Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds.

Accumulation of mutant proteins is a major cause of many diseases (collectively called proteopathies), and lowering the level of these proteins can be useful for treatment of these diseases. We hypothesized that compounds that interact with both the autophagosome protein microtubule-associated protein 1A/1B light chain 3 (LC3)1 and the disease-causing protein may target the latter for autophagic clearance. Mutant huntingtin protein (mHTT) contains an expanded polyglutamine (polyQ) tract and causes Huntington's disease, an incurable neurodegenerative disorder2. Here, using small-molecule-microarray-based screening, we identified four compounds that interact with both LC3 and mHTT, but not with the wild-type HTT protein. Some of these compounds targeted mHTT to autophagosomes, reduced mHTT levels in an allele-selective manner, and rescued disease-relevant phenotypes in cells and in vivo in fly and mouse models of Huntington's disease. We further show that these compounds interact with the expanded polyQ stretch and could lower the level of mutant ataxin-3 (ATXN3), another disease-causing protein with an expanded polyQ tract3. This study presents candidate compounds for lowering mHTT and potentially other disease-causing proteins with polyQ expansions, demonstrating the concept of lowering levels of disease-causing proteins using autophagosome-tethering compounds.

Suppression of toxicity of the mutant huntingtin protein by its interacting compound, desonide.

Identifying inhibitors of pathogenic proteins is the major strategy of targeted drug discoveries. This strategy meets challenges in targeting neurodegenerative disorders such as Huntington's disease (HD), which is mainly caused by the mutant huntingtin protein (mHTT), an "undruggable" pathogenic protein with unknown functions. We hypothesized that some of the chemical binders of mHTT may change its conformation and/or stability to suppress its downstream toxicity, functioning similarly to an "inhibitor" under a broader definition. We identified 21 potential mHTT selective binders through a small-molecule microarray­based screening. We further tested these compounds using secondary phenotypic screens for their effects on mHTT-induced toxicity and revealed four potential mHTT-binding compounds that may rescue HD-relevant phenotypes. Among them, a Food and Drug Administration­approved drug, desonide, was capable of suppressing mHTT toxicity in HD cellular and animal models by destabilizing mHTT through enhancing its polyubiquitination at the K6 site. Our study reveals the therapeutic potential of desonide for HD treatment and provides the proof of principle for a drug discovery pipeline: target-binder screens followed by phenotypic validation and mechanistic studies.

CDK inhibitor Palbociclib targets STING to alleviate autoinflammation.

Aberrant activation of stimulator of interferon genes (STING) is tightly associated with multiple types of disease, including cancer, infection, and autoimmune diseases. However, the development of STING modulators for the therapy of STING-related diseases is still an unmet clinical need. We employed a high-throughput screening approach based on the interaction of small-molecule chemical compounds with recombinant STING protein to identify functional STING modulators. Intriguingly, the cyclin-dependent protein kinase (CDK) inhibitor Palbociclib was found to directly bind STING and inhibit its activation in both mouse and human cells. Mechanistically, Palbociclib targets Y167 of STING to block its dimerization, its binding with cyclic dinucleotides, and its trafficking. Importantly, Palbociclib alleviates autoimmune disease features induced by dextran sulphate sodium or genetic ablation of three prime repair exonuclease 1 (Trex1) in mice in a STING-dependent manner. Our work identifies Palbociclib as a novel pharmacological inhibitor of STING that abrogates its homodimerization and provides a basis for the fast repurposing of this Food and Drug Administration-approved drug for the therapy of autoinflammatory diseases.

Nuclear cGAS suppresses DNA repair and promotes tumorigenesis.

