Genetically Encoded FRET Biosensor Detects the Enzymatic Activity of Prostate-Specific Antigen

Prostate cancer is the most common cancer among men beyond 50 years old, and ranked the second in mortality. The level of Prostate-specific antigen (PSA) in serum has been a routine biomarker for clinical assessment of the cancer development, which is detected mostly by antibody-based immunoassays. The proteolytic activity of PSA also has important functions. Here a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) technology was developed to measure PSA activity. In vitro assay showed that the biosensor containing a substrate peptide ‘RLSSYYSGAG’ had 400% FRET change in response to 1 µg/ml PSA within 90 min, and could detect PSA activity at 25 ng/ml. PSA didn’t show enzymatic activity toward the biosensor in serum solution, likely reflecting the existence of other inhibitory factors besides Zn2+. By expressing the biosensor on cell plasma membrane, the FRET responses were significant, but couldn’t distinguish well the cultured prostate cancer cells from non-prostate cancer cells under microscopy imaging, indicating insufficient speci- ficity to PSA. The biosensor with the previously known ‘HSSKLQ’ substrate showed little response to PSA in solution. In summary, we developed a genetically encoded FRET biosensor to detect PSA activity, which may serve as a useful tool for relevant applications, such as screening PSA activation substrates or inhibitors; the purified biosensor protein can also be an alternative choice for measuring PSA activity besides currently commercialized Mu-HSSKLQ-AMC substrate from chemical synthesis.     

Distinct genomic traits of acral and mucosal melanomas revealed by targeted mutational profiling

The incidence of melanoma is rising globally including China. Comparing to Caucasians, the incidence of non‐cutaneous melanomas is significantly higher in Chinese. Herein, we performed genomic profiling of 89 Chinese surgically resected primary melanomas, including acral (n = 54), cutaneous (n = 22), and mucosal (n = 13), by hybrid capture‐based next‐generation sequencing. We show that mucosal melanomas tended to harbor more pathogenic mutations than other types of melanoma, though the biological significance of this finding remains uncertain. Chromosomal arm‐level alterations including 6q, 9p, and 10p/q loss were highly recurrent in all subtypes, but mucosal melanoma was significantly associated with increased genomic instability. Importantly, 7p gain significantly correlated with unfavorable clinical outcomes in non‐cutaneous melanomas, representing an intriguing prognostic biomarker of those subtypes. Furthermore, focal amplification of 4q12 (KIT, KDR, and PDGFRα) and RAD51 deletion were more abundant in mucosal melanoma, while NOTCH2 amplification was enriched in acral melanoma. Additionally, cutaneous melanomas had higher mutation load than acral melanomas, while mucosal melanomas did not differ from other subtypes in mutation burden. Together, our data revealed important features of acral and mucosal melanomas in Chinese including distinctive driver mutation pattern and increased genomic instability. These findings highlight the possibilities of combination therapies in the clinical management of melanoma.     

The Effect of Streptococcus salivarius K12 on Halitosis: a Double-Blind,Randomized, Placebo-Controlled Trial

This study was to evaluate the effect of Streptococcus salivarius K12 on tongue coating–associated halitosis. Twenty-eight subjects having tongue coating–associated halitosis were randomly divided into either a test or control group. For each of the 30 days, the test subjects sucked S. salivarius K12 tablet while the control subjects sucked placebo tablets. All the subjects did not take physical (tongue scraping) and chemical (antiseptic mouth-rinse) oral cavity pretreatment prior to use of the tablets. At baseline, and on the 1st, 7th, and 14th day after completing the course of tablets, the subjects were assessed for their organoleptic test (OLT) scores, volatile sulfur compound (VSC) levels, and tongue coating scores (TCS). During the course, all subjects kept their routine oral care habits without scraping their tongue coating. Plaque index, probing depth, and bleeding index were recorded at baseline and at the completion of the trial. On the 1st day following the end of tablet use, the OLT scores and VSC levels had significantly decreased in the test group when compared with the baseline values (P = 0.001 and P = 0.012). The TCS in the test group were also significantly decreased (P = 0.05). At days 7 and 14, the OLT scores in the test group were still significantly lower than the baseline levels (P = 0.006 and P = 0.039 respectively). However, there were no statistical differences with OLT, VSC, and TCS between the test group and the placebo group by analysis of multi-level regression model. The use of S. salivarius K12 did not have significant effect on halitosis with tongue coating cause when the tongue coating was not physically or chemically pre-treated, which implies removing tongue coating is required before Streptococcus salivarius K12 use.

     

Active interfacial dynamic transport of fluid in a network of fibrous connective tissues throughout the whole body

Fluid in interstitial spaces accounts for ~20% of an adult body weight and flows diffusively for a short range. Does it circulate around the body like vascular circulations? This bold conjecture has been debated for decades. As a conventional physiological concept, interstitial space is a micron‐sized space between cells and vasculature. Fluid in interstitial spaces is thought to be entrapped within interstitial matrix. However, our serial data have further defined a second space in interstitium that is a nanosized interfacial transport zone on a solid surface. Within this fine space, fluid along a solid fibre can be transported under a driving power and identically, interstitial fluid transport can be visualized by tracking the oriented fibres. Since 2006, our data from volunteers and cadavers have revealed a long‐distance extravascular pathway for interstitial fluid flow, comprising at least four types of anatomic distributions. The framework of each extravascular pathway contains the longitudinally assembled and oriented fibres, working as a fibrorail for fluid flow. Interestingly, our data showed that the movement of fluid in a fibrous pathway is in response to a dynamic driving source and named as dynamotaxis. By analysis of previous studies and our experimental results, a hypothesis of interstitial fluid circulatory system is proposed.