Multiplexed sequential imaging in living cells with orthogonal fluorogenic RNA aptamer/dye pairs.

Detecting multiple targets in living cells is important in cell biology. However, multiplexed fluorescence imaging beyond two-to-three targets remains a technical challenge. Herein, we introduce a multiplexed imaging strategy, 'sequential Fluorogenic RNA Imaging-Enabled Sensor' (seqFRIES), which enables live-cell target detection via sequential rounds of imaging-and-stripping. In seqFRIES, multiple orthogonal fluorogenic RNA aptamers are genetically encoded inside cells, and then the corresponding cell membrane permeable dye molecules are added, imaged, and rapidly removed in consecutive detection cycles. As a proof-of-concept, we have identified in this study four fluorogenic RNA aptamer/dye pairs that can be used for highly orthogonal and multiplexed imaging in living bacterial and mammalian cells. After further optimizing the cellular fluorescence activation and deactivation kinetics of these RNA/dye pairs, the whole four-color semi-quantitative seqFRIES process can be completed in ∼20 min. Meanwhile, seqFRIES-mediated simultaneous detection of critical signalling molecules and mRNA targets was also achieved within individual living cells. We expect our validation of this new seqFRIES concept here will facilitate the further development and potential broad usage of these orthogonal fluorogenic RNA/dye pairs for multiplexed and dynamic live-cell imaging and cell biology studies.

Adaptive Unsupervised Learning-Based 3D Spatiotemporal Filter for Event-Driven Cameras.

In the evolving landscape of robotics and visual navigation, event cameras have gained important traction, notably for their exceptional dynamic range, efficient power consumption, and low latency. Despite these advantages, conventional processing methods oversimplify the data into 2 dimensions, neglecting critical temporal information. To overcome this limitation, we propose a novel method that treats events as 3D time-discrete signals. Drawing inspiration from the intricate biological filtering systems inherent to the human visual apparatus, we have developed a 3D spatiotemporal filter based on unsupervised machine learning algorithm. This filter effectively reduces noise levels and performs data size reduction, with its parameters being dynamically adjusted based on population activity. This ensures adaptability and precision under various conditions, like changes in motion velocity and ambient lighting. In our novel validation approach, we first identify the noise type and determine its power spectral density in the event stream. We then apply a one-dimensional discrete fast Fourier transform to assess the filtered event data within the frequency domain, ensuring that the targeted noise frequencies are adequately reduced. Our research also delved into the impact of indoor lighting on event stream noise. Remarkably, our method led to a 37% decrease in the data point cloud, improving data quality in diverse outdoor settings.

Polarization of macrophages to an anti-cancer phenotype through in situ uncaging of a TLR 7/8 agonist using bioorthogonal nanozymes.

Macrophages are plastic cells of the immune system that can be broadly classified as having pro-inflammatory (M1-like) or anti-inflammatory (M2-like) phenotypes. M2-like macrophages are often associated with cancers and can promote cancer growth and create an immune-suppressive tumor microenvironment. Repolarizing macrophages from M2-like to M1-like phenotype provides a crucial strategy for anticancer immunotherapy. Imiquimod is an FDA-approved small molecule that can polarize macrophages by activating toll-like receptor 7/8 (TLR 7/8) located inside lysosomes. However, the non-specific inflammation that results from the drug has limited its systemic application. To overcome this issue, we report the use of gold nanoparticle-based bioorthogonal nanozymes for the conversion of an inactive, imiquimod-based prodrug to an active compound for macrophage re-education from anti- to pro-inflammatory phenotypes. The nanozymes were delivered to macrophages through endocytosis, where they uncaged pro-imiquimod in situ. The generation of imiquimod resulted in the expression of pro-inflammatory cytokines. The re-educated M1-like macrophages feature enhanced phagocytosis of cancer cells, leading to efficient macrophage-based tumor cell killing.

