Heart failure with preserved ejection fraction (HFpEF) is associated with changes in cardiac metabolism that affect energy supply in the heart. However, there is limited research on energy metabolism-related genes (EMRGs) in HFpEF.The HFpEF mouse dataset  was sourced from the Gene Expression Omnibus database. Gene expression profiles in HFpEF and control groups were compared to identify differentially expressed EMRGs (DE-EMRGs), and the diagnostic biomarkers with diagnostic value were screened using machine learning algorithms. Meanwhile, we constructed a biomarker-based nomogram model for its predictive power, and functionality of diagnostic biomarkers were conducted using single-gene gene set enrichment analysis, drug prediction, and regulatory network analysis. Additionally, consensus clustering analysis based on the expression of diagnostic biomarkers was utilized to identify differential HFpEF-related genes (HFpEF-RGs). Immune microenvironment analysis in HFpEF and subtypes were performed for analyzing correlations between immune cells and diagnostic biomarkers as well as HFpEF-RGs. Finally, qRT-PCR analysis on the HFpEF mouse model was used to validate the expression levels of diagnostic biomarkers.We selected 5 biomarkers  that showed excellent diagnostic performance. The nomogram model we constructed demonstrated high predictive power. Single-gene gene set enrichment analysis revealed enrichment in aerobic respiration and energy derivation. Further, various miRNAs and TFs were predicted by Gng7, such as Gng7-mmu-miR-6921-5p, ETS1-Gng7. A lot of potential therapeutic targets were predicted as well. Consensus clustering identified two distinct subtypes of HFpEF. Functional enrichment analysis highlighted the involvement of DEGs-cluster in protein amino acid modification and so on. Additionally, we identified five HFpEF-RGs . Immune analysis revealed correlations between Macrophage M2, T cell CD4+ Th1 and diagnostic biomarkers, as well as an association between Macrophage and HFpEF-RGs. We further validated the expression trends of the selected biomarkers through experimental validation.Our study identified 5 diagnostic biomarkers and provided insights into the prediction and treatment of HFpEF through drug predictions and network analysis. These findings contribute to a better understanding of HFpEF and may guide future research and therapy development. In HFpEF, the cardiac ejection fraction remains within the normal range, but the condition is characterized by ventricular underfilling, left atrial dilatation, and elevated left atrial pressure . The preEnergy metabolism is essential for maintaining normal physiological functions in the body, including processes like glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. The heart’s energy supply primarily relies on the metabolism of glucose, fatty acids, and lactate . Genes aTo identify potential diagnostic biomarkers and therapeutic targets for HFpEF, this study conducted bioinformatics analysis using the mouse HFpEF transcriptome from the GEO database. Through screening and research, we identified five potential biomarkers and constructed a regulatory network based on energy metabolism-related genes. These biomarkers have the potential to serve as diagnostic biomarkers for HFpEF and may provide a foundation for discovering new therapeutic targets.22.1https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE180065). The GSE180065 dataset (GPL24247) comprises RNA-seq data obtained from heart tissue samples of 10 HFpEF mice and five control mice. To construct the HFpEF mouse model in this publicly available dataset, wide-type male C57BL/6J mice were treated with a combination of high-fat diet and N[w]-nitro-l-arginine methyl ester (L-NAME) at a concentration of 0.5g/L for five weeks. The 325 energy metabolism-related genes (EMRGs) were downloaded from the NCBI and MsigDB databases.The GSE180065 dataset was sourced from the GEO database  (DEGs between the HFpEF and control groups were chosen by using the DESeq2 package (v 1.36.1)  in the Gv 3.4.1) . Next, t2.3Three machine learning models were constructed based on DE-EMRGs by least absolute shrinkage and selection operator (LASSO), random forest (RF) and Support Vector Machine-Recursive Feature Elimination (SVM-RFE) algorithms to screen feature genes separately. LASSO regression profiling was carried out using the glmnet package (version 4.1-6)  to obtaiSubsequently, ROC curves were plotted using the pROC package (v 1.17.0.1)  to asses2.4In order to further explore the biomarkers related pathways and the functions they play, we performed a single-gene GSEA analysis. The single-gene GSEA analysis of biomarkers was carried out via clusterProfiler package (v 4.4.4) . The top2.5In order to find potential therapeutic small molecule drugs acting on biomarkers, we performed drug prediction. The drugs targeting the biomarkers (transformation into human genes) were predicted through the DrugBank database. A mRNA-drug network was constructed based on the predicted results. Then, NetworkAnalyst database was utilized to predict the targeting miRNAs and TFs of biomarkers. Lastly, the network was visualized using Cytoscape software (v 3.8.2) .2.6The consensus clustering analysis was performed on the GSE180065 dataset utilizing the ConsensusClusterPlus package (v 1.60.0)  on the b2.72FC| > 0.5. Gene Ontology (GO) enrichment analysis of DEGs-cluster was executed via clusterProfiler package (v 4.4.4) (DEGs-cluster between the subtypes were selected via the DESeq2 package (v 1.36.1)  with P <v 4.4.4)  . In this study, the topology of the PPI network was analyzed using the plugin cytoHubba, and the top 5 genes under the MCC algorithm were selected as HFpEF-RGs for subsequent analysis.This part of the analysis was carried out in order to obtain further information on the genes associated with the development of HFpEF. Firstly, the DEGs was intersected with the DEGs-cluster to obtain the intersected genes. Then, the protein-protein interaction (PPI) network was created on the basis of intersected genes via the STRING database  . In the 2.10Male wild-type (WT) C57BL/6 mice weighing about 20 g at the age of 8 weeks were obtained from the Hu’nan Silaikejingda Experimental Animal Co., Ltd, China. All applicable international and national guidelines for the care and use of animals were followed. Mice were divided into two treatment groups and exposed to a combination of a high-fat diet (HFD)  and Nω-nitro-L-arginine methyl ester (L-NAME) (0.5 g l−1 in drinking water) or a standard (chow) diet for 15 weeks . The con2.11-ΔΔCT method (Eight pairs of frozen left ventricle tissue of mouse heart (8 HFpEF and 8 control samples) were collected. Afterwards, 16 samples were lysed with TRIzol reagent and total RNA was isolated following the manufacturer’s instructions. The concentration of RNA was measured with a NanoPhotometer N50. Afterwards, RNA was reverse transcribed into cDNA using the SureScript First strand cDNA synthesis kit . The qRT-PCR reaction consisted of 3 µL of reverse transcription product, 5 µL of 2xUniversal Blue SYBR Green qPCR Master Mix, and 1 µL each of forward and reverse primer. All primer sequence information were shown in T method . Graphpa2.12P < 0.05 was in significant difference, where P < 0.05: *, P < 0.01: **, P < 0.001: ***, and P < 0.0001: ****.All bioinformatics analyses were carried out in R language. And then, the data of different groups were compared by rank sum test. It was a truism that 33.12FC| > 0.5; A total of 971 DEGs were identified through differential expression analysis based on the GSE180065 dataset  were screened by LASSO regression analysis , which had the discrimination between subtypes  were acquired by overlapping 971 DEGs (DEGs1) between the HFpEF and control groups and 464 DEGs (DEGs2) in different clusters . Furthermore, moderate negative correlations were observed with Gnb3 , Ddit4l , and Prss55 . As for T cell CD4+ Th1, it demonstrated strong and moderate positive correlations with Prss55  and Ddit4l , respectively, along with a moderate negative correlation with Chrna2 . Conversely, B cell only exhibited a significant correlation with Chrna2, indicating a moderate negative relationship .To obtain correlations between biomarkers and HFpEF-RGs with immune cells, we performed an immune-related analysis. The Xcell analysis demonstrated significant down-regulation of B cell and T cell CD4+ Th1 in the HFpEF group compared to the control group, whereas Hematopoietic stem cell and Macrophage M2 exhibited higher expression in the HFpEF group , left ventricular posterior wall thickness (LVPW), and mitral ratio of peak early to late diastolic filling velocity (E/A), in the HFpEF group compared to normal mice. However, both groups exhibited preserved left ventricular ejection fraction (LVEF) Figure 8To further investigate the expression changes of biomarkers in the HFpEF and control groups, eight pairs of HFpEF and control samples were collected and subjected to qRT-PCR. The results demonstrated that the expression levels of Gng7 and Prss55 were significantly lower in HFpEF samples compared to control tissues; conversely, the expression of Chrna2 was higher in HFpEF samples than in control tissues , cholinergic receptor nicotinic alpha 2 subunit (Chrna2), DNA damage-inducible transcript 4-like (Ddit4l), guanine nucleotide binding protein beta 3 (Gnb3), guanine nucleotide binding protein gamma 7 (Gng7), and serine protease 55 (Pess55) emerged as promising potential diagnostic biomarkers for HFpEF associated with energy metabolism. Subsequent qRT-PCR experiment on the our HFpEF mouse model that were collected from eight HFpEF and eight control samples validated the upregulation of Chrna2, and the downregulations of Gng7 and Prss55 in HFpEF, as did in public database.Chrna2, the gene encoding acetylcholine receptor subunit α2, is expressed primarily in the nervous system . It is iDdit4l is a gene that plays an important role in cellular stress and DNA damage response. The expression of Ddit4l is regulated by a variety of factors such as oxygen levels, nutritional status, and cellular stress , 28. BriGnb3 is a gene encoding the beta subunit of the G protein. It has been considered a candidate gene for hypertension, autonomic nervous system disorders, and coronary heart disease –33. A geBioGRID database might provide more theoretical support to study the underlying mechanism of Gng7 in HFpEF as well (Gng7 is a gene that encodes the gamma subunit of G protein, which plays a regulatory or translational role in various transmembrane signaling systems . Previou as well . These fPrss55 is a gene that encodes a protease known as “proteinase, serine 55”. It belongs to the serine family of proteases and is primarily expressed in testicular tissue . DespiteIn the development of HFpEF, inflammatory response plays a significant role . This reTo promote the clinical application of the five diagnostic biomarkers, we used the DrugBank database to predict potential target drugs. Among the predictions, Cianidanol emerged as a co-targeted drug for Chrna2, Gnb3, and Gng7. Cianidanol is a member of the polyphenolic brass subfamily known for its strong antioxidant properties . It has There are some shortcomings of this study that need to be noted. First, the study was based on bioinformatic analysis of HFpEF mouse transcriptome data from the GEO database. However, the sample size was relatively small, which may limit the statistical reliability and generalizability of the results. Second, the study identified five diagnostic biomarkers associated with energy metabolism, but expression validation in human clinical samples was not performed. It is crucial to perform clinical sample validation to determine the practical application and validity of these markers in patients with HFpEF. Although PCR validation was conducted using HFpEF mice, the results did not successfully validate the differential expression of Gnb3 and Ddit4l between the control and HFpEF groups. This lack of validation may be attributed to individual differences between samples, the variability of experimental techniques, and the complexity of gene regulation. Thirdly, although a consistent cluster analysis of HFpEF was performed based on five diagnostic biomarkers associated with energy metabolism, practical clinical