A Force Sensor that Converts Fluorescence Signal into Force Measurement Utilizing Short Looped DNA

A force sensor concept is presented where fluorescence signal is converted into force information via single-molecule Förster resonance energy transfer (smFRET). The basic design of the sensor is a ~100 base pair (bp) long double stranded DNA (dsDNA) that is restricted to a looped conformation by a nucleic acid secondary structure (NAS) that bridges its ends. The looped dsDNA generates a tension across the NAS and unfolds it when the tension is high enough. The FRET efficiency between donor and acceptor (D&A) fluorophores placed across the NAS reports on its folding state. Three dsDNA constructs with different lengths were bridged by a DNA hairpin and KCl was titrated to change the applied force. After these proof-of-principle measurements, one of the dsDNA constructs was used to maintain the G-quadruplex (GQ) construct formed by thrombin binding aptamer (TBA) under tension while it interacted with a destabilizing protein and stabilizing small molecule. The force required to unfold TBA-GQ was independently investigated with high-resolution optical tweezers (OT) measurements that established the relevant force to be a few pN, which is consistent with the force generated by the looped dsDNA. The proposed method is particularly promising as it enables studying NAS, protein, and small molecule interactions using a highly-parallel FRET-based assay while the NAS is kept under an approximately constant force

A patient with AL amyloidosis presenting with refractory tuberculosis, chest tightness and hypotension: case report

Abstract Introduction Immunoglobulin light chain (AL) amyloidosis presents a clinical spectrum characterized by diverse manifestations and involvement of multiple organs, posing a significant diagnostic challenge for physicians. Methods and results We present a case of a patient admitted to our hospital due to recurrent cough and sputum, which was initially diagnosed as refractory tuberculosis. Throughout his hospitalization, the patient experienced distressing symptoms, including uncontrollable chest tightness, hypotension, and fever. Noteworthy observations included a persistent elevation in cardiac biomarkers, indicative of cardiac damage. Bronchoalveolar lavage revealed the presence of various pathogenic microorganisms, while bone marrow flow cytometry demonstrated the existence of clonal plasma cells. Additionally, the urine free light chain assay detected the presence of M protein, and the positive congo red staining of the abdominal wall fat biopsy confirmed amyloid deposition in the tissues. Taking into account the patient’s clinical presentation and the examination findings, we reached a conclusive diagnosis of immunoglobulin light chain (AL) amyloidosis. Conclusion This case serves as a reminder for physicians to consider rare diseases like AL amyloidosis when patients present with symptoms involving multiple organ systems such as heart, lung and kidney that are unresponsive to conventional treatment options

Oncolytic Virus Therapy Alters the Secretome of Targeted Glioblastoma Cells

Oncolytic virus (OV) therapy, which is being tested in clinical trials for glioblastoma, targets cancer cells, while triggering immune cells. Yet OV sensitivity varies from patient to patient. As OV therapy is regarded as an anti-tumor vaccine, by making OV-infected cancer cells secrete immunogenic proteins, linking these proteins to transcriptome would provide a measuring tool to predict their sensitivity. A set of six patient-derived glioblastoma cells treated ex-vivo with herpes simplex virus type 1 (HSV1) modeled a clinical setting of OV infection. The cellular transcriptome and secreted proteome (separated into extracellular vesicles (EV) and EV-depleted fractions) were analyzed by gene microarray and mass-spectroscopy, respectively. Data validation and in silico analysis measured and correlated the secretome content with the response to infection and patient survival. Glioblastoma cells reacted to the OV infection in a seemingly dissimilar fashion, but their transcriptomes changed in the same direction. Therefore, the upregulation of transcripts encoding for secreted proteins implies a common thread in the response of cancer cells to infection. Indeed, the OV-driven secretome is linked to the immune response. While these proteins have distinct membership in either EV or EV-depleted fractions, it is their co-secretion that augments the immune response and associates with favorable patient outcomes

G‑Quadruplex-Enabling Sequence within the Human Tyrosine Hydroxylase Promoter Differentially Regulates Transcription

G-Quadruplexes (GQs) found within the promoter regions of genes are known to mostly act as repressors of transcription. Here we report a guanosine (G)-rich segment in the 3′-proximal promoter region of human tyrosine hydroxylase (<i>TH</i>), which acts as a necessary element for transcription. Tyrosine hydroxylase catalyzes the rate-limiting step in the catecholamine biosynthesis and is linked to several common neurological disorders such as Parkinson’s and schizophrenia. A 45 nucleotide (nt) sequence (wtTH49) within the human <i>TH</i> promoter contains multiple G-stretches that are extremely well conserved among the primates but deviate in rodents, which raises the possibility of variation in the GQ structures formed in the two orders with the potential for a distinctive functional outcome. Biochemical and biophysical studies, including single-molecule Förster resonance energy transfer, indicate that the wtTH49 sequence can adopt multiple GQ structures by using different combinations of G-stretches. A functional assay performed with 2.8 kb of the 3′-proximal end of the <i>TH</i> promoter and a mutated version (TH49fm; mutated wtTH49) that is unable to form any GQ structure indicates that overall the GQ-enabling wtTH49 sequence is functionally necessary and enhances human <i>TH</i> promoter activity by 5-fold compared to that of the mutant. Two additional mutants, each of which was designed to form distinct GQs, differentially affected reporter gene transcription. A cationic porphyrin TMPyP4 destabilizes the wtTH49 GQ and lowers the level of reporter gene expression, although its analogue, TMPyP2, fails to elicit any response. The 45 nt G-rich sequence within the human <i>TH</i> promoter can form multiple GQ structures, is a necessary element in transcription, and depending on the utilized combination of G-stretches affects transcription in different ways