Nanoparticles with intermediate hydrophobicity polarize macrophages to plaque-specific Mox phenotype via Nrf2 and HO-1 activation.

Mox macrophages were identified recently and are closely associated with atherosclerosis. Considering the potential health risks and the impact on macrophage modulation, this study investigated the Mox polarization of macrophages induced by nanoparticles (NPs) with tunable hydrophobicity. One nanoparticle (C4NP) with intermediate hydrophobicity efficiently upregulated the mRNA expression of Mox-related genes including HO-1, Srxn1, Txnrd1, Gsr, Vegf and Cox-2 through increased accumulation of Nrf2 at a nontoxic concentration in both resting and LPS-challenged macrophages. Additionally, C4NP impaired phagocytic capacity by 20% and significantly increased the secretion of cytokines, including TNFα, IL-6 and IL-10. Mechanistic studies indicated that intracellular reactive oxygen species (ROS) were elevated by 1.5-fold and 2.6-fold in resting and LPS-challenged macrophages respectively. Phosphorylated p62 was increased by 2.5-fold in resting macrophages and maintained a high level in LPS-challenged ones, both of which partially accounted for the significant accumulation of Nrf2 and HO-1. Notably, C4NP depolarized mitochondrial membrane potential by more than 50% and switched macrophages from oxidative phosphorylation-based aerobic metabolism to glycolysis for energy supply. Overall, this study reveals a novel molecular mechanism potentially involving ROS-Nrf2-p62 signaling in mediating macrophage Mox polarization, holding promise in ensuring safer and more efficient use of nanomaterials.

Recent advancements in the discovery of small-molecule non-nucleoside inhibitors targeting SARS-CoV-2 RdRp.

The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 plays a pivotal role in the life cycle of the novel coronavirus and stands as a significant and promising target for anti-SARS-CoV-2 drugs. Non-nucleoside inhibitors (NNIs), as a category of compounds directed against SARS-CoV-2 RdRp, exhibit a unique and highly effective mechanism, effectively overcoming various factors contributing to drug resistance against nucleoside inhibitors (NIs). This review investigates various NNIs, including both natural and synthetic inhibitors, that closely interacting with the SARS-CoV-2 RdRp with valid evidences from in vitro and in silico studies.

Machine Learning in Predicting Printable Biomaterial Formulations for Direct Ink Writing.

Three-dimensional (3D) printing is emerging as a transformative technology for biomedical engineering. The 3D printed product can be patient-specific by allowing customizability and direct control of the architecture. The trial-and-error approach currently used for developing the composition of printable inks is time- and resource-consuming due to the increasing number of variables requiring expert knowledge. Artificial intelligence has the potential to reshape the ink development process by forming a predictive model for printability from experimental data. In this paper, we constructed machine learning (ML) algorithms including decision tree, random forest (RF), and deep learning (DL) to predict the printability of biomaterials. A total of 210 formulations including 16 different bioactive and smart materials and 4 solvents were 3D printed, and their printability was assessed. All ML methods were able to learn and predict the printability of a variety of inks based on their biomaterial formulations. In particular, the RF algorithm has achieved the highest accuracy (88.1%), precision (90.6%), and F1 score (87.0%), indicating the best overall performance out of the 3 algorithms, while DL has the highest recall (87.3%). Furthermore, the ML algorithms have predicted the printability window of biomaterials to guide the ink development. The printability map generated with DL has finer granularity than other algorithms. ML has proven to be an effective and novel strategy for developing biomaterial formulations with desired 3D printability for biomedical engineering applications.

Neurectomy of the Nerve of Henle Associated with Periarterial Sympathectomy for Management of Intractable Raynaud's Phenomenon.