significance could not be assigned to these subclasses due to the current lack of human data. Additionally, bioinformatics analysis, while important in HFpEF research, still suffers from limitations such as the quality and consistency of data, data processing, and choice of analysis methods. Furthermore, this study used a mouse model to study HFpEF, but the mouse model cannot fully reflect the complex pathological process and biological characteristics of human HFpEF, while enough public data on human HFpEF samples could not be found for analysis. Therefore, caution should be exercised when applying these findings to clinical practice. To overcome these shortcomings, future studies should aim to increase the sample size, perform human clinical sample validation, and validate and confirm the clinical application potential of these analytical results by integrating other experimental models and methods.5The analysis of HFpEF mouse transcriptome data from the GEO database successfully identified five diagnostic biomarkers, namely Chrna2, Ddit4l, Gnb3, Gng7, and Prss55, which are associated with energy metabolism. These biomarkers demonstrated a strong ability to distinguish between HFpEF samples and control samples. Furthermore, the GSEA analysis revealed their potential involvement in crucial biological processes like ventricular development and cardiac contraction. These findings have significant implications for understanding the pathogenesis of HFpEF and identifying potential disease biomarkers. Moreover, these biomarkers hold promise as early diagnostic and predictive indicators for HFpEF, offering new prospects for personalized therapy.The original contributions presented in the study are included in the article/The animal study was approved by The Ethics Committee of Shaanxi Provincial People’s Hospital, Xi’an, Shaanxi, China. The study was conducted in accordance with the local legislation and institutional requirements.QG: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing. QZ: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing. MD: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review and editing. LL: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing. HY: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing.


Summative lab assessments probe student mastery overconcepts,but conventional ones often result in decreased student engagementand confidence. If conventional summative lab assessments are replacedby accessible gamified evaluations, such as online escape rooms, thisleads to improved student engagement and confidence. In this work,we adapted two sustainability themed online escape room activitiesto increase student engagement and confidence in data analyses inIntegrated Chemistry I (CHEM 381) over three semesters at CSU, Chico.Over 89.7% of students earned full credit. Further, 80.0% of the writtencomments included positive feedback. After the online escape roomassessments, 60.0% of the students rated their confidence as “high”or “very high” in all categories assessed, comparedto 25.6% before the experience. Students found that the online escaperoom assessments were more engaging than the traditional assessmentand increased their confidence as they worked toward solving two sustainabilitycrises and competed for the quickest time to complete the escape rooms. Several works6 found that escape rooms promote social interaction and communication.8 Further, students reported learning gains,7 increased motivation,10 and a more positive learningexperience11 through a gamified escaperoom.15Throughout academia, conventional assessments are often used to measure student learning. However, gamifiedassessments may increase student engagement and confidence.27 and augmented reality activities30 became necessaryto enhance learning and retention. Previous chemistry instructorsadopted various platforms, such as Google Forms12 and WhatsApp,21 to conduct virtualescape room activities and evaluate student understanding in differentchemistry subdisciplines. To address misconceptions in general chemistrywhile improving learning motivation and increasing student engagement,Cai designed a Harry Potter themed online escape room.22 Similarly, Vergne and co-workers evaluated studentunderstanding of fundamental organic chemistry in a chocolate factorythemed digital escape room.20 To help pharmacystudents learn the complex topic of stereochemistry, Abdul Rahim foundthat the students became more engaged when participating in onlineescape rooms to solve puzzles rather than listening to a traditionallecture.16 Lopez-Pernas et al. noted thatwhile in-person and remote educational escape rooms improved students’knowledge in computer programming, in-person escape room participationyielded slightly higher postactivity test scores compared to the remoteversions.24 Interestingly, unlike the previousstudies, another involved undergraduates designing their own digitalescape rooms with peers in an introductory ecology course, leadingto increased engagement.17Duringthe pandemic, virtual activities such as online escape rooms19 along with water filtration materials20 to prevent environmental crises. Second, thestudents require intradisciplinary knowledge to solve the puzzlesin the escape rooms created for this work. They relied on combiningprior knowledge in general, organic, inorganic, physical, analytical,and materials chemistry to be successful in data analyses. Specifically,the students needed to interpret various data types, such as X-raydiffraction (XRD), scanning electron microscopy (SEM), ultraviolet–visible(UV–vis) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy,steady-state fluorescence spectroscopy, adsorption data, and electricalcharacterization. We previously evaluated individual student’sskills acquisition through questions (Supporting Information) in the final paper written by each student.20 Third, the online escape rooms created in this work target dataanalyses in the upper division integrated undergraduate chemistrylaboratory (CHEM 381) at CSU, Chico. Since in-person learning opportunitieswere minimal during the pandemic, gaining valuable exposure to differentdata analyses was essential to chemistry students’ educationaland professional development.However,this work differs from previous ones in several ways.First, these escape rooms center on sustainability themes, such assolar energy conversion and water decontamination. The students weretasked with finding experimental methods to build the most efficientsolar cell20 during theCHEM 381 course. These questions are also modular in rigor, questiontype, and concepts tested. Consequently, the online escape rooms canbe adapted for high school and all levels of undergraduate chemistryclasses.In this work, we designed twoaccessible online escape room activitiesto increase student engagement and confidence in data analyses inthe CHEM 381 course at CSU, Chico. These online escape rooms wereadministered utilizing user-friendly Google Forms and moderated inan online-learning environment using Zoom breakout rooms. To increaseaccessibility to more students, these escape rooms were executed remotelywithout any use of paper. To increase engagement, the puzzles involveanswering questions to solve a sustainability themed crisis in underan hour. To evaluate changes in student confidence, students self-ratedtheir confidence levels in each form of data analysis before and afterthe online activity. The questions in the online escape rooms areall based on laboratory projects20 in an upper-level integrated chemistry laboratory course (CHEM 381)at CSU, Chico. CHEM 381 is designed for chemistry and biochemistrymajors to apply concepts and techniques from their inorganic, physical,and analytical chemistry courses into research-based projects. Foreach laboratory project, students spent 7–8 weeks familiarizingthemselves with analyzing structural , morphological, spectroscopic , elemental composition , and performance metric data of the nanomaterials). At the conclusion of each project,a virtual escape room activity was used to assess the student’sproficiency at interpreting data of each type (Table S1).For a semester-long course, studentsvirtually participated in two laboratory projectsDesigning the digitalescape room includes the following:Goals:(a)Increase accessibility of assessmentsto more students.(b)Increasestudent engagement throughthe activity.(c)Increasestudents’ confidencelevels in data analyses relevant to this course.Sustainability Themes:(a)Lab 1. Renewable Energy Crisis(b)Lab 2. Water DecontaminationCrisisPlayer Control: Players cannotadvance to the next puzzleuntil they enter the correct numerical or textual answer to solvethe current puzzle they are in.GroupSize: Three students per group maximizes the amountof work each player is required to complete during the escape room.Time Constraint: Triads of students willhave 60 minto complete the virtual escape room puzzles. Students who do not completethe puzzles within the hour only earn the percentage of total pointsbased on the number of puzzles completed.Difficulty: Players must have knowledge of undergraduate-levelgeneral, inorganic, organic, physical, analytical, and materials chemistry.Players in this study were composed of majors-level biochemistry andchemistry students.Feedback: Feedbackwas collected immediately after thestudents’ game experience through surveys and verbal responses.Figures S4 and S6). Each room consisted of a data type which emphasized understandingof structure–property relationships (Supporting Information). The data that students analyzed included thefollowing: room 1, relating absorption spectra to structure of materials;room 2, confirming crystal structure via XRD patterns; room 3, relatingmorphology  of nanomaterials with electron microscopyimages; room 4, correlating elemental composition to spectroscopysignals; room 5, quantifying performance of each nanomaterial. Aseach section of the Google Form was completed with correct answers,the groups moved forward to the next section or “room”. Incorrect answers preventedgroups from advancing to the next section or “room”. Full points were earned bytriads of students by completing the five rooms of the escape roomwithin 60 min; if students completed one room, they earned 20% ofthe total points. Thus, if all five rooms were successfully completed,students earned 100% of the points. Groups competed for completingthe escape rooms in the shortest time, which resulted in additionalexcitement and incentive for the students. Times were calculated fromthe start time to the finish time at which the groups submitted theirGoogle Form, i.e., successfully completed the assigned escape roomactivities.In total, 90 studentsvirtually participated in this study over three semesters. Specifically,14 triads of students completed the energy crisis escape room, while16 triads of students completed the water decontamination escape room.Once students were randomly divided into Zoom breakout rooms to formthese triads, a predesigned Google Form escape room was released toeach student simultaneously . First, characterizationof the MOFs needed to be completed to proceed through the escape room,including analysis using powder X-ray diffraction, scanning electronmicroscopy, energy dispersive X-ray spectroscopy, and Fourier transforminfrared data. To complete the escape room, the students were requiredto determine the adsorption capacity of dyes compared to oils. Whilegiven the same amount of time and same size groups, the escape roomquestions were easily changed to meet the needs of the new projectassessment.The escape room was adapted to applyto the second experiment performed during the semester. Students hadsynthesized metal–organic frameworks (MOFs) for use in waterdecontamination of dyes and oils.Of the groups, 85.7% completedthe energy crisis escape room inunder 1 h, and 93.7% of groups completed the water decontaminationcrisis escape room within the 1 h time limit 1. StudenGroup completion times decreased after prior exposureto the escaperooms. Approximately 35.7% of students completed the energy crisisescape room within 40 min, compared to 75.0% of students in the waterdecontamination escape room. After exposure to the first virtual escaperoom activity, the students were more prepared for the expectationsof the second virtual escape