Accurate repair of DNA double-stranded breaks by homologous recombination preserves genome integrity and inhibits tumorigenesis. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that activates innate immunity by initiating the STING-IRF3-type I IFN signalling cascade1,2. Recognition of ruptured micronuclei by cGAS links genome instability to the innate immune response3,4, but the potential involvement of cGAS in DNA repair remains unknown. Here we demonstrate that cGAS inhibits homologous recombination in mouse and human models. DNA damage induces nuclear translocation of cGAS in a manner that is dependent on importin-α, and the phosphorylation of cGAS at tyrosine 215-mediated by B-lymphoid tyrosine kinase-facilitates the cytosolic retention of cGAS. In the nucleus, cGAS is recruited to double-stranded breaks and interacts with PARP1 via poly(ADP-ribose). The cGAS-PARP1 interaction impedes the formation of the PARP1-Timeless complex, and thereby suppresses homologous recombination. We show that knockdown of cGAS suppresses DNA damage and inhibits tumour growth both in vitro and in vivo. We conclude that nuclear cGAS suppresses homologous-recombination-mediated repair and promotes tumour growth, and that cGAS therefore represents a potential target for cancer prevention and therapy.

Analysis of Fluid Replacement in Two Fluidic Chambers for Oblique-Incidence Reflectivity Difference (OI-RD) Biosensor.

Optical biosensors have a significant impact on various aspects of our lives. In many applications of optical biosensors, fluidic chambers play a crucial role in facilitating controlled fluid delivery. It is essential to achieve complete liquid replacement in order to obtain accurate and reliable results. However, the configurations of fluidic chambers vary across different optical biosensors, resulting in diverse fluidic volumes and flow rates, and there are no standardized guidelines for liquid replacement. In this paper, we utilize COMSOL Multiphysics, a finite element analysis software, to investigate the optimal fluid volume required for two types of fluidic chambers in the context of the oblique-incidence reflectivity difference (OI-RD) biosensor. We found that the depth of the fluidic chamber is the most crucial factor influencing the required liquid volume, with the volume being a quadratic function of the depth. Additionally, the required fluid volume is also influenced by the positions on the substrate surface bearing samples, while the flow rate has no impact on the fluid volume.

Emerging degrader technologies engaging lysosomal pathways.

Targeted protein degradation (TPD) provides unprecedented opportunities for drug discovery. While the proteolysis-targeting chimera (PROTAC) technology has already entered clinical trials and changed the landscape of small-molecule drugs, new degrader technologies harnessing alternative degradation machineries, especially lysosomal pathways, have emerged and broadened the spectrum of degradable targets. We have recently proposed the concept of autophagy-tethering compounds (ATTECs) that hijack the autophagy protein microtubule-associated protein 1A/1B light chain 3 (LC3) for targeted degradation. Other groups also reported degrader technologies engaging lysosomal pathways through different mechanisms including AUTACs, AUTOTACs, LYTACs and MoDE-As. In this review, we analyse and discuss ATTECs along with other lysosomal-relevant degrader technologies. Finally, we will briefly summarize the current status of these degrader technologies and envision possible future studies.

Tunable optofluidic microbubble lens.

Optofluidic microlenses are one of the crucial components in many miniature lab-on-chip systems. However, many optofluidic microlenses are fabricated through complex micromachining and tuned by high-precision actuators. We propose a kind of tunable optofluidic microbubble lens that is made by the fuse-and-blow method with a fiber fusion splicer. The optical focusing properties of the microlens can be tuned by changing the refractive index of the liquid inside. The focal spot size is 2.8 µm and the focal length is 13.7 µm, which are better than those of other tunable optofluidic microlenses. The imaging capability of the optofluidic microbubble lens is demonstrated under a resolution test target and the imaging resolution can reach 1 µm. The results indicate that the optofluidic microbubble lens possesses good focusing properties and imaging capability for many applications, such as cell counting, optical trapping, spatial light coupling, beam shaping and imaging.

Early Detection of Cervical Cancer by Fluorescence Lifetime Imaging Microscopy Combined with Unsupervised Machine Learning.

