BackgroundIdentification of non-sputum diagnostic markers for tuberculosis (TB) is urgently needed. This exploratory study aimed to discover potential serum protein biomarkers for the diagnosis of active pulmonary TB (PTB).MethodWe employed Proximity Extension Assay (PEA) to measure levels of 92 protein biomarkers related to inflammation in serum samples from three patient groups: 30 patients with active PTB, 29 patients with other respiratory diseases with latent TB (ORD with LTBI+), and 29 patients with other respiratory diseases without latent TB (ORD with LTBI-). To understand the functional mechanisms associated with differentially expressed proteins, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Least absolute shrinkage and selection operator (LASSO) regression was employed to identify potential TB diagnostic protein biomarkers. Network interactions among the identified candidate diagnostic markers were then analyzed, and their diagnostic performance was evaluated using logistic regression and receiver operating characteristic (ROC) analysis.ResultThe analysis revealed 37 differentially expressed proteins (DEPs) in the active PTB group compared to both ORD with LTBI + and ORD with LTBI- groups. Gene Ontology analysis indicated that these DEPs were primarily involved in the inflammatory response, while KEGG enrichment analysis highlighted the cytokine-cytokine receptor interaction pathway as the top significant hit. LASSO regression identified eight promising candidate protein biomarkers: IFN-gamma, LIF, uPA, CSF-1, SCF, SIRT2, 4E-BP1, and GDNF. The combined set of these eight proteins yielded an AUC of 0.943 for differentiating active PTB from ORD with LTBI+, and an AUC of 0.927 for distinguishing PTB from ORD with LTBI-.ConclusionWe have identified eight protein markers that reliably differentiate active PTB from ORD irrespective of LTBI presence. Further large-scale validation and translation of these protein markers into a user-friendly and affordable point-of-care test hold the potential to significantly enhance TB control in high-burden regions.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) ˂14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

The fluorescent molecule diphenylhexatriene (DPH) has been often used in combination with fluorescence anisotropy measurements, yet little is known regarding the non-linear optical properties. In the current work, we focus on them and extend the application to fluorescence, while paying attention to the conformational versatility of DPH when it is embedded in different membrane phases. Extensive hybrid quantum mechanics/molecular mechanics calculations were performed to investigate the influence of the phase- and temperature-dependent lipid environment on the probe. Already, the transition dipole moments and one-photon absorption spectra obtained in the liquid ordered mixture of sphingomyelin (SM)-cholesterol (Chol) (2:1) differ largely from the ones calculated in the liquid disordered DOPC and solid gel DPPC membranes. Throughout the work, the molecular conformation in SM:Chol is found to differ from the other environments. The two-photon absorption spectra and the ones obtained by hyper-Rayleigh scattering depend strongly on the environment. Finally, a stringent comparison of the fluorescence anisotropy decay and the fluorescence lifetime confirm the use of DPH to gain information upon the surrounding lipids and lipid phases. DPH might thus open the possibility to detect and analyze different biological environments based on its absorption and emission properties.