In Situ Intracellular Autocatalytic Hairpin Assembly of the Y-Shaped DNA Nanostructure for miR-155 Sensing and Gene Silencing.

miR-155 is a class of cancer markers closely related to cancer metastasis and invasion. Combining in situ detection with gene silencing not only helps to analyze the information on the abundance and spatial location of microRNA expression in the cell but also synergizes the therapy. In this work, we prepared HD@CM vesicles with three hairpin DNAs by using MCF-7 cell membranes. The hairpin DNAs can be triggered by endogenous miR-155, which opens the autocatalytic molecular circuit (ACHA) and obtains Y-shaped DNA nanostructures. This nanostructure not only detects endogenous miR-155 with high sensitivity for in situ imaging but also enables gene regulation of intracellular survivin mRNA. The levels of miR-155 in MDA-MB-231, MCF-7, Hela, and HEK-293T cells are found to be 7703, 3978, 1696, and 1229 copies/cell, respectively, as detected by HD@CMs. The fluorescence produced by HD@CM after coincubation with different cells is found to be proportional to the intracellular miR-155 content by confocal imaging. In addition, the gene regulatory function of the Y-shaped DNA structure resulted in significant inhibition of survivin protein expression and apoptosis rates of up to 83%. We look forward to the future application of our HD@CM platform for the precise diagnosis and programmable treatment of clinical cancers.

Garlic-Derived Exosome-like Nanovesicles-Based Wound Dressing for Staphylococcus aureus Infection Visualization and Treatment.

Garlic-derived exosome-like nanovesicles (GELNs) could function in interspecies communication and may serve as natural therapeutics to regulate the inflammatory response or as nanocarriers to efficiently deliver specific drugs. Staphylococcus aureus (S. aureus) is able to hide within host cells to evade immune clearance and antibiotics, leading to life-threatening infections. On-site detection and efficient treatment of intracellular S. aureus infection in wounds remain challenging. Herein, we report a thermosensitive, injectable, visible GELNs-based wound dressing, Van@GELNs/F127 hydrogel (gel Van@GELNs), which is H2O2-responsive and can slowly release vancomycin into host cells forS. aureus infection visualization and treatment in wounds. GELNs show inherent antibacterial activity, which is significantly enhanced after loading vancomycin. Both GELNs and Van@GELNs have the ability to be internalized by cells, so Van@GELNs are more effective than free vancomycin in killing S. aureus in RAW 264.7 macrophages. When applied to an S. aureus-infected wound on a mouse, the colorless HRP&ABTS/Van@GELNs/F127 solution immediately changes to a green hydrogel and shows better therapeutic effect than vancomycin. Thus, direct visualization by the naked eye and effective treatment of S. aureus infection in wounds are achieved by gel Van@GELNs. We anticipate gel Van@GELNs be applied for the theranostics of S. aureus infection diseases in the clinic in the near future.

A double tangential flow filtration-based microfluidic device for highly efficient separation and enrichment of exosomes.

Recently, exosomes have been recognized as important disease biomarkers due to the essential roles they played in disease development. Nevertheless, the highly efficient isolation and enrichment of exosomes from complex body fluids continues to hinder the research and application of exosomes for clinical use. In this work, we developed a double tangential flow filtration-based microfluidic device for exosome isolation from cell supernatants and human serum. The microfluidic device contained two modules. Each module included two polymethylmethacrylate (PMMA) plates with symmetrical serpentine channels and a nanoporous membrane with 200 nm or 30 nm pore diameter and was used to separate larger vesicles, exosomes and free biomolecules. The design of double tangential flow filtration in symmetrical serpentine channels largely increased the contact area between the filtrate and the nanoporous membranes, thus improved the separation efficiency and prevented the clogging of the membrane. Compared with standard separation method, i.e. ultracentrifugation (UC), the microfluidic chip-based separation (Chip) of exosomes showed the advantages of much lower instrumental cost, lower consumable cost, shorter time (<120 min), higher purity (82.8%) and significantly higher recovery rate (77.8%). In addition, due to the label-free separation, the microfluidic device-collected exosomes could be directly used for downstream analysis such as proteomics analysis. The proteomics analysis results of exosomes isolated from the sera of clinical patients with different diseases by the chip revealed richer disease-related information comparing with those exosomes isolated by UC, demonstrating the good practicability of this chip for future clinical research and applications.

Maternal Outcomes Among Pregnant Women With Congenital Heart Disease-Associated Pulmonary Hypertension.