Cervical cancer has high morbidity and mortality rates, affecting hundreds of thousands of women worldwide and requiring more accurate screening for early intervention and follow-up treatment. Cytology is the current dominant clinical screening approach, and though it has been used for decades, it has unsatisfactory sensitivity and specificity. In this work, fluorescence lifetime imaging microscopy (FLIM) was used for the imaging of exfoliated cervical cells in which an endogenous coenzyme involved in metabolism, namely, reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H], was detected to evaluate the metabolic status of cells. FLIM images from 71 participants were analyzed by the unsupervised machine learning method to build a prediction model for cervical cancer risk. The FLIM method combined with unsupervised machine learning (FLIM-ML) had a sensitivity and specificity of 90.9% and 100%, respectively, significantly higher than those of the cytology approach. One cancer recurrence case was predicted as high-risk several months earlier using this method as compared to using current clinical methods, implying that FLIM-ML may be very helpful for follow-up cancer care. This study illustrates the clinical applicability of FLIM-ML as a detection method for cervical cancer screening and a convenient tool for follow-up cancer care.

Application of Dual-Enhanced Surface-Enhanced Raman Scattering Probe Technology in the Diagnosis of Tumor Cells in Vitro.

With the development of precision medicine, antigen/antibody-targeted therapy has brought great hope to tumor patients; however, the migration of tumor cells, especially a small number of cells flowing into blood or other tissues, remains a clinical challenge. In particular, it is difficult to use functional gold nanomaterials for targeted clinical tumor diagnosis while simultaneously obtaining stable and highly sensitive Raman signals. Therefore, we developed a detection method for functional Au Nanostars (AuNSs) with dual signal enhancement that can specifically track location and obtain high-intensity surface-enhanced Raman scattering (SERS) signals. First, AuNSs with specific optical properties were synthesized and functionalized. The Raman dye 4-mercapto-hydroxybenzoic acid and polyethylene glycol were coupled with the tumor marker, epidermal growth factor receptor, to obtain the targeted SERS probes. In addition, a detection chip was prepared for Raman detection with physical enhancement, exhibiting a 40-times higher signal intensity than that of quartz glass. This study combines physical enhancement and SERS enhancement technologies to achieve dual enhancement, enabling the detection of a highly sensitive and stable Raman signal; this has potential clinical value for antigen/antibody-targeted tumor diagnosis and treatment.

Visible Particle Identification Using Raman Spectroscopy and Machine Learning.

Visible particle identification is a crucial prerequisite step for process improvement and control during the manufacturing of injectable biotherapeutic drug products. Raman spectroscopy is a technology with several advantages for particle identification including high chemical sensitivity, minimal sample manipulation, and applicability to aqueous solutions. However, considerable effort and experience are required to extract and interpret Raman spectral data. In this study, we applied machine learning algorithms to analyze Raman spectral data for visible particle identification in order to minimize expert support and improve data analysis accuracy. We manually prepared ten types of particle standard solutions to simulate the particle types typically observed during manufacturing and established a Raman spectral library with accurate peak assignments for the visible particles. Five classification algorithms were trained using visible particle Raman spectral data. All models had high prediction accuracy of >98% for all types of visible particles. Our results demonstrate that the combination of Raman spectroscopy and machine learning can provide a simple and accurate data analysis approach for visible particle identification.

Quantifying tagged mRNA export flux via nuclear pore complexes in single live cells.

The mRNA export flux through nuclear pore complexes (NPC) changes under DNA manipulation and hence affects protein translation. However, monitoring the flux of a specific mRNA in single live cell is beyond reach of traditional techniques. We developed a fluorescence-based detection method for measuring the export flux of mRNA through NPC in single live cell using a snapshot image, which had been tested on exogenous genes' expression in HeLa cells, with transfection or infection, and endogenous genes' expression in yeast cells, during incubation and carbon catabolite repression. With its speediness, explicitness and noninvasiveness, we believe that it would be valuable in direct monitoring of gene behavior, and the understanding of gene regulation at a single cell level.

cGAS facilitates sensing of extracellular cyclic dinucleotides to activate innate immunity.

