Genome Editing VEGFA Prevents Corneal Neovascularization In Vivo.

Corneal neovascularization (CNV) is a common clinical finding seen in a range of eye diseases. Current therapeutic approaches to treat corneal angiogenesis, in which vascular endothelial growth factor (VEGF) A plays a central role, can cause a variety of adverse side effects. The technology of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 can edit VEGFA gene to suppress its expression. CRISPR offers a novel opportunity to treat CNV. This study shows that depletion of VEGFA with a novel CRISPR/Cas9 system inhibits proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Importantly, subconjunctival injection of this dual AAV-SpCas9/sgRNA-VEGFA system is demonstrated which blocks suture-induced expression of VEGFA, CD31, and α-smooth muscle actin as well as corneal neovascularization in mice. This study has established a strong foundation for the treatment of corneal neovascularization via a gene editing approach for the first time.

Identification and validation of circulating biomarkers for detection of liver cancer with antibody array.

The aim of this study was to find new protein biomarkers that could be used to detect hepatocellular carcinoma (HCC) in the serum. We identified 11 proteins in the tissue that could be used to classify samples from HCC and control subjects. The 11 identified tissue biomarkers were combined with 10 commonly used serum HCC biomarkers for further verification in a large number of serum samples from HCC patients and healthy controls. 17 of the 21 prospective serum biomarkers were determined to be differentially expressed through collinearity and significance analysis. Through the method of supervised learning, a random forest model was constructed to reduce the dimensionality of the number of differentially expressed proteins, and finally, 4 differentially expressed proteins were identified: AFP, GDF15, CEACAM-1, and MMP-9, and suggested to have potential application in clinical diagnosis of HCC.

The spike-ACE2 binding assay: An in vitro platform for evaluating vaccination efficacy and for screening SARS-CoV-2 inhibitors and neutralizing antibodies.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 has become a worldwide pandemic, and there is a pressing need for the rapid development of novel therapeutic strategies. SARS-CoV-2 viral entry is mediated by interaction between the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein and host cellular receptor, human angiotensin converting enzyme 2 (ACE2). The lack of a high throughput screening (HTS) platform for candidate drug screening means that no targeted COVID-19 treatments have been developed to date. To overcome this limitation, we developed a novel, rapid, simple, and HTS binding assay platform to screen potential inhibitors of the RBD-ACE2 complex. Three "neutralizing" mouse monoclonal antibodies capable of blocking the RBD-ACE2 interaction were identified using our binding assay and pseudovirus neutralization assay followed by further validation with the Focus Reduction Neutralization Test (FRNT), which analyzes the neutralization capacity of samples in the presence of live SARS-CoV-2. Furthermore, the consistency of our binding assay and FRNT results (R2 = 0.68) was demonstrated by patients' serum, of which were COVID-19 positive (n = 34) and COVID-19 negative (n = 76). Several small molecules selected for their potential to inhibit the Spike-ACE2 complex in silico were also confirmed with the binding assay. In addition, we have evaluated vaccine efficacy using binding assay platform and validated through pseudovirus neutralization assay. The correlation between binding assay & psuedovirus assay of the post vaccinated serum showed well correlated (R2 = 0.09) Moreover, our binding assay platform successfully validated different Spike RBD mutants. These results indicate that our binding assay can be used as a platform for in vitro screening of small molecules and monoclonal antibodies, and high-throughput assessment of antibody levels after vaccination. When conducting drug screening, computer virtual screening lacks actual basis, construction of pseudoviruses is relatively complicated, and even FRNT requires a P3 laboratory. There are few methods to determine the competitiveness of the target drug and SRBD or ACE2. Our binding assay can fill this gap and accelerate the process and efficiency of COVID-19 drug screening.

Comprehensive Profiling of Inflammatory Factors Revealed That Growth Differentiation Factor-15 Is an Indicator of Disease Severity in COVID-19 Patients.