BACKGROUND: Studies focused on pregnant women with congenital heart disease (CHD)-associated pulmonary hypertension (PH) are scarce and limited by small sample sizes and single-center design. This study sought to describe the pregnancy outcomes in women with CHD with and without PH. METHODS: Outcomes for pregnant women with CHD were evaluated retrospectively from 1993 to 2016 and prospectively from 2017 to 2019 from 7 tertiary hospitals. PH was diagnosed on the basis of echocardiogram or catheterization. The incidence of maternal death, cardiac complications, and obstetric and offspring complications was compared for women with CHD and no PH, mild, and moderate-to-severe PH. RESULTS: A total of 2220 pregnant women with CHD had completed pregnancies. PH associated with CHD was identified in 729 women, including 398 with mild PH (right ventricle to right atrium gradient 30-50 mm Hg) and 331 with moderate-to-severe PH (right ventricle to right atrium gradient >50 mm Hg). Maternal mortality occurred in 1 (0.1%), 0, and 19 (5.7%) women with CHD and no, mild, or moderate-to-severe PH, respectively. Of the 729 patients with PH, 619 (85%) had CHD-associated pulmonary arterial hypertension, and 110 (15%) had other forms of PH. Overall, patients with mild PH had better maternal outcomes than those with moderate-to-severe PH, including the incidence of maternal mortality or heart failure (7.8% versus 39.6%; P<0.001), other cardiac complications (9.0% versus 32.3%; P<0.001), and obstetric complications (5.3% versus 15.7%; P<0.001). Brain natriuretic peptide >100 ng/L (odds ratio, 1.9 [95% CI, 1.0-3.4], P=0.04) and New York Heart Association class III to IV (odds ratio, 2.9 [95% CI, 1.6-5.3], P<0.001) were independently associated with adverse maternal cardiac events in pregnancy with PH, whereas follow-up with a multidisciplinary team (odds ratio, 0.4 [95% CI, 0.2-0.6], P<0.001) and strict antenatal supervision (odds ratio, 0.5 [95% CI, 0.3-0.7], P=0.001) were protective. CONCLUSIONS: Women with CHD-associated mild PH appear to have better outcomes compared with women with CHD-associated moderate-to-severe PH, and with event rates similar for most outcomes with women with CHD and no PH. Multimodality risk assessment, including PH severity, brain natriuretic peptide level, and New York Heart Association class, may be useful in risk stratification in pregnancy with PH. Follow-up with a multidisciplinary team and strict antenatal supervision during pregnancy may also help to mitigate the risk of adverse maternal cardiac events.

Three-Dimensional Microfluidic Chip for Efficient Capture of Secretory Autophagosomes and Sensitive Detection of Their Surface Proteins.

Recent studies on autophagy demonstrated a new extracellular secretion pathway for autophagosomes in addition to the routinely described intracellular degradation pathway. Besides, the secretory autophagosomes were found closely related to the occurrence and development of cancers. Therefore, analysis of the protein expression on secretory autophagosomes is a promising noninvasive strategy for cancer diagnosis and mechanism study. Herein, we constructed a three-dimensional (3D) microfluidic chip employing a fusiform micropillar array and layer-by-layer modification of gelatins, which obviously enhanced the mass transfer between reactants and increased the immobilization sites for capture antibody. As a result, the autophagosome capture efficiency of the 3D chip (74%) is significantly higher than that of the unmodified flat chip (47%). Using a two-step immunoreaction, ovarian cancer cell-secreted autophagosomes were successfully captured and detected. The results showed that two proteins, LC3B and HSP60 at the surface of autophagosomes, can be detected with limits of detection (LODs) of 141 particles µL-1 and 126 particles µL-1, respectively. In addition, both LC3B and HSP60 expressions on autophagosomes can be used to distinguish the serum samples between cancer patients and healthy people, with a p value less than 0.01 (statistically significant difference) or 0.05 (statistically different), respectively. Moreover, the summed signal of LC3B and HSP60 showed a p value less than 0.001 (extremely statistically significant difference), demonstrating the good potential of this chip for further application in cancer diagnosis.

Nano adamantane-conjugated BODIPY for lipase affinity and light driven antibacterial.

The increasing number of resistant bacterial strains has raised efforts in developing alternative treatment strategies. Lipase is highly expressed in most bacteria and lipase targeting dyes will be non-sacrificed materials for a sustainable method against microorganism. The combination of chemotherapy and antimicrobial photodynamic inactivation (aPDI) method will be an effective method due to enhanced antibacterial activity. Here we reported the spectroscopic features of five boron dipyrrolylmethene (BODIPY) derivatives with different functional groups for lipase affinity and antibacterial activity. Lipase affinity tests and antibacterial assays were conducted by spectroscopic methods. Adamantane-conjugated BODIPY (BDP-2) was found to be the active compound against E. coli. Next, BDP-2 was brominated, and then assembled with PEG resulting biocompatible BDP2-Br2@mPEG nanoparticles. The MTT assay indicated that BDP2-Br2@mPEG was less toxicity on BGC-823 cancer cells without irradiation. The BDP2-Br2@mPEG can inhibit the proliferation of E. coli and damage the membrane of bacterial cell under green LED light irradiation. The results proved BDP2-Br2@mPEG can be a very promising green LED light driven antibacterial material.

Determination of melamine and melamine-Cu(II) complexes in milk using a DNA-Ag hydrocolloid as the sensor.

Simple and sensitive methods are required for the detection of melamine since melamine is harmful to human health. To detect the content of melamine and metal-melamine species in milk products, a new luminescent DNA silver hydrocolloid (AgNC53) was synthesized using a modified C3A-rich aptamer as the template. The emission of AgNC53 at 617 nm is sensitive to both melamine-Cu(II) and melamine, while almost no response to the free Cu(II) ion was detected. Moreover, AgNC53 is used to detect precisely the content of melamine in milk with a detection limit as low as 2.7 × 10-8 M. Thus, the AgNC53-based fluorescence method is a "label-free" fluorescence technology that can be used for the determination of melamine or the melamine-Cu(II) complex in milk products.