Artemisinin attenuated ischemic stroke induced pyroptosis by inhibiting ROS/TXNIP/NLRP3/Caspase-1 signaling pathway.

BACKGROUND: To explore the neuroprotective mechanism of artemisinin against ischemic stroke from the perspective of NLRP3-mediated pyroptosis. METHODS: Serum metabolomics technology was used to analyze the serum samples of mice, and KEGG metabolic pathway was analyzed for the different metabolites in the samples. PIT model and OGD/R model were used to simulate ischemic stroke damage in vivo and in vitro. Hoechst 33342 staining, Annexin V-FITC/PI staining and TUNEL staining were used to detect the pyroptosis rate of cells. The contents of IL-1ß and IL-18 in PC12 cells and serum of mice were detected by ELISA. The expressions of NLRP3, ASC-1, Caspase-1 and TXNIP in PC12 cells and mouse brain tissue were detected by Western Blot. RESULTS: Serum metabolic profiles of animal models identified 234 different metabolites and 91 metabolic pathways. Compared with the Sham group and the Stroke+ART group, the KEGG pathway in the Stroke group was concentrated in the Necroptosis pathway associated with cell growth and death, and the NLRP3 inflammasome-mediated pyroptosis pathway was activated in the Necroptosis pathway after ischemic stroke. The results of in vivo and in vitro experiments showed that pretreatment with 10 µM artemisinin reduced ROS production, decreased Δψm, reduced pyroptosis, maintained neuronal cell morphology, and down-regulated the contents of IL-1ß and IL-18 as well as the expression of key proteins of NLRP3, ASC-1, Caspase-1 and TXNIP(p<0.01). CONCLUSION: Artemisinin can reduce neuronal pyroptosis induced by ischemic stroke by inhibiting ROS/TXNIP/NLRP3/Caspase-1 signaling pathway.

Unraveling the core symptoms of mental health in senior grade three students- a network analysis.

Background: Adolescence is not only an important transitional period of many developmental challenges, but also a high risk period for mental health problems. Psychotherapy is recommended for mental health problems in adolescents, but its effectiveness is not always satisfactory. One possible contributing factor may be the lack of clarity surrounding core symptoms. Methods: In this study, we investigated the mental health status of senior grade three students, a group of adolescents facing college entrance exams, by the Middle School Student Mental Health Test (MHT) and analyzed the core symptoms by network analysis. This study was conducted through an online survey platform (www.xiaodongai.com) from 15 February 2023 to 28 March 2024. The subjects scanned a QR code with their mobile phone to receive the questionnaire. Results: The mean age of these 625 students were 18.11 ± 2.90 years. There are 238 male participants and 387 female participants. 107 individuals scored above 56 (107/461, 23.2%), with individual scale scores over 8 up to over 60% of participating students. Notably, the top three prominent symptoms were "academic anxiety", "allergic tendency" and "somatic symptoms". However, upon conducting network analysis, it became evident that three strongest edges in this network were "somatic symptoms" and "impulsive tendency", "academic anxiety" and "social anxiety" as well as "social anxiety" and "Loneliness tendency". "somatic symptoms", "social anxiety" and "self-blame tendency" exerted the highest expected influence. This suggests that, statistically speaking, these three symptoms exhibited the strongest interconnections within the network. Limitation: Cross-sectional analysis; Bias in self-reported variables. Conclusion: These findings can deepen the knowledge of mental health among senior grade three students and provide some implications (i.e., targeting symptoms having highest expected influence) for clinical prevention and intervention to address the mental health needs of this particular group.

The Impact of Mobile Social Media Use on Depressive Mood Among College Students: A Chain Mediating Effect of Upward Social Comparison and Cognitive Overload.

