A mutated glycosaminoglycan-binding domain functions as a novel probe to selectively target heparin-like epitopes on tumor cells.

The high heterogeneity and mutation rate of cancer cells often lead to the failure of targeted therapy, and therefore, new targets for multitarget therapy of tumors are urgently needed. Aberrantly expressed glycosaminoglycans (GAGs) have been shown to be involved in tumorigenesis and are promising new targets. Recently, the GAG-binding domain rVAR2 of the Plasmodium falciparum VAR2CSA protein was identified as a probe targeting cancer-associated chondroitin sulfate A-like epitopes. In this study, we found that rVAR2 could also bind to heparin (Hep) and chondroitin sulfate E. Therefore, we used rVAR2 as a model to establish a method based on random mutagenesis of the GAG-binding protein and phage display to identify and optimize probes targeting tumor GAGs. We identified a new probe, VAR2HP, which selectively recognized Hep by interacting with unique epitopes consisting of a decasaccharide structure that contains at least three HexA2S(1-4)GlcNS6S disaccharides. Moreover, we found that these Hep-like epitopes were overexpressed in various cancer cells. Most importantly, our in vivo experiments showed that VAR2HP had good biocompatibility and preferentially localizes to tumors, which indicates that VAR2HP has great application potential in tumor diagnosis and targeted therapy. In conclusion, this study provides a strategy for the discovery of novel tumor-associated GAG epitopes and their specific probes.

miR-4732-5p promotes breast cancer progression by targeting TSPAN13.

MiR-4732-5p was previously found to be dysregulated in nipple discharge of breast cancer. However, the expression and function of miR-4732-5p in breast cancer remain largely unknown. Here, the expression of miR-4732-5p was detected using quantitative real-time PCR in breast cancer tissues and cell lines. Cell proliferation, apoptosis, migration and invasion assays were performed to examine the effects of miR-4732-5p in breast cancer. In addition, mRNA sequencing, bioinformatics analysis, Western blot and luciferase assays were performed to identify the target of miR-4732-5p. Overall, miR-4732-5p was significantly down-regulated in breast cancer tissues, especially in lymph node metastasis (LNM)-negative tissues, compared with adjacent normal tissues. However, it was more highly expressed in LNM-positive breast cancer tissues, compared with LNM-negative ones. Expression of miR-4732-5p was positively correlated with lymph node metastasis, larger tumour size, advanced clinical stage, high Ki-67 levels and poor prognosis. MiR-4732-5p promoted cell proliferation, migration and invasion in breast cancer. MiR-4732-5p directly targeted the 3'-UTR of tetraspanin 13 (TSPAN13) and suppressed TSPAN13 expression at the mRNA and protein levels. These results suggested that miR-4732-5p may serve as a tumour suppressor in the initiation of breast cancer, but as a tumour promoter in breast cancer progression by targeting TSPAN13.

An NIR light-responsive "on-off-on" photoelectrochemical aptasensor for carcinoembryonic antigen assay based on Y-shaped DNA.

This work develops a novel photoelectrochemical sensor for the detection of carcinoembryonic antigen (CEA) based on the composite of UCNPs with semiconductors and conformational changes in the DNA structure. Firstly, SnS2, ZnIn2S4 and UCNPs were assembled on the surface of the ITO electrode. Then Au NPs were dropped, which could facilitate the coupling of CdSe NPs modified DNA1 via Au-S bond, giving an ITO/SnS2/ZnIn2S4/UCNPs/CdSe heterojunction structure. When irradiated with 980 nm near-infrared (NIR) light, the UV-visible light emitted by the UCNPs could excite the nanocomposite, producing an enhanced photoelectric reaction. Subsequently, CEA aptamer and DNA2-modified SiO2 were added to form a Y-shaped DNA structure. At this time, the photocurrent was significantly reduced by the combination of the light-blocking effect of SiO2 and the departure of CdSe NPs from the electrode surface. When the target CEA was added, the recognition between CEA and the aptamer led to the collapse of the Y-shaped DNA structure, the restoration of hairpin DNA and the proximity of CdSe to the electrode. Accordingly, the photocurrent signals enhanced again. Under optimal experimental conditions, the detection limit as low as 0.3 pg mL-1 was obtained with good selectivity, achieving a sensitive "on-off-on" photoelectrochemical sensor for CEA detection.

CRISPR/Cas12a system responsive DNA hydrogel for label-free detection of non-glucose targets with a portable personal glucose meter.

In this work, personal glucose meter (PGM) as a portable electrochemical device was utilized for sensitive detection of non-glucose targets: N-gene and PCB77, respectively. DNA hydrogel, which can respond to CRISPR/Cas system, was prepared for label-free encapsulating invertase. In the presence of targets, the repeated sequence for the activation of Cas12a was obtained due to the performance of RCA. Unlike "one-to-one" recognition, activated Cas12a can efficiently cleave multiple single-stranded linker DNAs on DNA hydrogels, thus releasing many invertase that can be used for PGM detection. With the amplification of RCA and CRISPR/Cas system, high detection sensitivity can be obtained even using portable PGM. The detection limits for N-gene and PCB77 were 2.6 fM and 3.2 × 10-5 µg/L, respectively, with high specificity and good practical application performance. The developed biosensor can be used for online monitoring with the merit of low cost, easy operation and can be used for various targets analysis.

