Synthetic Promoter Design and Functional Evaluation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae has become a key microbial cell factory for producing biofuels, recombinant proteins, and natural products. The development of efficient cell factories relies on the precise control and fine-tuning of gene expression, underscoring the pivotal role of promoters in pathway engineering. However, natural promoters often have limited transcriptional capacity and thus fall short of the metabolic engineering requirements. This chapter provides protocols and guidelines for constructing and evaluating synthetic promoters in S. cerevisiae. Moreover, these protocols are applicable for creating and testing various synthetic promoters in other host systems.

Acoustothermal transfection for cell therapy.

Transfected stem cells and T cells are promising in personalized cell therapy and immunotherapy against various diseases. However, existing transfection techniques face a fundamental trade-off between transfection efficiency and cell viability; achieving both simultaneously remains a substantial challenge. This study presents an acoustothermal transfection method that leverages acoustic and thermal effects on cells to enhance the permeability of both the cell membrane and nuclear envelope to achieve safe, efficient, and high-throughput transfection of primary T cells and stem cells. With this method, two types of plasmids were simultaneously delivered into the nuclei of mesenchymal stem cells (MSCs) with efficiencies of 89.6 ± 1.2%. CXCR4-transfected MSCs could efficiently target cerebral ischemia sites in vivo and reduce the infarct volume in mice. Our acoustothermal transfection method addresses a key bottleneck in balancing the transfection efficiency and cell viability, which can become a powerful tool in the future for cellular and gene therapies.

Tailored UPRE2 variants for dynamic gene regulation in yeast.

Genetic elements are foundational in synthetic biology serving as vital building blocks. They enable programming host cells for efficient production of valuable chemicals and recombinant proteins. The unfolded protein response (UPR) is a stress pathway in which the transcription factor Hac1 interacts with the upstream unfolded protein response element (UPRE) of the promoter to restore endoplasmic reticulum (ER) homeostasis. Here, we created a UPRE2 mutant (UPRE2m) library. Several rounds of screening identified many elements with enhanced responsiveness and a wider dynamic range. The most active element m84 displayed a response activity 3.72 times higher than the native UPRE2. These potent elements are versatile and compatible with various promoters. Overexpression of HAC1 enhanced stress signal transduction, expanding the signal output range of UPRE2m. Through molecular modeling and site-directed mutagenesis, we pinpointed the DNA-binding residue Lys60 in Hac1(Hac1-K60). We also confirmed that UPRE2m exhibited a higher binding affinity to Hac1. This shed light on the mechanism underlying the Hac1-UPRE2m interaction. Importantly, applying UPRE2m for target gene regulation effectively increased both recombinant protein production and natural product synthesis. These genetic elements provide valuable tools for dynamically regulating gene expression in yeast cell factories.

Advances in recombinant protease production: current state and perspectives.

Proteases, enzymes that catalyze the hydrolysis of peptide bonds in proteins, are important in the food industry, biotechnology, and medical fields. With increasing demand for proteases, there is a growing emphasis on enhancing their expression and production through microbial systems. However, proteases' native hosts often fall short in high-level expression and compatibility with downstream applications. As a result, the recombinant production of proteases has become a significant focus, offering a solution to these challenges. This review presents an overview of the current state of protease production in prokaryotic and eukaryotic expression systems, highlighting key findings and trends. In prokaryotic systems, the Bacillus spp. is the predominant host for proteinase expression. Yeasts are commonly used in eukaryotic systems. Recent advancements in protease engineering over the past five years, including rational design and directed evolution, are also highlighted. By exploring the progress in both expression systems and engineering techniques, this review provides a detailed understanding of the current landscape of recombinant protease research and its prospects for future advancements.

Microglia sense and suppress epileptic neuronal hyperexcitability.