Background: The 18-24 age group has a much higher rate of depression risk than other age groups, and this age group has the highest proportion among users of mobile social media. The relationship between the use of mobile social media and depressive mood is inconsistent and the mechanism of action is controversial. Purpose: This study explored the relationship among the intensity of social media use, upward social comparison, cognitive overload and depressive mood. Methods: In this research, we used the Brief Self-rating Depression Scale (PHQ-9), the Social Media Usage Intensity Questionnaire, the Social Comparison Scale on Social Networking Sites and the Social Networking Site Cognitive Overload Scale to investigate the depressive mood and mobile social media use of 568 college students. Results: The intensity of mobile social media use, social networking site upward social comparison, and social networking site cognitive overload are all positively correlated with depressive mood. The intensity of mobile social media use has a positive predictive effect on depressive mood, with upward social comparison and cognitive overload acting as independent mediators in the relationship between mobile social media use intensity and depressive symptoms, as well as exhibiting a chained mediating effect of upward social comparison-cognitive overload. Conclusion: The upward social comparison and cognitive load that occur during the use of mobile social media are important predictive factors for the occurrence of depressive mood. This study is a supplement to the mechanism of the relationship between mobile social media use and depression, providing more evidence-based evidence and intervention directions for university teachers, mobile social media developers, and psychologists.

Effects of intermittent theta-burst transcranial magnetic stimulation on post-traumatic stress disorder symptoms: A randomized controlled trial.

Post-traumatic stress disorder (PTSD) is a prevalent and debilitating illness, which can be alleviated by transcranial magnetic stimulation (TMS). Intermittent theta burst stimulation (iTBS), a newer form of repetitive transcranial magnetic stimulation (rTMS), offers the advantage of shorter treatment sessions compared to the standard 10 Hz rTMS treatment. In order to compare the two forms of TMS, we enrolled 75 participants aged between 18 and 55 years who presented with (PCL-C) scale score of at least 50. Participants were randomly assigned to groups in a ratio of 1:1:1, receiving either 10 Hz rTMS, iTBS, or sham-controlled iTBS. Participants in the two treatment groups underwent 15 therapies which consisted of 1800 pulses and targeted the right dorsolateral prefrontal cortex (DLPFC). The main outcomes included changes in scores on the PCL-C and the Post-Traumatic Growth Inventory (PTGI). After intervention, the PCL-C and PTGI scores in iTBS and rTMS groups were significantly different from those in sham-controlled iTBS group. No significant differences in PCL-C and PTGI were found between the two active treatment groups. ITBS, with a shorter treatment duration, can effectively improve the symptoms of PTSD, with no significant difference in effect from that of rTMS. Future studies need to further elucidate the mechanisms, optimize the parameters and investigate the therapeutic potential and efficacy of iTBS in PTSD.

The relationship between obsessive-compulsive symptoms and depressive symptoms in patients with obsessive-compulsive disorder: A network analysis.

Aim: The network model suggests that the comorbidity of obsessive-compulsive disorder (OCD) and depression is due to direct interactions between OCD and depression symptoms. The study investigates the network structure of OCD and depressive symptoms in patients with OCD and explores the pathways that connect the OCD and depression symptoms. Materials and Methods: The items of Yale-Brown Obsessive-Compulsive Symptom (Y-BOCS) Scale and the Depression Self-Rating Scale of 445 patients with OCD were analyzed by network model. Statistical analysis and visualization of the network were conducted using R software. Results: Two bridge edges "uneasiness" and "time consumed by obsessions" and "low spirit" and "distress caused by obsessions" connected the OCD symptoms to depressive symptoms. Two closely related edges were between "interference due to obsessions" and "interference due to compulsions" and between "difficulty resisting obsessions" and "difficulty resisting compulsions." The symptoms "interference due to compulsions," "distress caused by obsessions," "time consumed by compulsions," and "uneasiness" had the highest expected influence centrality. Conclusions: This study highlighted the relationship between "uneasiness" and "time consumed by obsessions" and between "low spirit" and "distress caused by obsessions." In addition, "interference due to compulsions" is found as the core symptom in the network. Targeting these symptoms may help prevent and treat the comorbidity of obsession-compulsion and depression in patients with OCD.

