A streamlined method to generate endothelial cells from human pluripotent stem cells via transient doxycycline-inducible ETV2 activation.

The development of reliable methods for producing functional endothelial cells (ECs) is crucial for progress in vascular biology and regenerative medicine. In this study, we present a streamlined and efficient methodology for the differentiation of human induced pluripotent stem cells (iPSCs) into induced ECs (iECs) that maintain the ability to undergo vasculogenesis in vitro and in vivo using a doxycycline-inducible system for the transient expression of the ETV2 transcription factor. This approach mitigates the limitations of direct transfection methods, such as mRNA-mediated differentiation, by simplifying the protocol and enhancing reproducibility across different stem cell lines. We detail the generation of iPSCs engineered for doxycycline-induced ETV2 expression and their subsequent differentiation into iECs, achieving over 90% efficiency within four days. Through both in vitro and in vivo assays, the functionality and phenotypic stability of the derived iECs were rigorously validated. Notably, these cells exhibit key endothelial markers and capabilities, including the formation of vascular networks in a microphysiological platform in vitro and in a subcutaneous mouse model. Furthermore, our results reveal a close transcriptional and proteomic alignment between the iECs generated via our method and primary ECs, confirming the biological relevance of the differentiated cells. The high efficiency and effectiveness of our induction methodology pave the way for broader application and accessibility of iPSC-derived ECs in scientific research, offering a valuable tool for investigating endothelial biology and for the development of EC-based therapies.

Near-Infrared Photothermally Enhanced Photo-Oxygenation for Inhibition of Amyloid-ß Aggregation Based on RVG-Conjugated Porphyrinic Metal-Organic Framework and Indocyanine Green Nanoplatform.

Amyloid aggregation is associated with many neurodegenerative diseases such as Alzheimer's disease (AD). The current technologies using phototherapy for amyloid inhibition are usually photodynamic approaches based on evidence that reactive oxygen species can inhibit Aß aggregation. Herein, we report a novel combinational photothermally assisted photo-oxygenation treatment based on a nano-platform of the brain-targeting peptide RVG conjugated with the 2D porphyrinic PCN-222 metal-organic framework and indocyanine green (PCN-222@ICG@RVG) with enhanced photo-inhibition in Alzheimer's Aß aggregation. A photothermally assisted photo-oxygenation treatment based on PCN@ICG could largely enhance the photo-inhibition effect on Aß42 aggregation and lead to much lower neurotoxicity upon near-infrared (NIR) irradiation at 808 nm compared with a single modality of photo-treatment in both cell-free and in vitro experiments. Generally, local photothermal heat increases the instability of Aß aggregates and keeps Aß in the status of monomers, which facilitates the photo-oxygenation process of generating oxidized Aß monomers with low aggregation capability. In addition, combined with the brain-targeting peptide RVG, the PCN-222@ICG@RVG nanoprobe shows high permeability of the human blood-brain barrier (BBB) on a human brain-on-a-chip platform. The ex vivo study also demonstrates that NIR-activated PCN-222@ICG@RVG could efficiently dissemble Aß plaques. Our work suggests that the combination of photothermal treatment with photo-oxygenation can synergistically enhance the inhibition of Aß aggregation, which may boost NIR-based combinational phototherapy of AD in the future.

Porphyrinic Metal-Organic Framework Nanorod-Based Dual-Modal Nanoprobe for Sensing and Bioimaging of Phosphate.

Herein, a dual-modal fluorescent/colorimetric "Signal-On" nanoprobe based on PCN-222 nanorods (NRs) toward phosphate was proposed for the first time. Due to the high affinity of the zirconium node in PCN-222 NRs for phosphate, the structure collapse of PCN-222 NRs was triggered by phosphate, resulting in the release of the tetrakis(4-carboxyphenyl)porphyrin (TCPP) ligand from PCN-222 NRs as well as the enhancement of fluorescence and absorbance signals. The PCN-222 NR-based nanoprobe could be employed for phosphate detection over a wide concentration range with a detection limit down to 23 nM. The practical application of the PCN-222 NR-based nanoprobe in real samples was evaluated. Moreover, benefitting from the good biocompatibility and water dispersibility of PCN-222 NRs, this nanoprobe was successfully employed in the intracellular imaging of phosphate, revealing its promising application in the biological science. The present work would greatly extend the potential of nanostructured MOFs in the sensing and biological fields.

