The Keap1-Nrf2 signaling axis is a validated and promising target for cellular defense and survival pathways. This minireview discusses the potential off-target effects and their impact on future drug development originating from Keap1-targeting small molecules that function as displacement activators of the redox-sensitive transcription factor Nrf2. We argue that small-molecule displacement activators, similarly to electrophiles, will release both Nrf2 and other Keap1 client proteins from the ubiquitin ligase complex. This non-specificity is likely unavoidable and may result in off-target effects during Nrf2 activation by targeting Keap1. The small molecule displacement activators may also target Kelch domains in proteins other than Keap1, causing additional off-target effects unless designed to ensure specificity for the Kelch domain only in Keap1. A potentially promising and alternative therapeutic approach to overcome this non-specificity emerging from targeting Keap1 is to inhibit the Nrf2 repressor Bach1 for constitutive activation of the Nrf2 pathway and bypass the Keap1-Nrf2 complex. With the increase in the global population, the worldwide prevalence of neurodegenerative diseases is on the rise. They are among the leading causes of disability and death worldwide and will continue to grow in the coming decades due to increased life expectancy. Despite a considerable amount of basic and clinical research, most strategies to manage neurodegenerative diseases are palliative. A large body of evidence suggests that these diseases are multifactorial caused by genetic, environmental, and endogenous factors related to aging. An emerging target for neurodegenerative diseases that could modulate multiple etiological pathways involves drug-induced activation of a coordinated genetic program to maintain redox equilibrium through the expression of pro-survival proteins and cytoprotective genes . A key t1, the minimum recognition sequence in Neh2 domain includes 16–85 aa residues. Keap1 is a redox sensor protein -stress-induced 3-hydroxy-3-ethylglutaryl-CoA reductase degradation protein 1 (Hrd1) . UbiquitTwo principally different modes of Nrf2 stabilization in the constitutive pathway can be achieved: (a) irreversible, via Keap1 thiols modification followed by either dimer destabilization or Cul3 ubiquitin ligase detachment from Keap1, and (b) reversible, via competitive displacement of Nrf2 from the complex with Keap1 using Nrf2 peptides or small molecules specifically targeting Keap1 Kelch domain . The staThe strong binding peptide motif for the Keap1 Kelch domain has the sequence of ETGE or ESGE, and mutations in these sequences disrupts Nrf2 interaction with the Keap1 Kelch-domain and results in Nrf2 activation. A comprehensive proteomics study of Keap1 interaction network  identifiAs seen from The list of Keap1 interactors covers a panoply of cellular processes, from DNA replication and licensing to cytoskeletal dynamics, transcription, and apoptosis. Predicting the plausible side effects is difficult given the absence of detailed information on Keap1 clients. However, based on the differences in the mechanism of Keap1 targeting by alkylating and displacement activators, some general predictions can be made. If we focus on thiol modifying Nrf2 activators targeting Keap1, they either destroy its dimeric structure or detach Keap1 from the ubiquitin ligase complex. In both cases, Keap1 will be no longer active with respect to ubiquitination of a client protein or its 2-point stretching between Kelch domains. However, this treatment should have no effect on the ability of individual Kelch domains to bind Keap1 client proteins via ETGE-like sequences. Contrary to this, high-affinity displacement activators targeting the Kelch domain will be capable of displacing all Keap1 interactors even if they are “hanging” onto one of the Kelch domains in the Keap1 dimer or are bound to the Keap1 monomer. So, one may expect more additional pathways to be triggered with displacement activators due to their tight binding to the Kelch domain in Keap1, which results in the release of all Keap1 dimer and monomer interactors. Displacement with peptides will be more selective since the affinity of Keap1 clients for Kelch is different, and only less tightly bound Keap1 client proteins will be displaced with a peptide . So, peptide-targeted alkylating agents may turn out to be more specific than small-molecule displacement activators. However, there is an additional concern relevant to the small molecule displacement activator’s action, as we discuss below.Small molecule displacement activators of Nrf2 are designed to target the Kelch domain in Keap1. However, Keap1 is only one among 42 Kelch-like gene family member (KLHLs) proteins in humans. Only a small portion of these proteins are currently being studied concerning their structure and functions. KLHLs are closely related to the pathogenesis of various human diseases, and specifically cancer. KLHL members associated with inherited forms of the human disease include KLHL3, KLHL7, KLHL9, KLHL12, and GAN (KLHL16), whereas KLHL6, KEAP1 (KLHL19), KLHL20, and ENC1 (KLHL37) are associated with cancer . The KLHWe decided to pick available crystal structures of “6-blade propeller” Kelch domains and run docking for a well-known displacement activator N,N′-naphthalene-1,4-diylbis(4-methoxybenzenesulfonamide) or NMBSA, whose structure is shown in As we can see from the discussion on the molecular mechanisms of Nrf2 ubiquitination inhibition, there are principal concerns for using this approach to treat ongoing neurodegeneration. First, only one ubiquitination pathway is really well characterized at the molecular level – Keap1-mediated degradation of Nrf2 protein. Second, the existing thiol-modifying agents are potent inducers of the Nrf2-mediated transcriptional program and can be effectively used as preventive therapies. However, their use for existing and ongoing neurological conditions is problematic unless these agents are targeting Keap1 specifically, which is a big challenge. The excitement to use small molecule displacement activators to stabilize and activate Nrf2 by targeting the Kelch domain of Keap1 will likely diminish, as it becomes more and more evident that- (a) Keap1 has multiple client proteins, and they all will come to play upon Nrf2 displacement with high-affinity activators of this kind, and (b) there are dozens of Kelch-BTB protein adaptors for multiple ubiquitin ligase complexes having very similar structures of Kelch domains, and (c) working concentrations of displacement activators in the cells are high above their dissociation constant determined in fluorescent polarization Keap1 binding assay.Acute and chronic oxidative stress generates intracellular ROS and RNS, and they, as we recently learned from in silico modeling. DH, NA, MA, AP, and SS provided intellectual contribution and assisted in writing the manuscript. All authors approved the submitted version of the manuscript.BT and IG conceived and wrote the manuscript. DH performed 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.


Saprolegnia monoica, two chitin synthases have been found, and both contain a Microtubule Interacting and Trafficking (MIT) domain. The MIT domain has been implicated in lipid interaction, which in turn may be of significance for targeting of chitin synthases to the plasma membrane. In this work we have investigated the lipid interacting properties of the MIT domain from chitin synthase 1 in Saprolegnia monoica. We show by fluorescence spectroscopy techniques that the MIT domain interacts preferentially with phosphatidic acid (PA), while it does not interact with phosphatidylglycerol (PG) or phosphatidylcholine (PC). These results strongly suggest that the specific properties of PA are required for membrane interaction of the MIT domain. PA is negatively charged, binds basic side chains with high affinity and its small headgroup gives rise to membrane packing defects that enable intercalation of hydrophobic amino acids. We propose a mode of lipid interaction that involves a combination of basic amino acid residues and Trp residues that anchor the MIT domain specifically to bilayers that contain PA.Chitin synthases are vital for growth in certain oomycetes as chitin is an essential component in the cell wall of these species. In   •S. monoica interacts specifically with PA.MIT from chitin synthase 1 in •Interactions with lipids is does not depend only on charge.•MIT does not alter its structure in response to lipids.•Specific interaction with PA suggests a role for MIT in trafficking or insertion of chitin synthase 1. CDcircular dichroismCHSchitin synthaseCPEceramide phospatidylethanolamineMIT domainMicrotubule Interacting and Trafficking domainMMDMyosin motor-like domainMMD PAphosphatidic acidPAGEpolyacrylamide gel electrophoresisPCphosphatidylcholinePEphosphatidylethanolaminePGphosphatidylglycerolPOPA1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acidPOPC1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine;PCphosphatidylcholinePOPE1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolaminePOPG1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine;PSphosphatidylserineSECsize exclusion chromatographySmCHSSaprolegnia monoica chitin synthase1Saprolegnia monoica, also contain a small amount of chitin, which is synthesized by chitin synthases (CHS)  is the concentration of acrylamide. The quenching constant was used to determine the degree of association of the MIT domain to vesicles of different composition. Three independent experiments were recorded for each measurement.Fluorescence emission spectra were recorded with a Fluorolog fluorescence spectrometer  at 25 °C using a quartz cuvette with an optical path length of 10 mm. The samples were excited at 295 nm and spectra were recorded from 310 nm to 400 nm in 1 or 0.5 nm steps with 2 nm excitation and emission slit widths. The sample conditions were 10 μM MIT domain and 500 μM lipids in 50 mM phosphate buffer pH 7.5 and 150 or 300 mM NaCl, giving a protein/lipid ratio of 1:50. Five repetitions were carried out for each sample. Quenching studies were performed using a Varian Cary Eclipse Fluorescence Spectrophotometer . Aliquots from a 1 M stock solution of acrylamide  were titrated to the sample containing the MIT domain to concentrations ranging between 0 mM and 100 mM. Samples were mixed and incubated for 30 min before addition of acrylamide. Fluorescence was excited at 280 nm and scans were taken with a 5 nm excitation and emission slit width. The quenching constant (Ksv) was