Evaluation of the impact of the COVID-19 pandemic on health service utilization in China: A study using auto-regressive integrated moving average model.

Background: The outbreak of COVID-19 in early 2020 presented a major challenge to the healthcare system in China. This study aimed to quantitatively evaluate the impact of COVID-19 on health services utilization in China in 2020. Methods: Health service-related data for this study were extracted from the China Health Statistical Yearbook. The Auto-Regressive Integrated Moving Average model (ARIMA) was used to forecast the data for the year 2020 based on trends observed between 2010 and 2019. The differences between the actual 2020 values reported in the statistical yearbook and the forecast values from the ARIMA model were used to assess the impact of COVID-19 on health services utilization. Results: In 2020, the number of admissions and outpatient visits in China declined by 17.74 and 14.37%, respectively, compared to the ARIMA model's forecast values. Notably, public hospitals experienced the largest decrease in outpatient visits and admissions, of 18.55 and 19.64%, respectively. Among all departments, the pediatrics department had the greatest decrease in outpatient visits (35.15%). Regarding geographical distribution, Beijing and Heilongjiang were the regions most affected by the decline in outpatient visits (29.96%) and admissions (43.20%) respectively. Conclusion: The study's findings suggest that during the first year of the COVID-19 pandemic, one in seven outpatient services and one in six admissions were affected in China. Therefore, there is an urgent need to establish a green channel for seeking medical treatment without spatial and institutional barriers during epidemic prevention and control periods.

Directed Evolution of Laccase for Improved Thermal Stability Facilitated by Droplet-Based Microfluidic Screening System.

Laccases are attractive biocatalysts for industry due to their broad substrate spectrum, the use of oxygen as final electron acceptor, and water as the sole byproduct. Increasing efforts have been devoted to the engineering of laccases to improve their properties. The droplet-based microfluidic screening (DMFS) technology can accelerate the screening procedure and probe the large sequence space. In this study, a DMFS system including a heating step and picoinjection was used to sort large laccase libraries, yielding 12 variants with enhanced thermotolerance. All the obtained amino acid substitutions are distributed on the surface of the laccase. Interestingly, recombination of three identified substitutions of Asp to Asn on the surface resulted in the best variant M20, exhibiting 24.0-fold higher remaining activity at 58.8 °C and 1.9-3.4-fold higher remaining activity after incubation in organic solvents solution (20% (v/v) methanol and ethanol) and ionic liquid solution (20% (v/v) 1-ethyl-3-methylimidazolium ethyl sulfate) for 12 h. Furthermore, molecular dynamic simulations revealed that the recombination of the three beneficial substitutions, Asp98Asn, Asp474Asn, and Asp340Asn on the surface introduced more hydrogen bonds compared to the wild type, which made M20 more thermostable. This study highlighted the importance of the DMFS system for an efficient identification of beneficial long-distance amino acid substitutions.

Emerging advances in identifying signal transmission molecules involved in the interaction between Mycobacterium tuberculosis and the host.

Tuberculosis caused by Mycobacterium tuberculosis (MTB) is an ancient chronic infectious disease and is still the leading cause of death worldwide due to a single infectious disease. MTB can achieve immune escape by interacting with host cells through its special cell structure and secreting a variety of effector proteins. Innate immunity-related pattern recognition receptors (PPR receptors) play a key role in the regulation of signaling pathways. In this review, we focus on the latest research progress on related signal transduction molecules in the interaction between MTB and the host. In addition, we provide new research ideas for the development of new anti-tuberculosis drug targets and lead compounds and provide an overview of information useful for approaching future tuberculosis host-oriented treatment research approaches and strategies, which has crucial scientific guiding significance and research value.

Microfluidic screening and genomic mutation identification for enhancing cellulase production in Pichia pastoris.