An ultrasensitive and selective fluorescent nanosensor based on porphyrinic metal-organic framework nanoparticles for Cu2+ detection.

Detecting trace amounts of copper ions (Cu2+) is of high importance since copper is an essential element in the environment and the human body. Despite the recent advances in Cu2+ detection, the current approaches still suffer from insensitivity and lack of in situ detection in living cells. In the present work, a fluorescent nanosensor based on porphyrinic metal-organic framework nanoparticles (MOF-525 NPs) is proposed for sensitive and selective monitoring of Cu2+ in aqueous solution and living cells. The MOF-525 NPs with attractive properties, including ultrasmall size, good water dispersity and intense red fluorescence, are prepared via a facile and environment-friendly hydrothermal route. The fluorescence signal of MOF-525 NPs could be quenched statically by Cu2+ with high selectivity due to the strong affinity of Cu2+ to the porphyrin ligand in MOF-525. The proposed fluorescent nanosensor has a linear response in the range of 1.0-250 nM with a low detection limit of 220 pM. Furthermore, it is successfully employed for the detection of Cu2+ in water samples and the intracellular imaging of Cu2+ in living cells, demonstrating its great potential in the sensing and biological fields.

Porphyrinic Metal-Organic Framework PCN-224 Nanoparticles for Near-Infrared-Induced Attenuation of Aggregation and Neurotoxicity of Alzheimer's Amyloid-ß Peptide.

The aberrant aggregation of amyloid-ß peptide (Aß) in the brain has been considered as the major pathological hallmark of Alzheimer's diseases (AD). Inhibition of Aß aggregation is considered as an attractive therapeutic intervention for alleviating amyloid-associated neurotoxicity. Here, we report the near-infrared light (NIR)-induced suppression of Aß aggregation and reduction of Aß-induced cytotoxicity via porphyrinic metal-organic framework (MOF) PCN-224 nanoparticles. PCN-224 nanoparticles are hydrothermally synthesized by coordinating tetra-kis(4-carboxyphenyl)porphyrin (TCPP) ligands with zirconium. The PCN-224 nanoparticles show high photo-oxygenation efficiency, good biocompatibility, and high stability. The study reveals that the porphyrinic MOF-based nanoprobe activated by NIR light could successfully inhibit self-assembly of monomeric Aß into a ß-sheet-rich structure. Furthermore, photoexcited PCN-224 nanoparticles also significantly reduce Aß-induced cytotoxicity under NIR irradiation.

Simultaneous Extraction of DNA and RNA from Hepatocellular Carcinoma (Hep G2) Based on Silica-Coated Magnetic Nanoparticles.

Nucleic acid (NA) extraction from cancer cells is an essential step in molecular oncologic testing. The conventional NA extraction protocols, based on several ultracentrifugation steps, suffer from time-consuming and complex manipulation. Here, a magnetic nanoparticle (MNP) based method for simultaneous extraction of DNA and RNA from cancer cells is described. This MNP based technique has received great attention and significant interest due to its convenient manipulation, low cost and ease for automation. Different factors including lysis buffer, ethanol, MNPs and washing buffers which may affect the yield of nucleic acid were optimized. The average yield of DNA and RNA obtained from 1 mL Hep G2 (˜106 cells) ranged from 9.7 to 14.7 µg with A260/A280 values between 1.68 and 2.01. The isolated DNA and RNA, using this method, were suitable for downstream activities such as PCR and RT-PCR.

Integration of Nucleic Acid Extraction Protocol with Automated Extractor for Multiplex Viral Detection.

The isolation of nucleic acids (NA) is the preliminary step to carry out genetic studies and DNA biosensor development. The presence of inhibitors in the purified NA interferes with the downstream application. These salts and other organic contaminations particularly challenge the analytical sensitivity of DNA biosensors. The detailed study was carried out to optimize the factors which might affect viral nucleic acid purification. The results suggested that 6 M guanidinium hydrochloride salt concentration was critical for NA isolation. The inverse relation has been found in the pH of the lysis buffer and quality and quantity of NA. The NA yield was relatively stable at pH 4­5. It has been observed that the use of carrier RNA was indispensable for viral genome isolation. The addition of ethanol to lysate in 1:1 ratio greatly improved NA recovery. The elution efficiency of DNase and RNase free water, 1× TE buffer and 1× PCR buffer was compared. The carrier RNA was best eluted in DNase and RNase free water and 1× TE buffer. It was further demonstrated that this method can be automatized for high throughput detection. A simple experiment was conducted to optimize the different parameters of an automated NA extractor to simultaneously extract HBV DNA and HCV RNA. The purified NA was successfully amplified in PCR and RT-PCR to verify the reliability of the established protocol. Thus a semi-automated system for the simultaneous detection of multiple viruses has been demonstrated.

