundEnhancers are distal cis-regulatory elements required for cell-specific gene expression and cell fate determination. In cancer, enhancer variation has been proposed as a major cause of inter-patient heterogeneity - however, most predicted enhancer regions remain to be functionally tested. ResultsAnalyzing 128 epigenomic histone modification profiles of primary GC samples, normal gastric tissues, and GC cell lines, we report a comprehensive catalog of 75,730 recurrent predicted enhancers, the majority of which are tumor-associated in vivo (>50,000) and associated with lower somatic mutation rates inferred by whole-genome sequencing. Applying Capture-based Self-Transcribing Active Regulatory Region sequencing (CapSTARR-seq) to the enhancer catalog, we observed significant correlations between CapSTARR-seq functional activity and H3K27ac/H3K4me1 levels. Super-enhancer regions exhibited increased CapSTARR-seq signals compared to regular enhancers even when decoupled from native chromatin contexture. We show that combining histone modification and CapSTARR-seq functional enhancer data improves the prediction of enhancer-promoter interactions and pinpointing of germline single nucleotide polymorphisms (SNPs), somatic copy number alterations (SCNAs), and trans-acting TFs involved in GC expression. Specifically, we identified cancer-relevant genes (e.g. ING1, ARL4C) whose expression between patients is influenced by enhancer differences in genomic copy number and germline SNPs, and HNF4 as a master trans-acting factor associated with GC enhancer heterogeneity. ConclusionsOur study indicates that combining histone modification and functional assay data may provide a more accurate metric to assess enhancer activity than either platform individually, and provides insights into the relative contribution of genetic (cis) and regulatory (trans) mechanisms to GC enhancer functional heterogeneity.

cancer biology↗

bioRxiv2019

MutSpot: detection of non-coding mutation hotspots in cancer genomes

SummaryRecurrence and clustering of somatic mutations (hotspots) in cancer genomes may indicate positive selection and involvement in tumorigenesis. MutSpot performs genome-wide inference of mutation hotspots in non-coding and regulatory DNA of cancer genomes. MutSpot performs feature selection across hundreds of epigenetic and sequence features followed by estimation of position and patient-specific background somatic mutation probabilities. MutSpot is user-friendly, works on a standard workstation, and scales to thousands of cancer genomes.\n\nAvailability and implementationMutSpot is implemented as an R package and is available at https://github.com/skandlab/MutSpot/\n\nSupplementary informationSupplementary data are available at https://github.com/skandlab/MutSpot/

genomics↗

bioRxiv2019

Natural Amelioration of Mn-induced Chlorosis Facilitated by Mn Down-regulation, Ammonium and Rainwater in Sugarcane Seedlings

We had previously reported that manganese (Mn)-induced chlorosis is a serious problem in ratoon sugarcane seedlings grown in acidic soils. To further monitor the progression of chlorosis and elucidate the corresponding mechanism, both plant growth and nutrient status of sugarcane plants and soils were investigated in the growth seasons of ratoon cane seedlings in 2016 and 2018. The impacts of rainfall and ammonium on chlorosis were also investigated hydroponically. The results showed that the chlorotic seedlings could green in mid-summer; Mn content in the first expanded leaf decreased significantly, whereas iron (Fe) content increased significantly during the progression of greening. The leaf Mn content in the greened seedlings decreased by up to 78.1% when compared with that in the initial chlorotic seedlings. The seedlings also showed a significant increase in seedling height and weight of the expanded leaves, accompanied by a decrease in plant Mn content during the progression of greening. Moreover, young seedlings with less Mn content showed earlier greening than older seedlings with more Mn content. The exchangeable ammonium content in the soils increased significantly during the progression of greening, and the addition of 1 mM ammonium to the chlorotic seedlings resulted in a decrease in leaf Mn content by up to 80%. Furthermore, leaf SPAD value and Fe content increased by 2.0-fold and 1.4-fold, respectively, after rainwater was applied to the chlorotic seedling. These results indicate Mn-induced chlorotic seedlings can turn naturally green, and downregulation of plant Mn content, rainfall in summer, and soil ammonium contribute to the greening of chlorotic seedlings.

plant biology↗

bioRxiv2017

Dose-response surface fits to drought and nitrogen limitation applied together allow mapping of loci that exhibit nonlinear responses

Crop improvement must accelerate to feed an increasing human population in the face of environmental changes. Breeding programs can include anticipated climatic changes and genetic architecture to optimize improvement strategies. We analyzed the genetic architecture underlying the response of Zea mays to combinations of water and nitrogen stresses. Recombinant inbreds were subjected to nine combinations of the two stresses using an optimized response surface design, and their growth was measured. Three-dimensional dose response surfaces were fit globally and to each polymorphic allele to determine which genetic markers were associated with different response surfaces. Three quantitative trait loci that produced nonlinear surfaces were mapped. Alleles that performed better in combinations of mid-range stresses were typically not the alleles that performed best under combinations of extreme stresses. To develop physiologically relevant models for future genetic analyses, we modeled the network that explains the response surfaces. The network contains two components, an elliptical paraboloid and a plane, that each combine the nitrogen and water inputs. The relative weighting of the two components and the inputs is governed by five parameters. We estimated parameter values for the smoothed surfaces from the experimental lines using a set of points that covered the most distinctive regions of the three-dimensional surfaces. Surfaces computed using these values reproduced the smoothed experimental surfaces well, especially in the neighborhood of the peaks, as judged by three different criteria. The parameters exaggerated the amplitudes of the simulated surfaces. Experiments using single stresses could misestimate responses to their combinations and disguise loci that respond nonlinearly. The three-dimensional shape evaluation strategy used here more thoroughly compares nonlinear, nonplanar responses. We encourage the application of our findings and methods to experiments that mix crop protection measures, stresses, or both, on elite and landrace germplasm.

genetics↗