Cyclic dinucleotides (CDNs) are important second messenger molecules in prokaryotes and eukaryotes. Within host cells, cytosolic CDNs are detected by STING and alert the host by activating innate immunity characterized by type I interferon (IFN) responses. Extracellular bacteria and dying cells can release CDNs, but sensing of extracellular CDNs (eCDNs) by mammalian cells remains elusive. Here, we report that endocytosis facilitates internalization of eCDNs. The DNA sensor cGAS facilitates sensing of endocytosed CDNs, their perinuclear accumulation, and subsequent STING-dependent release of type I IFN Internalized CDNs bind cGAS directly, leading to its dimerization, and the formation of a cGAS/STING complex, which may activate downstream signaling. Thus, eCDNs comprise microbe- and danger-associated molecular patterns that contribute to host-microbe crosstalk during health and disease.

Hyperboloid-Drum Microdisk Laser Biosensors for Ultrasensitive Detection of Human IgG.

Whispering gallery mode (WGM) microresonators have been used as optical sensors in fundamental research and practical applications. The majority of WGM sensors are passive resonators that require complex systems, thereby limiting their practicality. Active resonators enable the remote excitation and collection of WGM-modulated fluorescence spectra, without requiring complex systems, and can be used as alternatives to passive microresonators. This paper demonstrates an active microresonator, which is a microdisk laser in a hyperboloid-drum (HD) shape. The HD microdisk lasers are a combination of a rhodamine B-doped photoresist and a silica microdisk. These HD microdisk lasers can be utilized for the detection of label-free biomolecules. The biomolecule concentration can be as low as 1 ag mL-1 , whereas the theoretical detection limit of the biosensor for human IgG in phosphate buffer saline is 9 ag mL-1 (0.06 aM ). Additionally, the biosensors are able to detect biomolecules in an artificial serum, with a theoretical detection limit of 9 ag mL-1 (0.06 aM ). These results are approximately four orders of magnitude more sensitive than those for the typical active WGM biosensors. The proposed HD microdisk laser biosensors show enormous detection potential for biomarkers in protein secretions or body fluids.

Generalized ellipsometry characterization of Ag nanorod arrays prepared by oblique angle deposition.

Silver nanorods arrays (AgNRs) prepared by oblique angle deposition were characterized by the generalized ellipsometry method in the spectral range from 370 to 950 nm. Three structure models were used to fit the ellipsometry data, the uniaxial model, the biaxial orthorhombic model, and the biaxial monoclinic model. Unlike the uniaxial model reported in most literature, the biaxial models are found to give better fitting results. The optical properties along the three principle axes are different: along long axis it displays predominantly metallic behavior; along one short axis it approaches to a lossless dielectric while along the other it behaves as an absorbance dielectric. The AgNRs also demonstrate epsilon-near-zero property with the real part of dielectric constant along the rod being very close to zero at wavelength of 416 nm, which is expected to be tuned with changing of the vapor incident angles.

Fast Focal Point Correction in Prism-Coupled Total Internal Reflection Scanning Imager Using an Electronically Tunable Lens.

Total internal reflection (TIR) is useful for interrogating physical and chemical processes that occur at the interface between two transparent media. Yet prism-coupled TIR imaging microscopes suffer from limited sensing areas due to the fact that the interface (the object plane) is not perpendicular to the optical axis of the microscope. In this paper, we show that an electrically tunable lens can be used to rapidly and reproducibly correct the focal length of an oblique-incidence scanning microscope (OI-RD) in a prism-coupled TIR geometry. We demonstrate the performance of such a correction by acquiring an image of a protein microarray over a scan area of 4 cm² with an effective resolution of less than 20 microns. The electronic focal length tuning eliminates the mechanical movement of the illumination lens in the scanning microscope and in turn the noise and background drift associated with the motion.

Characterization of protein expression levels with label-free detected reverse phase protein arrays.

In reverse-phase protein arrays (RPPA), one immobilizes complex samples (e.g., cellular lysate, tissue lysate or serum etc.) on solid supports and performs parallel reactions of antibodies with immobilized protein targets from the complex samples. In this work, we describe a label-free detection of RPPA that enables quantification of RPPA data and thus facilitates comparison of studies performed on different samples and on different solid supports. We applied this detection platform to characterization of phosphoserine aminotransferase (PSAT) expression levels in Acanthamoeba lysates treated with artemether and the results were confirmed by Western blot studies.

