Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing.

The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.

Fluorinated porphyrin-based theranostics for dual imaging and chemo-photodynamic therapy.

Convenient strategies to transform regular liposomes or nano-micelles into multifunctional theranostics would be highly valuable in cancer therapy. Herein, we developed an amphiphilic fluorinated porphyrin dendrimer as a multifunctional "add-on" module which would self-assemble onto liposomal drug delivery systems and conveniently transform the liposomes into novel theranostics. Through cancer cells and murine xenograft tumor model assays, the theranostics showed valuable fluorescence/19F magnetic resonance dual modal imaging and highly efficient chemo-photodynamic therapy. The modular strategy facilitates the convenient and standardized preparation of multifunctional theranostics.

Fluorinated cryptophane-A and porphyrin-based theranostics for multimodal imaging-guided photodynamic therapy.

Herein, we developed fluorinated nanoemulsions with significantly enhanced in vitro and in vivo129Xe hyper-CEST MRI, 19F MRI and fluorescence imaging signals for selective and sensitive tumor detection and NIR-activated photodynamic therapy.

Peptidic Monodisperse PEG "Comb" as Multifunctional "Add-On" Module for Imaging-Traceable and Thermo-Responsive Theranostics.

Monodisperse polyethylene glycols-modified (M-PEGylated) biomaterials exhibit high structural accuracy, biocompatibility, and fine-tunable physicochemical properties. To develop "smart" drug delivery systems in a controllable and convenient manner, a peptidic M-PEG "comb" with fluorinated L-lysine side chains and a fluorescent N-terminal is conveniently prepared as a 19 F magnetic resonance imaging (19 F MRI) and fluorescence dual-imaging traceable and thermo-responsive "add-on" module for liposomal theranostics in cancer therapy. The peptidic M-PEG "comb" has high biocompatibility, thermo-responsivity with a sharp lower critical solution temperature, an aggregation-induced emission fluorescence, and high 19 F MRI sensitivity. As a highly branched amphiphile, it self-assembles and firmly anchors on the doxorubicin-loaded liposomal nanoparticles, which M-PEGylates the liposomes and facilitates the thermo-responsive drug release and drug tracking with dual-imaging technologies. In a rodent xenograft model of human liver cancer HepG2 cells, the M-PEGylated liposomes exhibit long in vivo half time, low toxicity, high tumor accumulation, "hot spot" 19 F MRI, and therapeutic efficacy. With accurately programmable chemical structure, fine-tunable physicochemical and biological properties to meet the demands of diagnosis, drug delivery, and therapy, the M-PEG "comb" is promising as a versatile "add-on" module for rapid and convenient formulation of various "smart" theranostics.

New Butenolides and Cyclopentenones from Saline Soil-Derived Fungus Aspergillus Sclerotiorum.

Three new γ-hydroxyl butenolides (1-3), a pair of new enantiomeric spiro-butenolides (4a and 4b), a pair of enantiomeric cyclopentenones (5a new and 5b new natural), and six known compounds (6-11), were isolated from Aspergillus sclerotiorum. Their structures were established by spectroscopic data and electronic circular dichroism (ECD) spectra. Two pairs of enantiomers [(+)/(-)-6c and (+)/(-)-6d] obtained from the reaction of 6 with acetyl chloride (AcCl) confirmed that 6 was a mixture of two pairs of enantiomers. In addition, the X-ray data confirmed that 7 was also a racemate. The new metabolites (1-5) were evaluated for their inhibitory activity against cancer and non-cancer cell lines. As a result, compound 1 exhibited moderate cytotoxicity to HL60 and A549 with IC50 values of 6.5 and 8.9 µM, respectively, and weak potency to HL-7702 with IC50 values of 17.6 µM. Furthermore, compounds 1-9 were screened for their antimicrobial activity using the micro-broth dilution method. MIC values of 200 µg/mL were obtained for compounds 2 and 3 towards Staphylococcus aureus and Escherichia coli, while compound 8 exhibited a MIC of 50 µ/mL towards Candida albicans.

Peptidic Monodisperse PEG "combs" with Fine-Tunable LCST and Multiple Imaging Modalities.

Thermosensitive and imaging-traceable materials with fine-tunable lower critical solution temperature (LCST) around body temperature are highly valuable in biomedicine. However, such materials are rare because it is challenging to fine-tune the LCST and incorporate suitable imaging modalities. Herein, peptidic monodisperse polyethylene glycol (M-PEG) "combs" with fine-tunable LCST, "hot spot" fluorine-19 magnetic resonance imaging (19F MRI), thermoresponsive fluorescent imaging, and drug loading ability were developed through accurately programming their structures during solid phase peptide synthesis (SPPS). The easy availability, structural accuracy, biocompatibility, and versatility provide the M-PEG "combs" with promising prospects as thermoresponsive and imaging-traceable biomaterials for controlled drug delivery.