Simultaneous detection of multiple viruses based on chemiluminescence and magnetic separation.

Nucleic acid testing (NAT) based methods are more sensitive and specific, and are preferred over enzyme immunoassays. Different NAT based protocols have been designed to detect multiple pathogens in order to reduce the inherent high cost for detection. However, these assays do not reliably detect a large number of pathogens at once. In this report, a DNA hybridization based chemiluminescence detection method has been proposed for reliable detection of multiple pathogens. The idea was practically demonstrated by carrying out simultaneous extraction and amplification of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV-1 group M, herein referred to as HIV) through the one step multiplex reverse transcription polymerase chain reaction (RT-PCR). A detailed study was conducted to optimize the factors which could affect the chemiluminescence signal. The probes for HBV, HCV, and HIV demonstrated higher specificity by only capturing their respective target sequence. 10 viral copies per mL of serum were detected in the monoplex detection protocol for HBV, HCV, and HIV, respectively. However, when the sensitivity of each virus was analyzed in the presence of higher loads of other viruses in the multiplex detection assay, the assay finally detected 10 HBV copies, 10 HCV copies, and 100 HIV copies per microliter of serum. The use of silica modified magnetic nanoparticles during nucleic acid extraction and carboxyl coated magnetic nanoparticles during the chemiluminescence step can help modify this system into an automated platform for high throughput applications.

Rapid and Sensitive Detection of RNA Viruses Based on Reverse Transcription Loop-Mediated Isothermal Amplification, Magnetic Nanoparticles, and Chemiluminescence.

RNA viruses, particularly, the highly pathogenic avian influenza (HPAI) virus, pose serious health concerns, and cause huge economic losses worldwide. Diagnostic tools for the early detection of these deadly RNA viruses are urgently needed to implement treatment and disease control strategies. Conventional reverse transcription polymerase chain reaction (RT-PCR)-based chemiluminescent (RT-PCR-CL) detection is frequently used for the diagnosis of viral infections. However, the requirements for expensive PCR machines and longer thermocycling times are significant drawbacks. In this study, we propose a method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with chemiluminescence (CL) to detect H7N9 virus. The proposed method does not require any expensive instruments, and processing time is remarkably shortened compared to that of RT-PCR-CL. Since several factors including RT-LAMP temperature, probe concentration, hybridization temperature, and hybridization duration might affect the CL signal, each of these parameters was investigated and optimized. One thousand copies/mL of H7N9 RNA were detectable using the optimized RT-LAMP-CL method. The detection time was significantly reduced by using RT-LAMP, in comparison with conventional RT-PCR-CL. This technique holds great promise for viral detection and diagnosis, especially with regard to avian influenza virus.

Genetic Variation of BCL2 (rs2279115), NEIL2 (rs804270), LTA (rs909253), PSCA (rs2294008) and PLCE1 (rs3765524, rs10509670) Genes and Their Correlation to Gastric Cancer Risk Based on Universal Tagged Arrays and Fe3O4 Magnetic Nanoparticles.

With the help of Fe3O4 nagnetic nanoparticles as a solid carrier and an excellent tool for separation, six SNP loci (rs2279115 of BCL2 gene, rs804270 of NEIL2 gene, rs909253 of LTA gene, rs2294008 of PSCA gene, rs3765524 and rs10509670 of PLCE1 gene) were selected to evaluate their relation to gastric cancer risk. Using two kinds of functionalized magnetic nanoparticles and universal tagged arrays, the whole operation procedure including genome DNA extraction and SNP genotyping was performed. All genotypes and allele frequencies were calculated in the cases and controls respectively to analyze their association with gastric cancer risk. Totally 200 pathological samples and 134 normal control subjects were collected. The results demonstrated that four SNP loci (rs2279115, rs804270, rs909253 and rs3765524) showed a potential association with gastric cancer risk, and the other two (rs2294008, rs10509670) possessed no difference/association among cases and controls.