To systematically explore potential biomarkers which can predict disease severity in COVID-19 patients and prevent the occurrence or development of severe COVID-19, the levels of 440 factors were analyzed in patients categorized according to COVID-19 disease severity; including asymptomatic, mild, moderate, severe, convalescent and healthy control groups. Factor candidates were validated by ELISA and functional relevance was uncovered by bioinformatics analysis. To identify potential biomarkers of occurrence or development of COVID-19, patient sera from three different severity groups (moderate, severe, and critical) at three time points (admission, remission, and discharge) and the expression levels of candidate biomarkers were measured. Eleven differential factors associated with disease severity were pinpointed from 440 factors across 111 patients of differing disease severity. The dynamic changes of GDF15 reflect the progression of the disease, while the other differential factors include TRAIL R1, IGFBP-1, IGFBP-4, VCAM-1, sFRP-3, FABP2, Transferrin, GDF15, IL-1F7, IL-5Rα, and CD200. Elevation of white blood cell count, neutrophil count, neutrophil-lymphocyte ratio (NLR), Alanine aminotransferase and Aspartate aminotransferase, low lymphocyte and eosinophil counts in the severe group were associated with the severity of COVID-19. GDF15 levels were observed to be associated with the severity of COVID-19 and the dynamic change of GDF15 levels was closely associated with the COVID-19 disease progression. Therefore, GDF15 might serve as an indicator of disease severity in COVID-19 patients.

Enhanced Piezoelectric Performance of Various Electrospun PVDF Nanofibers and Related Self-Powered Device Applications.

The piezoelectric polymer poly(vinylidene fluoride) (PVDF) has been widely employed for energy harvesting or sensors. Its piezoelectricity originates from the unique crystal structure and the oriented electric dipoles. Generally, electrospinning (ES) is able to improve the crystallinity and piezoelectricity of PVDF. In this investigation, three types of ES, including far-field ES with random (R-PVDF-FFES) or aligned distribution (A-PVDF-FFES) and near-field ES (PVDF-NFES), are applied to fabricate a one-step polarized PVDF membrane. To compare the piezoelectricity of separated PVDF-NFES fibers, the array of PVDF-NFES fibers is encapsulated in polylactic acid (PLA). The result shows that the piezoelectricity of PVDF is dramatically enhanced by NFES and FFES. In d31 mode, the current density of PVDF-NFES-PLA is 75.63 pA cm-2 with a 2 Hz shear force, which is four times larger than A-PVDF-FFES (17.62 pA cm-2) or seven times larger than R-PVDF-FFES (10.63 pA cm-2). Due to its outstanding property, the PVDF-NFES-PLA membrane is designed to be applied as a self-powered device. The unimorph cantilevers are prepared to harvest the vibration energy and their output power density reaches 95.3 µW m-2 at a resonance frequency of 31 Hz. Meanwhile, the flexible bending sensor and pressure sensor are fabricated with a PVDF-NFES-PLA membrane, which shows the stable response for pressure with sensitivity values of 864.68 and 22.6 mV kPa-1, respectively.

Active-Sensing Epidermal Stretchable Bioelectronic Patch for Noninvasive, Conformal, and Wireless Tendon Monitoring.

Sensors capable of monitoring dynamic mechanics of tendons throughout a body in real time could bring systematic information about a human body's physical condition, which is beneficial for avoiding muscle injury, checking hereditary muscle atrophy, and so on. However, the development of such sensors has been hindered by the requirement of superior portability, high resolution, and superb conformability. Here, we present a wearable and stretchable bioelectronic patch for detecting tendon activities. It is made up of a piezoelectric material, systematically optimized from architectures and mechanics, and exhibits a high resolution of 5.8 × 10-5 N with a linearity parameter of R 2 = 0.999. Additionally, a tendon real-time monitoring and healthcare system is established by integrating the patch with a micro controller unit (MCU), which is able to process collected data and deliver feedback for exercise evaluation. Specifically, through the patch on the ankle, we measured the maximum force on the Achilles tendon during jumping which is about 16312 N, which is much higher than that during normal walking (3208 N) and running (5909 N). This work not only provides a strategy for facile monitoring of the variation of the tendon throughout the body but also throws light on the profound comprehension of human activities.

Comparison of a New Optical Biometer That Combines Scheimpflug Imaging With Partial Coherence Interferometry With That of an Optical Biometer Based on Swept-Source Optical Coherence Tomography and Placido-Disk Topography.

