Cascade System Research Articles

Structural basis for RNA-guided DNA degradation by Cas5-HNH/Cascade complex

Type I-E CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated proteins) system is one of the most extensively studied RNA-guided adaptive immune systems in prokaryotes, providing defense against foreign genetic elements. Unlike the previously characterized Cas3 nuclease, which exhibits progressive DNA cleavage in the typical type I-E system, a recently identified HNH-comprising Cascade system enables precise DNA cleavage. Here, we present several near-atomic cryo-electron microscopy (cryo-EM) structures of the Candidatus Cloacimonetes bacterium Cas5-HNH/Cascade complex, both in its DNA-bound and unbound states. Our analysis reveals extensive interactions between the HNH domain and adjacent subunits, including Cas6 and Cas11, with mutations in these key interactions significantly impairing enzymatic activity. Upon DNA binding, the Cas5-HNH/Cascade complex adopts a more compact conformation, with subunits converging toward the center of nuclease, leading to its activation. Notably, we also find that divalent ions such as zinc, cobalt, and nickel down-regulate enzyme activity by destabilizing the Cascade complex. Together, these findings offer structural insights into the assembly and activation of the Cas5-HNH/Cascade complex.

Efficient Escherichia coli Platform for Cannabinoid Precursor Olivetolic Acid Biosynthesis from Inexpensive Inputs.

Olivetolic acid (OLA), an initial precursor of cannabinoids, is catalyzed by type III polyketide synthase, which has a wide range of pharmacological activities, such as antimicrobial and cytotoxic effects. Here, we applied systematic metabolic engineering to develop a multienzyme cascade system to produce OLA via two low-cost inputs. The polyketide synthase (OLS) and cyclase enzymes (OAC), along with the best combination of hexanoyl-CoA and malonyl-CoA synthetases (AEE3 and MatB), were first introduced into the biocatalytic system to increase the supply of hexanoyl-CoA and malonyl-CoA as starting and extender units. To drive the catalysis smoothly, an ATP regeneration system and a CoA-sufficient supply system were incorporated into the biocatalysts to provide enough cofactors. Furthermore, malonyl-CoA flux was redirected to OLA biosynthesis through delicate control of the fatty acid biosynthesis (FAB) pathway via promoter engineering. Collectively, these strategies have led us to produce OLA at a titer of 102.1 mg/L with a productivity of 25.5 mg/L/h by using malonate and hexanoate as direct substrates. Our biocatalytic system provides an effective platform for the production of the cannabinoid precursor OLA in Escherichia coli and may be a valuable reference for the development of microbial cell factories that use hexanoyl-CoA and malonyl-CoA as important intermediates.

Compartmentalized co-immobilization of cellulase and cellobiose phosphorylase within zeolitic imidazolate framework efficiently synthesizes 1-p-Glc: Glycosylation of 18FDG.

Enzymatic glycosylation is an efficient and biocompatible approach to enhance natural product bioavailability. Cellobiose phosphorylase, a novel glycosyltransferase, utilizes 1-phospho-glucose (1-p-Glc) as a glycosyl donor for regioselective glycosylation of various natural substrates. However, the high cost of 1-p-Glc limits the economic feasibility of the process. Thus, a dual-enzyme cascade system involving cellulase AcCel9A and cellobiose phosphorylase CbCBP using a co-immobilization strategy was developed to overcome this challenge. The system utilizes low-cost carboxymethyl cellulose (CMC) for continuous 1-p-Glc production, which was then used in the fluorodeoxy glucose (FDG) glycosylation to generate fluorodeoxy cellobiose (FDC), which potentially traces fungal infections. The compartmentalized co-immobilization of the two enzymes within the internal and external regions of a porous zeolitic imidazolate framework-8 (ZIF-8) carrier enhanced the overall stability of the dual-enzyme system. The immobilized enzymes retained approximately 63.3% activity after seven reuse cycles and 74% catalytic efficiency after 12days of storage at room temperature. Therefore, the developed co-immobilized multi-enzyme system holds significant potential for industrial biocatalysis applications.