room activity. They gained familiaritywith using the Google Form application as well as the types of questionsthey would encounter. Students were also better able to build theskills of data analysis throughout the semester. Thus, they were moreexperienced with the analyses that they solved within the escape rooms.It was noted that students had commented that solving an efficiencyequation had taken them a long time to complete. This could have beena factor in the length of time that the students had taken to completethe energy crisis escape room as there was not an energy efficiencyequation within the water decontamination escape room.To monitorhow students felt about the escape rooms, a short discussionwas held once each was finished 2. The diFigure S7). The students didnot receive practice escape room questions and so did not know whatkind of questions to expect before the activity. These are capturedas more blue colors, denoting low confidence levels in In order to assess student confidence in specificlab techniquesused in this course, students completed the same survey comparingself-assessed confidence levels before and after the escape room wascompleted S7. The sCompared to the 25.6% of students who were confidentor very confidentin all lab techniques assessed in the energy crisis escape room, 60.0%of the students rated their confidence this way after the escape roomin all techniques except current density–voltage mapping. Thisis consistent with findings in In summary, online escape rooms using GoogleForms are an accessiblemethod for assessing students’ proficiency of data analysisin a majors-level chemistry laboratory at CSU, Chico. During the escaperoom, students were engaged, since an average of 89.7% completed theonline activities in under 60 min, and 80.0% of the written commentsabout the activity were positive. Since many of the groups completedthe entire escape rooms in under 60 min, the difficulty of the escaperooms was adequate for our participants at CSU, Chico. The studentswrote that they welcomed participating in the online escape roomsagain and found that the escape rooms were more fun than the traditionalexam. It helped that students worked toward solving sustainabilitythemed crises and competed for the shortest time to complete the escaperooms. After the activity, 60.0% of the students rated their confidenceas “high” or “very high” in all categoriesassessed, compared to 25.6% before the experience.These onlineescape rooms benefit from being highly adaptable.First, since students are returning to in-person classes, the onlineescape rooms can be adapted in a classroom setting. Students can besplit into breakout rooms on their devices through Zoom and work togetheras a group to complete the escape room while in the classroom. Thiscan increase student interaction and lead to increased student performance.Furthermore, the difficulty and time of completion may be modifiedto meet the demographic of the targeted students of other future work.For example, escape rooms using Google Forms can also be modifiedto meet the abilities of lower division courses or secondary schoolstudents. These escape rooms evaluated the student’s abilityto interpret data, but it could also be applied to other fields ofchemistry. As we have adjusted from an energy crisis themed escaperoom to a water decontamination themed escape room, we changed thetypes of analyses that students completed to reflect the knowledgethey had gained from the water decontamination data analysis.30 a web platform that allows teachers to conductremote and face-to-face escape rooms, would allow for a deeper quantitativeanalysis. This platform allows for teachers to obtain various learninganalytics such as progress graphs that show the puzzles each groupsolves and the moment in which each one was completed. This wouldbe useful information to understand which topics are more challengingthan others. Escapp also provides a hint chart which shows the numberof hints provided to each team for each puzzle. Monitoring the hintswould allow teachers to understand which specific topics studentsstruggle with the most.The activity could benefit from more controlvariables plannedduring the experimental setup. Future implementation of Escapp,Another limitation to the study includedtechnological challengeswith Google Forms. Some groups experienced difficulties with caps-sensitivityissues and using the incorrect number of decimal places, which slowedtheir progress. This could be improved in the future by changing theGoogle Form settings to be less sensitive prior to the escape roombeing conducted. Clarifying the number of decimal points before completingthe escape room would have saved students time with numerical answers.Finally, this study can be improved by evaluating the effect ofthe online escape room activities on teamwork skills and stress levelson students. By administering surveys pre- and post-activity to thestudents, researchers can further investigate essential teamwork skillssuch as active listening, cooperation, conflict resolution, coordination,creativity, feedback, and problem solving. To assess stress levelson students, a Likert scale questionnaire ranging from 1 “nostress” to 5 “extreme stress” before and afterthe activity can be implemented.


Central nervous system (CNS) diseases are currently a major challenge in medicine. One reason is the presence of the blood–brain barrier, which is a significant limitation for currently used medicinal substances that are characterized by a high molecular weight and a short half-life. Despite the application of nanotechnology, there is still the problem of targeting and the occurrence of systemic toxicity. Viral vectors and virus-like particles (VLPs) may provide a promising solution to these challenges. Their small size, biocompatibility, ability to carry medicinal substances, and specific targeting of neural cells make them useful in research when formulating a new generation of biological carriers. Additionally, the possibility of genetic modification has the potential for gene therapy. Among the most promising viral vectors are adeno-associated viruses, adenoviruses, and retroviruses. This is due to their natural tropism to neural cells, as well as the possibility of genetic and surface modification. Moreover, VLPs that are devoid of infectious genetic material in favor of increasing capacity are also leading the way for research on new drug delivery systems. The aim of this study is to review the most recent reports on the use of viral vectors and VLPs in the treatment of selected CNS diseases. Central nervous system (CNS) diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Canavan disease (CD), multiple sclerosis (MS) and those associated with the neoplastic process in astrocytic series cells, such as glioblastoma multiforme (GBM), are a major problem in modern medicine ,2. The rThe effectiveness of the delivery of medicinal substances to the CNS therefore depends on the size of the molecule, which should be below 200 nm, and the lipophilicity, polarity, and charge for optimal transport across the BBB . LipophiAccording to the literature, viral vectors and virus-like particles (VLP) may offer hope for new methods of delivering medicinal substances to treat CNS diseases ,20. TheiWith regard to this information, the purpose of this review is to present the latest developments, advantages, limitations and prospects for the use of virus-based drug delivery systems in the treatment of selected CNS diseases.l-lactide-co-glycolide-co-trimethylene carbonate) (P(l-LA:GA:TMC)), with aripiprazole (ARP) administered via a ampoule syringe as an alternative to small microparticles administered as suspensions ..E1 gene LV vectors are produced by the calcium cotransfection of the HEK293T cell line with three plasmids: one containing the genome of the produced vector, one containing element-encoding capsid proteins, and one containing genes and regulatory elements that are important for cell proliferation . The obtThe mechanism involved in the delivery of the medical substance or molecules, i.e., proteins and nucleic acids, largely depends on the virus-based system used. There are minor variations in the use of specific viruses or virus-like particles ,33. ThesIn addition, the delivery of genetic material by means of viral-based molecules provides considerable opportunities, since they are able to escape lysosomal degradation, which reduces the bioavailability of the transferred compound . The mecAD is a slowly progressive neurodegenerative disease that is a major cause of dementia. It involves the progressive loss of cognitive function, which includes memory loss and difficulty thinking, speaking, and solving logical problems . In addiAlthough the causes of most AD cases are not fully understood, the search for alternatives to classical pharmacotherapy has begun. Interleukin-2, which acts as a regulator of inflammation, was introduced into the AAV vector and used experimentally to alleviate AD symptoms in a mouse model of the disease . The AAVPD is an age-related neurodegenerative disease. Several specific motor symptoms are caused by the extensive and progressive loss of dopamine-containing neurons located in the black matter of the midbrain . CurrentNeurturin belongs to the family of neurotrophic factors of glial origin. As an endogenous trophic factor, it significantly increases dopamine activity in the midbrain, improves neuronal survival, and protects the integrity of neurons from neurotoxic damage, thus potentially inhibiting the progression of PD . CERE-12l-amino acid decarboxylase (AADC), an inhibitor of the premature conversion of levodopa to dopamine. A phase I clinical trial using adeno-associated virus serotype 2 delivering AADC (AAV2-AADC) has shown that such gene therapy is safe ..83].In addition, some VLPs derived from viruses exhibit natural tropism to central nervous system cells ,99. One VLPs, as nanometric protein structures that lack infectious genetic material, are formed by the spontaneous self-assembly reaction of individual proteins ,101. ThiNonenveloped VLPs do not have the aforementioned envelope in their structure ,105. TheEnveloped VLPs (eVLPs) have a lipid envelope derived from the host they infect. They can also be multilayered and contain several different capsid proteins ,105. HowThe production process and the selection of a suitable expression system for the desired VLP are crucial ,107. FirFor the production of VLPs as carriers of medicinal substances for the treatment of CNS diseases, those based on bacterial, plant, and mammalian systems are primarily used .Escherichia coli, Lactobacillus casei, and Pseudomonas fluorescens, whose doubling time is relatively short, making the process of VLP production potentially shorter [Bacterial expression systems are among the most widely used platforms for generating VLPs ,106. Thi shorter .Nicotiana tabacum and Nicotiana benthamiana [Eukaryotic cells, i.e., plant and mammalian cells, are also used to produce a VLP that can be used as a nanocarrier for the medicinal substance. Using plants as nanoreactors for VLPs is a more efficient system than using bacteria because it is possible to obtain a large amount of soluble bilayer at reduced production costs ,107,111.thamiana ,111. Expthamiana ,107. In thamiana ,106,107.VLPs, due to their distinctive properties, i.e., small size (20–200 nm), ability to carry medicinal substances to a specific site, ease of surface functionalization, and natural tropism to cells while being biodegradable and low in toxicity, have found application in CNS disease therapy research . FinblooAdditional examples of the application of VLPs as drug delivery systems for the treatment of CNS diseases are summarized in The process of producing viral carriers on a large scale usually involves introducing a vector structure containing the desired gene and the necessary, e.g., lentiviral elements, such as regulatory and structural sequences, into packaging cells such as HEK293T . After aA major problem in the production of new therapies based on viral vectors, especially lentiviruses and retroviruses, is the possibility of spontaneous recombination during the production process in packaging cells ,122. ThiICP34.5 gene, which is responsible for neurovirulence. The virus used with the deletion of ICP34.5 