Microglia are the resident immune cells of the central nervous system, undertaking surveillance role and reacting to brain homeostasis and neurological diseases. Recent studies indicate that microglia modulate epilepsy-induced neuronal activities, however, the mechanisms underlying microglia-neuron communication in epilepsy are still unclear. Here we report that epileptic neuronal hyperexcitability activates microglia and drives microglial ATP/ADP hydrolyzing ectoenzyme CD39 (encoded by Entpd1) expression via recruiting the cAMP responsive element binding protein (CREB)-regulated transcription coactivator-1 (CRTC1) from cytoplasm to the nucleus and binding to CREB. Activated microglia in turn suppress epileptic neuronal hyperexcitability in a CD39 dependent manner. Disrupting microglial CREB/CRTC1 signaling, however, decreases CD39 expression and diminishes the inhibitory effect of microglia on epileptic neuronal hyperexcitability. Overall, our findings reveal CD39-dependent control of epileptic neuronal hyperexcitability by microglia is through an excitation-transcription coupling mechanism.

Overexpression of genes by stress-responsive promoters increases protein secretion in Saccharomyces cerevisiae.

Recombinant proteins produced by cell factories are now widely used in various fields. Many efforts have been made to improve the secretion capacity of cell factories to meet the increasing demand for recombinant proteins. Recombinant protein production usually causes cell stress in the endoplasmic reticulum (ER). The overexpression of key genes possibly removes limitations in protein secretion. However, inappropriate gene expression may have negative effects. There is a need for dynamic control of genes adapted to cellular status. In this study, we constructed and characterized synthetic promoters that were inducible under ER stress conditions in Saccharomyces cerevisiae. The unfolded protein response element UPRE2, responding to stress with a wide dynamic range, was assembled with various promoter core regions, resulting in UPR-responsive promoters. Synthetic responsive promoters regulated gene expression by responding to stress level, which reflected the cellular status. The engineered strain using synthetic responsive promoters P4UPRE2 - TDH3 and P4UPRE2 - TEF1 for co-expression of ERO1 and SLY1 had 95% higher α-amylase production compared with the strain using the native promoters PTDH3 and PTEF1. This work showed that UPR-responsive promoters were useful in the metabolic engineering of yeast strains for tuning genes to support efficient protein production.

A Conway-Maxwell-Poisson-Binomial AR(1) Model for Bounded Time Series Data.

Binomial autoregressive models are frequently used for modeling bounded time series counts. However, they are not well developed for more complex bounded time series counts of the occurrence of n exchangeable and dependent units, which are becoming increasingly common in practice. To fill this gap, this paper first constructs an exchangeable Conway-Maxwell-Poisson-binomial (CMPB) thinning operator and then establishes the Conway-Maxwell-Poisson-binomial AR (CMPBAR) model. We establish its stationarity and ergodicity, discuss the conditional maximum likelihood (CML) estimate of the model's parameters, and establish the asymptotic normality of the CML estimator. In a simulation study, the boxplots illustrate that the CML estimator is consistent and the qqplots show the asymptotic normality of the CML estimator. In the real data example, our model takes a smaller AIC and BIC than its main competitors.

Comprehensive Analysis of Signal Peptides in Saccharomyces cerevisiae Reveals Features for Efficient Secretion.

Signal peptides (SPs) are N-terminus sequences on the nascent polypeptide for protein export or localization delivery, which are essential for maintaining cell function. SPs are also employed as a key element for industrial production of secreted recombinant proteins. Yet, detailed information and rules about SPs and their cellular interactions are still not well understood. Here, systematic bioinformatics analysis and secretion capacity measurement of genome-wide SPs from the model organism Saccharomyces cerevisiae is performed. Several key features of SPs, including region properties, consensus motifs, evolutionary relationships, codon bias, e.g., are successfully revealed. Diverse cell metabolism can be trigged by using different SPs for heterologous protein secretion. Influences on SPs with different properties by chaperones can cause different secretory efficiencies. Protein secretion by the SP NCW2 in SEC72 deletion strain is 10 times than the control. These findings provide insights into the properties and functions of SPs and contribute to both fundamental research and industrial application.

Fast identification and quantification of c-Fos protein using you-only-look-once-v5.