Cascade Sliding Mode Control for Linear Displacement Positioning of a Quadrotor

This paper contains an example of a simulation implementation of sliding mode control algorithms for the problem of adjusting the linear position of a quadrotor. A mathematical model of the drone was proposed, which was then implemented in a simulation environment. The method of designing sliding mode controllers using the Lyapunov method in order to improve stability was presented. A cascade system based entirely on sliding mode control algorithms is introduced. The article ends with a comparative analysis of simulation test results of classical control systems and controllers based on sliding mode control.

Enhanced Activity and Stability for Electrocatalytic Nitrate Reduction to Ammonia over Low-Coordinated Cobalt.

It is still challenging to develop an effective strategy to simultaneously enhance the activity and stability of electrocatalysts for electrocatalytic nitrate reduction reaction (eNO3RR). Herein, taking metallic cobalt as an example, it is demonstrated that the construction of low-coordinated cobalt nanosheets (L-Co NSs) by H2 plasma etching of electrodeposited cobalt nanosheets (Co NSs) can greatly enhance the activity and stability of metallic cobalt for eNO3RR. Compared with Co NSs, at -0.4V versus RHE, the nitrate removal rate, ammonia partial current density, and ammonia yield are increased by L-Co NSs from 82.14% to 98.57%, from 476 to 683mA cm-2, and from 2.11 to 2.54mmol h-1 cm-2, respectively. In addition, L-Co NSs demonstrate negligible activity decay after 30 cycles of stability test, while the Co NSs show significant activity decline. In situ electrochemical characterizations and theoretical calculations verify that the abundance of Co vacancies in L-Co NSs not only contribute to the optimized electronic structure and enhanced desorption of key intermediate to boost the activity but also facilitate the transformation of Co(OH)2 to Co0 to promote the stability. Furthermore, L-Co NSs exhibit favorable performance in removing nitrate from simulated wastewater and air plasma discharge-electrocatalytic reduction cascade system to produce ammonia.

Highly Efficient Biosynthesis of Rebaudioside M9 through Enzyme Screening and Structure-Guided Engineering.

Steviol glycosides (SGs) are highly valued for their sweetness, safety, and zero calories, but their bitter taste and low solubility limit their application. Modifying glycosyl units is a promising strategy to improve sensory qualities. In this study, we identified the enzyme UGT94E13 through phylogenetic analysis and enzyme screening, which catalyzes the glycosylation of rebaudioside M2 (Reb M2) at the C-13 position, producing the novel β-1,6-O-glycosylated product rebaudioside M9 (Reb M9). Subsequently, the catalytic activity of UGT94E13 toward Reb M2 was enhanced 12.0-fold through a structure-guided enzyme engineering strategy, and the mechanism behind this enhancement was analyzed. Finally, an enzymatic cascade system comprising the optimal mutant UGT94E13-F169A/I185A and sucrose synthase AtSuSy was constructed and optimized, achieving efficient synthesis of Reb M9 with a yield of 98.3% and a titer of 42.8 g·L-1. Overall, this study provides an effective method for enhancing glycosylation of SGs and a reference for the glycosylation modification of natural products.

Biocatalytic enantioselective formation and ring-opening of oxetanes

Although biocatalysis offers complementary or alternative approaches to traditional synthetic methods, the limited range of available enzymatic reactions currently poses challenges in synthesizing a diverse array of desired compounds. Consequently, there is a significant demand for developing novel biocatalytic processes to enable reactions that were previously unattainable. Herein, we report the discovery and subsequent protein engineering of a unique halohydrin dehalogenase to develop a biocatalytic platform for enantioselective formation and ring-opening of oxetanes. This biocatalytic platform, exhibiting high efficiency, excellent enantioselectivity, and broad scopes, facilitates the preparative-scale synthesis of chiral oxetanes and a variety of chiral γ-substituted alcohols. Additionally, both the enantioselective oxetane formation and ring-opening processes are proven scalable for large-scale transformations at high substrate concentrations, and can be integrated efficiently in a one-pot, one-catalyst cascade system. This work expands the enzymatic toolbox for non-natural reactions and will promote further exploration of the catalytic repertoire of halohydrin dehalogenases in synthetic and pharmaceutical chemistry.