BACKGROUND: Pichia pastoris is a widely used host organism for heterologous production of industrial proteins, such as cellulases. Although great progress has been achieved in improving protein expression in P. pastoris, the potential of the P. pastoris expression system has not been fully explored due to unknown genomic impact factors. Recently, whole-cell directed evolution, employing iterative rounds of genome-wide diversity generation and high-throughput screening (HTS), has been considered to be a promising strategy in strain improvement at the genome level. RESULTS: In this study, whole-cell directed evolution of P. pastoris, employing atmospheric and room temperature plasma (ARTP) mutagenesis and droplet-based microfluidic HTS, was developed to improve heterogenous cellulase production. The droplet-based microfluidic platform based on a cellulase-catalyzed reaction of releasing fluorescence was established to be suitable for methanol-grown P. pastoris. The validation experiment showed a positive sorting efficiency of 94.4% at a sorting rate of 300 droplets per second. After five rounds of iterative ARTP mutagenesis and microfluidic screening, the best mutant strain was obtained and exhibited the cellulase activity of 11,110 ± 523 U/mL, an approximately twofold increase compared to the starting strain. Whole-genome resequencing analysis further uncovered three accumulated genomic alterations in coding region. The effects of point mutations and mutant genes on cellulase production were verified using reconstruction of point mutations and gene deletions. Intriguingly, the point mutation Rsc1G22V was observed in all the top-performing producers selected from each round, and gene deletion analysis confirmed that Rsc1, a component of the RSC chromatin remodeling complex, might play an important role in cellulase production. CONCLUSIONS: We established a droplet-based microfluidic HTS system, thereby facilitating whole-cell directed evolution of P. pastoris for enhancing cellulase production, and meanwhile identified genomic alterations by whole-genome resequencing and genetic validation. Our approaches and findings would provide guides to accelerate whole-cell directed evolution of host strains and enzymes of high industrial interest.

Working Memory Capacity of Biological Motion's Basic Unit: Decomposing Biological Motion From the Perspective of Systematic Anatomy.

Many studies have shown that about three biological motions (BMs) can be maintained in working memory. However, no study has yet analyzed the difficulties of experiment materials used, which partially affect the ecological validity of the experiment results. We use the perspective of system anatomy to decompose BM, and thoroughly explore the influencing factors of difficulties of BMs, including presentation duration, joints to execute motions, limbs to execute motions, type of articulation interference tasks, and number of joints and planes involved in the BM. We apply the change detection paradigm supplemented by the articulation interference task to measure the BM working memory capacity (WMC) of participants. Findings show the following: the shorter the presentation duration, the less participants remembered; the more their wrist moved, the less accurate their memory was; repeating verbs provided better results than did repeating numerals to suppress verbal encoding; the more complex the BM, the less participants remembered; and whether the action was executed by the handed limbs did not affect the WMC. These results indicate that there are many factors that can be used to adjust BM memory load. These factors can help sports psychology professionals to better evaluate the difficulty of BMs, and can also partially explain the differences in estimations of BM WMC in previous studies.

Network structure of PTSD symptoms in Chinese male firefighters.

The network perspective of mental disorder offers a novel way of understanding the psychopathology of post-traumatic stress disorder (PTSD). In this framework, PTSD may arise from direct interactions between its symptoms. In the present study, we used the Post-Traumatic Stress Disorder Checklist-civilian Version (PCL-C) to investigate the network structure of PTSD symptoms in 994 Chinese male firefighters. We also calculated the micro (i.e., edges weight and node expected influence) and middle (i.e., community) indicators of the final network. Nine strongest edges existed in the final network were from the same dimension of PCL-C, like "avoidance of thoughts" and "avoidance of reminders". Symptoms "emotional reactivity", "avoidance of reminders" and "exaggerated startle response" had the highest expected influence. As for the results of community detection, the spinglass and walktrap algorithm detected the same three communities which are slightly different from the original dimensions of PCL-C (i.e., symptoms "avoidance of thoughts", "avoidance of reminders" and "trauma-related amnesia" of avoidance dimension of PCL-C were added to the intrusion dimension of PCL-C). The present study explored the network structure of PTSD symptoms in Chinese male firefighters and provided several implications for clinical prevention and intervention to address the mental health needs in this special group.