calculated with the Stern-Volmer equation:2.51H NMR spectra were acquired for samples containing 3.5 mM lipids, 28 μM MIT domain in 50 mM phosphate buffer pH 7.5, 150 mM NaCl, 5% D2O and 0.1 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid for chemical shift referencing. Spectra were acquired at 25 °C on a Bruker 700 MHz spectrometer equipped with a cryo-probe. The 90° pulse length was 19 μs and 8000 transients were acquired.33.1S. monoica interacts specifically with certain lipids, fluorescence spectroscopy was used to assess both changes in spectral features, as well as to study the degree of insertion of the protein into a bilayer (The MIT domain was expressed and purified as described previously  with the bilayer . To prod bilayer  and the  bilayer A. Simila−1 [−1), indicating an exposed location of the Trp side chains. Quenching by acrylamide became somewhat less effective as vesicles with either PC or PG were added to the protein (26 M−1 and 25 M−1 respectively), while a marked decrease in quenching efficiency was observed for vesicles containing PA (13 M−1). For Trp residues deeply immersed into a membrane the quenching constant can be close to 0 [Quenching of intrinsic Trp fluorescence by the hydrophilic quencher acrylamide was also measured B. The St−1 , and herose to 0 . While wTo further test the binding specificity of the MIT domain for PA, LUVs with different amounts of PA were used, ranging from PC only to 50 mol% PA. These were used to monitor changes in fluorescence intensity and blue-shift . In addi3.2To investigate if the structure of the MIT domain is affected by lipid interactions, far-UV CD spectroscopy was used to monitor changes in secondary structure A. As exp1H NMR spectra of the MIT domain in different vesicles were recorded. The amide region of the 1H NMR spectrum in buffer and in different vesicles is shown in To further test whether the MIT domain binds to vesicles and retains its structure, 1D 4S. monoica has a strong preference for interacting with phosphatidic acid over e.g. PG, which represents a generic negatively charged lipid. In previous work, we have established that the MIT domain interacts strongly with PA, while the protein does not interact with phosphatidylserine to a significant degree, although some degree of interaction with monophosphorylated phosphatidylinositols was observed [The presence of specific lipids varies greatly during the life cycle of a cell, and the lipid composition is also specific for certain types of vesicles, important for cell signaling and trafficking . The preobserved . These robserved ,33 and tobserved ,27.S. monoica membranes are not known, but it is likely that it is present primarily in curved regions in the membrane, such as at the apex of the hyphae where CHS is found [S. monoica CHS, including the MIT domain studied here [The high ratios of PA needed to detect an interaction in the present study indicate that the MIT domain interacts with the membrane weakly. Another possibility is that the local concentration of PA varies in the membrane. Local concentrations of PA in is found . Althougis found , but micied here . This suied here , but theied here .The MIT domain contains ten basic amino acid residues, an attribute that has been suggested to be important for lipid interactions. MD simulations have suggested that binding to PA is achieved through a “hot-spot” containing several Arg residues (mostly so in helix 1 and helix 2) . Here weTaken together, our data suggest that the interaction between the MIT domain and the lipid membrane are driven by specific properties of PA, such as strong electrostatic interactions between Lys residues and the phosphate headgroup as well as curvature stress induced by the small headgroup of PA. Our observations, specific binding to PA and shallow immersion of Trp residues, together with the structural properties of helix 3, exposed Lys, Arg, His and Trp residues, indicate that the binding of the MIT domain to PA is consistent with the "electrostatic/hydrogen bond switch model" . In this5in vivo. Interactions between the MIT domain and PA may be of relevance for the proper transport and localization of chitin synthase 1 to the plasma membrane, through fusion of transport vesicles to the membrane. Based on these and previous results we suggest a role for the MIT domain in insertion or in trafficking of the chitin synthase to the plasma membrane.We studied the interaction of the MIT domain of chitin synthase 1 with vesicles and found that it preferentially interacts with PA via electrostatic and hydrophobic interactions. The specific affinity of the MIT domain for PA suggests that this interaction serves a specific purpose The authors declare that there are no conflicts of interest.


UBE3A-ATS for the purpose of developing therapies for the imprinting disorders Prader–Willi syndrome and Angelman syndrome. In summary, a better understanding of long non-coding RNA-related mechanisms is key to the development of potential therapeutic targets for human imprinting disorders.Genomic imprinting is a term used for an intergenerational epigenetic inheritance and involves a subset of genes expressed in a parent-of-origin-dependent way. Imprinted genes are expressed preferentially from either the paternally or maternally inherited allele. Long non-coding RNAs play essential roles in regulating this allele-specific expression. In several well-studied imprinting clusters, long non-coding RNAs have been found to be essential in regulating temporal- and spatial-specific establishment and maintenance of imprinting patterns. Furthermore, recent insights into the epigenetic pathological mechanisms underlying human genomic imprinting disorders suggest that allele-specific expressed imprinted long non-coding RNAs serve as an upstream regulator of the expression of other protein-coding or non-coding imprinted genes in the same cluster. Aberrantly expressed long non-coding RNAs result in bi-allelic expression or silencing of neighboring imprinted genes. Here, we review the emerging roles of long non-coding RNAs in regulating the expression of imprinted genes, especially in human imprinting disorders, and discuss three strategies targeting the central long non-coding RNA  In diploid organisms, most genes are transcribed in an unbiased fashion from both alleles. However, in a small subset of genes, genetically identical alleles can be expressed differentially, a process referred to as ASE. In the mammalian genome, common epigenetic examples of ASE include random X-chromosome inactivation in females , genomicGenomic imprinting has been described in diverse organisms, including marsupials, flowering plants, and insects . In the Ube3a-ATS for the purpose of therapeutically correcting the PWS/AS locus imprinting disorders. We also discuss the functional mechanisms of imprinted lncRNAs in the regulation of mono-allelic imprinted gene expression and how it could help us understand ASE mechanisms and underlying pathological mechanisms of human imprinting disorders, hopefully inspiring additional efficient therapeutic strategies.Long non-coding RNAs are a subgroup of non-coding RNAs defined as having a length longer than 200 nucleotides, and are extensively expressed among the genome . The numAlong with more profound analysis of patient samples and well-established mouse reciprocal crossing models using high-throughput sequencing, the monoallelic expression of imprinted genes has been observed extensively in mice and humans . MethyloIn the human and mouse genome, imprinted genes often reside together within imprinted clusters . More thUBE3A and IGF2 show imprinted expression patterns in specific human brain cell types /Kcnq1 antisense transcript 1 (Kcnq1ot1) cluster and the antisense of Igf2r non-protein coding RNA (Airn)/Igf2r cluster /H19 locus are mechanistically regulated  cells that do not include the overlapping region are unable to silence the Igf2r paternal allele, thus demonstrating that inhibition of RNA polymerase II recruitment to Igf2r promoter region does not depend on the overlap between Airn transcription and the promoter  targeting Ube3a-ATS rescued the expression of Ube3a efficiently , with the embedded lncRNA Kcnq1ot1 promoter, is unmethylated on the paternal allele , EZH2 (PRC2), and histone methyltransferase euchromatic histone lysine N-methyltransferase-2 (EHMT2 or G9a) to neighboring gene regions, forming repressive histone modifications such as H3K27me3 and H3K9me2  and Slc22a3 (about 300 kb to Airn locus), are also silenced by Airn in the extra-embryonic lineage, where Airn mediates the recruitment of PRC1 and PRC2 to distal targets on the paternal alleles  on human chromosome 15q11-13   and lncRNAs including 116HG, 115HG, and the antisense transcript to UBE3A  and may participate in splicing, ribosomal RNA maturation, RNA modifications, and regulation of prohormone processing-related gene expression  . The neuto UBE3A . The mosne locus . SNORD11pression . Meanwhirecently . 116HG iion site . 116HG pt manner . Similarof UBE3A .SNORD116 non-coding gene locus, which regulates the maturation of the central nervous system. The overlap between the phenotype caused by SNORD116 microdeletion and MAGEL2 mutation suggests that transcripts from SNORD116 locus may modify MAGEL2 expression via long-range chromatin interactions , maternal UPD (36%), small microdeletion in SNORD116 locus (<1%), and epigenetic alternations in DNA methylation of the PWS-ICR region (4%)  was identified in a patient with typical PWS syndrome  . Rare mipatients . Moreovesyndrome . Since tpatients . Besides anxiety .SNORD116 host non-coding transcript has been described in human  processed from in human  III. Them of PWS . SNORD11ein ends . These sein ends . These l6 snoRNA . Since i nucleus . TherefoUBE3A gene expression , in which the expression of UBE3A is silenced by UBE3A-ATS as discussed previously  imprinted cluster on human chromosome 14q32 are associated with two human imprinting disorder-related diseases, KOS14 and TS14 , the Rtl1-antisense Rtl1as, the C/D-box snoRNA cluster Rian, and the microRNA cluster Mirg are transcribed from the maternal allele  Ezh2 and PRC2 in the maternal Dlk1 gene region , snoRNAs, and miRNAs are transcribed on the maternal allele. Importantly, the DLK1 gene plays essential functions in regulating development and metabolism. In KOS14 patients, gain of DNA methylation on the maternal ICR leads to MEG3 silencing  signaling pathway, a well-known growth-related pathway. Therefore, unraveling the roles of imprinted lncRNAs in cancer may reveal novel biomarkers and therapeutic