A Rapid and Ultrasensitive Thrombin Biosensor Based on a Rationally Designed Trifunctional Protein.

Rapid and sensitive detection of thrombin is imperative for the early diagnosis, prevention, and treatment of thrombin-related diseases. Here, an ultrasensitive and rapid thrombin biosensor is developed based on rationally designed trifunctional protein HTs, comprising three functional units, including a far-red fluorescent protein smURFP, hydrophobin HGFI, and a thrombin cleavage site (TCS). smURFP is used as a detection signal to eliminate any interference from the autofluorescence of sample matrix to increase detection sensitivity. HGFI serve as an adhesive unit to allow rapid immobilization of HTs on a multiwall plate. The TCS linking HGFI and smURFP function as a sensing element to recognize and detect thrombin. HTs immobilization is symmetrically optimized and characterized. Thrombin assay reveals the specific recognition of active thrombin in samples and the hydrolysis of the immobilized HTs, resulting in a decrease in the fluorescence intensity of the sample in a thrombin concentration-dependent manner. The limit of detection (LOD) is as low as 0.2 am in the serum. To the authors' knowledge, this is the lowest LOD ever reported for any thrombin biosensor. This study sheds light on the engineering of multifunctional proteins for biosensing.

Proteome analysis of biofilm produced by a Fusarium falciforme keratitis infectious agent.

Biofilms are structures that confer adaptive ability to and facilitate the virulence of fungal pathogens. Certain multi-functional proteins have been shown to be involved in fungal pathogenesis and these proteins may also be implicated in biofilm formation. The aim of this study was to identify a fungal agent isolated from the human cornea, to analyze the ability of this organism to form biofilms in vitro and to investigate protein expression in this condition. The fungus was identified by phylogenetic inference analysis. Biofilm formation and structure were evaluated by colorimetric methods and by optical and electron microscopy. We also resolved proteins obtained from biofilms and planktonic cultures by two-dimensional gel electrophoresis and identified those proteins by mass spectrometry. The fungus was identified as Fusarium falciforme. Colorimetric analysis and microscopy revealed that the highest level of biofilm formation was obtained at a concentration of 1 × 106 conidia/mL with 96 h of incubation at 28 °C. The biofilm architecture consisted of an extracellular matrix that embedded fungal filaments. We found nineteen proteins that were over-expressed in biofilms, as compared with planktonic cultures, and six proteins with unique expression in biofilms. Among the more abundant proteins identified were: transketolase, a putative antigen 1, enolase, phosphoglycerate kinase and ATP-citrate synthase. Some of these proteins are involved in basal metabolism, function as multi-functional proteins or have been described as potential virulence factors. We focused on the expression in biofilm of the enzyme, enolase, which was determined by real-time PCR. Our findings provide a perspective on the proteins associated with the formation of biofilms in vitro by an F. falciforme keratitis isolate.

Moonlighting proteins in cancer.

Since the 1980s, growing evidence suggested that the cellular localization of proteins determined their activity and biological functions. In a classical view, a protein is characterized by the single cellular compartment where it primarily resides and functions. It is now believed that when proteins appear in different subcellular locations, the cells surpass the expected activity of proteins given the same genomic information to fulfill complex biological behavior. Many proteins are recognized for having the potential to exist in multiple locations in cells. Dysregulation of translocation may cause cancer or contribute to poorer cancer prognosis. Thus, quantitative and comprehensive assessment of dynamic proteins and associated protein movements could be a promising indicator in determining cancer prognosis and efficiency of cancer treatment and therapy. This review will summarize these so-called moonlighting proteins, in terms of a coupled intracellular cancer signaling pathway. Determination of the detailed biological intracellular and extracellular transit and regulatory activity of moonlighting proteins permits a better understanding of cancer and identification of potential means of molecular intervention.