In neuroscience, protein activity characterizes neuronal excitability in response to a diverse array of external stimuli and represents the cell state throughout the development of brain diseases. Importantly, it is necessary to characterize the proteins involved in disease progression, nuclear function determination, stimulation method effect, and other aspects. Therefore, the quantification of protein activity is indispensable in neuroscience. Currently, ImageJ software and manual counting are two of the most commonly used methods to quantify proteins. To improve the efficiency of quantitative protein statistics, the you-only-look-once-v5 (YOLOv5) model was proposed. In this study, c-Fos immunofluorescence images data set as an example to verify the efficacy of the system using protein quantitative statistics. The results indicate that YOLOv5 was less time-consuming or obtained higher accuracy than other methods (time: ImageJ software: 80.12 ± 1.67 s, manual counting: 3.41 ± 0.25 s, YOLOv5: 0.0251 ± 0.0003 s, p < 0.0001, n = 83; simple linear regression equation: ImageJ software: Y = 1.013 × X + 0.776, R 2 = 0.837; manual counting: Y = 1.0*X + 0, R 2 = 1; YOLOv5: Y = 0.9730*X + 0.3821, R 2 = 0.933, n = 130). The findings suggest that the YOLOv5 algorithm provides feasible methods for quantitative statistical analysis of proteins and has good potential for application in detecting target proteins in neuroscience.

Performance Analysis of Electromyogram Signal Compression Sampling in a Wireless Body Area Network.

The rapid growth in demand for portable and intelligent hardware has caused tremendous pressure on signal sampling, transfer, and storage resources. As an emerging signal acquisition technology, compressed sensing (CS) has promising application prospects in low-cost wireless sensor networks. To achieve reduced energy consumption and maintain a longer acquisition duration for high sample rate electromyogram (EMG) signals, this paper comprehensively analyzes the compressed sensing method using EMG. A fair comparison is carried out on the performances of 52 ordinary wavelet sparse bases and five widely applied reconstruction algorithms at different compression levels. The experimental results show that the db2 wavelet basis can sparse EMG signals so that the compressed EMG signals are reconstructed properly, thanks to its low percentage root mean square distortion (PRD) values at most compression ratios. In addition, the basis pursuit (BP) reconstruction algorithm can provide a more efficient reconstruction process and better reconstruction performance by comparison. The experiment records and comparative analysis screen out the suitable sparse bases and reconstruction algorithms for EMG signals, acting as prior experiments for further practical applications and also a benchmark for future academic research.

Secretion of collagenases by Saccharomyces cerevisiae for collagen degradation.

BACKGROUND: The production and processing of animal-based products generates many collagen-rich by-products, which have received attention both for exploitation to increase their added value and to reduce their negative environmental impact. The collagen-rich by-products can be hydrolyzed by collagenases for further utilization. Therefore, collagenases are of benefit for efficient collagen materials processing. An alternative and safe way to produce secreted collagenases is needed. RESULTS: Two collagenases from Hathewaya histolytica, ColG and ColH, were successfully secreted by the yeast Saccharomyces cerevisiae. Compared with the native signal peptide of collagenase, the α-factor leader is more efficient in guiding collagenase secretion. Collagenase secretion was significantly increased in YPD medium by supplementing with calcium and zinc ions. Recombinant collagenase titers reached 68 U/mL and 55 U/mL for ColG and ColH, respectively. Collagenase expression imposed metabolic perturbations on yeast cells; substrate consumption, metabolites production and intracellular cofactor levels changed in engineered strains. Both recombinant collagenases from yeast could hydrolyze soluble and insoluble collagen materials. Recombinant ColG and ColH showed a synergistic effect on efficient collagen digestion. CONCLUSIONS: Sufficient calcium and zinc ions are essential for active collagenase production by yeast. Collagenase secretion was increased by optimization of expression cassettes. Collagenase expression imposed metabolic burden and cofactor perturbations on yeast cells, which could be improved through metabolic engineering. Our work provides a useful way to produce collagenases for collagen resource utilization.

Ultrasound Deep Brain Stimulation Modulates Body Temperature in Mice.