targets for cancer treatment.Another well-studied cancer-related imprinted lncRNA is gnancies . In pituntiation . Hypermepatients , while rpatients . One sucUbe3a/Ube3a-ATS imprinted cluster are understood best, attempts have been made to rescue Ube3a expression through modulating the collision between the transcriptional machinery of Ube3a and Ube3a-ATS in an allele-specific manner. Herein, three state-of-the art therapeutic strategies by targeting Ube3a-ATS lncRNA, editing Ube3a-ATS gene region, or modulating chromatin transcriptional state by small molecules are discussed along with recent preclinical studies of UBE3A/UBE3A-ATS imprinted cluster-related diseases.Although our understanding of the mechanisms of imprinting disorders has grown, efficient molecular diagnosis and effective treatments are limited to nonexistent . ModulatAntisense oligonucleotides are single-stranded DNA oligos designed using sequence homology with their RNA targets that hybridize with the targeted RNA region based on complementary base pairs, and induce subsequent RNA degradation at the ASO-RNA heteroduplex part . ASOs caUbe3a/Ube3a-ATS imprinted cluster, Ube3a-ATS represses Ube3a expression by prematurely terminating the elongation of Ube3a transcripts. Therefore, a potential strategy is to rescue the defective Ube3a transcription by targeting Ube3a-ATS transcripts using ASOs  inhibitors have been identified with the capacity to downregulate Ube3a-ATS expression and induce reactivation of UBE3A expression from the paternal allele . On the maternal chromosome, histone methyltransferase euchromatic histone lysine N-methyltransferase-2 (G9a) locates at the methylated PWS-ICR and recruits repressive histone modifications (H3K9me2) along the PWS-ICR in a bidirectional manner. This leads to condensed chromatin structure and silencing of PWS-associated genes , imprinted lncRNAs which play essential regulatory roles in silencing other imprinted genes are all expressed on the paternal allele. It has been reported that maternal expressed imprinted genes are prominent with protein-coding genes, while paternal expressed genes exhibit consistent distribution between non-coding and protein-coding sequences (Several imprinting gene clusters have been discovered and studied since the middle of the last century. These studies have shown that lncRNAs play crucial roles in regulating imprinted gene clusters and individual imprinted genes related to human health and diseases. However, from a genomic perspective, the characteristics of gene regulation among imprinting loci remain to be fully elucidated. This is despite the advancement in knowledge of the epigenetic regulatory mechanisms of a subset of genes in imprinted regions. In the three imprinted clusters (equences . HoweverHULC in mice, essential for phenylalanine metabolism (in vivo stability. Further translation of these strategies to real clinical tools will require further investigation to overcome related challenges. In vivo delivery of synthesized RNA molecules, cellular permeability, immunogenicity, and potential of organ toxicity also deserve further investigation (Pharmacological treatments for congenital imprinting disorders are limited to symptomatic therapies, which are inefficient in promoting the patients’ quality of life . Fortunatabolism . In additigation . AnotherTW and QM wrote and edited the manuscript and drew the pictures. JL, LY, and MW assisted in manuscript collation and review. QM provided critical inputs as the corresponding authors and obtained funds. All authors contributed to the article and approved the submitted version.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.


India follows a pluralistic system for strategic and focused health care delivery in which traditional systems of medicine such as Ayurveda, yoga and naturopathy, Unani, Siddha, Sowa Rigpa, and homoeopathy (AYUSH) coexist with contemporary medicine, and this system functions under the Ministry of AYUSH (MoA). The MoA developed a mobile app, called AYUSH Sanjivani, to document the trends of the use of AYUSH-based traditional and holistic measures by the public across India. Analysis of the data generated through this app can help monitor the extent of the use of AYUSH measures for maintenance of health during the COVID-19 pandemic and aid effective health promotion and communication efforts focused on targeted health care delivery during the pandemic.The purpose of the study was to determine the extent of use of AYUSH measures by the public in India for maintenance of health during the COVID-19 pandemic as reported through the AYUSH Sanjivani mobile app.Cross-sectional analysis of the data generated through the Ayush Sanjivani app from May 4 to July 31, 2020, was performed to study the pattern and extent of the use of AYUSH-based measures by the Indian population. The responses of the respondents in terms of demographic profile, use pattern, and benefits obtained; the association between the use of AYUSH-based measures and symptomatic status; and the association between the duration of use of AYUSH-based measures and the outcome of COVID-19 testing were evaluated based on bivariate and multivariate logistic regression analysis.Data from 723,459 respondents were used for the analysis, among whom 616,295 (85.2%) reported that they had been using AYUSH measures for maintenance of health during the COVID-19 pandemic. Among these 616,295 users, 553,801 (89.8%) either strongly or moderately agreed to have benefitted from AYUSH measures. Ayurveda and homeopathic measures and interventions were the most preferred by the respondents across India. Among the 359,785 AYUSH users who described their overall improvement in general health, 144,927 (40.3%) rated it as good, 30,848 (8.6%) as moderate, and 133,046 (40.3%) as slight. Respondents who had been using AYUSH measures for less than 30 days were more likely to be COVID-19–positive among those who were tested . The odds of nonusers of AYUSH measures being symptomatic if they tested positive were greater than those of AYUSH users .The findings of this cross-sectional analysis assert that a large proportion of the representative population practiced AYUSH measures across different geographic locations of the country during the COVID-19 pandemic and benefitted considerably in terms of general well-being, with a possible impact on their quality of life and specific domains of health. Coronaviruses, a large family of single-stranded RNA viruses, can infect animals and humans, causing respiratory, gastrointestinal, hepatic, and neurologic diseases . To dateClinical evidence from a study on the effects of Chinese traditional medicine in the treatment of SARS-CoV-2 demonstrated significant results, and the study proposed that herbal medicine has a beneficial effect in the treatment and prevention of epidemic diseases . A CochrThe government of India has taken the initiative to use and integrate the preventive, curative, and rehabilitative potential of AYUSH systems of medicine to strengthen the health care delivery system, and the AYUSH Sanjivani app was developed through a consultative process among experts in the field of AYUSH and information technology (IT) by the MoA to record the patterns and trends of the use of preventive measures adopted by the public to enhance immunity and maintain health during the COVID-19 pandemic. The AYUSH Sanjivani app was intended to motivate and persuade users to achieve a status of healthy well-being while thwarting the tendency of the masses to use untested and unproven remedies or over-the-counter or self-prescription measures, especially when faced with the threat of the pandemic and the physical, physiological, social and economic ramifications of the containment measures required of the public.Through recent initiatives in smart devices, mobile apps have become a convenient, easy-to-use, and less time-consuming method to generate data from the public. Self-reported health status and health care service use are indispensable indicators to assess the performance and attitude of any health system in the absence of recorded health administration data . An app-Hence, a cross-sectional analysis of the data generated from the app was performed to determine use trends of AYUSH measures by the public during the COVID-19 pandemic.The primary objective of the cross-sectional analysis was to determine the extent of use of AYUSH advocacies and measures by the public for maintenance of health during the COVID-19 pandemic, as reported through the AYUSH Sanjivani mobile app.Secondary objectives were to compare the self-reported incidence of COVID-19 and symptomatic status of the respondents affected with COVID-19 among the users of AYUSH measures as compared to nonusers and to determine the pattern of use of AYUSH measures by users across India. Perceived change in general well-being in terms of appetite, bowel habits, sleep, stamina, and mental well-being among users of AYUSH measures, the relationship between the duration of the use of AYUSH measures and the incidence of COVID-19, and the relationship between the symptomatic status of COVID-19–positive respondents among users and nonusers of AYUSH-based measures were also included as secondary objectives.This is a cross-sectional analysis of data generated through the AYUSH Sanjivani mobile app. The MoA launched the AYUSH Sanjivani app to generate data on the acceptance and use of AYUSH advocacies and measures by the population and its possible impact on the maintenance of health during the COVID-19 pandemic. The content of the app is a self-reporting questionnaire intended for the public to report their preferences, patterns, and trends of use of the measures circulated through the AYUSH advisory released by the MoA for maintenance of health during the pandemic. Self-perceived impact on improvement in general health and the benefits of using AYUSH measures during the COVID-19 pandemic by the respondents were also recorded in the app. We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines when reporting the findings [The study was approved by the Central Ethics Committee of Central Council for Research in Ayurvedic Sciences, Ministry of AYUSH, India (1-12/2020-CARICD/Tech/CEC). Upon downloading the app, before voluntary consent was obtained, the user was informed that the information they entered would only be used for research purposes and that their anonymity and confidentiality would be maintained. The users were also informed that by choosing to provide information in this app, they were making a valuable contribution to public