Although it is estimated that more than one million Americans have celiac disease (CD), it remains challenging to diagnose. CD, an autoimmune and inflammatory response following the ingestion of gluten-containing foods, has symptoms overlapping with other diseases and requires invasive diagnostics. The gold standard for CD diagnosis involves serologic blood tests followed by invasive confirmatory biopsies. Here, we propose a less invasive method using an electrochemical DNA (E-DNA) biosensor for CD-specific autoantibodies (AABs) circulating in blood. In our approach, CD-specific AABs bind a synthetic neoepitope, causing a conformational change in the biosensor, as well as a change in the environment of an attached redox reporter, producing a measurable current reduction. We assessed the biosensor’s ability to detect CD-specific patient-derived AABs in physiological buffer as well as buffer supplemented with bovine serum. Our biosensor was able to detect AABs in a dose-dependent manner; increased signal change correlated with increased AAB concentration with an apparent dissociation constant of 0.09 ± 0.03 units/mL of AABs. Furthermore, we found our biosensor to be target-specific, with minimal off-target binding of multiple unrelated biomarkers. Future efforts aimed at increasing sensitivity in complex media may build upon the biosensor design presented here to further improve CD AAB detection and CD diagnostic tools.

Aberrant biomolecular condensates are implicated in multiple incurable neurological disorders, including Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), and DYT1 dystonia. However, the role of condensates in driving disease etiology remains poorly understood. Here, we identify myeloid leukemia factor 2 (MLF2) as a disease-agnostic biomarker for phase transitions, including stress granules and nuclear condensates associated with dystonia. Exploiting fluorophore-derivatized MLF2 constructs, we developed a high-content platform and computational pipeline to screen modulators of NE condensates across chemical and genetic space. We identified RNF26 and ZNF335 as protective factors that prevent the buildup of nuclear condensates sequestering K48-linked polyubiquitinated proteins. Chemical screening identified four FDA-approved drugs that potently modulate condensates by resolving polyubiquitinated cargo and MLF2 accumulation. Our exploratory integrated chemical-genetics approach suggests that modulation of zinc, and potentially autophagy and oxidative stress, is critical for condensate modulation and nuclear proteostasis, offering potential therapeutic strategies for neurological disorders. Application of our platform to a genome-wide CRISPR KO screen identified strong enrichment of candidate genes linked to primary microcephaly and related neurodevelopmental disorders. Two hypomorphic microcephaly-associated alleles of ZNF335 failed to rescue nuclear condensate accumulation in ZNF335 KO cells, suggesting that aberrant condensates and impaired nuclear proteostasis may contribute to the pathogenesis of microcephaly. HIGHLIGHTS MLF2 emerges as a disease-agnostic condensate biomarker co-localizing with TDP-43 and G3BP1 FDA-approved drugs target condensates linked to perturbed proteostasis. RNF26 and ZNF335 are identified as modulators of nuclear phase transitions. Microcephaly patient disease alleles fail to counteract aberrant condensates.

Transient transfection is widely used for protein expression in heterologous systems, yet uncontrolled overexpression frequently introduces artifacts that confound functional analyses. Although stable cell lines can mitigate these issues, generating lines for multiple constructs or variants is often impractical. Common alternatives, such as DNA titration, altered transfection conditions, or promoter swapping, provide only coarse and inconsistent control of protein abundance. Here, we establish a panel of ten human 5’ untranslated regions (5’UTRs) as a modular strategy to tune protein expression during transient transfection. Across three soluble proteins and three membrane proteins, these 5’UTRs produce a reproducible dynamic range of expression, including fine-grained control of eYFP and the large sensory ion channel TRPA1. Notably, one 5’UTR consistently suppresses expression across all proteins tested and alleviates overexpression-associated artifacts, improving functional analysis of a hyperactive channel variant, substantially reducing background in proximity biotinylation assays, and enhancing the specificity of a stress granule marker. In contrast, most 5’UTRs enhance expression of the TRPV1 and TRPM8 sensory receptors, improving protein yield in heterologous systems. Together, this work identifies 5’UTRs as a compact, versatile, and broadly applicable tool to fine-tune protein abundance, enabling more physiologically relevant and assay-optimized expression in transient transfection experiments. Significance Statement: Protein overexpression remains a pervasive limitation in transient transfection experiments, as commonly used strategies provide coarse, labor-intensive, and often unpredictable control over protein abundance, frequently leading to artifacts or loss of physiological relevance. A compact, modular 5’UTR toolkit enables fine-grained and scalable control of protein abundance in transient transfection, providing a reproducible dynamic range across soluble and membrane proteins outperforming promoter swapping for continuous tuning of expression levels. Tunable expression improves experimental fidelity across diverse applications, allowing investigators to match protein abundance to specific assay needs, reducing overexpression artifacts, preserving physiological readouts, and enhancing yield when high expression is required.