Calibration of oblique-incidence reflectivity difference for label-free detection of a molecular layer.

Oblique-incidence reflectivity difference (OI-RD) is a form of polarization-modulation ellipsometry that measures properties of thin films on a solid surface through the change in polarization state of light upon reflection from the surface. The measurement accuracy depends on the precision of the phase modulation amplitude and azimuthal alignments of key polarizing optical elements and, thus, requires careful calibration. In the present work, we describe robust methods of such calibrations that enable precise determination of the modulation amplitude and static retardation of a phase modulator and azimuths of key polarizing optics in an OI-RD system.

Developing an Efficient and General Strategy for Immobilization of Small Molecules onto Microarrays Using Isocyanate Chemistry.

Small-molecule microarray (SMM) is an effective platform for identifying lead compounds from large collections of small molecules in drug discovery, and efficient immobilization of molecular compounds is a pre-requisite for the success of such a platform. On an isocyanate functionalized surface, we studied the dependence of immobilization efficiency on chemical residues on molecular compounds, terminal residues on isocyanate functionalized surface, lengths of spacer molecules, and post-printing treatment conditions, and we identified a set of optimized conditions that enable us to immobilize small molecules with significantly improved efficiencies, particularly for those molecules with carboxylic acid residues that are known to have low isocyanate reactivity. We fabricated microarrays of 3375 bioactive compounds on isocyanate functionalized glass slides under these optimized conditions and confirmed that immobilization percentage is over 73%.

Enhancing the photodynamic effect of curcumin through modification with TiO2 nanoparticles and cationic polymers.

Curcumin (CUR), a natural compound extracted from turmeric, has shown potential as a photosensitizer in photodynamic therapy (PDT). The aim of this work was to enhance the efficacy of CUR by modifying it using titanium dioxide (TiO2) nanoparticles and a cationic polymer called Sofast to create a nanocomposite TiO2-CUR-Sofast (TCS). Compared to unmodified CUR, TCS exhibited a broadening toward longer wavelength in the absorption wavelength within the 400-550 nm range, leading to improved CUR absorption. Cellular uptake efficiency of TCS was also enhanced, and it demonstrated nearly 4.7-fold higher reactive oxygen species (ROS) generation than CUR. Furthermore, TCS displayed the ability to attach to the cell membrane and enter cells within a 30-min incubation period. Upon irradiation, TCS exhibited remarkable cytotoxicity, resulting in a significant reduction in the viability of various cancer cells. Autofluorescence lifetime imaging of intracellular reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) enzymes indicated that cancer cells treated with TCS and irradiation undergo a metabolic pathway shift from oxidative phosphorylation to glycolysis. These findings highlight the potential of TCS as an effective PDT agent for cancer treatment.

Non-invasive screening of bladder cancer using digital microfluidics and FLIM technology combined with deep learning.

Non-invasive screening for bladder cancer is crucial for treatment and postoperative follow-up. This study combines digital microfluidics (DMF) technology with fluorescence lifetime imaging microscopy (FLIM) for urine analysis and introduces a novel non-invasive bladder cancer screening technique. Initially, the DMF was utilized to perform preliminary screening and enrichment of urine exfoliated cells from 54 participants, followed by cell staining and FLIM analysis to assess the viscosity of the intracellular microenvironment. Subsequently, a deep learning residual convolutional neural network was employed to automatically classify FLIM images, achieving a three-class prediction of high-risk (malignant), low-risk (benign), and minimal risk (normal) categories. The results demonstrated a high consistency with pathological diagnosis, with an accuracy of 91% and a precision of 93%. Notably, the method is sensitive for both high-grade and low-grade bladder cancer cases. This highly accurate non-invasive screening method presents a promising approach for bladder cancer screening with significant clinical application potential.