BACKGROUND: In periarterial sympathectomy for intractable Raynaud's phenomenon, the extent of adventitiectomy as well as postoperative outcomes and hand perfusion assessment tools remain debatable. We evaluated the outcome of neurectomy of the nerve of Henle combined with ulnar tunnel release and periarterial adventitiectomy in the treatment of refractory Raynaud's phenomenon using objective measurements and patient-reported outcomes. METHODS: Nineteen patients with 20 affected hands were prospectively enrolled and underwent the proposed procedures from 2015 to 2021. Relevant data, including Michigan Hand Outcomes Questionnaire and 36-Item Short Form health questionnaire scores, were documented for analysis during a 3-year follow-up. RESULTS: The average ingress value of the three measured fingers (index, long, and ring) on indocyanine green angiography increased after surgery (p=0.02). The median number of ulcers decreased (p<0.001) and the median digital skin temperature increased (p<0.001). Questionnaire scores showed improvement in physical aspects, such as overall hand function (p≤0.001), activities of daily living (p=0.001), work performance (p=0.02), pain (p<0.001), physical function (p=0.053), and general health (p=0.048), as well as mental aspects, such as patient satisfaction (p<0.001) and mental health (p=0.001). The average indocyanine green ingress value of the three measured fingers significantly correlated with the patient-reported outcomes, including overall hand function (r=0.46, p=0.04), work performance (r=0.68, p=0.001), physical function (r=0.51, p=0.02), and patient satisfaction (r=0.35, p=0.03). CONCLUSIONS: The proposed surgical procedures provided satisfactory outcomes, both subjectively and objectively, over a follow-up period of up to 3 years. Indocyanine green angiography may provide rapid and quantitative measurements for perioperative hand perfusion assessment.

Multiplexed Sequential Imaging in Living Cells with Orthogonal Fluorogenic RNA Aptamer/Dye Pairs.

Single-cell detection of multiple target analytes is an important goal in cell biology. However, due to the spectral overlap of common fluorophores, multiplexed fluorescence imaging beyond two-to-three targets inside living cells remains a technical challenge. Herein, we introduce a multiplexed imaging strategy that enables live-cell target detection via sequential rounds of imaging-and-stripping process, which is named as "sequential Fluorogenic RNA Imaging-Enabled Sensor" (seqFRIES). In seqFRIES, multiple orthogonal fluorogenic RNA aptamers are genetically encoded inside cells, and then the corresponding cell membrane permeable dye molecules are added, imaged, and rapidly removed in consecutive detection cycles. As a proof-of-concept, we have identified in this study five in vitro orthogonal fluorogenic RNA aptamer/dye pairs (>10-fold higher fluorescence signals), four of which can be used for highly orthogonal and multiplexed imaging in living bacterial and mammalian cells. After further optimizing the cellular fluorescence activation and deactivation kinetics of these RNA/dye pairs, the whole four-color semi-quantitative seqFRIES process can now be completed in ~20 min. Meanwhile, seqFRIES-mediated simultaneous detection of two critical signaling molecules, guanosine tetraphosphate and cyclic diguanylate, was also achieved within individual living cells. We expect our validation of this new seqFRIES concept here will facilitate the further development and potential broad usage of these orthogonal fluorogenic RNA/dye pairs for highly multiplexed and dynamic cellular imaging and cell biology studies.

Pioglitazone reduces cardiovascular events and dementia but increases bone fracture in elderly patients with type 2 diabetes mellitus: a national cohort study.

The prevalence of type 2 diabetes (T2DM) in elderly people has expanded rapidly. Considering cognitive impairment and being prone to hypoglycemia of the elder, the pros and cons of oral hypoglycemic agents (OHA) should be reassessed in this population. Pioglitazone might be appropriate for elderly DM patients because of its insulin-sensitizing effect and low risk of hypoglycemia. By using Taiwan's National Health Insurance Research Database, 191,937 types 2 diabetes patients aged ≥65 years under treatment between 2005 and 2013 were identified and further divided into two groups according to whether they received pioglitazone (pioglitazone group) or other OHAs (non-pioglitazone group) in the 3 months preceding their first outpatient visit date after 65 years of age, with a diagnosis of T2DM. Propensity score stabilization weight (PSSW) was used to balance the baseline characteristics. In results, the pioglitazone group (n = 17,388) exhibited a lower rate (per person-years) of major advanced cardiovascular events MACCE (2.76% vs. 3.03%, hazard ratio [HR]: 0.91, 95% confidence interval [CI]: 0.87-0.95), new- diagnosis dementia (1.32% vs. 1.46%, HR: 0.91, 95% CI: 0.84-0.98) but a higher rate of new-diagnosis bone fractures (5.37% vs. 4.47%, HR: 1.24, 95% CI: 1.19-1.28) than the non-pioglitazone group (n = 174,549). In conclusion, using pioglitazone may reduce the risks of MACCE and dementia but increases the probability of bone fractures in the elderly DM population.