showed a reduced ability to replicate and reactivate in the nervous system. However, there are studies showing that viruses with ICP34.5 deletion can cause neuronal destruction and produce a strong immune response in the brains of various strains of mice, which is associated with significant weight loss in symptomatic animals [It is worth mentioning the limitations of viral vectors and VLPs. Viral vectors and nonviral particles, by virtue of their biology, require host cellular machinery for their self-organization. This phenomenon makes the production of these vectors difficult due to the long, tedious process of cell culture and the risk of contamination and loss of raw material ,122. Bec animals . These r animals . TherefoFurther research is also needed on tropism, i.e., the ability to precisely deliver molecules to appropriate tissues and cells, and the development of VLPs specific to target cells, as well as controlled cargo release. This research can greatly enhance the effectiveness of the method and remains one of the main areas for improvement .https://epo.org (accessed on 18 June 2023).However, in addition to these limitations, new patents are being registered for modifications of viruses and VLPs as carriers of medicinal substances, mainly based on their functionalization or the process of obtaining mutants and new serotypes. Therefore, several patents related to the production of viruses and VLPs as carriers are presented in This review presents numerous studies based on the use of viral vectors and virus-like particles in CNS disease therapies. In the case of viral vectors, it is possible to modify capsid proteins, create chimeras, or manipulate the capsid surface to achieve the desired tissue tropism or migration across biological barriers, such as the BBB. With VLPs, the possibilities are even greater, as it is possible to transport chemotherapeutics, manipulate the shape and size of the vector, and modify its structure. Also, the advantages of viral vectors include their ability to express proteins or genes over the long term and their safety due to the low incidence of side effects and their ability to manipulate the immune profile. Viral vectors and VLPs are successfully used in gene therapies because they effectively protect the transgene and arming them with appropriate promoters facilitates their integration and expression in target cells. Due to the simplicity of viral genome modification, we can easily introduce a large group of transgenes into the aforementioned vectors. The properties of penetration through biological barriers or particle sizes allow them to reach their targets in high concentrations. However, some limitations of viral vectors and virus-like particles should not be forgotten.The data presented in this work indicate that viral vectors and VLPs are a promising strategy for delivering therapeutic agents in CNS diseases. Nevertheless, to strive for the most effective delivery of medicinal substances, further research on these vectors is needed to improve their tropism, stability, and ability to cross biological barriers or refine viral delivery methods.


Manorina melanocephala. We provide some support for two predictions of the “habitat connectivity hypothesis” in at least some taxa, and conclude it warrants further investigation across a broader range of taxa inhabiting contrasting landscapes. Increasing habitat connectivity within urban landscapes may reduce escape stress experienced by urban birds.Habitat destruction and fragmentation increasingly bring humans into close proximity with wildlife, particularly in urban contexts. Animals respond to humans using nuanced anti-predator responses, especially escape, with responses influenced by behavioral and life history traits, the nature of the risk, and aspects of the surrounding environment. Although many studies examine associations between broad-scale habitat characteristics  and escape response, few investigate the influence of fine-scale aspects of the local habitat within which escape occurs. We test the “habitat connectivity hypothesis,” suggesting that given the higher cost of escape within less connected habitats (due to the lack of protective cover), woodland birds should delay escape (tolerate more risk) than when in more connected habitat. We analyze flight-initiation distances (FIDs) of five species of woodland birds in urban Melbourne, south-eastern Australia. A negative effect of habitat connectivity (the proportion of the escape route with shrubs/trees/perchable infrastructure) on distance fled was evident for all study species, suggesting a higher cost of escape associated with lower connectivity. FID did not vary with connectivity at the location at which escape was initiated (four species), apart from a positive effect of habitat connectivity on FID for Noisy Miner  Many birds escape from humans as they do from predators, and it might be that birds in urban parks adjust their escape depending on the closeness of safe places such as trees. We show that birds flew further when disturbed where fewer trees occurred. This suggests that plantings in urban parks might influence how well people and birds coexist. Urban expansion and associated habitat fragmentation increasingly bring humans into close proximity with wildlife including birds . While uDuring an “encounter” . Specifically, we predict that lower connectivity is associated with longer escape flights  because suitable refuge is not available nearby. Consequently, because the costs of escape are comparatively higher where low connectivity exists, we predict that FIDs will be shorter in relatively poorly connected habitats.To index avian escape responses, we conducted “approaches” (which evoked escape) and enabled the quantification of escape initiation, distance fled, and connectivity of habitat. All approaches were conducted across 69 urban parks and reserves across metropolitan Melbourne  was then recorded. If the bird did not respond at the point where the observer was at the bird’s initial position, this was recorded as a “non-response” (6.2% of 499 approaches) and was excluded from analysis. In some cases , we measured the horizontal distance to the bird’s perch and subsequently calculated the straight-line distance accounting for observer height, as do distances measured by a rangefinder (sensuFlight-initiation distance (FID) measures were based on established protocols summarized by Blumstein and Cooper (2015) and der sensu. We did To measure habitat connectivity in relation to each bird’s escape response, we calculated the density of perches available to focal birds at their initial position prior to the commencement of an approach. We use this metric because birds rarely flew to the closest available perch  where possible (97.7% of 468 flight responses). Coordinates were taken by GPS at this point and at the bird’s initial position, except for birds that flew ≤5 m. For these birds, the initial position and refuge location were considered the same. The distance between the initial location and refuge location  was also measured.>5 m. In this way, the relevant measure of connectivity, as defined by the focal bird’s escape route, was measured rather than a general measure of connectivity at the bird’s initial location (which contained areas within which connectivity was irrelevant to the escape). Measurements of “route connectivity”, as defined by the proportion of vegetation/perchable infrastructure coverage along the escape route of each bird (which moved > 5 m), were calculated in ArcMap version 10.7.1 (World Imagery source: Maxar) and QGIS  were considered for statistical analysis.We removed instances when birds did not respond to the approach  and when the bird moved away by means other than flying (0.6% of 499 approaches). We also removed data for which the refuge location of the bird could not be determined in the field (2.3% of 468 flight responses). Species that subsequently had To test the “habitat connectivity hypothesis”, we built two generalized linear mixed models for each species pertaining to 1) escape response, and 2) post-initiation aspects of escape . The first model tested whether the FID of woodland birds is influenced by the level of habitat connectivity at their initial position (“perch density”) and along their escape route (“route connectivity”). Starting distance (StD) was also included for its known influence on FID . The parsensuAdj.”) are the expected FID at the mean StD for each species, derived by adjusting each FID value according to the slope of a linear regression between FID against StD (“SlopeFID/StD”). Specifically, FIDAdj. is calculated for each species as follows: FIDAdj. = FIDObserved + SlopeFID/StD*(x̄StD – StDObserved).The second set of generalized linear mixed models considered the post-initiation elements of escape, and therefore tested for an effect of habitat connectivity and FID on the distance fled when flushed, as well as an interaction between these variables. “Perch density” was included as the measure of habitat connectivity at the point from which escape was initiated, and “route connectivity” was included as the measure of habitat connectivity during escape. Since FID is highly positively correlated with StD, including both as separate predictor variables in a single model would likely result in unacceptably high variance inflation factors. We therefore adjusted each FID value to account for StD sensu. AdjusteAdj., and between “route connectivity” and FIDAdj. were included to test whether an effect of habitat connectivity pre- and post-initiation of escape is dependent on FID, regarding relation to the distance fled by the focal bird. Location was included as a random factor to account for locational differences that may influence escape response.Interaction terms between “perch density” and FIDP-value of <0.05; or as trends if the P-value is < 0.10 but greater than 0.05. P-values 0.05–0.10 do not meet the usual threshold for a “statistical difference”, but instead suggest a “trend” toward an effect which might be realized with increased sampling.Statistical analysis was implemented in R , using tManorina melanocephala (N = 116), Red Wattlebird Anthochaera carunculata (N = 80), Common Myna Acridotheres tristis (N = 52), Spotted Dove Spilopelia chinensis (N = 24), and New Holland Honeyeater Phylidonyris novaehollandiae (N = 25).We collected 499 FIDs from 28 species across 69 locations . Five spPerch density at the point of escape had a positive influence on FID of Noisy Miners with birds taking flight at longer distances from the observer where more perches where available in their immediate vicinity . Perch dAdj. on distance fled in Common Myna with longer FID Adj. resulting in longer distances fled , by placing more cover/obstacles between themselves and a predator, given that structure can impede predator pursuit . As suchAdj. on distance fled only in Common Myna, with longer FIDAdj. being associated with greater distances fled. It may be that heavier birds, realizing greater costs of escape, may exhibit positive relationships between FID and distance fled . Nevertheless, we also predicted negative association between FID and perch density. The only statistically significant association of FID and perch density (Noisy Miner) was in the opposite direction to that predicted; althougha trend in the expected direction existed for Red Wattlebird and Common Myna. For Common Myna, there was a trend towards a negative association between FID and route connectivity. While this requires verification from further study, it may suggest escape routes are known before escape commences in this species. The effect of nearest refuge on FID is clearly not universal among birds , and then only for perch density. Longer “distance from nearest refuge” may result in increased perceived predation risk, thereby increasing FID  which wong birds , and norIn general accordance with our results, We demonstrate an additional cost of living in poorly connected habitats. The mediation of the efficiency of effective predator escape  by habitat connectivity may be a mechanism which contributes to some of the larger scale patterns whereby depauperate avian diversity persists in poorly connected, fragmented contexts . Clearlyarac127_suppl_Supplementary_MaterialClick here for additional data file.