health research in the country. It was made explicitly clear that by participating in the survey, users were voluntarily giving their consent to use the data for research purposes.The app was released through the Google Play store in May 2020 and was available for download across India. The data generated from respondents across all States and Union Territories of India during the period from May 4 to July 31, 2020, were used for the analysis.All the residents of India who possessed a smartphone, tablet, or other such device and who were willing to download the app and voluntarily provide the requisite information to the questionnaire in either English or Hindi were eligible to participate in the study. The primary respondents were health seekers of AYUSH or their families, who preferred AYUSH systems for preventive or curative purposes; health seekers who sought consultation at the outpatient department of a national institution, research council, college, hospital, or primary or secondary health care facility across the country; and members of the public who were motivated to use AYUSH measures for maintenance of health during the COVID-19 pandemic. These beneficiaries were sensitized to the AYUSH measures through interaction with health professionals.The AYUSH Sanjivani app was conceived to motivate and persuade the public to achieve a state of general well-being during the COVID-19 pandemic while documenting the patterns and trends of the use of AYUSH systems in India. The app was also announced and promoted through social media platforms such as the Twitter accounts, Instagram, and Facebook pages of MoA, AYUSH institutions, and hospitals, as well as AYUSH professionals and students, which enabled a wider reach among the general public. The AYUSH Sanjivani app was available for the Android and iOS (Apple) platforms and was made available through the App Store and Google Play store, and the questionnaire was drafted in a simple, easily comprehensible manner, initially launched in English and later rolled out in Hindi according to the World Health Organization guidance for translation and adaptation of instruments . The appThe app contains three modules to report the desired data . The firThe questionnaire was subdivided into three different layers. The first layer captured basic sociodemographic characteristics, such as gender, and the geographical location of the respondents. A question on whether they were using AYUSH measures  or not using any AYUSH measures was also included to classify the respondents into two categories (users or nonusers of AYUSH measures). Those who responded that they are using or have used AYUSH measures during this pandemic were asked three additional questions. The first question was related to the duration of use of AYUSH measures, and the second question captured the opinions of the respondents on whether the practice of AYUSH measures had benefitted them. The third question was intended to capture the possible reasons for finding AYUSH beneficial, reported as per the respondent’s experiences.The second layer of the app contained another four questions, which could be answered by all respondents irrespective of whether they were using AYUSH measures. The questions were intended to capture the information such as the respondent’s occupation, presence or absence of any pre-existing disease, the risk of contracting COVID-19 . The COVID-19 test status was elicited through a separate question in which the respondent was required to select any of the options of tested positive and asymptomatic, tested positive and symptomatic, tested negative, and never tested. The respondents were required to furnish data related to their COVID-19 status if they underwent testing either on their own or on medical advice.The third layer of the app was accessible to only those respondents who were using AYUSH measures, and the questions included in it pertained to the use trends of measures advised under various AYUSH systems. This layer contained another set of six questions, namely duration of intake of AYUSH measures, the regularity of intake, self-perceived improvement in parameters of well-being  or no improvement after use of AYUSH, and the onset of any influenza-like-illness symptoms. The respondent’s self-perceived impact on their general health was also captured , where they were numerically coded. Numerical codes were assigned to all the options for each question in the questionnaire. This coded Excel file was then imported into STATA 16.1 (StataCorp LLC) and used for statistical analysis. Descriptive statistics for categorical data were reported using frequencies and percentages. Comparisons among users and nonusers of AYUSH measures were performed using the chi-square test in terms of respondents being tested or not for COVID-19, the outcome of COVID-19 testing, the symptomatic status of the COVID-19–positive respondents, the risk status of the respondents, and the presence or absence of comorbid conditions. Logistic regression analysis was performed to compute the crude odds ratio by measuring the association between the duration of use of AYUSH-based measures and the outcome of COVID-19 testing (positive or negative). The association between the use of AYUSH-based measures and the symptomatic or asymptomatic status of COVID-19–positive respondents was also evaluated. Adjusted odds ratios considering the risk status and presence of comorbidities as confounders were also computed. A Data from 723,459 participants who downloaded the app and furnished the required information were available for analysis. The majority of the respondents  reported having been using AYUSH measures for maintenance of health during the pandemic. Among the 616,295 respondents who were AYUSH users, 493,766 (80.1%) were male, and 467,047 (75.8%) lived in rural areas. Data for occupation was available for 359,745 users, of which the majority were self-employed ; 83,281 (23.2%) were students, and 67,230 (18.7%) were unemployed .Among the 616,295 AYUSH users, the maximum participants were from Uttar Pradesh , followed by Maharashtra ; Madhya Pradesh ; Gujarat ; and Chhattisgarh . A small proportion of the respondents were from the states of Rajasthan, Odisha, Haryana, Bihar, Karnataka, and West Bengal. The smallest number of respondents were from the states of Meghalaya and Manipur and the union territories of Ladakh and Lakshadweep .The number of respondents who reported using Ayurveda measures was 90,357/433,560 (21.0%), Homeopathy was used by 47,639/433,560 (11.0%), while a small proportion reported the use of Unani and Siddha Interventions. It is intriguing to note that 291,251/433,560 (67.0%) of the users reported having been using yoga, pranayama, meditation, or the use of home remedies such as spices in cooking, drinking warm water, steam inhalation, and other such practices for maintenance of health .Samshamani Vati and AYUSH-64 were the most popular among the Ayurveda interventions. Kaba Sura Kudineer, a decoction used in the Siddha system, and the Unani interventions of Behidana, Unnab, and Sapistan decoction were reported as the most commonly used, albeit by only a small proportion of users. Agastya Hareetaki (an Ayurvedic intervention); use of Anu Taila, coconut oil, or sesame oil for nasal instillation, or oil pulling with coconut or sesame oil; use of Chyavanprasha; turmeric milk; and herbal tea were the other frequently used interventions in the Ayurveda stream. Bryonia alba, Rhus toxicodendron, Belladonna, Gelsemium, and Eupatorium perfoliatum were the other commonly used homeopathic interventions. Nilavembu Kudineer decoction and Adathodai Manapagu were some other popular Siddha interventions  reported using them for more than 30 days, while 231,735 (37.6%) reported having used them for less than 15 days. A considerable proportion of respondents either strongly agreed  or moderately agreed  that the use of AYUSH measures had benefitted them. AYUSH measures were considered beneficial by 487466/616,295 (79.1%) of the respondents, as these measures gave them an overall feeling of good health. Most of the respondents  reported improvement in parameters of well-being in terms of their perception of change in appetite, bowel movements, sleep, stamina, and mental well-being. Improvement in appetite was reported by 20,618/359,785 respondents (5.7%), while improvement in bowel habits was reported by 42,017/359,785 respondents (11.7%). Moreover, improvement in more than one parameter was reported by 98,352/359,785 respondents (27.3%). Overall improvement in general health was rated as excellent by 30,848 of 359,785 respondents (8.6%), while 144,927 (40.3%) reported good improvement and 133,046 (36.9%) reported slight improvement .Data on pre-existing diseases (comorbidities) were furnished by 408,089 respondents, of whom 380,731 (93.3%) reported the absence of any pre-existing disease. Hypertension was the most common pre-existing disease (comorbidity), reported by 11,941/408,089 respondents (2.9%), followed by diabetes mellitus, heart disease, and asthma. The presence of more than one pre-existing disease was reported by 9266/408,089 respondents (2.3%) .Among 408,089 participants, only 27,661 (6.8%) underwent testing for COVID-19. Out of the 27,661 respondents who underwent testing, 13,320 (48.2%) tested positive for COVID-19, among whom 8545 (64.2%) reported themselves to be asymptomatic.Among the 12,002 respondents who tested positive for COVID-19 and used AYUSH measures, 8101 (67.5%) reported their duration of use of AYUSH measures as less than 30 days, and the others reported a longer duration of use. Among the 12,002 COVID-19–positive respondents using AYUSH measures, 8100 (67.5%) were asymptomatic.P<.001) for respondents who were using AYUSH measures for less than 30 days compared to those who were using these measures for more than 30 days. The adjusted OR considering the effects of confounders, namely the presence of comorbidities and the respondent being in a risk category, is 0.90   of testing positive for COVID-19 is 1.52  .The results of the logistic regression analysis revealed that the OR of being symptomatic was 4.01 (95% CI 3.61-4.59) for nonusers of AYUSH measures compared to users. The adjusted OR considering the effects of confounders, namely the presence of comorbidities and respondents being in a risk category, is 3.48 (95% CI 3.06-3.95) .A representative population of 723,459 people from different geolocations across the country downloaded the AYUSH Sanjivani app and reported the perceptions and practices they had adopted in the wake of the COVID-19 