The Establishment of Artificial RNA Cascade Circuits for Gene Regulation Based on Doxycycline-Induced Pre-mRNA Alternative Splicing

This study developed an artificial chimeric intron module with an RNA riboswitch and TetR aptamer that were integrated into essential gene exons. Doxycycline can modulate Pre-mRNA alternative splicing, modify the exon reading frame, and dynamically regulate gene expression. By shifting the aptamer 2 base pair within the switch, we unexpectedly obtained the “on-switch” CTM and “off-switch” C2ITetR>4A, which possess thoroughly contrasting regulatory functions. The CTM module can conditionally induce tumor cell apoptosis and regulate genes reversibly and sustainably following doxycycline induction. We integrated the C2ITetR>4A/CTM switches with the L7Ae/k-turn module to create an intron-spliced double-switched RNA cascade system. The system can both activate and inhibit the splicing mechanism utilizing the same ligand to minimize crosstalk among aptamer switching elements, control target gene leakage, and enhance the dynamic range of gene expression. We analyzed numerous factors affecting Pre-mRNA splicing to identify the optimal equilibrium point for switch regulation. This will enable precise predictions of dynamic regulatory efficiency and the rational design of genetic modules, thereby providing a valuable instrument for mammalian synthetic biology.

An Efficient Transferred Cascade System for COVID-19 Detection from Chest X-ray Images

An Efficient Transferred Cascade System for COVID-19 Detection from Chest X-ray Images

Tibetan–Chinese speech-to-speech translation based on discrete units

Speech-to-speech translation (S2ST) has evolved from cascade systems which integrate Automatic Speech Recognition (ASR), Machine Translation (MT), and Text-to-Speech (TTS), to end-to-end models. This evolution has been driven by advancements in model performance and the expansion of cross-lingual speech datasets. Despite the paucity of research on Tibetan speech translation, this paper endeavors to tackle the challenge of Tibetan-to-Chinese direct speech-to-speech translation within the multi-task learning framework, employing self-supervised learning (SSL) and sequence-to-sequence model training. Leveraging HuBERT model to extract discrete units of target speech, we develop a speech-to-unit translation (S2UT) model using an encoder-decoder architecture which subsequently generates speech output through a unit-based vocoder. By employing SSL and utilizing discrete representations as training targets, our approach effectively captures linguistic differences, facilitating direct translation between the two languages. We evaluate the performance of HuBERT model under various configurations to select the optimal setup based on Phone-unit Normalized Mutual Information (PNMI) values. After fine-tuning the chosen HuBERT model on specific corpora, we introduce auxiliary tasks to enhance translation performance. This underscores the pivotal role of multi-task learning in improving overall model efficacy. Experimental results validate the feasibility of Tibetan-to-Chinese S2ST, demonstrating promising translation quality and semantic content preservation, despite limited data availability.

Fabrication of Compartmentalized Multienzyme Reactor for Colorimetric Biosensing of Glucose and Phenol with High Sensitivity.

Enzymatic cascade reactions are widely utilized in food security, environmental monitoring, and disease diagnostics, whereas their practical application was hindered due to their limited catalytic efficiency and intrinsic fragility to environmental influences. Herein, a compartmentalized dual-enzyme cascade nanoreactor was constructed in metal-organic frameworks (ZIF-8) by a shell-by-shell growth method. ZIF-8 provided a good microenvironment to maintain the activity of enzymes and protected them against harsh conditions. Importantly, experimental results revealed that the encapsulation order and enzyme ratio affected the cascade catalytic activity. When the cascade enzyme ratio was 1:1 and horseradish peroxidase (HRP) was encapsulated in the inner layer with glucose oxidase (GOx) in the outer layer (H@ZIF-8@G@ZIF-8), the nanoreactor facilitated the mass transfer process of substrates and showed the highest cascade catalytic efficiency. The maximum reaction rate (Vmax) of H@ZIF-8@G@ZIF-8 was 294.96 nM s-1, which was 1.6 times greater than G@ZIF-8@H@ZIF-8 (182.84 nM s-1). Therefore, H@ZIF-8@G@ZIF-8 was effectively applied in glucose monitoring and phenol sensing. The glucose biosensor showed a low detection limit of 0.76 μM and a broad linear range of 5-300 μM. The phenol biosensor demonstrated a wide linear range (20-300 μM) with a detection limit of 0.60 μM. In addition, the spiked recovery experiments for glucose and phenol were carried out in serum (recovery: 95.26-100.04%) and tap water (recovery: 97.05-106.50%), respectively. The high accuracy demonstrated potential applications of the cascade system in biosensing and environmental detection.