Bioorthogonal nanozymes for breast cancer imaging and therapy.

Bioorthogonal catalysis via transition metal catalysts (TMCs) enables the generation of therapeutics locally through chemical reactions not accessible by biological systems. This localization can enhance the efficacy of anticancer treatment while minimizing off-target effects. The encapsulation of TMCs into nanomaterials generates "nanozymes" to activate imaging and therapeutic agents. Here, we report the use of cationic bioorthogonal nanozymes to create localized "drug factories" for cancer therapy in vivo. These nanozymes remained present at the tumor site at least seven days after a single injection due to the interactions between cationic surface ligands and negatively charged cell membranes and tissue components. The prodrug was then administered systemically, and the nanozymes continuously converted the non-toxic molecules into active drugs locally. This strategy substantially reduced the tumor growth in an aggressive breast cancer model, with significantly reduced liver damage compared to traditional chemotherapy.

Small change for big improvement in the preparation of the key intermediate N 1, N 3-disubstituted 1,3,5-triazone of ensitrelvir.

In this study, the key intermediate N 1, N 3-disubstituted 1,3,5-triazone of ensitrelvir fumaric acid, approved in Japan for the treatment of SARS-CoV-2 infection under the emergency regulatory approval system, was produced from S-ethylisothiourea hydrobromide and aminomethyl triazole with CDI by four-step telescoped strategy including CDI-activated, condensation, CDI-cyclization, and N 1-alkylation. The strategy with simple conditions and operations had a total yield of 53% on a gram scale. The strategy for synthesizing the key N 1, N 3-disubstituted 1,3,5-triazone intermediate of ensitrelvir might provide a new avenue for further research and development of ensitrelvir analogs.

Engineered Polymer-siRNA Polyplexes Provide Effective Treatment of Lung Inflammation.

Uncontrolled inflammation is responsible for acute and chronic diseases in the lung. Regulating expression of pro-inflammatory genes in pulmonary tissue using small interfering RNA (siRNA) is a promising approach to combatting respiratory diseases. However, siRNA therapeutics are generally hindered at the cellular level by endosomal entrapment of delivered cargo and at the organismal level by inefficient localization in pulmonary tissue. Here we report efficient anti-inflammatory activity in vitro and in vivo using polyplexes of siRNA and an engineered cationic polymer (PONI-Guan). PONI-Guan/siRNA polyplexes efficiently deliver siRNA cargo to the cytosol for highly efficient gene knockdown. Significantly, these polyplexes exhibit inherent targeting to inflamed lung tissue following intravenous administration in vivo. This strategy achieved effective (>70%) knockdown of gene expression in vitro and efficient (>80%) silencing of TNF-α expression in lipopolysaccharide (LPS)-challenged mice using a low (0.28 mg/kg) siRNA dosage.

Study of the mechanism of embolism removal in xylem vessels by using microfluidic devices.