129Xe Hyper-CEST/19F MRI Multimodal Imaging System for Sensitive and Selective Tumor Cells Detection.

Heteronuclear MRI offers broad potential for specific detection and quantification of molecularly targeted agents in diagnosis and therapy planning or monitoring. Here we report a novel method for simultaneous acquisition of dual-nuclei hyper-CEST 129Xe and 19F tumor targeting MRI. 129Xe hyper-CEST MRI, 19F MRI and fluorescent imaging were integrated into PFOB nanoemulsion as a imaging system for sensitive and selective tumor cells detection. As the complement to 1H MRI, 129Xe and 19F signals can also provide efficient and precise anatomical localization of tumors.

AuNP flares-capped mesoporous silica nanoplatform for MTH1 detection and inhibition.

The human mutT homologue MTH1, a nucleotide pool sanitizing enzyme, represents a vulnerability factor and an attractive target for anticancer therapy. However, there is currently a lack of selective and effective platforms for the detection and inhibition of MTH1 in cells. Here, we demonstrate for the first time a gold nanoparticle (AuNP) flares-capped mesoporous silica nanoparticle (MSN) nanoplatform that is capable of detecting MTH1 mRNA and simultaneously suppressing MTH1 activity. The AuNP flares are made from AuNPs that are functionalized with a dense shell of MTH1 recognition sequences hybridized to short cyanine (Cy5)-labeled reporter sequences and employed to seal the pores of MSN to prevent the premature MTH1 inhibitors (S-crizotinib) release. Just like the pyrotechnic flares that produce brilliant light when activated, the resulting AuNP flares@MSN (S-crizotinib) undergo a significant burst of red fluorescence enhancement upon MTH1 mRNA binding. This hybridization event subsequently induces the opening of the pores and the release of S-crizotinib in an mRNA-dependent manner, leading to significant cytotoxicity in cancer cells and improved therapeutic response in mouse xenograft models. We anticipate that this nanoplatform may be an important step toward the development of MTH1-targeting theranostics and also be a useful tool for cancer phenotypic lethal anticancer therapy.

Correlations of salivary biomarkers with clinical assessments in patients with cystic fibrosis.

RATIONALE: Monitoring clinical disease status in cystic fibrosis frequently requires invasive collection of clinical samples. Due to its noninvasive collection process and direct anatomic relationship with the lower airway, saliva shows great potential as a biological fluid for cystic fibrosis monitoring. OBJECTIVES: To measure the levels of multiple protein markers in human saliva supernatants and investigate the possibility of utilizing them to provide a more quantitative measure of disease state for use in research and monitoring of patients with cystic fibrosis clinically. METHODS: Whole saliva samples were collected and processed from cystic fibrosis patients at two distinct time points (2010 and 2013) and measured by two separate platforms. In this cross sectional study, a convenience sample of 71 participants were recruited with samples measured by multiplexed fluorescence microarray (fiber microarray) and another 117 participant samples were measured by an automated, point-of-care, analyzer (SDReader) using a microsphere-based array via fluorescence sandwich immunoassay. For comparison, saliva from 56 and 50 healthy subjects were collected, respectively. The levels of six target proteins were quantified. Various demographic and clinical data, including spirometry, medical history, and clinicians' assessments were also collected from patients with cystic fibrosis on the day of saliva collection. MEASUREMENTS AND MAIN RESULTS: Similar trends were observed with both platforms and compared with healthy subjects, cystic fibrosis patients had significantly elevated levels of VEGF, IP-10, IL-8, and EGF as well as lower levels of MMP-9 (P ≤ 0.005) using fiber microarray and significantly elevated levels of IP-10, IL-8 with lower levels of MMP-9 and IL-1ß (P ≤ 0.02) using the SDReader. The levels of the six proteins correlated with each other significantly, and in some cases, biomarker levels could be used to differentiate between subgroups of patients with different clinical presentations. For example, IP-10 levels significantly correlated with FEV1 and disease severity (as evaluated by clinicians) with both platforms (P < 0.05). CONCLUSIONS: Significant variations of the levels of six proteins in saliva supernatants, and the correlations of these levels with clinical assessments, demonstrated the potential of saliva for cystic fibrosis research and monitoring.

Targeting lysosomal membrane permeabilization to induce and image apoptosis in cancer cells by multifunctional Au-ZnO hybrid nanoparticles.

We have developed multifunctional Au-ZnO hybrid nanoparticles (NPs) for targeted induction lysosomal membrane permeabilization (LMP)-dependent apoptosis in cancer cells and real-time imaging.

Multiplexed salivary protein profiling for patients with respiratory diseases using fiber-optic bundles and fluorescent antibody-based microarrays.