Cascaded MXene/Metal-Organic framework system enables rapid gelation of ultrastretchable and high-toughness conductive hydrogels for multifunctional wearable electronics

Flexible conductive hydrogels have emerged as promising materials for the fabrication of advanced wearable electronics. Although various crosslinking strategies and functional materials have been introduced to prepare hydrogels with high resilience, conformal adhesion and fatigue resistance, the rapid, facile and energy-free gelation of conductive hydrogels with excellent mechanical strength remains a critical challenge. Herein, a facile and universal strategy for the rapid gelation of hydrogels is proposed via constructing a cascade catalytic system comprising MXene and Fe-MIL-88NH2 metal–organic framework to accelerate the gelation process through their synergistic enhancement effect. The cascade system not only initiates the gelation of hydrophilic monomers within 101 s, but also functions as the cross-linker and conductive filler to form a multibond network and multiple conductive pathways with oxidized sodium alginate (OSA) and Ca2+, which significantly improves the mechanical and electrical performance of the hydrogels, including high stretchability (2200 %), toughness (1.15 MJ/m−3(−|-)), tunable adhesion, stability, excellent fatigue resistance (1500 cycles) and superior conductivity (3.51 S m−1). With high strain sensitivity (gauge factor = 9.51) and low detection limit, hydrogel-based wearable sensors are capable of accurately and consistently monitoring of the movements and electrophysiological signals from the human body. By harmonizing the preparation and performance of hydrogels, this universal strategy sheds new light on the construction of next-generation of advanced wearable electronics.

Advanced systems for enhanced CO2 electroreduction.

Carbon dioxide (CO2) electroreduction has extraordinary significance in curbing CO2 emissions while simultaneously producing value-added chemicals with economic and environmental benefits. In recent years, breakthroughs in designing catalysts, optimizing intrinsic activity, developing reactors, and elucidating reaction mechanisms have continuously driven the advancement of CO2 electroreduction. However, the industrialization of CO2 electroreduction remains a challenging task, with high energy consumption, high costs, limited reaction products, and restricted application scenarios being the issues that urgently need to be addressed. To accelerate the progress of CO2 electroreduction towards practical application, this review shifts the research focus from catalysts to aspects such as reactions and systems, aiming to improve reaction efficiency, reduce technical costs, expand the range of products, and enhance selectivity, offering readers a new perspective. In particular, innovative and specific design strategies such as CO2 reduction coupled with alternative oxidation, co-reduction reaction of CO2 and C/N/O/S-containing species, cascade systems, and integrated CO2 capture and reduction systems are discussed in detail. Additionally, personal views on the opportunities and future challenges of the aforementioned innovative strategies are provided, offering new insights for the future research and development of CO2 electroreduction.

Sampled-data control of an unstable cascaded heat–heat system with different reaction coefficients

The present paper is devoted to sampled-data control design of a PDE–PDE cascade system. These PDEs are governed by heat equations with different reaction coefficients. In order to stabilize the cascaded heat–heat system, we start with the design of continuous-time feedback control law. Then sampled-data control is further proposed for practical reasons. Sufficient conditions are derived for guaranteeing the exponential stability and well-posedness of the corresponding closed-loop system via Lyapunov method and modal decomposition method. Numerical examples illustrate the efficiency of the proposed method.

Increasing the dual-enzyme cascade biocatalysis efficiency and stability of metal-organic frameworks via one-step coimmobilization for visual detection of glucose.