The rapid evolution of new SARS-CoV-2 variants poses a continuing threat to human health. Vaccination has become the primary therapeutic intervention. The goal of the current work was the construction of immunogenic virus-like particles (VLPs). Here, we describe a human cell line for cost-efficient and scalable production of immunogenic SARS-CoV-2 VLPs. The modular design of the VLP-production platform facilitates rapid adaptation to new variants. Methods: The N, M-, and E-protein genes were integrated into the genome of Expi293 cells (ExpiVLP_MEN). Subsequently, this cell line was further modified for the constitutive expression of the SARS-CoV-2 spike protein. The resulting cell line (ExpiVLP_SMEN) released SARS-CoV-2 VLP upon exposure to doxycycline. ExpiVLP_SMEN cells were readily adapted for VLP production in a 5 L bioreactor. Purified VLPs were quantified by Western blot, ELISA, and nanoparticle tracking analysis and visualized by electron microscopy. Immunogenicity was tested in mice. Results: The generated VLPs contained all four structural proteins, are within the size range of authentic SARS-CoV-2 virus particles, and reacted strongly and specifically with immunoserum from naturally infected individuals. The VLPs were stable in suspension at 4 °C for at least 10 weeks. Mice immunized with VLPs developed neutralizing antibodies against lentiviruses pseudotyped with the SARS-CoV-2 spike protein. The flexibility of the VLP-production platform was demonstrated by the rapid switch of the spike protein to a new variant of concern (BA.1/Omicron). The present study describes an efficient, scalable, and adaptable production method of immunogenic SARS-CoV-2 VLPs with therapeutic potential. Severe-Acute-Respiratory-Syndrome-Virus 2 (SARS-CoV-2) caused the disease COVID-19 and the recent pandemic ,4. The rStructurally, the positive-sense, single-stranded RNA virus consists of the spike glycoprotein (S), nucleoprotein (N), membrane (M) and envelope (E) proteins ,7. The NVirus-like particles (VLPs) are macromolecular protein assemblies that mimic the structure of a virus. VLPs are non-infectious, as they do not contain replication-competent genetic material (RNA or DNA). Because VLPs are highly immunogenic, they have attracted considerable attention as vaccine candidates and as research tools to model mechanisms of infection ,15,16. VCulturing of Expi293 cells: The Expi293 Expression System Kit, composed of Expi293 suspension adapted cell line, Expi293 transfection reagents, and Expi293 culture medium was purchased from Thermo Fisher Scientific . Cultivation and transfection of Expi293 cells were mostly performed according to the manufacturer’s instructions. Briefly, Expi293 cells were grown at 37 °C, 8% CO2 with 130–150 rpm on a Rotamax120 platform shaker  in Expi293 medium containing a final concentration of 100 units/mL penicillin and 100 µg/mL streptomycin. Cell diameter, the percentage of viable cells (viability), and the concentration of viable cells were measured using LUNA Cell Counter . Cells were seeded at a density between 0.3 × 106 and 0.5 × 106 cells/mL and subcultured at a concentration of 3 × 106 viable cells/mL after 3–5 days. For transfection, cells were precipitated for 5 min at 150× g and subsequently resuspended and adjusted in fresh media to a final concentration of approximately 3 × 106 viable cells/mL and a viability exceeding 95%.Generation of SARS-CoV-2 VLPs from genetically modified cell lines: As described previously   of VLPs generated by transient transfection  and by the ExpiVLP_SMEN cell line . The VLPs from both production methods were coated as a two-fold dilution series to the solid phase of a microtiter plate. ELISAs were performed to analyze the effect of VLP production method on the normalized absorption. In addition, 0.25 µg/mL of the spike protein or nucleoprotein was used as a reference. Two-way ANOVA revealed that VLPs from the ExpiVLP_SMEN cell line incorporated a significantly larger proportion of spike protein  = 19.73, p = 0.0002, n = 3) and nucleoprotein  = 6.133, p = 0.020, n = 3) than VLPs generated by transient transfection . Both purification steps significantly increased the proportion of the VLP peak which correlated to the purity of the sample  = 49.80, p = 0.0194) (p = 0.0261) in PEG-it precipitated VLP from the retentate as compared to a direct precipitation from the cleared cell culture supernatant . However, we still noticed that small but consistent peaks, which represent the Expi293 media alone, were present in the diafiltrated retentate and PEG-it precipitated VLP from the retentate. We concluded that larger diafiltration volumes would be beneficial.To improve the purification of VLPs harvested from the supernatant of ExpiVLP_SMEN cells exposed to doxycycline (1 µg/mL) for five days, we introduced a cross-flow filtration step using a Minimate tangential-flow filtration system. The cell culture supernatant was cleared by centrifugation and sterile-filtered prior to diafiltrating with PBS. Four times the initial volume was fed continuously to the supernatant. A 300 kDa Minimate tangential flow filtration capsule with an approximate pore size of 35 nm was used to separate the VLPs from smaller molecules in the supernatant. Filtration was performed at 0.124–0.165 kPa which generated a mean flow rate of 4.79 ± 1.01 mL/min (SD) across the three batches that were systematically analyzed. Subsequently, VLPs were precipitated with PEG-it Virus Precipitation Solution (System Biosciences) and pellets resuspended in PBS A. Interm 0.0194) B. The pup = 0.0113, n = 3 batches) between the median particle diameter of the cleared supernatant , the retentate , and the PEG-it precipitation from the retentate . Interestingly, we found that VLPs that were precipitated with PEG-it from the retentate incorporated significantly larger amounts of spike protein (nested t-test: p = 0.0441) and nucleoprotein (nested t-test: p = 0.0108) (p = 0.0023) (t-test: p = 0.7846). The demonstrated improvements in purity and reactivity of VLPs precipitated from diafiltrated supernatant established this method as our preferred preparation protocol.Next, we analyzed whether the purification steps had an impact on the size of the particles which was determined by nanoparticle tracking analysis C,D. Inde 0.0108) F. This w 0.0023) G. No difQuantification of N- and S-protein contend of purified SARS-CoV-2 VLPs9, SD = 3.14 × 108) and the predicted molecular weight of the N-protein (45.63 kDa) and S-protein (140.95 kDa) into account, we calculated the mean number of 1174 ± 275.50 N-proteins and 121.51 ± 36.15 trimeric S-proteins per VLP.The combination of nanoparticle tracking and a quantitative ELISA was used to further our understanding of the molecular composition of the VLPs that were purified by cross flow filtration and precipitated with PEG-it. Samples from three independent batches were subjected to an in-house ELISA to quantify the concentration of N- and S-protein against a two-fold serial dilution of the reference protein . The VLPLarge-scale production of VLPs in a 5 L bioreactor6 cells/mL and a viability of 91%. Subsequently, this starting culture was transferred to a 5 L bioreactor and cultivated with continuous supply of fresh serum containing media up to the maximal volume of the bioreactor, which was reached after four days. Cells had a density of 1.5 × 106 cells/mL and were fed continuously for another four days to further increase cell density. The induction of VLP production by the addition of doxycycline was initiated at a cell density of approximately 1 × 107 cell-agglomerations per milliliter. Of note, the exact concentration of cells could only be approximated because cells agglomerated into spherical units of five to ten cells. For another three days, cells were fed continuously with doxycycline containing fresh medium and harvested on day seven after induction. Using nanoparticle tracking analysis, we determined, that the total harvest of 4.7 L cell culture supernatant contained 7.68 × 1011 ± 3.16 × 1010 (SD) particles per milliliter. The purified VLP-containing samples contained 1.85 ± 0.01 (SD)% nucleoprotein and 1.99 ± 0.06 (SD)% spike protein, as determined by ELISA. The protein concentration was 1.35 ± 0.04 (SD) milligram per mL.The ExpiVLP_SMEN cell line was transferred to a roller bottle cell culture system and cultivated up to a density of 1.35 × 10SARS-CoV-2 VLPs are immunogenic in mice and elicit a neutralizing antibody responsep < 0.0001), the spike protein ELISA  = 11.75, p = 0.0019), and the nucleoprotein ELISA  = 4.215, p = 0.0495). The concentration of VLPs had a positive effect in the VLP-ELISA  = 52.59, p < 0.0001) and the spike protein ELISA  31.97, p < 0.0001). There was no significant interaction between the number of doses and the injected dose (p > 0.05). Subsequently, the serum samples of the eight animals that received two injections with 50 µg of VLPs were subjected to a neutralization assay . In contrast, samples stored at −80 °C or +4 °C showed only minor changes in the nanoparticle tracking analysis and were consistently detected by human convalescent serum .The distribution of a vaccine at the appropriate temperature is critical and caused delays at the beginning of the recent pandemic. Therefore, we next investigated the stability of VLPs produced from the ExpiVLP_SMEN cell line at different temperatures. In an initial experiment, VLPs were resuspended in sterile PBS to a concentration of 2.08 mg/mL, analyzed by nanoparticle tracking analysis and ELISA and then stored in cryogenic tubes at −80 °C, −20 °C, +4 °C, +20 °C or +36 °C . After fIn a follow-up stability test, we compared the storage in the fridge and a deep freezer and extended the storage time with a new batch of VLPs to 10 weeks . EncouraRapid adaptation of ExpiVLP_MEN with the BA.1 spike proteinThe VLP-producing cell line developed here was constructed as a modular system so that the base-cell line contains the M-, E-, and N-protein and lacks the S-protein. Therefore, we could rapidly add a new spike protein (Omicron BA.1) by infecting the parental cell line (ExpiVLP_MEN) with a newly constructed lentivirus for the dual expression of the BA.1 spike protein and puromycin selection marker. qPCR ,39 was uIn the current study, we describe a platform technology for the reproducible and adaptable production of VLP-based vaccine candidates for COVID19. We have generated a bioreactor-compatible mammalian cell line that produces SARS-CoV-2 VLPs upon exposure to doxycycline. The VLPs closely mimic the authentic SARS-CoV-2 and are immunogenic in mice. Furthermore, the design of our genetic construct allows the rapid adaption of the cell line without affecting down-stream processes.The SARS-CoV-2 VLPs produced by our