Over the past 40 years, the incidence and prevalence of respiratory diseases have increased significantly throughout the world, damaging economic productivity and challenging health care systems. Current diagnoses of different respiratory diseases generally involve invasive sampling methods such as induced sputum or bronchoalveolar lavage that are uncomfortable, or even painful, for the patient. In this paper, we present a platform incorporating fiber-optic bundles and antibody-based microarrays to perform multiplexed protein profiling of a panel of six salivary biomarkers for asthma and cystic fibrosis (CF) diagnosis. The platform utilizes an optical fiber bundle containing approximately 50,000 individual 4.5 µm diameter fibers that are chemically etched to create microwells in which modified microspheres decorated with monoclonal capture antibodies can be deposited. On the basis of a sandwich immunoassay format, the array quantifies human vascular endothelial growth factor (VEGF), interferon gamma-induced protein 10 (IP-10), interleukin-8 (IL-8), epidermal growth factor (EGF), matrix metalloproteinase 9 (MMP-9), and interleukin-1 beta (IL-1ß) salivary biomarkers in the subpicomolar range. Saliva supernatants collected from 291 individuals (164 asthmatics, 71 CF patients, and 56 healthy controls (HC)) were analyzed on the platform to profile each group of patients using this six-analyte suite. It was found that four of the six proteins were observed to be significantly elevated (p < 0.01) in asthma and CF patients compared with HC. These results demonstrate the potential to use the multiplexed protein array platform for respiratory disease diagnosis.

Autophagy inhibition promotes 5-fluorouraci-induced apoptosis by stimulating ROS formation in human non-small cell lung cancer A549 cells.

Chemotherapy is an important option for the treatment of various cancers including lung cancer. However, tumor resistance towards cytotoxic chemotherapy has become more common. It has been reported that autophagy is one of the processes contributing to this resistance. In the present study, we found that the anti-cancer drug 5-fluorouraci(5-FU) could induce autophagy in A549 cells. 5-FU treatment could lead to the conversion of LC3 I/II, the up-regulation of Beclin-1, the down-regulation of p62 and the formation of acidic vesicular organelles (AVOs) in A549 cells. Pre-treatment of cancer cells with 3-MA or siAtg7 could enhance 5-FU-induced apoptosis through the activation of caspases, and the caspase inhibitor z-VAD-fmk rescued the cell viability reduction. Furthermore, the inhibition of autophagy also stimulated ROS formation and scavenging of ROS by antioxidant NAC inhibited caspase-3 activity, prevented the release of cyt-c from mitochondria and eventually rescued cancer cells from 5-FU-mediated apoptosis. These results suggest that 5-FU-elicited autophagic response plays a protective role against cell apoptosis and the inhibition of autophagy could sensitize them to 5-FU-induced caspase-dependent apoptosis through the stimulation of ROS formation.

Oil-sealed femtoliter fiber-optic arrays for single molecule analysis.

This paper describes a novel method for fabricating and sealing high-density arrays of femtoliter reaction chambers. We chemically etch one end of a 2.3 mm diameter glass optical fiber bundle to create an array of microwells. We then use a contact printing method to selectively modify the surface of the material between microwells with a hydrophobic silane. This modification makes it possible to fill the wells with aqueous solution and then seal them with a droplet of oil, forming an array of isolated reaction chambers. Individual ß-galactosidase molecules trapped in these reaction chambers convert a substrate into a fluorescent product that can be readily detected because a high local concentration of product is achieved. This binary readout can be used for ultra-sensitive measurements of enzyme concentration. We observed that the percentage of wells showing enzyme activity was linearly dependent on the concentration of soluble ß-galactosidase in the picomolar range. A similar response was also observed for streptavidin-ß-galactosidase captured by biotinylated beads. These arrays are also suitable for performing single-molecule kinetics studies on hundreds to thousands of enzyme molecules simultaneously. We observed a broad distribution of catalytic rates for individual ß-galactosidase molecules trapped in the microwells, in agreement with previous studies using similar arrays that were mechanically sealed. We have further demonstrated that this femtoliter fiber-optic array can be integrated into a PDMS microfluidic channel system and sealed with oil on-chip, creating an easy to use and high-throughput device for single-molecule analysis.

The synthesis of highly water-dispersible and targeted CdS quantum dots and it is used for bioimaging by confocal microscopy.

Synthesis of a highly dispersed hydrophilic CdS nanocrystals and their use as fluorescence labeling for live cell imaging is reported here. By carefully manipulating the surface of CdS nanocrystals, the dispersions of CdS-MAA-PEI-FA nanocrystals with high photostability is prepared. The receptor-mediated delivery of folic acid conjugated quantum dots into folate-receptor-positive cell lines such as CBRH7919 liver cancer cells was demonstrated by confocal microscopy. In the future, the further modified CdS nanoparticles can be used for the tissue imaging in vivo studies.