In biosensing analysis, the activity of enzyme systems is limited by their fragility, and substrates catalyzed by monoenzymes tend to undergo spontaneous decomposition during ineffective mass transfer processes. In this study, we propose a novel strategy to encapsulate the glucose oxidase and horseradish peroxidase (GOx&HRP) cascade catalytic system within the hydrophilic zeolite imidazole framework ZIF-90. By leveraging the specific pore structure of ZIF-90, we effectively immobilized GOx and HRP molecules in their three-dimensional conformations, which improved the catalytic activity of the encapsulated enzymes compared with that of free GOx and HRP in various harsh environments. Additionally, our strategy reduced the occurrence of ineffective mass transfer and enhanced the sensitivity of the biosensor through an enzyme cascade system. When this biosensor was applied to serum samples containing complex biological matrices, the degradation of GOx&HRP by various proteases and the surface adsorption of diverse biomolecules were effectively prevented, thereby generating stable and reliable signals of glucose levels. The sensor shows remarkable sensitivity and selectivity for determining glucose concentrations ranging from 0 to 2.5 μg ml-1, with a detection limit as low as 0.034 μg ml-1. Furthermore, we developed a paper-based colorimetric sensor utilizing GOx&HRP@ZIF-90 integrated with a smartphone platform for the visual detection of blood glucose.

Efficient spermidine production using a multi-enzyme cascade system utilizing methionine adenosyltransferase from Lactobacillus fermentum with Reduced Product Inhibition and Acidic pH Preference.

Methionine adenosyltransferases (MATs; EC 2.5.1.6) are key enzymes that catalyze a crucial step in the spermidine biosynthesis pathway. Due to MAT's significant product inhibition, S-adenosylmethionine (SAM) and spermidine production faces challenges. We evaluated MATs from 20 lactic acid bacteria (LAB) to identify enzymes with acidic preference and lower susceptibility to product inhibition. Lactobacillus fermentum's MAT (LfMAT) emerged as a candidate with desirable characteristics. LfMAT exhibited strong activity in acidic environments, maintaining over 85 % activity between pH 6.0-8.5 for 60 min, with peak efficacy at pH 7.0. LfMAT produced 4.2 mM SAM from 5 mM substrate, indicating reduced product inhibition. Ultimately, using an in vitro multi-enzyme cascade system containing LfMAT, S-adenosylmethionine decarboxylase, and spermidine synthase, we successfully produced 12.9 g·L-1 of spermidine. This study establishes a cascade reaction platform, offering a novel approach for the efficient synthesis of spermidine and other polyamines.

Chitosan-derived drug free “artificial beacon” simulating immunogenetic cell death cascade effector to initiate immune response for cancer therapy

Immunogenetic cell death (ICD) is widely participated in tumor immune therapy. However, the stress responses triggered by individual ICD inducers are typically not strong enough to effectively kickstart an ICD effect and successful ICD necessitates a high level of ICD stimulus, which may be linked to dose-related toxicity. In this research, we developed a drug-free “artificial beacon” ATP/CSO@ECM that mimics the ICD cascade system to kickstart an immune response with cationic chitosan (CSO) as a bridge, which participated in integrating tumor antigens and functional damage-associated molecular patterns (DAMPs) into one effector by electrostatic interaction. This beacon is made up of ATP/CSO nanocomplexes covered by an engineered cell membrane (ECM), which is verified to enrich with high mobility group box 1 (HMGB1) and calreticulin (CRT). When exposed to the acidic tumor environment, the ATP/CSO@ECM underwent a morphological change by proton buffering capability of CSO. This resulted in the release of simulated DAMPs and the adjuvant CSO, all of which collaborated to activate dendritic cells and ultimately prolong the effectiveness of immunotherapy. This chitosan-derived “artificial beacon” ATP/CSO@ECM fully mobilizes the function of CSO and avoids the insufficient ICD effect by concentrating signaling molecules, providing a hopeful strategy for using the ICD process in targeted cancer therapy.

Construction of Compartmentalized Meso/Micro Spaces in Hierarchically Porous MOFs with Long-Chain Functional Ligands Inspired by Biological Signal Amplification.