Transient transfection is widely used for protein expression in heterologous systems, yet uncontrolled overexpression frequently introduces artifacts that confound functional analyses. Although stable cell lines can mitigate these issues, generating lines for multiple constructs or variants is often impractical. Common alternatives, such as DNA titration, altered transfection conditions, or promoter swapping, provide only coarse and inconsistent control of protein abundance. Here, we establish a panel of 10 human 5′ untranslated regions (5′UTRs) as a modular strategy to tune protein expression during transient transfection. Across three soluble proteins and three membrane proteins, these 5′UTRs produce a reproducible dynamic range of expression, including fine-grained control of eYFP and the large sensory ion channel TRPA1. Notably, one 5′UTR consistently suppresses expression across all proteins tested and alleviates overexpression-associated artifacts, improving functional analysis of a hyperactive channel variant, substantially reducing background in proximity biotinylation assays, and enhancing the specificity of a stress granule marker. In contrast, most 5′UTRs enhance expression of the TRPV1 and TRPM8 sensory receptors, improving protein yield in heterologous systems. Together, this work identifies 5′UTRs as a compact, versatile, and broadly applicable tool to fine-tune protein abundance, enabling more physiologically relevant and assay-optimized expression in transient transfection experiments.

Mycobacterium tuberculosis (M.tb) is one of the world’s most prevalent bacterial pathogens. However, Nontuberculous Mycobacterium (NTM) are less recognized than M.tb while remaining a pressing public health risk. Often, NTM lung disease is confused with that of a tubercle infection leading to unreliable diagnosis. Additionally, the time spent to fully diagnose a mycobacterial disease is extensive and dependent on the species growth rate due to the necessity of culturing for identification. Thus, designing a sensitive, electrochemical DNA‐based (E‐DNA) biosensor as a diagnostic tool for NTM would dramatically decrease the timeline of diagnosis and therefore could improve patient outcomes. In this work, we pursued detection of the cell envelope of NTM, which includes many complex glycolipids and glycopeptidolipids—many of which are believed to have immunopathogenic mechanisms. As mannose‐capped lipoarabinomannan (ManLAM) is one of the most prevalent of the glycolipids, we adapted it as a biomarker for the sensitive detection of NTM. An aptamer specific to ManLAM was incorporated into our E‐DNA biosensor, and exhibits significant current change under square wave voltammetric interrogation.

Mycobacterial infection and mortality rates remain very high; it is estimated that 10 million cases of Mycobacterium Tuberculosis ( M.Tb ) emerge each year. However, a less‐studied class of mycobacteria is becoming a growing concern: Nontuberculous Mycobacteria (NTM). Over the last 30 years, NTM‐based lung disease has grown to outnumber M.Tb infections in many regions. The gold standard for positive identification of NTM in a patient sample involves a lengthy microbiological culture‐based method which frequently takes weeks to months in order to make a diagnosis. In this work, we propose a novel electrochemical biosensor that can be used to detect NTM using a DNA aptamer. Our biosensor changes conformational shape when binding to the molecular target mannosylated lipoarabinomannan (ManLAM). ManLAM is a complex lipoglycan abundantly present in the cell envelope of many NTM and M.Tb. This conformational change is measured by voltammetric analysis, and we observe a significant difference in electrochemical current upon binding of ManLAM. We are also expanding our NTM detection efforts through the design of a biosensor targeting glycopeptidolipids (GPL). This strategy will increase our specificity, as GPL is absent from M.Tb . The expected outcome of this project is to greatly improve patient outcomes for NTM infection by allowing rapid, point‐of‐care diagnostics that eliminate invasive sampling and lengthy culturing requirements. This would significantly reduce the time between correct diagnosis and subsequent treatment.