PURPOSE: To evaluate measurement precision and to compare the Pentacam AXL (Oculus Optikgeräte, Wetzlar, German), a new optical biometer based on Scheimpflug imaging and partial coherence interferometry (PCI) with that of the OA-2000 biometer (Tomey, Nagoya, Japan), which combines swept-source optical coherence tomography (SS-OCT) and Placido-disk topography. METHODS: Axial length (AL), central corneal thickness (CCT), anterior chamber depth (ACD), aqueous depth (AQD), mean keratometry (Km), astigmatism vectors J0, J45, and corneal diameter (CD) were measured in triplicate by two technical operators. Within-subject standard deviation (Sw), repeatability and reproducibility (2.77 Sw), coefficient of variation (CoV), and intraclass correlation coefficient (ICC) were used to assess the Pentacam AXL intra-observer repeatability and inter-observer reproducibility. Paired t-test and Bland-Altman plots were used to determine the agreement between the two biometers. RESULTS: The new optical biometer had high intra-observer repeatability [all parameters evaluated had low CoV (<0.71%) and high ICC (>0.88)]. Inter-observer reproducibility was also excellent, with high ICC (>0.95) and low CoV (<0.52%). The 95% LoA between the new biometer and OA-2000 were insignificant for most of the parameters evaluated, especially for AL. However, the measurement agreement was moderate for CCT. CONCLUSIONS: Intra-observer repeatability and inter-observer reproducibility were excellent for all parameters evaluated using the new optical biometer based on Scheimpflug imaging and PCI. There was a high agreement between the two devices and hence could be clinically interchangeable for the measurement of most ocular parameters.

Imaging and Inhibiting: A Dual Function Molecular Flare for Cancer Cells.

The Wnt pathway is dysregulated and activated in many human malignancies. More than 90% of colon cancers have variations in the Wnt pathway. Sulindac, a drug that targets protein Dvl of the Wnt/Dvl/ß-catenin pathway, which regulates cancer gene expression, has been reported to significantly reduce the incidence and the risk of death from colorectal cancer and other types of cancer. Herein, a dual functional compound (SLN) containing Sulindac and a linked fluorophore is first reported, combining the functions of lighting up colon cancer cells as a flare and inhibiting colon tumors as a drug. SLN can not only mark the Dvl protein in the Wnt pathway to recognize tumors layer by layer but also achieve effective inhibition of colon cancer, providing a promising reagent for chemotherapy and a fluorescent indicator for surgery during the removal the colon tumors in situ.

Functional daidzein enhances the anticancer effect of topotecan and reverses BCRP-mediated drug resistance in breast cancer.

Topotecan (TPT), a semisynthetic derivative of camptothecin, has been used in cancer chemotherapy, but side effects and drug resistance limit its clinical application. Daidzein (DAI), a natural isoflavone and bioactive food component widely existing in fruits, nuts, soybeans and soy-based products, is a type of phytoestrogen. Combination treatment with DAI and TPT showed a strong synergistic effect on tumor cells, with a 0.10˜0.66 combined index, by increasing TPT inhibition on Topo Ⅰ, resulting in more cells arresting at the G2/M phase and inducing more cells to undergo apoptosis. In addition, the resistance of MCF7/ADR cells to TPT was reversed (the resistance index decreased from 7.17 to 0.77) by inhibiting the expression of ERα and BCRP to increase TPT accumulation intracellularly. Moreover, the combination of DAI and TPT showed a stronger inhibitory effect (P < 0.01) on tumor growth in both MCF7 and MCF7/ADR xenograft models than the 9 mg/kg TPT monotherapy group. Our results may provide a reasonable, new approach to develop safe and efficient nutrition components from foods for breast cancer combination treatment.

Celecoxib Conjugated Fluorescent Probe for Identification and Discrimination of Cyclooxygenase-2 Enzyme in Cancer Cells.

Cyclooxygenase-2 (COX-2) is an enzyme overexpressed in most types of cancers and has been used for an excellent targetable biomarker. Celecoxib is an effective inhibitor of COX-2, used in anti-inflammation. Herein we report a one and two-photon fluorescence probe (NP-C6-CXB) for COX-2, based on the conjugation of naphthalamide with Celecoxib, by using flexible hexylene linker. NP-C6-CXB is nonfluorescent in buffer solution and normal cells, while it shows bright fluorescence in solutions and cancer cells in the presence of COX-2 with an excellent selectivity. Interestingly, NP-C6-CXB can discriminate cancer cells (MCF-7) from normal cells (COS-7) in the single culture medium under confocal microscopy. Due to the selective binding affinity of NP-C6-CXB with a COX-2 enzyme, the intensity is proportional to the level of COX-2 enzyme in cancer cells. In vivo and in vitro experiments proved that NP-C6-CXB is a potential tool for identification of tumor and might be used in surgical resection of COX-2 expressed tumors.