pandemic that significantly altered their lifestyle. A majority of the respondents used AYUSH measures for maintenance of health and prevention of disease, and most of them reported having benefitted from the use of various interventions and practices. A positive association between the prolonged practice of AYUSH measures and symptomatic status could be observed in the respondents who were infected with COVID-19.In this study, the maximum representation was of AYUSH users, and it is expected that the willingness to use an app specifically targeting AYUSH users will be greater among health seekers who are familiar with these systems of medicine. Findings from a previous study revealed that even in a developing country such as India, 32% of the patients attending a medical care facility in urban settings used the internet, and 75% of them sought medical information through the internet; this would support the substantial amount of data generated through this app . In a naMaximum reporting was observed from the states of Uttar Pradesh, Maharashtra, and Madhya Pradesh, which can be attributed to the high population density in these states. The decision to seek health care is not only contingent upon the experience of illness but also depends on various social, economic, and demographic factors ; it was The majority of respondents reported having benefitted from using AYUSH measures; they rated the degree of improvement as mild, good, or excellent, and attributed this improvement to their perceived experience of overall well-being. The self-reported public experience of improvement in parameters of well-being, such as sleep, appetite, stamina, mental well-being, and sleep, is a good indicator for integrating AYUSH measures for well-being into the daily routine. Preliminary evidence on the impact of COVID-19 on the public reveals significant health-related anxiety, generalized anxiety, psychological stress, and sleep disorders, and government-implemented lockdowns inculcated many habits, such as decreased physical activity and exercise and increased snacking, with deleterious effects on vulnerable populations and especially on those with pre-existing comorbidities ,26.The improvements perceived in the level of well-being and the general aspects of health measured in terms of individual satisfaction with appetite, sleep, stamina, mental wellness, and bowel habits indicate a positive role for the use of traditional AYUSH interventions and practices in maintaining holistic health and preventing long-lasting adverse health outcomes.Samshamani Vati, and Ayush-64, which were deployed as frontline prophylactic interventions, were the most used interventions by the public to maintain health. Arsenicum Album 30C is a homeopathic intervention that is used for respiratory ailments; meanwhile, the other two interventions are Ayurvedic formulations (Samshamani Vati and Ayush-64), which are prescribed for the clinical management of pyrexia, influenza-like illness, cough, and dyspnea [Ayurveda and homeopathy were the systems of medicine that were preferred by the majority of the respondents; this can be attributed to the maximum number of hospitals and health care providers in India under these two AYUSH systems. Arsenicum Album 30C,  dyspnea .The practices that the public engaged in include the practice of yoga, pranayama, and meditation along with common home remedies such as using spices in cooking, drinking turmeric milk, drinking warm water, and steam inhalation. The practice of engaging the mind and body through meditation, pranayama, and yoga has attracted significant attention and has been extensively studied for its possible beneficial effects on physical and mental health outcomes . A growiRasayana activity  in healthy individuals reflect better outcomes when administered for 60 days or more, which implies that compliance with AYUSH measures would ideally require a longer duration to act in the macrochannels and microchannels of circulation to bring about optimal health; this underlines the pattern seen in this analysis, where the odds of being COVID-19–positive or symptomatic for respondents who tested positive are greater in respondents who have used the preventive measures for a lesser duration [The proportion of participants who underwent laboratory testing for COVID-19 among AYUSH users and nonusers could not be compared to arrive at meaningful outcomes, as the majority of the respondents were using AYUSH measures, and those not using it were few in number. A longer duration of use of AYUSH measures is more likely to produce better protection when compared with use for less than 15 days, as it was observed that the likelihood of being COVID-19–positive was lower in respondents who were using the AYUSH measures for more than 30 days. In AYUSH systems, the use of diet or medicine is targeted at producing an ideal state of homeostasis, which would reflect the inherent strength of the body in immunopotentiation and prevention of diseases. Clinical studies demonstrating The qualitative appraisal of the analyzed data reflects that a considerable majority of the respondents benefitted from the use of AYUSH measures, which were either traditional formulations with centuries of use in the maintenance of health or home remedies and are an integral part of Indian culture and cuisine. Due to the long history of use of many herbal remedies and the experiences that have passed from generation to generation, people are relying on herbal remedies and some simple home remedies for common diseases that are used across India irrespective of sociocultural, religious, and geographical differences . The useBecause this is a cross-sectional analysis of the data generated from a mobile app, the documented data are a representation of smartphone users only. A limitation of the study is the inability to capture generalizable data reflecting true health-seeking trends, as only people with access to smartphones and good internet connectivity responded to the questionnaire. Because the representation of nonusers of AYUSH measures was minimal, statistical comparison among users and nonusers of AYUSH measures could not be performed. Although AYUSH measures are generally practiced in many states for both curative as well as preventive aspects, representation from some of these states was meager; hence, a true representation of users or nonusers of AYUSH measures could not be captured. The incidence of COVID-19 among the respondents was self-reported, and it is difficult to determine the relationship between the use of AYUSH measures, duration of use, and incidence of disease or symptomatic status among the general public.Finally, this is the first study to document how time-tested indigenous systems of medicine are being used by the public during a pandemic of unprecedented spread, morbidity, and mortality. The large amount of data obtained is the greatest benefit of this analysis, as it would sweep outliers that may misrepresent the data and has enabled us to provide a realistic picture of the characteristic attributes and patterns of the population. This analysis offers a starting point for future researchers to initiate more interventional studies based on the use trends demonstrated in this study.The findings of the cross-sectional analysis assert that a good proportion of the representative population has practiced AYUSH advocacy across different geolocations of the country during the COVID-19 pandemic. Although anecdotally, people report that traditional systems of holistic healing are good for the maintenance of health and well-being, our study findings also support that use of AYUSH measures provided better health, improved parameters of well-being, and even helped prevent other illnesses. This pattern suggests possibilities of exploring the role of AYUSH care, considering its acceptance, accessibility, and possible benefits, in the area of pluralistic health care.To improve the use of a pluralistic health care delivery system, it is imperative to understand the acceptability, use trends, and possible impact on the quality of life and specific domains of health among the public. The response obtained in this study points to a possible functional integration and cross-hybridization of the merits of different systems to effectively generate a positive outcome on integrated health care delivery targeting universal health coverage. To assess the multiple levels of impact of AYUSH preventive measures on health, future studies need to apply diverse disciplines and methods, including intervention studies, longitudinal cohort studies, as well as qualitative observations to examine the nature of the benefits offered by these measures.


Here, a versatile condition of Knoevenagel polycondensation for constructing vinylene-linked 2D COFs was explored. Three new examples of vinylene-linked 2D COFs  containing benzobisthiazoles units as functional groups were successfully prepared under this versatile and mild condition. The electron-deficient benzobisthiazole units and cyano-vinylene linkages were both integrated into the π conjugated COFs skeleton and acted as acceptor moieties. Interestingly, we found the construction of a highly ordered and conjugated D-A system is favorable for photocatalytic activity. BTH-3 with benzotrithiophene as the donor with a strong D-A effect exhibited an attractive photocatalytic HER of 15.1 mmol h−1g−1 under visible light irradiation.Developing a facile strategy for the construction of vinylene-linked fully  π-conjugated covalent organic frameworks (COFs) remains still challenging. Here, the authors propose a versatile synthesis method based on Knoevenagel polycondensation for the synthesis of vinylene-linked benzobisthiazole-based 2D COFs.Synthesis of fully  The unique features of COFs such as the well-defined structure, high specific surface area, ordered pore channels, etc. endow them with potential applications in gas storage and separation15, catalysis19, sensor22, energy storage24, and optoelectronics25, etc. So far, the construction of highly ordered structures of COFs mainly relies on the reversibility of dynamic covalent bonds between the building blocks. Schiff base reaction and boronic acid dehydration reaction are frequently used and prone to achieve high crystallinity owing to their dynamic reversibility3. Although the reversibility of these dynamic covalent bonds endows the COFs good crystallinity, it weakens the π-conjugation and chemical stability of these materials28. On one hand, hydrolysis of these dynamic covalent bonds usually occurs under strongly acidic