Determining the mechanism that effects embolism repair in the xylem vessels of plants is of great significance in predicting plant distribution and the screening of drought-resistant plants. However, the mechanism of perforation plates of xylem vessels in the acceleration of embolism repair is still not clear by using conventional methods of anatomy and visualization technology. Microfluidic devices have shown their ability to simulate physiological environments and conduct quantitative experiments. This work proposes a biomimetic microfluidic device to study the mechanism of perforation plates of xylem vessels in the acceleration of embolism repair. The results proffered that the perforation plates increase the rate of embolism removal by increasing the pressure differential through the vessel, and the rate of embolism removal is related to the structural parameters of the perforation plate. A combination of the perforation size, the vessel diameter and the perforation plate angle can be optimised to generate higher pressure differentials, which can accelerate the process of embolism repair. This work provides a new method for studying the mechanism of microstructures of natural plants. Furthermore, the mechanism that perforation plates accelerate embolism repair was applied to an electrochemical flow sensor for online determination of heavy metal ions. Test results of this application indicate that the mechanism can be applied in the engineering field to solve the problems of reduced sensitivity of devices, inaccuracy of analysis results and poor reaction performance caused by bubbles that are generated or introduced easily in microdevices, which paves the way for applying the theory to engineering.

Two-stage one-pot synthetic strategy for the key triazone-triazole intermediate of ensitrelvir (S-217622), an oral clinical candidate for treating COVID-19.

Herein, the preparation of the key triazone-triazole intermediate of ensitrelvir (S-217622) via sequential cyclization and alkylation reaction is described. Firstly, chloromethyl triazole was synthesized through a one-pot tandem process (condensation and cyclization reaction) from commercially available chloroacetamide in a 72% yield. Then, the key triazone-triazole intermediate was obtained in a second one-pot process by N-alkylation with triazone followed by highly selective N 1-methylation with iodomethane in a 54% yield. In addition, two of the main process impurities were synthesized and identified. This novel alternative two-stage one-pot strategy for synthesizing the key triazone-triazole intermediate opens a new avenue for further research and development of ensitrelvir analogs.

Engineering a dynamic three-dimensional cell culturing microenvironment using a 'sandwich' structure-liked microfluidic device with 3D printing scaffold.

Most ofin vivotissue cells reside in 3D extracellular matrix (ECM) with fluid flow. To better study cell physiology and pathophysiology, there has been an increasing need in the development of methods for culturing cells inin vivolike microenvironments with a number of strategies currently being investigated including hydrogels, spheroids, tissue scaffolds and very promising microfluidic systems. In this paper, a 'sandwich' structure-liked microfluidic device integrated with a 3D printing scaffold is proposed for three-dimensional and dynamic cell culture. The device consists of three layers, i.e. upper layer, scaffold layer and bottom layer. The upper layer is used for introducing cells and fixing scaffold, the scaffold layer mimicking ECM is used for providing 3D attachment areas, and the bottom layer mimicking blood vessels is used for supplying dynamic medium for cells. Thermally assisted electrohydrodynamic jet (TAEJ) printing technology and microfabrication technology are combined to fabricate the device. The flow field in the chamber of device is evaluated by numerical simulation and particle tracking technology to investigate the effects of scaffold on fluid microenvironment. The cell culturing processes are presented by the flow behaviors of inks with different colors. The densities and viabilities of HeLa cells are evaluated and compared after 72 h of culturing in the microfluidic devices and 48-well plate. The dose-dependent cell responses to doxorubicin hydrochloride (DOX) are observed after 24 h treatment at different concentrations. These experimental results, including the evaluation of cell proliferation andin vitrocytotoxicity assessment of DOX in the devices and plate, demonstrate that the presented microfluidic device has good biocompatibility and feasibility, which have great potential in providing native microenvironments forin vitrocell studies, tissue engineering and drug screening for tumor therapy.

Degradable ZnS-Supported Bioorthogonal Nanozymes with Enhanced Catalytic Activity for Intracellular Activation of Therapeutics.