Ultrahigh-throughput screening of industrial enzyme-producing strains by droplet-based microfluidic system.

Droplet-based microfluidics has emerged as a powerful tool for single-cell screening with ultrahigh throughput, but its widespread application remains limited by the accessibility of a droplet microfluidic high-throughput screening (HTS) platform, especially to common laboratories having no background in microfluidics. Here, we first developed a microfluidic HTS platform based on fluorescence-activated droplet sorting technology. This platform allowed (i) encapsulation of single cells in monodisperse water-in-oil droplets; (ii) cell growth and protein production in droplets; and (iii) sorting of droplets based on their fluorescence intensities. To validate the platform, a model selection experiment of a binary mixture of Bacillus strains was performed, and a 45.6-fold enrichment was achieved at a sorting rate of 300 droplets per second. Furthermore, we used the platform for the selection of higher α-amylase-producing Bacillus licheniformis strains from a mutant library generated by atmospheric and room temperature plasma mutagenesis, and clones displaying over 50% improvement in α-amylase productivity were isolated. This droplet screening system could be applied to the engineering of other industrially valuable strains.

Establishment of a Biosensor-based High-Throughput Screening Platform for Tryptophan Overproduction.

With the flexibility to fold into complex structures, RNA is well-suited to act as a cellular sensor to recognize environmental fluctuations and respond to changes by regulating the corresponding genes. In this study, we established a high-throughput screening platform to screen tryptophan high-producing strains from a large repertoire of candidate strains. This platform consists of a tryptophan-specific aptamer-based biosensor and fluorescence-activated droplet sorting technology. One mutant strain, with a 165.9% increase in Trp titer compared with the parental strain, was successfully screened from a random mutagenesis library. Sequencing results revealed that a total of 10 single-nucleotide polymorphisms were discovered in the genome of the mutant strain, among which CRP(T29K) was proven to significantly increase Trp production through improving the strain's tolerance of the harsh environment during the stationary phase of the fermentation process. Our results indicate that this strategy has great potential for improving the production of other amino acids in Escherichia coli.

Recent advances in droplet microfluidics for enzyme and cell factory engineering.

Enzymes and cell factories play essential roles in industrial biotechnology for the production of chemicals and fuels. The properties of natural enzymes and cells often cannot meet the requirements of different industrial processes in terms of cost-effectiveness and high durability. To rapidly improve their properties and performances, laboratory evolution equipped with high-throughput screening methods and facilities is commonly used to tailor the desired properties of enzymes and cell factories, addressing the challenges of achieving high titer and the yield of the target products at high/low temperatures or extreme pH, in unnatural environments or in the presence of unconventional media. Droplet microfluidic screening (DMFS) systems have demonstrated great potential for exploring vast genetic diversity in a high-throughput manner (>106/h) for laboratory evolution and have been increasingly used in recent years, contributing to the identification of extraordinary mutants. This review highlights the recent advances in concepts and methods of DMFS for library screening, including the key factors in droplet generation and manipulation, signal sources for sensitive detection and sorting, and a comprehensive summary of success stories of DMFS implementation for engineering enzymes and cell factories during the past decade.

Li1.2Mn0.54Ni0.13Co0.13O2 nanosheets with porous structure as a high-performance cathode material for lithium-ion batteries.