system are structurally intact, are within the size range of the authentic virus, and contain all four major structural proteins without tags or deletions. The only modification is the destruction of the furin-cleavage site between the S1 and S2 subdomain to avoid loss of S1 during the purification steps. This is one of the few studies that directly validates the presence of the M- and E-protein in their VLPs and, to our knowledge, the only study that quantified the number of nucleoproteins and spike proteins per particle. We found approximately 1100 nucleoproteins and 120 spike trimers per VLP which are within the reported ranges for natural viruses of SARS-CoV-2 and the closely related SARS-CoV. From the structural data on ribonucleoprotein complexes reported by two groups, it was approximated that SARS-CoV-2 contains at least 300 to 600 nucleoproteins per virion ,40. The 14 particles per liter in the unpurified supernatant from the fed production, or 3.2 × 1013 purified particles and 33.7 mg protein per liter of cell culture from the shaker culture. The purified samples contain 160 mg/mL nucleoprotein and 150 mg/mL spike protein and 1.85 × 1012 particles per milliliter. With these specifications, our production system exceeds the yield of all, to our knowledge, reported production systems in mammalian or insect cells.The generated inducible cell line is the simplest production method for SARS-CoV-2 VLPs that has been described today. ExpiVLP_SMEN cells can be grown to the required volume and density. Subsequently, VLP production is induced in one simple step by the addition of doxycycline. The fact that SARS-CoV-2 proteins were not detected in the absence of doxycycline indicates low leak expression of the inducible promoter and the absence of the M- and E-proteins which are necessary for VLP assembly and release ,10,28. H5 infectious units per milliliter and that approximately 1.25 μg/mL S2 equivalent was integrated into their VLPs. Yilmaz et al. 2021 used transient transfection of HEK293 cells to generate VLPs that contained all four major structural proteins of SARS-CoV-2 [11 purified particles per liter of cell culture using a baculoviral system [13 particles per liter of unpurified cell culture supernatant using a baculovirus-free transient expression system [Mammalian cell lines have the advantage of the correct glycosylation, which is important for the spike protein, instead of the simpler glycosylation in insect cells or no glycosylation in bacterial cells. Roy et al. 2021 used a stable mammalian platform to generate chimeric VLPs from the Moloney murine leukemia virus (MLV) that were pseudotyped with the SARS-CoV-2 spike protein . They esRS-CoV-2 . The totl system  up to apWe could not compare the VLP yield to the reported production in yeast  or plantIn the future, it is necessary to use a uniform system to report the yield of VLPs to facilitate the comparison between production systems. We suggest a combination of nanoparticle tracking analysis of the unpurified cell culture supernatant and the purified sample. Furthermore, the total amount of purified protein per liter of cell culture as well as the concentration of specific protein  of the purified sample would be beneficial. Such data would allow to compare the productivity of the cell culture system itself and the loss of product due to the applied purification method.® in Canada. Suitable adjuvants for the VLPs produced by our system as well as the final concentration of VLPs have to be determined in future experiments.In a first proof-of-concept experiment, we show that the VLPs produced here are immunogenic. Mice that received two doses of VLPs had IgG antibodies against the nucleoprotein (50%) and the spike protein (100%). The serum neutralized lentiviruses pseudotyped with the SARS-CoV-2 spike protein. Of note, we have not yet tested the addition of adjuvants. Adjuvants drastically enhanced the immunogenicity of a plant-based VLP-vaccine candidate , which hWe also addressed the issue of inactivation of vaccine candidates over time. The stability of the compounds is important for the worldwide distribution of vaccines. Keeping a constant temperature can be particular challenging or sometimes even impossible in developing countries  if the pAs a result of the continuous evolution of new SARS-CoV-2 variants of concern, a vaccine production system has to be adaptable. This essential feature was demonstrated by the rapid exchange of the spike protein in the VLPs produced in our system. In future experiments, it is important to explore the potential of the VLPs produced here and their variants to induce antibodies that neutralize current variants of concern.In this study, we describe a stably modified mammalian SARS-CoV-2 VLP production system which minimizes economical and technical difficulties whilst achieving the highest particle yield reported so far in mammalian or insect culture systems. The produced VLPs are immunogenic and stable when stored at −80 °C or 4 °C.


Hnscr is crucial for metabolic regulation, but its roles in lipid metabolism during obesity are still unknown. In this article, we found that the expression of Hnscr gradually decreased in adipose tissues of diet-induced obese mice. Furthermore, the deletion of Hnscr promoted an increase in body weight and adipose tissue weight by upregulating the expression of lipogenesis genes and downregulating lipolysis genes in inguinal white adipose tissue (iWAT) and brown adipose tissue. In vitro knockdown of Hnscr in adipocytes resulted in reduced lipolysis of adipocytes. Overexpression of Hnscr by adenovirus or drug mimics showed the opposite. Mechanistically, Hnscr regulated adipose lipid metabolism by mediating the cyclic adenosine monophosphate/protein kinase A signaling pathway. This study identifies the initial characterization of Hnscr as a critical modifier that regulates lipid metabolism, suggesting that lncRNA Hnscr is a potential target for treating obesity.Obesity is a process of fat accumulation due to the imbalance between energy intake and consumption. Long noncoding RNA (lncRNA)  The worldwide obesity pandemic has dramatically increased the incidence of type 2 diabetes, cardiovascular disease, and other related complications , 2. ObesLong noncoding RNAs (lncRNAs), defined as nonprotein-coding RNA transcripts longer than 200 nucleotides, are emerging as crucial regulators of different cellular processes . AccumulThe cyclic adenosine monophosphate (cAMP)/PKA pathway is the most well-known mechanism that mediates lipolysis . IncreasHnscr (Gm31629) is crucial to glucose metabolism  and brown adipose tissue (BAT) was observed in high-fat diet (HFD)–induced obese mice. In in vivo and in vitro experiments, Hnscr reduction promotes adipogenesis and reduces lipolysis in adipocytes. In comparison, overexpression of Hnscr or supplementation with theaflavin 3-gallate (TF2A) stimulated adipocyte lipolysis. Mechanistically we found that Hnscr regulated lipid metabolism by mediating the cAMP/PKA signaling pathway. Taken together, our findings highlight Hnscr as a target for treating obesity by alleviating lipid metabolism disorders.Our previous studies discovered that lncRNA tabolism . HoweverHnscr-null mice were generated as described previously (Hnscr-deficient (Hnscr-null) mice, the genomic region of the mouse Gm31629 gene was knocked out by using TetraOne technology from Cyagen Biosciences (China). Hnscr genotyping was determined by extracting genomic DNA from the toes or tails of newborn mice and performing polymerase chain reaction (PCR) with the following PCR primers: forward 1:5′-GCTAAGCTCGAGGGACCTAATA-3′, forward 2:5′-CTGCTGCTCACCTCTAGTCATT-3′, reverse: 5′-AGGGATGTTCCTCACTGGCTGG-3′.Eight-week-old male C57BL/6J mice were obtained from the Hunan SJA Laboratory Animal Company. Male eviously . BrieflyAll mice were kept in a C57BL/6J background, maintained in the specific pathogen-free (SPF) class at the Experimental Animal Research Center of Central South University, with a constant temperature of 22 to 24 °C, constant humidity of 60% to 75%, 12-hour dark/light cycle, 4 or 5 mice per cage, free access to food and water, and regular change of bedding. All animal care protocols and experiments were reviewed and approved by the Animal Care and Use Committee of the Laboratory Animal Research Center, Xiangya School of Medicine, Central South University. The study complied with all relevant ethical regulations for animal research.Hnscr-null NCD group (n = 6), and Hnscr-null HFD group (n = 6), were fed rodent chow diet or HFD  for 3 months at 8-10 weeks age. Subsequently, mice were executed, and the tissues were isolated. Tissues were weighed, snap-frozen in liquid nitrogen, and then stored at −80 °C for subsequent studies.Four groups of male C57BL/6J mice, including wildtype (WT) normal chow diet (NCD) group, n = 6), WT HFD group (n = 6), 2 and 37 °C. After primary iWAT adipocytes or 3T3-L1 cells reached confluence (day 0), differentiation of the cells was induced in DMEM high-glucose medium supplemented with 0.5 mM isobutyl methylxanthine , 1 μM dexamethasone (Sigma), 10 μg/mL insulin (Sigma), and 1 μM rosiglitazone (Sigma) for 3 days and then the medium changed to 10 μg/mL insulin and 1 μM rosiglitazone every 2 days until day 8.Male WT mice were euthanized at 8 weeks of age, and stromal vascular fractions (SVFs) from iWAT pads were isolated as described in previous studies , 27. SVFHnscr (Ad-Hnscr) were generated from Obio Co . A 12-well plate was seeded with 3T3-L1 cells at 5 × 104 cells/well overnight (70-80% confluency), then transfected with siRNA or adenovirus at an multiplicity of infection (MOI) of 7 for 2 days before induction of adipocyte differentiation. Cells were treated with TF2A (80 μmol) for 72 hours before induction of adipocyte differentiation.Gm31629-targeted small interfering RNAs (siRNAs) were purchased. The siRNA was transfected using lipofectamine RNAiMAX (Invitrogen) according to the instructions. Knockdown efficiency was detected by qPCR 2 days after transfection. Adenovirus control (Ad-Scramble) and adenovirus After 2 to 3 rounds of induction, the fluid was replaced with growth medium. Then cells were treated with H-89  for 2 hours. Cells were collected for further analysis.The mice were placed in a temperature-controlled chamber  at 4 °C for 4 hours. The rectal temperature was measured with a rectal probe at a specified time after cold exposure. Body temperatures were read before and 2 hours after cold exposure using infrared thermography . Infrared images were analyzed with FLIR-Tools-Software (version 6.4).The following antibodies were purchased from Cell Signaling Technology: anti-ATGL , anti-Phospho-HSL , anti-HSL , and anti-Phospho-PKA . The following antibodies were purchased from Abcam: anti-cAMP  and anti-UCP1 .