Identification and Action Patterns of Two Chondroitin Sulfate Sulfatases From a Marine Bacterium Photobacterium sp. QA16.

Chondroitin sulfate (CS)/dermatan sulfate (DS) is a kind of sulfated polyanionic, linear polysaccharide belonging to glycosaminoglycan. CS/DS sulfatases, which specifically hydrolyze sulfate groups from CS/DS oligo-/polysaccharides, are potential tools for structural and functional studies of CD/DS. However, only a few sulfatases have been reported and characterized in detail to date. In this study, two CS/DS sulfatases, PB_3262 and PB_3285, were identified from the marine bacterium Photobacterium sp. QA16 and their action patterns were studied in detail. PB_3262 was characterized as a novel 4-O-endosulfatase that can effectively and specifically hydrolyze the 4-O-sulfate group of disaccharide GlcUAß1-3GalNAc(4-O-sulfate) but not GlcUAß1-3GalNAc(4,6-O-sulfate) and IdoUAα1-3GalNAc(4-O-sulfate) in CS/DS oligo-/polysaccharides, which is very different from the identified 4-O-endosulfatases in the substrate profile. In contrast, PB_3285 specifically hydrolyzes the 6-O-sulfate groups of GalNAc(6-O-sulfate) residues located at the reducing ends of the CS chains and is the first recombinantly expressed 6-O-exosulfatase to effectively act on CS oligosaccharides.

Increased Ileal Immunoglobulin A Production and Immunoglobulin A-Coated Bacteria in Diarrhea-Predominant Irritable Bowel Syndrome.

OBJECTIVES: Immune activation and intestinal microbial dysbiosis could induce diarrhea-predominant irritable bowel syndrome (IBS-D). We examined the roles of ileal immunoglobulin A (IgA) and IgA-coated bacteria in IBS-D pathogenesis. METHODS: Peripheral blood, fecal samples, and ileal and cecal biopsies were collected from 32 healthy volunteers and 44 patients with IBS-D. Quantitative reverse transcriptase polymerase chain reaction was used to assess differential gene expression. IgA levels in the blood and fecal samples were quantified by an enzyme-linked immunosorbent assay. IgA cells were assessed by immunofluorescence imaging. Flow-cytometry-based IgA bacterial cell sorting and 16S rRNA gene sequencing (IgA-SEQ) was used to isolate and identify fecal IgA bacteria. RESULTS: Fecal IgA, particularly IgA1, was upregulated in patients with IBS-D. IgA class switch and B cell-activating factor-receptor were increased in the terminal ileum of patients. The intestinal microbiota composition was altered in patients compared with that in controls. IgA-SEQ showed that the proportion of fecal IgA-coated bacteria was increased significantly in patients with IBS-D. IgA bacteria in patients with IBS-D showed higher abundances of Escherichia-Shigella, Granulicatella, and Haemophilus compared with healthy controls and IgA bacteria in patients with IBS-D. The Escherichia-Shigella IgA coating index was positively correlated with anxiety and depression. The Escherichia-Shigella relative abundance, luminal IgA activity, and some altered IgA-coated bacteria were positively associated with the clinical manifestations of IBS-D. DISCUSSION: Microbial dysbiosis may promote the terminal ileal mucosa to produce higher levels of IgA, increasing the proportion of IgA-coated bacteria by activating IgA class switching, which might regulate local inflammation and clinical manifestations in IBS-D. IgA may mediate the effects of microbial dysbiosis on the pathogenesis of IBS-D.

[Primary benign paroxysmal positional vertigo vestibular detection of evoked myogenic potential significance].

OBJECTIVE: To analyze the clinical significance of ocular vestibular evoked myogenic potentials (oVEMP) and cervical vestibular evoked myogenic potentials (cVEMP) in primary unilateral benign paroxysmal positional vertigo (BPPV). METHOD: Fifty-two patients with unilateral primary BPPV (BPPV group) and 38 normal subjects (control group) received ocular vestibular evoked myogenic potential (oVEMP) and cervical vestibular evoked myogenic potential (cVEMP) test using tone burst stimuli. The response rate, latency and amplitude were analyzed. RESULT: In BPPV group, the response rate of oVEMP was 46.15% in lesioned side and 48.08% in healthy side, respectively. The response rate of cVEMP was 67.31% in lesioned side and 65.38% in healthy side, respectively. In control group, the response rate on the left ear was 84.21% for oVEMP and 92.11% for cVEMP. On the right ear, was 81.58% for oVEMP and 94.74% for cVEMP in control group, there was no significant difference in cVEMP and oVEMP P1, N1 N1-P1 latency and amplitude between left and right ear. The interaural amplitude ratio and asymmetry of cVEMP and oVEMP was significantly different between BPPV group and control group (P<0.05). CONCLUSION: Unilateral primary BPPV with bilateral impaired vestibular otolith pathways function can be objectively evaluated by oVEMP and cVEMP detection. Abnormal oVEMP responses were more frequently detected than cVEMP.