The morphological and structural optimizations of electrode materials are efficient ways to enhance their electrochemical performance. Herein, we report a facile co-precipitation and subsequent calcination method to fabricate Li1.2Mn0.54Ni0.13Co0.13O2 nanosheets consisting of interconnected primary nanoparticles and open holes through the full thickness. By comparing the nanosheets and the agglomerated nanoparticles, the effects of the morphology and structure on the electrochemical performance are investigated. Specifically, the nanosheets exhibit a discharge capacity of 210 mA h g-1 at 0.5C with a capacity retention of 85% after 100 cycles. The improved electrochemical performance could be attributed to their morphological and structural improvements, which may facilitate sufficient electrolyte contacts, short diffusion paths and good structural integrity during the charge/discharge process. This work provides a feasible approach to fabricate lithium-rich layered oxide cathode materials with 2D morphology and porous structure, and reveals the relationships between their morphology, structure and electrochemical performance.

Controllable preparation of one-dimensional Li1.2Mn0.54Ni0.13Co0.13O2 cathode materials for high-performance lithium-ion batteries.

Lithium-rich layered oxides are attractive candidates of high-energy-density cathode materials for high-performance lithium ion batteries because of their high specific capacity and low cost. Nevertheless, their unsatisfactory rate capability and poor cycling stability have strongly hindered commercial applications in lithium ion batteries, mainly due to the ineffectiveness of the complicated synthesis techniques to control their morphologies and sizes. In this work, the Li1.2Mn0.54Ni0.13Co0.13O2 cathode materials with a one-dimensional rod-like morphology were synthesized via a facile co-precipitation route followed by a post-calcination treatment. By reasonably adding NH3·H2O in the co-precipitation reaction, the sizes of the metal oxalate precursors could be rationally varied. The electrochemical measurements displayed that the Li1.2Mn0.54Ni0.13Co0.13O2 short rods delivered a high capacity of 286 mA h g-1 at 0.1C and excellent capacity retention of 85% after 100 cycles, which could be contributed to the improvement of the electrolyte contact, Li+ diffusion, and structural stability of the one-dimension porous structure.

Biosensor-enabled droplet microfluidic system for the rapid screening of 3-dehydroshikimic acid produced in Escherichia coli.

Genetically encoded biosensors are powerful tools used to screen metabolite-producing microbial strains. Traditionally, biosensor-based screening approaches also use fluorescence-activated cell sorting (FACS). However, these approaches are limited by the measurement of intracellular fluorescence signals in single cells, rather than the signals associated with populations comprising multiple cells. This characteristic reduces the accuracy of screening because of the variability in signal levels among individual cells. To overcome this limitation, we introduced an approach that combined biosensors with droplet microfluidics (i.e., fluorescence-activated droplet sorting, FADS) to detect labeled cells at a multi-copy level and in an independent droplet microenvironment. We used our previously reported genetically encoded biosensor, 3-dehydroshikimic acid (3-DHS), as a model with which to establish the biosensor-based FADS screening method. We then characterized and compared the effects of the sorting method on the biosensor-based screening system by subjecting the same mutant library to FACS and FADS. Notably, our developed biosensor-enabled, droplet microfluidics-based FADS screening system yielded an improved positive mutant enrichment rate and increased productivity by the best mutant, compared with the single-cell FACS system. In conclusion, the combination of a biosensor and droplet microfluidics yielded a more efficient screening method that could be applied to the biosensor-based high-throughput screening of other metabolites.

[Rapid generation of double-layer emulsion droplets based on microfluidic chip].