conditions. On the other hand, the poor electron delocalization caused by the strongly polarized nature of the dynamic covalent bonds such as the imine bond will hamper the charge carrier mobility of the COFs28. Developing COFs with fully π-conjugated structures and excellent chemical stability is highly desirable. Enormous efforts have been done to explore the facile access to highly stable and fully π-conjugated COFs28. New chemistry reactions for constructing conjugated COFs with optimum electron delocalization have been explored. Oxazole31, thiazole33, vinylene42, pyrazine44, imidazole45, and thienopyridine46 as stable and conjugated linkages have been developed recently. These π-conjugated COFs have displayed exceptional properties and greatly broadened the application fields. However, the study of these fully π-conjugated COFs is still in its infancy stage either in structural diversities or applications. The synthesis of these non-imine COFs is usually tricky with a low success rate and bad generality. This is understandable in consideration of the bad reversibility of the coupling reactions. To simultaneously gain desirable conjugation and refined crystallinity within the COF structure is still highly challenging.Covalent organic frameworks (COFs), in which the building blocks were connected covalently and arranged precisely, are attracting increasing attention since the first report by Yaghi’s group in 2005π-conjugated vinylene-linked 2D COFs47. Benefiting from the extended π conjugation along with the 2D skeletons, these cyano-vinylene-linked 2D COFs have shown excellent performance in magnetic properties48, photoluminescence50, photocatalysis54, and energy storage55. Herein, we developed a facile way to prepare the cyano-vinylene-linked 2D COFs through Knoevenagel polycondensation. A new reaction mechanism was proposed. The neutral AcONH4 was used to catalyze the condensation, which was supposed to enhance the reversibility of the initial step of Knoevenagel polycondensation. This strategy shows better success rates and universality. Three new fully sp2-carbon π-conjugated COFs containing benzobisthiazole (BTH) as functional groups were constructed by Knoevenagel polycondensation under this new explored reaction condition. The integration of BTH unit and cyano-vinylene linkage provides an electron-deficient π-conjugated C2 symmetrical building block  and transmission electron microscopy (TEM)  crystal plane of BTH-1, 2 appeared at a similar position of 2θ = 2.3°. Pawley refinement of the AA stacking model based on the experimental profile gave unit cell with parameters  for BTH-1  and  for BTH-2 , consistent with hexagonal unit cell. The optimized PXRD of BTH-3 displayed a (100) reflection at 2θ = 2.8° due to the smaller pore size. Pawley refinement data exhibited cell parameters of a = b = 37.45 Å, c = 3.52 Å, α = β = 90°, γ = 120°, revealing minimal difference (Rp = 4.09% and Rwp = 5.57%). Furthermore, the porosities were further examined via nitrogen adsorption experiments at 77 K. The sharp rise in the low-pressure range, as a characteristic of the type I sorption isotherm, indicate their microporous characteristics. The Brunauer–Emmett–Teller (BET) surface areas were observed to be 644, 686, 1140 m2 g−1 for BTH-1, 2, 3, respectively. The pore size distribution curves clearly show two different pore limiting diameters (PLD) for BTH-1, 2, 3  of 10.5 mmol h−1 g−1 under visible light (λ > 420 nm), while BTH-3 reached a higher HER of 15.1 mmol h−1 g−1. Although BTH-1, 2 possess similar skeleton structures, BTH-2 only exhibited an HER of 1.2 mmol h−1g−1. The apparent quantum yields (AQYs) of BTH-1 (5 mg) were measured to be 1.569%, 1.089%, 1.925% and 0.75% at 420, 450, 500, and 550 nm, respectively. The narrow bandgap of BTH-3 endows its good photoactivity even at 600 nm. The AQYs of BTH-3 is 0.792%, 0.676%, 1.256%, 0.883% and 0.735% at 420, 450, 500, 550, and 600 nm, respectively. The stability test result suggests that BTH-3 could keep relatively good photocatalytic performance under irradiation  and the benzene-containing BTH-2 (1.2 mmol h−1 g−1). This research first provides a facile way for the construction of sp2-carbon conjugated COFs. The conventional intractable preparation of the sp2-carbon conjugated COFs was greatly simplified. Second, the fully π conjugated COFs materials with D-A system show excellent photocatalytic hydrogen evolution performance and will definitely promote the development of COFs in photocatalysis.In summary, a new condition of the Knoevenagel reaction was first explored for constructing the spTA , BTH-2CN , AcONH4 , tetrahydrofuran (2.0 mL). After being degassed by freeze-pump-thaw technique for three times and then sealed under vacuum, the tube was placed in an oven at 100 °C for 3 d. The resulting precipitate was filtered, washed with tetrahydrofuran and ethanol for 2 d, dried at 120 °C under vacuum for 12 h. The activated BTH-1 was obtained as a yellow powder insoluble in common organic solvents  , BTH , AcONH4 , tetrahydrofuran (2.0 mL). After being degassed by freeze-pump-thaw technique for three times and then sealed under vacuum, the tube was placed in an oven at 100 °C for 3 d. The resulting precipitate was filtered, washed with tetrahydrofuran and ethanol for 2 d, dried at 120 °C under vacuum for 12 h. The activated BTH-2was obtained as a yellow powder insoluble in common organic solvents  , BTH , AcONH4 , tetrahydrofuran (2.0 mL). After being degassed by freeze-pump-thaw technique for three times and then sealed under vacuum, the tube was placed in an oven at 100 °C for 3 d. The resulting precipitate was filtered, washed with tetrahydrofuran and ethanol for 2 d, dried at 120 °C under vacuum for 12 h. The activated BTH-3 was obtained as a dark red powder insoluble in common organic solvents  (13C CP/MAS spectra shown as Fig. A Pyrex tube was charged with Supplementary InformationPeer Review File


In addition, NL and ECL pizzas were characterized by higher contents of bioactive compounds, including anthocyanins, and by higher in vitro antioxidant activity  than the control pizza (1.07 µmol Trolox/g d.m.). However, NL and ECL pizzas also contained small amounts of undigestible oligosaccharides, typically present in lentils . No significant differences were observed in the liking level expressed by consumers between ECL and control pizzas.Extruded-cooked lentil (ECL) flour was used to fortify (10/100 g dough) gluten-free pizza, which was compared with rice/corn-based pizza (control), and with pizza containing native lentil (NL) flour. Viscoamylograph and Mixolab data evidenced the hydrocolloid properties of ECL flour , which contained pregelatinized starch. The use of ECL flour made it possible to eliminate hydroxymethylcellulose (E464), obtaining a clean label product. Both NL and ECL pizzas showed significantly ( In recent years, the market of gluten-free (GF) products has increased because these products are in demand, not only by celiac patients, but also by non-celiac consumers . The latLegumes are a sustainable crop rich in proteins, fibers, and micronutrients . Many stPizza is an ancient food with a rich history and a strong cultural significance: in 2010, the Neapolitan-style pizza received the “Traditional Specialty Guaranteed” mark at the European level ; in 2017In a previous study, the fortification with chickpea flour was found to be effective in improving the nutritional profile of conventional (gluten-containing) pizza without worsening its quality . No studSaccharomyces cerevisiae, Lievitalia SpA, Parma, Italy), and sea salt  were purchased from local retailers. Hydroxypropylmethylcellulose  was provided by Laboratori Bio Line . Psyllium (Plantago ovata Forsk) seed husk powder was provided by Erbavoglio .Rice flour , corn flour , corn starch , baker’s yeast (Lens culinaris Medik.). Lentils were divided into two batches, submitted to the processing steps schematized in −1 moisture and was processed by an industrial co-rotating twin-screw extruder-cooker  at a 500 kg/h feed rate  with a 3:1 compression ratio, a 220-rpm screw rotation speed, and 100 °C die head temperature. The extrusion-cooking conditions were selected from previous research based on the cold viscosity of the extruded-cooked flour obtained, which had to be acceptably high (69.3 Brabender units—BU) /D (or T) before baking × 100.The analyses were carried out in triplicate.L*, a*, and b* were measured by using a Chromameter CM-600d  with a D65 illuminant and a CIE 10 standard observer angle. Five replicated analyses were made. The total colour variation (∆E) was then calculated to compare the differences between the control pizza and the two types of lentil-enriched pizza, according to the following equation:The color indices The following scale was considered: ΔE = 0–0.5, which indicated a very low difference; 0.5–1.5, which indicated a slight difference; 1.5–3.0, which indicated a noticeable difference; 3.0–6.0, which indicated an appreciable difference; 6.0–12.0, which indicated a large difference; and >12.0, which indicated a very obvious difference .A quantitative descriptive analysis (QDA) was performed, according to the International Standardization Organization (ISO) standard 13299 , by a sePre-test sessions were carried out: (i) to define the list of descriptors to be evaluated; (ii) to define the intensity range of each descriptor; (iii) to fix the scale anchors of each descriptor; and (iv) to verify the reliability, consistency, and discriminating abilities of the panelists, according to the ISO standard 11132 . The stuSamples were presented in dishes coded with three-digit random numbers and were distributed simultaneously in a random order. Sensory evaluations took place in a conference room, where temporary partitions were used to set up isolated tasting booths to separate panelists during the analysis, in agreement with the ISO standard 8589 . TestingA total number of eight sensory descriptors were considered. In the order of evaluation, they were: olfactory descriptors , visual–tactile descriptors , taste descriptors  and flavor descriptors (lentil flavor). The descriptors were rated on a structured scale that provided a 0–9 score range . The scale anchors for global odor, lentil odor, sweetness, saltiness, and lentil flavor were: 0 c.u. = minimum intensity; 9 c.u. = maximum intensity. The scale anchors for color were: 0 c.u. = ivory; 9 c.u. = carrot orange with rust undertones. The scale anchors for consistency  were: 0 c.u. = very soft and tender, like a brioche; 9 c.u. = hard, not very compressible. The scale anchors for pliability