Body temperature plays a critical role in rehabilitation, and numerous studies proved that the regulation of body temperature contributes to the sensorimotor recovery of patients with brain diseases such as stroke. The hypothalamus plays a key role in thermoregulation. Ultrasound deep brain stimulation (UDBS) can noninvasively modulate deep brain nuclei and have potential applications in the treatment of Parkinson's disease, Alzheimer's disease, and depression, among others. The purpose of this study was to investigate whether ultrasound stimulation of the hypothalamus could regulate body temperature in free-moving mice. Results showed that thermoregulation was related to ultrasonic parameters (pulse repetition frequency (PRF), duty cycle, total time, and acoustic pressure). UDBS of the preoptic area of the anterior hypothalamus at 500 Hz PRF could significantly reduce body temperature ( [Formula: see text] at t = 5 min, [Formula: see text] at t = 10 min, [Formula: see text] at t = 15 min). Meanwhile, UDBS of the dorsomedial hypothalamus at 10 Hz PRF triggered a significant increase in body temperature ( [Formula: see text] at t = 5 min, [Formula: see text] at t = 10 min). These results suggest that UDBS, as a noninvasive neuromodulation tool, may play a key role in the future clinical treatment of malignant hyperthermia and hypothermia.

Inhibition of neuronal nitric oxide synthase protects against hippocampal neuronal injuries by increasing neuropeptide Y expression in temporal lobe epilepsy mice.

Neuronal nitric oxide synthase (nNOS) plays a pivotal role in the pathological process of neuronal injury in the development of epilepsy. Our previous study has demonstrated that nitric oxide (NO) derived from nNOS in the epileptic brain is neurotoxic due to its reaction with the superoxide radical with the formation of peroxynitrite. Neuropeptide Y (NPY) is widely expressed in the mammalian brain, which has been implicated in energy homeostasis and neuroprotection. Recent studies suggest that nNOS may act as a mediator of NPY signaling. Here in this study, we sought to determine whether NPY expression is regulated by nNOS, and if so, whether the regulation of NPY by nNOS is associated with the neuronal injuries in the hippocampus of epileptic brain. Our results showed that pilocarpine-induced temporal lobe epilepsy (TLE) mice exhibited an increased level of nNOS expression and a decreased level of NPY expression along with hippocampal neuronal injuries and cognition deficit. Genetic deletion of nNOS gene, however, significantly upregulated hippocampal NPY expression and reduced TLE-induced hippocampal neuronal injuries and cognition decline. Knockdown of NPY abolished nNOS depletion-induced neuroprotection and cognitive improvement in the TLE mice, suggesting that inhibition of nNOS protects against hippocampal neuronal injuries by increasing neuropeptide Y expression in TLE mice. Targeting nNOS-NPY signaling pathway in the epileptic brain might provide clinical benefit by attenuating neuronal injuries and preventing cognitive deficits in epilepsy patients.

Lactiplantibacillus plantarum P9 improved gut microbial metabolites and alleviated inflammatory response in pesticide exposure cohorts.

Multiple pesticide residue accumulations increase the probability of chronic metabolic diseases in humans. Thus, we applied multi-omics techniques to reveal how the gut microbiome responded to pesticide exposure. Then, we explored how probiotic Lactiplantibacillus plantarum P9 (P9) consumption impacted the gut microbiota and immune factors after high pesticide exposure. Multi-omics results indicated frequent exposure to pesticides did not alter the composition of the intestinal microbiota, but it did increase the abundance of Lipopolysaccharide in the gut, which might contribute to chronic inflammation. Supplementation with P9 maintained the homeostasis of the gut microbiota and reduced the abundance of pathogens in the high pesticide-exposed subjects. By detecting metabolites, we observed uridine and 5-oxoproline concentrations increased significantly after P9 consumption. Furthermore, P9 alleviated immune factors disorder and promoted pesticide residue excretion. Our findings provide new insights into the application of probiotics for pesticide detoxification, and suggest probiotics as daily supplements for pesticide exposure prevention.

Ultrasound Stimulation of Prefrontal Cortex Improves Lipopolysaccharide-Induced Depressive-Like Behaviors in Mice.

