Ifitm3 was previously identified as an endosomal protein that blocks viral infection 1 – 3 . Studying clinical cohorts of B-cell leukemia and lymphoma patients, we identified IFITM3 as a strong predictor of poor outcome. In normal resting B-cells, Ifitm3 was minimally expressed and mainly localized in endosomes. However, B-cell receptor (BCR) engagement induced expression of Ifitm3 and phosphorylation at Y20, resulting in accumulation at the cell surface. In B-cell leukemia, oncogenic kinases phosphorylate IFITM3-Y20, causing constitutive plasma membrane localization. Ifitm3 ˉ / ˉ naïve B-cells developed at normal numbers; however, germinal center formation and production of antigen-specific antibodies were compromised. Oncogenes that induce development of leukemia and lymphoma failed to transform Ifitm3 ˉ / ˉ B-cells. Conversely, the phospho-mimetic IFITM3-Y20E induced oncogenic PI3K-signaling and initiated transformation of pre-malignant B-cells. Mechanistic experiments revealed that Ifitm3 functions as PIP3-scaffold and central amplifier of PI3K signaling. PI3K signal-amplification depends on Ifitm3 scaffolding PIP3-accumulation via two lysine residues (K83 and K104) in its conserved intracellular loop. In Ifitm3 ˉ / ˉ B-cells, lipid rafts were depleted of PIP3, resulting in defective expression of >60 lipid raft-associated surface receptors, impaired BCR-signaling and cellular adhesion. We conclude that phosphorylation of IFITM3 upon B-cell antigen-encounter induces a dynamic switch from antiviral effector functions in endosomes to a PI3K-amplification loop at the cell surface. IFITM3-dependent amplification of PI3K-signaling in part downstream of the BCR is critical to enable rapid expansion of B-cells with high affinity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signaling complexes and amplify PI3K-signaling for malignant transformation.

Toward combating infectious diseases caused by pathogenic bacteria, there remains an unmet need for diagnostic tools that can broadly identify the causative bacteria and determine their antimicrobial susceptibilities from complex and even polymicrobial samples in a timely manner. To address this need, a microfluidic and machine-learning-based platform that performs broad bacteria identification (ID) and rapid yet reliable antimicrobial susceptibility testing (AST) is developed. Specifically, this platform builds on “pheno–molecular AST”, a strategy that transforms nucleic acid amplification tests (NAATs) into phenotypic AST through quantitative detection of bacterial genomic replication, and utilizes digital polymerase chain reaction (PCR) and digital high-resolution melt (HRM) to quantify and identify bacterial DNA molecules. Bacterial species are identified using integrated experiment–machine learning algorithm via HRM profiles. Digital DNA quantification allows for rapid growth measurement that reflects susceptibility profiles of each bacterial species within only 30 min of antibiotic exposure. As a demonstration, multiple bacterial species and their susceptibility profiles in a spiked-in polymicrobial urine specimen were correctly identified with a total turnaround time of ∼4 h. With further development and clinical validation, this platform holds the potential for improving clinical diagnostics and enabling targeted antibiotic treatments.

Although nucleic acid amplification testing (NAAT) has become the cornerstone for molecular diagnosis of diseases, expanding the multiplexed detection capacity of NAAT remains an important objective. To this end, encoding each nucleic acid target with a specific fluorescently labeled probe has been the most mature approach for multiplexed detection. Unfortunately, the number of targets that can be differentiated via this one-target-one-fluorophore multiplexed detection approach is restricted by spectral overlaps between fluorophores. In response, we present herein a new multiplexed detection approach termed ratiometric fluorescence coding, in which we encode each nucleic acid target with a specific ratio between two standard fluorophores. In ratiometric fluorescence coding, we employ the padlock probe chemistry to encode each nucleic acid target with a specific number of binding sites for two probes labeled with different fluorophores. Coupling the padlock probes with either rolling circle amplification (RCA) or hyperbranched rolling circle amplification (HRCA), we transform each nucleic acid target into a specific template that allows hybridization with the fluorescently labeled probes at predesigned ratios, thereby achieving multiplexed detection. For demonstration, we detected DNA targets from six infectious diseases and demonstrated the potential for further expanding the multiplexing capability of our approach. With further development, ratiometric fluorescence coding has the potential to enable highly multiplexed detection of nucleic acid targets and facilitate molecular diagnosis of diseases.

Polymerase chain reaction (PCR) is by far the most commonly used method of nucleic acid amplification and has likewise been employed for a plethora of diagnostic purposes. Nonetheless, multiplexed PCR-based detection schemes have hitherto been largely limited by technical challenges associated with nonspecific interactions and other limitations inherent to traditional fluorescence-based assays. Here, we describe a novel strategy for multiplexed PCR-based analysis called Ligation-eNabled fluorescence-Coding PCR (LiNC PCR) that exponentially enhances the multiplexing capability of standard fluorescence-based PCR assays. The technique relies upon a simple, preliminary ligation reaction in which target DNA sequences are converted to PCR template molecules with distinct endpoint fluorescence signatures. Universal TaqMan probes are used to create target-specific multicolor fluorescence signals that can be readily decoded to identify amplified targets of interest. We demonstrate the LiNC PCR technique by implementing a two-color-based assay for detection of 10 ovarian cancer epigenetic biomarkers at analytical sensitivities as low as 60 template molecules with no detectable target cross-talk. Overall, LiNC PCR provides a simple and inexpensive method for achieving high-dimensional multiplexing that can be implemented in manifold molecular diagnostic applications.