were: 0 c.u. = inflexible, rigid, and with a tendency to break; 9 c.u. = very pliable and elastic, easily bending without breaking. The sensory analysis was carried out in triplicate.Seventy consumers  that were not affected by food allergies or intolerances, and were regular consumers of pizza and lentils, evaluated the pizza samples. They were given information about study aims, and individual written informed consent was obtained from each participant. The consumers were asked to state if they liked or disliked each pizza sample, rating their disliking or liking levels on a structured scale from 1 to 5 c.u. .p < 0.05 by a one-way analysis of variance (ANOVA) followed by Tukey’s HSD test. The results of the consumers test were statistically analyzed by the Friedman test using XLStat software .Statistical analyses were carried out using the Minitab 17 Statistical Software . Significant differences were determined at Pizza formulation is reported in In setting up the formulation of the control pizza, corn and rice flours were used in a 1:4 ratio. The quantity of corn flour, typically bright yellow, was smaller than rice to obtain, in the mixture, a yellowish color similar to that of a conventional wheat-based dough. Moreover, the selected flour ratio assured that the intense smell of the corn flour did not prevail, as assessed in preliminary trials.As for the nutritional characteristics, rice and corn flours were richer in carbohydrates and presented lower contents of proteins and fibers compared to lentil flours (both NL and ECL). In particular, rice flour showed the lowest protein and fiber content. Lentil flour, instead, showed a relevant protein content, in agreement with the range of values reported in other studies ,46. The The gelation properties (viscoamylograph data) of the flours used for preparing the experimental GF pizza samples are reported in The ECL flour showed a significantly higher initial viscosity than the other flours. This result was expected, because the extrusion-cooking treatment was specifically aimed at gelatinizing the starch granules, while in the raw flours  the starch remained in its native form. The ECL flour showed the lowest peak viscosity, again indicating that the starch had already been completely pregelatinized during the extrusion-cooking process. The ECL flour also showed a very limited retrogradation, with significantly lower values of the cooling maximum viscosity and the setback compared to the other flours. A lower tendency towards retrogradation is appreciated in the production of bakery products, which should stay soft as long as possible. A limited setback, but greater than in ECL, was also observed in NL, confirming the results of . OverallThe gels from rice and corn flours showed remarkably high peak viscosity values, indicating a rapid and pronounced swelling of the native starch granules. The viscosity of gel from corn flour also remained high at its cooking temperature and under prolonged stirring (95 °C for 30 min), recording a very high value of minimum viscosity and a limited breakdown. During its subsequent cooling, the viscosity of both corn and rice gels noticeably increased, thus indicating a strong tendency towards starch retrogradation, which could be useful in noodle preparation  but is uRegarding the better evaluation of its suitability to the preparation of GF pizza, ECL flour was also analyzed by Mixolab, in comparison with NL flour . MixolabThe Mixolab profiles of the examined flours agreed with the viscoamylograph findings. ECL, in fact, showed a very high maximum torque in the first part of the analysis (C1). This result was due to the presence of pregelatinized starch, which increased the water absorption, producing a more consistent system that was already at 50 °C. On the contrary, NL showed an extremely low initial value of torque, because the contained starch, still native, could not gelatinize at 50 °C. Even at its minimum (C2), ECL showed a higher torque value than NL, indicating its ability to form a sufficiently structured gel to simulate a conventional (gluten-containing) dough. This ability, instead, was absent in NL which, before raising the temperature to 90 °C, never reached 0.4 Nm, which is the torque value below which the dough is considered not workable . The torViscoamylograph and Mixolab data clearly showed that rice, corn, and NL flours were not able to produce a viscous system after simple hydration, i.e., without heating at a sufficiently high temperature to achieve starch gelatinization (approximatively 95 °C). Therefore, hydrocolloids, namely HPMC (E464) and psyllium seed husk powder, were included in the formulation of pizza, to mime gluten and improve the dough structure. HPMC is known for its strong gelation capacity, which is able to increase dough viscosity . This adPsyllium has water-absorbing and gel-forming properties at room temperature. It is composed of arabinoxylans, whose numerous hydroxyl groups increase the capacity to bind to water and generate viscous solutions . While, Psyllium is also an effective anti-staling agent, appreciated in GF formulations . MoreoveIn particular, the NL and ECL pizza samples were able to provide >12% of the energy value by adding proteins and could, therefore, be labeled with the nutritional claim “a source of proteins” according to the Regulation (EC) No. 1924/06 . MoreoveThe raffinose family, oligosaccharides (RFOs), or α-galactosides, were also determined , becauseAs for bioactive compounds , high lep < 0.05) less pliable; therefore, it required a higher force to be bent  decrease in L* and an increase in a* and b*, compared with the control pizza. This result was due to the color of the lentil flour used: the NL flour was reddish and the ECL flour was amber yellow , the ECL pizza showed a perceptible difference in color, but this difference was even greater between the control pizza and the NL pizza, as can be observed in Baking involves physical changes in the bakery products, related to the increase in volume which, in turn, is due to the heat-induced expansion of the gases incorporated into the dough during the kneading and fermentation phases. This increase in volume resulted in an associated increase in the thickness of pizza samples (up to 111.1%), and a moderate decrease in diameter (10–13%). Similar values were observed in pizza prepared from blends of wheat and chickpea flour . The incAs for the sensorial profile , the addThe control pizza and the ECL pizza did not show significant differences in consistency, being both moderately soft, whereas the NL pizza was significantly harder and less compressible. NL pizza was also less pliable and more rigid than the control and ECL pizzas, which appeared more elastic and pliable. The control and ECL pizzas did not show significant differences between them, again in agreement with the instrumental determinations, highlighting the positive effect of ECL flour on the mechanical properties of pizza.Flavor notes, namely, saltiness and sweetness, instead, were not influenced by the addition of lentil flours. The lentil flavor was moderately perceived in both the NL and ECL pizzas, without significant differences between them. Therefore, in an overall sensory comparison with the control, the ECL pizza differed substantially only in the intensity of the lentil odor and flavor, and in the color.The consumer test indicated that no one expressed a dislike towards the three GF pizza types, highlighting that even the qualitative level of the NL pizza, despite being lower than the control and ECL pizzas, was not too low. The three types of pizza, therefore, were liked by all consumers, but to a different extent. The liked level of the NL pizza was lower than the control pizza, while between the ECL and control pizzas, there were no significant differences. The lower liked level of the NL pizza was probably imputable to its unusual orange color, harder consistency, and reduced pliability.The obtained results showed that lentil flour can be added to GF pizza with interesting nutritional and qualitative results, particularly if extruded flours are used. In fact, the use of ECL flour, containing pregelatinized starch, made it possible to substitute HPMC, obtaining a “clean label” product. Compared to the control, the ECL pizza differed substantially only in the intensity of the lentil odor and flavor, and in color, but with no significant differences in the liked level. These findings are of great significance for people suffering from celiac disease, because the quality defects of GF pizza may contribute to an occasional lack of adherence to a strict GF diet, which is relatively frequent in adults. Moreover, a high content of bioactive compounds, coupled with a high protein content, makes the ECL pizza a potentially functional food product.Pizza has spread all over the world and has adapted to the most diverse cultures and tastes, and it is still far from stopping in its path. The addition of extruded lentil flour is another example of its possible evolution.