−ΔΔCt method using β-actin as an internal control.Real-time PCR analysis was conducted as previously described , 29. TheParaformaldehyde-fixed and paraffin-embedded iWAT and BAT sections were examined histologically with hematoxylin and eosin (H&E) staining. Adipocyte diameters were quantified using ImageJ V1.42q .Primary iWAT adipocytes or 3T3-L1 cells that induce differentiation maturation were collected, the medium was discarded, and cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. The 4% paraformaldehyde was discarded and cells were immersed in 60% isopropanol for 5 minutes. Then the 60% isopropanol was discarded and cells were stained with Oil Red O.Glycerin was measured with a glycerin kit according to the manufacturer’s instructions. The glycerin kit was purchased from Elabscience Company .P < .05 was considered statistically significant.All data are expressed as means ± SEM. The statistical significance of the differences between various treatments or groups was measured by either Student's t-test or analysis of variance (ANOVA) followed by the Bonferroni post-test. Data analyses were performed using GraphPad Prism 7.0. Hnscr could regulate systematic aging  mice using TetraOne technology . The rectal temperature of the mice exposed to cold  and control siRNAs in primary adipocytes. The results show that si-Hnscr significantly inhibited the level of Hnscr in cells . As expected, the expression of Hnscr was dramatically increased compared with the control group  , 36. TheL1 cells . Consistt uptake., accompat uptake. and 5J. t uptake. and 5L. ol level . In concHnscr  or hypertrophy (increased volume), or even both. It is generally believed that adipocyte hypertrophy occurs before adipocyte hyperplasia and is the primary mechanism of fat mass expansion  and adipose tissue weight . One of  studies , we consxpansion . In thisHnscr in in vitro experiments and found that overexpression of Hnscr in adipocytes inhibited lipogenesis and promoted lipolysis. Furthermore, our results also showed that although the intervention of adipocytes with TF2A resulted in increased expression of genes related to lipogenesis and uptake, there were significantly increased levels of browning and lipolysis.Lipolysis is described as the metabolic process of catabolism of triacylglycerols stored in cellular lipid droplets . AbnormaHnscr regulated adipose lipid metabolism by mediating the cAMP/PKA signaling pathway. Overexpression of Hnscr in adipocytes increased the protein levels of cAMP/PKA signaling. Moreover, TF2A can mimic the action of Hnscr and promote cAMP/PKA activation. We also verified in Hnscr-null mice that Hnscr knockout downregulated the cAMP/PKA signaling activity in adipose tissues.The cAMP/PKA signaling pathway is a recognized mechanism for activating lipolysis in adipose tissue . The resHnscr expression in iWAT and BAT of obese mice, we constructed Hnscr-null mice to explore the role of Hnscr in adipose tissue. Meanwhile, we also found that overexpression of Hnscr promoted lipolysis in adipocytes. Mechanistically, Hnscr regulated lipid metabolism by activating the cAMP/PKA signaling pathway. We also intervened in adipocytes with TF2A, a compound that could mimic the activity of Hnscr to promote lipolysis .


Flexible work arrangements (FWA) are becoming increasingly widespread as an efficient means of coping with a dynamic and competitive business environment. Existing studies have primarily examined the impact of FWA as a management system; however, its impact on employee innovation behavior has not been fully explored. Based on the self-determination theory, this study constructed a moderated mediation model that empirically examined the influence of FWA on the innovation behavior of knowledge employees. Our findings are as follows: (1) FWA can activate innovation behavior among knowledge employees; (2) thriving at work plays a partial mediating role; (3) human resource policies that facilitate opportunities have a positive moderating effect. The findings fill a theoretical research gap and provide insights for managers on implementing FWA to promote the innovative behavior of knowledge employees. Flexible work arrangements (FWA), generally being referred to flexibility of spatial (where work is conducted) and/or temporal (when work is conducted) , have beResearch in developed countries have examined FWA-related topics, such as its antecedents  and inflThe theoretical base of this study is provided by the self-determination theory, which suggests that an individual’s environment can enhance their autonomy and competence, which can, in turn, enhance and stimulate their intrinsic motivation and facilitate the internalization of extrinsic motivation. Accordingly, this study suggests that FWA plays a role in stimulating employees’ intrinsic motivation through two mechanisms. First, FWA provides employees with more job autonomy in work practices, as shown by This study also examined the role of thriving at work as part of this process. According to Furthermore, organizational climate or conditions often influence innovation behavior, especially innovation-related human resource management (HRM) policies and practices. Based on the discussion above, this study makes two significant theoretical contributions. First, our study reveals that FWA is conducive to stimulating the innovative behavior of knowledge employees, which extends the research perspective of the consequences of FWA to some work behavior with certain job characteristics. Existing studies have focused on the impact of FWA on broad work behaviors common to all employees, such as absenteeism, turnover, and work satisfaction, or work-related attainment, such as work-family balance. These studies did not distinguish between the work characteristics of different jobs. Although some studies have suggested that FWA can stimulate employees’ proactive work behaviors , no studThe findings of this study have several important practical implications. First, it contradicts the negative perception of FWA (common in developing countries) as a means by which organizations can extend working hours without limitations. The actual application of FWA can improve employee satisfaction, as previous studies have revealed, and benefit their innovative behavior, as our study showed. Both of them will have a significantly positive effect on organizational performance in the long run. Furthermore, our study suggests that organizations can practice FWA in a planned manner. Job characteristics are the most important considerations when activating employees’ innovation potential. HRM policies should be implemented to support FWA.Continued adherence by companies to a fixed work system makes it more difficult for their employees to autonomously arrange their work time, especially for the knowledge employees who have difficulty working owing to their physiological condition or who have specific needs related to personal affairs or family life . Mental Compared to other employees, knowledge employees may have a stronger desire to realize their self-worth and enjoy challenging work. They prefer a pleasant, autonomous work atmosphere to stressful conditions . FWA sigOwing to their experience of job autonomy from FWA, knowledge employees have greater job autonomy and will to boost their innovation performance. We propose the following hypothesis:H1: FWA is positively related to knowledge employees’ innovation behavior.Especially for knowledge employees, FWA gives them more autonomy to coordinate their jobs with other affairs, which helps them to have more opportunities to learn and communicate. Additionally, they can easily maintain high vitality levels when they feel more control over their working hours or places. So, they maintain a good situation of thriving at work and have more innovative behaviors, ultimately contributing to the organization’s innovative development. Accordingly, we propose the following hypothesis:H2: Thriving at work mediates the relationship between FWA and knowledge employees’ innovation behavior.Knowledge employees’ innovation behavior is built on a continuous search for inspiration and innovation opportunities. HRM policies and practices that facilitate this kind of opportunity include job rotation, information sharing, employee engagement, teamwork, and strengthening employees’ social networks inside and outside organizations. HRM opportunity creates more opportunities for knowledge flows and information exchange, facilitating innovation among knowledge employees. For example, job rotation can expose employees to new work environments and job content; information sharing can enable employees to gain new knowledge; employee engagement and teamwork promote team innovation and collaboration; and external interactions may contain new knowledge to inspire innovation. Therefore, under a high HRM opportunity scenario, knowledge employees who thrive in the workplace may discover more possibilities for innovation. Additionally, opportunity-facilitating HRM methods help create and maintain emotional bonds within the organization by fostering connections and communication among employees, making it easier for employees to obtain support and assistance from colleagues .Thus, it can be expected that high HRM opportunity not only increases the individual incentive of employees with FWA to innovate but also increases team support for their innovations. Consequently, we propose the following hypothesis:H3: HRM opportunity moderates the indirect effect between FWA and knowledge employees’ innovation behavior through thriving at work.This study examined knowledge employees’ engagement in producing, creating, diffusing, and applying knowledge and bringing value-added intellectual capital to organizations. Considering the above literature review, the sample for this study includes knowledge employees who hold positions in R&D, design, engineering, quality, production management, marketing, procurement, finance, and human resources within organizations. Since we targeted knowledge employees, non-organization personnel  and non-knowledge employees  were excluded. We cooperated with organizations that have applied FWA even before the pandemic and are strongly interested in research, mainly from the manufacturing and IT industries, based on the following considerations: the application and development of FWA in developing countries such as China is still in its infancy, and is mainly concentrated in the manufacturing and IT industries. Furthermore, innovation is valued and common in these industries. Considering the common application of digital working platforms after the pandemic, we distributed electronic versions of the questionnaire in Chinese with the