The creation of spatially coupled meso-/microenvironments with biomimetic compartmentalized functionalities is of great significance to achieve efficient signal transduction and amplification. Herein, using a soft-template strategy, UiO-67-type hierarchically mesoporous metal-organic frameworks (HMMOFs) were constructed to satisfy the requirements of such an artificial system. The key to the successful synthesis of HMUiO-67 is rooted in the utilization of the preformed cerium-oxo clusters as metal precursors, aligning the growth of MOF crystals with the mild conditions required for the self-assembly of the soft template. The adoption of long-chain functional 2,2'-bipyridine-5,5'-dicarboxylic acid ligands not only resulted in larger microporous sizes, facilitating the transport of various cascade reaction intermediates, but also provided anchorages for the introduction of enzyme-mimicking active sites. A cascade amplification system was designed based on the developed HMUiO-67, in which enzyme cascade reactions were initiated and relayed by a target analyte in the separate but coupled meso/micro spaces. As a proof of concept, natural acetylcholinesterase (AChE) and Cu-based laccase mimetics were integrated into HMMOFs, establishing a spatially coupled nanoreactor. The activity of AChE was triggered by the target analyte of carbaryl, while the amplified products of AChE catalysis mediated the activity of biomimetic enzyme in the closely proximate microporous spaces, producing further amplification of detectable signal. This enabled the entire cascade system to respond to minimal carbaryl with a limit of detection as low as approximately 2 nM. Such a model of cascade amplification is expected to set a conceptual guideline for the rational design of various bioreactors, serving as a sensitive response system for quantifying numerous target analytes.

Design and Analysis of a Two-Stage Cascade System for Heating and Hot Water Production in Nearly Zero-Energy Buildings Using Thermoelectric Technology

This paper proposes an innovative system that integrates two thermoelectric heat pumps (one air–water and the other water–water) with two thermal storage tanks at different temperatures to provide heating and domestic hot water to a 73.3 m2 passive-house-certified dwelling in Pamplona (Spain). The air–water thermoelectric heat pump extracts heat from the ambient air and provides heat to a tank at intermediate temperature, which supplies water to a radiant floor. The water–water heat pump takes heat from this tank and provides heat to the other tank, at higher temperature, which supplies domestic hot water. The system performance and comfort conditions are computationally analyzed during the month of January under the climate of Pamplona and under different European climates. The COP of the system lays between 1.3 and 1.7, depending on the climate, because of the low COP of the air–water thermoelectric heat pump. However, it is able to provide water for the radiant floor and to maintain the temperature of the dwelling above 20 °C 99.8% of the time. Moreover, it provides domestic hot water at a temperature above 43 °C 99.9% of the time. Noteworthy is the fact that the water–water heat pump presents a COP close to 4, which opens up the possibilities of working in combination with more efficient heat pumps for the first stage.

Isolated and Cumulative Water Quality Effects of two Small Hydropower Plants of a Pantanal Tributary

The construction of small hydropower plants (SHP) can cause several changes in river ecosystems, especially when designed in cascade. Thus, this study aimed to quantify the effects of two SHP operating in cascade and evaluate the individual and cumulative water quality effects on the Ponte de Pedra Stream. The study was developed at the SHP José Gelázio da Rocha and Rondonópolis, both located in Rondonópolis county, state of Mato Grosso, about 15 water quality parameters from the environmental monitoring of the power plants, collected every six months between 2006 to 2013. To evaluate water quality effect, we used the paired Wilcoxon test, and the percentage of change in relation to the natural point, upstream of the hydroelectric plants. The Wilcoxon test showed that downstream of the José Gelázio PCH there was a significant change in all five parameters, causing a 3% increase in temperature, and a 28% reduction in color, 22% in dissolved solids, 20% in total solids and 12% in suspended solids. Downstream of the Rondonópolis PCH there was a significant change in only two parameters, , reducing the temperature by 3% and increasing pH by 1.5%. Regarding the cumulative effects, the reservoirs significantly changed two parameters, causing an 18% increase in total phosphorus and a 48% reduction in total solids. These effects are associated with increased exposure of water to solar radiation and the particle retention process, due to increased hydraulic retention time in the reservoirs. The cumulative effects of the cascade system were 15% greater than the sum of the individual effects, indicating that projects in cascade tend to potentiate the isolated effects.