Bioorthogonal catalysis using transition-metal catalysts (TMCs) provides a toolkit for the in situ generation of imaging and therapeutic agents in biological environments. Integrating TMCs with nanomaterials mimics key properties of natural enzymes, providing bioorthogonal "nanozymes". ZnS nanoparticles provide a platform for bioorthogonal nanozymes using ruthenium catalysts embedded in self-assembled monolayers on the particle surface. These nanozymes uncage allylated profluorophores and prodrugs. The ZnS core combines the non-toxicity and degradability with the enhancement of Ru catalysis through the release of thiolate surface ligands that accelerate the rate-determining step in the Ru-mediated deallylation catalytic cycle. The maximum rate of reaction (Vmax) increases ∼2.5-fold as compared to the non-degradable gold nanoparticle analogue. The therapeutic potential of these bioorthogonal nanozymes is demonstrated by activating a chemotherapy drug from an inactive prodrug with efficient killing of cancer cells.

BIM-based task and motion planning prototype for robotic assembly of COVID-19 hospitalisation light weight structures.

Fast transmission of COVID-19 led to mass cancelling of events to contain the virus outbreak. Amid lockdown restrictions, a vast number of construction projects came to a halt. Robotic platforms can perform construction projects in an unmanned manner, thus ensuring the essential construction tasks are not suspended during the pandemic. This research developed a BIM-based prototype, including a task planning algorithm and a motion planning algorithm, to assist in the robotic assembly of COVID-19 hospitalisation light weight structures with prefabricated components. The task planning algorithm can determine the assembly sequence and coordinates for various types of prefabricated components. The motion planning algorithm can generate robots' kinematic parameters for performing the assembly of the prefabricated components. Testing of the prototype finds that it has satisfactory performance in terms of 1) the reasonableness of assembly sequence determined, 2) reachability for the assembly coordinates of prefabricated components, and 3) capability to avoid obstacles.

A bioinspired bubble removal method in microchannels based on angiosperm xylem embolism repair.

It is difficult to remove and eliminate bubbles in microchannels in many devices used in various biomedical fields, such as those needed for microfluidic immunoassays, point-of-care testing, and cell biology evaluations. Accumulated bubbles are associated with a number of negative outcomes, including a decrease in device sensitivity, inaccuracy of analysis results, and even functional failure. Xylem conduits of angiosperm have the ability to remove bubbles in obstructed conduits. Inspired by such an embolism repair mechanism, this paper proposes a bioinspired bubble removal method, which exhibits a prominent ability to dissolve bubbles continuously within a large range of flow rates (2 µL/min-850 µL/min) while retaining the stability and continuity of the flow without auxiliary equipment. Such a method also shows significant bubble removal stability in dealing with Newtonian liquids and non-Newtonian fluids, especially with high viscosity (6.76 Pa s) and low velocity (152 nL/min). Such advantages associated with the proposed bioinspired method reveal promising application prospects in macro/microfluidic fields ranging from 3D printing, implantable devices, virus detection, and biomedical fluid processing to microscale reactor operation and beyond.

Nanotherapeutics using all-natural materials. Effective treatment of wound biofilm infections using crosslinked nanoemulsions.

Bacterial wound infections are a threat to public health. Although antibiotics currently provide front-line treatments for bacterial infections, the development of drug resistance coupled with the defenses provided through biofilm formation render these infections difficult, if not impossible, to cure. Antimicrobials from natural resources provide unique antimicrobial mechanisms and are generally recognized as safe and sustainable. Herein, an all-natural antimicrobial platform is reported. It is active against bacterial biofilms and accelerates healing of wound biofilm infections in vivo. This antimicrobial platform uses gelatin stabilized by photocrosslinking using riboflavin (vitamin B2) as a photocatalyst, and carvacrol (the primary constituent of oregano oil) as the active antimicrobial. The engineered nanoemulsions demonstrate broad-spectrum antimicrobial activity towards drug-resistant bacterial biofilms and significantly expedite wound healing in an in vivo murine wound biofilm model. The antimicrobial activity, wound healing promotion, and biosafety of these nanoemulsions provide a readily translatable and sustainable strategy for managing wound infections.