Increasing evidence indicates that inflammatory responses may influence brain neurochemical pathways, inducing depressive-like behaviors. Ultrasound stimulation (US) is a promising non-invasive treatment for neuropsychiatric diseases. We investigated whether US can suppress inflammation and improve depressive-like behaviors. Mice were intraperitoneally injected with lipopolysaccharide to induce depressive-like behaviors. Ultrasound wave was delivered into the prefrontal cortex (PFC) for 30 min. Depressive- and anxiety-like behaviors were evaluated through the forced swimming test (FST), tail suspension test (TST), and elevated plus maze (EPM). Biochemical analyses were performed to assess the expression of inflammatory cytokines in the PFC and serum. The results indicated that US of the PFC significantly improved depressive-like behaviors in the TST (p < 0.05) and FST (p < 0.05). Anxiety-like behaviors also improved in the EPM (p < 0.05). Furthermore, the lipopolysaccharide-mediated upregulation of IL-6, IL-1ß, and TNF-α in the PFC was significantly reduced (p < 0.05) by US. In addition, no tissue damage was observed. Overall, US of PFC can effectively improve lipopolysaccharide-induced depressive-like behaviors, possibly through the downregulation of inflammatory cytokines in the PFC. US may be a safe and promising tool for improvement of depression.

Rapid cell pairing and fusion based on oscillating bubbles within an acoustofluidic device.

Cell fusion is an essential event in many biological processes and has gained increasing attention in the field of biotechnology. In this study, we demonstrate an effective and convenient strategy for cell capture, pairing, and fusion based on oscillating bubbles within an acoustofluidic device. Multirectangular structures of the same size were fabricated at the sidewall of polydimethylsiloxane to generate monodisperse microbubbles. These microbubbles oscillated with a similar amplitude under single-frequency acoustic excitation. Cells were simultaneously captured and paired on the surface of the oscillating bubbles within 40 ms, and the efficiency reached approximately 90%. Homotypic or heterotypic cell membrane fusion was achieved within 15 and 20 min, respectively. More importantly, the homotypic fused cells enabled migration and proliferation at 24 h, indicating that the important biological functions were not altered.

Non-Cavitation Targeted Microbubble-Mediated Single-Cell Sonoporation.

Sonoporation employs ultrasound accompanied by microbubble (MB) cavitation to induce the reversible disruption of cell membranes and has been exploited as a promising intracellular macromolecular delivery strategy. Due to the damage to cells resulting from strong cavitation, it is difficult to balance efficient delivery and high survival rates. In this paper, a traveling surface acoustic wave (TSAW) device, consisting of a TSAW chip and a polydimethylsiloxane (PDMS) channel, was designed to explore single-cell sonoporation using targeted microbubbles (TMBs) in a non-cavitation regime. A TSAW was applied to precisely manipulate the movement of the TMBs attached to MDA-MB-231 cells, leading to sonoporation at a single-cell level. The impact of input voltage and the number of TMBs on cell sonoporation was investigated. In addition, the physical mechanisms of bubble cavitation or the acoustic radiation force (ARF) for cell sonoporation were analyzed. The TMBs excited by an ARF directly propelled cell membrane deformation, leading to reversible perforation in the cell membrane. When two TMBs adhered to the cell surface and the input voltage was 350 mVpp, the cell sonoporation efficiency went up to 83%.

The illness perception and health promotion behaviour of young and middle-aged patients with hyperuricaemia: A qualitative study.

AIM: The purpose of this qualitative study was to describe the health-promoting behaviours of patients with hyperuricaemia and influencing factors. DESIGN: A descriptive qualitative design was used to gain insight into the personal experience of health promotion behaviour in patients with hyperuricaemia. METHODS: Sixteen patients were sampled in face-to-face interviews with maximum variation, and the data were transcribed verbatim. The data analysis was based on the phrases of thematic analysis outlined by Braun and Clarke (2006). RESULTS: Four main themes were identified in the data: (a) Perception of disease; (b) Motivation to change health-promoting behaviour; (c) Strategies for health-promoting behaviour; and (d) Encounter obstacles to change health-promoting behaviour.