In Arabidopsis thaliana, the PDI-S subgroup contains only one member, AtPDI11, with an a-a′-D organization, which has no orthologs in mammals or yeast. However, the expression pattern of AtPDI11 and the functioning mechanism of AtPDI11 D domain are currently unclear. In this work, we found that PDI-S is evolutionarily conserved between land plants and algal organisms. AtPDI11 is expressed in various tissues and its induction by ER stress is disrupted in bzip28/60 and ire1a/b mutants that are null mutants of key components in the unfolded protein response (UPR) signal transduction pathway, suggesting that the induction of AtPDI11 by ER stress is mediated by the UPR signaling pathway. Furthermore, enzymatic activity assays and genetic evidence showed that the D domain is crucially important for the activities of AtPDI11. Overall, this work will help to further understand the working mechanism of AtPDI11 in catalyzing disulfide formation in plants.The formation and isomerization of disulfide bonds mediated by protein disulfide isomerase (PDI) in the endoplasmic reticulum (ER) is of fundamental importance in eukaryotes. Canonical PDI structure comprises four domains with the order of  Most newly synthesized secretory and membrane proteins are folded in the endoplasmic reticulum (ER), in which an oxidative environment facilitates the formation of intramolecular disulfide bonds . In eukabinding protein (BIP), calnexin (CNX), and PDI, so as to facilitate protein folding and alleviate ER stress [Once the demand for protein folding exceeds the capacity of the ER, especially under adverse environmental conditions, the unfold protein response (UPR) signaling pathways will be activated ,5,6. In R stress ,8.a-b-b′-a′, with two a-type thioredoxin domains containing an active site motif -CGHC- and two b-type thioredoxin domains lacking an active site motif [a-b-b′-a′ domain organization of canonical PDI [0a-a-b domain arrangement, which is required for pollen viability and normal exine formation in plants subjected to heat stress [PDI is a multifunctional enzyme as it can catalyze free-thiol oxidation reaction and reduction or isomerization of disulfide bonds depending on their redox states . Canonicte motif ,11,12. Ite motif ,14. Six ical PDI ,16,17,18ical PDI . It is nical PDI . PDI-M st stress . Althougt stress ,22,23. Rt stress ,25. A sit stress .a-a′-D domain arrangement [D domain is analogous to the C-terminal domain of human ERp29 which has a conserved 5-α-helix hydrophobic region serving as a protein binding site [AtPDI11, was significantly upregulated by ER stresses [a and a′ domains [D domain of AtPDI11 interacts with calreticulin 1 (CRT1) and calreticulin 2 (CRT2) and probably assists with the folding of glycoproteins in the ER [pdi1, pdi2, pdi5, pdi6, pdi9, pdi10, and pdi11), only pdi11 single mutants displayed obviously growth inhibition under reducing treatment [AtPDI11 expression remains unclear and its working mechanism is not fully understood.PDI-S is a unique plant subgroup with angement . The D ding site . In Arabstresses . The expstresses . GmPDIS- domains . The D dn the ER . Among treatment . All of AtPDI11 transcripts and its induction under ER stress were analyzed, and the function of D domain of AtPDI11 was studied. Our work provides a basis for further investigation on AtPDI11 function.In this work, we reported the evolutionary relationships of the PDI-S subgroup across Algae, Mosses, Gymnosperms, and Angiosperms. Tissue-specific expression of Chlorella variabilis, Coccomyxa subellipsoidea, Chlamydomonas reinhardtii, and Volvox carteri f. nagariensis. Such phylogenetic analyses are not surprising given the most ancient ancestor in the analysis. In addition, land plants possess an arrangement with a-a′-D, and algal organisms share an a-D domain arrangement, which is probably due to duplication of single domains during the evolutionary process. In sessile organism-formed clades, the moss and Fern AtPDI11 homolog (Selaginella moellendorffii) also clade separately in an outgroup on their own branch. After these outgroups at the base of the tree, the angiosperms branch off into their separate clades of monocotyledons  and dicotyledons , clearly separated from each other. Interestingly, dicotyledon Arabidopsis thaliana falls into the monocotyledon clade. Furthermore, amino acid sequence alignment showed that the active sites in two catalytic Trx domains are very well conserved in sessile organisms. We also found a conserved CysX6Cys motif between two type-a domains in various species except algae   B. In Araisulfide .AtPDI11, a transgenic plant with β-glucuronidase (GUS) gene driven by the promoter of AtPDI11  was generated. As shown in ProPDI11 was active in all tissues of one- or two-day-old plants, especially in cotyledons, hypocotyls, and root apex , and true leaf (two-week-old plant)  C–E. AtPDd plants F,G. It hn pollen K. Meanwh stamens L,M. TakeAtPDI genes was induced by UPR [AtPDI11 is changed when plants suffer from different degrees of ER stress. As shown in AtBIP3 transcripts measured by reverse-transcription PCR (RT–PCR) significantly increased under ER stress inducers Dithiothreitol (DTT) or Tunicamycin (Tm) treatment. Similarly, the transcript level of AtPDI11 upon activation of the UPR was markedly higher than control. In order to more intuitively show the expression trend of AtPDI11 under different DTT/Tm concentration stress, we conducted a more thorough quantitative RT-PCR (qRT–PCR) analyses of AtPDI11 expression. Similar with RT-PCR, the expression level of AtBIP3 and AtPDI11 gradually increased under DTT or Tm treatment in a dose-dependent manner. It should be noted that the expression of AtPDI11 under 10 mM DTT treatment decreased to the level observed under 0.1 mM DTT, and the AtBIP3 transcript level also decline to some extent. This could be because ER stress caused by DTT treatment exceeded the tolerance of plants  were dramatically inhibited compared to wild-type, especially in 2.0 mM DTT (pdi11 mutant lines and transgenic complementation lines (Com) were significantly inhibited in 50 ng/mL or 75 mg/mL, and positive control bzip28/60 was more sensitive to Tm treatment compared with wild-type Col-0. It is worth noting that the extent of both pdi11-1 and pdi11-2 growth inhibition was similar to that of Col-0   RNase A system, where AtPDIs relay the disulfide bonds from AtERO1 to Dred RNase A in vitro. The oxidative folding of Dred RNase A protein can be evaluated by examining changes of molecular weight (MV), as blocking of free thiols of Dred RNase A by 4-acetamido-4′-maleimidylstilbene -2, 2′-disulfonic acid (AMS) results in increases in its MV by 0.5 kDa per thiol residue [D was also able to catalyze the conversion of Dred RNase A into Pox and Fox states, the catalytic efficiency of AtPDI11ΔD was much lower than that of PDI11 (D domain seriously affects the activity of PDI11 to oxidize the substrates in vitro.AtPDI11 is the only member of the PDI family that possess a D domain . However domain) . To exam residue . As showof PDI11 A. This wof PDI11 B,C. ThesD domain genetically, we transformed pdi11-2 mutant plants with a construct containing DPDI11Δ driven by the native promoter of AtPDI11. As shown in pdi11-2, Com, and DAtPDI11Δ/pdi11-2 under normal growth conditions. When grown on medium containing 2 mM DTT, the Com plants displayed a similar growth phenotype to Col-0 plants. However, the expression of AtPDI11ΔD only partially rescued the growth inhibition of pdi11-2 under reducing conditions. Taken together, these results suggest that the D domain is required for AtPDI11′s function in vivo under reducing conditions.To further confirm the function of the a-b-b′-a′ arrangement, with an a-a′-D domain arrangement and lacking b′ domain, is a unique PDI [Dictyostelium discoideum [In eukaryotic cells, the ERO1-PDI protein folding pathway acts as an important oxidative protein folding system. PDI-S subgroup isoform, unlike canonical PDI sharing ique PDI ,30. It hscoideum ,30,35. HAtPDI11 was upregulated under different concentrations of DTT and Tm  to higher plants (gymnosperms and angiosperms) . In ArabT and Tm . The ind pathway . Both ox AtPDI11 . These r AtPDI11 . Recentlefolding . These rAtPDI11 gene was induced by ER stress [AtBIP3 is significantly lower in bzip28/60 in normal conditions in our work. This result is in line with previous reports that AtBIP1 was lower expression level in bzip28/60 than that in wild-type plants [AtBIP3 transcription predominately relies on bZIP28 and bZIP60. IRE1 is a transmembrane ER stress sensor. It was demonstrated in yeast and animals that BIP can bind to the luminal domain of IRE1 under normal conditions [AtBIP3 expression is higher in ire1a/b than that in wild type. However, according to Ruberti et al., there was no significant difference in the expression of AtBIP3 between ire1a/b mutant and wild-type plants [It was reported that the transcription of R stress . In thise plants  and suggnditions . In our e plants . This neb′xa′ fragment of HsPDI serves as the minimal element for its binding to HsEro1α [a-a′ region, but not D domain of AtPDI11, provides the binding site for AtERO1 [D domain is necessary for the ability of AtPDI11 to catalyze oxidative protein folding  fused in frame with 57 residue C-terminal amino acids of Dd-PDI was also localized to the ER [D domain in the localization of the ER [D driven by its native promoter largely rescued the growth inhibition of the pdi11 mutant under reducing conditions , pdi11-2 (SALK_148421), and proPDI111::PDI11-FLAG/pdi11-1 were reported previously [ire1a/b was generated by crossing ire1a-2 and ire1b-4 single mutant [w/v) sucrose, 0.8% (w/v) agar, and 2.5 mM MES at pH 5.7) with or without different concentration of DTT or Tm. Before moving to the growth chamber, the plates were first kept at 4 °C for 2 days to break dormancy. The plants eviously . ire1a/be mutant . All plaE.coli, His-FLAG-AtPDI11, DHis-FLAG-AtPDI11Δ , and GST-AtERO1 were generated as described previously [proPDI11::PDI11-FLAG was generated as described previously, and D-FLAGproPDI11::PDI11Δ was generated using the same method and vector. For GUS histochemical assay, 2.0 kb upstream of the start codon for AtPDI11 was cloned into the pCAMBIA1391 binary plasmid vector fused with the β-glucuronidase (GUS) reporter gene at the BamHI and HindIII sites.For the expression of recombinant proteins in eviously . proPDI1D proteins were expressed in E. coli BL21 (DE3) cells by the addition of 0.5 mM isopropyl thiogalactoside at 25 °C for 10 h and purified using Ni-NTA agarose  and following the manufacturer’s instruction. The recombinant GST-AtERO1 proteins were expressed in E. coli 21 (DE3) cells and purified as previously described [The recombinant AtPDI11 and AtPDI11Δescribed . Then GSescribed ,26.Phylogenetic Analysis and Sequence Alignment was performed as described previously .® Green qPCR SuperMix . AtGAPC was used for internal reference genes [Seven-day-old seedlings were treated with 2 mM DTT or 5 μg/mL Tm for 5 h. Total RNA of seedlings was isolated using TransZol  following the manufacturer’s instructions. In total, 1 μg DNase-treated total RNA was used for synthesis of first-strand cDNA in 20 μL reactions. qRT-PCR was performed on Bio-Rad CFX96 Real-Time PCR system using a PerfectStartce genes . The prid-glucuronide , and 0.1% (v/v) Triton X-100) at 37 °C in the dark for 1 h . Chlorophyll was removed by submerging samples in anhydrous ethanol two times and in 70% (v/v) ethanol three times. The samples were photographed using a Nikon AZ100 5X microscope.Histochemical staining for GUS activity was performed as described previously . Plants The oxygen consumption assay was performed as described previously . Oxygen D, 3 μM AtERO1, 100 μM FAD, and 8 μM denatured and reduced RNase A were freshly mixed in buffer B. At various time points, the same volume of sample from the reaction mixture was quenched in addition of 5 x SDS loading buffer with 10 mM AMS  and separated by 15% non-reducing SDS-PAGE. The proteins were stained with Coomassie Blue.Gel-based RNase A reoxidation analyses were performed as described previously . In tota