Fluorescence imaging of hepatocellular carcinoma with a specific probe of COX-2.

Hepatocellular carcinoma (HCC) is the major subtype of primary liver cancer. Although the standard treatment method based on surgery has generally extended life, it still causes the second and sixth most prevalent cancer-related death in men and women, respectively. The recurrence of cancer caused by unclear resection margins and any remaining undiscovered metastatic nodules should take a large proportion of responsibility for the poor prognosis after resective surgery. Therefore, a practical and effective method that can be used during hepatectomy to specifically identify HCC is a potentially significant area deserving attention. Tests involving fluorescence have been used in many biological systems. In this study, we use a probe that can combine with cyclooxygenase-2 (COX-2) and subsequently emit fluorescence to identify HCC cells and heteroplastic tumors in a mouse model. The results show that this specific probe can clearly differentiate HCC, with differences that could be observed with the naked eye in human samples. The biotechnology of knocking down COX-2 and its inhibitor were used on human HCC cell line SMMC7721, and the outcomes confirmed the above results. The toxic effect also showed that the probe had no harmful effect on normal liver cells. Taken together, our study demonstrates that a COX-2-specific fluorescence probe may be a new and effective method to identify HCC, especially during surgery.

Lighting up fluoride ions in cellular mitochondria using a highly selective and sensitive fluorescent probe.

We report a highly selective and sensitive fluorescent probe () for detecting fluoride ions, for the first time, lighting up the fluoride ions in mitochondria with a strong green fluorescence. could be easily prepared as fluoride paper test strips to detect fluoride ions in aqueous solutions with a detection limit as low as 19 ppb.

An off-on COX-2-specific fluorescent probe: targeting the Golgi apparatus of cancer cells.

Identifying cancer cells and quantifying cancer-related events in particular organelles in a rapid and sensitive fashion are important for early diagnosis and for studies on pathology and therapeutics of cancers. Herein a smart "off-on" cyclooxygenase-2-specific fluorescence probe (ANQ-IMC-6), able to report the presence of cancer cells and to image Golgi-related events, has been designed and evaluated. Cyclooxygenase-2 (COX-2) has been used as imaging target in the probe design, since this enzyme is a biomarker of virtually all cancer cell lines. In the free state in aqueous solution, ANQ-IMC-6 mainly exists in a folded conformation where probe fluorescence is quenched through photoinduced electron transfer between the fluorophore acenaphtho[1,2-b]quinoxaline (ANQ) and the recognition group, indomethacin (IMC). Fluorescence is turned on, by restraining the photoinduced electron transfer, when ANQ-IMC-6 is forced to adopt the unfolded state following binding to COX-2 in the Golgi apparatus of cancer cells. ANQ-IMC-6 provides high signal-to-background staining and has been successfully used to rapidly differentiate cancer cells from normal cells when using flow cytometry and one- and two-photon fluorescence microscopic imaging. Furthermore, ANQ-IMC-6 may be able to visualize dynamic changes of the Golgi apparatus during cancer cell apoptosis, with possible application to early diagnosis.

Fluorene-derived two-photon fluorescent probes for specific and simultaneous bioimaging of endoplasmic reticulum and lysosomes: group-effect and localization.

Two novel fluorene-derived two-photon fluorescent probes (TPFL) targeting the endoplasmic reticulum (TPFL-ER) and lysosomes (TPFL-Lyso) were synthesized by introducing a chlorine group and a morpholine group, respectively. They were shown to be suitable for specific and simultaneous imaging of the endoplasmic reticulum and lysosomes without affecting protein movements owing to their similar molecular structures. TPFL-ER and TPFL-Lyso were successfully used to visualize the changes of the endoplasmic reticulum and lysosomes during cancer cell apoptosis and they demonstrated high specificity and sensitivity, excellent photostability, and low phototoxicity.