In vitro compartmentalization (IVC) links genotype and phenotype by compartmentalizing individual genes (including expression system) or cells into a micro-droplet reaction region. Combined with fluorescence-activated cell sorting (FACS), it can detect and separate single droplets in ultra-high throughput. IVC-FACS screening method has been widely used in protein engineering, enzyme directed evolution, etc. However, it is difficult to control the homogeneity of droplet size by mechanical dispersion method in previous studies, which seriously affects the quantitative detection of droplets and reduces the efficiency and accuracy of this screening method. With the rapid development of microfluidic chip manufacturing technology, the microfluidic chip-based methods for droplet generation are becoming more efficient and controllable. In this study, firstly, the water-in-oil (W/O) single-layer droplet generation chip was used to prepare single-layer monodisperse W1/O droplets at a high generation frequency, and then the W1/O droplets were reinjected into water-in-oil-in-water (W/O/W) double-layer droplet generation chip to prepare uniform W1/O/W2 double-layer emulsion droplets. By optimizing the flow rate and ratio of the oil and water phases, a single-layer micro-droplet can be generated with a diameter range from 15.4 to 23.2 µm and remain stable for several days under normal incubation. Then the single-layer droplets were reinjected into the double emulsion generation chip. By adjusting the flow rate of drop phase, oil phase and water phase, the double-layer emulsion droplets with a diameter range from 30 to 100 µm at a rate of 1 000 droplets/s could be obtained. Escherichia coli embedded in the double-layer emulsion droplets could be cultured and induced for protein expression. This study lays a foundation for the establishment of a high-throughput screening method based on the droplet and flow cytometry.

Long noncoding RNA lnc-LOC645166 promotes adriamycin resistance via NF-κB/GATA3 axis in breast cancer.

Chemoresistance remains a significant obstacle for effective adriamycin (ADR) treatment in breast cancer. Recent efforts have revealed that long noncoding RNAs (lncRNAs) play a crucial role in cancer biology, including chemoresistance. We identified the lncRNA LOC645166 was upregulated in adriamycin resistant-breast cancer cells by Microarray analysis, which was further confirmed in the tissues of nonresponsive patients by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting, and immunohistochemical assays. Downregulation of lncRNA LOC645166 increased cell sensitivity to adriamycin both in vitro and in vivo. In contrast, upregulation of lncRNA LOC645166 strengthened the tolerance of breast cancer cells to adriamycin. Chromatin immunoprecipitation (ChIP) and RNA binding protein immunoprecipitation (RIP) demonstrated that lncRNA LOC645166 could increase the expression of GATA binding protein 3 (GATA3) via binding with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), leading to the activation of STAT3 and promoting chemoresistance in breast cancer. Together, the present study suggested that lncRNA LOC645166 mediated adriamycin chemoresistance in breast cancer by regulating GATA3 via NF-κB.

[Development and application of a droplet-based microfluidic high-throughput screening of Pichia pastoris].

Pichia pastoris is one of the most convenient and widely used heterologous protein expression systems. To further improve its ability to express heterologous proteins, we developed a high-throughput P. pastoris screening method based on droplet microfluidic and demonstrated the method by screening and obtaining mutants with enhanced xylanase expression and secretion abilities. We used PCR (Polymerase Chain Reaction) amplification to obtain a fusion fragment of xylanase xyn5 gene and green fluorescent protein gfp gene, and cloned this fragment into pPIC9K, the expression vector of Pichia pastoris, to construct the plasmid pPIC9K-xyn5-gfp that recombined the DNA fragments of xylanase and green fluorescent protein. After this plasmid entered P. pastoris GS115 by electroporation, the P. pastoris SG strain that could express xylanase and green fluorescent protein was obtained. The above-said strains were then mutagenized by atmospheric room temperature plasma and subsequently encapsulated to form single-cell droplets. After 24-hour cultivation of the droplets, microfluidic screening was carried out to obtain the mutant strain with high xylanase expression for further construction and screening of the next mutagenesis library. After five rounds of droplet microfluidic screening, a highly productive strain P. pastoris SG-m5 was obtained. The activity of the expressed xylanase was 149.17 U/mg, 300% higher than that of those expressed by the original strain SG. This strain's ability to secrete heterologous protein was 160% higher than that of the original strain. With a screening throughput of 100 000 strains per hour, the high-throughput P. pastoris screening system based on single-cell droplet microfluidic developed by the present study screens a library with million strains in only 10 hours and consumes only 100 µL of fluorescent reagent, thus reducing the reagent cost by millions of times compared with the traditional microplate screening and more importantly, providing a novel method to obtain P. pastoris with high abilities to express and secret heterologous proteins by efficient and low-cost screening.