help of these organizations’ human resource managers through their internal working systems. A letter attached before the questionnaire explained that the university conducted the investigation for research goals, and each participant had the right to refuse to give a response.A small-scale pilot study with a sample of 100 knowledge employees was conducted to further check and refine the measures. The investigation period lasted from October 2021 to December 2021, with 500 questionnaires distributed and 488 returned. The questionnaires were screened as follows: first, questionnaires with incomplete or incorrect answers  were excluded; second, questionnaires with a strong regularity of answers  were excluded; again, the answers to the reverse test questions were checked and excluded without consistent answers, and if the length of time to fill in the questionnaire was too short (for not thinking carefully about the implications of the questions) or too long (for the possible interruption in filling the electronic questionnaires), the questionnaire was regarded as unreliable and rejected. A total of 429 valid questionnaires were obtained, representing a response rate of 85.8%.As shown in We chose the established measures that are commonly adopted in existing studies. Measures of employee innovation, thriving at work, and HRM opportunity were originally developed in English and have Chinese versions. All items of the measures were scored on a 5-point Likert scale (from 1 = strongly disagree to 5 = strongly agree).Referring to We used a six-item scale to measure employee innovation; an example item is, ‘I am an innovative person at work’ . The CroWe used the 10-item scale that We used a six-item scale to measure the HRM opportunity . An examReferring to r = 0.440, p < 0.01), between the mediating variable of thriving at work and the outcome variable of employee innovation , and between the independent variable of FWA and the outcome variable of innovation behavior , which provided initial support for the proposed hypothesis and enabled us to proceed with the regression analysis and moderated mediation test.The variance inflation factor (VIF) values of each variable in the model were less than three, far lower than the critical value of 10, which is far lower than the critical value of 10, indicating that there were no serious multicollinearity issues. Thus, regression analysis is a feasible approach for testing the hypotheses.Regarding the common method variance, Harman’s single one-way analysis of factors was applied. Four factors (more than one) were analyzed using Harman’s one-way test, and the explanatory power of factor 1 was only 36.949% (less than the cutoff of 50%) , indicatχ2/df = 2.647). Therefore, the four-factor model was the most appropriate for this study.We conducted a series of confirmatory factor analyses using AMOS 26.0. A validated factor analysis was conducted on four variables to confirm the discriminant validity of the variables: FWA, employee innovation, thriving at work, and HRM opportunity. The closer the RMSEA and SRMR are to 0, the better the model fit . The cloWe used SPSS 25.0 for the following statistical analysis and PROCESS macro (Model = 4 for Hypothesis 2 and Model = 14 for Hypothesis 3) , which cb = 0.390, p < 0.001); thus, Hypothesis 1 is supported.Hypothesis 1 predicts that FWA is positively related to knowledge employees’ innovation behavior. In b = 0.336, p < 0.001). Model 4 examined the relationship between thriving at work and employee innovation behavior. The regression analysis showed that, after adding the mediating variable, there was still a significant positive relationship between FWA and innovation among knowledge employees; the absolute value of the coefficient became smaller . The mediating variable also has a significant positive relationship with knowledge employees’ innovation . Thus, the result supports the positive relationship between thriving at work and employee innovation behavior.Hypothesis 2 predicted that thriving at work mediates the relationship between FWA and knowledge employees’ innovation behavior. For robustness, we used stepwise regression and bootstrapping analyses to test the proposed mediation effect. In b = 0.09, CI = ) and did not contain zero  developed by ain zero , indicatb = 0.362, p < 0.001), HRM-opportunity , interaction term , and knowledge employees’ innovation all had significant positive relationships with each other. Thus, HRM-opportunity positively moderates the relationship between thriving at work and innovation among knowledge employees.Hypothesis 3 predicted that HRM opportunity would moderate the indirect effect between FWA and knowledge employees’ innovation through thriving at work. As shown in As shown in The moderating variable was then tested for its moderating effect on the mediating effect of thriving at work. The bootstrap test under different HRM opportunity conditions was conducted using the PROCESS macro (Model = 14), with 95% confidence intervals and 5,000 samples. In addition, based on the judgment index method , the indThe ongoing improvement in digital and telecommuting tools and systems has meant that the technical conditions for the practice of FWA have matured. An increasing number of businesses are providing more autonomy for employees through FWA to respond to the dynamic and competitive business environment . ExistinFirst, FWA stimulates innovation behavior among knowledge employees through intrinsic motivating mechanisms. FWA gives knowledge employees more autonomy in work practices and enhances their feelings of autonomy . This enSecond, thriving at work partially mediates between FWA and knowledge employees’ innovation behavior. Some studies have shown that FWA can relieve pressure and improve employees’ physical and mental states . We exteThird, HRM opportunity positively moderates the mediating role of thriving at work. Previous research has shown that promoting opportunity-oriented HRM policies provides more innovation opportunities for knowledge employees and makes it easier for employees to obtain innovation support and trust from colleagues . Thus, wThe primary theoretical contributions of this study are as follows. Firstly, our study reveals that FWA is conducive to stimulating the innovative behavior of knowledge employees, which extends the research perspective of the consequences of FWA to some work behavior with certain job characteristics. Existing studies have focused on the impact of FWA on broad work behaviors common to all employees, such as absenteeism, turnover, and work satisfaction, or work-related attainment, such as work-family balance. These studies did not distinguish between the work characteristics of different jobs. Although some studies have suggested that FWA can stimulate employees’ proactive work behaviors , no studThe findings also enrich research on the antecedents of employee innovation behavior. This field has focused more on specific antecedent factors , such asSecond, this study explored the underlying mechanism and theoretical boundary conditions between FWA and innovation behavior. Previous studies have provided plentiful discussions on the antecedents and outcomes of FWA but have not examined the internal mechanisms by which FWA plays a role in specific conditions. This study’s findings have two practical implications. First, the results may reduce public misunderstanding of FWA to some extent. It is wise for organizations to use FWA to facilitate new forms of work and ease psychological pressure on employees, which may have a range of positive effects and even mitigate labor conflicts, especially during difficult times, such as the COVID-19 pandemic. In industrialized nations, mostly in Europe and the U.S., FWA is a standard organizational management practice. In contrast, there is still a severe lack of awareness and comprehension in developing countries, especially China, where overtime work culture prevails. Leaving aside the complex causes of this variation, Chinese organizations are generally unenthusiastic about FWA and are thus missing valuable opportunities. This study shows that FWA, which allows employees to arrange their own working time or space to a certain extent, has a practical and positive effect on organizational development.Second, the results have implications for the optimal application of FWA. In many developing countries, small- and medium-sized businesses are frequently unable to innovate owing to insufficient capital and a dearth of innovative personnel. This study proposes that organizations consider the innovation potential of different management systems and pertinent HRM policies. The use of HRM to promote innovation opportunities can intensify the role of FWA in promoting employee innovation.Despite these contributions, this study had some limitations. First, it is based on cross-sectional survey data and thus lacks a longitudinal perspective. This research design can only reflect correlations between variables at one point in time; the intensity of the effect of FWA on thriving at work and employee innovation may change over time. Second, our results cannot necessarily be generalized to knowledge employees from other industries owing to their different job characteristic. The subjects in this study are mainly from the manufacturing and IT industries that knowledge employees need more work autonomy for improving their innovation performance to support the organizational innovative development. This job characteristic is different from the same work positions in other industries, e.g., the service industry.These limitations suggest future avenues for research that focus on the following aspects. First, the research could provide a richer consideration of the degree of flexibility and dynamic impact of FWA in different scenarios. For example, what degree of flexibility is necessary for enhancing innovation performance? Does FWA positively or negatively impact team  innovation performance? Second, future research should consider other job characteristics such as employee from the platform-based internet companies. We can extend the The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the patients/participants or patients/participants legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.LJ guided the research design and reviewed the manuscript. DK participated in the study design and data collection. LJ and DK prepared the manuscript. ZP, YL, and ZG designed the study, performed the statistical analyses, and revised the manuscript. All authors have contributed to the manuscript and approved the submitted version.This study was supported by the National Social Science Foundation of China (20BGL146).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.